Histone deacetylase inhibitors for immune modulation in the tumor microenvironment

Abstract

Class I HDAC inhibitor compounds, their preparation and applications are provided. The compounds have epigenetic immunomodulatory activity in the tumor microenvironment (TME), thus inhibiting tumor cell growth. TIFF2023524230000042.tif35144

Description

[Technical Field]

[0001] The present disclosure relates generally to Class I HDAC inhibitor compounds, their production, and applications. In particular, the compounds have epigenetic immunomodulatory activity in the tumor microenvironment (TME), thus inhibiting tumor cell growth. [Background technology]

[0002] Immunotherapy has become the standard of care for the treatment of several advanced cancers. A breakthrough in immunotherapy is the development and clinical application of immune checkpoint inhibitors (ICIs), such as anti-PD-1 / anti-PD-L1 / anti-CTLA-4 antibodies. However, ICIs can cause immune-related adverse events, and more importantly, only a small number of patients achieve therapeutic benefit (low response rates). The dynamic and complex tumor microenvironment (TME) is a key factor determining the immune response to tumors. The composition of the TME includes cancer cells and many different immune cells interwoven with normal tissue cells. Many growth factors, cytokines, and chemokines are secreted by different cells in the TME.

[0003] CTLs (cytotoxic T lymphocytes) are the primary immune cells of adaptive immunity that are specific for the direct killing of cancer cells. CTLs are susceptible to multiple immunosuppressive cells infiltrating the TME, which cause CTL inactivation. Well-known immunosuppressive cells include Tregs (regulatory T cells), M-MDSCs (monocytic myeloid-derived suppressor cells), PMN-MDSCs (polymorphonuclear myeloid-derived suppressor cells), and TAMs (tumor-associated macrophages). These immunosuppressive cells contribute to the inhibition of the cytotoxic effect of CTL-mediated cancer cell killing. There are different mechanisms implemented by these immunosuppressive cells that lead to CTL dysfunction. Summary of the Invention

[0004] Although ICI therapy has been shown to be effective in enhancing immune activation to eradicate cancer, these therapies still face the unresolved problem of primary and acquired drug resistance. Intrinsic factors driving primary and acquired resistance to these immunotherapies include genetic and epigenetic mechanisms that often cause downregulation of MHC I or loss of antigen expression through processes such as immunoediting, resulting in a total loss of antigen presentation. Therefore, there is a need to develop compounds with immunomodulatory activity in the TME to stimulate antitumor immunity by upregulating antigen processing and presentation mechanisms.

[0005] Briefly, embodiments of the present disclosure provide Class I HDAC inhibitor compounds (including pharmaceutically acceptable salts, hydrates, stereoisomers, solvates, or prodrugs thereof) capable of epigenetically immunomodulating in the TME. Methods for the use of such compounds for the treatment of various diseases or conditions, such as cancer, are also provided.

[0006] In one embodiment, the present disclosure provides a compound of formula (I):

[0007] [ka] wherein W and Y are each independently selected from CH and N; R1 is independently selected from hydrogen, halogen, C1-C3 alkyl, and halogenated C1-C3 alkyl, and may be mono-, di-, tri-, or tetra-substituted; C1 and C2 are C atoms connected by a single or double bond, Ar is the following:

[0008] [ka] wherein Ar is connected to C2 via a solid line; R2 has the same meaning as described for R1; R3 is hydrogen or C1-C3 alkyl. or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[0009] In one embodiment, the compound of Formula (I) is 6-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)pyridine-3-carboxamide and is designated GNTbm-01. In one embodiment, the compound of Formula (I) is 5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)pyridine-2-carboxamide and is designated GNTbm-02. In one embodiment, the compound of Formula (I) is 4-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)benzamide and is designated GNTbm-03. In one embodiment, the compound of Formula (I) is 5-((E)-4-(pyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)pyridine-2-carboxamide and is designated GNTbm-04. In one embodiment, the compound of Formula (I) is 5-((E)-4-(pyridin-3-yl)but-3-enamido)-N-(2-aminophenyl)pyridine-2-carboxamide and is designated GNTbm-05. In one embodiment, the compound of Formula (I) is 5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-aminophenyl)pyridine-2-carboxamide and is designated GNTbm-06. In one embodiment, the compound of Formula (I) is 5-(4-(6-methylpyridin-3-yl)butanamido)-N-(2-amino-4-fluorophenyl)pyridine-2-carboxamide and is designated GNTbm-08. In one embodiment, the compound of Formula (I) is 5-((E)-4-(pyridin-3-yl)but-3-enamido)-N-(2-amino-4-(trifluoromethyl)phenyl)pyridine-2-carboxamide and is designated GNTbm-11. In one embodiment, the compound of Formula (I) is 5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-(trifluoromethyl)phenyl)pyridine-2-carboxamide and is designated GNTbm-12.In one embodiment, the compound of Formula (I) is 5-(4-(6-methylpyridin-3-yl)butanamido)-N-(2-aminophenyl)pyridine-2-carboxamide and is designated GNTbm-19. In one embodiment, the compound of Formula (I) is 5-(4-(6-methylpyridin-3-yl)butanamido)-N-(2-amino-4-(trifluoromethyl)phenyl)pyridine-2-carboxamide and is designated GNTbm-25. In one embodiment, the compound of Formula (I) is 4-((E)-4-(pyridin-3-yl)but-3-enamido)-N-(2-amino-4-(trifluoromethyl)phenyl)benzamide and is designated GNTbm-33. In one embodiment, the compound of Formula (I) is 4-(4-(pyridin-3-yl)butanamido)-N-(2-amino-4-fluorophenyl)benzamide and is designated GNTbm-37. In one embodiment, the compound of Formula (I) is 4-((E)-4-(pyridin-3-yl)but-3-enamido)-N-(2-aminophenyl)benzamide and is designated GNTbm-38. In one embodiment, the compound of Formula (I) is 4-((E)-4-(pyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)benzamide and is designated GNTbm-39.

[0010] In other embodiments, the present disclosure provides pharmaceutical compositions or combinations comprising compounds described herein.

[0011] In other embodiments, the present disclosure provides a method for epigenetic immune modulation of the TME and / or treatment of cancer, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition or combination comprising any one or more of the compounds of Formula (I) or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof.

[0012] In some embodiments, the methods are for inducing cell cycle arrest of tumor cells, for inducing apoptosis of tumor cells, for inducing histone H3 acetylation, for inducing immune memory, for activating CTLs, for reducing immune suppressor cells.

[0013] In other embodiments, the present disclosure provides the use of an effective amount of a compound or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or combination in the manufacture of a medicament for epigenetic immunomodulation of the TME and / or treatment of cancer in a subject in need thereof.

[0014] In some embodiments, the medicament is for inducing cell cycle arrest in tumor cells, for inducing apoptosis in tumor cells, for inducing histone H3 acetylation, for inducing immunological memory, for activating CTLs, or for reducing immunosuppressive cells.

[0015] In other embodiments, the present disclosure provides a method for treating or preventing a class I HDAC-associated disease in a subject, comprising administering to a subject in need thereof an effective amount of a compound or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or combination.

[0016] In other embodiments, the disclosure provides the use of an effective amount of a compound or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or combination in the manufacture of a medicament for treating or preventing a disease associated with Class I HDAC in a subject in need thereof. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the structures of compounds GNTbm-01, GNTbm-02, and GNTbm-03. [Figure 2(a)]NMR and high-resolution MS spectra: (a) H-NMR spectroscopic data of compound GNTbm-01, (b) H-NMR spectroscopic data of compound GNTbm-02, (c) H-NMR spectroscopic data of compound GNTbm-03, (d) high-resolution MS spectroscopic data of compound GNTbm-01, (e) high-resolution MS spectroscopic data of compound GNTbm-02, and (f) high-resolution MS spectroscopic data of compound GNTbm-03. [Figure 2(b)] NMR and high-resolution MS spectra: (a) H-NMR spectroscopic data of compound GNTbm-01, (b) H-NMR spectroscopic data of compound GNTbm-02, (c) H-NMR spectroscopic data of compound GNTbm-03, (d) high-resolution MS spectroscopic data of compound GNTbm-01, (e) high-resolution MS spectroscopic data of compound GNTbm-02, and (f) high-resolution MS spectroscopic data of compound GNTbm-03. [Figure 2(c)] NMR and high-resolution MS spectra: (a) H-NMR spectroscopic data of compound GNTbm-01, (b) H-NMR spectroscopic data of compound GNTbm-02, (c) H-NMR spectroscopic data of compound GNTbm-03, (d) high-resolution MS spectroscopic data of compound GNTbm-01, (e) high-resolution MS spectroscopic data of compound GNTbm-02, and (f) high-resolution MS spectroscopic data of compound GNTbm-03. [Figure 2(d)] NMR and high-resolution MS spectra: (a) H-NMR spectroscopic data of compound GNTbm-01, (b) H-NMR spectroscopic data of compound GNTbm-02, (c) H-NMR spectroscopic data of compound GNTbm-03, (d) high-resolution MS spectroscopic data of compound GNTbm-01, (e) high-resolution MS spectroscopic data of compound GNTbm-02, and (f) high-resolution MS spectroscopic data of compound GNTbm-03. [Figure 2(e)]NMR and high-resolution MS spectra: (a) H-NMR spectroscopic data of compound GNTbm-01, (b) H-NMR spectroscopic data of compound GNTbm-02, (c) H-NMR spectroscopic data of compound GNTbm-03, (d) high-resolution MS spectroscopic data of compound GNTbm-01, (e) high-resolution MS spectroscopic data of compound GNTbm-02, and (f) high-resolution MS spectroscopic data of compound GNTbm-03. [Figure 2(f)] NMR and high-resolution MS spectra: (a) H-NMR spectroscopic data of compound GNTbm-01, (b) H-NMR spectroscopic data of compound GNTbm-02, (c) H-NMR spectroscopic data of compound GNTbm-03, (d) high-resolution MS spectroscopic data of compound GNTbm-01, (e) high-resolution MS spectroscopic data of compound GNTbm-02, and (f) high-resolution MS spectroscopic data of compound GNTbm-03. [Figure 3(a)] Figure 1 shows cell morphology observed by phase-contrast light microscopy after treatment: Changes in cell morphology were observed by phase-contrast light microscopy. (a) MDA-MB-231 cells, (b) SW48 cells, (c) M10 cells. [Figure 3(b)] Figure 1 shows cell morphology observed by phase-contrast light microscopy after treatment: Changes in cell morphology were observed by phase-contrast light microscopy. (a) MDA-MB-231 cells, (b) SW48 cells, (c) M10 cells. [Figure 3(c)] Figure 1 shows cell morphology observed by phase-contrast light microscopy after treatment: Changes in cell morphology were observed by phase-contrast light microscopy. (a) MDA-MB-231 cells, (b) SW48 cells, (c) M10 cells. [Figure 4(a)] Figure 1 shows results from the evaluation of GNTbm-02-induced cell cycle arrest at G0 / G1 phase in MDA-MB-231 cells: Evaluation was performed after treatment with GNTbm-02 and entinostat in MDA-MB-231 cells in a dose- and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 4(b)] Figure 1 shows results from the evaluation of GNTbm-02-induced cell cycle arrest at G0 / G1 phase in MDA-MB-231 cells: Evaluation was performed after treatment with GNTbm-02 and entinostat in MDA-MB-231 cells in a dose- and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 4(c)] Figure 1 shows results from the evaluation of GNTbm-02-induced cell cycle arrest at G0 / G1 phase in MDA-MB-231 cells: Evaluation was performed after treatment with GNTbm-02 and entinostat in MDA-MB-231 cells in a dose- and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 4(d)] Figure 1 shows results from the evaluation of GNTbm-02-induced cell cycle arrest at G0 / G1 phase in MDA-MB-231 cells: Evaluation was performed after treatment with GNTbm-02 and entinostat in MDA-MB-231 cells in a dose- and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 5(a)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell cycle arrest at G0 / G1 phase in SW48 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in SW48 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 5(b)]Figure 1 shows the results from the evaluation of GNTbm-02-induced cell cycle arrest at G0 / G1 phase in SW48 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in SW48 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 5(c)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell cycle arrest at G0 / G1 phase in SW48 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in SW48 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 5(d)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell cycle arrest at G0 / G1 phase in SW48 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in SW48 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 6(a)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell cycle arrest at G2 / M phase in M10 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in M10 cells in a dose- and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 6(b)]Figure 1 shows the results from the evaluation of GNTbm-02-induced cell cycle arrest at G2 / M phase in M10 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in M10 cells in a dose- and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 6(c)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell cycle arrest at G2 / M phase in M10 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in M10 cells in a dose- and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 6(d)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell cycle arrest at G2 / M phase in M10 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in M10 cells in a dose- and time-dependent manner. Cells were stained with PI and the percentage of cells in different cell cycle phases was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 7(a)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in MDA-MB-231 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in MDA-MB-231 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 7(b)]Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in MDA-MB-231 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in MDA-MB-231 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 7(c)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in MDA-MB-231 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in MDA-MB-231 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 7(d)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in MDA-MB-231 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in MDA-MB-231 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 8(a)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in SW48 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in SW48 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 8(b)]Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in SW48 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in SW48 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 8(c)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in SW48 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in SW48 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 8(d)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in SW48 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in SW48 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 9(a)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in M10 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in M10 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 9(b)]Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in M10 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in M10 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 9(c)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in M10 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in M10 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 9(d)] Figure 1 shows the results from the evaluation of GNTbm-02-induced cell apoptosis in M10 cells: The evaluation was performed after treatment with GNTbm-02 and entinostat in M10 cells in a dose-dependent and time-dependent manner. Cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed by using a flow cytometer. (a) and (b) Dose-dependent manner. (c) and (d) Time-dependent manner. [Figure 10(a)] Figure 1 shows the results of Western blot analysis of histone H3 acetylation levels in cells treated with GNTbm-02 and entinostat: Representative immunoblot analysis of acetyl histone H3 and β-actin in MDA-MB-231 or SW48 cells. Cells were treated with the indicated concentrations of GNTbm-02 and entinostat for 24 hours. Control cells were incubated with vehicle. (a) (c) Extracts of MDA-MB-231 or SW48 cells treated with GNTbm-02 or entinostat as indicated were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) (d) Quantification of AcH3 protein expression levels was normalized to β-actin and presented as fold change. [Figure 10(b)] Figure 1 shows the results of Western blot analysis of histone H3 acetylation levels in cells treated with GNTbm-02 and entinostat: Representative immunoblot analysis of acetyl histone H3 and β-actin in MDA-MB-231 or SW48 cells. Cells were treated with the indicated concentrations of GNTbm-02 and entinostat for 24 hours. Control cells were incubated with vehicle. (a) (c) Extracts of MDA-MB-231 or SW48 cells treated with GNTbm-02 or entinostat as indicated were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) (d) Quantification of AcH3 protein expression levels was normalized to β-actin and presented as fold change. [Figure 10(c)] Figure 1 shows the results of Western blot analysis of histone H3 acetylation levels in cells treated with GNTbm-02 and entinostat: Representative immunoblot analysis of acetyl histone H3 and β-actin in MDA-MB-231 or SW48 cells. Cells were treated with the indicated concentrations of GNTbm-02 and entinostat for 24 hours. Control cells were incubated with vehicle. (a) (c) Extracts of MDA-MB-231 or SW48 cells treated with GNTbm-02 or entinostat as indicated were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) (d) Quantification of AcH3 protein expression levels was normalized to β-actin and presented as fold change. [Figure 10(d)]Figure 1 shows the results of Western blot analysis of histone H3 acetylation levels in cells treated with GNTbm-02 and entinostat: Representative immunoblot analysis of acetyl histone H3 and β-actin in MDA-MB-231 or SW48 cells. Cells were treated with the indicated concentrations of GNTbm-02 and entinostat for 24 hours. Control cells were incubated with vehicle. (a) (c) Extracts of MDA-MB-231 or SW48 cells treated with GNTbm-02 or entinostat as indicated were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) (d) Quantification of AcH3 protein expression levels was normalized to β-actin and presented as fold change. [Figure 11(a)] Figure 1 shows the time course of induction of histone H3 acetylation by the class I HDAC inhibitor GNTbm-02: MDA-MB-231 or SW48 cells were treated with GNTbm-02 at a concentration of 1 μM for 2, 6, 24, 48, and 72 hours. (a) (c) Extracts of MDA-MB-231 or SW48 cells treated with GNTbm-02, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) (d) Quantification of AcH3 protein expression levels was normalized to β-actin and presented as fold change. [Figure 11(b)] Figure 1 shows the time course of induction of histone H3 acetylation by the class I HDAC inhibitor GNTbm-02: MDA-MB-231 or SW48 cells were treated with GNTbm-02 at a concentration of 1 μM for 2, 6, 24, 48, and 72 hours. (a) (c) Extracts of MDA-MB-231 or SW48 cells treated with GNTbm-02, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) (d) Quantification of AcH3 protein expression levels was normalized to β-actin and presented as fold change. [Figure 11(c)]Figure 1 shows the time course of induction of histone H3 acetylation by the class I HDAC inhibitor GNTbm-02: MDA-MB-231 or SW48 cells were treated with GNTbm-02 at a concentration of 1 μM for 2, 6, 24, 48, and 72 hours. (a) (c) Extracts of MDA-MB-231 or SW48 cells treated with GNTbm-02, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) (d) Quantification of AcH3 protein expression levels was normalized to β-actin and presented as fold change. [Figure 11(d)] Figure 1 shows the time course of induction of histone H3 acetylation by the class I HDAC inhibitor GNTbm-02: MDA-MB-231 or SW48 cells were treated with GNTbm-02 at a concentration of 1 μM for 2, 6, 24, 48, and 72 hours. (a) (c) Extracts of MDA-MB-231 or SW48 cells treated with GNTbm-02, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) (d) Quantification of AcH3 protein expression levels was normalized to β-actin and presented as fold change. [Figure 12(a)]Figure 1 shows the results of Western blot analysis of histone H3 acetylation levels in cells treated with GNTbm-04, GNTbm-05, GNTbm-06, GNTbm-11, GNTbm-38, GNTbm-39, and chidamide (as a positive control). Representative immunoblot analysis of acetyl histone H3 and β-actin in SW48 cells. Cells were treated with the indicated concentrations of compounds for 24 hours. Control cells were incubated with vehicle. (a) Extracts of SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-11, and chidamide as indicated were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) Extracts from SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-06, and chidamide, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (c) Extracts from SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-38, GNTbm-39, and chidamide, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). All data represent quantification of AcH3 protein expression levels normalized to β-actin and shown as fold change. [Figure 12(b)]Figure 1 shows the results of Western blot analysis of histone H3 acetylation levels in cells treated with GNTbm-04, GNTbm-05, GNTbm-06, GNTbm-11, GNTbm-38, GNTbm-39, and chidamide (as a positive control). Representative immunoblot analysis of acetyl histone H3 and β-actin in SW48 cells. Cells were treated with the indicated concentrations of compounds for 24 hours. Control cells were incubated with vehicle. (a) Extracts of SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-11, and chidamide as indicated were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) Extracts from SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-06, and chidamide, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (c) Extracts from SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-38, GNTbm-39, and chidamide, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). All data represent quantification of AcH3 protein expression levels normalized to β-actin and shown as fold change. [Figure 12(c)]Figure 1 shows the results of Western blot analysis of histone H3 acetylation levels in cells treated with GNTbm-04, GNTbm-05, GNTbm-06, GNTbm-11, GNTbm-38, GNTbm-39, and chidamide (as a positive control). Representative immunoblot analysis of acetyl histone H3 and β-actin in SW48 cells. Cells were treated with the indicated concentrations of compounds for 24 hours. Control cells were incubated with vehicle. (a) Extracts of SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-11, and chidamide as indicated were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (b) Extracts from SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-06, and chidamide, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). (c) Extracts from SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-38, GNTbm-39, and chidamide, as indicated, were separated by SDS-PAGE, followed by Western blotting and immunostaining after detection with an antibody against histone H3 acetylation (AcH3). All data represent quantification of AcH3 protein expression levels normalized to β-actin and shown as fold change. [Figure 13(a)]Figure 1 shows results from an evaluation of the therapeutic response of various doses of GNTbm-02 plus celecoxib in combination with an anti-PD-1 antibody in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated. IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (12.5, 25 mg / kg). Total tumor volume (a) and (b), individual tumor volume (c), mouse weight (d), and survival rate (e) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volume reached 3000 mm3. Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA with Tukey's test. Gehan-Breslow-Wilcoxon test (e). *, compared with IgG control. #, compared with PD-1 group. [Figure 13(b)] Figure 1 shows results from an evaluation of the therapeutic response of various doses of GNTbm-02 plus celecoxib in combination with an anti-PD-1 antibody in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated. IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (12.5, 25 mg / kg). Total tumor volume (a) and (b), individual tumor volume (c), mouse weight (d), and survival rate (e) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volume reached 3000 mm3. Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA with Tukey's test. Gehan-Breslow-Wilcoxon test (e). *, compared with IgG control. #, compared with PD-1 group. [Figure 13(c)]Figure 1 shows results from an evaluation of the therapeutic response of various doses of GNTbm-02 plus celecoxib in combination with an anti-PD-1 antibody in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated. IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (12.5, 25 mg / kg). Total tumor volume (a) and (b), individual tumor volume (c), mouse weight (d), and survival rate (e) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volume reached 3000 mm3. Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA with Tukey's test. Gehan-Breslow-Wilcoxon test (e). *, compared with IgG control. #, compared with PD-1 group. [Figure 13(d)] Figure 1 shows results from an evaluation of the therapeutic response of various doses of GNTbm-02 plus celecoxib in combination with an anti-PD-1 antibody in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated. IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (12.5, 25 mg / kg). Total tumor volume (a) and (b), individual tumor volume (c), mouse weight (d), and survival rate (e) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volume reached 3000 mm3. Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA with Tukey's test. Gehan-Breslow-Wilcoxon test (e). *, compared with IgG control. #, compared with PD-1 group. [Figure 13(e)]Figure 1 shows results from an evaluation of the therapeutic response of various doses of GNTbm-02 plus celecoxib in combination with an anti-PD-1 antibody in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated. IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (12.5, 25 mg / kg). Total tumor volume (a) and (b), individual tumor volume (c), mouse weight (d), and survival rate (e) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volume reached 3000 mm3. Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA with Tukey's test. Gehan-Breslow-Wilcoxon test (e). *, compared with IgG control. #, compared with PD-1 group. [Figure 14(a)] Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(b)] Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(c)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(d)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(e)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(f)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(g)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(h)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(i)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(j)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(k)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(l)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(m)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(n)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(o)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(p)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(q)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(r)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(s)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(t)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 14(u)]Figure 1 shows evaluation of combination therapy response of the GNTbm compound series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); chidamide (50 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (5, 10, 20, 25, 50 mg / kg); GNTbm-03 (50 mg / kg); GNTbm-04 (50 mg / kg); GNTbm-06 (50 mg / kg); regorafenib (30 mg / kg). Total tumor volume (a), (b), (f), (j), (n), (r), individual tumor volumes (c), (g), (k), (o), (s), mouse weights (d), (h), (l), (p), (t), and survival rates (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and euthanized after tumor implantation when tumor volumes reached 3000 mm3. Data are shown as mean ± SEM; one-way ANOVA with Tukey's test (*P<0.05, **P<0.01, ***P<0.001 vs. anti-IgG control). Gehan-Breslow-Wilcoxon test (e). [Figure 15(a)] Figure 1 shows treatment results from BALB / c nude mice bearing CT26 tumors treated with various therapeutic modalities: anti-IgG control (2.5 mg / kg); anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (10 mg / kg). (a) Scheme of subcutaneous injection of CT26 tumors and different treatment groups (n = 6 mice per group). (b) Total tumor volume. (c) Tumor volume fold change. (d) Mouse body weight. (e) Individual tumor volume. CT26 tumor-bearing nude mice were treated as indicated and euthanized after tumor implantation when the tumor volume reached 3000 mm3. [Figure 15(b)]Figure 1 shows treatment results from BALB / c nude mice bearing CT26 tumors treated with various therapeutic modalities: anti-IgG control (2.5 mg / kg); anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (10 mg / kg). (a) Scheme of subcutaneous injection of CT26 tumors and different treatment groups (n = 6 mice per group). (b) Total tumor volume. (c) Tumor volume fold change. (d) Mouse body weight. (e) Individual tumor volume. CT26 tumor-bearing nude mice were treated as indicated and euthanized after tumor implantation when the tumor volume reached 3000 mm3. [Figure 15(c)] Figure 1 shows treatment results from BALB / c nude mice bearing CT26 tumors treated with various therapeutic modalities: anti-IgG control (2.5 mg / kg); anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (10 mg / kg). (a) Scheme of subcutaneous injection of CT26 tumors and different treatment groups (n = 6 mice per group). (b) Total tumor volume. (c) Tumor volume fold change. (d) Mouse body weight. (e) Individual tumor volume. CT26 tumor-bearing nude mice were treated as indicated and euthanized after tumor implantation when the tumor volume reached 3000 mm3. [Figure 15(d)] Figure 1 shows treatment results from BALB / c nude mice bearing CT26 tumors treated with various therapeutic modalities: anti-IgG control (2.5 mg / kg); anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (10 mg / kg). (a) Scheme of subcutaneous injection of CT26 tumors and different treatment groups (n = 6 mice per group). (b) Total tumor volume. (c) Tumor volume fold change. (d) Mouse body weight. (e) Individual tumor volume. CT26 tumor-bearing nude mice were treated as indicated and euthanized after tumor implantation when the tumor volume reached 3000 mm3. [Figure 15(e)]Figure 1 shows treatment results from BALB / c nude mice bearing CT26 tumors treated with various therapeutic modalities: anti-IgG control (2.5 mg / kg); anti-PD-1 monoclonal antibody (2.5 mg / kg); celecoxib (50 mg / kg); GNTbm-02 (10 mg / kg). (a) Scheme of subcutaneous injection of CT26 tumors and different treatment groups (n = 6 mice per group). (b) Total tumor volume. (c) Tumor volume fold change. (d) Mouse body weight. (e) Individual tumor volume. CT26 tumor-bearing nude mice were treated as indicated and euthanized after tumor implantation when the tumor volume reached 3000 mm3. [Figure 16(a)] Figure 1 shows the effect of GNTbm-02 on inhibiting HDAC3 enzymatic activity: (a) Evaluation was performed after incubation of 2 μM GNTbm-02, chidamide, or entinostat (including assay buffer) with HDAC3 enzyme for 20, 40, and 60 minutes. GNTbm-02 was shown to bind to HDAC3 and inhibit it more potently than entinostat. (b) Evaluation was performed after incubation of 2 μM GNTbm-02, GNTbm-03, or GNTbm-01 (including assay buffer) with HDAC3 enzyme for 20, 40, and 60 minutes. GNTbm-02 was shown to bind to HDAC3 and inhibit it more potently than GNTbm-03 and GNTbm-01. [Figure 16(b)]Figure 1 shows the effect of GNTbm-02 on inhibiting HDAC3 enzymatic activity: (a) Evaluation was performed after incubation of 2 μM GNTbm-02, chidamide, or entinostat (including assay buffer) with HDAC3 enzyme for 20, 40, and 60 minutes. GNTbm-02 was shown to bind to HDAC3 and inhibit it more potently than entinostat. (b) Evaluation was performed after incubation of 2 μM GNTbm-02, GNTbm-03, or GNTbm-01 (including assay buffer) with HDAC3 enzyme for 20, 40, and 60 minutes. GNTbm-02 was shown to bind to HDAC3 and inhibit it more potently than GNTbm-03 and GNTbm-01. [Figure 17(a)] Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(b)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(c)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(d)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(e)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(f)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(g)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(h)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(i)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(j)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). [Figure 17(k)]Figure 1 shows that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear and T cell responses in a CT26-bearing model: BALB / c mice bearing CT26 tumors were treated with the indicated therapeutic modalities, followed by FACS analysis to assess circulating immune cells. Mean and SD are shown, and P values ​​are indicated. Blood samples were isolated 16 days after treatment in CT26-bearing mice. (a) FACS results for circulating lymphocyte cells. (b) FACS results for circulating monocytic cells. (c) FACS results for circulating granulocyte cells. (d) FACS results for circulating CD3+ T cells. (e) FACS results for circulating CD4+ T cells. (f) FACS results for circulating CD8+ T cells. (g) FACS results for circulating Treg cells. (h) FACS results for circulating CD11b+ cells. (i) FACS results for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS results for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS results for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells. Mean ± SD is shown for n = 8-12 mice per group. One-way ANOVA and Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 vs. IgG control). DETAILED DESCRIPTION OF THE INVENTION

