HDAC7-targeted therapeutic agents for human malignancies

Abstract

Provided herein are HDAC-7 inhibitors and their uses, including their use as therapeutic agents for treating brain cancer and / or solid tumors, such as glioblastoma multiforme (GBM). In some embodiments, provided herein are HDAC-7 inhibitors selected from small molecules, siRNAs, or HDAC class IIa inhibitors, such as TMP269, or pharmaceutically acceptable salts thereof. These HDAC-7 inhibitors or their pharmaceutically acceptable salts are useful in treating brain cancer and / or solid tumors, such as glioblastoma multiforme (GBM).

Description

[Technical Field]

[0001] (Sequence Listing) This application contains a Sequence Listing having the file name "GLIOBLASTOMA MULTIFORME TREATMENT.xml", which is 4,589 bytes in size and was created on September 26, 2022. The entire contents of this Sequence Listing are incorporated herein by reference.

[0002] (Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 254,826, filed October 12, 2021, the entire contents of which are incorporated herein by reference.

[0003] (Field) The present disclosure relates to the treatment of brain cancer and / or solid tumors (e.g., glioblastoma multiforme (GBM)) using inhibitors of histone deacetylase 7 (HDAC-7). [Background technology]

[0004] (background) Glioblastoma multiforme (GBM) is the most malignant and aggressive primary brain tumor. GBM has a poor prognosis, with a survival rate of 14 to 15 months after diagnosis. Despite global efforts to optimize therapeutic approaches, GBM remains one of the most difficult diseases to treat and the most rapidly recurring in clinical oncology. The treatment resistance of GBM and its inevitable tumor recurrence are primarily attributed to the presence of tumor-initiating cells, or glioma stem cells (GSCs).

[0005] Due to the above drawbacks, there is an urgent need for effective GBM treatments. Summary of the Invention [Means for solving the problem]

[0006] (overview) Based on the findings that histone deacetylase 7 (HDAC-7) is highly expressed in glioblastoma compared with normal brain tissue and that inhibiting HDAC-7 has a significant effect on GBM growth and inhibits GSC characteristics, transcriptome expression, and cancer cell invasion, inhibitors of histone deacetylase 7 (HDAC-7) were selected as potential therapeutic targets.

[0007] A small molecule inhibitor library that exhibits remarkable specificity for HDAC-7 over other class IIa histone deacetylases has been designed and screened to identify potential drugs for human brain tumors and other solid tumors. These small molecules, or pharmaceutically acceptable salts thereof, are disclosed herein.

[0008] In addition to these small molecules, inhibitors selected from the following siRNAs are disclosed herein: GACAAGAGCAAGCGAAGUG (SEQ ID NO: 1), GCAGAUACCCUCGGCUGAA (SEQ ID NO: 2), GGUGAGGGCUUCAAUGUCA (SEQ ID NO: 3), or UGGCUGCUCUUCUGGGUAA (SEQ ID NO: 4). Other HDAC-7 inhibitors include TMP269, trichostatin A, and vorinostat.

[0009] These HDAC-7 inhibitors, or pharmaceutically acceptable salts thereof, are useful in treating brain cancer and / or solid tumors (eg, glioblastoma multiforme (GBM)).

[0010] BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are included to illustrate certain aspects of the present disclosure and should not be construed as exclusive embodiments. The disclosed subject matter is capable of considerable modification, permutation, combination, and equivalents in form and function, as will occur to those skilled in the art having the benefit of this disclosure. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows a graphical abstract of one histone HDAC-7 inhibition model approach as a potential treatment for GBM, including a schematic of the elucidated HDAC-7 interactome and post-translational modifications following HDAC-7 inhibition to inhibit specific cell cycle processes and cell division signaling to regulate GSC self-renewal.

[0012] [Figure 2-1] Figures 2A-2F show the five-gene epigenetic signature for IDH wild-type glioblastoma. Overview of the in vivo pooled overexpression epigenetic screen (Figure 2A). Differentially represented genes in tumors versus baseline controls (Figure 2B). Selection of clinically relevant genes in three GBM cohorts (Figure 2C). Waterfall plot showing similar cutoffs from TCGA applied to the Gravendeel cohort (Figure 2D). Overall survival analysis of high-risk (HR) and low-risk (LR) subgroups stratified using the five-gene signature in the training and validation sets (Figure 2E). Subclass mapping analysis of HR and LR subgroups (SubMap software). Significant p-values ​​confirmed that the HR subgroups in the TCGA cohort had significant correspondence with the HR subgroups in Gravendeel; the same was true for LR (Figure 2F). [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above.

[0013] [Figure 3-1]Figures 3A-3F. NanoString data summary. Summary of NanoString analysis of 84 GBM patient samples (Figure 3A). Overall survival (Figure 3B) and progression-free survival (Figure 3C) analyses of HR vs. LR subgroups stratified by the 5-gene signature. Heatmap showing differentially expressed genes in HR vs. LR tumors (Figure 3D). DAVID analysis of differentially expressed genes in HR vs. LR tumors (Figure 3E). Volcano plot display of differential expression analysis of genes expressed in HR vs. LR tumors (Figure 3F). [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above.

[0014] [Figure 4-1] Figures 4A-4B show HDAC-7 expression in the Chinese Glioma Genome Atlas (CGGA). HDAC-7 expression in various forms of glial tumors for patients derived from this CGGA data is shown, with GBM showing the highest HDAC-7 expression (Figure 4A). Kaplan-Meyer survival analysis for all GBM patients derived from the CGGA data was divided into two groups ("high HDAC-7 expression" and "low HDAC-7 expression"), with the HDAC-7 "high expression" group showing reduced survival compared to the HDAC-7 "low expression" group (Figure 4B). [Figure 4-2] Same as above.

[0015] [Figure 5-1]Figures 5A-5D show HDAC-7 expression in The Cancer Genome Atlas (CGGA). This TCGA data-derived data shows the expression of HDAC-7 in GBM tumors versus non-tumors, with GBM showing significantly higher HDAC-7 expression compared to non-tumors (Figure 5A). Kaplan-Meyer survival analysis of all GBM patients from TCGA data was divided into two groups ("high HDAC-7 expression" and "low HDAC-7 expression"), with the "high" HDAC-7 risk group showing reduced survival compared to the "low" HDAC-7 risk group (Figure 5B). Figures 5C-5D show heatmap and volcano blot representations, respectively, of differential expression analysis of genes expressed in the high HDAC-7 expression group versus the low HDAC-7 expression group in the TCGA-derived GBM cohort. [Figure 5-2] Same as above.

[0016] [Figure 6] Figures 6A-6D show that GSCs treated with HDAC class IIa inhibitors are unable to form neurospheres in a 2-week limiting dilution assay. Images of GSC cultures treated with DMSO (control) and HDAC class IIa inhibitors in a limiting dilution assay (LDA) are shown. Here, compared to control GSCs, which showed sphere-forming ability at very early time points, GSCs treated with HDAC class IIa inhibitors are unable to form neurospheres in a 2-week LDA (Figures 6A and 6C). Graphs using an online algorithm to score and evaluate the self-renewal capacity of DMSO-treated GSCs and HDAC class IIa inhibitor-treated GSCs are shown (Figures 6B and 6D).

