EZH2-HSP90 dual inhibitor

Abstract

This disclosure relates to a first-in-class EZH2-HSP90 dual inhibitor, a method for preparing the same, and its use in the treatment of diseases / disorders mediated by EZH2, HSP90, or both, particularly glioblastoma.

Description

[Technical Field]

[0001] <Priority Information> This application claims the interests and priority of U.S. Provisional Patent Application No. 63 / 501,939, filed on 12 May 2023, entitled “First-in-Class EZH2-HSP90 Dual Inhibitor,” the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] This disclosure relates to EZH2-HSP90 inhibitors, methods for preparing the same, and their use in the treatment of diseases / disorders mediated by EZH2, HSP90, and / or both, particularly glioblastoma.

[0003] Glioblastoma (GBM), also known as grade IV astrocytoma, is characterized by a genetically unstable and highly invasive population of cells with high invasiveness or invasiveness. GBM is considered the most destructive intracranial cancer and is classified into two types: primary (or de novo) GBM (the most common and aggressive form) and secondary GBM (rare and less aggressive). GBM tumors primarily arise from abnormal astrocytes, are overwhelmingly found in the frontal lobe, and can metastasize to other parts of the brain via the ventricular system or corpus callosum, with sporadic cases of spinal cord metastasis also reported.

[0004] Surgical debulking of the tumor with radiotherapy and chemotherapy is the currently recommended multimodal approach to treating GBM. However, this trimodal therapy has not achieved the expected survival benefits.

[0005] Of particular note is the limited number of available anti-GBM chemotherapy options, which has hindered GBM treatment. Factors that impede the clinical success of currently recommended chemotherapy regimens include spontaneous resistance to temozolomide (an oral alkylating agent, the only first-line drug for GBM) via methylguanine DNA methyltransferase, cytological heterogeneity of GBM, and the presence of highly tumorigenic cell populations (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. Drug delivery strategies to enhance the permeability of the blood-brain barrier for treatment of glioma. Drug Des. Devel. Ther. 2015, 9, 2089). [Overview of the project]

[0006] This disclosure provides an EZH2-HSP90 dual inhibitor for the prevention and / or treatment of diseases / disorders mediated by EZH2, HSP90, and / or both (such as glioblastoma).

[0007] In one embodiment, the present disclosure relates to a compound of formula (I), The present invention provides JPEG2026516102000002.jpg87170 or its pharmaceutically acceptable salts, hydrates, solvates, prodrugs, or isomers. In the formula, L is a linker selected from the group consisting of -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, A and B are independently selected from the group consisting of direct bonds, C1-C3 alkyl, and C1-C3 alkoxy. R is selected from the group consisting of H, linear or branched C1-C6 alkyl, linear or branched C1-C6 alkoxy, C6-C 10 aryl and C5-C 10 heteroaryl, Y is selected from the group consisting of O, S and N, R 1 and R 2 are independently selected from H, halo, C1-C6 alkyl and C 16 -C 20 (poly)unsaturated fatty acid, alkyl and alkoxyl may be optionally substituted with halogen, hydroxy, amino or C6-C 10 aryl or C5-C 10 heteroaryl, aryl and heteroaryl may be optionally substituted with halogen, hydroxy, amino, C1-C3 alkyl or C1-C3 alkoxy.

[0008] In some embodiments, L is -A-NR-C(=Y)-B- and -A-C(=Y)-NR-B-, Y is O, S or N, A and B are independently selected from the group consisting of direct bond, C1-C3 alkyl and C1-C3 alkoxy, and R is H, linear or branched C1-C6 alkyl, linear or branched C1-C6 alkoxy, C6-C 10 aryl and C5-C 10 heteroaryl. In some embodiments, C 16 -C 20 (poly)unsaturated fatty acid is α-linolenic acid, stearidonic acid, eicosapentaenoic acid, cervonic acid, linoleic acid, linoleic elaidic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, oleic acid, elaidic acid, erucic acid, nervonic acid, mead acid or paulic acid; preferably including but not limited to oleic acid and linolenic acid (such as α- or γ-linolenic acid). In some further embodiments, L and C 16 -C 20 (poly)unsaturated fatty acid are the species of L and C 16 -C 20It is any combination of (polyunsaturated) fatty acids.

[0009] In some embodiments, L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, R is H, R 1 and R 2 H is H.

[0010] In some embodiments, L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, R is H, R 1 and R 2 These are, independently, oleic acid and linolenic acid (such as α- or γ-linolenic acid).

[0011] In some embodiments, L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, and R is a linear or branched C1-C6 alkyl, R 1 and R 2 These are H.

[0012] In some embodiments, L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, and R is a linear or branched C1-C6 alkyl, R 1 and R 2 These are, independently, oleic acid and linolenic acid (such as α- or γ-linolenic acid).

[0013] In some embodiments, L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, and R is C6-C 10 It is an aryl (such as phenyl or benzyl), R 1 and R 2 These are H.

[0014] In some embodiments, L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, and R is C6-C 10 It is an aryl (such as phenyl or benzyl), R 1 and R 2 These are, independently, oleic acid and linolenic acid (such as α- or γ-linolenic acid).

[0015] In some embodiments, L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, and R is C6-C 10 Linear or branched C1-C6 alkyl groups optionally substituted with aryl groups, R 1 and R 2 These are H.

[0016] In some embodiments, L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, and R is C6-C 10 Linear or branched C1-C6 alkyl groups optionally substituted with aryl groups, R 1 and R 2 These are, independently, oleic acid and linolenic acid (such as α- or γ-linolenic acid).

[0017] In one embodiment, the present disclosure relates to a compound of formula (Ia) or (Ib), The following are provided: JPEG2026516102000003.jpg80170, JPEG2026516102000004.jpg80170, or pharmaceutically acceptable salts, hydrates, solvates, prodrugs, or isomers thereof. During the ceremony, Y is either O or S, R is H, a linear or branched C1-C4 alkyl group, phenyl, or benzyl. R 1 and R 2 Each of these is independently H, halo, C1-C6 alkyl, and C 16 -C 20(Polyunsaturated) fatty acids, C 16 -C 20 Selected from (polyunsaturated) fatty acids, preferably oleic acid and alpha-linolenic acid, Optionally, linear or branched C1-C4 alkyl groups may be substituted with halogens, hydroxyls, or aminos. Optionally, phenyl or benzyl may be substituted with a C1-C2 alkyl, halogen, hydroxyl, or amino group.

[0018] In one embodiment, the disclosure also relates to compounds of formula (Ia-p) or (Ib-p), The following are provided: JPEG2026516102000005.jpg83170, JPEG2026516102000006.jpg81170, or pharmaceutically acceptable salts, hydrates, solvates, prodrugs, or isomers thereof. During the ceremony, Y is either O or S, R is H, a linear or branched C1-C4 alkyl group, phenyl, or benzyl. R 1 and R 2 These are independently H, halo, C1-C6 alkyl and C 16 -C 20 (Polyunsaturated) fatty acids, preferably selected from oleic acid and alpha-linolenic acid, Optionally, linear or branched C1-C4 alkyl groups may be substituted with halogens, hydroxyls, or aminos. Optionally, phenyl or benzyl may be substituted with a C1-C2 alkyl, halogen, hydroxyl, or amino group.

[0019] In one embodiment, the disclosure also relates to compounds of formula (Ia-m) or (Ib-m), The following are provided: JPEG2026516102000007.jpg84170, JPEG2026516102000008.jpg82170, or pharmaceutically acceptable salts, hydrates, solvates, prodrugs, or isomers thereof. During the ceremony, Y is either O or S, R is H, a linear or branched C1-C4 alkyl group, phenyl, or benzyl. R 1 and R 2 These are independently H, halo, C1-C6 alkyl and C 16 -C 20 (Poly)unsaturated fatty acids, preferably selected from oleic acid and alpha-linolenic acid, Optionally, linear or branched C1-C4 alkyl groups may be substituted with halogens, hydroxyls, or aminos. Optionally, phenyl or benzyl may be substituted with a C1-C2 alkyl, halogen, hydroxyl, or amino group.

[0020] In some embodiments of any formula described herein, Y is O, and R is H, a linear or branched C1-C4 alkyl, phenyl, or benzyl.

[0021] In some embodiments of any formula described herein, Y is O, R is H, a linear or branched C1-C4 alkyl, phenyl or benzyl, and R 1 and R 2 Each of the following is H. The linear or branched C1-C4 alkyl is optionally substituted with a halogen, hydroxyl, amino, or phenyl. Phenyl or benzyl as R is optionally substituted with a C1-C2 alkyl, halogen, hydroxyl, or amino. In some embodiments of any formula described herein, Y is O, R is H, linear or branched C1-C4 alkyl, phenyl, or benzyl, and R 1 and R 2 Independently, C 16 -C 20 Selected from (polyunsaturated) fatty acids. Linear or branched C1-C4 alkyl groups are optionally substituted with halogens, hydroxyl groups, amino groups, or phenyl groups. Phenyl or benzyl as R is optionally substituted with C1-C2 alkyl groups, halogens, hydroxyl groups, or amino groups. In some further embodiments of any formula described herein, Y is O, R is H, linear or branched C1-C4 alkyl group, phenyl group, or benzyl group, and R 1 and R 2Each of these is H. Linear or branched C1-C4 alkyl groups are optionally substituted with halogens, hydroxyl groups, or amino groups. Phenyl or benzyl groups are optionally substituted with C1-C2 alkyl groups, halogens, hydroxyl groups, or amino groups. In some further embodiments of any formula described herein, Y is O, R is H, linear or branched C1-C4 alkyl group, phenyl or benzyl group, and R 1 and R 2 The following are independently selected from linolenic acid (such as α- or γ-linolenic acid), stearidonic acid, eicosapentaenoic acid, cervicic acid, linoleic acid, linole-elaidic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, oleic acid, elaidic acid, erucic acid, nervic acid, meadic acid, or pauric acid, preferably selected from oleic acid and α-linolenic acid. Linear or branched C1-C4 alkyl groups are optionally substituted with halogens, hydroxyl groups, or amino groups. Phenyl or benzyl groups are optionally substituted with C1-C2 alkyl groups, halogens, hydroxyl groups, or amino groups. In some further embodiments of any formula described herein, Y, R, R 1 and R 2 This is any combination of the species listed above.

[0022] In some embodiments of any formula described herein, Y is S and R is H, a linear or branched C1-C4 alkyl, phenyl, or benzyl.

[0023] In some embodiments of any formula described herein, Y is S, R is H, a linear or branched C1-C4 alkyl, phenyl, or benzyl, 1 and R 2 Each of these is H. Linear or branched C1-C4 alkyl groups are optionally substituted with halogens, hydroxyl groups, or amino groups. Phenyl or benzyl groups are optionally substituted with C1-C2 alkyl groups, halogens, hydroxyl groups, or amino groups. In some embodiments of any formula described herein, Y is S, R is H, linear or branched C1-C4 alkyl group, phenyl or benzyl group, and R 1 and R 2 Independently, C 16 -C20 Selected from (polyunsaturated) fatty acids. Linear or branched C1-C4 alkyls are optionally substituted with halogens, hydroxy, or aminos. Phenyl or benzyl is optionally substituted with C1-C2 alkyls, halogens, hydroxy, or aminos. In some further embodiments of any formula described herein, Y is S, R is H, linear or branched C1-C4 alkyl, phenyl, or benzyl, and R 1 and R 2 Each of the following is H. Phenyl or benzyl is optionally substituted with a C1-C2 alkyl, halogen, hydroxyl or amino. In some further embodiments of any formula described herein, Y is S, R is H, linear or branched C1-C4 alkyl, phenyl or benzyl, and R 1 and R 2 The following are independently selected from linolenic acid (such as α- or γ-linolenic acid), stearidonic acid, eicosapentaenoic acid, cervicic acid, linoleic acid, linole-elaidic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, oleic acid, elaidic acid, erucic acid, nervic acid, meadic acid, or pauric acid, preferably selected from oleic acid and α-linolenic acid. Linear or branched C1-C4 alkyl groups are optionally substituted with halogens, hydroxyl groups, or amino groups. Phenyl or benzyl groups are optionally substituted with C1-C2 alkyl groups, halogens, hydroxyl groups, or amino groups. In some further embodiments of any formula described herein, Y, R, R 1 and R 2 This is any combination of the species listed above.

[0024] This disclosure also provides methods for preparing compounds of formula (I), formula (Ia), formula (Ib), formula (Ia-p), formula (Ib-p), formula (Ia-m), or formula (Ib-m).

[0025] Specific examples of compounds described herein are: JPEG2026516102000009.jpg83170JPEG2026516102000010.jpg81170JPEG2026516102000011.jpg88170JPEG2 026516102000012.jpg80170JPEG2026516102000013.jpg88170JPEG2026516102000014.jpg88170JPEG2026516 102000015.jpg85170JPEG2026516102000016.jpg84170JPEG2026516102000017.jpg90170JPEG2026516102000018.jpg72170JPEG2026516102000019.jpg80170 or its pharmaceutically acceptable salts, hydrates, solvates, prodrugs or isomers, including but not limited to these.

[0026] This disclosure provides pharmaceutical compositions / formulations comprising the compounds described herein and pharmaceutically acceptable carriers.

[0027] This disclosure also provides a method for treating diseases or disorders mediated by EZH2 and / or HSP90. This method comprises administering a therapeutically effective amount of a compound of formula (I), formula (Ia), formula (Ib), formula (Ia-p), formula (Ib-p), formula (Ia-m), or formula (Ib-m), or any pharmaceutically acceptable salt, hydrate, solvate, or prodrug, to a subject particularly in need thereof. This disclosure also provides a pharmaceutical composition / formulation for use in a method for treating diseases or disorders mediated by EZH2 and / or HSP90. This pharmaceutical composition / formulation comprises a compound of formula (I), formula (Ia), formula (Ib), formula (Ia-p), formula (Ib-p), formula (Ia-m), or formula (Ib-m), or any pharmaceutically acceptable salt, hydrate, solvate, or prodrug.

[0028] In one embodiment, the disease or disorder mediated by EZH2 and / or HSP90 is brain cancer.

[0029] In further embodiments, brain cancer is a primary brain tumor or metastatic brain cancer. In some embodiments, brain cancer is a glioma or meningioma.