[0018] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and apply the terms in the context of their use in describing this disclosure. The terminology used in this description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0019] Where a range of values ​​is expressed, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed within the invention, unless the context clearly dictates otherwise (e.g., in the case of a group containing a number of carbon atoms, when each number of carbon atoms within the range is recited). The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0020] The articles "a" and "an," as used in this specification and the appended claims, are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless the context clearly dictates otherwise. By way of example, "an element" means one element or more than one element.

[0021] The term "and / or," as used in the specification and claims, should be understood to mean "one or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or not to those elements specifically identified.

[0022] The terms "halo" and "halogen," as used herein, refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0023] The term "alkyl" refers to a straight or branched hydrocarbon chain radical, consisting solely of carbon and hydrogen atoms, containing no unsaturation, and having from 1 to 15 carbon atoms (e.g., C1-C15 In certain embodiments, alkyl includes 1 to 13 carbon atoms (e.g., C1 to C 13 In certain embodiments, alkyl contains 1 to 8 carbon atoms (e.g., C1-C8 alkyl). In other embodiments, alkyl contains 1 to 5 carbon atoms (e.g., C1-C5 alkyl). In other embodiments, alkyl contains 1 to 4 carbon atoms (e.g., C1-C4 alkyl). In other embodiments, alkyl contains 1 to 3 carbon atoms (e.g., C1-C3 alkyl). In other embodiments, alkyl contains 1 to 2 carbon atoms (e.g., C1-C2 alkyl). In other embodiments, alkyl contains 1 carbon atom (e.g., C1 alkyl). In other embodiments, alkyl contains 5 to 15 carbon atoms (e.g., C5-C6 alkyl). 15 alkyl). In other embodiments, alkyl contains 5 to 8 carbon atoms (e.g., C5-C8 alkyl). In other embodiments, alkyl contains 2 to 5 carbon atoms (e.g., C2-C5 alkyl). In other embodiments, alkyl contains 3 to 5 carbon atoms (e.g., C3-C5 alkyl). In other embodiments, alkyl groups are selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), and 1-pentyl (n-pentyl). An alkyl is attached to the remainder of the molecule by a single bond. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted with one or more substituents. The term "alkenyl," as used herein, refers to a monovalent group derived from a hydrocarbon moiety, in certain embodiments, containing 2 to 6 or 2 to 8 carbon atoms and having at least one carbon-carbon double bond. The double bond may or may not be the point of attachment to another group. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl, and the like.

[0024] The term "alkoxy" refers to a group attached through an oxygen atom of the formula --O-alkyl, where alkyl is an alkyl chain as defined above.

[0025] The term "alkenyl" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and having from 2 to 12 carbon atoms. In certain embodiments, an alkenyl contains from 2 to 8 carbon atoms. In other embodiments, an alkenyl contains from 2 to 4 carbon atoms. An alkenyl is attached to the remainder of the molecule by a single bond and is, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted with one or more substituents.

[0026] The term "cycloalkyl," as used herein, refers to a monovalent group derived from a monocyclic or polycyclic saturated or partially unsaturated carbocyclic ring compound. Examples of C3-C8-cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, and cyclooctyl.

[0027] The term "aryl," as used herein, refers to a fused or non-fused, monocyclic or polycyclic carbocyclic ring system having one or more aromatic rings, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl, and the like.

[0028] The term "heteroaryl," as used herein, refers to a monocyclic or polycyclic (e.g., bicyclic, or tricyclic or higher polycyclic), fused or unfused, group or ring system having at least one aromatic ring and having 5 to 10 ring atoms, one of which is selected from S, O, and N, zero, one, or two ring atoms being an additional heteroatom independently selected from S, O, and N, and the remaining ring atoms being carbon. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like.

[0029] The term "heterocycloalkyl," as used herein, refers to a fused or non-fused system of non-aromatic 3-, 4-, 5-, 6-, or 7-membered rings or bicyclic or tricyclic groups, in which (i) at least one ring contains between 1 and 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen; (ii) each 5-membered ring has 0 to 1 double bond and each 6-membered ring has 0 to 2 double bonds; (iii) the nitrogen and sulfur heteroatoms are optionally oxidized; (iv) the nitrogen heteroatom is optionally quaternized; and (iv) any of the above rings are optionally fused to a benzene ring. Representative heterocycloalkyl groups include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0030] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases and inorganic or organic acids. Salts of basic compounds encompassed by the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of the present invention, which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts of basic compounds of the present disclosure include, but are not limited to, acetate, ascorbate, adipate, alginate, aspirate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, clavulanate, citrate, cyclopentanepropionate, diethylacetic acid, digluconate, dihydrochloride, dodecylsulfanate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptanoate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, and hydrabamate. (hydrabamate), hydrobromide, hydrochloride, 2-hydroxyethanesulfonate, hydroxynaphthoate, hydroiodide, iodide, isonicotinic acid, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl nitrate, methyl sulfate, methanesulfonate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, oleate, oxalate Acid salts, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, phosphate / diphosphate, pimelate, phenylpropionate, polygalacturonate, propionate, salicylate, stearate, sulfate, monoacetate, succinate, tannate, tartrate, teoclate, thiocyanate, tosylate, triethiodide, trifluoroacetate, undecanoate, valerate, and the like.Additionally, when the compound of the present invention contains an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases such as aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, cyclic amines, dicyclohexylamine, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. Also included are basic nitrogen-containing groups that can be quaternized with agents such as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, e.g., dimethyl sulfate, diethyl sulfate, dibutyl sulfate; and diamyl sulfate; long chain halides, e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides, e.g., benzyl and phenethyl bromides; and the like.

[0031] The term "subject" includes living organisms, such as humans, monkeys, cows, sheep, horses, pigs, cattle, goats, dogs, cats, mice, rats, cultured cells thereof, and transgenic species. In a preferred embodiment, the subject is a human.

[0032] The term "administering" includes routes of administration that allow the active ingredients of the present invention to perform their intended function.

[0033] The term "treat" or "treatment" refers to a method of reducing the effects of a disease or condition. Treatment can refer to a method of reducing not only the symptoms but also the underlying cause of the disease or condition itself. Treatment can be any reduction from natural levels, including, but not limited to, complete elimination of the disease, condition, or symptoms of the disease or condition.

[0034] The terms "prevent", "prevention" or "preventing" refer to the inhibition or avoidance of symptoms associated with a target disease.

[0035] The phrase "therapeutically effective amount" refers to an amount of a compound, substance, or composition, including a compound of the present disclosure, that is effective for producing a desired therapeutic effect at a reasonable benefit / risk ratio applicable to any medical treatment.

[0036] Class I HDAC inhibitor compounds Epigenetic therapies for cancer, such as histone deacetylase inhibitors, can stimulate antitumor immunity by upregulating antigen processing and presentation mechanisms. Epigenetic modifications play an important role in controlling tumor initiation and progression. Epigenetic regulation is primarily achieved through two major mechanisms that affect gene expression: DNA methylation / demethylation, which occurs by the addition / removal of methyl groups to DNA, and histone acetylation / deacetylation, which occurs by the enzymatic addition / removal of acetyl groups to histone proteins wrapped around DNA. Histone deacetylase inhibitors (HDACi) are considered promising targets for new drug development. The fundamental mechanism by which HDACs play an important role in cancer is by controlling the degree of acetylation in histone or nonhistone proteins, which are involved in the regulation of cell cycle, differentiation, apoptosis, DNA damage response, angiogenesis, metastasis, and other cellular processes.

[0037] Class I HDACs are primarily located in the cell nucleus, are ubiquitously expressed in human tissues, and play an important role in regulating cell proliferation, differentiation, and cell cycle progression. Class I HDACs are highly expressed in certain cancers. For example, HDAC1 is highly expressed in prostate cancer, gastric cancer, colon cancer, breast cancer, lung cancer, and esophageal cancer; HDAC2 is highly expressed in gastric cancer, cervical cancer, and colorectal cancer; and HDAC3 is highly expressed in colon cancer and breast cancer. Uncontrolled expression of HDACs leads to the silencing of many genes that inhibit cell growth, thereby causing the loss of cell growth surveillance and the control of cell differentiation, cell cycle arrest, and apoptosis. Dysregulation of HDAC overexpression has been significantly correlated with tumor malignancy and poor prognosis. Many class I HDAC inhibitors have epigenetic immunomodulatory properties.

[0038] The mechanism of immunomodulation by HDAC inhibitors in the TME has been reported to involve both soluble factors and immune cell components. By inhibiting specific HDAC isoforms, HDAC inhibitors alter the expression of various genes and proteins through epigenetic regulation, resulting in a shift in the TME state to a mode favorable for cancer cell killing. Previously published studies have shown that some HDAC inhibitors have immunomodulatory properties, such as regulating cytokine / chemokine secretion, antigen-presenting cells, reducing the number or function of Tregs, and inducing NK cell activation. Other studies have demonstrated mechanisms for enhancing cancer antigen expression and modulating the activity of immune suppressor cells such as MDSCs. Selective class I HDAC inhibitors can increase PD-L1 and MHC I expression on cancer cells. Furthermore, class I HDAC inhibitors downregulate myeloid-derived suppressor cells (MDSCs) that infiltrate the tumor microenvironment.