[0017] [Figure 7-1]Figures 7A-7D show transcriptome analysis for HDAC-7 siRNA. Heatmap and volcano blot representations, respectively, of differentially expressed genes in HDAC-7 knockdown versus control derived from RNASeq applied after siRNA knockdown in primary GSCs (Figures 7A-7B). Gene enrichment analysis of differentially expressed genes in HDAC-7 knockdown versus control derived from RNASeq applied after siRNA knockdown in primary GSCs (Figure 7C). Overlap radar chart showing overlap between input downregulated genes in various stem cell types (Figure 7D). [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0018] [Figure 8-1] Figures 8A-8B show that HDAC-7 inhibition suppresses the mesenchymal and proneural phenotypes of GSCs. GSEA enrichment plots of HDAC-7 knockdown GSCs versus controls using two different signature gene sets show that HDAC-7 inhibition inhibits the mesenchymal and proneural phenotype signature genes (Figure 8A). Normalized enrichment scores (NES) are shown for each plot. HDAC-7 expression is relatively similar in various GSC subtypes derived from publicly available data from TCGA (Figure 8B). [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0019] [Figure 9] Figures 9A-9B show the relative protein levels of HDAC-7 in wild-type (WT) GSCs and after siRNA knockdown. Relatively low HDAC-7 protein levels compared to housekeeping genes in GSCs (Figure 9A). Relative protein levels of HDAC-7 in GSCs after HDAC-7 siRNA versus control (Figure 9B).

[0020] [Figure 10] Figure 10 shows that HDAC-7 inhibition results in specific changes to histone post-translational modifications (PTMs) in GSCs. Heatmap representation of changes in the abundance of histone post-translational modifications (PTMs) in HDAC-7 knockdown versus control, as detected via ModSpec. The experiment was performed in three independent trials as technical replicates.

[0021] [Figure 11-1] Figure 11 shows that HDAC-7 inhibition results in specific changes to other HDAC members in GSCs. The plot shows normalized counts from RNASeq data for the effect on other HDACs in GSCs versus controls after knocking down HDAC-7 using siRNA, adjusted P<0.05. [Figure 11-2] Same as above.

[0022] [Figure 12] Figure 12 shows that class IIa HDAC inhibitors have comparable inhibitory effects on cell viability of GSCs. Dose-response curves for GSCs treated with the pan-HDAC inhibitors TSA (trichostatin A) and SAHA (vorinostat) compared with GSCs treated with the HDAC class IIa inhibitor TMP269. Cell viability assays were determined by MTS after treatment with increasing doses of each corresponding drug at 72 hours, with a P value of <0.0001.

[0023] [Figure 13] Figures 13A-13B. Kaplan-Meier survival analysis for GBM patients from RIH. Data are divided into two groups, with HDAC-7 "high" patients showing poorer survival compared to "low" patients (Figure 13A). HDAC-7 expression in GBM tumors versus non-tumors from TCGA, with GBM showing higher HDAC-7 expression compared to non-tumors (Figure 13B).

[0024] [Figure 14-1]Figures 14A-14E. Transcriptome analysis for HDAC-7 siRNA. Heatmap (Figure 14A) and volcano blot display (Figure 14B) of 4963 differentially expressed genes in HDAC-7 siRNA knockdown versus control in primary GSCs. Gene Ontology enrichment analysis of differentially expressed genes in HDAC-7 knockdown versus control (Figure 14C). A grid shows the presence of each knockdown gene and its presence in various stemness signature databases (Figure 14D). Radar chart showing overlap between genes downregulated by si-HDAC-7 in various stem cell types (Figure 14E). [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above.

[0025] [Figure 15-1] Figures 15A-15B. Proteomic analysis of HDAC-7 siRNA. Heatmap representation of changes in the abundance of histone post-translational modifications in HDAC-7 knockdown versus control detected via Mod Spec (mass spectrometry) (Figure 15A). Schematic representation of HDAC-7 and its DNA-binding and chromatin-associated protein partners identified via RIME (Figure 15B). The identified proteins were in close proximity to HDAC-7 in GSCs (2.3 Å to 2.7 Å). [Figure 15-2] Same as above. [Figure 15-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0026] (Detailed explanation) One of the difficulties in treating GBM is the lack of a treatment that can overcome the inter- and intratumor heterogeneity and plasticity exhibited in GBM. Here, it has been discovered that HDAC-7 is highly expressed in glioblastoma compared to normal brain tissue. It has also been found that inhibiting HDAC-7 (histone deacetylase 7) has a significant effect on GBM growth and inhibits GSC characteristics, transcriptome expression, and cancer cell invasion.

[0027] Furthermore, it was discovered that inhibiting HDAC-7 or CLOCK (Circadian Locomotor Output Cycles Kaput) or downregulating ASF1A (Anti-Silencing Function 1A Histone Chaperone), SUV39H2 (Suppressor Of Variegation 3-9 Homolog 2), or WHSC1L1 (Wolf-Hisrchhorn Syndrome Candidate 1-Like 1) resulted in phenotypic changes in in vitro GSC cultures and reduced survival in an in vivo GBM human xenograft mouse model.

[0028] Therefore, in one embodiment, the GBM inhibitor provided herein is HDAC-7 inhibitor.In some embodiments, the HDAC-7 inhibitor selected from small molecule, siRNA, their pharmaceutically acceptable salt or their combination is provided herein.In some embodiments, the inhibitor includes HDAC class IIa inhibitor (such as TMP269) or their pharmaceutically acceptable salt.In some embodiments, the inhibitor includes HDAC-7 targeting siRNA or their pharmaceutically acceptable salt. In some embodiments, the inhibitors include siRNAs targeting one or more of the following transcripts of HDAC7: NM_001098416; NM_001308090; NM_015401; XM_011538478; XM_011538479; XM_011538480; XM_011538481; XM_011538482; XM_011538483; XM_017019455; XM_017019456; or XR_001748761. In some embodiments, the siRNA comprises one or more of GACAAGAGCAAGCGAAGUG (SEQ ID NO: 1), GCAGAUACCCUCGGCUGAA (SEQ ID NO: 2), GGUGAGGGCUUCAAUGUCA (SEQ ID NO: 3), or UGGCUGCUCUUCUGGGUAA (SEQ ID NO: 4).

[0029] In addition to the above inhibitors, small molecule inhibitor libraries have been designed that exhibit remarkable specificity for HDAC-7 over other Class IIa histone deacetylases. These small molecules are disclosed in Table 1. Thus, in some embodiments, provided herein is a compound selected from Table 1, or a pharmaceutically acceptable salt thereof. [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

[0030] HDAC-7 has been crystallized, and like other members of class I, class II, and class IV, HDAC-7 binds to its substrates through a zinc-mediated charge relay system. Class IIa HDACs possess another specific zinc-binding motif adjacent to their active site, which directs recognition and protein-protein interactions, is most likely a site for docking of cofactors and regulatory proteins, and provides a site for regulation of activity. Remarkable structural diversity in HDAC-7 is found in a loop region near the active site entrance, which may mediate monodentate substrate coordination rather than the conventional bidentate substrate coordination of other HDACs, thus mediating substrate binding and specificity.

[0031] To design novel small molecule inhibitors (e.g., those in Table 1) with increased specificity for HDAC-7 relative to other HDAC class IIa members, we performed structure-based virtual screening of structurally related compounds of HDAC IIa inhibitors. This screening was performed against a 1.5 billion compound library, and binding to HDAC-7 was compared with binding to HDAC-4 and HDAC-5. The hits were also compared to the inhibitory binding of TMP269, a commercially available pan-class IIa HDAC inhibitor. By superimposing their crystal structures together, we compared the chemical binding of various binding groups with TMP269 to assess whether any potential binding groups increased affinity for HDAC-7 and decreased affinity for HDAC-4 and HDAC-5. The latter approach was applied in a sequential pipeline by examining structural differences in inhibitor binding during superimposition of input HDAC structures. We then used the Schrodinger computational ligand designer software to integrate physics-based prediction methods with machine learning techniques in combinatorial chemical binding, docking, and affinity calculations to model potential compounds for HDAC-7 versus HDAC-4 and HDAC-5. Thus, instead of manually predicting sites in TMP269 that allow for extension of binding groups, this software provides reliable predictions. By testing multiple binding groups at these predicted sites, compounds with higher affinity for HDAC-7 were successfully docked. Finally, we used a more complex library for the Schrodinger software, which incorporates more complex binding considerations (such as dynamic simulations and interactions with water molecules, neighboring ions, and required cofactors) into the binding to calculate the physical interactions and energy perturbations for the affinity calculations for these compounds against HDAC-7. This approach resulted in more applicable, "druggable," compounds that may exhibit better pharmacokinetics and pharmacodynamics when used biologically.

