Pyridine and dihydropyridine compounds and their uses

Abstract

The present disclosure provides compounds of Formula I that are inhibitors of the histone methyltransferase polycomb repressive complex 2 (PRC2) and its subunits, including embryonic ectoderm development (EED) proteins, and are therefore useful for treating diseases treatable by inhibiting dysregulation of PRC2 and EED, such as cancer. Pharmaceutical compositions containing such compounds and methods for preparing such compounds are also provided. In certain embodiments, the subject matter described herein is directed to compounds of Formula I, including all of the subformulas of Formula I, or pharmaceutically acceptable salts or solvates thereof. JPEG2026504602000095.jpg5157

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 483,480, filed February 6, 2023. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support awarded by the National Institutes of Health (NIH) under R01 CA256058 and R42 CA217491. The federal government has certain rights in this invention. [Background technology]

[0003] background Cancer remains one of the deadly threats to human health. In the United States, an estimated 1,958,310 new cancer cases and 609,820 cancer-related deaths will occur in 2023 (ACS Cancer Statistics 2023). Globally, cancer contributed to nearly 10 million deaths in 2020. By 2040, the global cancer burden is projected to increase to 27.5 million new cancer cases and 16.3 million cancer-related deaths due to population growth and aging. Furthermore, cancers often develop multidrug resistance, further complicating cancer treatment. The development of novel therapeutic agents that overcome this chemotherapy resistance is essential. However, developing novel therapeutic agents incurs enormous development costs and lengthy timelines. Reformulation of existing approved or generic drugs for alternative use in cancer treatment is an attractive approach to generate drugs or drug combinations suitable for new medical indications, reducing clinical development costs and shortening timelines. Effective reformulation of drugs for the treatment of cancer and modification of existing drugs to improve their performance as anticancer agents that overcome chemotherapy resistance are highly desirable. The subject matter described herein addresses this need. Summary of the Invention [Means for solving the problem]

[0004] Quick summary In certain embodiments, the subject matter described herein is a compound of Formula I, including all of the subformulas of Formula I: [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein [ka] is independently a single bond or a double bond; n is 0 or 1, p is 0, 1 or 2; -ADX is attached to carbon (a) or (b), A is selected from the group consisting of a bond, —C(O)—, —C(O)—, —O—, —S—, —S(O)— and —S(O)—, or A is selected from the group consisting of a bond, —C(O)—, —C(O)—, —CHO—, —O—, —S—, —S(O)— and —S(O)—; D is an optionally substituted C1-C8 alkylene; [ka] etc., X is [ka] and R 5 and R 6 are each independently selected from the group consisting of optionally substituted C1-C6 alkyl, optionally substituted cycloalkyl; optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl, or R 5 and R6 and the nitrogen to which each is attached, together form a heterocycloalkyl; R 7 , R 8 , R 9 and R 10 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, and C1-C6 alkyl; R 7 , R 8 , R 9 and R 10 Two of these, or R 7 , R 8 , R 9 and R 10 One of the R 5 and R 6 together with the atom to which each is attached form a C3-C7 cycloalkyl or a 4-7 membered heterocycloalkyl; m, o, q, and r are each independently 0 or 1; R 1 H, -CN, -OH, halo, oxo, optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 10 Alkylamino, optionally substituted C1-C 10 Alkoxy, optionally substituted C1-C 10 Cycloalkyl, optionally substituted C1-C 10 selected from the group consisting of heterocycloalkyl and unsubstituted aryl, or R 1 is H, -CN, -OH, halo, oxo, optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 10 Alkylamino, optionally substituted C1-C 10 Alkoxy, optionally substituted C1-C 10 Cycloalkyl, optionally substituted C1-C 10 selected from the group consisting of heterocycloalkyl and optionally substituted aryl; R 2-H, -OH, -NR 2A R 2B , -OR 2A , -SR 2A , optionally substituted C1-C6 alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; R 2A and R 2B are each independently H or C1-C6 alkyl, R 3 is, if present, H or optionally substituted C1-C6 alkyl; R 4 is selected from the group consisting of H, -CN, -OH, halo, -C(O)-(C1-C6 alkyl), -C(O)2-(C1-C6 alkyl), -S(O)-(C1-C6 alkyl), -S-(C1-C6 alkyl), -S(O)2-(C1-C6 alkyl), optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkylamino, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl). The target is.

[0005] In certain embodiments, the subject matter described herein is directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I, including all of the subformulas of Formula I, and a pharmaceutically acceptable carrier.

[0006] In certain embodiments, the subject matter described herein is directed to a method for treating a histone methyltransferase-associated disorder, comprising administering to a subject an effective amount of at least one compound of formula I, including at least one compound of formula I that includes all of the subformulas of formula I.

[0007] In certain embodiments, the subject matter described herein is directed to a method of treating a histone methyltransferase-associated disorder, comprising administering to a subject an effective amount of at least one compound of Formula I, including at least one compound of Formula I that includes all of the subformulas of Formula I.

[0008] Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale. [Brief explanation of the drawings]

[0009] [Figure 1](±)-Nicardipine (hereafter referred to as nicardipine) selectively and potently inhibits chemotherapy-resistant PCa cells. (A) In vitro cytotoxicity of nicardipine in the ARCaPE and C4-2B models (72 hours). (B) Flow cytometry results for cell cycle in C4-2B-TaxR cells treated with the indicated concentrations of nicardipine (48 hours). ***p<0.001 for all pairwise comparisons between the percentage of cells from the control and nicardipine-treated groups in each cell cycle phase. (C) Left: Flow cytometry analysis for Annexin V staining in C4-2B-TaxR cells treated with the indicated concentrations of nicardipine (72 hours). For all pairwise comparisons between control and nicardipine-treated groups, ****p<0.0001; Right: Western blot analysis of apoptotic marker expression in C4-2B and C4-2B-TaxR cells treated with nicardipine (2.1 μM) at the indicated time points. β-actin was used as a loading control. (D) In ​​vitro cytotoxicity of nicardipine in C4-2, PC-3, ARCaPE, and CWR22Rv1 cells (72 h).