[0039] In one aspect, the present disclosure provides a compound of formula (I):

[0040] [ka] wherein W and Y are each independently selected from CH and N; each R1 is independently selected from hydrogen, halogen, C1-C3 alkyl, and halogenated C1-C3 alkyl, and may be mono-, di-, tri-, or tetra-substituted; C1 and C2 are C atoms connected by a single or double bond, Ar is the following:

[0041] [ka] wherein Ar is connected to C2 via a solid line; R2 has the same meaning as described for R1; R3 is hydrogen or C1-C3 alkyl. or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[0042] In one embodiment, the compound of formula (I) has the following formula (Ia):

[0043] [ka] wherein W, Y, R1, C1, C2 and Ar have the same meanings as described above. or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[0044] In one embodiment, Ar is selected from a 6-membered ring. In one embodiment, R2 and the atom of Ar that is connected to C2 are in the para position.

[0045] In one embodiment, Ar is:

[0046] [ka] is selected from the group consisting of:

[0047] In one embodiment, W and Y are selected from the following combinations: (1) W is N and Y is CH, (2) W is CH and Y is N, and (3) W and Y are CH. In a preferred embodiment, W is N or CH and Y is CH.

[0048] In one embodiment, R1 is F or fluorinated C1-C3 alkyl. In one embodiment, the fluorinated C1-C3 alkyl is CF3.

[0049] In one embodiment, R1 is hydrogen.

[0050] In one embodiment, C1 and C2 are C atoms connected by a double bond. In another embodiment, C1 and C2 are C atoms connected by a single bond.

[0051] In one embodiment, R2 is C1-C3 alkyl or fluorinated C1-C3 alkyl. In one embodiment, C1-C3 alkyl is CH3. In one embodiment, fluorinated C1-C3 alkyl is CF3.

[0052] In one embodiment, R2 is hydrogen.

[0053] In one embodiment, Ar is

[0054] [ka] where R2 and the atom of Ar connected to C2 are in the para position, and C1 and C2 are C atoms connected by a double bond.

[0055] In one embodiment, Ar is

[0056] [ka] wherein R2 and the atom of Ar connected to C2 are in the para position, and R1 is hydrogen or F.

[0057] In one embodiment, Ar is

[0058] [ka] wherein R2 and the atom of Ar connected to C2 are in the para position, and R2 is hydrogen or CH3.

[0059] In one embodiment, R1 is hydrogen or F and R2 is hydrogen or CH3.

[0060] In one embodiment, R1 is hydrogen or F, R2 is hydrogen or CH3, and C1 and C2 are C atoms connected by a double bond.

[0061] In one embodiment, Ar is

[0062] [ka] wherein R2 and the atom of Ar connected to C2 are in the para position, R1 is hydrogen or F, R2 is hydrogen or CH3, and C1 and C2 are C atoms connected by a double bond.

[0063] In one embodiment, the compound of formula (I) is the following compound:

[0064] [ka] TIFF0007748392000012.tif149162 or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[0065] The present disclosure encompasses all stereoisomeric forms of the compounds of formula (I). All asymmetric centers present in the compounds of formula (I) may, independently of one another, have either the (R) or the (S) configuration. When a bond to a chiral carbon is depicted as a straight line in a structural formula of the present invention, it is understood that both (R) and (S) are configurations of the chiral carbon, and thus both enantiomers and mixtures thereof are encompassed within the formula. When a specific configuration is depicted, that enantiomer (either (R) or (S) at that center) is intended. Similarly, when a compound name is given without a chiral designation for a chiral carbon, it is understood that both (R) and (S) are configurations of the chiral carbon, and thus individual enantiomers and mixtures thereof are encompassed within the name.

[0066] The present invention includes all possible enantiomers, regioisomers, and diastereomers, as well as mixtures of two or more stereoisomers in all ratios, for example, mixtures of enantiomers and / or diastereomers. Accordingly, enantiomers are subject of the present invention in enantiomerically pure form, as both levorotatory and dextrorotatory antipodes, in the form of racemates, and in the form of mixtures of the two enantiomers in all ratios. In the case of cis / trans isomerism, the present invention includes both the cis and trans forms, as well as mixtures of these forms in all ratios. The preparation of individual stereoisomers, if desired, can be carried out by conventional methods, for example, by separation of the mixture by chromatography or crystallization, by using stereochemically uniform starting materials for synthesis, or by stereoselective synthesis. Optionally, derivatization can be carried out before the separation of stereoisomers. Separation of the mixture of stereoisomers can be carried out at the stage of intermediates during the synthesis of the compound of formula (I) or on the final racemic product. Absolute stereochemistry can be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereocenter of known configuration. Where the compounds of the invention are capable of tautomerization, all individual tautomers and mixtures thereof are included within the scope of the invention. The present disclosure includes all such isomers, as well as salts, solvates (including hydrates) and solvated salts of such racemates, enantiomers, diastereomers and tautomers and mixtures thereof.

[0067] As used herein, the symbols and conventions used in these processes, schemes, and examples are consistent with those used in modern scientific literature, e.g., the Journal of the American Chemical Society or the Journal of Biological Chemistry, regardless of whether a particular abbreviation is specifically defined. Specifically, but not by way of limitation, the following abbreviations may be used in the examples and throughout the specification: g (gram); mg (milligram); mL (milliliter); μL (microliter); mM (millimolar); M (micromolar); Hz (hertz); MHz (megahertz); mmol (millimolar); hr or hrs (hours); min (minutes); MS (mass spectrometry); ESI (electrospray ionization); TLC (thin layer chromatography); and HPLC (high pressure liquid chromatography). Standard workup and purification methods known to those skilled in the art can be utilized for all of the following examples. Unless otherwise indicated, all temperatures are in °C (degrees Celsius). Unless otherwise noted, all reactions are performed at room temperature. The synthetic methodologies presented herein are intended to illustrate the applicable chemistry through the use of specific examples and are not indicative of the scope of the disclosure.

[0068] The compounds of formula (I) of the present disclosure are prepared according to general chemical synthesis procedures. Exemplary synthetic routes are shown below:

[0069] [ka] [In the formula, R A is in formula (I).

[0070] [ka] corresponds to the part R B is in formula (I).

[0071] [ka] corresponding to the part].

[0072] In this route, N,N'-dicyclohexylcarbodiimide (DCC) and dichloromethane (DCM) can be used under condition a, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HOBt), and N,N-dimethylformamide (DMF) can be used under condition b.

[0073] Other appropriate modifications to the process, such as using suitable protecting / deprotecting agents for groups sensitive to certain reaction conditions during the synthesis, isolating and purifying intermediates for subsequent reactions, selecting appropriate solvents, etc., can also be introduced by those skilled in the art as needed. For example, the —OH group of compound (β) in the above pathway may be protected before reacting with compound (α), and the resulting product may be deprotected to give compound (γ).

[0074] Pharmaceutical Compositions / Combinations In another aspect, the present invention provides a pharmaceutical composition / combination comprising a compound of any of formula (I) or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, together with a pharmaceutically acceptable carrier.

[0075] The pharmaceutical composition / combination may further comprise one or more second agents. In one embodiment, the second agent is an immune checkpoint inhibitor, an NSAID, a tyrosine kinase inhibitor (TKI), or an anti-cancer agent. In a further embodiment, the pharmaceutical composition / combination comprises a compound described herein and an immune checkpoint inhibitor and / or an NSAID, or optionally a tyrosine kinase inhibitor (TKI).

[0076] In one embodiment, immune checkpoint inhibitors can be used in combination with the pharmaceutical combinations described herein to stimulate the immune system against cancer cells and treat cancer. The immune checkpoint inhibitor is an anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibody or agent, an anti-programmed cell death protein 1 (PD-1) antibody or agent, an anti-programmed death-ligand 1 (PD-L1) antibody or agent, an anti-T-cell immunoglobulin and mucin domain-3 (TIM-3) antibody or agent, an anti-B-lymphocyte and T-lymphocyte attenuator (BTLA) antibody or agent, an anti-V-domain Ig-containing suppressor of T-cell activation (VISTA) antibody or agent, an anti-lymphocyte-activation gene-3 (LAG-3) antibody or agent, a KIR (killer cell immunoglobulin-like receptor) inhibitor or antibody, an A2AR (adenosine A2A receptor) inhibitor or antibody, a CD276 inhibitor or antibody, or a VTCN1 inhibitor or antibody. More preferably, the immune checkpoint inhibitor is pembrolizumab, lambrolizumab, pidilizumab, nivolumab, durvalumab, avelumab, or atezolizumab. Examples of PD-1 or PD-L1 inhibitors include, but are not limited to, humanized antibodies that block human PD-1, such as lambrolizumab (anti-PD-1 Ab, Keytruda™) or pidilizumab (anti-PD-1 Ab), Bavencio (anti-PD-L1 Ab, avelumab), Imfinzi (anti-PD-L1 Ab, durvalumab), and Tecentriq (anti-PD-L1 Ab, atezolizumab), and fully human antibodies, such as nivolumab (anti-PD-1 Ab, Opdivo™) and cemiplimab-rwlc (anti-PD-1 Ab, Libtayo™). Other PD-1 inhibitors may include soluble PD-1 ligand presentations, including, but not limited to, B7-DC-Ig or the PD-L2 Fc fusion protein also known as AMP-244, as well as other PD-1 inhibitors currently under investigation and / or development for use in therapy.Additionally, immune checkpoint inhibitors may include, but are not limited to, humanized or fully human antibodies that block PD-L1, such as durvalumab and MIH1 (an anti-CD274 (PD-L1, B7-H1) monoclonal antibody), as well as other PD-L1 inhibitors currently under investigation.

[0077] NSAIDs are a class of drugs that relieve pain, reduce fever, and, at higher doses, reduce inflammation. Most NSAIDs inhibit the activity of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), thereby inhibiting the synthesis of thromboxanes and prostaglandins. While COX-2 inhibition leads to anti-inflammatory, analgesic, and antipyretic effects, NSAIDs that also inhibit COX-1, particularly aspirin, are thought to cause gastrointestinal bleeding and ulcers at high doses. COX-2 inhibitors are widely used to treat autoimmune and inflammatory diseases. Cyclooxygenase (COX) has two isoforms, COX-1 and COX-2, and is the rate-limiting enzyme in the synthesis of prostanoids, the bioactive lipids consisting of prostaglandin D2 (PGD2), PGE2, PGF2α, prostacyclin PGI2, and thromboxane TXA2. COX-1 is constitutively expressed in body tissues to maintain homeostatic prostanoids and is involved in several biological functions, such as angiogenesis, vasodilation, and tissue maintenance. However, COX-2 is expressed at low levels under normal conditions. COX-2 is rapidly induced by stimuli, such as infection, injury, and pain, and initiates proinflammatory processes. Selective COX-2 inhibitors are a type of nonsteroidal anti-inflammatory drug (NSAID). In some embodiments, NSAIDs include, but are not limited to, aspirin, ibuprofen, indomethacin, naproxen, and COX-2 inhibitors. In some embodiments of the present disclosure, the NSAID is a COX-2 inhibitor. In some embodiments, COX-2 inhibitors include, but are not limited to, Celebrex (generic name celecoxib), rofecoxib, imrecoxib, and etoricoxib. Preferably, the COX-2 inhibitor is celecoxib.

[0078] Tyrosine kinase inhibitors (TKIs) are a family of small molecules that inhibit either cytosolic tyrosine kinases or receptor tyrosine kinases. TKIs inhibit these growth factor signaling pathways through various mechanisms. They compete for ATP, substrates, or dimerization sites, and can also act allosterically. Inhibition of cytosolic or receptor tyrosine kinases has been demonstrated by several different classes of TKIs, for example, through direct competition for ATP binding to the tyrosine kinase, allosteric inhibition of the tyrosine kinase, and inhibition of ligand binding to the receptor tyrosine kinase. TKIs, particularly VEGFR inhibitors such as axitinib, lenvatinib, cabozantinib, and regorafenib, are playing an increasingly important role in treating cancer. In some embodiments of the present disclosure, the TKI is a receptor tyrosine kinase inhibitor. Preferably, the TKI is a vascular endothelial growth factor receptor (VEGFR) inhibitor. More preferably, the TKI is cabozantinib, regorafenib, axitinib, afatinib, nintedanib, crizotinib, alectinib, trametinib, dabrafenib, sunitinib, ruxolitinib, vemurafenib, sorafenib, ponatinib, encorafenib, brigatinib, pazopanib, dasatinib, imatinib, lenvatinib, vandetanib, surufatinib or sitravatinib.

[0079] The additional anticancer agent is any anticancer agent described herein or known in the art. In one embodiment, the additional anticancer agent is chemotherapy or platinum-based doublet chemotherapy. In certain embodiments, the additional anticancer agent is a tyrosine kinase inhibitor (TKI). In one embodiment, the additional anticancer agent is an anti-VEGF or anti-VEGFR antibody or compound. In other embodiments, the anticancer agent is a platinum agent (e.g., cisplatin, carboplatin), a mitotic inhibitor (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel, taxotere, docecad), a fluorinated vinca alkaloid (e.g., vinflunine, javlor), vinorelbine, vinblastine, etoposide, or pemetrexed gemcitabine. In one embodiment, the additional anticancer agent is 5-fluorouracil (5-FU). In certain embodiments, the additional anti-cancer agent is any other anti-cancer agent known in the art.

[0080] To prepare the pharmaceutical compositions / combinations of the present invention, one or more compounds of the present disclosure as active ingredients are thoroughly mixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral, e.g., intramuscular, administration. Any of the usual pharmaceutical media may be used to prepare the compositions into oral dosage forms. Thus, for liquid oral preparations, e.g., suspensions, elixirs, and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc.; for solid oral preparations, e.g., powders, capsules, caplets, gelcaps, and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. Tablets and capsules are the most advantageous oral dosage unit forms due to their ease of administration, and in this case, a solid pharmaceutical carrier is naturally used. If desired, tablets may be sugar-coated or enteric-coated using standard techniques. In parenteral formulations, the carrier will usually comprise sterile water, although other ingredients may be included, for example, to aid solubility or for preservative purposes. Injectable suspensions can also be prepared, in which case appropriate liquid carriers, suspending agents, and the like can be used. The pharmaceutical compositions herein will contain the amount of active ingredient required to deliver an effective dose, as described above, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful, etc.

[0081] Liquid forms into which the novel compositions of the present disclosure may be incorporated for oral or injectable administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions containing edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone, or gelatin.

[0082] Tablets and capsules for oral administration are usually provided in unit dose form and contain conventional excipients such as binding agents, fillers (including cellulose, mannitol, lactose), diluents, tableting agents, lubricants (including magnesium stearate), detergents, disintegrants (e.g., polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate), colorants, flavoring agents, and wetting agents (e.g., sodium lauryl sulfate).

[0083] Oral solid compositions can be prepared by conventional methods of blending, filling, or tabletting. Blending operations can be repeated to distribute the active ingredient throughout compositions containing large amounts of filler. Such operations are conventional.

[0084] For parenteral administration, a fluid unit dose can be prepared containing the compound and a sterile vehicle. The compound can be suspended or dissolved, depending on the vehicle and concentration. Parenteral solutions are usually prepared by dissolving the compound in a vehicle, sterilizing by filtration, filling a suitable vial, and sealing it. Advantageously, adjuvants such as local anesthetics, preservatives, and buffering agents can also be dissolved in the vehicle. To increase stability, the composition can be frozen after filling the vial and removing the water under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound can be suspended in the vehicle instead of dissolved, and can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent can be included in the composition to promote uniform distribution of the compound of the present application.

[0085] Pharmaceutical preparations for administration by inhalation can be delivered from insufflator or nebulizer pressurized packs.

[0086] therapeutic application In another aspect, the present disclosure provides a method for epigenetic immune modulation of the TME, comprising administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition / combination described herein.

[0087] In another aspect, the present disclosure provides a method for treating or preventing a class I HDAC-associated disease in a subject, comprising administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition / combination described herein.

[0088] In one embodiment, the method further comprises administering one or more second agents. In some embodiments, the second agent is an immune checkpoint inhibitor, an NSAID, a TKI, or an anti-cancer agent. In further embodiments, the pharmaceutical composition / combination comprises a compound described herein and an immune checkpoint inhibitor and / or an NSAID, or optionally a TKI. Embodiments of the immune checkpoint inhibitor, NSAID, TKI, or anti-cancer agent are those described herein.

[0089] The compounds of the present invention are useful for treating or preventing any disease and / or condition in which inhibition of class I HDAC is desired. In particular, the compounds of the present invention have epigenetic immunomodulation of the TME, thereby improving immunotherapy. Inhibition of HDAC enzyme activity may lead to a reduction in tumor growth. Thus, the present invention provides a method for treating or preventing tumors or cancer.

[0090] Examples of cancers that can be treated according to the present teachings include, but are not limited to, invasive breast cancer, adenocarcinoma, lung cancer (non-small cell, squamous cell, adenocarcinoma, and large cell lung cancer), liver cancer, colorectal cancer, brain, head and neck cancer (e.g., neuro / glioblastoma), breast cancer, ovarian cancer, bladder transitional cell carcinoma, prostate cancer, oral squamous cell carcinoma, osteosarcoma, adrenocortical carcinoma, gastrointestinal tumors including colorectal cancer, biliary tract cancers, e.g., gallbladder cancer (GBC), and others. , bladder cancer, esophageal cancer, gastric cancer, cervical cancer, salivary gland cancer, diarrheal benign neoplasms, ductal carcinoma in situ, paronychia, bile duct carcinoma, kidney cancer, pancreatic cancer, medulloblastoma, glioblastoma, luminal, HER2-positive and triple-negative breast tumors, hematological malignancies and leukemias (acute myeloid leukemia (AML), B-cell precursor acute lymphoblastic leukemia (ALL), T-cell fraction ALL, and chronic myeloid leukemia (CML)).

[0091] The compound, or a pharmaceutically acceptable salt thereof, may be administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrathoracically, intrathecally, and parenterally. In one embodiment, the compound is administered orally. Those skilled in the art will recognize the advantages of certain routes of administration.

[0092] The dosage regimen utilizing the compound is selected according to a variety of factors, including, for example, the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition being treated; the route of administration; the patient's renal and hepatic function; and the particular compound or salt thereof being used. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.

[0093] Although the invention has been described herein by way of written description, those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the above description and the following examples are intended to be illustrative, and not limiting, of the appended claims. [Example]

[0094] Materials and methods for preparing exemplary compounds of the invention are described below.