[0032] In some embodiments, the compounds described herein (eg, HDAC-7 inhibitors or salts thereof) exist as tautomers.

[0033] The compounds described herein also include isotopically labeled compounds in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from that predominantly found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include: 2 H, 3 H, 11 C. 13 C. 14 C. 36 Cl, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O. 17 O. 18 O. 32 P, and 35 In some embodiments, isotope-labeled compounds are useful in drug or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes (e.g., deuterium) provides for greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements). In yet another embodiment, positron-emitting isotopes (e.g., 11 C. 18 F, 15 O and 13 Substitution at N) is useful in positron emission tomography (PET) studies to examine receptor occupancy of substrates. Isotopically labeled compounds are prepared by any suitable method or process that employs an appropriate isotopically labeled reagent in place of an unlabeled reagent used in other methods.

[0034] In some embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0035] The compounds described herein, and other related compounds having various substituents, are described herein and can be found in, for example, Fieser and Fieser's Reagents for Organic Synthesis, Vols. 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Vols. 1-5 and Supplements (Elsevier Science Publishers, 1989); Organic Reactions, Vols. 1-40 (John Wiley and Sons, 1991); Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989); March, Advanced Organic Chemistry, 4th Edition (Wiley, 1992); Carey and Sundberg, Advanced Organic Chemistry, 4th Edition, Vols. A and B (Plenum, 2000, 2001); and Green and Wuts, Protective Groups in Organic Chemistry Synthesis, 3rd Edition (Wiley, 1999), all of which are incorporated by reference for such disclosures. The general methods for the preparation of the compounds described herein are modified by the use of appropriate reagents and conditions for the introduction of the various moieties found in the formulas provided herein.

[0036] The compounds described herein are synthesized using any suitable procedure starting from compounds available from commercial sources or prepared using the procedures described herein.

[0037] In some embodiments, reactive functional groups (e.g., hydroxyl, amino, imino, thio, or carboxy groups) are protected to avoid their unwanted participation in reactions. Protecting groups are used to block some or all of these reactive moieties, preventing such groups from participating in chemical reactions until the protecting group is removed. In other embodiments, each protecting group is removable by various means. Protecting groups that are cleaved under distinct reaction conditions satisfy the requirement for differential removal.

[0038] In some embodiments, protecting groups are removed by acid, base, reducing conditions (e.g., by hydrogenolysis), or oxidizing conditions. Groups such as trityl, dimethoxytrityl, acetal, and t-butyldimethylsilyl are acid-labile, and these groups are used to protect carboxy- and hydroxy-reactive moieties in the presence of amino groups protected with Cbz groups (which are removable by hydrogenolysis) and Fmoc groups (which are base-labile). Carboxylic acid- and hydroxy-reactive moieties are blocked with base-labile groups (e.g., t-butyl carbamate) or amines blocked with carbamates, which are stable to both acid and base but hydrolytically removable.

[0039] Thus, in some embodiments, provided herein is Compound 1 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0040] In some embodiments, provided herein is Compound 2, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, Compound 2, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0041] In some embodiments, provided herein is Compound 3, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, Compound 3, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0042] In some embodiments, provided herein is Compound 4, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, Compound 4, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0043] In some embodiments, provided herein is Compound 5, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, Compound 5, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0044] In some embodiments, provided herein is Compound 6, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, Compound 6, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0045] In some embodiments, provided herein is Compound 7, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, Compound 7, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0046] In some embodiments, provided herein is Compound 8, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, Compound 8, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0047] In some embodiments, provided herein is Compound 9 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, Compound 9 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0048] In some embodiments, provided herein is compound 10, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 10, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0049] In some embodiments, provided herein is compound 11 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, compound 11 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0050] In some embodiments, provided herein is compound 12, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 12, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0051] In some embodiments, provided herein is compound 13, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 13, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0052] In some embodiments, provided herein is compound 14, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 14, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0053] In some embodiments, provided herein is compound 15, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 15, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0054] In some embodiments, provided herein is compound 16, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 16, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0055] In some embodiments, provided herein is compound 17, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 17, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0056] In some embodiments, provided herein is compound 18, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 18, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0057] In some embodiments, provided herein is compound 19, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 19, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0058] In some embodiments, provided herein is compound 20, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 20, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0059] In some embodiments, provided herein is compound 21 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, compound 21 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0060] In some embodiments, provided herein is compound 22, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 22, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0061] In some embodiments, provided herein is compound 23, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 23, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0062] In some embodiments, provided herein is compound 24, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 24, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0063] In some embodiments, provided herein is compound 25, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 25, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0064] In some embodiments, provided herein is compound 26, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 26, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0065] In some embodiments, provided herein is compound 27, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 27, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0066] In some embodiments, provided herein is compound 28, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 28, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0067] In some embodiments, provided herein is compound 29, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 29, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0068] In some embodiments, provided herein is compound 30, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 30, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0069] In some embodiments, provided herein is compound 31, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 31, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0070] In some embodiments, provided herein is compound 32, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 32, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0071] In some embodiments, provided herein is compound 33, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 33, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0072] In some embodiments, provided herein is compound 34, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 34, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0073] In some embodiments, provided herein is compound 35, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 35, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0074] In some embodiments, provided herein is compound 36, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 36, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0075] In some embodiments, provided herein is compound 37, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 37, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0076] In some embodiments, provided herein is compound 38, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 38, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0077] In some embodiments, provided herein is compound 39, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 39, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0078] In some embodiments, provided herein is compound 40, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 40, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0079] In some embodiments, provided herein is compound 41, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 41, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0080] In some embodiments, provided herein is compound 42, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 42, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0081] In some embodiments, provided herein is compound 43, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 43, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0082] In some embodiments, provided herein is compound 44, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 44, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0083] In some embodiments, provided herein is compound 45, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 45, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0084] In some embodiments, provided herein is compound 46, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 46, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0085] In some embodiments, provided herein is compound 47, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 47, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0086] In some embodiments, provided herein is compound 48, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 48, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0087] In some embodiments, provided herein is compound 49, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 49, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0088] In some embodiments, provided herein is compound 50, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 50, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0089] In some embodiments, provided herein is compound 51, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 51, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0090] In some embodiments, provided herein is compound 52, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 52, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0091] In some embodiments, provided herein is compound 53, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 53, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0092] In some embodiments, provided herein is compound 54, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 54, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0093] In some embodiments, provided herein is compound 55, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 55, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0094] In some embodiments, provided herein is compound 56, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 56, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0095] In some embodiments, provided herein is compound 57, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 57, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0096] In some embodiments, provided herein is compound 58, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 58, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0097] In some embodiments, provided herein is compound 59, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 59, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0098] In some embodiments, provided herein is compound 60 of Table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, compound 60, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0099] In some embodiments, provided herein is compound 61 in Table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, compound 61, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0100] In some embodiments, provided herein is compound 62, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 62, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0101] In some embodiments, provided herein is compound 63, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 63, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0102] In some embodiments, provided herein is compound 64, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 64, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0103] In some embodiments, provided herein is compound 65 of Table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, compound 65, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0104] In some embodiments, provided herein is compound 66, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 66, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0105] In some embodiments, provided herein is compound 67, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 67, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0106] In some embodiments, provided herein is compound 68, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 68, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0107] In some embodiments, provided herein is compound 69 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, compound 69 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0108] In some embodiments, provided herein is compound 70, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 70, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0109] In some embodiments, provided herein is compound 71 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, compound 71 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0110] In some embodiments, provided herein is compound 72, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 72, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0111] In some embodiments, provided herein is compound 73, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 73, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0112] In some embodiments, provided herein is compound 74, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 74, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0113] In some embodiments, provided herein is compound 75, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 75, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0114] In some embodiments, provided herein is compound 76, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 76, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0115] In some embodiments, provided herein is compound 77, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 77, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0116] In some embodiments, provided herein is compound 78, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 78, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0117] In some embodiments, provided herein is compound 79 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, compound 79 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0118] In some embodiments, provided herein is compound 80, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 80, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0119] In some embodiments, provided herein is compound 81 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, compound 81 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0120] In some embodiments, provided herein is compound 82, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 82, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0121] In some embodiments, provided herein is compound 83, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 83, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0122] In some embodiments, provided herein is compound 84, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 84, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0123] In some embodiments, provided herein is compound 85, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 85, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0124] In some embodiments, provided herein is compound 86, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 86, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0125] In some embodiments, provided herein is compound 87, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 87, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0126] In some embodiments, provided herein is compound 88, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 88, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0127] In some embodiments, provided herein is compound 89, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 89, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0128] In some embodiments, provided herein is compound 90, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 90, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancers, including, for example, GBM.

