Histone deacetylase inhibitors for immune modulation in the tumor microscopic environment

Abstract

The present disclosure generally relates to class I HDAC inhibitor compounds, their production and uses. These compounds have epigenetic immune modulatory activities in the tumor microenvironment (TME) and thus inhibit tumor cell growth.

Description

Histone deacetylase inhibitors for immunomodulation in the tumor microenvironment. Field of invention [1] The present disclosure generally relates to class I HDAC inhibitor compounds, their production and uses. In particular, these compounds have epigenetic immunomodulatory activities in the tumor microenvironment (TME) and thus inhibit tumor cell growth. History of the invention [2] Immunotherapy is the standard of care for the treatment of several advanced cancers. Advances in immunotherapy include 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 have immune-related side effects and, more importantly, only a small proportion of patients experience 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, which are intertwined with normal tissue cells. Many growth factors, cytokines, and chemokines are secreted by various cells in the TME. [3] CTLs (cytotoxic T lymphocytes) are primary adaptive immune cells that are dedicated to directly killing cancer cells. CTLs are susceptible to the infiltration of several immunosuppressive cells into the TME, which inactivates CTLs. Well-known immunosuppressive cells include Treg (regulatory T cells), M-MDSC (monocyte-myeloid-derived suppressor cells), PMN-MDSC (polymorphonuclear-myeloid-derived suppressor cells), and TAM (tumor-associated macrophages). These immunosuppressive cells help to inhibit the cytotoxic effect of CTLs on cancer cells. Various mechanisms are implemented by these immunosuppressive cells that lead to dysfunction of CTLs. [4] Although ICI therapies have been shown to be effective in enhancing immune activation to eradicate cancer, these therapies still face the unresolved issues of primary and acquired drug resistance. Intrinsic factors driving primary and acquired resistance to these immunotherapies include genetic and epigenetic mechanisms, which through processes such as immune editing often result in downregulation of MHC I or loss of antigen expression, resulting in a total loss of antigen presentation. Therefore, there is a need to develop compounds with immunomodulatory activities in the TME to stimulate antitumor immunity by modulating the antigen processing and presentation machinery. Summary of the invention [5] In summary, embodiments of the present disclosure provide HDAC class I inhibitor compounds, including pharmaceutically acceptable salts, hydrates, stereoisomers, solvates, or prodrugs thereof, that are capable of modulating epigenetic immunity in the TME. Methods of using such compounds to treat various diseases or conditions, such as cancer, are also provided. [6] In one embodiment, the present disclosure provides a compound of formula (I): (I) 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 can be mono-, di-, tri-, or tetra-substituted; C1 and C2 are C atoms connected by a single bond or a double bond; Ar is selected from the group consisting of: , , , and wherein Ar is connected to C2 through a solid line; R2 has the same meaning as explained for R1. and R3 is hydrogen or C1-C3 alkyl; or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof. [7] 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, designated GNTbm-01. 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, designated GNTbm-02. 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, 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, 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, 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, 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, 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, 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, designated GNTbm-11. In one embodiment, the compound of formula (I) is 5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(22-amino-4-(trifluoromethyl)phenyl)pyridine-2-carboxamide, 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, 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, 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, designated GNTbm-33. In one embodiment, the compound of formula (I) is 4-(4-(pyridin-3-yl)butanamido)-N-(2-amino-4-fluorophenyl)benzamide, 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, 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, designated GNTbm-39. [8] In other embodiments, the present disclosure provides a pharmaceutical formulation or composition comprising a compound as described herein. [9] In other embodiments, the present disclosure provides a method for epigenetic immunomodulation of the TME and / or treating cancer, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical formulation or composition comprising any one or more compounds of Formula (I) or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate, or prodrug thereof.

[10] In some embodiments, the method 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 CTL, for reducing immunosuppressive cells.

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

[12] In some embodiments, the drug 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 CTL, for reducing immunosuppressive cells.

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

[14] In other embodiments, the present disclosure provides the use of an effective amount of the compound or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof or a pharmaceutical formulation or composition in the manufacture of a medicament for treating or preventing a Class I HDAC-associated disease in a subject in need thereof. Brief description of the maps

[15] Figure 1 shows the structures of compounds GNTbm-01, GNTbm-02, and GNTbm-03.

[16] Figure 2 shows the NMR and high-resolution MS spectra: (a) 1H-NMR spectroscopic data of compound GNTbm-01, (b) 1H-NMR spectroscopic data of compound GNTbm-02, (c) 1H-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, (f) high-resolution MS spectroscopic data of compound GNTbm-03.

[17] Figure 3 shows the cell morphology observed by phase-contrast light microscopy after treatment: The change in cell morphology was observed by phase-contrast light microscopy. (a) MDA-MB-231 cells, (b) SW48 cells, (c) M10 cells.

[18] Figure 4 shows the results of the evaluation of GNTbm-02-induced cell cycle arrest in 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 using flow cytometry. (a) and (b) Dose-dependent assay. (c) and (d) Time-dependent assay.

[19] Figure 5 shows the results of the evaluation of GNTbm-02-induced cell cycle arrest in G0 / G1 phase in SW48 cells: 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 phases of the cell cycle was analyzed using flow cytometry. (a) and (b) Dose-dependent method. (c) and (d) Time-dependent method.

[20] Figure 6 shows the results of the evaluation of GNTbm-02-induced cell cycle arrest in G2 / M phase in M10 cells: 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 phases of the cell cycle was analyzed using flow cytometry. (a) and (b) Dose-dependent assay. (c) and (d) Time-dependent assay.

[21] Figure 7 shows the results of the evaluation of GNTbm-02-induced cell apoptosis 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 sub-G1 phase was analyzed using flow cytometry. (a) and (b) Dose-dependent assay. (c) and (d) Time-dependent assay.

[22] Figure 8 shows the results of 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. The cells were stained with PI and the percentage of cells in sub-G1 phase was analyzed using flow cytometry. (a) and (b) Dose-dependent method. (c) and (d) Time-dependent method.

[23] Figure 9 shows the results of the evaluation of GNTbm-02-induced cell apoptosis in M10 cells: 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 sub-G1 phase was analyzed using flow cytometry. (a) and (b) Dose-dependent assay. (c) and (d) Time-dependent assay.

[24] Figure 10 shows the results of Western blot analysis of histone H3 acetylation levels in cells treated with GNTbm-02 and entinostat: Representative immunoblot analysis of acetylated histone H3, β-actin in MDA-MB-231 or SW48 cells. Cells were treated with the indicated concentrations of GNTbm-02 and entinostat for 24 h. Control cells were incubated with vehicle. (a) (c) Extracts from MDA-MB-231 or SW48 cells treated with GNTbm-02 or entinostat were resolved by SDS-PAGE as indicated, followed by Western blot and immunohistochemistry after detection with an antibody for acetylated histone H3 (AcH3). (b) (d) Quantification of AcH3 protein expression levels normalized to β-actin, shown as fold change.

[25] Figure 11 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, 72 h. Extracts of MDA-MB-231 or SW48 cells treated with GNTbm-02 were resolved as indicated by SDS-PAGE followed by Western blotting and immunostaining after detection with an antibody for histone H3 acetylation (AcH3). (b) (d) Quantification of AcH3 protein expression levels normalized to β-actin, shown as fold change.

[26] Figure 12 shows the results of Western blot analysis of the level of histone H3 acetylation in cells treated with GNTbm-04, GNTbm-05, GNTbm-06, GNTbm-11, GNTbm-38, GNTbm-39 and chidamide (as positive control): Representative immunoblot analysis of acetylated histone H3, β-actin in SW48 cells. Cells were treated with the indicated concentrations of the compound for 24 h. Control cells were incubated with a vehicle. (a) Extracts of SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-11 and chidamide were resolved as indicated by SDS-PAGE followed by Western blotting and immunostaining after detection with an antibody for histone H3 acetylation (AcH3). (B) Extracts of SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-06 and chidamide as indicated were resolved by SDS-PAGE followed by Western blotting and immunostaining after detection with an antibody for histone H3 acetylation (AcH3).(c) Extracts of SW48 cells treated with GNTbm-04, GNTbm-05, GNTbm-38, GNTbm-39 and chidamide were resolved using SDS-PAGE, followed by western blotting and staining after detection with an antibody for histone H3 acetylation (AcH3). All these data represent the quantification of AcH3 protein expression levels normalized to β-actin, expressed as fold change.

[27] Figure 13 shows the results of the evaluation of the therapeutic response of GNTbm-02 plus celecoxib at different doses in combination with anti-PD-1 antibody in CT26 tumor-bearing mice: BALB / c mice bearing CT26 tumors were treated with the various treatments 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 (25 mg / kg). Total tumor volume (a) and (b), individual tumor volume (c), mouse body weight (d), and survival rate (e) were recorded. CT26 tumor-bearing mice were treated as indicated and sacrificed when tumor volume reached 3000 mm3 after tumor implantation. Data are given as mean ± SEM; *P< 0.05, **P< 0.01, ***P< 0.001; One-way ANOVA with Tukey test. Gehan-Breslow-Wilcoxon test (e). * Compared to IgG control #, compared to PD-1 group.

[28] Figure 14 shows the evaluation of the combined therapeutic response of the GNTbm series in CT26 tumor-bearing mice. BALB / c mice bearing CT26 tumors were treated with the various therapeutic regimens 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 volume (c), (g), (k), (o), (s), body weight of mice (d), (h), (l), (p), (t), and survival rate (e), (i), (m), (q), (u) were recorded. CT26 tumor-bearing mice were treated as indicated and sacrificed when tumor volume reached 3000 mm3 after tumor implantation. Data are given as mean ± SEM. One-way ANOVA with Tukey test (*P< 0.05, **P< 0.01, ***P< 0.001 versus anti-IgG control). Gehan-Berlow-Wilcoxon test (e).

[29] Figure 15 shows the treatment results of neutral BALB / c mice bearing CT26 tumors treated with different therapeutic regimens: 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) Subcutaneous injection schedule of CT26 tumors and different treatment groups (n = 6 mice per group). (b) Total tumor volume. (c) Folds of tumor volume changes. (d) Mouse body weight. (e) Individual tumor volumes. Neutral mice bearing CT26 tumors were treated as indicated and sacrificed when tumor volume reached 3000 mm3 after tumor implantation.

[30] Figure 16 shows the effect of GNTbm-02 on inhibiting HDAC3 enzyme activity: (a) The assay was performed after incubation of 2 μM GNTbm-02, chidamide or entinostat with HDAC3 enzyme (including assay buffer) for 20 min, 40 min and 60 min. It was shown that GNTbm-02 binds to HDAC3 and inhibits HDAC3 more potently than entinostat. (b) The assay was performed after incubation of 2 μM GNTbm-02, GNTbm-03 orGNTbm-01 with HDAC3 enzyme (including assay buffer) for 20 min, 40 min and 60 min. It was shown that GNTbm-02 binds to HDAC3 and inhibits HDAC3 more potently than GNTbm-03 and GNTbm-01.

[31] Figure 17 GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) modulates mononuclear cell and T cell responses in CT26-bearing models: BALB / c mice bearing CT26 tumors were treated with the indicated treatments, followed by FACS analysis to assess circulating immune cells. Means and SDs are shown with P values. Blood samples were collected on day 16 after treatment in CT26-bearing mice. (a) FACS result for circulating lymphocytes. (b) FACS result for circulating monocytes. (c) FACS result for circulating granulocytes. (d) FACS result for circulating CD3+ T cells. (e) FACS result for circulating CD4+ T cells. (f) FACS result for circulating CD8+ T cells. (g) FACS result for circulating Treg cells. (h) FACS result for circulating CD11b+ cells. (i) FACS result for circulating M-MDSC (CD11b+Ly6C+) cells. (j) FACS result for circulating CD11b+Ly6G+Ly6C+ cells. (k) FACS result for circulating PMN-MDSC (CD11b+Ly6G+Ly6C-) cells.Mean ± SD for n = 8–12 mice per group are shown. 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

[32] Definitions

[33] All technical and scientific terms used herein, unless otherwise defined, have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains, the terms being used in the context of their use in describing the present disclosure. The terms used in the description are for the purpose of describing particular embodiments only and are not intended to limit the invention.

[34] Where a range of values ​​is given, it is understood that any intermediate value, up to one tenth of the lower limit—unless the context clearly dictates otherwise (such as a group containing a number of carbon atoms in which each number of carbon atoms falling within the range is given)—is between the upper and lower limits of that range, and any stated value or other intermediate value within that stated range is included within the invention. The upper and lower limits of such smaller ranges may be independently included within smaller ranges and also be included within the invention, subject to any limitations specifically excluded in the stated range. Where the stated range includes one or both limits, ranges that do not include one or both of those limitations are also included within the invention.

[35] The words "an" as used herein and in the appended claims are used to refer to one or more than one (i.e., at least one) of the grammatical object, unless the context clearly indicates otherwise. For example, "an element" means one element or more than one element.

[36] The term "and / or" as used herein in the specification and in the claims shall be understood to mean "one or both" of the elements that are connected in some cases and in other cases, disconnected. Multiple elements listed with "and / or" shall be construed in the same manner, i.e., "one or more" of the elements that are connected. Other elements may optionally be present in addition to those specifically identified by the "and / or" clause, whether related or unrelated to those specifically identified.

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

[38] The term "alkyl" refers to a straight or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms and lacking unsaturation, having one to fifteen carbon atoms (e.g., C1-C15 alkyl). In certain embodiments, an alkyl contains one to thirteen carbon atoms (e.g., C1-C13 alkyl). In certain embodiments, an alkyl contains one to eight carbon atoms (e.g., C1-C8 alkyl). In other embodiments, an alkyl contains one to five carbon atoms (e.g., C1-C5 alkyl). In other embodiments, an alkyl contains one to four carbon atoms (e.g., C1-C4 alkyl). In other embodiments, an alkyl contains one to three carbon atoms (e.g., C1-C3 alkyl). In other embodiments, an alkyl contains one to two carbon atoms (e.g., C1-C2 alkyl). In other embodiments, an alkyl comprises one carbon atom (e.g., C1 alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., C5-C8 alkyl).In other embodiments, an alkyl group contains two to five carbon atoms (e.g., C2-C5 alkyl). In other embodiments, an alkyl group contains three to five carbon atoms (e.g., C3-C5 alkyl). In other embodiments, the alkyl group is 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), 1-pentyl (n-pentyl). The alkyl group is attached to the rest of the molecule by a bond. Unless specifically stated 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 that in certain embodiments contains two to six or two to eight carbon atoms with 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, ethynyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl, and the like.

[39] The term "alkoxy" refers to a radical bonded through an oxygen atom with the formula -O-alkyl, where alkyl is an alkyl chain as defined above.

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

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

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

[43] The term "heteroaryl" as used herein refers to a single or multiple ring system (such as two or three or more rings), fused or unfused, radical or ring, having at least one aromatic ring, having five to ten ring atoms, one of which is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

[44] The term "heterocycloalkyl", as used herein, refers to a 3-, 4-, 5-, 6-, or 7-membered non-aromatic ring or a fused two- or three-ring group of the unfused system, wherein (i) at least one ring contains between one and three 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 may be optionally oxidized, (iv) the nitrogen heteroatom may be optionally quaternized, and (iv) any of the foregoing rings may be 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.