[0030] In a further embodiment, the brain cancer is glioblastoma (GBM). [Brief explanation of the drawing]

[0031] [Figure 1] This exhibits EZH2 inhibitory activity that is anti-GBM. [Figure 2] This shows molecular docking based on the structure of compound 7. [Figure 3] This study demonstrates the effect of Epz-6438-derived inhibitors on the viability of Pt3R cells. [Figure 4A-4C] This study demonstrates the effect of compound 7 on the gene expression profile in TMZ-resistant Pt3R cells. [Figures 5A-5E] This shows that compound 7 suppresses the gene expression of the CENP family. [Figure 6A-6H] This shows that compound 7 suppresses DNA repair-related genes. [Figure 7A-7C] This shows that compound 7 induces ROS production from mitochondria. [Figure 8A-8B] This demonstrates the effect of compound 7 on the proliferation of in vivo TMZ-resistant Pt3R cells. [Modes for carrying out the invention]

[0032] To facilitate understanding of the disclosures herein, the terms used herein are defined below.

[0033] In the context of the specification and claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise specified. Unless otherwise specified, any examples or illustrative expressions provided herein (e.g., "etc.") are not intended to limit the scope of the invention and are used solely to more clearly illustrate the invention.

[0034] It should be understood that any numerical range listed herein is intended to include all partial ranges contained therein. For example, the range "50-70°C" includes all partial ranges between the stated minimum value of 50°C and the stated maximum value of 70°C, as well as specific values ​​such as 58°C-67°C, 53°C-62°C, 60°C, or 68°C. Since the disclosed numerical ranges are continuous, they include each value between the minimum and maximum values. Unless otherwise specified, the various numerical ranges shown herein are approximate values.

[0035] The term "approximately" refers to the acceptable deviation of a given value measured by a person skilled in the art, depending in part on how that value is measured or determined.

[0036] As used herein, the term "hydrocarbyl" refers to a monovalent radical derived from a hydrocarbon. As used herein, the term "hydrocarbon" refers to a molecule consisting only of carbon and hydrogen atoms. Examples of hydrocarbons include, but are not limited to, (cyclo)alkanes, (cyclo)alkenes, alkadienes, and aromatics. If the hydrocarbyl is further substituted as described above, the substituents may be halogens, amino groups, hydroxyl groups, thiol groups, etc. If the hydrocarbyl is interrupted by a heteroatom as described above, the heteroatom may be S, O, or N. In the present invention, the hydrocarbyl preferably contains 1 to 30 carbon atoms.

[0037] The term "alkyl" refers to a saturated linear or branched alkyl group, preferably containing 1 to 10 carbon atoms, more preferably 1 to 4 carbon atoms. Examples of alkyl groups 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, and others.

[0038] As used herein, the terms "alkoxyl" or "alkoxy" mean a group having the formula "-O-alkyl," and the definition of "alkyl" in the formula is as defined above.

[0039] As used herein, the term "cycloalkyl" means a saturated or partially unsaturated cyclic carbon radical containing 3 to 10 cyclic carbon atoms, more preferably 3 to 8 cyclic carbon atoms, and optionally an alkyl substituent on the ring. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclopropenyl, cyclobutyl, cyclopentyl, cyclohexyl, and 2-cyclohexen-1-yl.

[0040] As used herein, the term "aryl" 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 multiple fused rings. Examples of aryls include, but are not limited to, phenyl, benzyl, tolyl, xylyl, indenyl, naphthyl, mesityrenyl, and durenyl. Preferably, the aryl is phenyl or benzyl, more preferably benzyl. As used herein, the term "heteroaryl" means a radical comprising at least one aromatic moiety having at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, preferably having 5 to 10 carbon atoms. Examples of heteroaryl compounds include, but are not limited to, furanyl, pyrrolyl, diazolyl (e.g., imidazolyl, pyrazolyl), thiophenyl, pyranyl, pyridinyl, diazinyl (e.g., pyridazinyl, pyrimidinyl, pyrazinyl), oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, isobenzofuranyl, quinolinyl, isoquinolinyl, indazolyl, indolyl, isoindolyl, etc.

[0041] The terms "halogen" or "halo" refer to fluorine, chlorine, bromine, or iodine.

[0042] As used herein, the term "amino" refers to formula -NR a R b It refers to the functional group, and in the formula, R a and R b Each of these independently represents either a hydrogen atom or a hydrocarbyl group as defined above.

[0043] As used herein, the term "(polyunsaturated) fatty acid" means a fatty acid that contains at least one double bond in its main chain. 16 -C 20Examples of (polyunsaturated) fatty acids include, but are not limited to, hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA), stearidonic acid (SDA), linoleic acid (LA), gamma-linolenic acid (GLA), oleic acid, rumenic acid, alpha-calendic acid, beta-calendic acid, jacalic acid, alpha-eleostearic acid, beta-eleostearic acid, catalpic acid, punicic acid, rumelenic acid, alpha-parinalic acid, beta-parinalic acid, pinolenic acid, eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA), eicosenoic acid, and meadic acid. In certain embodiments, C 16 -C 20 (Polyunsaturated) fatty acids are selected from oleic acid and linolenic acid.

[0044] The term "pharmaceutically acceptable salt" refers to a salt or zwitterion of a medicinal compound that is soluble or dispersible in water or oil, suitable for the treatment of a disorder, and effective for its intended use. These salts can be prepared, for example, during the final isolation and purification of the compound, or separately by reacting the amino group of the compound with a suitable acid.

[0045] As used herein, the term “treatment” includes a medical condition, disease, disorder, process, situation, or event such as diabetes, or the alleviation, prevention, reversal, improvement, or control of the symptoms of such a medical condition, disease, disorder, process, situation, or event.

[0046] The term "prodrug" refers to a compound that is converted into a pharmaceutical compound in the body, for example, by hydrolysis in the blood. The term "prodrug" refers to a compound that contains, but is not limited to, substituents known as "therapeutably appropriate esters." In certain embodiments, a "prodrug" is a compound that is modified with a (polyunsaturated) fatty acid moiety in its structure, or has a (polyunsaturated) fatty acid moiety, for example, R 1 and / or R 2 C 16 -C20 This concerns compounds that are (poly)unsaturated fatty acids.

[0047] Numerous studies have shown that overexpression of EZH2 in cancer stem cells of malignant tumors plays a crucial role in the proliferation and maintenance of these stem cells (Wen, Y.; Cai, J.; Hou, Y.; Huang, Z.; Wang, Z. Role of EZH2 in cancer stem cells: from biological insight to a therapeutic target. Oncotarget 2017, 8 (23), 37974; Zhang, H.; Qi, J.; Reyes, JM; Li, L.; Rao, PK; Li, F.; Lin, CY; Perry, JA; Lawlor, MA; Federation, A. Oncogenic Deregulation of EZH2 as an Opportunity for Targeted Therapy in Lung Cancer; An Oncogenic Role for EZH2 in Lung Cancer. Cancer Discov. 2016, 6 (9), 1006-1021).EZH2, the most important subunit of the Polycomb Repressive Complex (PRC2), is responsible for the methylation (monomethylation, dimethylation, and trimethylation) of lysine 27 of histone H3 (H3K27), and H3K27me3 is more frequently associated with transcriptional repression (Zhuang, S. Histone methyltransferase EZH2: a potential therapeutic target for kidney diseases. Front. Physiol. 2021, 12, 640700; Hill, MA; Kollak, I.; Keck, M.; Schroeder, V.; Wirth, J.; Skronska‐Wasek, W.; Schruf, E.; Strobel, B.; Stahl, H. An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodeling after injury. EMBO reports 2021, 22 (8)). e52785). As a result, several important findings have been obtained. For example, i) the involvement of the miR-206 / Twist axis in malignant tumors of GBM cells regulated by EZH2, ii) the mediation of E2's oncogenic effects (proliferation, migration, and invasion) in GBM cells by EZH2, and iii) the direct transcriptional regulation of c-myc by EZH2 leading to the maintenance of GSCs.

[0048] Despite the well-established role of EZH2 in various malignancies, only one EZH2 inhibitor (tazemetostat) was approved by the FDA in 2020 for the treatment of epithelioid sarcoma and follicular lymphoma (Huang, N.; Liao, P.; Zuo, Y.; Zhang, L.; Jiang, R. Design, Synthesis, and Biological Evaluation of a Potent Dual EZH2-BRD4 Inhibitor for the Treatment of Some Solid Tumors. J. Med. Chem. 2023).

[0049] This disclosure aims to overcome the pharmacodynamic and physicochemical shortcomings associated with HDAC inhibitors by modifying a histone deacetylase (HDAC) inhibitory structural template to elicit an anti-GBM effect (Hsu, C.-C.; Chang, W.-C.; Hsu, T.-I.; Liu, J.-J.; Yeh, S.-H.; Wang, J.-Y.; Liou, J.-P.; Ko, C.-Y.; Chang, K.-Y.; Chuang, J.-Y. Suberoylanilide hydroxamic acid represses glioma stem-like cells. J. Biomed. Sci. 2016, 23 (1), 1-12; Nepali, K.; Wu, A.-C.; Lo, W.-L.; Chopra, B.; Lai, M.-J.; Chuang, J.-Y.; Liou, J.-P. Rationally designed donepezil-based Hydroxamates modulate Sig-1R and HDAC isoforms to exert anti-glioblastoma effects. Eur. J. Med. Chem. 2023, 115054). Therefore, this disclosure relates to EZH2 as an epigenetic target and a candidate for constructing novel anti-GBM chemical structures. Tracking the progress of cocktails of EZH2 inhibitors in GBM at the clinical level has not provided or revealed a starting point for selecting alternative targets. Therefore, this disclosure aims to “run a campaign” to evaluate the anticancer effects of cocktails of epigenetic inhibitors and drugs with different mechanisms of action. Notably, this campaign will support the design of dual-target adducts based on the demonstrated cell proliferation inhibitory effects of the evaluated combinations.

[0050] Fortunately, the results of this campaign drew the inventors' attention to the remarkable and noteworthy cell proliferation inhibitory effect achieved against GBM cell lines by the combination of tazemetosat (an EZH2 inhibitor) and STA9090 (an HSP90 inhibitor), which initiated this initiative. The tendency for tazemetosat to not exhibit cytotoxicity to GBM cell lines was interesting and appealing. Despite this unfortunate tendency observed with tazemetosat, the precedent evidence (preclinical studies) of eliciting the anti-GBM effect of tazemetosat via co-administration with a PI3K inhibitor provided sufficient motivation to adopt a strategy of designing a bifunctional EZH2 inhibitor adduct (dual inhibitor). This approach is based on the expectation that, as with combination therapy, simulation tuning of additional targets along with EZH2 via the dual inhibitor may activate the chemical structure of tazemetosat to exert an anti-GBM effect.

[0051] Unlike tazemetostat, STA9090 as an individual component of the cocktail showed moderate cytotoxicity to GBM cell lines. However, its cell proliferation inhibitory effect was several times weaker compared to that observed with the cocktail. Thus, Hsp90 is an ATP-dependent molecular chaperone that regulates the conformation, stability, and degradation of proteins. Several HSP90 inhibitors were evaluated at a preliminary level for their anti-GBM effects. For example, the resorcinol-based HSP90 inhibitor AUY922 showed a significant and impressive anti-GBM effect, inducing GBM cell death via apoptosis and autophagy. Treatment with AUY922 also resulted in decreased mRNA and protein expression of EGFR, PDGFRA, CDK4, and NF1 in heterologous GBM cells. Another HSP90 inhibitor, YZ129, promoted apoptosis, halted the GBM cell cycle at the G2 / M phase, and inhibited tumor cell proliferation and migration. Furthermore, NW457, a pochoxime-based HSP90 inhibitor with a favorable neuropharmacokinetic profile, demonstrated the ability to improve treatment outcomes of fractionated CBCT-based irradiation in an orthotopic homogeneous GBM mouse model. Another study reported that BIIB021, an HSP90 inhibitor, may overcome resistance to molecularly targeted therapy in brafv600e mutant GBM.

[0052] Therefore, this disclosure assumes that inhibition of EZH2 and HSP90 may act through unique pathways involved in the observed antiproliferative effects on GBM cell lines. However, rather than presenting speculation about biochemical correlations between the two targets, in line with the current perspective of medicinal chemists, this disclosure plans to highlight the positive aspects of a preliminary work program and to verify preliminary evidence through the design of an EZH2-HSP90 dual inhibitor chemoprobe.

[0053] This disclosure generally relates to small molecule compounds of EZH2-HSP90 biinhibitors and their effective therapeutic use. These compounds can be administered to treat / control / alleviate cancer, preferably brain cancer, disease and condition.

[0054] Embodiments of the compounds are described in the summary section of the invention, and methods for their preparation are shown in the examples. Those skilled in the art can prepare the compounds according to the teachings in the examples.

[0055] The compounds of this disclosure can be prepared as pharmaceutical compositions or formulations for administration to subjects for the treatment of cancers such as brain cancer. Examples of brain cancers include, but are not limited to, primary or metastatic brain cancer, glioma, meningioma, and glioblastoma. In one embodiment, the pharmaceutical composition of the invention comprises a second anticancer agent.

[0056] While the compounds of this disclosure may be administered as raw chemical substances, they may also be presented as pharmaceutical compositions or formulations. Accordingly, this disclosure provides pharmaceutical formulations or compositions comprising a compound or a pharmaceutically acceptable salt thereof, a prodrug, or a solvate thereof, together with one or more pharmaceutically acceptable carriers and optionally one or more other therapeutic components. The carrier must be “acceptable” in the sense that it is compatible with the other components of the composition or formulation and is not harmful to its recipient. The appropriate composition / formulation depends on the chosen route of administration. The composition / formulation may take the form of a tablet, pill, capsule, semi-solid, powder, sustained-release formulation, solution, suspension, elixir, aerosol, or any other appropriate composition, and may contain at least one compound of the present invention in combination with at least one pharmaceutically acceptable excipient. Suitable excipients are well known to those skilled in the art, and methods for formulating these excipients and compositions / formulations can be found in standard references such as Remington: The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, Pa. Suitable liquid carriers include water, aqueous saline solution, aqueous dextrose solution, and glycol, particularly for injectable solutions. The pharmaceutical compositions / formulations of this disclosure can be manufactured by methods known in themselves, such as conventional mixing, dissolution, granulation, sugar-coated tablet manufacturing, wet grinding, emulsification, encapsulation, encapsulation, or compression processes.

[0057] The most appropriate route may depend, for example, on the recipient's condition and disability, but compositions / formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intra-articular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal, and topical (including cutaneous, buccal, sublingual, and intraocular) administration. Oral administration is the preferred route. Compositions / formulations are conveniently provided in unit dosage forms and can be prepared by any method well known in the field of pharmacy. All methods involve the step of binding the compound of the present invention or a pharmaceutically acceptable salt, prodrug, or solvate thereof ("active ingredient") to a carrier constituting one or more auxiliary components. Generally, compositions / formulations are prepared by uniformly and tightly binding the active ingredient to a liquid carrier or a pulverized solid carrier or both, and then, if necessary, shaping the product into the desired composition / formulation.