[0129] In some embodiments, provided herein is compound 91 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, compound 91 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0130] In some embodiments, provided herein is compound 92, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 92, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0131] In some embodiments, provided herein is compound 93, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 93, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0132] In some embodiments, provided herein is compound 94, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 94, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0133] In some embodiments, provided herein is Compound 95 of Table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, Compound 95, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer, including, for example, GBM.

[0134] In some embodiments, provided herein is compound 96, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 96, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0135] In some embodiments, provided herein is compound 97, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 97, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0136] In some embodiments, provided herein is compound 98, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 98, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0137] In some embodiments, provided herein is Compound 99 or a pharmaceutically acceptable salt thereof in Table 1. In some embodiments, Compound 99 or a pharmaceutically acceptable salt thereof is useful in treating brain cancer (including, for example, GBM).

[0138] In some embodiments, provided herein is compound 100, or a pharmaceutically acceptable salt thereof, of Table 1. In some embodiments, compound 100, or a pharmaceutically acceptable salt thereof, is useful in treating brain cancer (including, for example, GBM).

[0139] Embodiments of the present disclosure relate to a method of treating brain cancer and / or solid tumors in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a histone deacetylase 7 (HDAC-7) inhibitor.

[0140] Another aspect of the present disclosure relates to a method of treating glioblastoma multiforme in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a histone deacetylase 7 (HDAC-7) inhibitor.

[0141] In some embodiments, the HDAC-7 inhibitor comprises one or more of the compounds in Table 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more of the compounds in Table 1 or a pharmaceutically acceptable salt thereof.

[0142] In some embodiments, the HDAC-7 inhibitor comprises one or more of TMP269, trichostatin A, vorinostat, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more of TMP269, trichostatin A, vorinostat, or a pharmaceutically acceptable salt thereof. [ka]

[0143] In some embodiments, the HDAC-7 inhibitor comprises one or more siRNAs or pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising one or more of the siRNAs or pharmaceutically acceptable salts thereof.

[0144] In some embodiments, the siRNA comprises GACAAGAGCAAGCGAAGUG (SEQ ID NO: 1), GCAGAUACCCUCGGCUGAA (SEQ ID NO: 2), GGUGAGGGCUUCAAUGUCA (SEQ ID NO: 3), or UGGCUGCUCUUCUGGGUAA (SEQ ID NO: 4).

[0145] In some embodiments, the glioblastoma multiforme is at least partially present as a tumor.

[0146] In some embodiments, the HDAC-7 inhibitor is administered before, after, or simultaneously with surgical removal of at least a portion of the tumor.

[0147] In some embodiments, the HDAC-7 inhibitor is administered in an amount sufficient to cause one or more of the following: a decrease in tumor growth, a decrease in tumor cell proliferation, an increase in tumor cell apoptosis, an inhibition of metastatic dissemination of glioblastoma multiforme, an improvement in the survival of the subject, a decrease in the ability of tumor cells to form colonies, or a decrease in the ability of tumor cells to migrate.

[0148] In some embodiments, the HDAC-7 inhibitor is administered before, after, or simultaneously with the antibody therapy.

[0149] In some embodiments, the HDAC-7 inhibitor is administered before, after, or simultaneously with radiation therapy.

[0150] A further aspect of the present disclosure relates to pharmaceutical compositions for treating brain cancer and / or solid tumors. In some embodiments, the compositions comprise one or more of the compounds in Table 1 or pharmaceutically acceptable salts thereof.

[0151] Another aspect of the present disclosure relates to pharmaceutical compositions for treating glioblastoma multiforme. In some embodiments, the compositions comprise one or more of the compounds in Table 1 or pharmaceutically acceptable salts thereof.

[0152] Another aspect of the present disclosure relates to a pharmaceutical composition for treating brain cancer and / or solid tumor.In some embodiments, the composition comprises one or more siRNAs or their pharmaceutically acceptable salts.In some embodiments, the siRNAs comprise SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0153] Another aspect of the present disclosure relates to a pharmaceutical composition for treating glioblastoma multiforme.In some embodiments, the composition comprises one or more siRNAs or their pharmaceutically acceptable salts.In some embodiments, the siRNAs comprise SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0154] As used herein, " pharmaceutically acceptable salt " refers to the ionizable therapeutic agent that is combined with counterion to form a neutral complex.The list of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., 1985, p.1418; Journal of Pharmaceutical Science, 66, 2 (1977); and " Handbook of Pharmaceutical Salts: Properties, Selection, and Use " (P. Henrich Stahl and Camille G. Wermuth (eds.), VHCA & Wiley-VCH, 2002).

[0155] The terms "pharmaceutical" and "pharmaceutically acceptable" can refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0156] As used herein, the term "cell" is meant to refer to a cell that is in vitro, ex vivo, or in vivo. In some embodiments, an ex vivo cell may be part of a tissue sample excised from an organism (e.g., a mammal). In some embodiments, an in vitro cell may be a cell in cell culture. In some embodiments, an in vivo cell is a cell that is living in an organism (e.g., a mammal).

[0157] As used herein, the terms "individual," "patient," or "subject," used interchangeably, refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, and most preferably humans.

[0158] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to that amount of an active compound or agent that elicits the biological or medical response being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, physician, or other clinician.

[0159] The term "effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic agent (i.e., dose) administered to a subject (e.g., a mammalian subject, i.e., a human subject), either in a single dose or as part of a series, effective to produce the desired therapeutic effect (e.g., effective to affect, reduce, or inhibit the activity of, or prevent the activation of, a kinase, or to produce a desired in vivo effect (e.g., reduced intraocular pressure) in an animal (preferably a human).

[0160] As used herein, the term "treating" or "treatment" refers to 1) inhibiting a disease (e.g., inhibiting a disease, condition, or disorder in an individual experiencing or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., halting further development of the pathology and / or symptoms)); or 2) alleviating a disease (e.g., alleviating a disease, condition, or disorder in an individual experiencing or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., reversing the pathology and / or symptoms)).