[45] The term "pharmaceutically acceptable salt" 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 falling within the term "pharmaceutically acceptable salt" refer to non-toxic salts of the compounds of this invention, generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts of the 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, diethylacetate, digluconate, dihydrochloride, dodecylsulfane, edetate, adesylate, acetylate, isylate, ethanesulfonate, formate, fumarate, glucoseptate, glucoheptanoate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamate, hydrobromide, hydrochloride, 2-hydroxyethanesulfonate, hydroxynaphthoate, hydroiodide,Iodide, isonicotinate, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl nitrate, methyl sulfate, methane sulfonate, mucate, 2-naphthalene sulfonate, napsylate, nicotinate, nitrate, oleate, oxalate, pamoate (ambonate), palmitate, pantothenate, pectinate, persulfate, phosphate / diphosphate, pimelate, phenylpropanoate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, theochlate, thiocyanate, tosylate, triethiodide, trifluoroacetate, undecunate, valerate, and the like. In addition, where the compounds of the invention have an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese, manganeseous, potassium, sodium, zinc and the like. Salts derived from pharmaceutically acceptable non-toxic organic bases include salts of primary, secondary and tertiary amines, cyclic amines, dicyclohexylamines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine,amine, 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 includes nitrogen-containing basic groups which may be reacted with agents such as lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides such as benzyl and phenethyl bromides, etc., can be quaternized.

[46] The term "individual" includes living organisms such as humans, monkeys, cattle, sheep, horses, pigs, cows, goats, dogs, cats, mice, rats, cultured cells, and transgenic strains thereof. An individual is a human being.

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

[48] ​​The term "treatment" or "treatment" refers to a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the underlying cause of the disease or condition itself, not just the symptoms. Treatment can be any reduction at the local level and can be, but is not limited to, complete eradication of the disease, condition, or symptoms of the disease or condition.

[49] The terms "prevention", "inhibition", or "inhibition" mean the inhibition or prevention of symptoms associated with the target disease.

[50] The term "therapeutically effective amount" refers to that amount of a compound, substance, or combination, including a compound of the present disclosure, that is effective to produce a desired therapeutic effect, with a reasonable benefit / risk ratio applicable to any medical treatment.

[51] Class I HDAC inhibitor compounds

[52] Epigenetic therapies for cancer, such as histone deacetylase inhibitors, can stimulate antitumor immunity by regulating the transformation of the antigen processing and presentation machinery. Epigenetic modifications play an important role in controlling the initiation and progression of tumors. Epigenetic regulation is mainly mediated by two major mechanisms affecting gene expression: DNA methylation / demethylation, which occurs by the addition / removal of a methyl group to DNA, and histone acetylation / deacetylation, which occurs by the enzymatic addition / removal of an acetyl group to histone proteins complexed with DNA. Histone deacetylase inhibitors (HDACis) are thought to be promising targets for the development of new drugs. The underlying mechanism for the important role of HDACs in cancer is the control of the degree of acetylation on histones or non-histone proteins, which are involved in the regulation of the cell cycle, differentiation, apoptosis, DNA damage response, proliferation, metastasis, and other cellular processes.

[53] Class I HDACs are predominantly located in the nucleus and are ubiquitously expressed in human tissues, playing an important role in the control of cell proliferation, differentiation, and cell cycle progression. Class I HDACs are highly expressed in some cancers; for example, HDAC1 is highly expressed in prostate, gastric, colon, breast, lung, and esophageal cancers; HDAC2 is highly expressed in gastric, cervical, and colorectal malignancies; and HDAC3 is highly expressed in colon and breast cancers. Uncontrolled expression of HDACs results in the silencing of many genes that inhibit cell growth, and thus loss of cell growth surveillance and control of cell differentiation, cell cycle arrest, and apoptosis. Impaired HDAC overexpression was significantly associated with tumor malignancy and poor prognosis. Many class I HDAC inhibitors have epigenetic immunomodulatory properties.

[54] The mechanisms of immunomodulation by HDAC inhibitors in the TME have been reported to involve components of soluble factors and immune cells. The expression of a variety of genes and proteins is altered through epigenetic regulation by HDAC inhibitors by inhibiting specific HDAC isoforms, which shifts the state of the TME to one that favors the killing of cancer cells as a result. Previously published studies have shown that some HDAC inhibitors have immunomodulatory properties that control the secretion of cytokines / chemokines, antigen-presenting cells, and reduce the number or function of Tregs and induce the activation of NK cells. Other studies have shown mechanisms that increase the expression of cancer antigens and modulate the activity of immunosuppressive cells such as MDSCs. Selective class I HDAC inhibitors can increase the expression of PD-L1 and MHC I on cancer cells. In addition, class I HDAC inhibitors reduce myeloid-derived suppressor cells (MDSCs) that infiltrate the tumor microenvironment.

[55] In one aspect, the present disclosure provides a composition of formula (I): (I) 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 can be mono-, di-, tri-, or tetra-substituted; C1 and C2 are C atoms connected by a single bond or a double bond; Ar is selected from the group consisting of: , , , and wherein Ar is connected to C2 through a solid line; R2 has the same meaning as explained for R1. and R3 is hydrogen or C1-C3 alkyl; or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[56] In one embodiment, the compound of formula (I) has the following formula (Ia): (Ia), wherein W, Y, R1, C1, C2 and Ar have the same meaning as described; or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[57] In one embodiment, Ar is selected from six-membered rings. In one embodiment, R2 and the Ar atom attached to C2 are in the para positions.

[58] In one embodiment, Ar is selected from the group consisting of: , and

[59] 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 preferred embodiments, W is N or CH and Y is CH.

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

[61] In one embodiment, R1 is hydrogen.

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

[63] 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.

[64] In one embodiment, R2 is hydrogen.

[65] In one embodiment, Aris, R2 and the Ar atom attached to C2 are in para positions, and C1 and C2 are C atoms connected by a double bond.

[66] In one embodiment, Aris, R2 and the Ar atom attached to C2 are in the para positions and R1 is hydrogen or F.

[67] In one embodiment, Aris, R2 and the Ar atom attached to C2 are in the para positions and R2 is hydrogen or CH3.

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

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

[70] In one embodiment, Aris, R2, and the Ar atom attached to C2 are in the para positions, R1 is hydrogen or F, R2 is hydrogen or CH3, and C1 and C2 are C atoms linked by a double bond.

[71] In one embodiment, the compound of formula (I) can be the following compounds: or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[72] The present disclosure encompasses all stereoisomeric forms of compounds of formula (I). The asymmetric centers present in compounds of formula (I) can independently have the (R) or (S) configuration. When bonds to chiral carbons are shown as straight lines in the structural formulas of the invention, it is understood that both (R) and (S) are chiral carbon configurations, and thus both enantiomers and mixtures thereof are included in the formula. When a particular configuration is depicted, that enantiomer (either (R) or (S), at that center)) is intended. Similarly, when a compound name is recited without a chiral name for a chiral carbon, it is understood that both (R) and (S) are chiral carbon configurations, and thus the individual enantiomers and mixtures thereof are embraced by that name.

[73] The invention includes all enantiomers, region isomers, and diastereomers and mixtures of two or more stereoisomers, i.e., mixtures of enantiomers and / or diastereomers, in all ratios. Thus, the enantiomers are subject to the invention in pure enantiomer form, both as levorotatory antipodes and as rotatory antipodes, in racemate form and in the form of mixtures of two enantiomers in all ratios. In the case of cis / trans isomerism, the invention includes both the cis form and the trans form as well as mixtures of these forms in all ratios. The preparation of the individual stereoisomers can be carried out, if desired, by separating the mixture by conventional methods, for example by chromatography or crystallization, using stereochemically uniform starting materials for the synthesis or by stereoselective synthesis. If desired, the separation of the stereoisomers can be preceded by derivatization. The separation of a mixture of stereoisomers can be carried out at an intermediate stage during the synthesis of a compound of formula (I) or on a racemic final product.Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates, if necessary derivatized with a reagent containing a stereogenic center of known configuration. Where the compounds of the invention are capable of tautomerization, all individual tautomers as well as 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 thereof, including racemates, enantiomers, diastereomers and tautomers and mixtures thereof.

[74] As used herein, the symbols and conventions used in these processes, schemes, and examples, unless a particular abbreviation is specifically defined, are consistent with those used in contemporary scientific works, e.g., the Journal of the American Chemical Society or the Journal of Biological Chemistry. In particular, but without 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 (millimole); hr or hrs (hours); min (minutes); MS (mass spectrometry); ESI (electrospray ionization); TLC (thin layer chromatography); and HPLC (high pressure liquid chromatography). For all of the following examples, standard work-up and purification procedures known to those skilled in the art may be used. Unless otherwise noted, all temperatures are expressed in °C. (degrees Celsius). All reactions are performed at room temperature unless otherwise noted.The synthetic methods illustrated herein are intended to exemplify applied chemistry through the use of specific examples and are not indicative of the scope of the disclosure.

[75] The compounds of formula (I) of the present disclosure are prepared according to general chemical synthetic methods. An exemplary synthetic route is shown below: Where RA refers to the –C-C1C2-Ar moiety and RB refers to the moiety in formula (I).

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

[77] Other suitable modifications to the process, e.g., use of appropriate protecting / deprotecting agents for groups sensitive to specific reaction conditions during synthesis, isolation and purification of intermediates for subsequent reactions, selection of appropriate solvents, etc., can also be introduced by a person skilled in the art as needed. For example, the -OH group of compound (β) in the route shown above may be protected prior to reaction with compound (α), and the resulting product may be protected to provide compound (γ), etc.

[78] Pharmaceutical formulas / compounds

[79] In another aspect, the invention provides a pharmaceutical formulation / composition comprising a combination of any of Formula (I) or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, together with a pharmaceutically acceptable carrier.

[80] The pharmaceutical formulation / composition can 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 anticancer agent. In another embodiment, the pharmaceutical composition / formulation comprises a compound described herein and an immune checkpoint inhibitor and / or an NSAID or optionally a tyrosine kinase inhibitor (TKI).

[81] In one embodiment, the immune checkpoint inhibitor can be used in a formulation with the pharmaceutical composition described herein to stimulate the immune system against cancer cells and to treat cancer. The immune checkpoint inhibitor is an antibody or agent against cytotoxic T lymphocyte antigen-4 (CTLA-4), an antibody or agent against cell death protein 1 (PD-1), an antibody or agent against programmed death ligand 1 (PD-L1), an antibody or agent against T-cell immunoglobulin and mucin domain 3 (TIM-3), an antibody or agent against B and T lymphocyte attenuating (BTLA). The anti-V domain Ig comprises a T cell activation suppressor (VISTA) antibody or agent, a lymphocyte anti-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. Preferably, the immune checkpoint inhibitor is pembrolizumab, lambrolizumab, pidilizumab, nivolumab, durvalumab, avelumab, or atezolizumab.Examples of PD-1 or PD-L1 inhibitors include, , humanized antibodies that block human PD-1 without restriction, such as lambrolizumab (anti-PD-1 Ab, brand name Keytruda), or pidilizumab (anti-PD-1 Ab), Boencio (anti-PD-L1 Ab, avelumab), Imfinzi (anti-PD-L1 Ab, durvalumab), and Tecentriq (anti-PD-L1 Ab, atezolizumab), as well as fully human antibodies such as nivolumab (anti-PD-1 Ab, brand name Opdivo) and cemiplimab-rwlc (anti-PD-1 Ab, brand name Libtayo). Other PD-1 inhibitors may include soluble PD-1 ligand displays, including, PD-L2 Fc-free fusion protein also known as B7-DC-Ig or AMP-244, and other PD-1 inhibitors currently under investigation and / or development for use in therapy. In addition, immune checkpoint inhibitors may include, without limitation, human or fully human PD-L1-blocking antibodies such as durvalumab and MIH1 (anti-CD274 (PD-L1, B7-H1) monoclonal antibody) and other PD-L1 inhibitors currently under investigation.

[82] NSAIDs are a class of drugs that reduce pain and fever and, at higher doses, reduce inflammation. Most NSAIDs inhibit the activity of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), and thus the synthesis of thromboxanes and prostaglandins. Inhibition of COX-2 is thought to result in anti-inflammatory, analgesic, and antipyretic effects, whereas those NSAIDs that inhibit COX-1, particularly aspirin, may cause gastrointestinal bleeding and ulceration at high doses. COX-2 inhibitors are widely used to treat autoimmune and inflammatory diseases. Cyclooxygenase (COX), which has two isoforms, COX-1 and COX-2, is an enzyme responsible for the rate-determining step in the synthesis of the bioactive lipids prostanoids, including 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 to initiate inflammatory processes. Selective COX-2 inhibitors are a type of nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the NSAID includes, but is not limited to, aspirin, ibuprofen, indomethacin, naproxen, and a COX-2 inhibitor. In some embodiments of the present disclosure, the NSAID is a COX2 inhibitor. In some embodiments, the COX2 inhibitor includes, but is not limited to, Celebrex (generic name celecoxib), Rofecoxib, Imrecoxib, and Etoricoxib. Preferably, the COX2 inhibitor is celecoxib.

[83] Tyrosine kinase inhibitors (TKIs) are a family of small molecules with cytosolic or receptor tyrosine kinase inhibitory activity. TKIs inhibit these growth factor signaling pathways by various mechanisms. They compete with ATP, substrates, or sites for dimerization (pairing or bifurcation), and can also act allosterically. Cytosolic or receptor tyrosine kinase inhibition has been demonstrated by several different classes of TKIs, such as direct competition for ATP binding to tyrosine kinase, allosteric inhibition of tyrosine kinase, and inhibition of ligand binding to receptor tyrosine kinase. TKIs are increasingly playing an important role in the treatment of cancers, particularly VEGFR inhibitors such as Axitinib, Lenvatinib, Cabozantinib, and Regorafenib. In some embodiments of the disclosure, the TKI is a receptor tyrosine kinase inhibitor. Preferably, the TKI is a vascular endothelial growth factor receptor (VEGFR) inhibitor.Preferably, the TKI is Cabozantinib, Regorafenib, Axitinib, Afatinib, Ninetedanib, Crizotinib, Alectinib, Trametinib, Dabrafenib, Sunitinib, Ruxolitinib, Vemurafenib, Sorafenib, Ponatinib, Encorafenib, Brigatinib, Pazopanib, Dasatinib, Imatinib, Lenvatinib, Vandetanib, Surufatinib or Sitravatinib.

[84] The additional anticancer agent is any anticancer agent described herein or known in the art. In one embodiment, the additional anticancer agent is platinum-based chemotherapy or 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, duscad), a vinca alkaloid, vinflurane (vinflurane (Javlor), vinorelbine, vinblastine, etoposide, or pemetrexed, cytarabine). In one embodiment, the additional anticancer agent is 5-fluorouracil (5-FU). In certain embodiments, the additional anticancer agent is any other anticancer agent known in the art.

[85] To prepare the pharmaceutical compositions / formulations of the invention, one or more compounds of this disclosure as the active ingredient are intimately mixed with a pharmaceutical carrier in accordance with conventional pharmaceutical compounding techniques, which carrier may take a variety of forms depending on the form of preparation intended for administration, for example, oral or injectable, such as intramuscular. In preparing the compositions in oral dosage form, any of the conventional pharmaceutical excipients may be used. Thus, for liquid oral preparations, such as suspensions, elixirs, and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavors, preservatives, colors, and the like; For solid edible preparations such as powders, capsules, caplets, gelcaps, and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrants, and the like. Because of their ease of administration, tablets and capsules are the most convenient oral dosage unit form, in which case solid pharmaceutical carriers are obviously used.If desired, the tablets may be sugar-coated or enteric-coated by standard methods. For injectable drugs, the carrier usually comprises sterile water, although other ingredients may be included, for example, for purposes such as aiding in solubility or preservation. Injectable suspensions may also be prepared, in which case suitable liquid carriers, suspending agents and the like may be used. The pharmaceutical compositions herein contain, in each dosage unit, for example, a tablet, capsule, powder, injection, teaspoonful and the like, an amount of the active ingredient necessary to provide an effective dosage as described above.

[86] Types of liquids into which the novel compounds 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 with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical carriers. Suitable dispersing or suspending agents for aqueous suspensions include: synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethyl cellulose, methyl cellulose, polyvinyl pyrrolidone, or gelatin.