[0095] GNTbm-01, GNTbm-02, GNTbm-03, GNTbm-04, GNTbm-05, GNTbm-06, GNTbm-08, GNTbm-11, GNTbm-12, GNTbm-19, GNTbm-25, GNTbm-33, GNTbm-37, GNTbm-38, GNTbm-39, entinostat-API (active pharmaceutical ingredient), and chidamide-API were provided by GNTbm (GNT Biotech & Medicals Co. Ltd, Taiwan). Celecoxib capsule product (Celebrex®, 200 mg) was purchased from Pfizer (Taiwan). Regorafenib (HY-1031, 30 mg / kg, daily, p.o., MedChemExpress, USA). The following antibodies and reagents were used in animal experiments: mouse anti-PD-1 (CD279) monoclonal antibody (RMP1-14; Bio X Cell) and rat anti-IgG2a isotype monoclonal antibody (2A3; Bio X Cell). Electrospray ionization mass spectra were recorded on a Bruker microTOF, and electrospray mass spectra (ESMS) were recorded as m / z values ​​using a Waters mass spectrometer. All commercial chemicals and solvents were reagent grade and used without further purification unless otherwise noted. All reactions were monitored for completion by thin-layer chromatography using Merck 60 F254 silica gel glass-backed plates (20 × 20 cm). The resulting chromatograms were visualized by visual detection under UV irradiation (254 nm). 1 H NMR and 13 C NMR was recorded on a Bruker AVANCE 400 MHz PLUS and a Bruker AVANCE III HD 600 MHz mass spectrometer and instrument, and chemical shifts were reported in parts per million (ppm, δ). Multiplicities are reported as s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), td (triplet of doublets), and m (multiplet). Coupling constants (J) are expressed in Hertz. The purity of the final compounds was determined by a C18 The determination was performed using a Waters ACQUITY Arc system with a Waters XSelect HSS T3 5 μm column (4.6 mm × 250 mm). Elution was performed using water containing 0.1% trifluoroacetic acid as mobile phase A and methanol as mobile phase B. The elution conditions were: 90% Phase A + 10% Phase B at 0 min; 70% Phase A + 30% Phase B at 6 min; 50% Phase A + 50% Phase B at 12 min; 10% Phase A + 90% Phase B at 18 min; and 90% Phase A + 10% Phase B at 23 min. The mobile phase flow rate was 1 mL / min, the sample injection volume was 10 μL, and the run time was 30 min. Peaks were detected at 254 nm. The purity of the final compound was found to be >90%.

[0096] Preparation example

[0097] [Example 1] GNTbm-01 The synthetic route is shown below:

[0098] [ka]

[0099] 6-Aminopyridine-3-carboxylic acid (1). To a solution of methyl 6-aminopyridine-3-carboxylate (1.2 g) was added LiOH (3.309 g) in MeOH, and the mixture was stirred at 40-65° C. for 4-8 hours. After cooling to room temperature, the mixture was acidified with 10% HCl (aq), filtered by suction, and the product was dried in an oven for approximately 24 hours to obtain the solid product, Compound 1.

[0100] 2-(Trimethylsilyl)ethyl 6-aminopyridine-3-carboxylate (2). To a solution of compound 1 (1.5 g) and triphenylphosphine (2.848 g) in THF, 2-(trimethylsilyl)ethanol (1.84 mL mmol) and diisopropyl azodicarboxylate (DIAD, 2.56 mL) were added at −5 to 10° C. The mixture was stirred at room temperature for approximately 8 hours. The mixture was concentrated and purified by silica gel column chromatography to give compound 2.

[0101] 2-(Trimethylsilyl)ethyl 6-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)pyridine-3-carboxylate (3). To a solution of DCC (86.9 mg) in DCM, compound 2 (50 mg) and (E)-4-(6-methylpyridin-3-yl)but-3-enoic acid (67.2 mg) in DCM were added in an ice bath. The mixture was stirred at room temperature for approximately 8 hours. The product was extracted using ethyl acetate, and the organic layer was washed with water. The combined organic layer was dried over MgSO, concentrated, and purified by silica gel column chromatography to give compound 3.

[0102] 6-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)pyridine-3-carboxylic acid (4). To a solution of compound 3 (50 mg) in THF (11 mL) was added 12 N HCl (11 mL), and the mixture was stirred at room temperature for 4-10 hours. The mixture was concentrated and purified by silica gel column chromatography to give compound 4.

[0103] 6-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)pyridine-3-carboxamide (5). A solution of 4-fluorobenzene-1,2-diamine (72.9 mg), EDC (89.7 mg), and HOBt (46.8 mg) in DMF was stirred at −10 to 10° C. for 20 to 60 minutes. Compound 4 (85.9 mg) in DMF and EtN (161 μL) was added, and the mixture was stirred at room temperature for approximately 72 hours. The mixture was diluted with water and extracted with EtOAc. The combined organic layers were dried over MgSO, concentrated, and purified by silica gel column chromatography to produce compound 5. 1 H NMR (400 MHz, acetone-d): δ 2.46 (3H, s), 3.52 (2H, d), 4.95 (2H, br), 6.39 (1H, td), 6.59 (3H, m), 7.21 (1H, t), 7.77 (1H, dd), 8.38 (3H, m), 9.08 (1H, s), 9.75 (1H, s). 13 C NMR (100 MHz, DMSO-d6): δ23.77, 40.47, 101.16, 101.41, 101.80, 102.03, 123.05, 124.73, 125.54, 128.76, 128.86, 129.21, 129.56, 132.84, 136.68, 138.02, 145.71, 147.16, 148.12, 156.82, 163.76, 170.27; + ].

[0104] [Example 2] GNTbm-02 The synthetic route is shown below:

[0105] [ka]

[0106] 5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)pyridine-2-carboxylic acid (6). A solution of (E)-4-(6-methylpyridin-3-yl)but-3-enoic acid (769 mg) and DCC (895 mg) in DCM was stirred at −10 to 10° C. for 20 to 60 minutes. 6-Aminopyridine-3-carboxylic acid (500 mg) in DCM was added, and the mixture was stirred at room temperature for approximately 48 hours. The mixture was filtered to collect the solid powder. The solid powder was dissolved in MeOH, filtered, and concentrated by rotavapor to give crude compound 6.

[0107] 5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)pyridine-2-carboxamide (7). A solution of 4-fluorobenzene-1,2-diamine (42.4 mg), EDC (52.2 mg), and HOBt (26 mg) in DMF was stirred at −10 to 10° C. for 20 to 60 minutes. 5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)pyridine-2-carboxylic acid (compound 6) (50 mg) in DMF was added, and the mixture was stirred at room temperature for approximately 16 hours. The mixture was diluted with water and extracted with EtOAc. The combined organic layer was dried over MgSO4, concentrated, and purified by silica gel column chromatography to give compound 7. 1 H NMR (400 MHz, acetone-d6): δ2.46(3H, s), 3.45(2H, d), 4.90(1H, br), 6.45(1H, m), 6.55(2H, m), 6.66(1H, dd), 7.19(1H, d), 7.53(1H, dd), 7.76(1H, dd), 8.15(1H, d), 8.31(1H, dd), 8.47(1H, d), 8.91(1H, s), 9.66(1H, s), 9.72(1H, s). 13C NMR (100 MHz, MeOD-d): δ41.93, 104.06, 104.32, 105.16, 105.39, 124.08, 125.12, 125.94, 128.14, 128.41, 128.65, 130.93, 132.17, 135.42, 139.91, 141.19, 146.07, 147.83, 158.38, 165.26, 168.08, 172.56.ESI-MS m / z: 428.1479[M+Na + ].

[0108] [Example 3] GNTbm-03 The synthetic route is shown below:

[0109] [ka]

[0110] 4-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)benzoic acid (8). A solution of (E)-4-(6-methylpyridin-3-yl)but-3-enoic acid (671.9 mg) and DCC (782.4 mg) in DCM was stirred at -10 to 10°C for 20 to 60 minutes. 4-Aminobenzoic acid (400 mg) in DCM was added, and the mixture was stirred at room temperature for an additional 5 to 10 hours. The mixture was filtered to collect the solid powder. The solid powder was dissolved in MeOH, filtered, and concentrated by rotavapor to produce crude compound 8.

[0111] 4-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)benzamide (9). A solution of 4-fluorobenzene-1,2-diamine (255.4 mg), EDC (314.4 mg), and HOBt (164 mg) in DMF was stirred at −10 to 10° C. for 20 to 60 minutes. 4-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)benzoic acid (compound 8) (300 mg) in DMF was added, and the mixture was stirred at room temperature for approximately 24 hours. The mixture was diluted with water and extracted with EtOAc. The combined organic layers were dried over MgSO4, concentrated, and purified by silica gel column chromatography to produce compound 9. 1 H NMR (400 MHz, acetone-d6): δ2.46(3H, s), 3.39(2H, d), 4.90(1H, br), 6.39(1H, td), 6.56(3H, m), 7.20(2H,m), 7.77(3H, m), 8.00(2H, m), 8.46(1H, s), 8.95(1H, s), 9.46(1H, s). 13 C NMR (100 MHz, DMSO-d6): δ23.75, 40.77, 101.48, 102.55, 118.23, 119.42, 123.07, 124.98, 128.51, 128.62, 128.76, 128.90, 129.05, 129.59, 132.85, 141.97, 145.51, 147.14, 156.79, 159.82, 162.19, 165.03, 169.36. + ].

[0112] [Example 4] GNTbm-04, GNTbm-05, GNTbm-11, GNTbm-33, GNTbm-37, GNTbm-38, and GNTbm-39 The synthetic route is shown below:

[0113] [ka]

[0114] Ethyl (E)-4-(pyridin-3-yl)but-3-enoate (11). A solution of nicotinaldehyde 10 (10 g, 93 mmol), PPh3 (36.7 g, 140 mmol), and ethyl acrylate (15.3 mL, 140 mmol) in n-hexanol (50 mL) was stirred at 120-160 °C for 12-18 h. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 11 (6 g, 34%) as a yellow liquid. 1 H NMR (600 MHz, CDCl3) δ 8.58 (d, J = 1.8 Hz, 1H), 8.46 (dd, J = 4.8, 1.5 Hz, 1H), 7.70 (dt, J = 7.9, 1.8 Hz, 1H), 7.24 (dd, J = 7.9, 4.9 Hz, 1H), 6.48 (d, J = 16.0 Hz, 1H), 6.38 (dt, J = 15.9, 7.0 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.27 (dd, J = 7.0, 1.3 Hz, 2H), 1.29 (t, J = 7.1Hz, 3H).

[0115] (E)-4-(Pyridin-3-yl)but-3-enoic acid (12). To a solution of 11 (6 g, 31 mmol) in THF (100 mL), LiOH (2.25 g, 94 mmol in 50 mL of HO) was added and stirred at RT for 1-4 h. The mixture was concentrated to remove THF. The aqueous solution was acidified with 1 N HCl (aq). The mixture was concentrated to dryness, and the crude product was purified by column chromatography to give compound 12 (3.7 g, 72%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 8.58 (d, J = 2.0 Hz, 1H), 8.42 (dd, J = 4.7, 1.5 Hz, 1H), 7.87 (dt, J = 8.0, 1.9 Hz, 1H), 7.34 (dd, J = 8.0, 4.7 Hz, 1H), 6.52 (d, J = 16.0 Hz, 1H), 6.45 (dt, J = 16.0, 6.5 Hz, 1H), 3.21 (d, J = 6.5 Hz, 2H).

[0116] Procedure for the synthesis of GNTbm-04, GNTbm-05, GNTbm-11, GNTbm-33, GNTbm-38, and GNTbm-39. To a solution of compound 12 (1 equivalent), compound 13 (1.1 equivalents), and HATU (1.1 equivalents) in DMF was added DIPEA (1-2.5 equivalents). The mixture was stirred at room temperature for 1-4 hours (monitored by LCMS). Aniline (1.1 equivalents), HATU (1.1 equivalents), and DIPEA (1-2.5 equivalents) were added to the reaction mixture. The mixture was stirred at room temperature for an additional 1-4 hours (monitored by LCMS). The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give the desired product.

[0117] GNTbm-04, yield: 45 mg, 40%. 1H NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.86 (s, 1H), 8.92 (d, J =1.8 Hz, 1H), 8.63 (d, J = 1.8 Hz, 1H), 8.44 (d, J = 4.8 Hz, 1H), 8.25 (dd, J = 8.7, 2.1 Hz, 1H), 8.09 (d, J = 9 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.38-7.32 (m, 2H), 6.62-6.55 (m, 3H), 6.39(td, J = 8.7, 2.4 Hz, 1H), 5.20 (s, 2H), 3.42 (d, J = 6 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 40.48, 101.95, 102.12, 102.41, 102.56, 119.74, 122.81, 123.69, 125.78, 126.52, 126.73, 126.79, LCMS (ESI) m / z 392.4 [M+H] + HPLC purity: 96.12%.

[0118] GNTbm-05, yield: 88mg, 53%. 11H NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.94 (s, 1H), 8.92 (d, J = 1.6 Hz, 1H), 8.63 (d, J = 1.5 Hz, 1H), 8.44 (d, J = 4.7 Hz, 1H), 8.26 (dd, J = 8.5, 2.0 Hz, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.36 (dd, J = 7.9, 4.8 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.82 (d, J = 7.9 Hz, 1H), 6.65 (t, J = 7.9 Hz, 1H), 6.61 - 6.57 (m, 2H), 4.88 (s, 2H), 3.42 (d, J = 6.0 Hz, 2H). 13 13C NMR (100 MHz, DMSO-d6): δ 40.49, 116.75, 117.01, 122.76, 123.68, 124.16, 124.29, 125.67, 125.78, 126.58, 129.29, 132.28, 132.56, 138.26, 138.97, 141.�6, 144.44, 147.80, 148.39, 161.86, 169.76. LCMS (ESI) m / z 374.3 [M+H] + . HPLC purity: 99.32%.

[0119] GNTbm-11, Yield: 48 mg, 22%. 11H NMR (600 MHz, DMSO-d6) δ 10.62 (s, 1H), 10.01 (s, 1H), 8.94 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.6 Hz, 1H), 8.44 (d, J = 4.6 Hz, 1H), 8.26 (dd, J = 8.6, 2.3 Hz, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.77 (s, 1H), 7.36 (dd, J = 7.9, 4.7 Hz, 1H), 7.27 (d, J = 8.5 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.62 - 6.55 (m, 2H), 5.63 (s, 2H), 3.42 (d, J = 6.0 Hz, 2H). 13 13C NMR (100 MHz, DMSO-d6): δ 40.49, 115.71, 121.95, 122.90, 122.94, 123.68, 125.76, 126.55, 129.30, 132.28, 132.57, 138.42, 138.97, 144.13, 145.75, 147.8, 162.50, 169.79. LCMS (ESI) m / z 442.4 [M+H] + . HPLC purity: 93.63%.

[0120] Yield of GNTbm-33: 63 mg, 16%. 11H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.60, (s, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.44 (dd, J = 4.7, 1.5 Hz, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.91 (dt, J = 8.0, 1.8 Hz, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 1.2 Hz, 1H), 7.36 (dd, J = 7.9, 4.8 Hz, 1H), 7.27 (dd, J = 8.5, 1.7 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.62 - 6.55 (m, 2H), 5.65 (s, 2H), 3.38 (d, J = 5.5 Hz, 2H). 13 13C NMR (100 MHz, DMSO-d6): δ 40.70, 115.22, 115.42, 115.63, 118.21, 122.44, 123.33, 123.69, 123.79, 124.10, 125.89, 126.19, 128.71, 128.82, 129.06, 132.34, 132.54, 142.05, 146.76, 147.78, 148.35, 165.15, 169.21. LCMS (ESI) m / z 441.4 [M+H] + . HPLC purity: 96.40%.

[0121] GNTbm-33, Yield: 108 mg, 47%. 11H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H), 9.56 (s, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.44 (dd, J = 4.7, 1.4 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.91 (dt, J = 8.0, 1.9 Hz, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.36 (dd, J = 7.9, 4.7 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H), 6.96 (dt, J = 7.9, 1.4 Hz, 1H), 6.78 (dd, J = 8.0, 1.2 Hz, 1H), 6.62 - 6.57 (m, 3H), 4.87 (s, 2H), 3.37 (d, J = 5.5 Hz, 2H). 13 13C NMR (100 MHz, DMSO-d6): δ 40.69, 116.10, 116.24, 118.23, 118.34, 123.46, 123.68, 126.20, 126.32, 126.60, 128.66, 129.0, 129.04, 132.33, 132.53, 141.87, 143.08, 147.78, 148.34, 164.67, 169.17. LCMS (ESI) m / z 373.4 [M+H] + . HPLC purity: 94.41%.

[0122] GNTbm - 39, Yield: 60 mg, 25%. 1H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H), 9.49 (s, 1H), 8.62 (d, J = 2.0 Hz, 1H), 8.44 (dd, J = 4.7, 1.6 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.90 (dt, J = 8.0, 1.9 Hz, 1H), 7.72 (d, J = 8.7 Hz, 2H), 7.36 (dd, J = 7.9, 4.8 Hz, 1H), 7.11 (dd, J = 8.4, 6.6 Hz, 1H), 6.62-6.57 (m, 2H), 6.54 (dd, J = 11.2, 2.9 Hz, 1H), 6.35 (td, J = 8.5, 2.8 Hz, 1H), 5.20 (s, 2H), 3.37 (d, J = 5.5 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 40.69, 101.35, 101.52, 101.92, 102.07, 118.20, 118.32, 119.40, 123.68, 126.20, 128.42, 128.49, 128.68, 128.89, 129.05, 132.33, 132.53, 141.89, 145.38, 145.45, 147.78, 148.34, 160.14, 161.73, 164.95, 169.17.LCMS (ESI) m / z 391.4 [M+H] + . HPLC purity: 94.66%.

[0123] Synthesis of GNTbm-37 To a solution of GNTbm-39 (0.11 g, 0.3 mmol) in MeOH (2 mL), Pd / C (22 mg) was added and stirred at room temperature for 8–16 h. The mixture was filtered through a Celite pad, and the filtrate was concentrated to dryness to give GNTbm-37 (95 mg, 86%) as a white solid.

[0124] GNTbm-37, yield: 110mg, 49%. 1H NMR (600 MHz, DMSO-d6) δ 10.20 (s, 1H), 9.52 (s, 1H), 8.45 (s, 1H), 8.41 (d, J = 4.0 Hz, 1H), 7.93 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.1 Hz, 2H), 7.66 (d, J = 7.6 Hz, 1H), 7.32 (dd, J = 7.3, 4.9 Hz, 1H), 7.10 (t, J = 7.1 Hz, 1H), 6.54 (dd, J = 11.1, 2.0 Hz, 1H), 6.35 (t, J = 7.2 Hz, 1H), 5.21 (s, 2H), 2.66 (t, J = 7.4 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 1.93 (m, J = 7.4 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 26.20, 31.56, 35.63, 99.13, 101.36, 101.52, 101.91, 102.06, 118.07, 119.44, 123.45, 128.44, 128.64, 135.84, 136.95, 142.06, 145.39, 145.47, 147.22, 149.63, 160.13, 161.71, 164.98, 171.20. LCMS (ESI) m / z 393.4 [M+H] + HPLC purity: 95.87%.