[0058] For oral administration, suitable pharmaceutical compositions / formulations of this 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 contain antioxidants, flavoring agents, preservatives, suspending agents, thickeners and emulsifiers, colorants, flavoring agents and other pharmaceutically acceptable additives. Formulations for oral administration may be formulated to be immediate-release or enhanced-release. Enhanced-release includes delayed-release, sustained-release, pulsed-release, controlled-release, targeted-release and programmed-release.

[0059] For parenteral administration, the compounds or compositions / formulations of this disclosure are administered directly into the bloodstream, muscle, or viscera via intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, or other injection or infusion. Parenteral formulations may be prepared as aqueous injection solutions that, in addition to the compounds of the present invention, may contain buffers, antioxidants, bacteriostatic agents, salts, carbohydrates, and other additives commonly used in such solutions. Parenteral administration may be immediate-release or modified-release (e.g., injection or implantation depot).

[0060] The compounds or compositions / formulations of the present disclosure can also be administered topically to the skin or mucosa, into the skin (intradermally), or transdermally. Typical formulations include gels, hydrogels, lotions, solutions, creams, ointments, bandages, foams, skin patches, wafers, implants, and microemulsions. The compounds or compositions / formulations of the present invention can also be administered by inhalation or intranasally, for example, as dry powders, aerosol sprays, or drops. Further routes of administration of the compounds of the present invention include intravaginally and rectally (by suppositories, pessaries, or enemas), eyes, and ears.

[0061] The following examples are provided to make the present disclosure more understandable to those skilled in the art to which the present disclosure pertains, but are not intended to limit the scope of the present invention.

Examples

[0062] <Experiments and Materials>

[0063] Chemical nuclear magnetic resonance ( 1 1H NMR and 13 13C NMR) spectra were measured using a Bruker DRX-500 spectrometer operating at 300 MHz. Chemical shifts are reported in parts per million (ppm, δ) from tetramethylsilane (TMS) as an internal standard to lower magnetic fields. High-resolution mass spectra (HRMS) were measured using a JEOL (JMS-700) electron impact (EI) mass spectrometer. The purity of the final compound was determined using a Hitachi 2000 series HPLC system using a C-18 column (Agilent ZORBAX Eclipse XDB-C18 5 mm, 4.6 mm × 150 mm). Column chromatography was performed using silica gel (Merck Kieselgel 60, No. 9385, 230 - 400 mesh ASTM). All reactions were carried out under an atmosphere of dry nitrogen. 36> <EZH2 Inhibition Assay>

[0065] The EZH2 inhibitory activity of the synthesized adducts was evaluated by Malvern, PA, Reaction Biology Corporation (http: / / www.reactionbiology.com).

[0066] <In vitro Hsp90 assay>

[0067] The HSP90 inhibitory activity of the synthesized adducts was evaluated by Reaction Biology Corporation, Malvern, PA (http: / / www.reactionbiology.com).

[0068] <Cell culture>

[0069] Pt3 GBM cells were isolated from male GBM patients, as described in the previous study. The temozolomide (TMZ) resistance phenotype of Pt3R cells has already been validated. Pt3 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 μg / ml streptomycin, and 100 μg / ml penicillin G. Pt3R cells were cultured in the presence of 100 μM TMZ (MilliporeSigma Corporate, St. Louis, MO, USA).

[0070] <Cell viability CCK8 assay>

[0071] The CCK8 reagent was purchased from TargetMOI (Wellesley Hills, MA, USA) and used according to the manufacturer's instructions.

[0072] <Molecular docking>

[0073] To screen MPT1A059 based on binding energy, structure-based molecular docking was performed. The docking process was carried out between the protein and the compound using iGEMDOCK version 2.1 with a population size of 1500, a generation number of 150, and a solution number of 10. The protein structures of HSP90 and EZH2 were obtained from the Protein Data Bank (8AGI and 4W2R).

[0074] <RNA-seq and Proteomics Assays>

[0075] RNA was isolated using an RNA extraction kit (Zymo Research, Irvine, CA, USA) and subjected to RNA-seq by BIOTOOLS Co., Ltd (New Taipei City, Taiwan). The cell pellet was collected and subjected to proteomics analysis by BIOTOOLS Co., Ltd. RNA seq was used to compare the gene expression of Pt3 and Pt3R. This result has already been reported

[60] .

[0076] <ROS Assay and MitoSOX Assay>

[0077] Dihydroethidium (DHR; Thermo Fisher Scientific, Waltham, MA, USA) was used to label cellular ROS detected by flow cytometry

[54] , and the MitoSOX reagent (Thermo Fisher Scientific) was used to label mitochondrial ROS

[55] .

[0078] <Western Blotting>

[0079] Details of the procedure have been described in previous studies [54, 55, 58, 59]. The primary antibodies, anti-CENPA, anti-CENPE, anti-CENPF, and anti-CENPI antibodies, were purchased from abcam (Cambridge, UK). The anti-CDK1 and anti-cyclin B1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The anti-EZH2 and anti-H3K27 me3 antibodies were purchased from GeneTex International Corporation (Hsinchu, Taiwan).

[0080] <Bioinformatics analysis>

[0081] Expression and survival plots were generated using the websites of Gepia and GlioVis.

[0082] <Animal experiments>

[0083] NOD.CB17-Prkdc scid / NCrCrl mice (8 weeks old) were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). Pt3R cells (1x10 6 ) in 50 ml of DMEM were injected into the back of the mice. Ten days later, the tumors became palpable. The mice were injected with DMSO (control group) or MPT1A059 (5 mg / kg) twice a week.

[0084] <Statistical analysis>

[0085] The experiments were independently performed three times, and the data were shown as mean ± s.e.m. A P value < 0.05 was determined to be significant.

[0086] <Synthesis example 1>

[0087] Scheme 1 shown below is a synthesis example of the compounds disclosed in this specification. JPEG2026516102000020.jpg181170

[0088] This pathway began with the high-yield esterification of 5-bromo-2-methyl-3-nitrobenzoic acid 11 to produce ester 12. Next, the aromatic ester containing a nitro group was subjected to Fe / NH4Cl-mediated reduction to obtain primary amine 13. This was then subjected to continuous reductive amination, first producing intermediate 14 containing pyran, and then tertiary amine 15 containing an N-ethyl substituent. Suzuki arylation of intermediate 15 with (4-nitrophenyl)boronic acid and (3-nitrophenyl)boronic acid using an organopalladium catalyst produced biphenyl skeletons 16 and 17. Fe / NH4Cl-mediated nitro reduction converted the nitro-containing biphenyls 16 and (17) into biphenyls containing an amino group, 18 and 19. Biphenyls 18 and 19 were further amidated with 2,4-dihydroxy-5-isopropylbenzoic acid using a carbodiimide-mediated method to obtain intermediates 20 and 21. Next, lithium hydroxide was used to hydrolyze the ester groups located on the trisubstituted phenyl rings of biphenyls 20 and 21 to produce carboxylic acids 22 and 23. Subsequently, 3-(aminomethyl)-4,6-dimethylpyridine-2(1H)-one was used as the amine, and these acids were subjected to EDC / HOBt-assisted amidation to obtain intermediates 24 and 25. Next, in a hydrogenation vessel, intermediates 24 and 25 were debenzylated with 10% Pd / C in methanol at 40-42 psi of H2 to obtain hybrid skeletons 1-2.

[0089] In particular, the reagents and conditions used in Scheme 1 are as follows: a) CH3I, K2CO3, DMF, room temperature, overnight; b) Fe, NH4Cl, EtOH:H2O (9:1), 100°C, 2 hours; c) Tetrahydro-4H-pyran-4-one, CH3COOH, NaBH3CN, MeOH, reflux, overnight; d) Acetaldehyde, CH3COOH, Na(CH3COO)3BH, DCE, room temperature, 5 hours; e) 4-nitrophenylboronic acid or 3-nitrophenylboronic acid, Pd(PPh3)3, Na2CO3, Dioxane:Water (9:1), 60℃, 2 hours; f) Fe, NH4Cl, EtOH:H2O (9:1), 100℃, 2 hours, g) 2,4-bis(benzyloxy)-5-isopropylbenzoic acid, EDC, HCl, HOBT, DIPEA, DMF, room temperature, 3 hours; h) LiOH (aq), dioxane, room temperature, 3 hours; i) 3-(aminomethyl)-4,6-dimethylpyridine-2(1H)-one, EDC, HCl, HOBT, DIPEA, DMF, room temperature, 3 hours; j) Pd / C, H2, MeOH, room temperature, 4 hours.

[0090] 5-Bromo-2-methyl-3-nitrobenzoate methyl(12)

[0091] In a 250 ml round-bottom flask (RBF), compound 11 (5 gm, 0.019 mol) and potassium carbonate (5.31 gm, 0.038 mol) were dissolved in 50 ml of DMF, and iodomethane (3.54 gm, 0.024 mol) was added dropwise to the reaction mixture. The resulting mixture was stirred overnight at room temperature, and the progress of the reaction was monitored using TLC. After the reaction was complete, 100 ml of cold water was added, and the resulting precipitate was filtered by suction, washed with water, and dried. The precipitate was not further purified and proceeded to the next step. The yield was 90%. 1 H NMR (300 MHz, DMSO-d6): δ 7.31(s, 1H), 7.27 (s, 1H), 3.87 (s, 3H), 2.26 (s, 3H).

[0092] 3-amino-5-bromo-2-methylbenzoate methyl(13)

[0093] Compound 12 (5 gm, 0.020 mol), iron powder (5.69 gm, 0.102 mol), and ammonium chloride (2.17 gm, 0.040 mol) were weighed and placed in 100 ml of RBF, and a mixture of ethanol and water (18:2 ml) was added as the solvent. The reaction mixture was refluxed at 100°C for 2 hours, and the progress of the reaction was monitored using TLC. After the reaction was complete, the mixture was diluted with methanol and passed through Celite. The solvent was evaporated under vacuum, and the residue was purified by silica gel column chromatography (ethyl acetate:hexane:1:5). The yield was 91%. 1 H NMR (300 MHz, CDCl3): δ 7.29 (d, J = 2.4 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 3.88 (s, 3H), 3.80 (s, 2H), 2.24 (s, 3H).

[0094] 5-Bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl)amino)methyl benzoate (14)

[0095] In 100 ml of RBF, compound 13 (5 gm, 0.020 mol) and tetrahydro-4H-pyran-4-one (4.10 gm, 0.040 mol) were dissolved in 100 ml of methanol, and acetic acid (1.22 gm, 0.020 mol) was added. The mixture was stirred at room temperature for 30 minutes, and then sodium borohydride cyanohydride (3.83 gm, 0.60 mol) was added in small amounts. The reaction was refluxed overnight, and the progress of the reaction was monitored using TLC. After the reaction was complete, the resulting precipitate was filtered by suction, washed with methanol, and dried. The compound was proceeded to the next step without further purification. The yield was 88%. 1 H NMR (300 MHz, CDCl3): δ 7.23 (d, J = 1.2 Hz, 1H), 6.85 (s, 1H), 3.97-4.03 (m, 2H), 3.86 (s, 3H), 3.46-3.56 (m, 3H), 2.23 (s, 3H) 2.03 (d, J = 11.4 Hz, 2H), 1.48-1.54 (m, 2H).

[0096] 5-Bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate methyl(15)

[0097] In 100 ml of RBF, compound 14 (3 gm, 0.009 mol), acetaldehyde (0.8 gm, 0.018 mol), and acetic acid (0.54 gm, 0.009 mol) were dissolved in 60 ml of dichloroethane. The reaction was stirred at room temperature for 30 minutes, and then sodium triacetoxyborohydride (5.72 gm, 0.027 mol) was added in small amounts under ice-cold conditions. The reaction was continued at room temperature for 5 hours. After the reaction was complete (TLC), the reaction mixture was diluted with cold water (100 ml), and the compounds were 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 silica gel column chromatography (ethyl acetate:hexane: 1:5) with a yield of 90%. 1 H NMR (300 MHz, CDCl3): δ 7.73 (d, J = 1.8 Hz, 1H), 7.38 (d, J = 1.8 Hz, 1H), 3.94-4.00 (m, 2H), 3.91 (s, 3H), 3.30-3.38 (m, 2H), 3.04-3.11 (m, 2H), 2.81-3.00 (m, 1H), 2.47 (s, 3H), 1.63-1.74 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H).

[0098] 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-nitro-[1,1'-biphenyl]-3-carboxylate methyl(16)

[0099] In 100 mL of RBF, compound 15 (4 g, 0.011 mol), the corresponding 4-nitrophenylboronic acid (2.24 g, 0.015 mol), palladium triphenylphosphine (1.15 g, 0.001 mol), and sodium carbonate (3.49 g, 0.033 mol) were dissolved in 40 mL of DMF, and the reaction was refluxed at 100 °C for 4 hours. After completion of the reaction (TLC), the reaction mixture was diluted with water (100 mL), and the compound was extracted using ethyl acetate (50 mL × 3). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was further purified by silica gel column chromatography (ethyl acetate:hexane:1:3), and the yield was 71%. 1 H NMR (300 MHz, DMSO-d6): δ 8.31 (d, J = 7.2 Hz, 2H), 7.85 (s, 1H), 7.74 (d, J = 7.2 Hz, 2H), 7.52 (s, 1H), 3.93 - 3.95 (m, 5H), 3.02 - 3.39 (m, 5H), 2.57 (s, 3H), 1.69 - 1.74 (m, 4H), 0.95 (t, J = 6.9 Hz, 3H).

[0100] Methyl 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-3'-nitro-[1,1'-biphenyl]-3-carboxylate (17)

[0101] Intermediate 17 was synthesized in 70% yield using the same experimental procedure as described for the synthesis of compound 16. 1H NMR (300 MHz, CDCl3): δ 8.41 (t, J = 2.1 Hz, 1H), 8.21-8.25 (m, 1H), 7.91-7.93 (m, 1H), 7.86 (d, J = 1.8 Hz, 1H), 7.65 (t, J = 8.1 Hz, 1H), 7.53 (d, J = 1.8 Hz, 1H), 4.02 (s, 1H), 3.97 (s, 4H), 3.33-3.41 (m, 2H), 3.15-3.22 (m, 2H), 2.85-3.10 (m, 1H), 2.60 (s, 3H), 1.69-1.76 (m, 4H), 0.95 (t, J = 7.2 Hz, 3H).