[87] Tablets and capsules for oral administration are usually presented in unit dosage form and contain common excipients such as binders, fillers (including cellulose, mannitol, lactose), diluents, tabletting agents, lubricants (including magnesium stearate), detergents, disintegrants (such as polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate), colorants, flavors, and wetting agents (e.g., sodium lauryl sulfate).

[88] Solid oral compositions can be prepared by conventional methods of compounding, filling, or tabletting. The compounding operation can be repeated to distribute the active principle throughout compositions containing large amounts of filler. Such operations are conventional.

[89] For injectable administration, unit doses of liquid can be prepared containing the compound and a sterile carrier. Depending on the type of carrier and concentration, the compound can be suspended or dissolved. Injectable solutions are usually prepared by dissolving the compound in a carrier, sterilizing by filtration, filling into suitable vials, and sealing. Advantageously, excipients such as local anesthetics, preservatives, and buffering agents can also be dissolved in the carrier. To increase stability, the compound can be frozen after filling the vials and the water removed under vacuum. Injectable suspensions are prepared in much the same manner, except that the compound can be suspended in the carrier instead of being dissolved, and sterilized by exposure to ethylene oxide before suspension in the sterile carrier. Advantageously, a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the compound used.

[90] Pharmaceutical preparations for administration by inhalation can be delivered from a nebulizer or a pressurized pack.

[91] Therapeutic applications

[92] In another aspect, the present disclosure provides a method of modulating epigenetic immunity of the TME comprising administering to a subject in need thereof an effective amount of a mixture or a pharmaceutical composition / formulation described herein.

[93] In another aspect, the present disclosure provides a method of treating or preventing a Class I HDAC-associated disease in a subject, comprising administering an effective amount of a mixture or a pharmaceutical composition / pharmaceutical formulation as provided herein to a subject in need thereof.

[94] In one embodiment, the methods further comprise administering one or more secondary agents. In some embodiments, the secondary agent is an immune checkpoint inhibitor, an NSAID, a TKI, or an anticancer agent. In another embodiment, the pharmaceutical composition / composition 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 anticancer agent are as described herein.

[95] The compounds of the invention are useful for the treatment or prevention of any disease and / or condition in which inhibition of class I HDAC is desired. In particular, the compounds of the invention have epigenetic immunomodulation of the TME, thereby improving immunotherapy. Inhibition of HDAC enzyme activity can result in reduced tumor growth. Accordingly, the invention provides methods for the treatment or prevention of tumors or cancers.

[96] Examples of cancers that can be treated according to current teachings include, invasive breast cancer, adenocarcinoma, lung cancer (non-small cell, squamous cell carcinoma, adenocarcinoma, and large cell lung cancer), liver cancer, colorectal cancer, brain and head and neck cancer (e.g., neuro / glioblastoma), breast cancer, ovarian cancer, transitional cell bladder cancer, prostate cancer, oral squamous cell carcinoma, bone sarcoma, adrenocortical cancer, gastrointestinal tumors including colorectal cancer, bile duct cancer such as gallbladder carcinoma (GBC), bladder cancer, esophageal cancer, gastric cancer, cervical cancer, salivary gland cancer, benign neoplasm diarrhea, ductal carcinoma in situ, paronychia, cholangiocarcinoma, kidney cancer, pancreatic cancer, medulloblastoma, glioblastoma, mammary ductal tumors, HER2 positive and triple negative, Hematologic malignancies and leukemias (acute myelogenous leukemia (AML), B-cell-precursor acute lymphoblastic leukemia (ALL), T-cell-deficient ALL, and chronic myelogenous leukemia (CML)) are included, but are not limited to.

[97] The compounds or pharmaceutically acceptable salts thereof are administered orally, nasally, dermally, pulmonaryly, inhaledly, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally, and injectablely. In one embodiment, the compound is administered orally. One skilled in the art will recognize the advantages of particular methods of administration.

[98] The dosage regimen for the use of the compounds is selected in accordance with various factors including the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt being used. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, combat, or arrest the progression of the disease.

[99] Now that the invention has been described by means of the written description, those skilled in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and the following examples are intended to illustrate and not to limit the claims that follow. Examples

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

[101] GNTbm-01, GNTbm-02, GNTbm-03, GNTbm-04, GNTbm-05, GNTbm-06, GNTbm-08, GNTbm-11, GNTbm-12, GNTbm-19, GNTbm-25, GNTb, 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, MedChemExpress USA). The following antibodies and reagents were used for animal experiments: mouse anti-PD-1 (CD279) monoclonal antibody (RMP1-14; Bio X Cell), and mouse anti-IgG2a isotype monoclonal antibody (2A3; Bio X Cell). Electrospray ionization mass was 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 of reagent grade and used without further purification unless otherwise noted.All reactions were monitored by thin layer chromatography using Merck silica gel 60 F254 glass-backed plates (20 x 20 cm) for completeness. The resulting chromatograms were visualized under UV (254 nm) illumination. 1H NMR and 13C NMR were recorded on a Bruker AVANCE 400MHz PLUS and Bruker AVANCEIII HD 600 MHz spectrometer and instruments, and chemical shifts were recorded in parts per million (ppm, ). Pluripotencies are reported as s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quadruplet), dd (doublet binary), td (triplet binary), and m (multiplet). Coupling constants (J) are expressed in Hz. The purity of the final compound was determined with the Waters ACQUITY Arc system using a C18 column (Waters XSelect HSS T3 5 μm, 4.6 mm x 250 mm) at 40°C. Elution was performed using water containing 0.1% trifluoroacetic acid as mobile phase A and methanol as mobile phase B.Washing conditions: at 0 min, phase A 90% + phase B 10%; at 6 min, phase A 70% + phase B 30%; at 12 min, phase A 50% + phase B 50%; at 18 min, phase A 10% + phase B 90%; at 23 min, phase A 90% + phase B 10%. 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 more than 90%.

[102] Preparation examples

[103] Example 1 GNTbm-01

[104] The combined route is shown below:

[105] 6-Aminopyridine-3-carboxylic acid (1).

[106] 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 for 4–8 h at 40–65 °C. After cooling to RT, it was acidified with 10% HCl(aq), filtered by suction and the product was dried in an oven for approximately 24 h to give solid product compound 1.

[107] 2-(Trimethylsilyl)ethyl 6-aminopyridine-3-carboxylate (2).

[108] 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 h. The mixture was concentrated and purified by silica gel column chromatography to give compound 2.

[109] 2-(Trimethylsilyl)ethyl 6-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)pyridine-3-carboxylate (3).

[110] To a solution of DCC (86.9 mg) in DCM was added compound 2 (50 mg) and (E)-4-(6-methylpyridin-3-yl)but-3-enoic acid (67.2 mg) in DCM in an ice bath. The mixture was stirred at room temperature for approximately 8 h. The product was extracted using ethyl acetate and the organic layer was washed with water. The combined organic layers were dried over MgSO4, concentrated, and purified by silica gel column chromatography to give compound 3.

[111] 6-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)pyridine-3-carboxylic acid (4).

[112] To a solution of compound 3 (50 mg) in THF (11 mL) was added 12N hydrochloride (11 mL), and the mixture was stirred at room temperature for 4–10 h. The mixture was concentrated and purified by silica gel column chromatography to give compound 4.

[113] 6-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)pyridine-3-carboxamide (5).

[114] To a solution of 4-fluorobenzene-1,2-diamine (72.9 mg), EDC (89.7 mg), HOBt (46.8 mg) in DMF was stirred at 10–10 °C for 20–60 min. Compound 4 (85.9 mg) in DMF and Et3N (161 μL) were added and the mixture was stirred at room temperature for approximately 72 h. 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 yield compound 5. 1H NMR (400 MHz, acetone-d6): 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). 13C NMR (100 MHz, DMSO-d6): δ 23.77, 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, 147.81, 147.16, 147.16, 147.16; ESI-MS m / z: 428.1496 [M+Na+].

[115] Example 2 GNTbm-02

[116] The combined route is shown below:

[117] 5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)pyridine-2-carboxylic acid

[118] A solution of E)-(4-(6-methylpyridin-3-yl)but-3-enoic acid (769 mg) and DCC (895 mg) in DCM was stirred at 10–10 °C for 20–60 min. 6-Aminopyridine-3-carboxylic acid (500 mg) in DCM was added and the mixture was stirred at room temperature for approximately 48 h. The mixture was filtered to collect a solid powder. The solid powder was dissolved in MeOH, filtered, and concentrated by rotapur to give crude compound 6.

[119] 5-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)pyridine-2-carboxamide (7).

[120] A solution of 4-fluorobenzene-1,2-diamine (42.4 mg) and EDC (52.2 mg, HOBt (26 mg)) in DMF was stirred at 10–10 °C for 20–60 min. 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 h. 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 give compound 7. 1H NMR (400 MHz, acetone-d6): δ 2.46 (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): δ 41.93, 104.06, 104.32, 105.16, 105.39, 124.08, 125.12, 125.94, 128.14. 128.41, 128.65, 130.93, 1313.14, 132.14, 139.91, 141.19, 146.07, 147.83, 158.38, 165.26, 168.08, 172.56. ESI-MS m / z: 428.1479 [M+Na+].

[121] Example 3 GNTbm-03

[122] The combined route is shown below:

[123] 4-((E)-4-(6-methylpyridin-3-yl)but-3-enamido)benzoic acid (8).

[124] A solution of 4(E)-4-(6-methylpyridin-3-yl)but-3-enoic acid (671.9 mg) and DCC (782.4 mg) in DCM was stirred at 10–10 °C for 20–60 min. 4-Aminobenzoic acid (400 mg) in DCM was added and the mixture was stirred at room temperature for another 5–10 h. The mixture was filtered to collect the solid powder. The solid powder was dissolved in MeOH, filtered, and concentrated by rotary evaporator to give crude compound 8.

[125] 4-((E)- ​​4-(6-methylpyridin-3-yl)but-3-enamido)-N-(2-amino-4-fluorophenyl)benzamide (9).

[126] A solution of 4-fluorobenzene-1,2-diamine (255.4 mg), EDC (314.4 mg), and HOBt (164 mg) in DMF was stirred at 10–10 °C for 20–60 min. 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 h. 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 yield compound 9. 1H NMR (400 MHz, acetone-d6): δ 2.46 (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). 13C NMR (100 MHz, DMSO-d6): δ 23.75, 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, 1615.03, 1615.03. ESI-MS m / z: 405.1731 [M+H+].

[127] Example 4 GNTbm-04, GNTbm-05, GNTbm-11, GNTbm-33, GNTbm-37, GNTbm-38, and GNTbm-39

[128] The combined route is shown below:

[129] Ethyl (E)-4-(pyridin-3-yl)but-3-enoate (11). To a solution of nicotinaldehyde 10 (10 g, 93 mmol), PPh3 (36.7 g, 140 mmol), 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. 1H NMR (600 MHz, CDCl3) δ 8.56 (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 =7.1 Hz, 3H).

[130] (E)-4-(Pyridin-3-yl)but-3-enoic acid (12). To a solution of 11 (6 g, 31 mmol) in THF (100 mL) was added LiOH (2.25 g in 50 mL H2O, 94 mmol) and stirred at RT for 1–4 h. The mixture was concentrated to remove THF. The aqueous solution was acidified with 1N 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 8.56 (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).

[131] Synthesis of GNTbm-04, GNTbm-05, GNTbm-11, GNTbm-33, GNTbm-38, and GNTbm-39. To a solution of compound 12 (1eq), compound 13 (1.1 eq), and HATU (1.1 eq) in DMF was added DIPEA (1~2.5 eq). The mixture was stirred at RT for 1~4 h (monitored by LCMS). Aniline (1.1 eq), HATU (1.1 eq), and DIPEA (1~2.5eq) were added to the reaction mixture. The mixture was stirred for another 1~4 h at RT (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.

[132] GNTbm-04, yield: 45 mg, 40%.1H NMR (600 MHz, DMSO-d6) δ10.60(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, 2.1Hz, 1H), 8.09(d, J=9Hz, 1H), 7.91(d, J = 7.8Hz, 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=6Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ40.48, 101.95, 102.12, 102.41, 102.56, 119.74, 122.81, 123.69, 125.78, 126.52, 126.73, 126.79, 129.29, 132.28, 132.57, 138.25, 138.95, 144.37, 144.41, 147.80, 148.40, 159.74, 161.33 162.27, 169.76. LCMS (ESI) m / z 392.4 [M+H] + , HPLC purity: 96.12%.

[133] GNTbm-05, yield: 88 mg, 53%.1H NMR (600 MHz, DMSO-d6) δ10.60(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, 2.1Hz, 1H), 8.11 (d, J = 8.6Hz, 1H), 7.91 (d, J = 8Hz, 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 = 7.9Hz, 1H), 6.61-6.57 (m, 2H), 4.88 (s, 2H), 3.42 (d, J=6.0Hz, 2H).13C NMR (100 MHz, DMSO-d6): δ40.49, 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, 141.56, 144.44, 147.80, 148.39, 161.86, 169.76. LCMS (ESI) m / z 374.3 [M+H]+.. HPLC purity: 99.32%.

[134] GNTbm-11, yield: 88 mg, 22%.1H NMR (600 MHz, DMSO-d6) δ10.62(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, 2.3Hz, 1H), 8.11 (d, J=8.6Hz, 1H), 7.91 (d, J = 8Hz, 1H), 7.77 (s, 1H), 7.36 (dd, J = 7.9, 4.7 Hz, 1H), 7.27 (d, J=8.5Hz, 1H), 6.91 (d, J=8.4Hz, 1H), 6.62-6.55 (m, 2H), 5.63(s, 2H), 3.42 (d, J=6Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ40.49, 115.71, 121.95, 122.90, 122.94, 123.68, 125.76, 126.55. 129.30, 132.28, 132.57, 138.42, 138.42, 138.97, 145.75, 147.80, 162.50, 169.79. LCMS (ESI) m / z 442.4 [M+H]+.. HPLC purity: 93.63%.

[135] GNTbm-33, yield: 63 mg, 16%.1H NMR (600 MHz, DMSO-d6) δ10.32(s, 1H), 9.60 (s, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.44 (dd, J = 1.5,4.7 Hz, 1H), 7.97 (d, J = 8.7 Hz, 1H), 7.91(dt, J=8.0, 1.8Hz, 1H), 7.74 (d, J=8.7Hz, 1H), 7.51 (d, J = 1.2Hz, 1H), 7.36 (dd, J = 7.9, 4.8 Hz, 1H), 7.27 (dd, J=1.7, 8.5Hz, 1H), 6.88 (d, J=8.4Hz, 1H), 6.62-6.55 (m, 2H), 5.65(s, 2H), 3.38 (d, J=5.5Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ40.70, 115.22, 115.42, 115.63, 118.21, 122.44, 123.33. 123.69, 123.79, 124.10, 125.89, 126.19, 112.78, 128.78, 128.78, 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%.

[136] GNTbm-33, yield: 108 mg, 47%.1H NMR (600 MHz, DMSO-d6) δ10.32(s, 1H), 9.56 (s, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.44 (dd, J = 1.4,4.7 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.91(dt, J=8.0, 1.9Hz, 1H), 7.73 (d, J=8.7Hz, 1H), 7.36 (dd, J = 7.9, 4.7 Hz, 1H), 7.16 (d, J=7.5Hz, 1H), 6.96 (dt, J=7.9, 1.4Hz, 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=5.5Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ40.69, 116.10, 116.24, 118.23, 118.34, 123.46, 123.68. 126.20, 126.32, 126.60, 128.66, 129.0, 129.04, 123.04, 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%.

[137] GNTbm-39, yield: 60 mg, 25%.1H NMR (600 MHz, DMSO-d6) δ10.30(s, 1H), 9.49 (s, 1H), 8.62 (d, J = 2.0 Hz, 1H), 8.44 (dd, J = 1.6,4.7 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.90(dt, J=8.0, 1.9Hz, 1H), 7.72 (d, J=8.7Hz, 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=5.5Hz, 2H).13C NMR(100 MHz, DMSO-d6): δ40.69, 101.35, 101.52, 101.92, 102.07, 118.20, 118.32, 119.40, 123.68, 126.20, 128.42, 128.49, 112.68, 128.68, 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%.