[0125] GNTbm-06 and GNTbm-12. The synthetic path is as follows:

[0126]

change

[0127] (E)-4-(6-Methyl-3-pyridyl)but-3-enoic acid (15). To a dry round-bottom flask containing 2-carboxyethyl(triphenyl)phosphonium bromide (37.7 g, 90.8 mmol), anhydrous THF (200 mL) was added, and the solution was cooled to -20 to 40 °C. To the white suspension, 2.00 M NaHMDS in THF (82.6 mL) was added dropwise. The resulting orange solution was stirred at -20 to 40 °C for 1 to 5 h. 6-Methylpyridine-3-carbaldehyde (10.0 g, 82.6 mmol) was added, and the resulting mixture was stirred at room temperature for 8 to 20 h. The reaction mixture was quenched with water (10 mL) and concentrated to dryness. The mixture was added to water (300 mL) and washed with EA (200 mL) and DCM (200 mL). The organic layer was removed, and the aqueous layer was acidified with 6N HCl (aq) and washed with EA (200 mL) and DCM (200 mL). The organic layer was removed, and the aqueous layer was pH adjusted with 4N NaOH (aq) and concentrated to dryness. The residue was purified by column chromatography to give (£)-4-(6-methyl-3-pyridyl)but-3-enoic acid (5.10 g, 35%) as a white solid.

[0128] Procedure for the synthesis of GNTbm-06 and GNTbm-12. To a solution of compound 15 (1 equivalent), compound 13b (1.1 equivalents), and HATU (1.1 equivalents) in DMF was added DIPEA (1-2.5 equivalents). The mixture was stirred at room temperature for 1-4 hours (monitored by LCMS). Aniline (1.1 equivalents), HATU (1.1 equivalents), and DIPEA (1-2.5 equivalents) were added to the reaction mixture. The mixture was stirred at room temperature for an additional 1-4 hours (monitored by LCMS). The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give the desired product.

[0129] GNTbm-06, yield: 95 mg, 43%. 11H NMR (600 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.94 (s, 1H), 8.91 (s, 1H), 8.47 (s, 1H), 8.26 (dd, J = 8.7, 2.0 Hz, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.82 (d, J = 7.9 Hz, 1H), 6.65 (t, J = 7.6 Hz, 1H), 6.57 (d, J = 16.1 Hz, 1H), 6.50 (dt, J = 15.5, 7.0 Hz, 1H), 4.88 (s, 2H), 3.40 (d, J = 6.7 Hz, 2H), 2.45 (s, 3H). 13 13C NMR (100 MHz, DMSO-d6): δ 23.71, 40.49, 116.75, 117.01, 122.75, 122.98, 124.16, 124.29, 124.48, 125.66, 126.56, 129.25, 129.47, 132.80, 138.28, 138.96, 139.05, 141.55, 144.43, 147.12, 156.80, 161.86, 169.87. LCMS (ESI) m / z 388.4 [M+H] + . HPLC purity: 94.56%.

[0130] GNTbm-12, Yield: 45 mg, 14%. 1H NMR (600 MHz, DMSO-d6) δ 10.61 (s, 1H), 10.01 (s, 1H), 8.94 (d, J = 2.2 Hz, 1H), 8.48 (d, J = 1.7 Hz, 1H), 8.26 (dd, J = 8.6, 2.3 Hz, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.80 (dd, J = 8.1, 2.0 Hz, 1H), 7.77 (s, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 16.1 Hz, 1H), 6.50 (dt, J = 15.8, 6.9 Hz, 1H), 5.63 (s, 2H), 3.40 (d, J = 6.8 Hz, 2H), 2.45 (s, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 23.70, 40.49, 115.71, 121.92, 122.90, 122.94, 122.98, 124.46, 126.53, 129.26, 129.47, 132.80, 138.43, 138.96, 144.11, 145.74, 147.12, 156.81, 162.49, 169.89. LCMS (ESI) m / z 456.5 [M+H] + HPLC purity: 92.94%.

[0131] [Example 6] GNTbm-08, GNTbm-19, and GNTbm-25 The synthetic path is as follows:

[0132]

change

[0133] 4-(6-Methylpyridin-3-yl)butanoic acid (17). To a solution of 15 (1 g, 5.6 mmol) in MeOH (10 mL), Pd / C (200 mg) was added and stirred at room temperature for 1-8 h. The mixture was filtered through a Celite pad, and the filtrate was concentrated to dryness to give compound 17 (1 g, 99%) as a white solid. 1 H NMR (600 MHz, DMSO-d6) δ 12.07 (s, 1H), 8.26 (d, J = 2.0 Hz, 1H), 7.49 (dd, J = 7.9, 2.3 Hz, 1H), 7.16 (d, J = 7.9 Hz, 1H), 2.55 (t, J = 7.7 Hz, 1H), 2.41 (s, 1H), 2.20 (t, J = 7.4 Hz, 1H), 1.77 (quintet, J = 7.5 Hz, 1H).

[0134] Procedure for the synthesis of GNTbm-08, GNTbm-19, and GNTbm-25. To a solution of compound 17 (1 eq.), compound 13b (1.1 eq.), and HATU (1.1 eq.) in DMF, DIPEA (1–2.5 eq.) was added. The mixture was stirred at room temperature for 1–4 h (monitored by LCMS). Aniline (1.1 eq.), HATU (1.1 eq.), and DIPEA (1–2.5 eq.) were added to the reaction mixture. The mixture was stirred at room temperature for an additional 1–4 h (monitored by LCMS). The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give the designed product.

[0135] GNTbm-8, yield: 35 mg, 25%. 1H NMR (600 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.85 (s, 1H), 8.86 (d, J = 2.3 Hz, 1H), 8.31 (d, J = 1.9 Hz, 1H), 8.23 ​​(dd, J = 8.6, 2.4 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.53 (dd, J = 7.9, 2.1 Hz, 1H), 7.34 (dd, J = 8.6, 6.4 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H), 6.58 (dd, J = 11.1, 2.9 Hz, 1H), 6.39 (td, J = 12.8, 2.8 Hz, 1H), 5.19 (s, 2H), 2.62 (t, J = 7.5 Hz, 2H), 2.42 (s, 3H), 2.40 (t, J = 7.5 Hz, 2H), 1.92 (quintet, J = 7.5 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 23.54, 26.90, 31.10, 35.47, 101.97, 102.14, 102.42, 102.57, 119.78, 122.68, 126.31, 126.67, LCMS (ESI) m / z 408.5 [M+H] + HPLC purity: 93.92%.

[0136] GNTbm-19, yield: 43mg, 20%. 1H NMR (600 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.92 (s, 1H), 8.86 (s, 1H), 8.31 (s, 1H), 8.25 (d, J = 8.5 Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.82 (d, J = 7.9 Hz, 1H), 6.65 (t, J = 7.6 Hz, 1H), 4.88 (s, 2H), 2.62 (t, J = 7.4 Hz, 2H), 2.42 (s, 3H), 2.40 (t, J = 7.4 Hz, 2H), 1.92 (quintet, J = 7.4 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 23.53, 26.09, 31.10, 35.47, 116.76, 117.02, 122.68, 124.20, 124.23, 125.63, 126.36, 133.25, 136.14, 138.36, 136.85, 141.51, 144.20, 148.77, 155.28, 161.87, 171.77. LCMS (ESI) m / z 390.4 [M+H] + HPLC purity: 99.12%.

[0137] GNTbm-25, yield: 30mg, 12%. 1H NMR (600 MHz, DMSO-d6) δ 10.43 (s, 1H), 10.00 (s, 1H), 8.88 (d, J = 1.7 Hz, 1H), 8.31 (s, 1H), 8.24 (dd, J = 8.7, 1.8 Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.77 (s, 1H), 7.53 (dd, J = 8.1, 1.8 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 5.62 (s, 2H), 2.62 (t, J = 7.5 Hz, 2H), 2.42 (s, 3H), 2.40 (t, J = 7.1 Hz, 2H), 1.92 (quintet, J = 7.4 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 23.54, 26.09, 31.11, 35.48, 115.73, 116.07, 116.28, 131.89, 122.69, 122.89, 122.94, 124.06, LCMS (ESI) m / z 458.5 [M+H] + . HPLC purity: 92.03%.

[0138] [Example 7] Determination of saturated solubility of chidamide, GNTbm-02, GNTbm-03, GNTbm-04, and GNTbm-06 A 5 mg sample of compound was added to a 5 ml volumetric flask containing ddH2O and shaken at 100 rpm in an incubator at 25°C for 90 minutes. The resulting suspension was filtered through a 0.22 μm filter. The concentration of compound was determined spectrophotometrically at 256 nm. The saturated solubility of each sample was determined in triplicate, and the mean and standard deviation were reported.

[0139] [Example 8] In Vitro Cytotoxicity Assay Human breast cancer cell line MDA-MB-231 (6 × 10 3 ), MDA-MB-453 (2.4×10 4 pieces), SK-BR-3(6×10 3 cells), human breast epithelial cell line M10 (6 × 10 3 cells), human gastric cancer NCI-N87 (2.4 × 10 4 cells), and human colorectal adenocarcinoma SW48 (2.4 × 10 4 Six different cell lines, including 1000 cells / ml, were used and seeded in 96-well plates. The cell lines were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan. All cell lines were treated with compounds, including the GNTbm compound series, chidamide (as a positive control), and entinostat (as a positive control), at doses ranging from 50 μM to 0.39 μM, and then incubated at 37°C under 5% CO2 for 72 hours. After 72 hours, cell viability was determined using an MTT assay (Cayman™). MDA-MB-231, MDA-MB-453, and SK-BR-3 cell lines were maintained in DMEM / F12 supplemented with 10% FBS and 0.2% antibiotics (MycoZap™, Pluse-CL). The M10 cell line was maintained in MEM Alpha (Gibco™) supplemented with 10% FBS, 0.2% antibiotics (MycoZap™, Pluse-CL). The NCI-N87 cell line was maintained in RPMI 1640 (CORNING™) supplemented with 10% FBS, 0.2% antibiotics (MycoZap™, Pluse-CL). The SK-BR-3 cell line was maintained in DMEM (CORNING™) supplemented with 10% FBS, 0.2% antibiotics (MycoZap™, Pluse-CL).

[0140] [Example 9] IC for HDAC1, 2, and 3 enzyme inhibition 50 The measured values ​​of HDAC assays were performed according to standard protocols (Fluorgenic HDAC 1, 2, and 3 Assay Kit, BPS Bioscience™). All compounds, including chidamide and entinostat as positive controls, at doses ranging from 20 μM to 1.28 nM were mixed with kit buffer and incubated at 37°C for 1 hour. After 1 hour, assay developer was added to the samples, and absorbance was read at the fluorogenic wavelength. Relative inhibition of HDAC 1, 2, and 3 activity in each sample was determined.

[0141] [Example 10] The kinetics of HDAC3 enzyme inhibition by GNTbm-02 were determined. HDAC3 enzyme kinetics assay was performed according to standard protocols (Fluorgenic HDAC3 Assay Kit, BPS Bioscience™). A 2 μM dose of the GNTbm-01, GNTbm-02, and GNTbm-03 compound series, chidamide, and entinostat was mixed with kit buffer and incubated at 37°C for 20, 40, and 60 minutes. After incubation, assay developer was added to the samples, and absorbance was read at the fluorescence emission wavelength. Relative inhibition of HDAC3 activity in each sample was determined.

[0142] [Example 11] IC between GNTbm-02 and entinostat (MS-275) for HDAC1-11 enzyme inhibition 50 It was decided to compare The completed assay report was provided by BPS Bioscience Inc. (6042 Cornerstone Court West, Ste. B, San Diego, CA 92121, USA). The purpose of the study was to determine the effects of two compounds, GNTbm-02 and the positive control entinostat (MS-275), on the activity of recombinant HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, and HDAC11 using an in vitro enzyme assay. HDAC assays were performed according to standard protocols (Fluorgenic HDAC1-11 Assay Kit, BPS Bioscience™). Doses of GNTbm-02 and entinostat (positive control) ranging from 10 μM to 0.51 nM were mixed with kit buffer and incubated at 37°C for 0.5 h. After 0.5 h, assay developer was added to the samples, and absorbance was read at the fluorogenic wavelength. Relative inhibition of HDAC1, 2, 3, 4, 5, 6, 7, 8, 9, and 11 activity was determined for each sample. Further details are described below. All compounds were dissolved in DMSO. Serial dilutions of compounds were initially performed in 100% DMSO with a top concentration of 1 mM. Each intermediate compound dilution (in 100% DMSO) was then diluted 10-fold directly into assay buffer for intermediate dilutions of 10% DMSO in HDAC assay buffer, and 5 μl of the dilution was added to a 50 μl reaction so that the final DMSO concentration in all reactions was 1%. Enzyme reactions for HDAC enzymes were performed in duplicate for 30 minutes at 37°C in a 50 μl mixture containing HDAC assay buffer, 5 μg BSA, HDAC substrate, HDAC enzyme, and test compound. After the enzymatic reaction, 50 μl of 2× HDAC developer was added to each well for HDAC enzyme, and the plate was incubated at room temperature for an additional 15 minutes. Fluorescence intensity was measured using a Tecan Infinite M1000 microplate reader at an excitation of 360 nm and an emission of 460 nm. HDAC activity assays were performed in duplicate at each concentration. Fluorescence intensity data were analyzed using the computer software Graphpad Prism.In the absence of compound, the fluorescence intensity (Ft) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescence intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following formula: % activity = (F - Fb) / (Ft - Fb), where F = fluorescence intensity in the presence of compound. The percent activity values ​​across a range of compound concentrations were then plotted using nonlinear regression analysis of a sigmoidal dose-response curve generated by the formula Y = B + (TB) / 1 + 10 ((LogEC50 - X) × Hill slope), where Y = percent activity, B = minimum percent activity, T = maximum percent activity, X = logarithm of the compound, and Hill slope = slope coefficient or Hill coefficient. IC. 50 Values ​​were determined by the concentration that caused half-maximal percent activity.

[0143] [Example 12] Cell apoptosis and cell cycle arrest were analyzed by flow cytometry. A PI / RNase staining assay (BD Bioscience™) was performed to reveal the presence of cell cycle arrest and apoptotic cells after treatment with GNTbm compounds, chidamide, and entinostat. Human breast cancer cell line MDA-MB-231 (1.5 × 10 5 cells) and human breast epithelial cell line M10 (1.5 × 10 5 Human colorectal adenocarcinoma SW48 cells (5 × 10 cells) were treated with GNTbm compounds, chidamide, and entinostat (1.625–25 μM) for 72 hours, or with the indicated doses for 3–72 hours. 5 Cells were treated with the GNTbm compound series, chidamide, and entinostat (at the indicated doses) for 72 hours, or with the indicated concentrations for 3 to 72 hours. After treatment, cells were harvested, fixed in 80% ethanol for 24 hours, washed with 1x PBS, and stained with PI / RNase I for 15 minutes at room temperature. Cells were then analyzed using a flow cytometer within 1 hour.

[0144] [Example 13] Western blot assay Human breast cancer MDA-MB-231 cells and human colorectal adenocarcinoma SW48 cells were analyzed. MDA-MB-231 and SW48 were obtained from the Bioresource Conservation and Research Center (BCRC, Taiwan). MDA-MB-231 and SW48 were grown in Leibovitz L-15 (catalog number 11415114, Thermo Fisher Scientific) containing 10% heat-inactivated fetal bovine serum (Thermo Scientific) and 1x MycoZap antibiotic (catalog number VZA-2011, Lonza) at 37°C under humidified air supplemented without CO2. Cells were treated with the GNTbm compound series, chidamide, or entinostat for different periods or at various doses. Cells were treated for 24 hours at the indicated doses, or for different periods at the indicated doses. Cell pellets were lysed in RIPA buffer (catalog no. 20-188, Merck) containing protease and phosphatase inhibitors (catalog no. K272, BioVision) and clarified by centrifugation. Equal amounts of total protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (catalog no. 1620177, BIO-RAD). Blots were incubated with primary antibodies against β-actin (catalog no. sc-47778, Santa Cruz Biotechnology), histone 3ac (catalog no. 61637, Active Motif), and HRP secondary antibodies anti-rabbit (ab6721, Abcam) and anti-mouse (sc-2005, Santa Cruz). Blots were developed using ECL Western blotting substrate (catalog no. sc-2048, Santa Cruz Biotechnology). Image blots were analyzed with an iBright FL1000 (Thermo Fisher Scientific) imaging system.

[0145] [Example 14] Anticancer activity in animal models Animal studies were approved and supervised by the Taipei Medical University Institutional Animal Care and Use Committee (TMU IACUC, NO: LAC-2019-0286, LAC-2020-0306). Male BALB / c mice aged 6–8 weeks (National Laboratory Animal Center, Taiwan) were used in each treatment group for all animal experiments. Tumors were grown at a density of 1 × 10 6 or 5 x 10 6 Tumors were established by sc injection of CT26 cells (CRL-2638; murine colorectal adenocarcinoma). The CT26 cell line was purchased from ATCC. CT26 tumor cells were grown in McCoy's 5A medium supplemented with 10% (vol / vol) FBS at 37°C and 5% CO2. CT26 cells were mixed with Matrigel (Cat. No. 354248, Corning®) and inoculated into the left flank of mice. Tumor growth was determined by measuring two perpendicular diameters. Tumors were allowed to grow for 8-11 days (tumor size approximately 150-250 mm). 3 ) followed by randomization and treatment. 3CT26-bearing mice received 2.5 mg / kg of anti-IgG (Cat. No. BE0089, Lot No. 716719J3, Bio X Cell) and anti-PD-1 (Cat. No. BE0146, Lot No. 735019J3, Bio X Cell) antibodies by i.p. administration on days 8, 11, 14, 17, 20, and 23 after tumor implantation. All antibodies were diluted to the appropriate concentration in 100 μL of sterile PBS (pH 7.4) (Invitrogen Life Technologies). Tumor-bearing mice were orally treated daily for 16 days from day 8 to day 23 with various doses of regorafenib (HY-1031, 30 mg / kg, daily po, MedChemExpress, USA), celecoxib (50 mg / kg, daily po, capsules / Celebrex®), chidamide-K30 (50 mg / kg, daily po, GNTbm Manufacturing, Taipei, Taiwan), and GNTbm-02 / k30, GNTbm-03 / k30, GNTbm-04 / k30, GNTbm-05 / k30, GNTbm-06 / k30, GNTbm-11 / k30, GNTbm-38 / k30, and GNTbm-39 / k30 compounds (50, 25, or 12.5 mg / kg, dissolved in water to form a stock solution, daily po). Anticancer activity was assessed by determining whether tumor volume increased from the start of treatment to 3,000 mm 3 The tumor volume was measured as length × width. 2 The calculation was made as ×0.5.

[0146] [Example 15] Survival in animal models Antibody or drug administration was carried out for 16 days from day 8 to day 23. Tumors continued to grow in the tumor-bearing mice. The tumor volumes of the mice were measured every 3 or 4 days (twice a week). When the tumor volume reached 3,000 mm 3 Tumor-bearing mice were considered dead when the tumor-bearing time reached 0. All treatment groups were recorded and analyzed.