[0161] The term "treatment" can refer to the application of one or more specific procedures used to alleviate disease. In certain embodiments, the specific procedure is the administration of one or more pharmaceutical agents. "Treatment" of an individual (e.g., a mammal, e.g., a human) or cell is any type of intervention used in an attempt to alter the natural course of that individual or cell. Treatment includes, but is not limited to, the administration of a therapeutic agent or pharmaceutical composition, and treatment can be performed either prophylactically or after the onset of a pathological event or contact with a pathogenic agent. Treatment includes any desired effect on the symptoms or pathology of a disease or condition; treatment can include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition being treated. "Preventive" treatment is also included, which can involve reducing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset.

[0162] As used herein, the terms "preventing" a disease, condition, or disorder or "prevention" of a disease, condition, or disorder refer to reducing the risk of developing the disease, condition, or disorder in a subject or a group of subjects (e.g., a subject or a group of subjects predisposed to or susceptible to the disease, condition, or disorder). In some embodiments, preventing a disease, condition, or disorder refers to reducing the chance of acquiring the disease, condition, or disorder and / or its associated symptoms. In some embodiments, preventing a disease, condition, or disorder refers to completely or nearly completely halting the disease, condition, or disorder from occurring.

[0163] Treatment of the brain cancer and / or solid tumor, glioblastoma multiforme, may further include any known therapy for treating cancer, including, but not limited to, surgical removal of the cancer, administration of chemotherapy, administration of radiation, administration of antibody therapy, and administration of anti-cancer drugs.

[0164] The term "chemotherapy" refers to the treatment of cancer, or diseases or disorders caused by viruses, bacteria, or other microorganisms, or inappropriate immune responses, using specific chemicals, drugs, or radioactive agents that are selectively toxic and destructive to malignant cells and tissues, viruses, bacteria, or other microorganisms. Preferred are chemotherapeutic agents or drugs (e.g., antifolates (e.g., methotrexate)) or any other agent or drug that is useful in treating cancer, inflammatory disease, or autoimmune disease. Suitable chemotherapeutic agents and drugs include actinomycin D, adriamycin, altretamine, azathioprine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, liposomal doxorubicin, lomustine, melamine, methylprednisolone ... These include, but are not limited to, cyclosporine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pentostatin, procarbazine, raltitrexed, steroids, streptozocin, taxol, taxotere, temozolomide, thioguanine, thiotepa, tomudex, topotecan, treosulfan, uft (uracil-tegufur), vinblastine, vincristine, vindesine, and vinorelbine.

[0165] As used herein, in such methods, the term "biological sample" refers to a bodily fluid or tissue. The bodily fluid may include, but is not limited to, whole blood, serum, plasma, peripheral blood, synovial fluid, cerebrospinal fluid, saliva, urine, semen, or other liquid secretions. The term "tissue" may include, but is not limited to, bone marrow and lymph node, as well as other tissue samples.

[0166] The present disclosure also provides pharmaceutical compositions, comprising an effective amount of the HDAC-7 inhibitor compound disclosed herein or its pharmaceutically acceptable salt, and pharmaceutically acceptable carrier.In certain embodiments, the present disclosure also provides pharmaceutical compositions and dosage forms, comprising any one of the additional therapeutic agents described herein.The carrier(s) is / are "acceptable" in the sense that it is compatible with other components of the formulation, and if it is a pharmaceutically acceptable carrier, it is not harmful to the recipient in the amount used in the pharmaceutical.

[0167] "Pharmaceutically acceptable carrier" means a carrier useful for the preparation of a pharmaceutical composition, which is generally compatible with the other ingredients of the composition, not harmful to the recipient, and not biologically or otherwise objectionable. "Pharmaceutically acceptable carrier" includes both one carrier and more than one carrier. Embodiments include carriers for topical administration, carriers for intraocular administration, carriers for parenteral administration, carriers for intravenous administration, carriers for intraperitoneal administration, carriers for intramuscular administration, carriers for sublingual administration, carriers for nasal administration, and carriers for oral administration. "Pharmaceutically acceptable carrier" also includes agents for the preparation of aqueous dispersions and sterile powders for injection or dispersion.

[0168] Pharmaceutically acceptable carriers, adjuvants, and vehicles that may be used in the pharmaceutical compositions of the present disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block copolymers, polyethylene glycol, and sheep wool oil.

[0169] The compositions or dosage forms may contain any one of the compounds and therapeutic agents described herein in the range of 0.005% to 100%, with the balance consisting of suitable pharmaceutically acceptable excipients. Contemplated compositions may contain any one of the compounds and therapeutic agents provided herein in the range of 0.001% to 100%, in one embodiment 0.1% to 95%, in another embodiment 75% to 85%, and in a further embodiment 20% to 80%, with the balance consisting of any pharmaceutically acceptable excipient described herein, or any combination of these excipients.

[0170] (Route of administration and dosage form) Pharmaceutical compositions of the present disclosure include those suitable for any acceptable route of administration, including buccal, cutaneous, intracervical, endosinusial, intratracheal, enteral, epidural, interstitial, intraperitoneal, intraarterial, intrabronchial, intravesical, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intranasal, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, and intraantral. These include, but are not limited to, intravenous, intrasinal, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, nasogastric, oral, parenteral, percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral, ​​and intravaginal.

[0171] The compositions and formulations described herein can be conveniently provided in unit dosage forms (e.g., tablets), sustained-release capsules, and liposomes, and can be prepared by any method well known in the art of pharmacy. See, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore, MD (20th ed., 2000). Such preparation methods include combining ingredients, such as carriers, which constitute one or more accessory ingredients, with the molecule to be administered. In general, the compositions are prepared by uniformly and intimately combining the active ingredient with liquid carriers, liposomes, or finely divided solid carriers, or both, and then, if necessary, shaping the product. See also, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Wolters Kluwer Health (11th ed., 2018).

[0172] In some embodiments, any one of the compounds and therapeutic agents disclosed herein is orally administered.The compositions of the present disclosure suitable for oral administration can be provided as separate units (for example, capsules, sachets, granules or tablets), each containing a predetermined amount (for example, effective amount) of active ingredient; as powder or granules; as a solution or dispersion in aqueous liquid or non-aqueous liquid; as oil-in-water liquid emulsion; as water-in-oil liquid emulsion; in liposome; or as bolus.Soft gelatin capsules can be useful for containing such suspension, which can beneficially increase the absorption rate of compound.For tablets for oral use, commonly used carriers include lactose, sucrose, glucose, mannitol, and silicic acid and starch. Other acceptable excipients may include a) fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), b) binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and gum arabic, and the like), c) humectants (e.g., glycerol), d) disintegrating agents (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), e) solution retarders (e.g., paraffin), f) absorption accelerators (e.g., quaternary ammonium compounds), g) wetting agents (e.g., cetyl alcohol and glycerol monostearate, and the like), h) absorbents (e.g., kaolin clay and bentonite clay), and i) lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof). For oral administration in capsule form, useful diluents include lactose and dried corn starch.When aqueous suspension is administered orally, its active ingredient is combined with emulsifying and suspending agent.If desired, certain sweeteners and / or flavoring agents and / or coloring agents can be added.Suitable compositions for oral administration include lozenges, which contain the ingredient in a flavored base (usually sucrose and gum arabic or tragacanth); and pastilles, which contain the active ingredient in an inert base (e.g., gelatin and glycerin, or sucrose and gum arabic).

[0173] Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection or infusion solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The preparations may be provided in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier (e.g., water for injection, saline (e.g., 0.9% saline solution) or 5% dextrose solution) immediately before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. The injection solutions may be in the form of, for example, sterile injectable aqueous or oily suspensions. The suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (for example, as a solution in 1,3-butanediol). Acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland, fixed oil, including synthetic monoglycerides or diglycerides, can be used. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants.