[138] Synthesis of GNTbm-37

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

[140] GNTbm-37, yield: 110 mg, 49%.1H NMR (600 MHz, DMSO-d6) δ10.20(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), 7.76(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 = 11.1, 2.0 Hz, 1H). 7.2, Hz, 1H), 5.21(s, 2H), 2.38 (t, J=7.2Hz, 2H) 1.93 (m, J=7.4Hz, 2H). 13C NMR(100 MHz, DMSO-d6): δ26.20, 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%.

[141] GNTbm-06 and GNTbm-12.

[142] The combined route is shown below:

[143] (E)-4-(6-Methyl-3-pyridyl)but-3-enoic acid (15). To a dried round-bottom flask was added 2-carboxyethyl(triphenyl)phosphonium bromide (37.7 g, 90.8 mmol) anhydrous THF (200 mL) 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 1–5 h. 6-Methylpyridine-3-carbaldehyde (10.0 g, 82.6 mmol) was added and the resulting mixture was stirred at room temperature for 8–20 h. The reaction mixture was quenched with water (10 mL) and concentrated to dryness. To the mixture was added 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 adjusted to pH with 4N NaOH (aq.) and concentrated to dryness. The residue was purified by column chromatography to give (E)-4-(6-methyl-3-pyridyl)but-3-enoic acid (5.10 g, 35%) as a white solid.

[144] Synthesis procedure of GNTbm-06 and GNTbm-12. To a solution of compound 15 (1eq), compound 13b (1.1 eq), and HATU (1.1 eq) in DMF was added DIPEA (1~2.5eq). The mixture was stirred at RT for 1 to 4 h (monitored by LCMS). Aniline (1.1 eq), HATU (1.1 eq), and DIPEA (1 to 2.5 eq) were added to the reaction mixture. The mixture was stirred at RT for another 1 to 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 desired product.

[145] GNTbm-37, yield: 95 mg, 43%.1H NMR (600 MHz, DMSO-d6) δ10.59(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, 1H), 7.50(d, J=7.9Hz, 1H), 7.22(d, J =7.3, 8.0 Hz, 1H), 6.94(t, J =7.6, 1H), 6.82(d, J =7.9, 1H), 6.65 (t, J = 7.6, Hz, 1H), 6.57 (d, J = 16.1, Hz, 1H), 6.50 (dt, J = 7.0,15.5, Hz, 1H), 4.88 (s, 2H), 3.40 (d, J=6.7Hz, 2H) 2.45 (s, 3H) . 13C NMR(100 MHz, DMSO-d6): δ23.71, 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, 1616.88, 161.98. LCMS (ESI) m / z 388.4 [M+H]+. HPLC purity: 94.56%.

[146] 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=8.6Hz, 1H), 7.80 (dd, J = 2.0, 8.1 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 = 6.9,15.8, Hz, 1H), 5.63 (s, 2H), 3.40 (d, J=6.8Hz, 2H), 2.45 (s, 3H).13C NMR(100 MHz, DMSO-d6): δ23.70, 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%.

[147] Example 6 GNTbm-08, GNTbm-19, and GNTbm-25

[148] The combined route is shown below:

[149] 4-(6-Methylpyridin-3-yl)butanoic acid (17) was added to a solution of 15 (1 g, 5.6 mmol) in MeOH (10 mL) of Pd / C (200 mg) and stirred at RT for 1–8 h. The mixture was filtered through a pad of Celite and the filtrate was concentrated to dryness to give compound 17 (1 g, 99%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 12.7(s, 1H), 8.26 (d, J = 2.0 Hz, 1H), 7.49 (dd, J=7.9,2.3, HZ 1H), 7.16 (d, J=7.9Hz, 1H), 2.55 (t, J = 2.0, 7.7 Hz, 1H). 2.41 (s, 1H), 2.20 (t, J = 7.4, Hz, 1H) 1.77 (quint, J = 7.5 Hz, 1H).

[150] Synthesis procedure of GNTbm-08, GNTbm-19, and GNTbm-25. To a solution of compound 17 (1eq), compound 13b (1.1 eq), and HATU (1.1 eq) in DMF was added DIPEA (1 to 2.5 eq). The mixture was stirred at RT for 1 to 4 h (monitored by LCMS). Aniline (1.1 eq), HATU (1.1 eq), and DIPEA (1 to 2.5 eq) were added to the reaction mixture. The mixture was stirred for another 1 to 4 h at RT (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 title product.

[151] GNTbm-8, yield: 35 mg, 25%.1H NMR (600 MHz, DMSO-d6) δ10.41(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 = 8.5Hz, 1H), 7.53 (dd, J = 7.9,2.1 Hz, 1H), 7.34 (dd, J =6.4,8.6,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=7.5Hz, 2H), 2.42 (s, 3H), 2.40(t, J = 7.5, Hz, 1H), 1.92 (quint, J = 7.5Hz, 2H).13C NMR(100 MHz, DMSO-d6): δ23.54, 26.90, 31.10, 35.47, 101.97, 102.14, 102.42, 102.57, 119.78, 122.68, 126.31, 126.67, 126.74, 133.58, 136.13, 138.53, 138.83, 144.16, 144.33, 148.78, 155.29, 159.72, 161.31, 162.29, 171.76. LCMS (ESI) m / z 408.5 [M+H]+. HPLC purity: 93.92%.

[152] GNTbm-19, base: 43 mG, 20%.1H NMR (600 MHz, DMSO-d6) δ10.42(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 = 7.4Hz, 2H), 2.42(s, 3H), 2.40(t, J = 7.4, Hz, 1H), 1.92(t, J = 7.4 Hz, 2H).13C NMR(100 MHz, DMSO-d6): δ23.54, 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 clearance: 99.12%.

[153] GNTbm-25, yield: 30 mg, 12%.1H NMR (600 MHz, DMSO-d6) δ10.43(s, 1H), 10.00 (s, 1H), 8.88 (d, J = 1.7 Hz, 1H), 8.31 (s, 1H), 8.24 (d, J = 8.7,1.8 Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.77 (s, 1H), 7.53(dd, J=8.1,1.8,1H), 7.26 (d, J=8.3Hz, 1H), 7.17 (d, J = 7.9, Hz, 1H), 6.91 (t, 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 (quint, J = 7.4 Hz, 2H). 13C NMR(100 MHz, DMSO-d6): δ23.54, 26.09, 31.11, 35.48, 115.73, 116.07, 116.28, 131.89, 122.69, 122.89, 122.94, 124.06, 125.85, 126.34, 133.58, 136.14, 138.52, 138.86, 143.88, 145.71, 148.78, 155.30, 162.52, 171.80. LCMS (ESI) m / z 458.5 [M+H]+. HPLC purity: 92.03%.

[154] Example 7 Determination of the saturation solubility of chidamide, GNTbm-02, GNTbm-03, GNTbm-04, and GNTbm-06

[155] 5 mg samples of the compounds were added to 5 mL volumetric flasks containing ddH2O and shaken at 100 rpm in an incubator at 25 °C for 90 min. The resulting suspension was filtered through a 0.22 μm filter. The concentration of the compounds was determined spectrophotometrically at 256 nm. The saturation solubility of each sample was determined in triplicate and the mean value and standard deviation were reported.

[156] Example 8 In vitro cytotoxicity assay

[157] Six different cell lines, including human breast cancer cell lines MDA-MB-231 (6x103), MDA-MB-453 (2.4x104), SK-BR-3 (6x103), human breast epithelial cell line M10 (6x103), human gastric carcinoma NCI-N87 (2.4x104), and human colorectal adenocarcinoma SW48 (2.4x104), were used and cultured in a 96-well plate. The cell lines were obtained from the Bioresources Research and Collection Center, BCRC, Taiwan. All cell lines were treated with compounds including GNTbm compound complex, chidamide (as positive control) and entinostat (as positive control), at doses ranging from 50 μM to 0.39 μM, then incubated at 37°C under 5% CO2 for 72 hours. After 72 hours, the MTT assay (CaymanTM) was used to determine cell viability. MDA-MB-231, MDA-MB-453, SK-BR-3 cell lines were maintained in DMEM / F12 supplemented with 10% FBS, 0.2% antibiotics (MycoZapTM, Pluse-CL). M10 cell line was maintained in MEM Alpha (gibcoTM) supplemented with 10% FBS, 0.2% antibiotics (MycoZapTM, Pluse-CL).The NCI-N87 cell line was maintained in RPMI 1640 (CORNINGTM) supplemented with 10% FBS, 0.2% antibiotics (MycoZapTM, Pluse-CL). The SK-BR-3 cell line was maintained in DMEM (CORNINGTM) supplemented with 10% FBS, 0.2% antibiotics (MycoZapTM,Pluse-CL).

[158] Example 9 IC50 measurements were determined on HDACs 1, 2, and 3 enzyme inhibition

[159] HDACs assays were performed according to standard protocols (HDACs 1, 2, and 3 Fluorogenic Assay Kit, BPS BioscienceTM). All compounds were mixed with positive controls chidamide and entinostatas at doses ranging from 20 μM to 1.28 nM in kit buffer and incubated for 1 h at 37 °C. After 1 h, assay developer was added to the samples and the absorbance at the fluorogenic wavelength was read. The relative inhibition of HDAC 1, 2, and 3 activity in each sample was determined.

[160] Example 10 Kinetics of HDAC3 enzyme inhibition determined by GNTbm-02

[161] HDAC3 enzyme kinetic assays were performed according to standard protocols (HDAC3 Fluorogenic Assay Kit, BPS BioscienceTM). A set of compounds GNTbm-01, GNTbm-02 and GNTbm-03, chidamide and entinostat at 2 μM were mixed with kit buffer and incubated at 37°C for 20 min, 40 min and 60 min. After incubation, assay developer was added to the samples and the absorbance at the fluorogenic wavelength was read. The relative inhibition of HDAC3 activity in each sample was determined.

[162] Example 11 Comparison of IC50 between GNTbm-02 and Entinostat (MS-275) in HDACs 1-11 enzyme inhibition was determined.

[163] The completed assay report is from BPS Bioscience (6042 Cornerstone Court West, Ste. B, San Diego, CA 92121, USA). The aim of this study was to determine the effect 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 enzymatic method. The HDAC assay was performed according to standard protocols (Fluorgenic HDACs 1-11 assay kit, BPS BioscienceTM). GNTbm-02 and entinostat (positive control) were mixed in kit buffer at doses ranging from 10 μM to 0.51 nM and incubated at 37°C for 0.5 h. After 0.5 h, assay developer was added to the samples and the absorbance at the fluorogenic wavelength was read. The relative inhibition of HDACs 1, 2, 3, 4, 5, 6, 7, 8, 9, and 11 activity was determined in each sample. Further details are described below. All compounds were dissolved in DMSO. Serial dilutions of compounds were first performed in 100% DMSO with the highest concentration being 1 mM.Each dilution of the intermediate compound (in 100% DMSO) was then directly diluted 10-fold in assay buffer to a mean dilution 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 at 37°C for 30 min in a 50 μL mixture containing HDAC assay buffer, 5 μg BSA, an HDAC substrate, an HDAC enzyme, and a test compound. After the enzyme reactions, 50 μL of 2x HDAC Developer for HDAC enzymes was added to each well, and the plate was incubated for an additional 15 min at room temperature. Fluorescence intensity was measured at excitation at 360 nm and emission at 460 nm using a Tecan Infinite M1000 microplate reader. HDAC activity assays were performed at each replicate concentration. Fluorescent intensity data were analyzed using Graphpad Prism computer software. In the absence of compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity.In the absence of HDAC, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percentage activity in the presence of each compound was calculated based on the following relationship: % activity =. (F-Fb) / (Ft-Fb), where F = fluorescent intensity in the presence of compound. The percent activity values ​​were then plotted against a series of compound concentrations using nonlinear regression analysis of a sigmoid dose-response curve generated by the equation Y=B+(TB) / 1+10((LogEC50-X)×Hill Slope), where Y = percent activity, B = minimum percent activity, T = maximum percent activity, X = logarithm of compound, and Hill Slope = slope coefficient or Hill coefficient. The IC50 value was determined as the concentration that caused half of the maximum percent activity.

[164] Example 12 Cell apoptosis and cell cycle arrest analyzed by flow cytometry

[165] PI / RNase (BD BioscienceTM) staining was performed to demonstrate the presence of cell cycle arrest and safer cell apoptosis treatment with GNTbm, chidamide, and entinostat. Human breast cancer cell line MDA-MB-231 (5×105) and human breast epithelial cell line M10 (5×105) were treated with GNTbm, chidamide, and entinostat (1.625 to 25 μM), respectively, for 72 h or at the indicated doses from 3 h to 72 h. Human colorectal adenocarcinoma SW48 cells (5×105) were treated with a series of GNTbm, chidamide, and entinostat (as indicated doses) for 72 h or at the indicated dose concentrations from 3 h to 72 h. After collection of treated cells, they were fixed with 80% ethanol for 24 hours, washed with 1XPBS, and stained with PI / RNase for 15 minutes at room temperature. Then, the cells were analyzed using a flow cytometer for 1 hour.

[166] Example 13 Western Blot Assay

[167] 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 Collection and Research Center (BCRC, Taiwan). MDA-MB-231 and SW48 were grown at 37 °C under humidified CO2-free atmosphere in Leibovitz L-15 (cat. #11415114, Thermo Fisher Scientific) containing 10% heat-inactivated fetal calf serum (Thermo Scientific), 1X concentration of MycoZap antibiotic (cat.# VZA-2011, Lonza). Cells were treated with a series of compounds GNTbm, chidamide, or entinostat for different periods of time or at different doses. Cell pellets were lysed with RIPA buffer (Cat. #20-188, Merck) with protease and phosphatase inhibitors (Cat. #K272, BioVision) and clarified by centrifugation. Equal amounts of total protein were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Cat. #1620177, BIO-RAD).Blots were incubated with primary antibodies against β-actin (catalog #sc-47778, Santa Cruz Biotechnology), histone 3ac (catalog #61637, Active Motif), and anti-rabbit (ab6721, Abcam) and anti-mouse (sc-2005, Santa Cruz) HRP secondary antibodies. Blots were developed using ECL Western blotting substrate (catalog #sc-2048, Santa Cruz Biotechnology). Image blots were analyzed with iBright FL1000 imaging systems (Thermo Fisher Scientific).

[168] Example 14 Anticancer activity in animal models

[169] The animal study was approved and monitored by the Institutional Animal Care and Use Committee of Taipei Medical University (TMU IACUC, NO: LAC-2019-0286, LAC-2020-0306). Six- to eight-week-old male BALB / c mice (National Laboratory Animal Center, Taiwan) in each treatment group were used for all animal experiments. Tumors were induced by sc injection of 1×106 or 5×106 CT26 (CRL-2638; murine colorectal adenocarcinoma) cells. The CT26 cell line was purchased from ATCC. CT26 tumor cells were grown in McCoy's 5A supplemented with 10% (vol / vol) FBS at 37 °C, 5% CO2. CT26 cells were mixed with Matrigel (lot #354248, Corning®) and inoculated into the left flank of mice, and tumor growth was determined by measuring two perpendicular diameters. Tumors were allowed to grow for 8–11 days (tumor size approximately 150–250 mm3) before randomization and treatment. Animals were sacrificed when the diameter of the tumors exceeded 3000 mm3. CT26-bearing mice received 2.5 mg / kg of anti-IgG (cat. #BE0089, Lot# 716719J3, Bio X Cell) and anti-PD-1 antibody (cat. #BE0146, Lot# 735019J3, Bio X Cell) were administered ip on days 8, 11, 14, 17, 20, and 23 after tumor implantation, and all antibodies were diluted to appropriate concentrations in 100 μL of sterile PBS (pH 7.4) (Invitrogen Life Technologies). Regorafenib (HY-1031, 30 mg / kg, daily, MedChemExpress USA), celecoxib (50 mg / kg, daily capsule / Celebrex®), chidamide-K30 (50 mg / kg, daily, manufactured by GNTbm, Taipei, Taiwan), and compounds GNTbm-02 / k30, GNTbm-03 / k30, GNTbm-04 / k30, GNTbm-05 / k30, GNTbm-06 / k30, GNTbm-11 / k30, GNTbm-38 / k30, GNTbm-39 / k30 (50, 25 or 12.5 mg / kg, dissolved in water to make stock solutions, daily) were orally administered to treat tumor-bearing mice at different doses daily from day 8 to 23 for 16 days. Anticancer activity was measured from the start of treatment until the tumor volume reached 3000 mm3. Tumor volume was calculated as length × width × 2 × 0.5.