[0147] [Example 16] Tumor rechallenge studies in tumor-bearing mouse animal models All mice with a PR / CR response after treatment were re-challenged with CT26 cells on the contralateral side (see Table 6). Re-challenge with CT26 was performed on day 33, 7 days after the first tumor assessment (day 26), with 5x10 cells in the right flank of each mouse. 6 Injections of CT26 cells were administered. After rechallenge with CT26 cells, tumors were allowed to grow for an additional 7 days (day 40) to determine a baseline of 1×. After an additional 10 days (day 50), tumor growth was assessed for rechallenge. A response was considered a regression if it met both of the following criteria: first, tumor size was more than doubled compared to baseline tumor size; second, tumor volume at day 50 was 300 mm 3 If immune memory activity is not sufficiently activated, relapse (recurrence) occurs. If tumor growth is inhibited, it means that immune memory is activated.

[0148] [Example 17] Flow cytometry The following antibodies and reagents were used for flow cytometry: CD8a PerCP-Cy5.5 (53-6.7; BioLegend), CD4 PE (GK 1.5; BioLegend), CD25 PerCP-Cy5.5 (PC61; BioLegend), Foxp3 PE (MF14; BioLegend), CD3 APC (17A2; BioLegend), CD11b APC (M1 / 70; BioLegend), Ly-6C PerCP-Cy5.5 (HK 1.4; BioLegend), Ly-6G PE (1A8; BioLegend), MHC-11-11 PE (BM8; BioLegend), and CD45 FITC (30-F11; BioLegend). Flow cytometry was performed on a FACS Caliber flow cytometer (BD Biosciences), and data were analyzed using FACS Diva software (BD Biosciences). To assess the levels of circulating cell populations, blood samples were collected from mice on days 8, 12, and 16 after the initiation of treatment with GNTbm-02 (12.5–50 mg / kg) or anti-PD-1 antibody (2.5 mg / kg) with or without chidamide (50 mg / kg, as a positive control) plus celecoxib (50 mg / kg). 150 microliters of blood was collected from either the right or left facial vein into a K2EDTA BD Microtainer (BD Biosciences). RBCs from the anticoagulated blood samples were immediately lysed using 2 mL of 1x RBC lysis buffer (Qiagen, Valencia, CA) for 10 minutes, and the samples were washed twice in ice-cold PBS (BD Biosciences). Samples were stained with the appropriate antibodies. For the analysis, we compared the already established phenotypic criteria of these cells with CD45 + CD11b + Ly6G + Ly6C - (PMN-MDSC), CD45 + CD11b + Ly6G - Ly6C + cells (M-MDSC), CD45 + CD3 + CD25 + Foxp3 +cells (Treg), CD45 + CD11b + MHC-II + Ly6C + cells (TAM), and CD45 + CD3 + CD4 + / CD45 + CD3 + CD8 + cells (CD4 + or CD8 + T cells) were used as the common denominator. Total mononuclear cells were used as the common denominator. To assess the level of tumor-infiltrating lymphocytes in tumors, intratumoral CD8+ / - cells were collected from tumor samples excised from mice 12 days after the initiation of GNTbm-02 or anti-PD-1 antibody treatment with or without chidamide plus celecoxib. + , CD4 + We first purified regulatory T cells (Tregs), PMN-MDSCs, M-MDSCs, and TAM cells. Briefly, primary tumor tissues were harvested, weighed, and minced into fine fragments. 1 mg / mL collagenase IV (Sigma-Aldrich) in HBSS (Invitrogen Life Technologies) was added to each sample at a ratio of 1 mL per 200 mg of tumor tissue. Samples were incubated at 37°C for 150 minutes on an end-over-end shaker. The resulting tissue homogenates were filtered through a 0.4 μm filter, washed three times in PBS (BD Biosciences), and separated by Percoll gradient to isolate mononuclear cells, 1 × 10 per sample. 6 CD8 cells were used for antibody labeling. + T cell levels were assessed by CD45 + CD3 + CD8 + Treg cell levels were assessed using previously established phenotypic criteria. + CD3 + CD25 + Foxp3 + PMN-MDSC / M-MDSC cell levels were assessed using previously established phenotypic criteria. + CD11b + Ly6G + Ly6C - / CD45+ CD11b + Ly6G - Ly6C + TAM cell levels were assessed using previously established phenotypic criteria. + CD11b + MHC-II + Ly6C + were assessed using previously established phenotypic criteria, with total mononuclear cells used as the common denominator.

[0149] [Example 18] Anti-cancer activity in nude mouse models Animal studies were approved and supervised by the Taipei Medical University Animal Care and Use Committee (TMU IACUC, NO: LAC-2019-0086). Male BALB / C nude mice aged 6-8 weeks (National Laboratory Animal Center, Taiwan) were used in each treatment group for all animal experiments. Tumors were implanted into the left flank of the mice, with 5 × 10 tumor cells containing Matrigel (Cat. No. 354248, Corning®). 6 Tumors were established by sc injection of CT26 cells, and growth was determined by measuring two perpendicular diameters. Tumors were allowed to grow for 8 days (tumor size approximately 100-150 mm). 3 ) followed by randomization and treatment. 3Upon reaching a median age of 10 days, the animals were euthanized. CT26-bearing mice received 2.5 mg / kg of anti-IgG (Cat. No. BE0089, Lot No. 716719J3, Bio X Cell) and anti-PD-1 (Cat. No. BE0146, Lot No. 735019J3, Bio X Cell) antibodies via ip administration on days 8, 11, 14, 17, 20, and 23 post-implantation. All antibodies were diluted to the appropriate concentrations in 100 μL of sterile PBS (pH 7.4) (Invitrogen Life Technologies). GNTbm compound and celecoxib (200 mg capsules / Celebrex®) were administered orally on day 8 post-implantation. The GNTbm compound (dissolved in DMSO to form a stock solution) was diluted or suspended in water and orally administered to tumor-bearing mice at various doses daily from days 8 to 23. Celecoxib was administered orally from a capsule to tumor-bearing mice at 50 mg / kg on days 8–23. Anticancer activity was assessed by the time tumor volumes increased from the start of treatment to 3,000 mm. 3 The tumor volume was measured as length × width. 2 The calculation was made as ×0.5.

[0150] result A series of synthetic picolinamide and benzamide derivatives of potent and novel class I HDAC inhibitors (termed the GNTbm compound series) GNTbm has developed a series of novel class I HDAC inhibitors with potent epigenetic immunomodulatory properties that can inhibit the enzymatic activity of HDACs 1, 2, and 3. Our research has revealed that class I HDAC inhibitors with potent regulatory capabilities in the tumor microenvironment (TME) significantly boost the immune response against tumor growth. Therefore, designing and synthesizing such novel class I HDAC inhibitors has been an intriguing challenge for enhancing the therapeutic efficacy of immunotherapy. Benzamide-based class I HDAC inhibitors, such as entinostat (MS-275), tucidinostat (chidamide / HBI-8000), and mocetinostat, have been investigated in the field of regulating the TME. In this study, we designed and synthesized a series of potent and novel class I HDAC inhibitors based on the picolinamide structure. GNTbm-01 [6-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)pyridine-3-carboxamide] was the first synthesized novel compound based on the carboxamide core structure, as shown in Figure 1 and Table 1. Compound GNTbm-01 was assayed for enzymatic inhibition of HDACs 1, 2, and 3, as shown in Table 4. The results indicated that the GNTbm-01 compound is a weaker Class I HDAC inhibitor compared to entinostat or chidamide. We optimized the structure and changed the position of the N atom to generate the novel compound GNTbm-02 [5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)picolinamide], as shown in Figure 1 and Table 1. The GNTbm-02 compound has the same molecular formula (C 22 H 20FN5O2), but only with a change in the position of the N atom in the picolinamide core. As shown in Table 4, the GNTbm-02 compound was highly potent in inhibiting HDAC1, 2, and 3 enzyme activity when compared to entinostat or chidamide. The results also showed that GNTbm-02 was more potent than GNTbm-01 in inhibiting HDAC1, 2, and 3 enzyme activity. Next, we designed a benzamide-based compound, GNTbm-03, by removing the N atom (i.e., replacing it with a C atom) and tested the difference in inhibition of HDAC1, 2, and 3 enzyme activity compared to GNTbm-02. The synthetic benzamide-based class I HDAC inhibitor GNTbm-03 [[4-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)benzamide]] is shown in Figure 1 and Table 1. As shown in Table 4, GNTbm-03 was shown to inhibit the enzymatic activity of HDACs 1, 2, and 3. The results showed that GNTbm-03 was more potent in inhibiting class I HDACs 1, 2, and 3 enzymatic activity than entinostat or chidamide. It was also shown that GNTbm-03 had similar inhibition of HDACs 1, 2, and 3 enzymatic activity compared to GNTbm-02. In summary, the picolinamide-based derivative GNTbm-02 was the first class I HDAC inhibitor in its chemical class. The present inventors were very interested in designing potent and novel class I HDAC inhibitor picolinamide- and benzamide-based derivatives. A novel series of GNTbm compounds, such as GNTbm-04, GNTbm-05, GNTbm-06, GNTbm-08, GNTbm-11, GNTbm-12, GNTbm-19, GNTbm-25, GNTbm-33, GNTbm-37, GNTbm-38, and GNTbm-39, were synthesized and assayed.

[0151] Analyze the saturated solubility of GNTbm-02, GNTbm-03, GNTbm-04, and GNTbm-06 Solubility is a very important determining parameter for oral bioavailability. The saturated solubility analysis of GNTbm-02, GNTbm-03, GNTbm-04, and GNTbm-06 is shown in Table 2. The results showed that chidamide had a lower saturated solubility compared to GNTbm-02 and GNTbm-04. The saturated solubilities of GNTbm-02 and GNTbm-04 were 33.6 and 7.2 μg / mL, respectively. These results suggested that GNTbm-02 and GNTbm-04 may have better oral bioavailability compared to chidamide.

[0152] In Vitro Cytotoxicity Assay of the GNTbm Compound Series We evaluated the cytotoxic effects of the GNTbm compound series in several cancer cell lines, including three human breast cancer cell lines (SK-BR-3, MDA-MB-453, and MDA-MB-231), human colorectal adenocarcinoma SW48, human gastric cancer NCI-N87, and human breast epithelial cell line M10 (a normal cell line). The results showed that chidamide or entinostat, as positive controls, induced significant cytotoxic effects, particularly in SK-BR-3 and MDA-MB-453 cells. Overall, six cell lines were sensitive to treatment, as shown in Tables 3, 8, and 9. Compound GNTbm-01 partially induced cytotoxic effects compared with entinostat. As shown in Table 3, the results indicated that GNTbm-02 was significantly more potent than GNTbm-01 in inducing cytotoxic effects, particularly in SK-BR-3, MDA-MB-453, and SW48 cells. This result indicated that the picolinamide core-containing structure in GNTbm-02 is crucial. Replacing the picolinamide core with a benzamide would prevent the cytotoxic effect. As shown in Table 3, compound GNTbm-03 was weaker than GNTbm-02 in inducing cytotoxic effects in SK-BR-3, MDA-MB-231, and SW48 cells. This result suggested that GNTbm-02, which has a picolinamide core structure, is superior to GNTbm-03, which has a benzamide core structure, in inducing cytotoxicity. Collectively, these results suggest that GNTbm-02 is a potent and novel class I HDAC inhibitor with a strong ability to induce cytotoxicity in several human cancer cells. Furthermore, we were interested in evaluating the cytotoxic effects of several newly synthesized picolinamide-based and benzamide-based derivatives. As shown in Table 8, the cytotoxic effects of picolinamide-based compounds were analyzed. GNTbm-04, GNTbm-05, GNTbm-06, and GNTbm-11 were more potent than chidamide or entinostat in inducing cytotoxic effects in six cell lines. Among the benzamide-based compounds, GNTbm-33, GNTbm-38, and GNTbm-39 showed the highest cytotoxic effects. These data indicated that these novel picolinamide- and benzamide-based derivatives were more potent in inducing cytotoxic effects than the well-known class I HDAC inhibitors, chidamide or entinostat.

[0153] Picoliamide-based GNTbm compound series for the inhibition of HDAC1, 2, and 3 The GNTbm compound series was shown to inhibit the enzymatic activity of HDAC1, 2, and 3. As shown in Tables 4 and 10, entinostat, as a positive control, was a potent class I HDAC inhibitor that selectively inhibited HDAC1, 2, and 3 enzyme activity. Tsidamide (tsidinostat) was approved for the treatment of relapsing or refractory peripheral T-cell lymphoma (PTCL) and advanced ER by NMPA in China. + / Her-2 -Another potent HDAC inhibitor approved for breast cancer is chidamide, a subtype-selective inhibitor for inhibiting HDAC1, 2, 3, and 10 enzyme activity. Both entinostat and chidamide demonstrated potent inhibition of HDAC1, 2, and 3 enzyme activity in Table 4. Next, GNTbm-01 was evaluated and, as shown in Table 4, was shown to have moderate potency in inhibiting HDAC1, 2, and 3 enzyme activity compared to entinostat. Dramatically, GNTbm-02 had highly potent activity inhibiting HDAC1, 2, and 3 enzyme activity at nanomolar levels. Comparison of GNTbm-02 with entinostat or chidamide in inhibiting HDAC1, 2, and 3 enzyme activity showed similar inhibitory effects. These results suggested that GNTbm-02 is a potent and selective class I HDAC inhibitor. As shown in Table 4, GNTbm-03 was a potent HDAC inhibitor with an inhibitory effect similar to that of GNTbm-02. Next, we investigated the inhibition of HDAC3 enzyme kinetics. As shown in Figure 16a, chidamide and GNTbm-02 exhibited stronger inhibition of HDAC3 enzyme activity than entinostat. As shown in Figure 16b, GNTbm-02 and GNTbm-03 exhibited stronger inhibition of HDAC3 enzyme activity than GNTbm-01. Collectively, these results suggest that GNTbm-02, which contains a picolinamide core structure, may have a more potent ability to inhibit HDAC1, 2, and 3 enzyme activity. Furthermore, we were interested in evaluating all newly synthesized picolinamide-based GNTbm compounds, as shown in Table 10. GNTbm-04, GNTbm-05, GNTbm-06, GNTbm-08, and GNTbm-11 were more potent than chidamide or entinostat in inhibiting HDAC3 enzyme activity. GNTbm-05 and GNTbm-06 were more potent than chidamide or entinostat in inhibiting HDAC1 enzyme activity. However, we also evaluated novel synthetic benzamide-based GNTbm compounds, as shown in Table 11. GNTbm-38 and GNTbm-39 appear to be less potent than chidamide or entinostat in inhibiting the activity of HDAC1, 2, and 3.

[0154] GNTbm-02 is a picolinamide-based subtype-selective class I HDAC inhibitor To further confirm the subtype-selective inhibition of HDAC1-11 enzyme activity, GNTbm-02 was tested by BPS Bioscience Inc. (6042 Cornerstone Court West, Ste. B, San Diego, CA 92121, USA). As shown in Table 5, the inhibition of HDAC1-11 (except HDAC10) enzyme activity was analyzed together with entinostat (MS-275) as a positive control. The results showed that GNTbm-02 was more potent than entinostat in inhibiting HDAC1, 2, and 3 under the same conditions. GNTbm-02 inhibited class I HDAC1, HDAC2, and HDAC3 with IC values ​​of 0.39, 0.91, and 0.73 μM, respectively. 50 However, entinostat inhibited class I HDAC1, HDAC2, and HDAC3 with IC of 0.95, 2.3, and 4.6 μM, respectively. 50 GNTbm-02 inhibits HDACs 1, 2, and 3 at concentrations up to 10 μM. Other HDACs, including 4, 5, 6, 7, 8, 9, and 11, were not inhibited by GNTbm-02 or entinostat at concentrations up to 10 μM. These results suggested that GNTbm-02 is a potent and subtype-selective class I HDAC inhibitor. GNTbm-02 is a picolinamide-based class I HDAC inhibitor. However, entinostat is a benzamide-based class I HDAC inhibitor. GNTbm-02 is more potent than entinostat in inhibiting HDACS 1, 2, and 3 enzyme activity.

[0155] The GNTbm compound series significantly affects human cancer cell proliferation and morphology. The inhibitory effect of GNTbm-02 on human cancer cell proliferation is shown in Figure 3. MDA-MB-231 cells were treated for 72 hours with various concentrations of GNTbm-02 and entinostat. As shown in Figure 3a, the potency of the inhibitory effect was similar for GNTbm-02 and entinostat, significantly inhibiting cell proliferation at a concentration of 12.5 μM. As shown in Figure 3b, the potency of the inhibitory effect was more pronounced for SW48 cells when treated with GNTbm-02 or entinostat at a concentration of 3.125 μM for 72 hours. Next, M10 cells were treated with GNTbm-02 or entinostat at a concentration of 12.5 μM, which significantly inhibited cell proliferation, as shown in Figure 3c. Collectively, these results suggested that GNTbm-02 has a potent ability to inhibit cell proliferation.

[0156] The GNTbm compound series induced cell cycle arrest at G0 / G1 or G2 / M phase in human cancer MDA-MB-231 and SW48 cells. To investigate the mechanism of cell proliferation inhibition, cell cycle arrest was analyzed using flow cytometry. As shown in Figure 4a, MDA-MB-231 cells were treated with GNTbm-02 and entinostat at various concentrations (1.625–25 μM) for 72 hours. The results indicated that GNTbm-02 and entinostat have similar mechanisms, which significantly induce cell cycle arrest at the G0 / G1 phase at a concentration of 3.125 μM, as shown in Figures 4a and 4b. As shown in Figures 4c and 4d, treatment with GNTbm-02 and entinostat at a concentration of 12.5 μM resulted in cell cycle arrest at the G0 / G1 phase in a time-dependent manner. The results indicated that treatment with GNTbm-02 or entinostat for 1 day significantly induced cell cycle arrest at the G0 / G1 phase. A similar mechanism was also observed in SW48 cells. As shown in Figures 5a and 5b, SW48 cells were treated with GNTbm-02 and entinostat at various concentrations ranging from 0.39 to 6.25 μM for 72 hours. The results, shown in Figures 5a and 5b, indicated that GNTbm-02 and entinostat significantly induced cell cycle arrest at the G0 / G1 phase at a concentration of 3.125 μM. The results also indicated that GNTbm-02 (76.9%) appeared to be more potent than entinostat (72.1%) at the same concentration of 3.125 μM in inducing cell cycle arrest at the G0 / G1 phase. As shown in Figures 5c and d, treatment with GNTbm-02 and entinostat at a concentration of 6.25 μM resulted in cell cycle arrest at the G0 / G1 phase in a time-dependent manner. The results showed that two-day treatment with GNTbm-02 or entinostat significantly induced cell cycle arrest at the G0 / G1 phase. Collectively, all these data indicated that GNTbm-02 and entinostat have a similar mechanism of inhibiting human cancer cell proliferation through the induction of cell cycle arrest at the G0 / G1 phase. Furthermore, we were interested in evaluating several potent picolinamide- and benzamide-based novel synthetic derivatives, such as GNTbm-04, GNTbm-05, GNTbm-38, and GNTbm-39. As shown in Table 12, GNTbm-04 significantly induced cell cycle arrest at the G0 / G1 phase in SW48 cells.This was similar to that of chidamide, which induced cell cycle arrest at the G0 / G1 phase. However, the similar chemical structures of GNTbm-05, GNTbm-38, and GNTbm-39 significantly induced cell cycle arrest at the G2 / M phase in SW48 cells. Thus, although the chemical structures of these potent compounds are very similar, their mechanisms of cell cycle arrest are very different.