[0102] 4'-amino-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(18)

[0103] Intermediate 16 (1 gm, 0.0025 mol), iron powder (0.69 gm, 0.012 mol), and ammonium chloride (0.267 gm, 0.005 mol) were weighed into a 100 ml round-bottom flask (RBF), and a mixture of ethanol and water (27:3 ml) was added as the solvent. The reaction mixture was refluxed at 100°C for 2 hours, and the progress of the reaction was monitored using TLC. After the reaction was complete, the reaction mixture was diluted with methanol and passed through Celite. The resulting solvent mixture was evaporated under vacuum, and the residue was purified by silica gel column chromatography (ethyl acetate:hexane:1:4). The yield was 80%. 1 H NMR (300 MHz, CD3OD): δ 7.22 - 7.26 (m, 3H), 7.08 (d, J = 1.5 Hz, 1H), 6.61 (d, J = 8.5 Hz, 2H), 3.91 - 3.94 (m, 5H), 3.06 - 3.34 (m, 5H), 2.54 (s, 3H), 1.63 - 1.69 (m, 4H), 0.93 (t, J = 6.9 Hz, 3H).

[0104] 3'-amino-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(19)

[0105] Using the same experimental procedure as described for the synthesis of compound 18, intermediate 19 was synthesized in 74% yield, with intermediate 17 as the starting material. 1 H NMR (300 MHz, CDCl3): δ 7.83 (d, J = 2.1 Hz, 1H), 7.51 (s, 1H), 7.24-7.30 (m, 1H), 6.99-7.02 (m, 1H), 6.93 (t, J = 1.8 Hz, 1H), 6.72-6.76 (m, 1H), 3.95-4.02 (m, 2H), 3.94 (s, 3H), 3.32-3.40 (m, 2H), 3.17 (d, J = 6.9 Hz, 2H), 3.13 (s, 1H), 2.58 (s, 3H), 1.69-1.77 (m, 4H), 0.91-0.96 (m, 3H).

[0106] 4'-(2,4-bis(benzyloxy)-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(20)

[0107] In 100 ml of RBF, the corresponding intermediate 18 (3.2 gm, 0.0086 mol), 2,4-bis(benzyloxy)-5-isopropylbenzoic acid (3.27 gm, 0.0086 mol), EDC.HCl (3.3 gm, 0.017 mol), and HOBT (1.7 gm, 0.013 mol) were dissolved in 20 ml of DMF and DIPEA (2.7 gm, 0.022 mol) and added dropwise to the reaction mixture. The reaction mixture was allowed to continue for 3 hours, and the progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was diluted with water (100 ml), and the compounds were 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 silica gel column chromatography (ethyl acetate:hexane:1:3). The yield was 65%. 1 H NMR (300 MHz, CDCl3): δ 7.72 (s, 1H), 7. 21 - 7.45 (m, 16H), 6.62 (s, 1H), 5.19 (s, 4H), 4.15 (m, 2H), 3.96 (s, 3H), 3.08 - 3.31 (m, 5H), 3.29 (m, 1H), 2.54 (s, 3H), 1.61 - 1.67 (m, 4H), 1.26 (d, J = 7.2 Hz, 6H), 0.95 (t, J = 6.9 Hz, 3H).

[0108] 3'-(2,4-bis(benzyloxy)-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(21)

[0109] Using the same experimental procedure described for the synthesis of compound 20, intermediate 21 was synthesized in 70% yield, with intermediate 19 as the starting material. 1H NMR (300 MHz, CDCl3): δ 9.97 (s, 1H), 8.24 (s, 1H), 7.72 (s, 1H), 7.19-7.53 (m, 15H), 6.67 (s, 1H), 5.20 (s, 2H), 5.18 (s, 2H), 3.96-4.15 (m, 5H), 3.33-3.41 (m, 3H), 3.14 (s, 3H), 2.59 (s, 3H), 1.72-1.76 (m, 4H), 1.27-1.32 (m, 6H), 0.91-0.96 (m, 3H).

[0110] 4'-(2,4-bis(benzyloxy)-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid(22)

[0111] Intermediate 20 (1 gm, 0.0014 mol) was dissolved in 20 ml of dioxane, and lithium hydroxide (0.29 gm, 0.0068 eq) was slowly added to the reaction mixture. The reaction was continued for 3 hours, and the progress of the reaction was monitored by TLC. After the reaction was complete, the pH was adjusted to 5 using 3N HCl, and the resulting precipitate was filtered, washed, and dried. The resulting acid was proceeded to the next step without further purification. The yield was 80%.

[0112] 3'-(2,4-bis(benzyloxy)-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid(23)

[0113] Intermediate 23 was synthesized from starting material 21 in 91% yield in the same manner as described for the synthesis of intermediate 22. The resulting acid was not further purified before proceeding to the next step.

[0114] 4'-(2,4-bis(benzyloxy)-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(24)

[0115] Intermediate 22 (1 gm, 0.0014 mol), 3-(aminomethyl)-4,6-dimethylpyridine-2(1H)-one (0.21 gm, 0.0014 mol), EDC.HCl (0.53 gm, 0.0028 mol), and HOBT (0.28 gm, 0.0021 mol) were dissolved in 10 ml of DMF, and DIPEA (0.45 gm, 0.0035 mol) was added dropwise to the reaction mixture. The reaction mixture was allowed to continue for 3 hours, and the progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was diluted with water (100 ml), and the compounds were 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 silica gel column chromatography (ethyl acetate:hexane:1:2) with a yield of 74%. 1 H NMR (300 MHz, CD3OD): δ 7.79 (s, 1H), 7. 09 - 7.38 (m, 16H), 6.62 (s, 1H), 6.11 (s, 1H), 5.19 (s, 4H), 4.49 (s, 2H), 4.15 (m, 2H), 3.08 - 3.31 (m, 5H), 3.29 (s, 1H), 2.53 (s, 3H), 2.33 (s, 3H), 2.28 (s, 3H), 1.61 - 1.67 (m, 4H), 1.26 (d, J = 7.2 Hz, 6H), 0.95 (t, J = 6.9 Hz, 3H).

[0116] 3'-(2,4-bis(benzyloxy)-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(25)

[0117] Intermediate 25 was synthesized from starting material 23 in 82% yield in the same manner as described for the synthesis of intermediate 23. 1 H NMR (300 MHz, CD3OD): δ 7.98 (s, 1H), 7.79 (s, 1H), 7.34 - 7.65 (m, 15 H), 6.54 (s, 1H), 6.17 (s, 1H), 5.11 (s, 2H), 5.07 (s, 2H), 4.66 (s, 2H), 3.95 (bs, 2H), 3.12- 3.33 (m, 6H), 2.51 (s, 3H), 2.35 (s, 3H), 2.26 (s, 3H), 1.59-1.76 (m, 4H), 1.29 (d, J = 6.6 Hz, 6H), 0.97 (t, J = 7.2 Hz, 3H).

[0118] 4'-(2,4-dihydroxy-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(1)

[0119] In 100 ml of RBF, intermediate 24 (0.5 gm, 0.0006 mol) was dissolved in 20 ml of methanol, and the catalyst Pd / C was added. The reaction mixture was allowed to continue under hydrogen gas conditions at room temperature for 4 hours, and the progress of the reaction was monitored using TLC. After the reaction was complete, the mixture was diluted with methanol and passed through Celite. The solvent was evaporated under vacuum, and the residue was purified by silica gel column chromatography (ethyl acetate:hexane:1:1). The yield was 50%. HPLC purity: 98.73%; mp: 166~168°C; 11H NMR (600 MHz, CD3OD): δ 7.82 (s, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 9 Hz, 2H), 7.41 (d, J = 1.2 Hz, 1H), 7.23 (d, J = 1.8 Hz, 1H), 6.34 (s, 1H), 6.10 (s, 1H), 4.49 (s, 2H), 3.90 (d, J = 10.8 Hz, 2H), 3.45 - 3.50 (m, 1H), 3.32 - 3.62 (m, 2H), 3.19 - 3.24 (m, 1H), 3.10 - 3.14 (m, 2H), 2.42 (s, 3H), 2.30 (s, 3H), 2.25 (s, 3H), 1.73 (d, J = 12 Hz, 2H), 1.58 - 1.65 (m, 2H), 1.26 (d, J = 7.2 Hz, 6H), 0.87 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CD3OD): δ 168.23, 160.13, 159.49, 152.05, 149.21, 143.46, 138.92, 137.98, 137.42, 136.04, 132.84, 127.32, 126.53, 126.09, 123.60, 121.69, 120.65, 109.64, 107.43, 102.24, 71.79, 70.64, 66.85, 60.82, 58.45, 41.78, 35.38, 31.45, 30.47, 26.56, 21.68, 18.87, 18.33, 17.18, 13.78, 12.81, 11.73. C 39 H 47 N4O6(M + H + ) HRMS (ESI): Calculated value; 667.3496, Measured value, 667.3474(M + H + ).

[0120] 3'-(2,4-dihydroxy-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(2)

[0121] Title compound 2 was synthesized from starting material 25 in 62% yield, in the same manner as described for the synthesis of title compound 1. HPLC purity: 97.25%; mp: 170~172℃; 1 H NMR (600 MHz, CD3OD): δ 7.89 (s, 1H), 7.76 (s, 1H), 7.58 (dd, J = 8.4 and 1.2 Hz, 1H), 7.48 (d, J = 1.8 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.34 (m, 2H), 6.34 (s, 1H), 6.09 (s, 1H), 4.48 (s, 2H), 3.90 (d, J = 10.2 Hz, 2H), 3.33-3.36 (m, 2H), 3.09-3.21 (m, 4H), 2.38 (s, 3H), 2.32 (s, 3H), 2.22 (s, 3H), 1.74 (d, J = 11.4 Hz, 2H), 1.59-1.66 (m, 2H), 1.24 (d, J = 6.6 Hz, 6H), 0.89 (t, J = 7.2 Hz, 3H). 13 C NMR (150 MHz, CD3OD): δ 171.65, 168.42, 164.21, 160.16, 159.54, 152.20, 149.33, 143.43, 140.88, 139.05, 138.68, 138.38, 133.19, 128.91, 127.28, 126.12, 123.99, 122.42, 121.32, 120.86, 120.36, 119.68, 109.67, 107.54, 102.31, 66.84, 58.36, 41.80, 35.19, 30.50, 26.61, 21.64, 18.30, 17.16, 13.69, 11.71.C 39 H47 N4O6[M + H + HRMS(ESI): Calculated value: 667.3496; Measured value: 667.3489.

[0122] <Synthesis Example 2>

[0123] Scheme 2, shown below, is another example of the synthesis of the compounds disclosed herein. JPEG2026516102000021.jpg106170JPEG2026516102000022.jpg117170

[0124] This synthetic route can be used for target skeletons 3-10. Adducts 20 and 21 were used as general starting materials to obtain N-substituted hybrid templates 3-10. N-alkylation / benzylation of intermediates 20 and 21 using alkyl iodide and benzyl bromide was achieved by utilizing 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 carboxylic acids 34-41, which were then amidated with 3-(aminomethyl)-4,6-dimethylpyridine-2(1H)-one. To obtain the desired hybrids 3-5 and 7-9, amides 42-44 and 46-48 were debenzylated using 10% Pd / C. Notably, debenzylation of intermediates 45 and 49 was attempted via a palladium-mediated hydrogenation protocol, but this method proved to be too unstable. Interestingly, competition between N-debenzylation and O-debenzylation was observed even at room temperature (not shown in the scheme). Therefore, we optimized different strategies for selectively debenzylating the O-benzyl group and used BCl3 to achieve such selectivity. Fortunately, both the palladium-based and BCl3-based methods yielded satisfactory yields of the target compound.

[0125] In particular, the reagents and conditions used in Scheme 2 were as follows: a) Alkyl iodide and benzyl bromide, NaH, DMF, room temperature, 4 hours; b) LiOH(aq), dioxane, room temperature, 3 hours; c) 3-(aminomethyl)-4,6-dimethylpyridine-2(1H)-one, EDC.HCl, HOBT, DIPEA, DMF, room temperature, 3 hours; d) For 42-44 and 46-48, Pd / C, H2, MeOH, room temperature, 4 hours; for 45 and 49, BCl3, DCM, room temperature, 3 hours.

[0126] 4'-(2,4-bis(benzyloxy)-5-isopropyl-N-methylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(26)

[0127] In 100 ml of RBF, intermediate 20 (6 gm, 0.0082 mol) and methyl iodide (3.49 gm, 0.025 eq) were dissolved in 30 ml of DMF, and sodium hydride (0.39 gm, 0.016 eq) was added in small increments. The reaction was stirred under nitrogen conditions at room temperature for 4 hours, and the progress of the reaction was monitored using TLC. After the reaction was complete, the reaction mixture was diluted with water (100 ml), and the compounds were 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 silica gel column chromatography (ethyl acetate:hexane:1:1). The yield was 51%. 1 H NMR (300 MHz, CD3OD): δ 7.75 (s, 1H), 7. 29 - 7.38 (m, 16H), 6.29 (s, 1H), 5.19 (s, 2H), 5.16 (s, 2H), 4.18 (m, 2H), 3.91 (s, 3H), 3.41 (s, 3H), 3.29 (s, 1H), 3.11-3.21 (m, 5H), 2.51 (s, 3H), 1.58 - 1.63 (m, 4H), 1.29 (d, J = 7.2 Hz, 6H), 0.92 (t, J = 6.9 Hz, 3H).

[0128] 4'-(2,4-bis(benzyloxy)-N-ethyl-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(27)

[0129] Intermediate 27 was synthesized in 69% yield from starting material 20 using ethyl iodide in the same manner as described for compound 26. 1 H NMR (300 MHz, CD3OD): δ 7.68 (s, 1H), 7. 32 - 7.39 (m, 16H), 6.59 (s, 1H), 5.11 (s, 2H), 5.09 (s, 2H), 3.98 (m, 4H), 3.87 (s, 3H), 3.11 - 3.27 (m, 6H), 2.46 (s, 3H), 1.58 - 1.63 (m, 4H), 1.29 (d, J = 7.2 Hz, 6H), 1.21 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H).

[0130] 4'-(2,4-bis(benzyloxy)-5-isopropyl-N-propylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(28)

[0131] Intermediate 28 was synthesized from starting material 20 in 58% yield using propyl iodide, in the same manner as described for compound 26. 1H NMR (300 MHz, CD3OD): δ 7.59 (s, 1H), 7. 32 - 7.39 (m, 16H), 6.64 (s, 1H), 5.14 (s, 2H), 5.11 (s, 2H), 3.88 - 3.92 (m, 4H), 3.89 (s, 3H), 3.17 - 3.22 (m, 6H), 2.41 (s, 3H), 1.47 - 1.62 (m, 6H), 1.29 (d, J = 7.2 Hz, 6H), 1.18 (t, J = 7.2 Hz, 3H), 0.94 (t, J = 6.9 Hz, 3H).