[0174] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, optionally encapsulated in liposomes, adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be, for example, a solvent or liquid dispersion medium, including water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include isotonic agents (e.g., sugars, buffers, or sodium chloride). Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0175] The pharmaceutical composition of the present disclosure can be administered in the form of suppositories for rectal administration.These compositions can be prepared by mixing the compound of the present disclosure with suitable non-irritating excipients that are solid at room temperature but liquid at rectal temperature, and therefore melt in rectum to release its active ingredient.Such materials include but are not limited to cocoa butter, beeswax and polyethylene glycol.

[0176] The pharmaceutical compositions of the present disclosure can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, and can be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. See, for example, U.S. Patent No. 6,803,031. Further formulations and methods for intranasal administration can be found in Ilium, L., J Pharm Pharmacol, 56:3-17, 2004 and Ilium, L., Eur J Pharm Sci, 11:1-18, 2000.

[0177] The topical compositions of the present disclosure can be prepared and used in the form of aerosol spray, cream, emulsion, solid, liquid, dispersion, foam, oil, gel, hydrogel, lotion, mousse, ointment, powder, patch, pomade, solution, pump spray, stick, towelette, soap, or other forms commonly used in topical administration and / or cosmetic and skin care formulations.The topical composition can be in emulsion form.Topical administration of the pharmaceutical compositions of the present disclosure is particularly useful when the desired treatment involves areas or organs that are easily accessible by topical application. In some embodiments, the topical composition comprises a combination of any one of the compounds and therapeutic agents disclosed herein with one or more additional ingredients, carriers, excipients, or diluents, including, but not limited to, absorbents, anti-irritants, anti-acne agents, preservatives, antioxidants, colorants / pigments, emollients (moisturizers), emulsifiers, film formers / film retention agents, fragrances, leave-on exfoliants, prescription medications, preservatives, scrubs, silicones, skin-identical / skin repair agents, slip agents, sunscreen actives, surfactants / detergents, penetration enhancers, and thickeners.

[0178] Examples of useful dermatological compositions that can be used to deliver the compounds to the skin are known in the art, see, for example, Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157), and Wortzman (U.S. Pat. No. 4,820,508).

[0179] The compounds and therapeutic agents of the present disclosure can be incorporated into compositions for coating implantable medical devices (such as prostheses, artificial valves, vascular implants, stents, or catheters).Suitable coatings and the general preparation of coated implantable devices are known in the art and are exemplified in U.S. Patent Nos. 6,099,562; 5,886,026; and 5,304,121.The coating is typically a biocompatible polymer material (such as hydrogel polymers, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof).The coating can optionally be further coated with a suitable topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids, or combinations thereof to impart sustained-release characteristics to the composition.Coatings for invasive devices should be included within the definition of pharmaceutically acceptable carrier, adjuvant, or vehicle when these terms are used herein.

[0180] According to another embodiment, the present disclosure provides an implantable drug release device impregnated with or containing a compound or therapeutic agent, or a composition comprising a compound or therapeutic agent of the present disclosure, such that the compound or therapeutic agent is released from the device to be therapeutically active.

[0181] (Dosage and Regimen) In the pharmaceutical compositions of the present disclosure, the compound of formula (I) is present in an effective amount (eg, a therapeutically effective amount).

[0182] The useful dose may vary depending on the condition being treated, the severity of the condition, the route of administration, the subject's sex, the subject's age, and the subject's general health, the use of excipients, and the possibility of co-administration with other therapeutic treatments (e.g., the use of other drugs), and the judgment of the treating physician.

[0183] In some embodiments, an effective amount of an HDAC-7 inhibitor is, for example, about 0.001 mg / kg to about 500 mg / kg (e.g., about 0.001 mg / kg to about 200 mg / kg; about 0.01 mg / kg to about 200 mg / kg; about 0.01 mg / kg to about 150 mg / kg; about 0.01 mg / kg to about 100 mg / kg; about 0.01 mg / kg to about 50 mg / kg; about 0.01 mg / kg to about 10 mg / kg; about 0.01 mg / kg to about 5 mg / kg; about 0.01 mg / kg to about 1 mg / kg; about The dose may be in the range of 0.01 mg / kg to about 0.5 mg / kg; about 0.01 mg / kg to about 0.1 mg / kg; about 0.1 mg / kg to about 200 mg / kg; about 0.1 mg / kg to about 150 mg / kg; about 0.1 mg / kg to about 100 mg / kg; about 0.1 mg / kg to about 50 mg / kg; about 0.1 mg / kg to about 10 mg / kg; about 0.1 mg / kg to about 5 mg / kg; about 0.1 mg / kg to about 2 mg / kg; about 0.1 mg / kg to about 1 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg).

[0184] In some embodiments, the effective amount of the HDAC-7 inhibitor is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, or about 5 mg / kg.

[0185] The above dosages can be administered daily (e.g., as a single dose or as two or more divided doses, e.g., once daily, twice daily, three times daily) or non-daily (e.g., every other day, every two days, every three days, once a week, twice a week, once every two weeks, once a month).

[0186] (kit) In some embodiments, provided herein is a packaged dosage form that includes a container holding a therapeutically effective amount of an HDAC-7 inhibitor or salt thereof provided herein, and instructions for using the dosage form according to one or more of the methods provided herein.

[0187] The dosage forms and related materials can be completed into commercial products by conventional processes practiced in the art (e.g., by appropriate sterilization and packaging). For example, the materials can be treated with UV / visible light irradiation (200 nm to 500 nm) using, for example, photoinitiators with various absorption wavelengths (e.g., Irgacure 184, 2959), preferably water-soluble initiators (e.g., Irgacure 2959). Such irradiation is typically carried out for a period of 1 minute to 60 minutes, although longer irradiation times may be applied depending on the particular method. Materials according to the present disclosure can be terminally sterilized and wrapped, and packaged in an appropriate container (such as a box) (e.g., by adding specific product information printouts) to maintain sterility until use.

[0188] According to further embodiments, the described dosage forms can also be provided in kit form with other components necessary for administration of the substance to a patient. The disclosed kits, such as for use in the treatments described herein, can further include, for example, administration substances.

[0189] The kits can be designed in a variety of forms based on the particular deficiency they are designed to treat.

[0190] The dosage forms provided herein can be prepared and placed in a container for storage at ambient or elevated temperatures.This is beneficial because the commercially viable transport of the dosage form can benefit from stability at temperatures higher than those that require a refrigerated or sub-freezing environment during transport and storage at the point of use.

[0191] When the dosage forms provided herein are stored in a polyolefin plastic container compared to a polyvinyl chloride plastic container, for example, discoloration of the dosage form may be reduced. Without wishing to be bound by theory, the container may reduce the exposure of the contents of the container to electromagnetic radiation, whether it be visible light (e.g., having a wavelength of about 380 nm to about 780 nm) or ultraviolet (UV) light (e.g., having a wavelength of about 190 nm to about 320 nm (UV B light) or about 320 nm to about 380 nm (UV A light)). Some containers also include the ability to reduce adsorption or absorption of active ingredients to the container surface, which can effectively dilute the concentration of the active ingredient in the contained solution. Some containers also include the ability to reduce the exposure of the contents of the container to infrared light, or a second component with such ability. Some containers further include the ability to reduce the exposure of the contents of the container to heat or humidity. Containers that can be used include those made of polyolefins (e.g., polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, polymethylpentene, polybutene, or combinations thereof (particularly polyethylene, polypropylene, or combinations thereof)). In some embodiments, the container is a glass container. The container can be further placed in a second container (e.g., a paper container, a cardboard container, a cardboard container, a metal film container, or a foil container, or combinations thereof) to further reduce the exposure of the contents of the container to UV light, visible light, or infrared light. Manufactured articles that benefit from reduced discoloration, reduced degradation, or both during storage include dosage forms comprising one or more of the HDAC-7 inhibitors or salts thereof provided herein. The dosage forms provided herein may require storage lasting up to three months or longer, and in some cases up to one year or longer. The container can be in any form (e.g., a bag, a bottle, or a box) suitable for containing the contents.