[0157] GNTbm-02 induced cell cycle arrest at the G2 / M phase in M10 cells The human breast epithelial cell line M10 was treated with different doses of GNTbm-02 or entinostat (1.625-25.0 μM) for 72 hours, as shown in Figure 6a and b. The results showed that GNTbm-02 and entinostat at a concentration of 12.5 μM significantly induced cell cycle arrest of M10 cells at the G2 / M phase. Entinostat (20.2%) was more potent than GNTbm-02 (16.2%) in inducing cell cycle arrest at the G2 / M phase, as shown in Figure 6a and b. As shown in Figure 6c and d, treatment with GNTbm-02 and entinostat at a concentration of 12.5 μM resulted in cell cycle arrest at the G2 / M phase in a time-dependent manner. The results showed that treatment with GNTbm-02 and entinostat for 2 days significantly induced cell cycle arrest at the G2 / M phase in M10 cells. These results suggest that treatment of human cancer cells with GNTbm-02 and entinostat significantly inhibits cancer cell proliferation through the induction of cell cycle arrest at the G0 / G1 phase; however, treatment of human normal cells with GNTbm-02 and entinostat significantly inhibits cell proliferation through the induction of cell cycle arrest at the G2 / M phase.

[0158] The GNTbm compound series induced apoptosis in several cell lines To investigate whether GNTbm-02 induced apoptosis in cancer cells, the results after treatment of MDA-MB-231 cells with GNTbm-02 and entinostat (as a positive control) at various concentrations ranging from 1.625 to 25.0 μM for 72 hours are shown in Figures 7a and 7b. The results indicated that GNTbm-02 and entinostat at a concentration of 6.25 μM significantly induced apoptosis (increased the percentage of sub-G1 phase), as shown in Figures 7a and 7b. As shown in Figures 7c and 7d, cell apoptosis was induced by treatment of MDA-MB-231 cells with GNTbm-02 and entinostat in a time-dependent manner. The results indicated that GNTbm-02 and entinostat at a concentration of 12.5 μM for 72 hours (3 days) significantly induced apoptosis in MDA-MB-231 cells. Entinostat was significantly more potent than GNTbm-02 in inducing apoptosis in a dose- and time-dependent manner, as shown in Figure 7. Next, cell apoptosis was also evaluated in SW48 cells. As shown in Figures 8a and 8b, GNTbm-02 and entinostat induced apoptosis in a dose-dependent manner. The results showed that treatment with GNTbm-02 and entinostat at various concentrations ranging from 0.39 to 6.25 μM for 72 hours induced cell apoptosis in SW48 cells. GNTbm-02 and entinostat significantly induced apoptosis at a concentration of 6.25 μM for 72 hours, as shown in Figures 8a and 8b. As shown in Figures 8c and 8d, treatment with entinostat and GNTbm-02 was shown to induce apoptosis at a fixed concentration of 6.25 μM in a time-dependent manner. GNTbm-02 and entinostat at a concentration of 6.25 μM for 72 hours (3 days) significantly induced apoptosis in SW48 cells. Entinostat was much more potent than GNTbm-02 in inducing apoptosis in SW48 cells in a dose- and time-dependent manner, as shown in Figure 8. Finally, the induction of cell apoptosis by GNTbm-02 and entinostat in the normal cell line M10 was also investigated.As shown in Figures 9a and 9b, treatment with GNTbm-02 and entinostat at various concentrations ranging from 1.625 to 25.0 μM for 72 hours induced cell apoptosis. GNTbm-02 and entinostat at a concentration of 12.5 μM for 72 hours significantly induced apoptosis in M10 cells. As shown in Figures 9c and 9d, treatment with GNTbm-02 and entinostat at a fixed concentration of 12.5 μM for 72 hours (3 days) in M10 cells significantly induced apoptosis. Entinostat induced apoptosis much more potently in M10 cells in a dose-dependent and time-dependent manner compared to GNTbm-02, as shown in Figure 9c and 9d. However, M10 cells appeared to be more resistant to induced apoptosis when treated with GNTbm-02 and entinostat at a concentration of 25.0 μM for 72 hours, as shown in Figure 7, compared with the results for MDA-MB-231 cells. Next, we were interested in investigating the activity of GNTbm-04, GNTbm-05, GNTbm-38, and GNTbm-39 in inducing apoptosis in SW48 cells. Apoptosis induced by these compounds was evaluated in SW48 cells treated at the indicated doses for 72 hours. As shown in Table 13, GNTbm-04, GNTbm-05, GNTbm-38, and GNTbm-39 were potent in inducing apoptosis in SW48 cells.

[0159] The GNTbm compound series induced histone H3 acetylation in several human cancer cell lines GNTbm-02 and entinostat have been demonstrated to be potent class I HDAC inhibitors. The effects of GNTbm-02 and entinostat on histone H3 acetylation in MDA-MB-231 and SW48 cells were investigated in a dose- or time-dependent manner. As shown in Figure 10a and b, treatment of MDA-MB-231 cells with GNTbm-02 and entinostat at concentrations ranging from 0.1 to 10.0 μM for 24 hours induced histone H3 acetylation. The results showed that GNTbm-02 and entinostat at a concentration of 1.0 μM significantly increased the level of histone H3 acetylation. As shown in Figure 10c and d, SW48 cells were more sensitive to the induction of histone H3 acetylation by treatment with GNTbm-02 and entinostat at concentrations ranging from 0.1 to 10.0 μM for 24 hours. As shown in Figures 11a and 11b, treatment with GNTbm-02 at a concentration of 1.0 μM for 2, 6, 24, 48, and 72 hours induced histone H3 acetylation in MDA-MB-231 cells in a time-dependent manner. The results showed that GNTbm-02 potently induced histone H3 acetylation in MDA-MB-231 cells after 6 hours of treatment. Similar results were also shown in SW48 cells, as shown in Figures 11c and 11d. Treatment with GNTbm-02 at a concentration of 1.0 μM for 2, 6, 24, 48, and 72 hours in SW48 cells showed histone H3 acetylation in a time-dependent manner. GNTbm-02 potently induced histone H3 acetylation levels in SW48 cells after 6 hours of treatment. Taken together, all these data suggested that GNTbm-02 is a potent class I HDAC inhibitor and induces histone H3 acetylation in several human cancer cell lines. Furthermore, we were interested in analyzing whether novel compounds have a more potent activity in increasing histone 3 acetylation in SW48 cells, as shown in Figure 12. As shown in Figure 12a, SW48 cells were treated with the same doses of GNTbm-04, GNTbm-05, and GNTbm-11, as well as chidamide as a positive control, for 24 hours.GNTbm-05 and GNTbm-04 at a dose of 0.25 μM were significantly more potent than the positive control, chidamide, in inducing histone 3 acetylation in SW48 cells. Similar results, shown in Figure 12b, also indicated that GNTbm-04, GNTbm-05, and GNTbm-06 at a dose of 0.25 μM were also highly potent in inducing histone 3 acetylation in SW48 cells. Furthermore, four potent compounds (GNTbm-04, GNTbm-05, GNTbm-38, and GNTbm-39) that induce histone 3 acetylation were evaluated, as shown in Figure 12c. The results indicated that GNTbm-05, GNTbm-04, GNTbm-38, and GNTbm-39 were more potent than chidamide in inducing histone 3 acetylation in SW48 cells.

[0160] The GNTbm compound series possessed epigenetic immunomodulatory properties in a CT26-bearing mouse model To investigate whether GNTbm-02 has epigenetic immunomodulatory properties, an in vivo animal model of BALB / c CT26 colon tumor-bearing mice was used for evaluation. BALB / c mice bearing murine CT26 colon tumors were treated with various therapeutic modalities as indicated: IgG, anti-IgG control (vehicle, 2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); GNTbm-02 12.5 and 25.0 mg / kg; and celecoxib capsules 50 mg / kg (Celebrex®). Tumor size in CT26 tumor-bearing mice was approximately 150-200 mm on day 8. 3The total tumor volume and fold change in tumor size are shown in Figures 13a and 13b. The results showed that the regimen of anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (12.5 mg / kg) in combination with celecoxib (50 mg / kg) had a significantly greater inhibitory effect on tumor growth than the regimen of anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (25.0 mg / kg) in combination with celecoxib (50 mg / kg) or the regimen of GNTbm-02 25 mg / kg plus celecoxib 50 mg / kg in the absence of anti-PD-1 antibody. Therefore, the tumor growth inhibitory effect was as follows: anti-PD-1 antibody plus GNTbm-02 (12.5 mg / kg) regimen combined with celecoxib > anti-PD-1 antibody plus GNTbm-02 (25.0 mg / kg) regimen > GNTbm-02 (25.0 mg / kg) regimen combined with celecoxib > anti-PD-1 antibody > anti-IgG regimen. However, the results also showed that GNTbm-02 combined with celecoxib had a potent inhibitory effect on tumor growth. Previously, our research showed that an HDAC inhibitor combined with a COX-2 inhibitor significantly regulated the TME, thus improving tumor growth inhibitory effects and immune response rates. These results indicated that GNTbm-02 is a potent and novel epigenetic immunomodulator. Individual tumor volumes were analyzed as shown in Figure 13c. In this study, we defined the following to evaluate treatment efficacy: complete response (CR, tumor growth of ≦0.5-fold in tumor-bearing mice 3 days after the end of treatment); partial response (PR, tumor growth of >0.5-fold but ≦2-fold in tumor-bearing mice 3 days after the end of treatment); stable disease (SD, tumor growth between 2-fold and 5-fold in tumor-bearing mice 3 days after the end of treatment); and progressive disease (PD, tumor growth of ≧5-fold in tumor-bearing mice 3 days after the end of treatment).The results showed that the anti-PD-1 antibody (2.5 mg / kg) group achieved an ORR (objective response rate) of 35.3%, with 5 CRs, 1 PR, 3 SDs, and 8 PDs; the anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (25 mg / kg) group combined with celecoxib (50 mg / kg) achieved an ORR of 66.7%, with 3 CRs, 3 PRs, 2 SDs, and 1 PD; the anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (12.5 mg / kg) group combined with celecoxib (50 mg / kg) achieved an ORR of 87.5%, with 5 CRs, 2 PRs, 1 SD, and 0 PDs; and the GNTbm-02 (25 mg / kg) group combined with celecoxib (50 mg / kg) achieved an ORR of 66.7%, with 2 CRs, 4 PRs, 2 SDs, and 1 PD. These results suggested that 12.5 mg / kg of GNTbm-02 was the optimal dose and that GNTbm-02 had potent immunomodulatory activity. The weight of CT26 tumor-bearing mice was shown in Figure 13d, indicating that these regimens did not have obvious toxicity that caused weight loss. Finally, survival rates were analyzed as shown in Figure 13e. After tumor implantation, tumor volumes were 3000 mm. 3Once the tumor size reached 100 mg / kg, the CT26 tumor-bearing mice were euthanized. Results showed that the anti-PD-1 antibody group achieved a 30% survival rate; the anti-PD-1 antibody (2.5 mg / kg) combined with celecoxib (50 mg / kg) plus GNTbm-02 (25 mg / kg) group achieved a 33% survival rate; the GNTbm-02 (25 mg / kg) combined with celecoxib (50 mg / kg) group achieved a 56% survival rate; and the anti-PD-1 antibody (2.5 mg / kg) combined with celecoxib (50 mg / kg) plus GNTbm-02 (12.5 mg / kg) group achieved a 63% survival rate. Collectively, these data suggested that GNTbm-02 plus celecoxib, or GNTbm-02 plus celecoxib in combination with an anti-PD-1 antibody, significantly improved ORR and survival compared with the anti-PD-1 antibody alone. Our data also demonstrated that a 12.5 mg / kg dose of GNTbm-02 showed better efficacy than a 25.0 mg / kg dose in the combination regimen of an anti-PD-1 antibody plus GNTbm-02 in combination with celecoxib. Next, we were interested in evaluating the regulation of tumor microenvironment activity using novel synthetic compounds, such as GNTbm-02, GNTbm-03, GNTbm-04, and GNTbm-06, as well as chidamide as a positive control. Using a solid dispersion of chidamide prepared by coating on PVP-K30, we improved the water solubility of chidamide-API, which ultimately improved its pharmacokinetic (PK) profile. Therefore, the present inventors used common preparation techniques in the art to produce solid dispersions of test compounds, such as GNTbm-02, GNTbm-03, GNTbm-04, and GNTbm-06, as well as chidamide as a positive control. All test compounds were coated onto PVP-K30 to prepare solid dispersions designated GNTbm-02 / k30, GNTbm-03 / k30, GNTbm-04 / k30, GNTbm-06 / k30, and chidamide / k30. Previous studies have demonstrated that chidamide / k-30 in combination with regorafenib exhibits highly potent anticancer activity through immunomodulatory mechanisms in CT-26 tumor-bearing mice.The anticancer activity of GNTbm-02 / k-30 in combination with regorafenib was further investigated to confirm its efficacy in CT26 tumor-bearing mice. We defined stricter criteria for evaluating treatment efficacy: CR (tumor growth ≤0.5-fold in tumor-bearing mice 3 days after the end of treatment); PR (tumor growth >0.5-fold but ≤1-fold in tumor-bearing mice 3 days after the end of treatment); SD (tumor growth between 1-fold and 5-fold in tumor-bearing mice 3 days after the end of treatment); and PD (tumor growth ≥5-fold in tumor-bearing mice 3 days after the end of treatment). As shown in Figures 14(f) to 14(i), GNTbm-02 / k-30 in combination with regorafenib versus chidamide / k-30 in combination with regorafenib was evaluated. The results showed that GNTbm-02 / k-30 (50 mg / kg) combined with regorafenib (30 mg / kg) had potent inhibition of tumor growth, but it was weaker than that of chidamide / k-30 combined with regorafenib (ORR: 10% vs. 30%). However, GNTbm-03 / k-30 showed similar anticancer activity compared to chidamide / k-30 combined with regorafenib (ORR: 40% vs. 30%), as shown in Figure 14(i) to (m). GNTbm-04 / k-30 in combination with regorafenib was more potent in inhibiting tumor growth than chidamide / k-30 in combination with regorafenib (ORR: 50% vs. 30%), as shown in Figures 14(n) to 14(q). GNTbm-06 / k-30 in combination with regorafenib had similar anticancer activity compared to chidamide / k-30 in combination with regorafenib (ORR: 50% vs. 30%), as shown in Figures 14(r) to 14(u). After 16 days of treatment, we continued to monitor tumor size until day 60. In mice with a CR or PR response after the first tumor evaluation, recurrence was defined as tumor growth reappearing and tumor size increasing by at least 5 times.As shown in Table 14, chidamide / k-30 combined with regorafenib showed 0% tumor recurrence, GNTbm-02 / k-30 combined with regorafenib showed 100% tumor recurrence, GNTbm-03 / k-30 combined with regorafenib showed 25% tumor recurrence, GNTbm-04 / k-30 combined with regorafenib showed 20% tumor recurrence, and GNTbm-06 / k-30 combined with regorafenib showed 0% tumor recurrence. Except for the GNTbm-02 / k-30 combined with regorafenib group, which had only one mouse with PR in the study, the results suggested that the GNTbm compound combined with regorafenib may have more potent activity in activating the immune system to avoid regression. Additionally, we investigate the epigenetic immunomodulatory properties of a series of GNTbm compounds, including GNTbm-05 / k-30, GNTbm-11 / k-30, GNTbm-38 / k-30, and GNTbm-39 / k-30. As shown in Table 14, the comparative efficacy of GNTbm-05 / k-30, GNTbm-11 / k-30, GNTbm-38 / k-30, GNTbm-39 / k-30, and chidamin / k-30 in combination with regorafenib was evaluated. The results showed that the chidamide / k30 (50 mg / kg) group combined with regorafenib (30 mg / kg) achieved an ORR of 60% with 2 CRs, 4 PRs, 4 SDs, and 0 PDs; the GNTbm-05 / k30 (50 mg / kg) group combined with regorafenib (30 mg / kg) achieved an ORR of 30% with 3 CRs, 0 PRs, 4 SDs, and 3 PDs; and the GNTbm-11 / k30 (50 mg / The GNTbm-38 / k30 (50 mg / kg) combined with regorafenib (30 mg / kg) group achieved an ORR of 80% with 8CR, 0PR, 2SD, and 0PD; and the GNTbm-39 / k30 (50 mg / kg) combined with regorafenib (30 mg / kg) group achieved an ORR of 30% with 2CR, 1PR, 5SD, and 2PD.Taken together, these in vivo animal data showed that when comparing all of the GNTbm compounds with the positive control chidamide, in combination with regorafenib, GNTbm-38 / k-30 exhibited superior epigenetic immunomodulatory activity, achieving an ORR of 80%, and without combination with regorafenib, GNTbm-38 / k-30 alone achieved an ORR of 56%.