[0132] '-(N-benzyl-2,4-bis(benzyloxy)-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(29)

[0133] Intermediate 29 was synthesized from starting material 20 in 65% yield using benzyl iodide, in the same manner as described for compound 26. 1 H NMR (300 MHz, CD3OD): δ 7.52 (s, 1H), 7. 24 - 7.41 (m, 21H), 6.62 (s, 1H), 5.11 (s, 2H), 5.08 (s, 2H), 5.04 (s, 2H), 3.93 (m, 2H), 3.91 (s, 3H), 3.11 - 3.20 (m, 6H), 2.39 (s, 3H), 1.43 - 1.57 (m, 4H), 1.29 (d, J = 7.2 Hz, 6H), 0.94 (t, J = 6.9 Hz, 3H).

[0134] 3'-(2,4-bis(benzyloxy)-5-isopropyl-N-methylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(30)

[0135] Intermediate 30 was synthesized from starting material 21 in 70% yield in the same manner as described for the synthesis of compound 26. 1 H NMR (300 MHz, CDCl3): δ 7.46 (s, 1H), 7.13-7.28 (m, 14H), 6.90 - 6.96 (m, 2H), 6.20 (s, 1H), 4.83 (s, 2H), 4.79 (s, 2H), 3.85 (m, 2H), 3.81 (s, 3H), 3.38 (s, 3H), 2.88 - 3.20 (m, 6H), 2.42 (s, 3H), 1.55-1.67 (m, 4H), 0.95 (d, J = 5.7 Hz, 6H), 0.88 (t, J = 3.6 Hz, 3H).

[0136] 3'-(2,4-bis(benzyloxy)-N-ethyl-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(31)

[0137] Intermediate 31 was synthesized from starting material 21 in 74% yield using ethyl iodide, in the same manner as described for the synthesis of compound 26. 1 H NMR (300 MHz, CDCl3): δ 7.49 (s, 1H), 7.17-7.22 (m, 14H), 6.93 - 6.95 (m, 2H), 6.16 (s, 1H), 4.87 (s, 2H), 4.71 (s, 2H), 3.98 (m, 4H), 3.91 (s, 3H), 3.15 - 3.32 (m, 6H), 2.42 (s, 3H), 1.51 - 1.68 (m, 4H), 1.32 (d, J = 7.2 Hz, 6H), 1.34 (t, J = 7.2 Hz, 3H), 0.91 (t, J = 6.9 Hz, 3H).

[0138] 3'-(2,4-bis(benzyloxy)-5-isopropyl-N-propylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(32)

[0139] Intermediate 32 was synthesized from starting material 21 in 62% yield using propyl iodide, in the same manner as described for the synthesis of compound 26. 1 H NMR (300 MHz, CDCl3): δ 7.44 (s, 1H), 7.19-7.25 (m, 14H), 6.97 - 6.99 (m, 2H), 6.21 (s, 1H), 4.97 (s, 2H), 4.95 (s, 2H), 3.95 - 3. 97 (m, 4H), 3.89 (s, 3H), 3.21 - 3.29 (m, 6H), 2.49 (s, 3H), 1.52 - 1.67 (m, 6H), 1.31 (d, J = 7.2 Hz, 6H), 1.21 (t, J = 7.2 Hz, 3H), 0.97 (t, J = 6.9 Hz, 3H).

[0140] 3'-(N-benzyl-2,4-bis(benzyloxy)-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylate methyl(33)

[0141] Intermediate 33 was synthesized from starting material 21 in 69% yield using benzyl bromide, in the same manner as described for compound 26. 1H NMR (300 MHz, CDCl3): δ 7.42 (s, 1H), 7.21-7.28 (m, 19H), 6.98 - 7.01 (m, 2H), 6.25 (s, 1H), 5.01 (s, 2H), 4.98 (s, 2H), 4.95 (s, 2H), 3. 99 (m, 2H), 3.89 (s, 3H), 3.18 - 3.29 (m, 6H), 2.39 (s, 3H), 1.49 - 1.51 (m, 4H), 1.32 (d, J = 7.2 Hz, 6H), 0.98 (t, J = 6.9Hz, 3H).

[0142] 4'-(2,4-bis(benzyloxy)-5-isopropyl-N-methylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid(34)

[0143] Intermediate 26 (6 gm, 0.0081 mol) was dissolved in 30 ml of dioxane, and lithium hydroxide solution (1.66 gm, 0.04 mol) was added dropwise to the reaction mixture. The reaction was continued at room temperature for 3 hours, and the progress of the reaction was monitored using TLC. After the reaction was complete, the pH was adjusted to 5 with 3N HCl, and the resulting precipitate was filtered, washed, and dried. The resulting acid was proceeded to the next step without further purification. The yield was 79%.

[0144] 4'-(2,4-bis(benzyloxy)-N-ethyl-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid(35)

[0145] Intermediate 35 was synthesized from starting material 27 in 56% yield in the same manner as described for compound 34. The carboxylic acid was used in the next step without purification.

[0146] 4'-(2,4-bis(benzyloxy)-5-isopropyl-N-propylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid(36)

[0147] Intermediate 36 was synthesized from the starting material 28% in yield 49% in the same manner as described for compound 34. The carboxylic acid was used in the next step without purification.

[0148] 4'-(N-benzyl-2,4-bis(benzyloxy)-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid(37)

[0149] Intermediate 37 was synthesized from starting material 29 in 57% yield in the same manner as described for compound 34. The carboxylic acid was used in the next step without purification.

[0150] 3'-(2,4-bis(benzyloxy)-5-isopropyl-N-methylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid(38)

[0151] Intermediate 38 was synthesized from starting material 30 in 48% yield in the same manner as described for compound 34. The carboxylic acid was used in the next step without purification.

[0152] 3'-(2,4-bis(benzyloxy)-N-ethyl-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid (39)

[0153] Intermediate 39 was synthesized from starting material 31 in 71% yield in the same manner as described for compound 34. The carboxylic acid was used in the next step without purification.

[0154] 3'-(2,4-bis(benzyloxy)-5-isopropyl-N-propylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid(40)

[0155] Intermediate 40 was synthesized from starting material 32 in 43% yield in the same manner as described for compound 34. The carboxylic acid was used in the next step without purification.

[0156] 3'-(N-benzyl-2,4-bis(benzyloxy)-5-isopropylbenzamide)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxylic acid (41)

[0157] Intermediate 41 was synthesized from starting material 33 in 55% yield in the same manner as described for compound 34. The carboxylic acid was used in the next step without purification.

[0158] 4'-(2,4-bis(benzyloxy)-5-isopropyl-N-methylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(42)

[0159] In 100 ml of RBF, intermediate 34 (5.3 gm, 0.0073 mol), 3-(aminomethyl)-4,6-dimethylpyridine-2(1H)-one (1.37 gm, 0.0073 mol), EDC.HCl (2.79 gm, 0.0145 mol), and HOBT (1.48 gm, 0.011 mol) were dissolved in 30 ml of DMF, and DIPEA (2.35 gm, 0.018 mol) was added dropwise to the reaction mixture. The reaction was continued for 3 hours, and the progress was monitored by TLC. After the reaction was complete, the reaction mixture was diluted with water (100 ml), and the compounds were 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 silica gel column chromatography (ethyl acetate:hexane:1:1) with a yield of 62%. 1 H NMR (300 MHz, CD3OD): δ 7.73 (s, 1H), 7. 11 - 7.29 (m, 16H), 6.66 (s, 1H), 6.15 (s, 1H), 5.23 (s, 2H), 5.15 (s. 2H), 4.53 (s, 2H), 3.99 (q, J = 6.6 Hz, 2H), 3.31 (m, 1H), 3.11 - 3.21 (m, 5H), 2.49 (s, 3H), 2.31 (s, 3H), 2.22 (s, 3H), 1.66 - 1.71 (m, 4H), 1.23 (d, J = 7.2 Hz, 6H), 0.91 (t, J = 6.9 Hz, 3H).

[0160] 4'-(2,4-bis(benzyloxy)-N-ethyl-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(43)

[0161] Intermediate 43 was obtained from the starting material 35 in a yield of 56% in the same manner as described for the synthesis of compound 42. 1H NMR (300 MHz, CD3OD): δ 7.69 (s, 1H), 7. 22 - 7.38 (m, 16H), 6.51 (s, 1H), 6.11 (s, 1H), 5.15 (s, 2H), 5.11 (s, 2H), 4.51 (bs, 2H) 3.91 - 3.95 (m, 4H), 3.29 (s, 3H), 3.15 - 3.25 (m, 6H), 2.49 (s, 3H), 2.32 (s, 3H), 2.24 (s, 3H), 1.63 - 1.69 (m, 4H), 1.31 (d, J = 7.2 Hz, 6H), 1.25 (t, J = 7.2 Hz, 3H), 0.92 (t, J = 6.9 Hz, 3H).

[0162] 4'-(2,4-bis(benzyloxy)-5-isopropyl-N-propylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(44)

[0163] Intermediate 44 was obtained from starting material 36 in 58% yield in the same manner as described for the synthesis of compound 42. 1 H NMR (300 MHz, CD3OD): δ 7.61 (s, 1H), 7. 32 - 7.39 (m, 16H), 6.68 (s, 1H), 6.12 (s, 1H), 5.17 (s, 2H), 5.18 (s, 2H), 4.43 (bs, 2H), 3.92 - 3.95 (m, 4H), 3.21 - 3.29 (m, 6H), 2.49 (s, 3H), 2.31 (s, 3H), 2.25 (s, 3H), 1.45 - 1.67 (m, 6H), 1.31 (d, J = 7.2 Hz, 6H), 1.16 (t, J = 7.2 Hz, 3H), 0.92 (t, J = 6.9 Hz, 3H).

[0164] 4'-(N-benzyl-2,4-bis(benzyloxy)-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(45)

[0165] Intermediate 45 was obtained from the starting material 37 in 60% yield in the same manner as described for the synthesis of compound 42. 1 H NMR (300 MHz, CD3OD): δ 7.56 (s, 1H), 7. 21 - 7.38 (m, 22H), 6.64 (s, 1H), 5.13 (s, 2H), 5.09 (s, 2H), 5.08 (s, 2H), 4.51 (bs, 2H), 3.92 (m, 2H), 3.15 - 3.25 (m, 6H), 2.49 (s, 3H), 2.29 (s, 3H), 2.21 (s, 3H), 1.48 - 1.54 (m, 4H), 1.25 (d, J = 7.2 Hz, 6H), 0.91 (t, J = 6.9 Hz, 3H).

[0166] 3'-(2,4-bis(benzyloxy)-5-isopropyl-N-methylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(46)

[0167] Intermediate 46 was obtained from starting material 38 in 63% yield in the same manner as described for the synthesis of compound 42. 1H NMR (300 MHz, DMSO-d6) δ 11.46 (s, 1H), 8.20 (s, 1H), 7.33-7.45 (m, 13H), 7.21 (s, 1H), 7.09 (d, J = 7.2 Hz, 1H), 7.04 (s, 1H), 6.88 (s, 1H), 6.77 (s, 1H), 5.85 (s, 1H), 5.11 (s, 2H), 5.05 (s, 2H), 4.33 (d, J = 4.5 Hz, 2H), 3.81 (d, J = 10.5 Hz, 2H), 3.22 (t, J = 10.8 Hz, 2H), 3.04 (m, 4H), 2.22 (d, J = 4.2 Hz, 5H), 2.10 (s, 3H), 2.07 (s, 3H), 1.62 (d, J = 11.1 Hz, 2H), 1.52 (d, J = 11.1 Hz, 2H), 1.25 (s, 2H), 0.93 (s, 5H), 0.79 (t, J = 6.6 Hz, 3H).

[0168] 3'-(2,4-bis(benzyloxy)-N-ethyl-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(47)

[0169] Intermediate 47 was obtained from the starting material 39 in 36% yield in the same manner as described for the synthesis of compound 42. 1H NMR (300 MHz, CD3OD): δ 7.51 (s, 1H), 7.19-7.23 (m, 14H), 6.94 (m, 2H), 6.16 (s, 1H), 6.09 (s, 1H), 5.03 (s, 2H), 5.01 (s, 2H), 4.68 (s, 2H), 3.95 - 3.98 (m, 4H), 3.89 (s, 3H), 3.19 - 3.21 (m, 6H), 2.49 (s, 3H), 2.33 (s, 3H), 2.21 (s, 3H), 1.58 - 1.71 (m, 4H), 1.38 (d, J = 7.2 Hz, 6H), 1.31 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H).

[0170] 3'-(2,4-bis(benzyloxy)-5-isopropyl-N-propylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(48)

[0171] Intermediate 48 was synthesized from starting material 40 in 62% yield in the same manner as described for compound 42. 1 H NMR (300 MHz, CDCl3): δ 7.49 (s, 1H), 7.21-7.27 (m, 14H), 6.94 - 6.97 (m, 2H), 6.11 (s, 1H), 4.97 (s, 2H), 4.95 (s, 2H), 4.51 (d, J = 5.4 Hz, 2H), 3.96 - 3. 99 (m, 4H), 3.91 (s, 3H), 3.25 - 3.31 (m, 6H), 2.51 (s, 3H), 2.31 (s, 3H), 2.21 (s, 3H), 1.55 - 1.72 (m, 6H), 1.23 (d, J = 7.2 Hz, 6H), 1.21 (t, J = 7.2 Hz, 3H), 0.97 (t, J = 6.9 Hz, 3H).

[0172] 3'-(N-benzyl-2,4-bis(benzyloxy)-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(49)

[0173] Intermediate 49 was synthesized from the starting material 41 in 38% yield in the same manner as described for compound 42. 1 H NMR (300 MHz, CDCl3): δ 7.51 (s, 1H), 7.28-7.31 (m, 19H), 7.03 (m, 2H), 6.27 (s, 1H), 5.02 (s, 2H), 4.99 (s, 2H), 4.97 (s, 2H), 4.44 (d, J = 5.4 Hz, 2H), 4.01 (s, 2H), 3.92 (s, 3H), 3.21 - 3.25 (m, 6H), 2.41 (s, 3H), 2.31 (s, 3H), 2.25 (s, 3H), 1.52 - 1.56 (m, 4H), 1.29 (d, J = 7.2 Hz, 6H), 0.99 (t, J = 6.9 Hz, 3H).