[0192] The following examples further illustrate embodiments of the present disclosure, but in no way limit the teachings or disclosure set forth herein. [Example]

[0193] (Example) Through viral library screening of all epigenetic regulators in GBM human xenograft mouse models and GSC cultures, we identified epigenetic regulators that, when overexpressed, reduced or increased survival in GBM mouse models or altered the self-renewal phenotype of GSC in vitro cultures. We identified five key epigenetic regulators in vitro and in vivo: HDAC-7, CLOCK, ASF1A, SUV39H2, and WHSC1L1. Here, upregulation of HDAC-7 and CLOCK and downregulation of ASF1A, SUV39H2, and WHSC1L1 resulted in phenotypic changes in vitro and reduced survival in GBM mouse models (Figure 2B).

[0194] Next, RNA was collected from paraffin-embedded samples from GBM patients and run against a 700-gene PanCancer progression panel using NonoString technology. Data for 33 patients were curated and classified as high-risk (HR) or low-risk (LR) based on risk derived from the expression of these five genes as epigenetic signatures. Differential gene expression analysis was applied to the HR vs. LR groups, and 293 / 700 genes were found to be differentially expressed between the HR and LR groups (Figures 3D and 3F). The functional significance of these differentially expressed genes showed enrichment for the biological processes of wound healing and cell proliferation (Figure 3E). Kaplan-Meier survival analysis was performed on this patient's data using the log-rank test and Gehan-Breslow-Wilcoxon method based on the same HR / LR stratification. We found a significant reduction in both overall survival (Figure 3B) and progression-free survival (Figure 3C) in these patients based on risk derived from the expression of five genes (HDAC-7, CLOCK, ASF1A, SUV39H2, and WHSC1L1) as an epigenetic signature.

[0195] RNA was collected from paraffin-embedded samples from GBM patients at Rhodesland Hospital (RIH), and survival analysis was performed according to HDAC-7 and mRNA expression. We found a significant decrease in patient survival for the HDAC-7 high-risk (HR) group (Figure 13A). HDAC-7 mRNA expression in GBM was compared with non-tumor samples from TCGA data using the GlioVis web tool (Bowman, RL et al., Neuro. Oncol., 19, 139-141 (2017)). We found that HDAC-7 expression in GBM was higher only in GBM compared with non-tumor samples (Figure 13A).

[0196] RNA sequencing was performed on primary GSCs after HDAC-7 siRNA knockdown, followed by transcriptome analysis. Differential gene expression analysis was performed between HDAC-7 siRNA knockdown and control siRNA cells with a false discovery rate of <0.01. Gene Ontology and KEGG enrichment pathways were used for gene enrichment analysis (Smith, RN et al., Bioinformatics, 28, 3163–3165 (2012)). Furthermore, we investigated whether genes downregulated by HDAC-7 knockdown have a role in stemness using the web tool Stem Checker (Pinto, JP et al., Nucleic Acids Res., 43, W72–W77 (2015)). Mass spectrometry was used on GSCs after HDAC-7 knockdown to determine the role of HDAC-7 in regulating post-translational modifications of histones at a global level. To elucidate the impact of HDAC-7 inhibition on the global chromatin landscape, we knocked down HDAC-7 in GSCs using siRNA and then performed mass spectrometry using ModSpec. This showed minimal deacetylation of histone marks in GSCs. To elucidate the protein partners of HDAC7 in the GSC nuclei, we performed rapid immunoprecipitation-mass spectrometry of endogenous proteins (RlME) on the GSCs using an HDAC-7 antibody.

[0197] HDAC-7 was investigated as one of two epigenetic regulators upregulated in the viral screening described above and to suggest a novel target in GBM. Using the GlioVis web tool6 to analyze data from the Chinese Glioma Genome Atlas (CGGA), we compared HDAC-7 mRNA expression in GBM with other forms of malignant glioma and found that HDAC-7 expression in GBM was the most prominent among all other glial tumors (Figure 4A).

[0198] Subsequently, survival analysis was performed on two cohorts of GBM patients (HDAC-7 high-expressing patients and HDAC-7 low-expressing patients), in which patients were divided into these two groups based on HDAC-7 expression and maximally ranked statistics using the log-rank test and Gehan-Breslow-Wilcoxon method. HDAC-7 expression was positively correlated with decreased overall survival in GBM patients (Figure 4B).

[0199] Similarly, when analyzing data from The Cancer Genome Atlas (TCGA), we compared HDAC-7 mRNA expression in GBM with non-tumor samples and found that HDAC-7 expression was higher in GBM than in non-tumor samples (Figure 5A). Survival analysis was also performed on two GBM patient cohorts (patients with high HDAC-7 expression and patients with low HDAC-7 expression). Patients were divided into these two groups according to HDAC-7 expression and maximum ranking statistics using the log-rank test and Gehan-Breslow-Wilcoxon method. This showed that HDAC-7 expression was positively correlated with decreased overall survival in GBM patients (Figure 5B). Furthermore, we performed differential gene expression analysis on the TCGA-derived RNASeq data and found that nearly 1600 differentially expressed genes were clustered into two distinct categories based on HDAC-7 expression levels (Figure 5C), with fold changes of up to 4 for upregulation and down to -2.5 for downregulation (Figure 5D). These data from CGGA and TCGA support the conclusion that HDAC-7 plays an important role in GBM pathology.

[0200] To elucidate whether HDAC-7 inhibition results in phenotypic changes in primary GSCs, we investigated its effect on altering GSC self-renewal. Because no small molecule drugs are currently available as selective HDAC-7 inhibitors, we used the class IIa HDAC inhibitor TMP269, which inhibits HDAC-4, HDAC-5, HDAC-7, and HDAC-9. We applied a 2-week limiting dilution assay (LDA) to examine the frequency of self-renewing cells within a population. Cells were treated with the inhibitor versus DMSO-treated control cells, and we observed a significant decrease in self-renewal of TMP269-treated cells. This was assessed by the ability of the GSCs to form neurospheres and through an online algorithmic scoring system. Furthermore, the same striking results were observed when GSCs derived from various genotypes were tested (Figures 6A-D).

[0201] To examine the genetic effects of HDAC-7 selective inhibition on GSCs, we knocked down HDAC-7 in GSCs using siRNA (SEQ ID NOs: 1-4) and then performed transcriptome analysis using RNASeq. Differential gene expression analysis with a false discovery rate (FDR) of less than 0.05 showed that 6,270 genes were differentially expressed between HDAC-7 siRNA knockdown and the siRNA negative control, with up to three-fold changes in up- and down-regulated genes (Figures 7A-7B). To understand the functional significance of these genes, we performed gene enrichment analysis on these differentially expressed genes using various gene enrichment tools, including Gene Ontology and KEGG pathway enrichment. We found that the predominantly suppressed enriched pathways were for biological processes related to cell cycle activity and cell division (Figure 7C). Furthermore, we investigated whether genes downregulated by HDAC-7 knockdown play a role in stemness using the web tool Stem Checker. We found that 653 of the 3150 down-regulated genes have a role in stemness, and these genes are more similar to the embryonic stem cell phenotype and embryonic carcinoma phenotype (Figure 7D).