[0161] Identifying the epigenetic immunomodulatory properties of the GNTbm compound series The optimal dose of GNTbm-02 in combination with celecoxib (a selective COX-2 inhibitor) was analyzed and confirmed. IgG, anti-IgG control (vehicle, 2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); GNTbm-02, 5, 10, 20, and 25.0 mg / kg; and celecoxib capsules 50 mg / kg (Celebrex®). Tumor size in CT26 tumor-bearing mice was approximately 150-200 mm on day 8. 3The total tumor volume and fold change in tumor size, as shown in Figures 14a and 14b, indicated that the GNTbm-02 (10 mg / kg) combined with celecoxib (50 mg / kg) group was more potent in inhibiting tumor growth than the GNTbm-02 (20 mg / kg) combined with celecoxib (50 mg / kg) group or the GNTbm-02 (5 mg / kg) combined with celecoxib (50 mg / kg) group. These results also suggested that combining GNTbm-02 with celecoxib at an optimal ratio was essential for controlling the TME and improved the tumor growth inhibitory effect in the CT26-bearing mouse model. As shown in Figure 14c, the individual tumor volumes and ORRs were as follows: the anti-PD-1 antibody (2.5 mg / kg) group achieved 5 CR, 1 PR, 3 SD, and 8 PD, with an ORR (objective response rate) of 35.3%; the GNTbm-02 (5 mg / kg) combined with celecoxib (50 mg / kg) group achieved 2 CR, 1 PR, 1 SD, and 5 PD, with an ORR of 33.3%; the GNTbm-02 (10 mg / kg) combined with celecoxib (50 mg / kg) group achieved 2 CR, 1 PR, 1 SD, and 5 PD, with an ORR of 33.3%. The GNTbm-02 (20 mg / kg) combined with celecoxib (50 mg / kg) group achieved an ORR of 55.6% with 2 CR, 3 PR, 1 SD, and 3 PD; and the GNTbm-02 (25 mg / kg) combined with celecoxib (50 mg / kg) group achieved an ORR of 66.7% with 2 CR, 4 PR, 2 SD, and 1 PD. These data suggested that the GNTbm-02 (10 mg / kg) combined with celecoxib (50 mg / kg) group achieved the best ORR resulting from an optimal ratio for TME control. This result was also observed in Figure 13, where the anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (12.5 mg / kg) regimen combined with celecoxib (50 mg / kg) achieved a better ORR. These data suggest that GNTbm-02 10 mg / kg combined with celecoxib 50 mg / kg has potent activity in regulating the TME, thus improving the immune response rate.The body weight of CT26 tumor-bearing mice was shown in Figure 14d, indicating that these regimens did not have significant toxicity that caused weight loss. Finally, survival rates were analyzed as shown in Figure 14e. Tumor volumes were 3000 mm after tumor implantation. 3 Once the tumor size reached 100 mg / kg, the CT26 tumor-bearing mice were euthanized. Results showed that the anti-PD-1 antibody group achieved a 30% survival rate; the GNTbm-02 (5 mg / kg) combined with celecoxib (50 mg / kg) group achieved a 22% survival rate; the GNTbm-02 (10 mg / kg) combined with celecoxib (50 mg / kg) group achieved a 44% survival rate; the GNTbm-02 (20 mg / kg) combined with celecoxib (50 mg / kg) group achieved a 33% survival rate; and the GNTbm-02 (25 mg / kg) combined with celecoxib (50 mg / kg) group achieved a 56% survival rate. Collectively, these data suggest that GNTbm-02 plus celecoxib significantly improved ORR and survival compared with anti-PD-1 antibody alone. Our data also showed that when GNTbm-02 was combined with celecoxib, a dose of 10 mg / kg of GNTbm-02 was better than other doses.

[0162] GNTbm-02 plus celecoxib with or without anti-PD-1, or the GNTbm compound series in combination with regorafenib, significantly induced immunological memory. The immune memory induced after treatment with different regimens as shown in Figures 13 and 14 was investigated for the status as shown in Tables 6 and 7. Mice were treated with the different regimens for 16 days, then the first tumor evaluation was performed (day 26). Mice with CR or PR entered a washout stage for 7 days (until day 33) without further treatment. Then, allogeneic cancer cells (CT26; 5x10 6The rechallenge was carried out for another 7 days (day 40) by inoculating the mice with 1000 or more vaccinated mice with 1000 or more vaccinated mice into the opposite flank, and then the tumor volume was determined as baseline (1x). The rechallenge tumors were allowed to grow for 10 days (day 50), and then the tumor size was measured to assess immune memory as positive or negative. If the assessment was negative, it had to meet both of two conditions: the tumor volume was 300 mm or more; 3The tumor size must exceed 100% and the tumor size must be more than doubled compared to baseline. If the immune memory induced after the previous treatment is active and specific to the recognition of cancer cells by the same antigen, the growth of the inoculated tumor during rechallenge will be inhibited, and immune memory was therefore defined as positive. If immune memory is not induced or is not fully activated, the growth of the inoculated tumor during rechallenge will not be inhibited. This evaluation process investigated the GNTbm-02 plus celecoxib regimen with or without an anti-PD-1 antibody to determine whether the regimen has the property of inducing immune memory. As shown in Table 6, the anti-PD-1 antibody group had only two mice that achieved CR, which showed 0% tumor progression after rechallenge. The results showed that these CR mice achieved 100% active immune memory. The GNTbm-02 (25 mg / kg) plus celecoxib (50 mg / kg) regimen combined with an anti-PD-1 antibody (2.5 mg / kg) achieved a CR / PR in four mice, which also showed 0% tumor progression after rechallenge. This also indicated active immune memory of 100%. The GNTbm-02 (12.5 mg / kg) plus celecoxib (50 mg / kg) regimen combined with an anti-PD-1 antibody (2.5 mg / kg) achieved a CR / PR in seven mice, which showed 29% tumor progression after rechallenge. This indicated active immune memory of 71%. The GNTbm-02 (25 mg / kg) regimen combined with celecoxib (50 mg / kg) achieved a CR / PR in six mice, which showed 17% tumor progression after rechallenge. This also indicated active immune memory of 83%. However, as shown in Table 7, the regimen of GNTbm-02 (10 mg / kg) combined with celecoxib (50 mg / kg) achieved CR / PR in 7 mice, which showed 14% tumor progression after rechallenge. This represented 86% active immune memory. From these data, mice with CR had stronger immune memory activity than mice with PR. In summary, GNTbm-02 plus celecoxib, with or without ICI, induced potent immune memory activity.The same phenomenon was reflected in other GNTbm compounds combined with regorafenib. As shown in Table 14, the GNTbm-02 / k-30 (50 mg / kg) group combined with regorafenib (30 mg / kg) achieved CR / PR in 1 mouse, which also showed 0% tumor progression after rechallenge; the GNTbm-03 / k-30 (50 mg / kg) group combined with regorafenib (30 mg / kg) achieved CR / PR in 4 mice, which also showed 0% tumor progression after rechallenge. The GNTbm-04 / k-30 (50 mg / kg) group combined with regorafenib (30 mg / kg) achieved CR / PR in 5 mice, which also showed 0% tumor progression after rechallenge; the GNTbm-06 / k-30 (50 mg / kg) group combined with regorafenib (30 mg / kg) achieved CR / PR in 5 mice, which also showed 0% tumor progression after rechallenge. These results indicated that the GNTbm compound combined with regorafenib was significant in inducing immunological memory.

[0163] Antitumor activity after treatment with GNTbm-02 plus celecoxib was mediated through immunomodulatory effects, resulting in CTL activation The treated mice were normal mice with intact immune systems, as shown in Figures 13 and 14. The regimen of GNTbm-02 plus celecoxib, with or without anti-PD-1 antibody, achieved a significantly high overall response rate (ORR) in wild-type normal mice. Next, we investigated treatment with the regimen of GNTbm-02 plus celecoxib, with or without anti-PD-1 antibody, in a BALB / C nude mouse model (with insufficient T cell function). As shown in Figure 15a, nude mice were inoculated with CT26 cells by sc injection. After 8 days, the average tumor volume was approximately 123.8 mm. 3Once the mice reached the target tumor size, they were then randomized into four groups and treated for 15 days with an anti-IgG antibody, an anti-PD-1 antibody, GNTbm-02 plus celecoxib in combination with an anti-PD-1 antibody, and GNTbm-02 plus celecoxib. As shown in Figures 15b and 15c, none of these treatment groups significantly inhibited tumor growth in nude mice with insufficient T cell function. These results indicated that GNTbm-02 plus celecoxib has potent activity in inhibiting tumor growth by regulating CTL (cytotoxic T lymphocyte) activation in the TME. As shown in Figure 15d, none of the treatment groups showed significant weight loss. As shown in Figure 15e, all mice in the treatment groups were shown to have low anti-cancer activity in nude mice, and none of them achieved ORR. These results demonstrated that an immune system with functional T cells is essential for achieving significant tumor growth inhibition with the combination regimen of GNTbm-02 plus celecoxib, with or without an anti-PD-1 antibody (Figures 13, 14, and 15). This also indicated that GNTbm-02 plus celecoxib inhibits tumor growth by regulating the activation of T cells (CTLs) in the TME for cancer cell killing. This anticancer activity was mediated through immunomodulatory effects rather than cytotoxic effects. In summary, we confirmed that GNTbm-02 has potent epigenetic immunomodulatory activity and, when combined with celecoxib, is more potent at regulating the TME than GNTbm-02 alone.

[0164] The antitumor activity of GNTbm-02 plus celecoxib is associated with a reduction in immunosuppressive cells HDACi treatment has been shown to alter the TME by reducing Treg cell activity and enhancing CD8 T cell infiltration. To determine whether the tumor growth inhibition resulting from treatment with GNTbm-02 plus celecoxib is associated with an enhanced immune response, we examined circulating leukocyte populations. On the final day of treatment (i.e., on day 16 of the treatment period), blood samples were collected from CT26 tumor-bearing mice and studied by FACS analysis. We observed a significant increase in lymphocytes and a decrease in granulocytes in the circulating blood after treatment with GNTbm-02 plus celecoxib (Figures 17a and 17c). However, there was no significant difference in circulating monocytic cells (Figure 17b). We also observed a significant increase in CD3 T cells in the circulating blood after treatment with GNTbm-02 plus celecoxib (Figure 17c). + A significant increase in T cells was also observed (Fig. 17d). + A modest increase in T cells was observed after treatment with anti-PD-1 or GNTbm-02 plus celecoxib (Fig. 17f). However, circulating CD4 + There were no significant differences in T cells and Tregs (Fig. 17e and g). + In addition to Tregs, there are other immunosuppressive myeloid cells that are recruited to the TME, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). When immature myeloid cells migrate into tumors, these cells are often primed to become TAMs in response to chemokines and cytokines released by cancer cells. MDSCs arise from immature myeloid cells and contribute to immunosuppression in the TME by inhibiting anti-cancer T cell activity. MDSCs are divided into two phenotypically defined subpopulations: granulocytic and Ly6G + Ly6C - (PMN-MDSC) and monocytic Ly6C + Ly6G - Treatment with GNTbm-02 plus celecoxib increased the phenotypically defined CD11b expression in circulating MDSCs (M-MDSCs). + Ly6G + Ly6C + and M-MDSCs, whereas treatment with anti-PD-1 alone resulted in a slight decrease in CD11b+ This led to a reduction in the PMN-MDSC population (Fig. 17h, i, and j). There was no reduction in PMN-MDSC after treatment with GNTbm-02 plus celecoxib (Fig. 17k).

[0165] In summary, because immunotherapy is an important and promising field for anti-cancer therapy, particularly for the treatment of advanced cancers, the claimed invention was evaluated for its potential application in immunotherapy. It was found that GNTbm-02 combined with celecoxib had more potent immunomodulatory activity in inhibiting tumor growth in the tumor microenvironment (TME) compared to GNTbm-02. Furthermore, when GNTbm-02 plus celecoxib was used in combination with immune checkpoint inhibitors, such as anti-PD-1 / anti-PD-L1 / anti-CTLA-4 antibodies, it was shown to have more potent anti-cancer activity, significantly boosting response rates through a synergistic effect resulting from the blockade of inhibitory signals to CTLs (cytotoxic T lymphocytes) by anti-PD-1 / anti-PD-L1 / anti-CTLA-4 antibodies and the immunomodulatory activity of GNTbm-02 plus celecoxib in the TME. Based on this study, GNTbm-02 is a novel epigenetic immunomodulator with great potential for cancer treatment. Furthermore, the inventors were interested in the immunomodulatory activity of the GNTbm compound series. Our data showed that GNTbm-02, GNTbm-03, GNTbm-04, GNTbm-06, and GNTbm-38, when combined with celecoxib or regorafenib, were highly potent in possessing epigenetic immunomodulatory activity to regulate the TME. These results suggested that the GNTbm compound series is a novel and potent epigenetic immunomodulator.

[0166] The above table is shown below:

[0167] [Table 1]

[0168] [Table 2]

[0169]

Table 3

[0170]

Table 4

[0171]

Table 5

[0172]

Table 6

[0173]

Table 7

[0174]

Table 8

[0175]

Table 9

[0176]

Table 10

[0177]

Table 11

[0178]

Table 12

[0179] [Table 13]

[0180] [Table 14]

[0181] It should be understood by those skilled in the art that variations and modifications can be made to the teachings and disclosures of the present invention without departing from the spirit and scope of the present application. Based on the above, the present application is intended to cover all variations and modifications thereof, provided that the variations or modifications fall within the scope defined in the appended claims or their equivalents. Aspects of the present disclosure include the following. [1] Compounds of formula (I): [ka] wherein W and Y are each independently selected from CH and N; R 1 are each independently hydrogen, halogen, or C 1 ~C 3 Alkyl and halogenated C 1 ~C 3 alkyl, which may be mono-, di-, tri- or tetra-substituted; C 1 and C 2 is a C atom connected by a single or double bond, Ar is the following: [ka] wherein Ar is selected from the group consisting of C through a solid line 2 It is connected to R 2 is R 1 has the same meaning as described above for R 3 is hydrogen or C 1 ~C 3 alkyl] or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof. [2] Formula (Ia): [ka] [Wherein W, Y, R 1 、C 1 、C 2 and Ar have the same meaning as described in formula (I). or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof. [3] Ar is selected from a six-membered ring; R 2 And, C 2 and the atom of Ar linked to is in the para position, or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof. [4] or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof, of the compound of Aspect 1, wherein W and Y are selected from the following combinations: (1) W is N and Y is CH, (2) W is CH and Y is N, and (3) W and Y are CH. [5] A compound according to embodiment 1, or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof, wherein W is N and Y is CH. [6] R 1 F or fluorinated C 1 ~C 3 The compound of embodiment 1, or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof, wherein R is alkyl. [7] C 1 and C 2 is a C atom that is linked by a double bond, or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof. [8] C 1 and C 2 is a C atom linked by a single bond, or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof. [9] R 2 But C 1 ~C 3 Alkyl or Fluorinated C 1 ~C 3 The compound of embodiment 1, or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof, wherein R is alkyl.

[10] The compound

change

[11] A pharmaceutical composition or combination comprising a compound according to any one of aspects 1 to 10, or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier.

[12] 12. The pharmaceutical composition or combination according to aspect 11, further comprising one or more second pharmaceutical agents.

[13] 13. The pharmaceutical composition or combination according to aspect 12, wherein the second agent is an immune checkpoint inhibitor, an NSAID, a TKI or an anticancer agent, or a combination thereof.

[14] 12. Use of a pharmaceutical composition or combination according to aspect 11 in the manufacture of a medicament for epigenetic immunomodulation of the tumor microenvironment (TME) and / or treatment of cancer in a subject in need thereof.

[15] 15. The use according to aspect 14, wherein the pharmaceutical composition or combination further comprises one or more second pharmaceutical agents.

[16] 16. The use of aspect 15, wherein the second agent is an immune checkpoint inhibitor, an NSAID, a TKI, or an anticancer agent, or a combination thereof.

[17] The use according to aspect 14, wherein the medicament is for inducing cell cycle arrest of tumor cells, for inducing apoptosis of tumor cells, for inducing histone H3 acetylation, for inducing immune memory, for activating CTLs, or for reducing immunosuppressive cells.

[18] 12. Use of a pharmaceutical composition or combination according to aspect 11 in the manufacture of a medicament for treating or preventing a disease associated with class I HDAC in a subject in need thereof.

[19] 19. The use according to aspect 18, wherein the pharmaceutical composition or combination further comprises one or more second pharmaceutical agents.

[20] 20. The use of aspect 19, wherein the second agent is an immune checkpoint inhibitor, an NSAID, a TKI, or an anticancer agent, or a combination thereof.

Claims

1. Compounds of formula (I): 【Chemical 1】 wherein W is selected from CH and N, and Y is CH; R 1 are each independently hydrogen, halogen, or C 1 ~C 3 Alkyl and halogenated C 1 ~C 3 alkyl, which may be mono-, di-, tri- or tetra-substituted; C 1 and C 2 is a C atom connected by a double bond, Ar is the following: 【Chemistry 2】 wherein Ar is selected from the group consisting of C through a solid line 2 It is connected to R 2 is R 1 has the same meaning as described above for R 3 is hydrogen or C 1 ~C 3 alkyl] (However, the compound 【Chemistry 3】 (Excluding or a pharmaceutically acceptable salt, hydrate, stereoisomer or solvate thereof.

2. Formula (Ia): 【Chemistry 4】 [Wherein W, Y, R 1 , C 1 , C 2 and Ar have the same meaning as described in formula (I).

10. The compound of claim 1, or a pharmaceutically acceptable salt, hydrate, stereoisomer or solvate thereof, having the formula:

3. Ar is selected from a six-membered ring; R 2 And, C 2 and the atom of Ar linked to is in the para position, or a pharmaceutically acceptable salt, hydrate, stereoisomer, or solvate thereof.

4. 2. The compound of claim 1, or a pharmaceutically acceptable salt, hydrate, stereoisomer, or solvate thereof, wherein W and Y are each CH.

5. 2. The compound of claim 1, or a pharmaceutically acceptable salt, hydrate, stereoisomer, or solvate thereof, wherein W is N and Y is CH.

6. R 1 F or fluorinated C 1 ~C 3 2. The compound of claim 1, or a pharmaceutically acceptable salt, hydrate, stereoisomer, or solvate thereof, wherein R is alkyl.

7. R 2 But C 1 ~C 3 Alkyl or Fluorinated C 1 ~C 3 2. The compound of claim 1, or a pharmaceutically acceptable salt, hydrate, stereoisomer, or solvate thereof, wherein R is alkyl.

8. The compound 【Chemistry 5】 【change】 2. The compound of claim 1, wherein:

9. 9. A pharmaceutical composition or pharmaceutical combination comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, hydrate, stereoisomer or solvate thereof, and a pharmaceutically acceptable carrier.

10. 10. The pharmaceutical composition or pharmaceutical combination of claim 9, further comprising one or more second pharmaceutical agents.

11. 11. The pharmaceutical composition or pharmaceutical combination according to claim 10, wherein the second drug is an immune checkpoint inhibitor, an NSAID, a TKI, or an anticancer drug, or a combination thereof.

12. 10. The pharmaceutical composition or pharmaceutical combination of claim 9 for use in a method for epigenetic immunomodulation of the tumor microenvironment (TME) and / or treatment of cancer in a subject in need thereof.

13. 13. The pharmaceutical composition or pharmaceutical combination of claim 12, wherein the pharmaceutical composition or pharmaceutical combination further comprises one or more second pharmaceutical agents.

14. 14. The pharmaceutical composition or pharmaceutical combination of claim 13, wherein the second drug is an immune checkpoint inhibitor, an NSAID, a TKI, or an anticancer drug, or a combination thereof.

15. 13. The pharmaceutical composition or pharmaceutical combination according to claim 12, wherein the medicament is for inducing cell cycle arrest in tumor cells, inducing apoptosis in tumor cells, inducing histone H3 acetylation, inducing immunological memory, activating CTLs, or reducing immunosuppressive cells.

16. 10. The pharmaceutical composition or pharmaceutical combination of claim 9 for use in a method for treating or preventing a disease associated with class I HDAC in a subject in need thereof.

17. 17. The pharmaceutical composition or pharmaceutical combination of claim 16, wherein the pharmaceutical composition or pharmaceutical combination further comprises one or more second pharmaceutical agents.

18. 18. The pharmaceutical composition or pharmaceutical combination of claim 17, wherein the second drug is an immune checkpoint inhibitor, an NSAID, a TKI, or an anticancer drug, or a combination thereof.

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