[0174] 4'-(2,4-dihydroxy-5-isopropyl-N-methylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(3)

[0175] In 100 ml of RBF, intermediate 42 (0.6 gm, 0.0007 mol) was dissolved in 20 ml of methanol, and the catalyst Pd / C was added. The reaction was continued under hydrogen gas conditions at room temperature for 4 hours, and the progress of the reaction was monitored using TLC. After completion, the mixture was diluted with methanol and passed through Celite. The solvent was evaporated under vacuum, and the residue was purified by silica gel column chromatography (ethyl acetate:hexane:1:1). The yield was 47%. HPLC purity: 96.16%; mp: 184~186°C; 1 H NMR (600 MHz, DMSO-d6): δ 11.43 (s, 1H), 10.69 (s, 1H), 9.72 (s, 1H), 8.15 (t, J = 5.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 1.2 Hz, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 1.8 Hz, 1H), 6.51 (s, 1H), 6.18 (s,1H), 5.82 (s, 1H), 4.25 (d, J = 5.4 Hz, 2H), 3.79 (d, J = 10.2 Hz, 2H), 3.33 (s, 3H), 3.21 (t, J = 10.8 Hz, 2H), 3.01-3.04 (m, 2H), 2.95-3.00 (m, 1H), 2.77-2.82 (m, 1H), 2.21 (s, 3H), 2.17 (s, 3H), 2.07 (s, 3H), 1.62 (d, J = 12.6 Hz, 2H), 1.44-1.51 (m, 2H), 0.78 (t, J = 7.2 Hz, 3H), 0.68 (d, J = 6.6 Hz, 6H). 13C NMR (150 MHz, DMSO-d6): δ 170.76, 170.67, 169.39, 163.44, 158.22, 157.97, 149.94, 149.27, 144.84, 143.18, 140.06, 138.55, 136.85, 133.30, 128.22, 127.84, 127.43, 125.09, 123.47, 122.05, 121.21, 109.76, 107.78, 102.72, 66.75, 60.18, 58.14, 41.56, 38.47, 35.29, 30.69, 25.46, 22.64, 21.19, 19.36, 18.62, 14.96, 14.52, 12.98.C 40 H 49 N4O6[M + H + HRMS(ESI): Calculated value; 681.3652; Measured value; 681.3625.

[0176] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4'-(N-ethyl-2,4-dihydroxy-5-isopropylbenzamide)-4-methyl-[1,1'-biphenyl]-3-carboxamide(4)

[0177] Title compound 4 was synthesized from starting material 43 in 42% yield, in the same manner as described for compound 3. HPLC purity: 98.38%; mp: 191~193°C; 1H NMR (600 MHz, DMSO-d6): δ 11.44 (s, 1H), 10.89 (s, 1H), 9.74 (s, 1H), 8.15 (t, J = 4.8 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 1.2 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 1.2 Hz, 1H), 6.48 (s, 1H), 6.18 (s,1H), 5.82 (s, 1H), 4.26 (d, J = 4.8 Hz, 2H), 3.81-3.85 (m, 2H), 3.79 (d, J = 10.2 Hz, 2H), 3.21 (t, J = 11.4 Hz, 2H), 3.01-3.05 (m, 2H), 2.77 (t, J = 6.6 Hz, 1H), 2.21 (s, 3H), 2.17 (s, 3H), 2.07 (s, 3H), 1.56 (d, J = 12 Hz, 2H), 1.44-1.51 (m, 2H), 1.09 (t, J = 7.2 Hz, 4H), 0.78 (t, J = 7.2 Hz, 3H), 0.66 (d, J = 6.6 Hz, 6H). 13 C NMR (150 MHz, DMSO-d6): δ 170.39, 169.39, 163.45, 158.06, 149.96, 149.28, 143.19, 143.08, 140.05, 138.82, 136.83, 133.36, 128.40, 128.26, 127.93, 125.03, 123.51, 122.05, 121.26, 107.80, 102.71, 66.75, 58.14, 45.42, 41.58, 35.29, 30.70, 25.43, 23.16, 22.62, 19.36, 18.62, 14.97, 13.05, 12.97. C 41 H 51 N4O6[M + H + ]のHRMS(ESI): calculated value; 695.3809; measured value, 695.3757(M + H + ).

[0178] 4'-(2,4-dihydroxy-5-isopropyl-N-propylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(5)

[0179] Title compound 5 was synthesized from starting material 44% in a yield of 55% in the same manner as described for compound 3. HPLC purity: 95.75%; mp: 155~157°C; 1 H NMR (600 MHz, CD3OD): δ 7.54 (d, J = 8.4 Hz, 2H), 7.38 (s, 1H), 7.26 (s, 1H), 7.21 (d, J = 7.8 Hz, 2H), 6.55 (s, 1H), 6.19 (s, 1H), 6.10 (s, 1H), 4.48 (s, 2H), 3.87-3.92 (m, 4H), 3.35 (t, J = 11.4 Hz, 2H), 3.11-3.14 (m, 2H), 3.06-3.09 (m, 1H), 2.83-2.88 (m, 1H), 2.38 (s, 3H), 2.31 (s, 3H), 2.23 (s, 3H), 1.73 (d, J = 12 Hz, 2H), 1.60-1.68 (m, 4H), 0.94 (t, J = 7.8 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H), 0.70 (d, J = 6.6 Hz, 6H). 13C NMR (150 MHz, CD3OD): δ 171.53, 164.21, 159.03, 158.12, 152.14, 149.29, 143.44, 143.41, 139.17, 139.08, 137.66, 133.27, 128.17, 127.71, 127.55, 125.36, 124.02, 121.33, 120.73, 109.63, 108.62, 101.89, 66.83, 58.27, 51.88, 41.70, 35.16, 30.42, 25.35, 21.46, 20.41, 18.27, 17.14, 13.62, 11.59, 10.22.C 42 H 53 N4O6[M + H + HRMS(ESI): Calculated value, 709.3965; Measured value, 709.3976.

[0180] 4'-(N-benzyl-2,4-dihydroxy-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(6)

[0181] Intermediate 45 (0.5 gm, 0.0006 mol) was dissolved in 20 ml of DCM, and 1 M BCl3 solution (0.27 gm, 0.0023 mol) in 2.3 ml of DCM was added dropwise to the solution. The 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 extracted with DCM (50 ml x 3). The separated organic layer was evaporated using a rotary evaporator. The residue was purified by silica gel column chromatography (ethyl acetate:hexane:2:1) to obtain target compound 6 in 51% yield. HPLC purity: 97.24%; mp: 135~137°C; 1H NMR (600 MHz, DMSO-d6): δ 11.41 (s, 1H), 10.54 (s, 1H), 8.10 (t, J = 4.8 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.27-7.30 (m, 4H), 7.24 (s, 2H), 7.19 (t, J = 7.2 Hz, 1H), 7.13 (d, J = 8.4.2 Hz, 2H), 7.10 (d, J = 1.8 Hz, 1H), 6.57 (s,1H), 6.20 (s, 1H), 5.81 (s, 1H), 5.07 (s, 2H), 4.24 (d, J = 4.8 Hz, 2H), 3.77 (d, J = 10.8 Hz, 2H), 3.34 (m, 1H), 3.19 (t, J = 10.8 Hz, 2H), 2.98-3.02 (m, 2H), 2.77-2.82 (m, 1H), 2.19 (s, 3H), 2.15 (s, 3H), 2.07 (s, 3H), 1.59 (d, J = 11.4 Hz, 2H), 1.42-1.46 (m, 2H), 0.75 (t, J = 7.8 Hz, 3H), 0.69 (d, J = 6.6 Hz, 6H). 13 C NMR (150 MHz, DMSO-d6): δ 170.71, 169.36, 163.42, 157.97, 149.92, 149.27, 143.17, 139.99, 138.35, 137.99, 136.64, 133.33, 128.79, 128.16, 127.95, 127.53, 125.20, 123.39, 122.04, 121.17, 107.75, 102.74, 72.44, 70.41, 66.73, 60.69, 58.11, 53.25, 35.28, 31.79, 30.71, 29.45, 25.52, 22.88, 22.65, 22.52, 19.36, 19.26, 18.62, 14.96, 14.23, 12.97. C 46 H 53 N4O6[M + H + ]のHRMS (ESI): calculated value, 757.3965; measured value, 757.3932.

[0182] 3'-(2,4-dihydroxy-5-isopropyl-N-methylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(7)

[0183] Title compound 7 was synthesized from starting material 46% in 48% yield, in the same manner as described for compound 3. HPLC purity: 95.14%; mp: 161°C~163°C; 1 H NMR (600 MHz, CD3OD): δ 7.46 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 6.6 Hz, 1H), 7.16 (d, J = 1.8 Hz, 1H), 7.11 (t, J = 1.8 Hz, 1H), 6.98 (d, J = 1.8 Hz, 1H), 6.57 (s, 1H), 6.27 (s,1H), 6.10 (s, 1H), 4.47 (s, 2H), 3.90 (d, J = 10.8 Hz, 2H), 3.47 (s, 3H), 3.33-3.37 (m, 2H), 3.02-3.06 (m, 2H), 2.37 (s, 3H), 2.28 (s, 3H), 2.23 (s, 3H), 1.68 (d, J = 11.4 Hz, 2H), 1.57-1.62 (m, 2H), 1.29 (d, J = 5.4 Hz, 2H) 0.82 (t, J = 7.2 Hz, 3H), 0.68 (d, J = 6.6 Hz, 6H). 13C NMR (150 MHz, CD3OD): δ 171.98, 171.52, 164.21, 158.72, 158.21, 152.23, 149.26, 146.14, 143.47, 141.67, 139.08, 137.76, 133.41, 129.76, 128.31, 125.86, 125.54, 124.67, 124.27, 124.16, 121.26, 120.68, 109.70, 108.59, 101.99, 66.81, 58.22, 41.71, 37.24, 35.20, 30.42, 25.32, 21.50, 18.27, 17.15, 13.58, 11.60.C 40 H 49 N4O6[M + H + HRMS (ESI): Calculated value; 681.3652; Measured value; 681.3623.

[0184] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-3'-(N-ethyl-2,4-dihydroxy-5-isopropylbenzamide)-4-methyl-[1,1'-biphenyl]-3-carboxamide(8)

[0185] The title compound 8 was synthesized from the starting material in the same manner as described for compound 3, with a yield of 47% and 68%. HPLC purity: 95.02%; mp: 151~153℃; 1H NMR (600 MHz, CD3OD): δ 7.46 (t, J = 7.8 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.16 (d, J = 1.8 Hz, 1H), 7.08 (s, 1H), 6.98 (s, 1H), 6.57 (s, 1H), 6.26 (s,1H), 6.10 (s, 1H), 4.47 (s, 2H), 4.00 (s, 2H), 3.91 (d, J = 10.8 Hz, 2H), 3.35 (t, J = 10.2 Hz, 2H), 3.03-3.06 (m, 2H), 2.97-3.00 (m, 1H), 2.83-2.87 (m, 1H), 2.37 (s, 3H), 2.28 (s, 3H), 2.23 (s, 3H), 1.68 (d, J = 11.4 Hz, 2H), 1.56-1.62 (m, 2H), 1.21 (t, J = 6.6 Hz, 3H) 0.82 (t, J = 7.2 Hz, 3H), 0.68 (d, J = 7.2 Hz, 6H). 13 C NMR (150 MHz, CD3OD): δ 171.57, 158.90, 158.14, 152.23, 149.28, 144.42, 143.47, 141.70, 139.07, 137.78, 133.43, 129.75, 129.42, 128.25, 126.88, 125.48, 124.83, 124.76, 124.29, 120.72, 109.69, 108.72, 101.95, 66.82, 58.25, 45.05, 41.72, 35.19, 35.12, 31.63, 30.42, 25.32, 22.30, 21.50, 18.26, 17.14, 13.58, 13.00, 11.63, 11.61. C 41 H 51 N4O6[M + H + ]のHRMS (ESI): calculated value; 695.3809; measured value, 695.3773.

[0186] 3'-(2,4-dihydroxy-5-isopropyl-N-propylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(9)

[0187] Title compound 9 was synthesized from starting material 48% in 71% yield, in the same manner as described for compound 3. HPLC purity: 97.52%; mp: 172~175°C; 1 H NMR (600 MHz, DMSO-d6): δ 11.43 (s, 1H), 10.56 (s, 1H), 9.69 (s, 1H), 8.11 (t, J = 4.8 Hz, 1H), 7.36-7.40 (m, 2H), 7.26 (s, 1H), 7.16 (d, J = 7.2 Hz, 1H), 7.10 (d, J = 1.2 Hz, 1H), 7.03 (s, 1H), 6.50 (s,1H), 6.21 (s, 1H), 5.83 (s, 1H), 4.26 (d, J = 4.8 Hz, 2H), 3.79 (t, J = 7.8 Hz, 4H), 3.22 (t, J = 10.8 Hz, 2H), 2.97-3.01 (m, 2H), 2.89-2.94 (m, 1H), 2.74-2.79 (m, 1H), 2.19 (s, 3H), 2.17 (s, 3H), 2.07 (s, 3H), 1.59 (d, J = 11.4 Hz, 2H), 1.46-1.53 ​​(m, 4H), 0.83 (t, J = 7.2 Hz, 3H), 0.76 (t, J = 7.2 Hz, 3H), 0.65 (d, J = 7.2 Hz, 6H). 13C NMR (150 MHz, DMSO-d6): δ 170.55, 169.37, 163.45, 157.80, 150.03, 149.28, 144.65, 141.62, 140.09, 137.05, 133.41, 130.16, 128.00, 126.44, 126.05, 125.22, 125.03, 123.65, 122.02, 121.34, 110.13, 107.85, 102.70, 66.73, 65.34, 58.18, 51.56, 41.56, 35.31, 30.69, 25.41, 22.70, 20.84, 19.37, 18.62, 15.60, 14.95, 13.05, 11.64.C 42 H 53 N4O6[M + H + HRMS(ESI): Calculated value; 709.3965; Measured value; 709.3956.

[0188] 3'-(N-benzyl-2,4-dihydroxy-5-isopropylbenzamide)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-[1,1'-biphenyl]-3-carboxamide(10)

[0189] Title compound 10 was synthesized from the starting material in the same manner as described for compound 6, with a yield of 49% to 53%. HPLC purity: 95.26%; mp: 146-148℃; 11H NMR (600 MHz, CD3OD): δ 7.36 (t, J = 7.2 Hz, 1H), 7.32 (t, J = 9 Hz, 3H), 7.25 (t, J = 7.2 Hz, 2H), 7.19 (t, J = 7.2 Hz, 2H), 7.05 (d, J = 0.6 Hz, 1H), 6.91 (d, J = 9Hz, 2H), 6.64 (s, 1H), 6.27 (s,1H), 6.10 (s, 1H), 5.16 (s, 2H), 4.47 (s, 2H), 3.90 (d, J = 11.4 Hz, 2H), 3.34 (t, J = 10.8 Hz, 2H), 3.01 - 3.04 (m, 2H), 2.94 - 2.98 (m, 1H), 2.83 - 2.88 (m, 1H), 2.37 (s, 3H), 2.26 (s, 3H), 2.23 (s, 3H), 1.65 (d, J = 12 Hz, 2H), 1.54 - 1.61 (m, 2H), 0.80 (t, J = 7.2 Hz, 3H), 0.68 (d, J = 6.6 Hz, 6H). 13 13C NMR (150 MHz, CD3OD): δ 171.79, 171.50, 164.22, 158.78, 158.25, 152.23, 149.20, 144.41, 143.47, 141.41, 139.04, 137.70, 137.43, 133.34, 129.54, 128.13, 128.10, 128.04, 127.05, 126.85, 125.57, 124.84, 124.81, 124.13, 121.28, 120.65, 109.71, 108.80, 102.00, 66.81, 58.22, 54.64, 52.91, 41.67, 35.17, 30.40, 28.11, 25.36, 21.50, 18.29, 17.15, 13.56, 11.60. C 46 H 53 N4O6[M + H + HRMS (ESI): Calculated value: 757.3965; Measured value: 757.3998.