[170] Example 15 Survival rates in animal models

[171] Antibody or drug administration was performed for 16 days from day 8 to day 23. Tumors continued to grow in tumor-bearing mice. Tumor volumes of mice were measured every three to four days (twice a week). Tumor-bearing mice were considered dead when tumor volumes reached 3000 mm3. All treatment groups were recorded and analyzed.

[172] Example 16 Tumor re-study in a tumor-bearing mouse animal model

[173] All mice with a PR / CR response were re-challenged with CT26 cells on the contralateral side after treatment (see Table 6). CT26 re-challenge was performed on day 33, which was 7 days (day 33) after the first tumor assessment (day 26), by injecting 5 × 106 CT26 cells into the right flank of each mouse. After re-challenge with CT26 cells, the tumor was allowed to grow for another 7 days (day 40) to establish a 1-fold baseline. After another 10 days (day 50), tumor growth was assessed for re-challenge. A response was considered a tumor relapse if both of the following criteria were met: first, the tumor size was more than 2-fold compared to the initial size; second, the tumor volume on day 50 was more than 300 mm3. Relapse occurs when immune memory activity has not been sufficiently activated. If tumor growth is inhibited, it means that immune memory has been activated.

[174] Example 17 Flow cytometry

[175] 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-ll-PE (BM8; BioLegend), CD45 FITC (30-F11; BioLegend). Flow cytometry was performed on a FACS Caliber flow cytometer (BD Biosciences) and data were analyzed with FACS Diva software (BD Biosciences). To assess the level of circulating cell populations, blood samples were collected from mice on days 8, 12, and 16 after the initiation of anti-PD-1 antibody treatment (2.5 mg / kg) with or without GNTbm-02 (12.5–50 mg / kg) or chidamide (50 mg / kg, as a positive control) plus celecoxib (50 mg / kg). One hundred and fifty microliters of blood were collected in a BD K2EDTA microtiter (BD Biosciences) from the right or left facial vein.Red blood cells from anticoagulated blood samples were immediately lysed using 2 ml of 1× RBC lysis buffer (Qiagen, Valencia, CA) for 10 min, and the samples were washed twice in ice-cold PBS (BD Biosciences). The samples were stained with appropriate antibodies. For analysis, we used previously established phenotypic criteria for these cells as CD45+CD11b+Ly6G+Ly6C-(PMN-MDSC), CD45+CD11b+Ly6G-Ly6C+cells (M-MDSC), CD45+CD3+CD25+Foxp3+(Treg), CD45+CD11b+MHC-ll+Ly6C+cells (TAM), and CD45+CD3+CD4+ / CD45+CD3+CD8+cells (CD4+or CD8+T cells). Total mononuclear cells were used as the common denominator. To assess the level of tumor-infiltrating lymphocytes in the tumor, intratumoral CD8+, CD4+ cells, regulatory T cells (Treg), PMN-MDSC, M-MDSC, and TAM cells were first purified from tumor samples isolated from mice 12 days after the start of anti-PD-1 antibody treatment with or without GNTbm-02 or chidamide plus celecoxib.Briefly, primary tumor tissues were removed, weighed, and minced into small pieces. Collagenase IV (Sigma-Aldrich) at a concentration of 1 mg / ml in HBSS (Invitrogen Life Technologies) was added to each sample at a ratio of 1 ml per 200 mg of tumor tissue. The samples were incubated on a shaker for 150 min at 37°C. The resulting tissue homogenates were 0.4 μm filtered, washed three times in PBS (BD Biosciences), and separated through a Percoll gradient to isolate mononuclear cells, and 1 × 106 cells per sample were used for antibody labeling. T+CD8+ cell levels were assessed using the predefined phenotypic criteria of CD45+CD3+CD8+. Treg cell levels were assessed using the predefined phenotypic criteria of D45+CD3+CD25+Foxp3+. PMN-MDSC / M-MDSC cell levels were assessed using pre-established phenotypic criteria of CD45+CD11b+Ly6G+Ly6C- / CD45+CD11b+Ly6G-Ly6C+.TAM cell levels were assessed using pre-established phenotypic criteria of CD45+CD11b+MHC-ll+Ly6C+, and total mononuclear cells were used as the common denominator.

[176] Example 18 Anticancer activity in an ineffective mouse model

[177] The animal study was approved and monitored by the Institutional Animal Care and Use Committee of Taipei Medical University (TMU IACUC, NO: LAC-2019-0086). Six- to eight-week-old male BALB / C naïve mice (National Laboratory Animal Center, Taiwan) in each treatment group were used for all animal experiments. Tumors were induced by sc injection of 5×106 CT26 cells with Matrigel (Batch #354248, Corning®) into the left flank of the mice, and growth was determined by measuring two perpendicular diameters. Before randomization and treatment, tumors were allowed to grow for 8 days (tumor size approximately 100–150 mm3). Animals were sacrificed when tumor volume reached 3000 mm3. CT26-bearing mice were given 2.5 mg / kg of anti-IgG (Group #BE0089, Lot# 716719J3, Bio X Cell) and anti-PD-1 (Group #BE0146, Lot# 735019J3, Bio X Cell) antibodies by intraperitoneal administration on days 8, 11, 14, 17, 20, and 23 after implantation, and the antibodies were diluted to appropriate concentrations in 100 μL of sterile PBS (pH 7.4) (Invitrogen Life Technologies).GNTbm and celecoxib (capsules / Celebrex®, 200 mg) were administered orally on day 8 after implantation. GNTbm (dissolved in DMSO to make stock solutions) were diluted or suspended in water and administered orally to treat tumor-bearing mice at various daily doses from days 8 to 23. Celecoxib from capsules was administered orally to treat tumor-bearing mice at 50 mg / kg from days 8 to 23. Anticancer activity was measured from the start of treatment until the tumor volume reached 3000 mm3. Tumor volume was calculated as length × width × 2 × 0.5.

[178] Results

[179] A series of synthetic picolinamide and benzamide derivatives of potent and novel class I HDAC inhibitors (called the GNTbm series of compounds)

[180] GNTbm has developed a series of novel class I HDAC inhibitors with potent epigenetic immunomodulatory properties, which can inhibit the enzymatic activity of HDACs 1, 2, and 3. Our research showed that class I HDAC inhibitors with strong regulatory ability in the tumor microenvironment (TME) greatly enhance the immune response against tumor growth. Therefore, the design and synthesis of such class I HDAC inhibitors was an interesting task to enhance the therapeutic effect in immunotherapy. Benzamide-based class I HDAC inhibitors have been studied in the field of TME control, such as entinostat (MS-275), tucidinostat (chidamide / HBI-8000), and mocetinostat, etc. In the present 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] is the first new synthetic compound based on the carboxamide core structure, as shown in Figure 1 and Table 1 .As shown in Table 4, compound GNTbm-01 was assayed for enzymatic inhibition of HDACs 1, 2, and 3. The results showed that compound GNTbm-01 was a weaker class I HDAC inhibitor compared to entinostat or chidamide. We optimized the structure and changed the position of one N atom, which created a new 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. Compound GNTbm-02 has the same molecular formula (C22H20FN5O2) as compound GNTbm-01 but with only a change in the position of one N atom in the picolinamide core. As shown in Table 4, the GNTbm-02 compound was very potent in inhibiting the enzymatic activity of HDACs 1, 2, and 3 compared to entinostat or chidamide. The results also showed that GNTbm-02 was more potent than GNTbm-01 in inhibiting the enzymatic activity of HDACs 1, 2, and 3.Next, we designed the benzamide-based compound GNTbm-03 by removing one N atom (i.e., replaced with a C atom) and tested the difference in inhibition of the enzymatic activity of HDACs 1, 2, and 3 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 is potent in inhibiting the enzymatic activity of class I HDACs 1, 2, and 3 compared to entinostat or chidamide. GNTbm-03 was also shown to have similar inhibition of the enzymatic activity of HDACs 1, 2, and 3 compared to GNTbm-02. Overall, the picolinamide-based derivative of GNTbm-02 was the first product in its chemical class to be a class I HDAC inhibitor. We were very interested in designing picolinamide- and benzamide-based derivatives of potent and novel class I HDAC inhibitors.A new series of GNTbm compounds were synthesized and assayed, 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.

[181] for saturation solubility analysis of GNTbm-02, GNTbm-03, GNTbm-04, and GNTbm-06

[182] Solubility is a very important determining parameter for oral bioavailability. The saturation solubility analysis of GNTbm-02, GNTbm-03, GNTbm-04, and GNTbm-06 is shown in Table 2. The results showed that chidamide had a reduced saturation solubility compared to GNTbm-02 and GNTbm-04. The saturation solubility of GNTbm-02 and GNTbm-04 was 33.6 and 7.2 μg / mL, respectively. These results indicated that GNTbm-02 and GNTbm-04 may have better oral bioavailability compared to chidamide.

[183] ​​In-Lab Cytotoxicity Testing of GNTbm Compound Complex

[184] We evaluated the cytotoxic effect 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 carcinoma NCI-N87, and human breast epithelial cell line M10 (normal cell line). The results showed that chidamide or entinostat as a positive control significantly induced cytotoxic effects, especially in SK-BR-3 and MDA-MB-453 cells. In total, six cell lines were sensitive to the treatment, as shown in Tables 3, 8, and 9. The GNTbm-01 compound induced a partially cytotoxic effect compared to entinostat. As shown in Table 3, the result showed that GNTbm-02 was more potent in inducing cytotoxic effect, especially in SK-BR-3, MDA-MB-453 and SW48 cells compared to GNTbm-01. This result indicated that the structure containing the picolinamide core in GNTbm-02 is very important. Replacing the picolinamide core structure with benzamide prevented the cytotoxic effect.As shown in Table 3, the compound GNTbm-03 was weaker in inducing cytotoxic effects than GNTbm-02 in SK-BR-3, MDA-MB-231 and SW48 cells. This result indicated that GNTbm-02 with a picolinamide core structure was superior in inducing cytotoxicity compared to GNTbm-03 with a benzamide core structure. Taken together, these results indicated that GNTbm-02 is a potent and novel class I HDAC inhibitor with a strong capacity to induce cytotoxicity in several human cancer cells. In addition, we are interested in evaluating the cytotoxicity of several new synthetic picolinamide-based and benzamide-based derivatives. As shown in Table 8, the cytotoxicity of picolinamide-based compounds was 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 benzamide-based compounds, GNTbm-33, GNTbm-38, and GNTbm-39 were more potent than chidamide or entinostat in inducing cytotoxic effects.These data showed that these novel picolinamide-based and benzamide-based derivatives are more potent in inducing cytotoxic effects than the well-known HDAC class I inhibitor chidamide or entinostat.

[185] Picoliamide-based GNTbm compound series for inhibiting HDACs 1, 2, and 3

[186] The GNTbm complex was shown to inhibit the enzymatic activity of HDACs 1, 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 the enzymatic activity of HDACs 1, 2, and 3. Chidamide (Tucidinostat) is another potent HDAC inhibitor that has been approved by the NMPA in China for relapsed or refractory peripheral T-cell lymphoma (PTCL) and ER+ / Her-2-positive advanced breast cancer. Chidamide is a subselective inhibitor for inhibiting the enzymatic activity of HDACs 1, 2, 3, and 10. Both entinostat and entinostat showed potent inhibition of the enzymatic activity of HDACs 1, 2, and 3 as shown in Table 4. Next, GNTbm-01 was evaluated and shown to have mild potency for inhibiting the enzymatic activity of HDACs 1, 2, and 3 compared to entinostat, as shown in Table 4. Remarkably, GNTbm-02 had very potent activities for inhibiting the enzymatic activity of HDACs 1, 2, and 3 at the nanomolar level.Comparison of GNTbm-02 with entinostat or chidamide in inhibiting the enzymatic activity of HDACs 1, 2, and 3 showed similar inhibitory effects. These results indicated 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 similar inhibitory effects to GNTbm-02. Next, the inhibition kinetics of HDAC 3 enzyme were examined. As shown in Figure 16a, chidamide and GNTbm-02 showed more potent inhibition of HDAC 3 enzyme activity than entinostat. As shown in Figure 16b, GNTbm-02 and GNTbm-03 showed more potent inhibition of HDAC 3 enzyme activity than GNTbm-01. Taken together, all these results indicated that the picolinamide core structure containing GNTbm-02 may have a stronger capacity to inhibit the activity of HDAC 1, 2, and 3 enzymes. Furthermore, we were interested in evaluating all the new synthetic GNTbm compounds based on picolinamide 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 HDAC 3 activity. GNTbm-05 and GNTbm-06 were more potent than chidamide or entinostat in inhibiting HDAC 1 activity. However, we also evaluated the new synthetic benzamide-based GNTbm compound as shown in Table 11. GNTbm-38 and GNTbm-39 appeared to be weaker than chidamide or entinostat in inhibiting HDACs 1, 2, and 3 activity. GNTbm-01 induced a partially cytotoxic effect compared to entinostat. As shown in Table 3, the result showed that GNTbm-02 was more potent in inducing cytotoxic effect, especially in SK-BR-3, MDA-MB-453 and SW48 cells compared to GNTbm-01. This result indicated that the structure containing the picolinamide core in GNTbm-02 is very important. Replacing the picolinamide core structure with benzamide prevents the cytotoxic effect. As shown in Table 3, the compound GNTbm-03 was weaker in inducing cytotoxic effect than GNTbm-02 in SK-BR-3, MDA-MB-231 and SW48 cells.This result showed that GNTbm-02 with a picolinamide core structure was superior in inducing cytotoxicity compared to GNTbm-03 with a benzamide core structure. Taken together, these results indicated that GNTbm-02 is a potent and novel class I HDAC inhibitor with strong capacity to induce cytotoxicity in several human cancer cells. In addition, we are interested in evaluating the cytotoxicity of several new synthetic picolinamide-based and benzamide-based derivatives. As shown in Table 8, the cytotoxicity of picolinamide-based compounds was analyzed. GNTbm-04, GNTbm-05, GNTbm-06, and GNTbm-11 were more potent in inducing cytotoxicity in six cell lines than chidamide or entinostat. Among the benzamide-based compounds, GNTbm-33, GNTbm-38, and GNTbm-39 were more potent in inducing cytotoxic effects than chidamide or entinostat. These data indicated that these novel picolinamide-based and benzamide-based derivatives were more potent in inducing cytotoxic effects than the well-known class I HDAC inhibitor chidamide or entinostat.