[0202] Similar to many other cancers, where a mesenchymal-to-epithelial phenotype is typically associated with minimally invasive tumors, GBM also exhibits this characteristic. To understand whether HDAC-7 inhibition plays a role in this phenotypic transition, we used the Broad Institute's Gene Set Enrichment Analysis web tool GSEA, which fed gene set classifiers for mesenchymal and proneural signature phenotypes according to TCGA and Carro signature stratification. By calculating enrichment scores according to the rank in each gene set, we found that HDAC-7 inhibition inhibited both the mesenchymal and proneural gene set signatures (Figure 8A). To confirm these results, we also examined the expression of signature genes for various GSC subtypes in publicly available data from TCGA using the GlioVis web tool 6 and found that the four GSC subtypes exhibited similar ratios of HDAC-7 mRNA expression (Figure 8B). This data supports the position that HDAC-7 inhibition can suppress all GSC subtypes, a pathway not involved in mesenchymal to proneural differentiation.

[0203] The interactome of HDAC-7 is not well understood. To elucidate the effects of HDAC-7 inhibition on the global chromatin landscape, HDAC-7 was knocked out in GSCs using siRNA and verified by Western blot, resulting in an average of 73.4% protein inhibition (Figure 9A). We noticed that HDAC-7 protein levels in GSC lysates were relatively low compared to housekeeping genes (Figure 9B). Mass spectrometry using ModSpec technology was then applied to HDAC-7 knockdown GSCs to determine the post-translational histone modification targets for HDAC-7 at a global level. Interestingly, the knockdown showed many histone modifications compared to the control. These changes included decreases or increases in acetylation and methylation of lysine, arginine, and glutamine amino acids in the N-terminal histone tail. Of the more than 80 histone modifications tested with ModSpec, significant changes in the relative abundance of mainly 15 histone modifications were found between the knockdown and control. The relative increase in acetylation of H3:K122AC upon HDAC-7 knockdown was particularly intriguing (Figure 10). Recently, it has been shown that H3K122ac is sufficient to stimulate transcription, and mutation of H3K122 inhibits its transcriptional activation, which has been shown to be a desirable effect of H3:K122AC on histone-DNA binding.

[0204] At the transcriptome level, a significant effect of inhibiting HDAC-7 without affecting other HDACs was observed (Figure 7). From the RNA-Seq data, we found that knocking down HDAC-7 upregulated HDAC-1, HDAC-5, HDAC-6, and HDAC-11, while downregulated HDAC-2 and HDAC-9 (Figure 11). Therefore, without being bound by theory, this may explain the increased acetylation of many post-translational modification (PTM) targets after HDAC-7 siRNA inhibition. Among the histone targets that showed decreased acetylation was that for H4:K20AC upon HDAC-7 knockdown. H4:K20AC is a unique acetylation mark associated with gene repression, and H4:K20AC is enriched near the transcription start sites (TSSs) of minimally expressed and silent genes. H4K20 can be acetylated in highly active genes, while its acetylation is associated with less expressed genes. Another target is H3R2UN:K4AC.

[0205] We found that an HDAC class IIa inhibitor (TMP269) reduced GSC viability with the same inhibitory tendency observed with the pan-HDAC inhibitor "vorinostat," an FDA-approved anticancer drug (Figure 12). Preliminary results from phenotypic experiments (Figures 6 and 12) and genotypic experiments (Figure 7) suggest that narrowing the scope of HDACs provides comparable inhibitory efficacy to pan-HDACs and therefore fewer off-target effects and side effects. HDAC-7 has been crystallized, and like other members of class I, class II, and class IV, HDAC-7 binds to its substrates through a zinc-mediated charge relay system. Class IIa HDACs have another specific zinc-binding motif adjacent to their active site, which directs recognition and protein-protein interaction. However, there is significant structural diversity in HDAC-7 found in the loop region near the active site entrance, which poses challenges for inhibitor generation.

[0206] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (e.g., molecular weight), reaction conditions, and the like used in the specification and claims should be understood to be modified in all instances by the term "about." As used herein, the terms "about" and "approximately" mean within 10% to 15%, preferably within 5% to 10%. Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0207] As used in the context of describing the present invention (particularly in the context of the appended claims), the terms "a," "an," and "the" should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or illustrative phrases (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the otherwise claimed invention. No phrase herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0208] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. The elements of each group may be referred to and claimed individually or in any combination with other elements of the group or other elements found herein. It is anticipated that one or more elements of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is considered to include the modified group, thereby satisfying the recitation requirements of all Markush groups used in the appended claims.

[0209] Specific embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Naturally, variations on these described embodiments will become apparent to those of skill in the art upon reading the following description. The inventors anticipate that such variations will be employed by those of skill in the art as appropriate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described features in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein or clearly contradicted by context.

[0210] Certain embodiments disclosed herein may be further limited in the claims using the phrases "consisting of" or "consisting essentially of." When used in the claims, whether as filed or added by amendment, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and to materials or steps that do not materially affect its basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or explicitly described and enabled herein.

[0211] Additionally, numerous references are made to patents and publications throughout this specification. Each of the above references and publications is herein individually incorporated by reference in its entirety.

[0212] Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

【Request 1】 【Chemical 2-1】 【Chemistry 2-2】 【Chemistry 2-3】 【Chemistry 2-4】 【Chemistry 2-5】 【Chemistry 2-6】 【Chemistry 2-7】 【Chemistry 2-8】 【Chemistry 2-9】 【Chemistry 2-10】 【Chemistry 2-11】 【Chemistry 2-12】 【Chemistry 2-13】 or a pharmaceutically acceptable salt thereof.

2. 10. A composition comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof.

3. 10. A composition comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. A composition for treating cancer or a tumor in a subject in need thereof, the composition comprising a histone deacetylase 7 (HDAC-7) inhibitor.

5. 5. The composition of claim 4, wherein the HDAC-7 inhibitor comprises one or more of the compounds of claim 1 or a pharmaceutically acceptable salt thereof, or the compositions of claims 2 or 3.

6. 5. The composition of claim 4, wherein the HDAC-7 inhibitor comprises one or more of TMP269, trichostatin A, vorinostat, or a pharmaceutically acceptable salt thereof.

7. 5. The composition of claim 4, wherein the HDAC-7 inhibitor comprises one or more siRNAs or pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising one or more of the siRNAs or pharmaceutically acceptable salts thereof.

8. The composition of claim 7 , wherein the siRNA comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4.

9. The composition of claim 4 , wherein the cancer or tumor comprises glioblastoma multiforme.

10. 10. The composition of claim 9, wherein the HDAC-7 inhibitor is administered before, after, or simultaneously with surgical removal of at least a portion of the cancer or tumor.

11. 10. The composition of claim 9, wherein the HDAC-7 inhibitor is administered in an amount sufficient to cause one or more of the following: a decrease in the growth of the cancer or tumor, a decrease in cell proliferation of the cancer or tumor, an increase in apoptosis of one or more cells of the cancer or tumor, an inhibition of metastatic dissemination of the glioblastoma multiforme, an improvement in survival of the subject, a decrease in the ability of cells of the cancer or tumor to form colonies, or a decrease in the ability of cells of the cancer or tumor to migrate.

12. 5. The composition of claim 4, wherein the HDAC-7 inhibitor is administered before, after, or simultaneously with antibody therapy.

13. 5. The composition of claim 4, wherein the HDAC-7 inhibitor is administered before, after, or simultaneously with radiation therapy.

14. A pharmaceutical composition for treating glioblastoma multiforme, comprising: 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 【Chemistry 3-4】 【Chemistry 3-5】 【3-6】 【3-7】 【Chemistry 3-8】 【Chemistry 3-9】 【Chemistry 3-10】 【Chemistry 3-11】 【Chemistry 3-12】 【Chemistry 3-13】 or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition for treating glioblastoma multiforme, comprising one or more of: siRNA or a pharmaceutically acceptable salt thereof; TMP269 or a pharmaceutically acceptable salt thereof; trichostatin A or a pharmaceutically acceptable salt thereof; or vorinostat or a pharmaceutically acceptable salt thereof, wherein the siRNA comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

Images