[0190] Scheme 3, shown below, is another example of the synthesis of the compounds disclosed herein. JPEG2026516102000023.jpg107170

[0191] This synthesis route can be used for target skeletons 50 and 51. In particular, the reagents and conditions used in Scheme 3 are as follows: d) Pd / C, H2, MeOH, room temperature, 4 hours; e) For 50, oleic acid, DCC, DMAP, DCM, room temperature, 4 hours; for 51, linolenic acid, DCC, DMAP, DCM, room temperature, 4 hours.

[0192] 4-((3'-(((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)carbamoyl)-5'-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4'-methyl-[1,1'-biphenyl]-3-yl)(methyl)carbamoyl)-6-isopropyl-1,3-phenylenedioleate(50)

[0193] In 100 ml of RBF, intermediate 7 (0.5 gm, 0.00073 mol), oleic acid (0.4 gm, 0.0014 mol), and DCC (0.75 gm, 0.0036 mol) were dissolved in 20 ml of DCM, and DMAP (0.089 gm, 0.0007 mol) was added to the reaction mixture in small amounts. The reaction was continued under nitrogen conditions at room temperature for 4 hours, and the progress of the reaction was monitored using TLC. After the reaction was complete, the reaction mixture was diluted with water (100 ml), and the compounds were 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 silica gel column chromatography (ethyl acetate:hexane:1:1). The yield was 68%. HPLC purity: XX%; mp: 133~135°C; 1H NMR (300 MHz, CDCl3) δ 11.60 (s, 1H), 7.31-7.39 (m, 3H), 7.24 (d, J = 6.3 Hz, 2H), 7.10 (d, J = 16.5 Hz, 2H), 6.90 (s, 1H), 6.81 (s, 1H), 5.95 (s, 1H), 5.34-5.38 (m, 4H), 4.58 (d, J = 5.7 Hz, 2H), 3.98 (d, J = 11.1 Hz, 2H), 3.52 (s, 4H), 3.29-3.38 (m, 2H), 3.15 (s, 2H), 2.79 (t, J = 5.7 Hz, 1H), 2.59 (d, J = 7.5 Hz, 2H), 2.52 (d, J = 7.5 Hz, 2H), 2.42 (s, 3H), 2.20 (s, 3H), 2.00-2.04 (m, 7H), 1.92-1.98 (m, 3H), 1.68-1.75 (m, 9H), 1.29-1.33 (m, 40 H), 1.09-1.18 (m, 3H), 0.88-0.92 (m, 8H), 0.78 (s, 4H).C 76 H 112 N4O8Na [M + Na + LRMS (ESI): Calculated value; 1231.83; Measured value; 1231.80.

[0194] 4-((3'-(((4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-yl)methyl)carbamoyl)-5'-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4'-methyl-[1,1'-biphenyl]-3-yl)(methyl)carbamoyl)-6-isopropyl-1,3-phenylene(9Z,9'Z,12Z,12'Z,15Z,15'Z)-bis(octadeca-9,12,15-trienoate)(51)

[0195] In 100 ml of RBF, intermediate 7 (0.5 gm, 0.00073 mol), linolenic acid (0.4 gm, 0.0014 mol), and DCC (0.75 gm, 0.0036 mol) were dissolved in 20 ml of DCM, and DMAP (0.089 gm, 0.0007 mol) was added to the reaction mixture in small amounts. The reaction was continued under nitrogen conditions at room temperature for 4 hours, and the progress of the reaction was monitored using TLC. After the reaction was complete, the reaction mixture was diluted with water (100 ml), and the compounds were 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 silica gel column chromatography (ethyl acetate:hexane:1:1) with a yield of 52%. HPLC purity: XX%; mp: 122~124°C; 1 H NMR (300 MHz, CDCl3) δ 11.47 (s, 1H), 8.07 (s, 1H), 7.47 (s, 1H), 7.39 (s, 2H), 7.30 (s, 1H), 7.13 (s, 1H), 7.00 (d, J = 9.9 Hz, 2H), 6.92 (s, 1H), 5.86 (s, 1H), 5.58 (d, J = 3.6 Hz, 1H), 5.32 (s, 11H), 4.30 (s, 2H), 3.84 (d, J = 9.9 Hz, 2H), 3.41 (s, 2H), 3.25 (t, J = 11.1 Hz, 2H), 3.02 (d, J = 5.1 Hz, 2H), 2.93 (s, 1H), 2.77 (s, 7H), 2.55 (s, 2H), 2.22 (t, J = 1.8 Hz, 6H), 2.11 (s, 3H), 2.03 (d, J = 4.8 Hz, 6H), 1.73 (d, J = 12 Hz, 2H), 1.60 (s, 6H), 1.52 (d, J = 12.3 Hz, 3H), 1.29 (s, 19H), 1.07 (t, J = 10.8 Hz, 2H), 0.90-0.95 (m, 5H), 0.77 (d, J = 6.3 Hz, 9H).C 76 H 104 N4O8Na [M + Na +LRMS (ESI): Calculated value; 1223.77; Measured value; 1223.75.

[0196] Example 3: EZH2 Inhibition

[0197] Next, having successfully constructed the target adducts (1-10), we performed profiling of their EZH2 inhibitory activity. The results of this study are summarized in Table 1 below. The EZH2 inhibition assay was performed by Reaction Biology Corporation, Malvern, PA.

[0198] By carefully observing the results, some insights were gained regarding the structure-EZH2 inhibition relationship. Five adducts (1-4, 7) showed IC25 50 The values ​​were in the single-order nanomolar range, demonstrating excellent EZH2 inhibitory activity. An important finding obtained from the correlation between the structural characteristics of the adduct and its EZH2 inhibitory activity was that compound 2 (resorcinol fragment, 3' position) had an IC50 of 2.06 nM. 50The tetheration of the resorcinol fragment via the amide bond at the 3' position is preferable to tetheration at the 4' position because it exhibits outstanding EZH2 inhibitory activity with a significant value. Notably, compound 2 was three times more potent in EZH2 inhibition than its counterpart compound 1 (resorcinol fragment, 4' position). To test resistance to N-alkylation / benzylation (amide NH), the resulting adducts 3-10 were also evaluated. Notably, the effect of N-alkylation / benzylation is clearly detrimental to EZH2 inhibitory activity, and a trend towards relatively reduced enzyme inhibitory activity was demonstrated in the regio-counterparts (resorcinol fragments at the 4' and 3' positions). A deeper examination of the enzyme inhibition profiles of compounds 3-10 clearly shows the aforementioned trend, as compound 1 is superior to its N-alkylated / benzylated derivatives (3-6). Similarly, compound 2 was observed to be remarkably more effective in inhibiting the enzymatic catalytic subunit of Polycomb repression complex 2 than its N-alkylated / benzylated derivatives (7-10). Other important structural findings from the assay were the greatest decrease in EZH2 inhibitory activity observed with N-benzylation (amide with N, resorcinol fragment) and the large downward trend observed with N-substitution in the hybrid skeleton adjacent to the resorcinol fragment at the 3' position, in both cases (amide-linked at the 4' and amide-linked hybrids). While several potent EZH2 inhibitors were identified through this evaluation study, relative comparisons with the EZH2 inhibitory activity of tazemetostat (I) showed that even the four best of the 10 hybrids supplied had slightly inferior EZH2 inhibitory profiles.Based on our preliminary screening results, several prior literature studies, and a comparative analysis report of tazemetostat(I) versus hybrid scaffolds (1-10, Table 6 below), it is important to note that despite being a very potent EZH2 inhibitor, tazemetostat(I) failed to exert cell proliferation inhibitory activity against GBM cell lines. Therefore, this observation did not hinder our development strategy. Accordingly, we hypothesized that balanced simultaneous regulation of EZH2 and HSP90 through the supply of a hybrid scaffold based on the structural commonality of the pharmacophores of their targets could activate the chemical structure of tazemetostat to exert an anti-GBM effect. Based on this hypothesis, our research group aimed to identify EZH2-HSP90 dual inhibitors and proceeded accordingly to evaluate the HSP90 inhibitory activity of adducts (1-10).

[0199] Example 4: HSP90 Inhibition

[0200] In vitro experiments were conducted to evaluate the inhibitory effect of the synthesized adduct on heat shock protein 90 (HSP90). Geldanamycin was used as a standard HSP90 inhibitor for comparison. The results of this study are summarized in Table 2 below. The HSP90 inhibition assay was performed by Reaction Biology Corporation, Malvern, PA.

[0201] It was somewhat interesting to observe that substitution at the amide NH (containing the resorcinol fragment) had a significant impact on the ability of the resulting adduct to inhibit chaperone proteins. Therefore, this assay demonstrated the opposite pattern compared to the EZH2 inhibition assay, with N-alkylation / benzylation increasing HSP90 inhibitory activity. Hybrid structures 1 and 2 did not exhibit HSP90 inhibitory activity, but methylation and ethylation at the amide nitrogen conferred HSP90 inhibitory activity to the skeleton (3, 4, 7, and 8). Unfortunately, N-propyl and -benzyl substitutions (5, 6, 9, and 10) failed to replicate the tendency to confer activity observed with the N-methyl and ethyl groups, suggesting that placing bulkier substituents at the amide NH (containing the resorcinol fragment) was unacceptable. Notably, hybrid templates featuring an amide bond connector at the 3' position (for the resorcinol fragment) were the most favorable in terms of activating the chemical structure toward HSP90 inhibitory activity. N-methyl and ethyl substitutions at the amide NH of the target skeleton, which contains the resorcinol fragment at the 3' position, result in two substantially potent HSP90 inhibitors, compound 7(IC) 50 Value = 60.1nM) and 8(IC 50 A value of 63.6 nM was obtained. As observed in the EZH2 inhibition evaluation test, the adducts tended to be less potent than commonly used standards (geldanamycin in the HSP90 inhibition assay and tazemettostat in the EZH2 inhibition assay). Undeterred by the inferior profiles of the adducts observed in both assays, IC values ​​of 6.29 nM (EZH2 inhibition) and 60.1 nM (HSP90 inhibition) were obtained, respectively. 50 Identifying seven balanced EZH2-HSP90 dual inhibitors with specific values ​​was a source of delight for our research group.

[0202] Example 5: Itraq-based proteomics analysis

[0203] After identifying the desired chemical probe 7, global protein expression was assessed using Itraq-based proteomics analysis to estimate whether 7 suppresses HSP90 (Pt3R cell line). As shown in Tables 3-5, proteins of the HSP family, including HSPA1A, HSPA8, HSP90AA1, HSPB1, HSPH1, and HSPA4, significantly increased in response to treatment with the dual inhibitor 7. These observations are consistent with those obtained with geldanamycin treatment (a well-known HSP90 inhibitor) (Cheung, CHA; Chen, H.-H.; Cheng, L.-T.; Lyu, KW; Kanwar, JR; 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. Cancer 2010, 9 (1), 1-11; Kuroyanagi, G.; Tokuda, H.; Fujita, K.; Kawabata, T.; Sakai, G.; Kim, W.; Hioki, T.; Tachi, J.; Matsushima-Nishiwaki, R.; Otsuka, T. Upregulation of TGF-β-induced HSP27 by HSP90 inhibitors in osteoblasts. BMC Musculoskelet. Disord. In 2022, 23 (1), 495), the HSP90 inhibitory activity of compound 7 in GBM cells was confirmed. JPEG2026516102000026.jpg84170JPEG2026516102000027.jpg159170JPEG2026516102000028.jpg210170

[0204] Example 6: Structural Molecular Docking

[0205] To rationally explain the experimental results of the enzymatic assay, structure-based molecular docking was performed (Figure 2). The docking process was performed between proteins and compounds using iGEMDOCK version 2.1 with a population size of 1500, 150 generations, and 10 solutions. The protein structures of HSP90 and EZH2 were obtained from the Protein Data Bank (8AGI and 4W2R). Notably, compound 7 was found to be associated with the ATP-binding pocket of HSP90, which is the target of the well-known HSP90 inhibitor ganetespib (STA9090)

[51] . Furthermore, similar to the EZH2 inhibitor tazemetostat, compound 7 was observed to bind to a site that plays a role in mediating the methyltransfer reaction of EZH2

[52] . These results demonstrate that compound 7 possesses high specificity targeting ability for HSP90 and EZH2.

[0206] Example 7: In vitro cytotoxicity study

[0207] To verify whether the hypothesis that the anti-GBM effect is derived through balanced dual regulation of EZH2 and HSP90 is experimentally confirmed, in vitro cytotoxicity studies were performed (Table 6, Figure 3). In this way, the ability of adducts (1-10) to inhibit the cell viability of TMZ-resistant cells (Pt3-R) was evaluated. Fortunately, the most potent and balanced EZH2-HSP90 dual inhibitor 7 showed an IC50 of 1.015 μM. 50The compounds showed significant cell proliferation inhibitory activity against Pt3-R cell lines. The correlation between the results in Tables 1, 2, and 6 indicates that the adducts (1, 2, 4-6, 9, and 10) and tazemetostat acting alone as an EZH2 inhibitor were unable to exert cell proliferation inhibitory effects, suggesting that the modulation of both targets (EZH2 and HSP90) was essential for the anti-GBM effect. Notably, compounds 3, 7, and 8, which can be classified as dual inhibitors, were the only ones to exert cell viability inhibitory activity. Among these, compound 7 [the most balanced and potent dual inhibitor of the targets (EZH2-HSP90)] was the most effective against Pt3R cell lines. These results clearly indicate that the superior enzyme inhibition profile of compound 7 was reflected in its cytotoxicity against TMZ-resistant (Pt3-R) GBM cell lines. In short, the cell proliferation inhibitory effect of compound 7 is presumed to stem from its ability to modulate the EZH2 and HSP90 chaperones in a balanced manner. JPEG2026516102000029.jpg109170

[0208] Figure 4A shows RNA extracts collected after 48 hours of treatment and subjected to RNA-seq. Left: Cell morphology; Right: Genes significantly affected by compound 7. Figure 4B shows genes functionally grouped by GSEA. Figure 4C shows clustering of genes involved in apoptosis / necrosis and M phase / kinetochore / spindle in a heatmap.