[185] Picoliamide-based GNTbm compound series for inhibiting HDACs 1, 2, and 3

[186] The GNTbm series of compounds were shown to inhibit the enzymatic activity of HDACs 1, 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 the enzymatic activity of HDACs 1, 2, and 3. Chidamide (Tucidinostat) is another potent HDAC inhibitor that has been approved by the NMPA in China for relapsed or refractory peripheral T-cell lymphoma (PTCL) and ER+ / Her-2-positive advanced breast cancer. Chidamide is a subselective inhibitor for inhibiting the enzymatic activity of HDACs 1, 2, 3, and 10. Both entinostat and entinostat showed potent inhibition of the enzymatic activity of HDACs 1, 2, and 3 as shown in Table 4. Next, GNTbm-01 was evaluated and shown to have mild potency for inhibiting the enzymatic activity of HDACs 1, 2, and 3 compared to entinostat, as shown in Table 4. Remarkably, GNTbm-02 had very potent activities for inhibiting the enzymatic activity of HDACs 1, 2, and 3 at the nanomolar level.Comparison of GNTbm-02 with entinostat or chidamide in inhibiting the enzymatic activity of HDACs 1, 2 and 3 showed similar inhibitory effects. These results indicated 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 similar inhibitory effects to GNTbm-02. Next, the inhibition kinetics of HDAC 3 enzyme were examined. As shown in Figure 16a, chidamide and GNTbm-02 showed more potent inhibition of HDAC 3 enzyme activity than entinostat. As shown in Figure 16b, GNTbm-02 and GNTbm-03 showed more potent inhibition of HDAC 3 enzyme activity than GNTbm-01. Taken together, all these results indicated that the picolinamide core structure containing GNTbm-02 may have a stronger capacity to inhibit the activity of HDAC 1, 2, and 3 enzymes. Furthermore, we are interested in evaluating all the new synthetic GNTbm compounds based on picolinamide, 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 HDAC 3 activity. GNTbm-05 and GNTbm-06 were more potent than chidamide or entinostat in inhibiting HDAC 1 activity. However, we also evaluated the new synthetic benzamide-based GNTbm compound as shown in Table 11. GNTbm-38 and GNTbm-39 appeared to be weaker than chidamide or entinostat in inhibiting HDACs 1, 2, and 3 activity.

[187] GNTbm-02 is a picoliamide-based, selective class I HDAC inhibitor

[188] To further confirm the selective inhibition of HDACs 1-11 enzyme activity subgroup, 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 HDACs 1-11 enzyme activity (except HDAC 10) was analyzed with entinostat (MS-275) as a positive control. This result showed that GNTbm-02 was more potent in inhibiting HDACs 1, 2, and 3 than entinostat under the same conditions. GNTbm-02 inhibited class I HDAC1, HDAC2, and HDAC3 with IC50s of 0.39, 0.91, and 0.73 μM, respectively. However, entinostatin inhibits class I HDAC1, HDAC2, and HDAC3 with IC50s of 0.95, 2.3, and 4.6 μM, respectively. 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 indicated that GNTbm-02 is a potent and subselective 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 the activity of HDACS 1, 2, and 3 enzymes.

[189] The GNTbm series of compounds significantly affects the proliferation and morphology of human cancer cells.

[190] The inhibitory effect of GNTbm-02 on the proliferation of human cancer cells is shown in Figure 3. Different concentrations of GNTbm-02 and entinostat were used to treat MDA-MB-231 cells for 72 hours. As shown in Figure 3a, the potency of the inhibitory effect was similar for GNTbm-02 and entinostat, with a concentration of 12.5 μM significantly inhibiting cell proliferation. As shown in Figure 3b, the potency of the inhibitory effect was more obvious 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. Taken together, these results demonstrated that GNTbm-02 has a strong capacity to inhibit cell proliferation.

[191] Cell cycle arrest induced by GNTbm series compounds in G0 / G1 or G2 / M phase in human cancer cells MDA-MB-231 and SW48

[192] To investigate the mechanism of cell proliferation inhibition, flow cytometry was used to analyze cell cycle arrest. As shown in Figure 4a, GNTbm-02 and entinostat were used at different concentrations from 1.625 to 25 μM to treat MDA-MB-231 cells for 72 h. The results showed that GNTbm-02 and entinostat have a similar mechanism, significantly inducing cell cycle arrest in the G0 / G1 phase at a concentration of 3.125 μM as shown in Figures 4a and b. As shown in Figures 4c and d, the cell cycle was arrested in the G0 / G1 phase by treatment with GNTbm-02 and entinostat at a concentration of 12.5 μM in a time-dependent manner. The results showed that treatment with GNTbm-02 or entinostat for 1 day caused cell cycle arrest in the G0 / G1 phase. A similar mechanism was also demonstrated in SW48 cells. As shown in Figure 5a and b, GNTbm-02 and entinostat were used at different concentrations from 0.39 to 6.25 μM to treat SW48 cells for 72 hours.The results showed that GNTbm-02 and entinostat significantly induced cell cycle arrest in the G0 / G1 phase at a concentration of 3.125 μM, as shown in Figure 5a and b. The results showed that GNTbm-02 (76.9%) appeared to be more potent in inducing cell cycle arrest in the G0 / G1 phase than entinostat (72.1%) at the same concentration of 3.125 μM. As shown in Figure 5c and d, the cell cycle in the G0 / G1 phase was arrested by treatment with GNTbm-02 and entinostat concentration of 6.25 μM in a time-dependent manner. The results showed that treatment with GNTbm-02 or entinostat for 2 days induced cell cycle arrest in the G0 / G1 phase. Taken together, all these data indicated that GNTbm-02 and entinostat have a similar mechanism to inhibit human cancer cell proliferation through induced cell cycle arrest in the G0 / G1 phase. In addition, we were interested in evaluating several new synthetic derivatives based on picolinamide and benzamide, such as GNTbm-04, GNTbm-05, GNTbm-38, and GNTbm-39.As shown in Table 12, GNTbm-04 caused significant cell cycle arrest at G0 / G1 phase in SW48 cells. This was similar to the cell cycle arrest induced by chidamide at G0 / G1 phase. However, the similar chemical structures GNTbm-05, GNTbm-38, and GNTbm-39 caused significant cell cycle arrest at G2 / M phase in SW48 cells. Therefore, the chemical structures of these potent compounds were very similar, but the mechanisms of cell cycle arrest were very different.

[193] GNTbm-02-induced cell cycle arrest in G2 / M phase in M10 cells

[194] The human breast epithelial cell line M10 was treated with various doses ranging from 1.625 to 25.0 μM of GNTbm-02 or entinostat for 72 h 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. As shown in Figure 6a and b, 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 6c and d, cell cycle arrest at the G2 / M phase was induced by treatment with GNTbm-02 and entinostat at a concentration of 12.5 μM in a time-dependent manner. The results showed that treatment with GNTbm-02 and entinostat for 2 days caused cell cycle arrest in the G2 / M phase in M10 cells.These results showed that GNTbm-02 and entinostat treatment for human cancer cells significantly inhibited cancer cell proliferation through induced cell cycle arrest in the G0 / G1 phase; however, GNTbm-02 and entinostat treatment for human normal cells significantly inhibited cell proliferation through induced cell cycle arrest in the G2 / M phase.

[195] Apoptosis induced by a series of GNTbm compounds in several cell lines

[196] To investigate whether GNTbm-02 induces apoptosis in cancer cells, the results after treatment with GNTbm-02 and entinostat (as positive control) for MDA-MB-231 cells at various concentrations from 1.625 to 25.0 μM for 72 h are shown in Figure 7a and b. The results showed that GNTbm-02 and entinostat at a concentration of 6.25 μM significantly induced apoptosis (increased the percentage of G1 subphase) as shown in Figure 7a and b. As shown in Figure 7c and d, cell apoptosis was induced by treatment with GNTbm-02 and entinostat for MDA-MB-231 cells in a time-dependent manner. The results showed that GNTbm-02 and Entionstat at a concentration of 12.5 μM for 72 hours (3 days) significantly induced apoptosis in MDA-MB-231 cells. Compared with GNTbm-02, Entionstat was more potent in inducing apoptosis in a dose-dependent or time-dependent manner, as shown in Figure 7. Cellular apoptosis was then also examined in SW48 cells.As shown in Figure 8a and b, GNTbm-02 and entinostat induced apoptosis in a dose-dependent manner. The results showed that treatment with GNTbm-02 and entinostat at different concentrations from 0.39 to 6.25 μM for 72 h induced cell apoptosis in SW48 cells. GNTbm-02 and entinostat significantly induced apoptosis at a concentration of 6.25 μM for 72 h, as shown in Figure 8a and b. As shown in Figure 8c and d, 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 more potent in inducing apoptosis in SW48 cells in a dose-dependent or time-dependent manner compared to GNTbm-02 as shown in Figure 8. Finally, the induction of cellular apoptosis by GNTbm-02 and entinostat in the normal M10 cell line was also examined.As shown in Figure 9a and b, treatment with GNTbm-02 and entinostat at different concentrations ranging from 1.625 to 25.0 μM for 72 h induced cell apoptosis. GNTbm-02 and entinostat at a concentration of 12.5 μM for 72 h significantly induced apoptosis in M10 cells. As shown in Figure 9c and d, treatment with GNTbm-02 and entinostat induced apoptosis at a concentration of 12.5 μM in a time-dependent manner. The results showed that treatment with GNTbm-02 and entinostat at a constant concentration of 12.5 μM in M10 cells for 72 h (3 days) significantly induced apoptosis, as shown in Figure 9c and d. As shown in Figure 9, entinostat induced significantly more apoptosis in M10 cells in a dose-dependent and time-dependent manner compared to GNTbm-02. However, as shown in Figure 7, M10 cells appeared to be more susceptible to apoptosis compared to MDA-MB-231 cells when treated with GNTbm-02 and entinostat at a concentration of 25.0 μM for 72 h, are more resistant to induced apoptosis. Next, we were interested in examining the apoptosis-inducing activity of GNTbm-04, GNTbm-05, GNTbm-38, and GNTbm-39 in SW48 cells. The apoptosis induced by these compounds was examined in SW48 cells treated with the indicated doses for 72 h. As shown in Table 13, GNTbm-04, GNTbm-05, GNTbm-38, and GNTbm-39 were potent in inducing apoptosis in SW48 cells.

[197] The GNTbm series of compounds induced histone H3 acetylation in several human cancer cell lines.

[198] GNTbm-02 and entinostat are potent class I HDAC inhibitors. The induction of histone H3 acetylation by GNTbm-02 and entinostat was examined in a dose-dependent and time-dependent manner in MDA-MB-231 and SW48 cells. As shown in Figure 10a and b, treatment of MDA-MB-231 cells with GNTbm-02 and entinostat at various concentrations ranging from 0.1 to 10.0 μM for 24 h 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 h. As shown in Figure 11a and b, treatment with GNTbm-02 at a concentration of 1.0 μM for 2, 6, 24, 48, and 72 h induced histone H3 acetylation in MDA-MB-231 cells in a time-dependent manner.The results showed that GNTbm-02 strongly induced histone H3 acetylation in MDA-MB-231 cells after 6 h of treatment. Similar results were also shown in SW48 cells, as shown in Fig. 11c and d. Treatment with GNTbm-02 at a concentration of 1.0 μM in SW48 cells for 2, 6, 24, 48, and 72 h induced histone H3 acetylation in a time-dependent manner. GNTbm-02 strongly induced the level of histone H3 acetylation in SW48 cells after 6 h of treatment. Taken together, all these data indicate that GNTbm-02 is a potent class I HDAC inhibitor and induces histone H3 acetylation in several human cancer cell lines. In addition, we were interested in analyzing whether the new compounds had stronger activity to increase the expression of histone 3 acetylation in SW48 cells, as shown in Figure 12. Cells were treated with the same doses of GNTbm-04, GNTbm-05, GNTbm-11, and chidamide as a positive control for 24 h in SW48 cells, as shown in Figure 12a. GNTbm-05 and GNTbm-04 at a dose of 0.25 μM were more potent in inducing histone 3 acetylation than the positive control chidamide in SW48 cells. Similar results also showed that GNTbm-04, GNTbm-05, and GNTbm-06 at a dose of 0.25 μM were more potent in inducing histone 3 acetylation in SW48 cells, as shown in Figure 12b. In addition, four potent compounds (GNTbm-04, GNTbm-05, GNTbm-38, GNTbm-39) that induce histone 3 acetylation and are shown in Figure 12c were evaluated. The results showed that GNTbm-05, GNTbm-04, GNTbm-38, and GNTbm-39 were more potent than chidamide in inducing the expression of histone 3 acetylation in SW48 cells.

[199] GNTbm series of compounds possess epigenetic immunomodulatory properties in CT26-bearing mouse model

[200] To investigate whether GNTbm-02 has epigenetic immunomodulatory properties, an in vivo and in vivo animal model of BALB / c CT26 colon tumor-bearing mice was used for evaluation. BALB / c mice bearing CT26 colon tumors were treated with various therapeutic regimens 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. Celecoxib-capsule 50 mg / kg (Celebrex®). Tumor size in CT26 tumor-bearing mice increased to approximately 150–200 mm3 by day 8. Total tumor volume and fold change in tumor size are shown in Figure 13a and b. The results showed that the regimen of anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (12.5 mg / kg) plus celecoxib (50 mg / kg) was more effective than the regimen of anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (25.0 mg / kg) combined with celecoxib (50 mg / kg) or GNTbm-02 25 mg / kg plus celecoxib 50 mg / kg regimen had a significant inhibitory effect on tumor growth in the absence of anti-PD-1 antibody. Therefore, the tumor growth inhibitory effect was anti-PD-1 antibody plus GNTbm-02 (12.5 mg / kg) combined with celecoxib regimen > anti-PD-1 antibody, plus GNTbm-02 (25.0 mg / kg) combined with celecoxib regimen > GNTbm-02 (25.0 mg / kg) combined with celecoxib regimen > anti-PD-1 antibody > anti-IgG regimen. However, the results also showed that GNTbm-02 combined with celecoxib had a strong inhibitory effect on tumor growth suppression. Previously, our research showed that HDAC inhibitor combined with COX-2 inhibitor significantly regulated TME and thus improved tumor growth inhibition effect and immune response rate. These results indicated that GNTbm-02 was a potent and novel epigenetic immunomodulator. Tumor volume was analyzed as shown in Figure 13c.In this study, we defined complete response (CR, ≦0.5 tumor growth time in tumor-bearing mice at three days after the end of treatment); partial response (PR, tumor size > 0.5 tumor growth time, but ≦ 2-fold tumor growth in tumor-bearing mice at three days after the end of treatment); stable disease (SD, between two and five-fold tumor growth in tumor-bearing mice at three days after the end of treatment); progressive disease (PD, equal to or greater than five-fold tumor growth in tumor-bearing mice at three days after the end of treatment) to evaluate the efficacy of treatment. The results showed that the anti-PD-1 antibody group (2.5 mg / kg) achieved 5CR, 1 PR, 3 SD, and 8 PD, with an ORR (objective response rate) of 35.3%; The anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (25 mg / kg) plus celecoxib (50 mg / kg) group achieved 3CR, 3 PR, 2 SD and 1 PD, with an ORR of 66.7%; the anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (12.5 mg / kg) plus celecoxib (50 mg / kg) group achieved 5CR, 2PR, 1 SD and 0PD, with an ORR of 87.5%; GNTbm-02 (25 mg / kg) combined with celecoxib (50 mg / kg) group achieved 2 CR, 4 PR, 2 SD and 1PD with an ORR of 66.7%. These results indicated that GNTbm-02 at 12.5 mg / kg was the optimal dose and GNTbm-02 had potent immunomodulatory activity. As shown in Figure 13d, the body weight of CT26 tumor-bearing mice showed that these regimens had no obvious toxicity to reduce body weight. Finally, the survival rate was analyzed as shown in Figure 13. When the tumor volume after tumor implantation reached 3000 mm3, CT26 tumor-bearing mice were euthanized. The results showed that the anti-PD-1 antibody group achieved a survival rate of 30%; Anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (25 mg / kg) plus celecoxib (50 mg / kg) achieved a survival rate of 33%; GNTbm-02 (25 mg / kg) plus celecoxib (50 mg / kg) achieved a survival rate of 56%; anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (12.5 mg / kg) plus celecoxib (50 mg / kg) group achieved a survival rate of 63%. Collectively, these data suggest that GNTbm-02 plus celecoxib or GNTbm-02 plus celecoxib plus anti-PD-1 antibody significantly improves ORR and survival rates compared with anti-PD-1 antibody alone. Our data also showed that GNTbm-02 at a dose of 12.5 mg / kg showed better efficacy than 25 mg / kg in the combination regimen of anti-PD-1 antibody plus GNTbm-02 plus celecoxib. Next, we were interested in evaluating the regulation of tumor microenvironment activities using novel synthetic compounds such as GNTbm-02, GNTbm-03, GNTbm-04, GNTbm-06 and Chidamide as a positive control. Solid dispersion of Chidamide prepared by coating with PVP-K30 was used to improve the water solubility of Chidamide-API, which will ultimately improve the PK (pharmacokinetic) profile.Therefore, we used a common preparation technique in the art to produce solid dispersions of test compounds such as GNTbm-02, GNTbm-03, GNTbm-04, GNTbm-06, and Chidamide as positive controls. All test compounds were coated on PVP-K30 to prepare solid dispersions named GNTbm-02 / k30, GNTbm-03 / k30, GNTbm-04 / k30, GNTbm-06 / k30, and Chidamide / k30. A previous study had demonstrated that Chidamide / k-30 combined with regorafenib had very potent anticancer activity in CT-26 tumor-bearing mice through an immunomodulatory mechanism. The anticancer activity of GNTbm-02 / k-30 combined with regorafenib was further studied to confirm its potency in CT26 tumor-bearing mice. We defined more stringent criteria for CR (≦0.5 tumor growth time in tumor-bearing mice at three days after the end of treatment); PR (tumor size > 0.5 tumor growth time, but ≦ 1-fold tumor growth in tumor-bearing mice at three days after the end of treatment); SD (between one and five-fold tumor growth in tumor-bearing mice at three days after the end of treatment); PD (equal to or greater than five-fold tumor growth in tumor-bearing mice at three days after the end of treatment) to evaluate the efficacy of the treatment. As shown in Figure 14(f) to Figure 14(i), GNTbm-02 / k-30 combined with regorafenib was evaluated versus Chidamide / k-30 combined with regorafenib. The results showed that GNTbm-02 / k-30 (50 mg / kg) combined with regorafenib (30 mg / kg) had strong tumor growth inhibition, but weaker than Chidamide / k-30 combined with regorafenib (ORR: 10% vs. 30%). However, as shown in Figure 14(i) to Figure (m), GNTbm-03 / k-30 showed that GNTbm-03 / k-30 combined with regorafenib had similar anticancer activity compared to Chidamide / k-30 combined with regorafenib (ORR: 40% vs. 30%).GNTbm-04 / k-30 combined with regorafenib was more potent in inhibiting tumor growth than Chidamide / k-30 combined with regorafenib as shown in Figure 14(n) to Figure 14(q) (ORR: 50% vs. 30%). GNTbm-06 / k-30 combined with regorafenib had similar anticancer activity compared to Chidamide / k-30 combined with regorafenib as shown in Figure 14(r) to Figure 14(u) (ORR: 50% vs. 30%). After 16 days of treatment, we continued to monitor tumor size until day 60. Relapse was defined as tumor growth restarted and tumor size increased by at least 5-fold in mice with CR or PR response after the first tumor assessment. 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, GNTbm-06 / k-30 combined with regorafenib showed 0% tumor recurrence.With the exception of GNTbm-02 / k-30 combined with regorafenib group, which had only 1 mouse with PR in the study, the results indicated that GNTbm compounds combined with regorafenib may have stronger activity in activating the immune system to prevent relapse. In addition, we also investigated 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 Chidamide / k-30 combined with regorafenib was evaluated. The results showed that Chidamide / k30 (50 mg / kg) combined with regorafenib (30 mg / kg) group achieved 2CR, 4 PR, 4 SD, and PD0 with an ORR of 60%.GNTbm-05 / k30 (50 mg / kg) plus regorafenib (50 mg / kg) achieved 3CR, 0 PR, 4 SD and 3PD, with an ORR of 30%; GNTbm-11 / k30 (50 mg / kg) plus regorafenib (30 mg / kg) achieved 1CR, 1 PR, 4 SD and 4PD, with an ORR of 20%; GNTbm-38 / k30 (50 mg / kg) plus regorafenib (30 mg / kg) achieved 8CR, 0 PR, 2 SD and 0PD, with an ORR of 80%. GNTbm-39 / k30 (50 mg / kg) plus regorafenib (30 mg / kg) achieved 2 CR, 1 PR, 5 SD and 2PD, with an ORR of 30%. Collectively, these in vivo animal data demonstrated that when comparing all GNTbm compounds with the positive control chidamide, in combination with regorafenib, GNTbm-38 / k-30 demonstrated superior epigenetic immunomodulatory activity, achieving an ORR of 80%, and without combination with regorafenib, GNTbm-38 / k-30 alone achieved an ORR of 56%.

[201] Confirmation of the epigenetic immunomodulatory properties of the GNTbm series of compounds

[202] 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; celecoxib-capsule 50 mg / kg (Celebrex®). Tumor size in mice bearing CT26 tumors increased to approximately 150–200 mm3 by day 8. The total tumor volume and fold change of tumor size as shown in Fig. 14a and b showed that GNTbm-02 (10 mg / kg) combined with celecoxib (50 mg / kg) group was more potent in inhibiting tumor growth than GNTbm-02 (20 mg / kg) combined with celecoxib (50 mg / kg) group or GNTbm-02 (5 mg / kg) combined with celecoxib (50 mg / kg). This result also indicated that GNTbm-02 combined with celecoxib at an optimal ratio is essential for controlling TME and improves the tumor growth inhibitory effect in CT26-bearing mouse model.The tumor volumes and unit ORRs shown in Figure 14c showed that the anti-PD-1 antibody group (2.5 mg / kg) achieved 5CR, 1 PR, 3 SD, and 8PD, with an ORR (objective response rate) of 35.3%; GNTbm-02 (5 mg / kg) combined with celecoxib (50 mg / kg) achieved 2CR, 1PR, 1 SD, and 5PD, with an ORR of 33.3%; GNTbm-02 (10 mg / kg) combined with celecoxib (50 mg / kg) group achieved 2 CR, 6 PR, 0 SD, and 1PD, with an ORR of 88.9%; GNTbm-02 (20 mg / kg) plus celecoxib (50 mg / kg) achieved 2CR, 3 PR, 1 SD and 3PD, with an ORR of 55.6%; GNTbm-02 (25 mg / kg) plus celecoxib (50 mg / kg) achieved 2 CR, 4 PR, 2 SD and 1PD, with an ORR of 66.7%. These data indicate that GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) achieved the best ORR, which is due to the optimal ratio for TME control. This result was also observed in Figure 13, where anti-PD-1 antibody (2.5 mg / kg) plus GNTbm-02 (12.5 mg / kg) combined with celecoxib (50 mg / kg) regimen achieved better ORR. From these data, it was suggested that GNTbm-02 10 mg / kg combined with celecoxib 50 mg / kg has potent activities in regulating TME and thus improves the immune response rate. As shown in Figure 14d, the body weight of CT26 tumor-bearing mice showed that these regimens had no obvious toxicity in terms of body weight loss. Finally, the survival rate was analyzed as shown in Figure 14. CT26 tumor-bearing mice were sacrificed when the tumor volume reached 3000 mm3 after tumor implantation.The results showed that the anti-PD-1 antibody group achieved a 30% survival rate, GNTbm-02 (5 mg / kg) plus celecoxib (50 mg / kg) achieved a 22% survival rate; GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) achieved a 44% survival rate; GNTbm-02 (20 mg / kg) plus celecoxib (50 mg / kg) achieved a 33% survival rate; GNTbm-02 (25 mg / kg) plus celecoxib (50 mg / kg) achieved a 56% survival rate. Taken together, these data indicate that GNTbm-02 plus celecoxib significantly improves ORR and survival compared with anti-PD-1 antibody alone. Our data also showed that GNTbm-02 at a dose of 10 mg / kg was better than other doses when GNTbm-02 was combined with celecoxib.

[203] GNTbm-02 plus celecoxib with or without a series of anti-PD-1 compounds or GNTbm plus regorafenib significantly induced immune memory.

[204] As shown in Figures 13 and 14, the immune memory induced after treatment with different regimens was examined for the condition as shown in Tables 6 and 7. Mice were treated with the different regimens for 16 days and then the first tumor assessment was performed (day 26). Mice with CR or PR were given a 7-day washout period (until day 33) without any further treatment. They were then re-challenged with the same type of cancer cells (CT26; 5×106) inoculated on the contralateral side for another 7 days (day 40) and then the tumor volume was determined as baseline (1-fold). The challenge tumor was again allowed to grow for 10 days (day 50) and then the tumor size was measured to assess positive or negative immune memory. If the assessment was negative, it had to meet both conditions: tumor volume was greater than 300 cubic millimeters and tumor size was more than 2 times compared to baseline.If the immune memory induced after the previous treatment was active and specific to recognize cancer cells with the same antigen, the growth of the inoculated tumors was inhibited during rechallenge and thus positive immune memory was defined. If the immune memory was not induced or not fully activated, the growth of the inoculated tumors was not inhibited during rechallenge. With this evaluation process, GNTbm-02 plus celecoxib with or without anti-PD-1 antibody regimens was examined to answer whether the regimens had immune memory-inducing properties. As shown in Table 6, the anti-PD-1 antibody group achieved only 2 mice in CR, which showed 0% tumor progression after rechallenge. The results showed that these CR mice achieved 100% immune memory. The GNTbm-02 regimen (25 mg / kg) plus celecoxib (50 mg / kg) plus anti-PD-1 antibody (2.5 mg / kg) group resulted in 4 CR / PR mice. It also showed 100% immune activation with active memory. The GNTbm-02 regimen (12.5 mg / kg) plus celecoxib (50 mg / kg) plus anti-PD-1 antibody (2.5 mg / kg) resulted in 7 CR / PR mice, which showed 29% tumor progression after rechallenge. 71% showed active memory immunity. GNTbm-02 (25 mg / kg) plus celecoxib (50 mg / kg) regimen resulted in 6 CR / PR mice, which showed 17% tumor progression after rechallenge. It also showed 83% active memory immunity. However, as shown in Table 7, GNTbm-02 (10 mg / kg) plus celecoxib (50 mg / kg) regimen resulted in 7 CR / PR mice, which showed 14% tumor progression after rechallenge. It showed 86% active memory immunity. CR mice had stronger immune memory activity than PR mice from these data. Overall, GNTbm-02 plus celecoxib with or without ICI induced strong immune memory activity. The same phenomenon was also reflected in other GNTbm combinations combined with regarofenib.As shown in Table 14, GNTbm-02 / k-30 (50 mg / kg) combined with regarofenib (30 mg / kg) resulted in 1 CR / PR mouse, which also showed 0% tumor progression after rechallenge. GNTbm- 03 / k-30 (50 mg / kg) combined with regarofenib (30 mg / kg) resulted in 4 CR / PR mice, which also showed 0% tumor progression after rechallenge; GNTbm-04 / k-30 (50 mg / kg) combined with regarofenib (30 mg / kg) resulted in 5 CR / PR mice, which also showed 0% tumor progression after rechallenge. GNTbm-06 / k-30 (50 mg / kg) combined with regarofenib (30 mg / kg) resulted in 5 CR / PR mice, which also showed 0% tumor progression after rechallenge. These results indicated that GNTbm combinations combined with regarofenib are important in inducing immune memory.

[205] Antitumor activity after treatment with GNTbm-02 in combination with celecoxib was through an immunomodulatory effect, leading to CTL activation.

[206] As shown in Figures 13 and 14, the treated mice were normal, immunocompetent mice. The GNTbm-02 regimen plus celecoxib with or without anti-PD-1 antibody achieved a significantly high overall response rate (ORR) in normal wild-type mice. Next, treatment with the GNTbm-02 regimen plus celecoxib with or without anti-PD-1 antibody was investigated in a BALB / C (T-cell-deficient) naive mouse model. As shown in Figure 15a, naive mice were inoculated with CT26 cells by sc injection. After 8 days, when the average tumor volume reached approximately 123.8 mm3, the mice were then randomly divided into four groups and treated with anti-IgG antibody, anti-PD-1 antibody, GNTbm-02 plus celecoxib plus anti-PD-1 antibody, and GNTbm-02 plus celecoxib for 15 days. As shown in Figure 15b and c, all of these treatment groups did not significantly inhibit tumor growth in naive mice with defective T cell function.These results demonstrated that GNTbm-02 in combination with celecoxib had potent activity in inhibiting tumor growth by regulating the activation of CTL (cytotoxic T lymphocytes) in the TME. As shown in Figure 15d, no significant reduction in body weight was observed in all treatment groups. As shown in Figure 15e, all mice in the treatment groups had low antitumor activity in the naive mice, and none of them achieved ORR. These results demonstrated that a functional T-cell immune system is essential for achieving significant inhibition of tumor growth with the combination regimen of GNTbm-02 in combination with celecoxib with or without anti-PD-1 antibody (Figures 13, 14, and 15). It was also shown that GNTbm-02 in combination with celecoxib inhibited tumor growth by regulating the activation of T-cells (CTL) in the TME to kill cancer cells. This anticancer activity was through an immunomodulatory effect rather than a cytotoxic effect.Overall, we confirmed that GNTbm-02 has potent epigenetic immunomodulatory activity, and when combined with celecoxib, it was more potent in regulating time compared with GNTm-02 alone.

[207] Antitumor activity of GNTbm-02 plus celecoxib is associated with a reduction in immunosuppressive cells

[208] HDACi treatment has been shown to alter the TME by reducing Treg cell activity and increasing CD8 T cell infiltration. To determine whether the inhibition of tumor growth induced by GNTbm-02 plus celecoxib treatment was associated with an enhanced immune response, we examined the circulating white blood cell population. On the last day of treatment (i.e., 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 circulating lymphocytes and a decrease in granulocytes after treatment with GNTbm-02 plus celecoxib (Figure 17a and c). However, there was no significant difference in circulating monocytes (Figure 17b). We also observed a significant increase in circulating CD3+ T cells after treatment with GNTbm-02 plus celecoxib (Figure 17d). A moderate increase in CD8+ T cells was observed after treatment with anti-PD-1 or GNTbm-02 plus celecoxib (Figure 17f). However, there was no significant difference in circulating CD4+ T cells and Tregs (Figure 17e and g).In addition to FoxP3+ Tregs, there are other immunosuppressive myeloid cells that have been recruited to the TME, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). As immature myeloid cells migrate to the tumor, 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 the activity of anti-cancer T cells. MDSCs exist in two phenotypically defined subgroups: granulocytic Ly6G+Ly6C-(PMN-MDSCs) and monocyte Ly6C+Ly6G-MDSCs (M-MDSCs). Treatment with GNTbm-02 plus celecoxib resulted in a slight reduction in CD11b+Ly6G+Ly6C+ and M-MDSC in the circulation, while treatment with anti-PD-1 alone resulted in a reduction in the CD11b+ population (Figure 17h, i, and j). No reduction in PMN-MDSC was observed after treatment with GNTbm-02 plus celecoxib (Figure 17k).

[209] In summary, since immunotherapy is an important promising field for anticancer therapy, especially for the treatment of advanced cancers, the claimed invention was evaluated for potential applications in immunotherapy. In combination with celecoxib, GNTbm-02 has more potent immunomodulatory activity to inhibit tumor growth in the tumor microenvironment (TME) compared to GNTbm-02 alone. Furthermore, when GNTbm-02 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 anticancer activity, significantly increasing the response rate through a synergistic effect attributed to the blocking of inhibitory signals to CTL (cytotoxic T lymphocyte) by anti-PD-1 / anti-PD-L1 / anti-CTLA-4 antibodies and the immunomodulatory activities of GNTbm-02 plus celecoxib in the TME. Based on the studies, GNTbm-02 is a novel epigenetic immunomodulator with great potential for cancer treatment. Furthermore, we were interested in the immunomodulatory activities of the GNTbm series of compounds.Our data showed that GNTbm-02, GNTbm-03, GNTbm-04, GNTbm-06 and GNTbm-38 were highly potent in possessing epigenetic immunomodulatory activities to control TME when combined with celecoxib or regorafenib. These results indicate that the GNTbm series of compounds are novel and potent epigenetic immunomodulators.