[0209] Example 8: Repression of kinetochore and DNA repair-related gene expression by dual inhibitor 7.

[0210] To gain insight into the ability of the dual inhibitor 7 to suppress the viability of TMZ-resistant GBM cells, RNA-seq was performed to analyze gene expression profiles. As shown in Figure 4A, treatment of Pt3R with 2 μM 7 for 48 hours significantly inhibited cell proliferation. RNA-seq also revealed that compound 7 increased and decreased the expression of 297 and 498 genes, respectively. Furthermore, the results of gene set enrichment analysis (GSEA) of compound 7 showed that cell division and DNA repair were highly suppressed (Figure 4B). Notably, treatment with compound 7 increased the expression of apoptosis / necrosis-related genes, while decreasing the expression of M-phase / kinetochore / spindle-related genes (Figure 4C).

[0211] Example 9: Decreased CENP protein expression

[0212] Furthermore, the effects of compound 7 treatment on centromere proteins (CENPs) were evaluated. Thus, CENPs are major constituent proteins of the kinetochore, which are essential for cell division. CENP expression is elevated in GBM tissue and correlates with unfavorable overall survival in glioma patients

[53] . Before evaluating the effects of the dual inhibitor 7, a study was conducted to assess CENP expression levels. As a result, CENPE and CENPI expression was upregulated in TMZ-resistant GBM cells (Pt3R) compared with patient-derived GBM cells (Pt3) (Figure 5A). CENPE and CENPI were observed to be significantly correlated with poor prognosis in GBM patients (Figure 5B), suggesting that CENPs are important for GBM progression. Encouragingly, further investigations revealed that centromere proteins (CENPs), including CENPF, CENPE, CENPA, and CENPI, which are crucial for regulating kinetochore assembly and mitosis, were reduced by treatment with compound 7 (Figure 5C). Western blot analysis also showed that the hybrid scaffold 7 also downregulated the expression of CDK1 and cyclin B1, which are necessary for M phase progression (Figure 5D). Furthermore, CENPs protein expression was also reduced by 7 in Pt3R cells (Figure 5D). In addition, the dual inhibitor 7 induced cell cycle arrest in M ​​phase (Figure 5E).

[0213] Figure 5A shows the CENP family suppressed by compound 7. Figure 5B shows the gene expression of the CENP family in Pt3 and TMZ-resistant Pt3R cells. Figure 5C shows that prognostic values ​​were analyzed using the GlioVis website. Figure 5D shows that Western blotting was used to confirm that compound 7 reduced CENP expression. Figure 5E shows the following cell cycle analysis. After 24 hours of treatment, cells were stained with propidium iodide (PI) and flow cytometry analysis was performed.

[0214] Example 9: Downregulation of DNA repair-related gene expression

[0215] In addition to cell division, the DNA repair ability was highly suppressed by compound 7. In particular, the expression of genes involved in the processing of DNA double-strand breaks and homologous recombination was decreased by 7 (Figure 6A). Further exploration carried out to establish the gene expression profile in TMZ-resistant Pt3R cells revealed that the expression of RB-binding protein (RBBP8) and BRCA1 was increased in TMZ-resistant Pt3R cells according to the relative comparison of the expression levels in Pt3 cells (Figure 6B). Also, these two genes (RBPP8 and BRCA1) were significantly correlated with poor prognosis in GBM patients (Figure 6C-D). Notably, both were suppressed by treatment with compound 7 (Figure 6A). Among the genes regulating homologous recombination, DNA topoisomerase II (TOP2A), DNA repair and recombination protein RAD54B, RAD21, crossover junction endonuclease EME1, and BRIP1 were found to be increased in TMZ-resistant Pt3R cells compared with Pt3 cells (Figure 6E). In particular, TOP2A and BRCA1-interacting helicase (BRIP) 1 were significantly correlated with poor prognosis (Figure 6F-G). Fortunately, the above genes regulating homologous recombination were suppressed by compound 7 (Figure 6H). Taken together, these findings strongly highlighted the possibility that compound 7 downregulates DNA repair-related gene expression, which is assumed to contribute to the anti-GBM effect of compound 7.

[0216] Figure 6A shows the effect of compound 7 on gene expression involved in the processing of DNA double-strand breaks. Figure 6B shows the comparison of gene expression between Pt3 cells and Pt3R cells. Figures 6C-6D show the prognostic values of RBBP8 and BRCA1 in GBM patients. Figure 6E shows the comparison of gene expression between Pt3 cells and Pt3R cells. Figures 6F-6G show the prognostic values of TOP2A and BRIP1 in GBM patients. Figure 6H shows the effect of compound 7 on gene expression involved in homologous recombination.

[0217] Example 10: Disruption of mitochondrial redox homeostasis and increased accumulation of ROS by dual inhibitors

[0218] Previous literature has shown that TMZ-resistant GBM cells can survive in the presence of TMZ due to enhanced reactive oxygen species (ROS) scavenging ability [54-57]. Therefore, the ability of compound 7 to disrupt redox homeostasis in TMZ-resistant cells and achieve amplified therapeutic efficacy against GBM was evaluated. In both Pt3 and Pt3R cells, 48-hour treatment with compound 7 increased ROS levels (Figure 7A). Furthermore, proteins involved in the removal of ROS, including catalase, superoxide dismutase (SOD)1, glutathione reductase, and glutathione peroxidase (GPX)1, were also decreased by compound 7 (Figure 7B). In parallel, the expression of EZH2 and EZH2-regulated H3K27 trimethylation were also inhibited by compound 7, indicating its inhibitory effect on EZH2 (Figure 7B). These results suggest that compound 7 suppresses the ROS catabolism pathway and causes the death of TMZ-resistant GBM cells. Furthermore, it was also observed that compound 7 increased mitochondrial-derived ROS. To evaluate the possibility of compound 7 increasing mitochondrial-derived ROS, fluorescent MitoSOX-labeled mitochondrial ROS was used, and it was found that compound 7 significantly enhanced the red signal representing ROS generated from mitochondria (Figure 7C).

[0219] Figure 7A shows that after 48-hour treatment, cells were stained with DHR reagent and the fluorescence signal was estimated by flow cytometry. The treatment concentrations of MPT1A059 are shown. Figure 7B shows that protein lysates were subjected to Western blotting using the indicated antibodies. Figure 7C shows that after staining with MitoSOX (red) and Hoechst33342 (blue), cell signals were photographed and quantified.

[0220] Example 11: In vivo growth inhibitory performance of TMZ-resistant GBM by dual inhibitors

[0221] This study also evaluated the in vivo anti-GBM effect of compound 7. In the in vivo study, experimental NOD.CB17-Prkdc scid / NCrCrl mice (8 weeks old) were used to collect Pt3R cells (1x10) in 50 ml of DMEM. 6 Compound 7 was injected into the back of mice. After 10 days, tumors became detectable. Mice were injected with either DMSO (control group) or compound 7 (5 mg / kg) twice a week. As shown in Figure 8, tumors in mice treated with compound 7 were significantly smaller in size and weight compared to those in mice treated with DMSO, suggesting the in vivo therapeutic efficacy of compound 7 (Figure 8A). Furthermore, the results presented in Figure 8B demonstrate the tumor growth inhibitory ability of compound 7. In short, the results of the in vivo study confirmed that the remarkable in vitro anti-GBM activity profile of the dual inhibitor 7 was reflected in its in vivo potential.

[0222] Figure 8A shows a typical photograph of a Pt3R tumor. Figure 8B shows the tumor growth curve and tumor weight.

[0223] Numerous precedents, combined with our investigation to evaluate EZH2 expression levels in tissue, suggest its overexpression in GBM tissue. Furthermore, high EZH2 expression significantly correlates with shorter survival times in GBM patients, confirming its role as a key oncogene in GBM pathogenesis. Interestingly, tazemetostat, the only FDA-approved EZH2 inhibitor, did not exhibit anti-GBM effects; however, the idea of ​​suturing another antitumor pharmacophore to the core structure of tazemetostat was considered a cautious strategy to activate its chemical structure and exert anti-GBM effects. Notably, this idea or implementation stemmed primarily from knowledge gained from our previous dual inhibitor development campaign. Considering numerous reports verifying the efficacy of HSP90 inhibitors in GBM, and our previous findings confirming the possible achievement of significant anti-GBM effects through the combined use of EZH2 and HSP90 inhibitors, candidate HSP90 chaperone protein inhibitors were deemed suitable for tetheration to the tazemettostat structural template. Therefore, to derive significant anti-GBM effects from the balanced regulation of EZH2 and HSP90, a hybrid template with structural commonalities between EZH2 and HSP90 inhibitors was constructed via a multi-step synthetic pathway. Encouragingly, the remarkably balanced dual inhibitor 7 was identified through in vitro enzyme assays, and the effect of dual inhibition was demonstrated in cytotoxicity studies. Hybrid template 7 showed substantial cell proliferation inhibitory activity against Pt3R, which was thought to be due to its EZH2-HSP90 dual inhibitory ability. Further in-depth exploration of chemical probe 7 confirmed its ability to i) suppress kinetochore and DNA repair-related gene expression, ii) increase ROS accumulation by disrupting mitochondrial redox homeostasis, and iii) inhibit the proliferation of TMZ-resistant GBM in vivo. Overall, this study leads to the identification of an easy-to-use dual inhibitor that encompasses the necessary characteristics of emerging therapeutic agents for treatment-resistant brain tumors.

[0224] Those skilled in the art should understand that changes and modifications can be made to the teachings and disclosures of this disclosure without departing from the spirit and scope of this application. Based on the foregoing, this application is intended to cover such variations and modifications, provided that the changes and modifications are within the scope defined in the appended claims or their equivalents.

Claims

1. Compound of formula (I), or its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs. In the formula, L is a linker selected from the group consisting of -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, A and B are independent, directly joined, C 1 -C 3 Alkyl and C 1 -C 3 Selected from the group consisting of alkoxys, R is selected from the group consisting of H, straight-chain or branched C 1 -C 6 alkyl, straight-chain or branched C 1 -C 6 alkoxy, C 6 -C 10 aryl and C 5 -C 10 heteroaryl, and is selected from the group consisting of Y is selected from the group consisting of O, S, and N. R 1 and R 2 These are H, Halo, and C, independently. 1 -C 6 Alkyl and C 16 -C 20 Selected from (polyunsaturated) fatty acids, The alkyl and alkoxyl are halogens, hydroxyls, aminos, or C 6 -C 10 Aryl or C 5 -C 10 It may be optionally substituted with a heteroaryl compound. The aryl and heteroaryl compounds include halogen, hydroxyl, amino, and C. 1 -C 3 Alkyl or C 1 -C 3 It may be optionally substituted with an alkoxy.

2. L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, R is H, R 1 and R 2 These are H, Halo, and C, respectively, and are independent of each other. 1 -C 6 The compound according to claim 1, characterized in that it is alkyl, oleic acid, and linolenic acid (such as α- or γ-linolenic acid), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

3. L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, and R is a linear or branched C 1 -C 6 It is alkyl, R 1 and R 2 The compound according to claim 1, characterized in that each of the elements is independently H, oleic acid, and linolenic acid (such as α- or γ-linolenic acid), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

4. L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, where Y is O, A is a direct bond, B is a direct bond, and R is C 6 -C 10 It is an aryl (such as phenyl or benzyl), R 1 and R 2 The compound according to claim 1, characterized in that each of the elements is independently H, oleic acid, and linolenic acid (such as α- or γ-linolenic acid), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

5. L is -A-NR-C(=Y)-B- and -AC(=Y)-NR-B-, Y is O, A is a direct bond, B is a direct bond, and R is C 6 -C 10 Linear or branched C molecules optionally substituted with aryl groups. 1 -C 6 It is alkyl, R 1 and R 2 The compound according to claim 1, characterized in that each of the elements is independently H, oleic acid, and linolenic acid (such as α- or γ-linolenic acid), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

6. Equation (I) is either equation (Ia) or (Ib), During the ceremony, Y is either O or S, R is H, straight chain or branched C 1 -C 4 Alkyl, phenyl, or benzyl, R 1 and R 2 These are H, Halo, and C, independently. 1 -C 6 Alkyl and C 16 -C 20 Selected from (polyunsaturated) fatty acids, The aforementioned straight-chain or branched C 1 -C 4 Alkyl can be optionally substituted with halogen, hydroxyl, or amino. The phenyl or benzyl is C 1 -C 2 The compound according to claim 1, characterized by being optionally substituted with alkyl, halogen, hydroxy, or amino, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

7. Equation (I) is either equation (Ia-p) or (Ib-p), During the ceremony, Y is either O or S, R is H, straight chain or branched C 1 -C 4 Alkyl, phenyl, or benzyl, R 1 and R 2 These are H, Halo, and C, independently. 1 -C 6 Alkyl and C 16 -C 20 Selected from (polyunsaturated) fatty acids, The aforementioned straight-chain or branched C 1 -C 4 Alkyl can be optionally substituted with halogen, hydroxyl, or amino. The phenyl or benzyl is C 1 -C 2 The compound according to claim 1, characterized by being optionally substituted with alkyl, halogen, hydroxy, or amino, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

8. Equation (I) is either equation (Ia-m) or (Ib-m), During the ceremony, Y is either O or S, R is H, straight chain or branched C 1 -C 4 Alkyl, phenyl, or benzyl, R 1 and R 2 These are H, Halo, and C, independently. 1 -C 6 Alkyl and C 16 -C 20 Selected from (polyunsaturated) fatty acids, The aforementioned straight-chain or branched C 1 -C 4 Alkyl can be optionally substituted with halogen, hydroxyl, or amino. The phenyl or benzyl is C 1 -C 2 The compound according to claim 1, characterized by being optionally substituted with alkyl, halogen, hydroxy, or amino, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

9. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, characterized in that formula (Ia) is formula (Ia-p) as described in claim 3, or formula (Ia-m) as described in claim 4.

10. A compound according to Claim 1, selected from Claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

11. The compound according to claim 1 having the following formula, or its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs.

12. A pharmaceutical composition / formulation comprising a compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

13. A method for treating a disease or disorder mediated by EZH2, HSP90, or both in a patient, comprising administering a compound described in any one of claims 1 to 11 or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof to a subject in need.

14. The method according to 13, characterized in that the disease or disorder is brain cancer.

15. The method according to 13, characterized in that the brain cancer is a primary brain tumor or a metastatic brain cancer.

16. The method according to claim 13, characterized in that the brain cancer is a glioma or a meningioma.

17. The method according to claim 13, characterized in that the brain cancer is glioblastoma (GBM).

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