[210] The tables mentioned above are presented below:

[211] Table 1. 1H-NMR and 13C-NMR spectroscopic study (400 MHz, d6-acetone) for compounds GNTbm-01, GNTbm-02 and GNTbm-03.

[212] Table 2. Saturated solubility analysis of GNTbm-02, GNTbm-03, GNTbm-04, and GNTbm-06 Compounds Saturation Solubility (μg / mL) Chidamide BDL GNTbm-02 33.6 ± 6.4 GNTbm-03 BDL GNTbm-04 7.2 ± 2.9 GNTbm-06 BDL *BDL: Below detection limit

[213] Table 3. IC50 values ​​of Entinostat, GNTbm-01, GNTbm-02 and GNTbm-03 against various cancer and normal cell lines. IC50, half of the maximal cytotoxic concentration. SD, standard deviation ±, IC50 interval estimated

[214] Table 4. Inhibitory effect of Chidamide, Entinostat, GNTbm-01, GNTbm-02 and GNTbm-03 on individual HDAC1-3 isoforms.

[215] Table 5. Inhibitory effect of entinostat and GNTbm-02 on HDAC1-11 isoforms (without HDAC10)

[216] Table 6. Treatment with GNTbm-02 plus celecoxib with Anti-PD-1 antibody significantly induced immune memory. +: Tumor size was greater than 300 mm3 and ≤ 2 times (normalized to tumor size measured 7 days after tumor rechallenge)

[217] Table 7. Treatment with GNTbm-02 combined with celecoxib significantly induced immune memory. +: Tumor size was greater than 300 mm3 and ≤ 2 times (normalized to tumor size measured 7 days after tumor rechallenge)

[218] Table 8. IC50 values ​​of picolinamide-based HDAC inhibitors against various cancer cell lines. Compound IC50 (μM) Cell Lines N CI- N87 M10 MDA-MB-453 MDA-MB-231 SW48 SK-BR-3 Chidamide (positive control) 0.2 ± 3.09 0.87 ± 4.94 0.12 ± 1.36 17.4 ± 1.98 1.057 ± 5.43 0.98 ± 3.24 Entinostat (positive control) - 0.01 ± 4.34 0.02 ± 1.82 13.16 ± 1.34 0.37 ± 5.21 1.94 ± 0.1 GNTbm-01 - 1.15 ± 24.8 4.44 ± 20.9 4.9 ± 49.8 > 50 1.85 ± 28.5 GNTbm-02 4.01 ± 0.49 4.31 ± 0.16 1.69 ± 1.78 13.5 ± 2.51 ± 2.51 0.02 ± 2 GNTbm-04 0.34 ± 0.04 1.81 ± 0.06 1.81 ± 1.59 0.003 1.64 ± 0.09 ± 0.6 - GNTbm-05 ± 0.28 ± 0.01 0.43 ± 0.003 1.58 ± 0.02 1.66 ± 0.02 1.64 ± 0.02 - GNTbm-06 2.2 ± 0.03 1.95 ± 0.01 1.59 ± 0.7 3.87 GNTbm-08 - 2.32 ± 0.08 15.5 ± 1.7 20.8 ± 0.14 3.78 ± 0.5 > 50 18.44 ± 1.0 - GNTbm-11 1.87 ± 0.04 1.65 ± 0.2 2.28 ± 0.06 1.71 ± 0.04 GNTbm-12 > 50 ± 0.45 38.48 ± 5.9 3.9 ± 15.8 > 50 > 50 - GNTbm-19 1.4 ± 4.3 2.89 ± 0.12 1.81 ± 0.02 5.69 ± 1.7 8.73 ± 0.24 GNTbm-25 > 50 > 50 19.12 ± 2.07 > 50 > 50 - IC50, half of the maximal cytotoxic concentration. SD, standard deviation Compounds IC50 (μM) Cell Lines N CI- N87 M10 MDA-MB-453 MDA-MB-231 SW48 SK-BR-3 Chidamide (positive control) 0.2 ± 3.09 0.87 ± 4.94 0.12 ± 1.36 17.4 ± 1.98 1.06 ± 5.43 0.98 ± 3.24 Entinostat (positive control) - 0.01 ± 4.34 0.024 ± 1.82 13.16 ± 1.34 0.37 ± 5.21 1.94 ± 0.1 GNTbm-03 - 0.13 ± 6.1 0.01 ± 1.81 26.7 ± 5.8 0.9 ± 5.26 0.68 ± 4.07 GNTbm-33 1.75 ± 0.02 2.01 ± 0.09 1.94 ± 0.22 1.62 ± 1.79 ± 0.03 - GNTbm-37 10.65 ± 0.6 8.85 ± 0.59 4.89 ± 0.34 8.07 21.3 ± 1.64 - GNTbm-38 0.42 ± 0.08 ± 1.99 0.06 ± 1.67 3.55 ± 0.45 ± 1.64 - GNTbm-39 0.64 ± 0.03 1.96 ± 0.02 1.84 ± 0.02 0.01 ± 2.93 0.45 ± 1.73 - ±, IC50 interval estimated

[219] Table 9. IC50 values ​​of benzamide-based HDAC inhibitors against various cancer cell lines. IC50, half of the maximal cytotoxic concentration. SD, standard deviation ±, IC50 interval estimated

[220] Table 10. Inhibitory effect of picolinamide-based compounds on individual HDAC1-3 isoforms. Compounds IC50 (μM) HDAC 1 HDAC 2 HDAC 3 Chidamide (positive control) 0.004 ± 0.14 0.04 ± 0.22 0.06 ± 0.62 Entinostat (positive control) 0.05 ± 0.25 0.06 ± 0.21 0.98 ± 0.19 GNTbm-01 0.76 ± 6.77 2.68 ± 0.2 0.13 ± 2.75 GNTbm-02 0.02 ± 0.52 0.06 ± 0.55 0.005 ± 0.67 GNTbm-04 0.04 ± 0.38 0.007 ± 0.54 0.20 ± 0.1 GNTbm-05 0.002 ± 0.14 GNTbm-06 0.001 ± 0.0004 ± 0.02 GNTbm-06 0.02 ± 0.02 0.02 ± 0.39 0.01 ± 0.009 GNTbm-08 0.37 ± 0.31 1.54 ± 0.28 0.0002 ± 0.001 GNTbm-11 > 20 > 0.001 GNTbm-12 > 20 > 20 > 20 GNTbm-19 0.48 ± 0.03 1.42 ± 0.19 2.98 ± 0.08 GNTbm-25 > 20 > 20 > 20

[221] Table 11. Inhibitory effect of benzamide-based compounds on individual HDAC1-3 isoforms. Compounds IC50 (μM) HDAC 1 HDAC 2 HDAC 3 Chidamide (positive control) 0.004 ± 0.14 0.037 ± 0.22 0.056 ± 0.62 Entinostat (positive control) 0.05 ± 0.25 0.06 ± 0.21 0.98 ± 0.19 GNTbm-03 0.04 ± 0.56 0.05 ± 0.51 0.06 ± 0.67 GNTbm-33 > 20 > 20 > 20 GNTbm-37 0.6 ± 9.04 3.15 ± 0.31 0.29 ± 1.74 GNTbm-38 0.03 ± 0.69 0.01 ± 0.28 0.01 ± 1.2 GNTbm-39 3.7 ± 0.04 0.68 ± 0.03 0.13 ± 0.89

[222] Table 12. Cell cycle arrest induced by GNTbm compound series in G0 / G1 or G2 / M phase in SW48 cells. Compounds Therapeutic Doses (micromolar) Percentage Cell Cycle Distribution (%) G0 / G1 S G2 / M Chidamide (Positive Control) 0 64.7 17.9 17.4 0.3125 65.7 17.6 16.7 0.625 68.1 14.4 17.5 1.25 70.6 14.1 15.3 2.5 76.5 10.8 12.7 5 79.9 8.5 11.6 GNTbm-04 0 59.5 14.7 25.8 0.125 56.9 17.9 25.2 0.25 57.9 18.2 23.9 0.5 63.2 17.5 19.3 1 67.2 13.9 18.9 2 70.5 10.8 18.7 GNTbm-05 0 61.6 17.2 21.2 0.125 61.5 19.8 18.7 0.25 57.8 19.3 22.9 0.5 59.2 18.6 22.2 1 55.4 12.8 31.8 2 53.6 11.8 34.6 GNTbm-38 0 64.7 17.9 17.4 0.125 63.3 19.3 17.4 0.25 62.4 15.7 21.9 0.5 61.8 10.4 27.8 1 57.7 12.5 29.8 2 53.9 13.6 32.5 GNTbm-39 0 64.7 17.9 17.4 0.125 57.2 20.5 22.3 0.25 57.8 17.7 24.5 0.5 58.8 16.5 24.7 1 46.5 20.7 32.8 2 42.3 18.8 38.9

[223] Table 13. Cell apoptosis induced by GNTbm compound series in SW48 cells. Compounds Therapeutic Doses (micromolar) Percentage of Cell Apoptosis (%) Chidamide (Positive Control) 0 3.2 0.3125 9.0 0.625 10.2 1.25 15.1 2.5 21.2 5 35.4 GNTbm-04 0 2.6 0.125 2.9 0.25 4.4 0.5 10.8 1 20.8 2 32 GNTbm-05 0 7.9 0.125 9.0 0.25 14.4 0.5 17.9 1 27.2 2 42.4 GNTbm-38 0 3.2 0.125 8.5 0.25 9.1 0.5 18.3 1 24.4 2 37.3 GNTbm-39 0 3.2 0.125 9.5 0.25 10.8 0.5 17.8 1 25.4 2 37.8

[224] Table 14. Efficacy of GNTbm compound series combined with the tyrosine kinase inhibitor regorafenib in a CT26 tumor-bearing mouse model. Trial regimens Initial tumor volume (mm3) ORR (%) PD SD PR CR ORR (%)& PD& SD& PR& CR& Survival rate (%) Relapse* (relapse) Immunity# (rechallenge) Exp1 245 Carrier 0% 7 1 0 0 0% 8 0 0 0 0% - - Regorafenib 11% 1 7 0 1 22% 5 2 0 2 11% 0% (0 / 1) 100% (1 / 1) Chidamide / k-30 11% 7 1 1 0 0% 7 2 0 0 0% 100% (1 / 1) - Chidamide / k-30 in combination with regorafenib 30% 0 7 1 2 40% 2 4 0 4 40% 0% (0 / 3) 100% (3 / 3) GNTbm-02 / k-30 0% 8 1 0 0 0% 9 0 0 0 0% 0% (0 / 0) - GNTbm-02 / k-30 in combination with regorafenib 10% 1 8 1 0 10% 6 3 1 0 10% 100% (1 / 1) 100% (1 / 1) GNTbm-03 / k-30 0% 9 0 0 0 0% 9 0 0 0 0% 0% (0 / 0) - GNTbm-03 / k-30 in combination with regorafenib 40% 1 5 1 3 30% 5 2 0 3 30% 25% (1 / 4) 100% (3 / 3) GNTbm-04 / k-30 10% 8 1 0 1 10% 9 0 0 1 10% 0% (0 / 1) 100% (1 / 1) GNTbm-04 / k-30 in combination with regorafenib 50% 1 4 1 4 40% 4 2 0 4 40% 20% (1 / 5) 100% (4 / 4) GNTbm-06 / k-30 0% 8 2 0 0 0% 10 0 0 0% 0% (0 / 0) - GNTbm-06 / k-30 in combination with regorafenib 50% 2 3 1 4 50% 5 0 0 5 50% 0% (0 / 5) 100% (5 / 5) Exp2 192 Carrier 0% 8 1 0 0 0% 9 0 0 0 - - - Regorafenib 0% 8 1 0 0 0% 9 0 0 0 - -- Chidamide / k-30 44% 4 1 1 3 33% 5 1 0 3 - - - Chidamide / k-30 in combination with regorafenib 60% 0 4 4 2 60% 1 3 0 6 - - - GNTbm-05 / k-30 0% 8 1 0 0 0% 8 1 0 0 - - - GNTbm-05 / k-30 in combination with regorafenib 30% 3 4 0 3 20% 7 1 0 2 - - - GNTbm-11 / k-30 11% 7 1 0 1 0% 8 1 0 0 - - - GNTbm-11 / k-30 in combination with regorafenib 20% 4 4 1 1 20% 7 1 1 1 - - - GNTbm-38 / k-30 56% 3 1 0 5 56% 4 0 0 5 - - - GNTbm-38 / k-30 in combination with regorafenib 80% 0 2 0 8 100% 0 0 1 9 - - - GNTbm-39 / k-30 0% 7 2 0 0 0% 9 0 0 0 - - - GNTbm-39 / k-30 in combination with regorafenib 30% 2 5 1 2 30% 4 3 1 2 - - - *: Recurrence / relapse was defined when there was at least 5-fold tumor growth in mice with a CR or PR response after the first tumor assessment. #: Mice resistant to CT26 rechallenge. &: Second tumor assessment on day 40 A person of ordinary skill in the art should recognize that changes and modifications may be made to the teaching and disclosure of the subject invention without departing from the spirit and scope of the subject invention. Based on the foregoing, the subject application is intended to cover any changes and modifications thereof, provided that the changes or modifications fall within the scope defined in the appended claims or their equivalents.

Claims

What is claimed is:

1. A compound of formula (I): wherein W and Y are each independently selected from CH and N; R1 is each independently selected from hy drogen, halogen, C1 -C3 alkyl and halogenated C1 -C3 alkyl, and can be mono-, di-, tri- or tetra-substitution; C1 and C2 are C atoms linked by a double bond; Ar is selected from the group consisting of the following: , , , , and wherein Ar is linked to C2 via the solid line; R2 has the same meaning as described for R1 ; and R3 is hydrogen or C1 -C3 alkyl; or a pharmaceutically acceptable salt, hydrate , stereoisomer or solvate thereof .

2. The compound of claim 1, which has the formula (Ia): (Ia) wherein W, Y, R1 , C1 , C2 and Ar have the same meaning as described in formula (I); or a pharmaceutically acceptable salt, hydrate , stereoisomer or solvate thereof .

3. The compound of claim 1 or a pharmaceutically acceptable salt, hydrate , stereoisomer or solvate thereof , wherein Ar is selected from the six-membered rings, and R2 and the atom of Ar linked to C2 are at para-positions.

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

6. The compound of claim 1 or a pharmaceutically acceptable salt, hydrate , stereoisomer or solvate thereof , wherein R1 is F or fluorinated C1 -C3 alkyl.

7. The compound of claim 1 or a pharmaceutically acceptable salt, hydrate , stereoisomer or solvate thereof , wherein R2 is C1 -C3 alkyl or fluorinated C1 -C3 alkyl.

8. The compound of claim 1 or a pharmaceutically acceptable salt, hydrate , stereoisomer or solvate thereof , wherein the compound is: GNTbm-01; GNTbm-02; GNTbm-03;GNTbm-04; GNTbm-05;GNTbm-06; GNTbm-07; GNTbm-09;GNTbm-10; GNTbm-11;GNTbm-12; GNTbm-13; GNTbm-14;GNTbm-15; GNTbm-16; GNTbm-17; GNTbm-18; GNTbm-20;GNTbm-21; GNTbm-24; GNTbm-26;GNTbm-27; GNTbm-28; GNTbm-29;GNTbm-30; GNTbm-31; GNTbm-33; GNTbm-38; or GNTbm-39 .

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

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