[0010] [Figure 2] Nicardipine is a putative EED inhibitor. (A) Left and center: docking poses of nicardipine and EED; right: schematic representation of the two-dimensional ligand-protein interactions of nicardipine in the binding site of human EED protein (PDB ID: 5WUK). Pink arrows indicate hydrogen bonds; blue-red lines indicate salt bridges; red lines represent pi cations. (B) Left: CETSA analysis of EED expression in C4-2B-TaxR cells treated with DMSO or nicardipine (50 μM, 1 h). β-actin was used as a loading control; right: melting temperature curves of EED protein in C4-2B-TaxR cells treated with DMSO or nicardipine.

[0011] [Figure 3-1]Nicardipine targets noncanonical EZH2 survival signaling in chemotherapy-resistant PCa cells. (A) Western blot analysis of p-EZH2, EZH2, EED, and SUZ12 expression in C4-2B and C4-2B-TaxR cells treated with nicardipine (2.1 μM) at the indicated time points. (B) Upper panel: EZH2 expression in C4-2B-TaxR cells treated with DMSO or nicardipine (2.1 μM) in the presence of CHX (50 μg / ml); Lower panel: Calculated half-life of EZH2 protein in C4-2B-TaxR cells treated with DMSO or nicardipine (2.1 μM). (C) Western blot analysis of H3K27 methylation expression in C4-2B and C4-2B-TaxR cells treated with nicardipine (2.1 μM) at the indicated time points. (D) Western blot analysis of p-Stat3, Stat3, SKP2, ABCB1, and survivin expression in C4-2B and C4-2B-TaxR cells treated with nicardipine (2.1 μM) at the indicated time points. (E) Fluorescence microscopy images of Oregon Green 488-paclitaxel cellular uptake at the indicated time points in C4-2B-TaxR cells treated with DMSO or nicardipine (2.1 μM) for 72 hours before incubation with paclitaxel. Scale bar: 50 μm. (F) Schematic of the proposed mechanism of action of nicardipine in chemotherapy-resistant PCa cells. [Figure 3-2] Same as above.

[0012] [Figure 4]RNA-seq analysis of potential target genes of nicardipine in C4-2B-TaxR cells. (A) Left: Heatmap of genes in C4-2B-TaxR cells treated with vehicle control (DMSO) or nicardipine (2.1 μM; 24 h). Right: Top genes affected by nicardipine treatment in C4-2B-TaxR cells. (B) IPA analysis of major gene clusters affected by nicardipine treatment in C4-2B-TaxR cells. (C) GESA of related gene signatures for EED, G1 / S checkpoint, and EZH2 in C4-2B-TaxR cells treated with nicardipine. Positive (red) and negative (blue) enrichment scores indicate enrichment in vehicle- and nicardipine-treated cells, respectively. Normalized enrichment scores (NES) and false discovery rates (FDR) are displayed for each gene set. The Y-axis shows the enrichment score (top) and the ranked list metric (bottom). The X-axis bars represent the individual genes in the gene set shown.

[0013] [Figure 5] Nicardipine monotherapy inhibits skeletal growth of chemotherapy-resistant PCa in male athymic nude mice. (A) Left: Serum PSA levels of C4-2B-TaxR tumor-bearing mice treated with vehicle control (n=4), docetaxel (5 mg / kg, i.p., once a week; n=3), or nicardipine (5 mg / kg, i.p., three times a week; n=5). Right: Two-way ANOVA analysis of PSA levels between different treatment groups. *p<0.05. (B) Left: Mean body weight of C4-2B-TaxR tumor-bearing mice in different treatment groups. Center: Pairwise comparison of body weight between different treatment groups. Right: Percentage body weight change of C4-2B-TaxR tumor-bearing mice in different treatment groups. **p<0.01.

[0014] [Figure 6-1]Nicardipine synergistically enhances the in vivo efficacy of docetaxel against skeletal growth of C4-2 tumors in male athymic nude mice. (A) Left: In vitro cytotoxicity of docetaxel in C4-2 cells at various concentrations of nicardipine (72 h); Right: CompuSyn analysis of the synergy between docetaxel and nicardipine in C4-2 cells. Fa: fraction affected; CI: combination index. (B) Left: Serum PSA levels in C4-2-Luc tumor-bearing mice treated with vehicle control (n = 5), docetaxel (5 mg / kg, i.p., once weekly; n = 5), nicardipine (10 mg / kg, i.p., three times weekly; n = 6), or a combination of docetaxel and nicardipine (n = 5); Right: Two-way ANOVA analysis of PSA levels among different treatment groups. *p<0.05, **p<0.01, ****p<0.0001. (C) Left: average body weight of C4-2-Luc tumor-bearing mice in different treatment groups; Middle: pairwise comparison of body weight between different treatment groups; Right: percentage change in body weight of C4-2-Luc tumor-bearing mice in different treatment groups. **p<0.01. [Figure 6-2] Same as above.

[0015] [Figure 7] 2D and 3D interactions of the (R)- and (S)-enantiomers of nicardipine and the (S)-enantiomer of compound 27 at the binding site of the human EED protein. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description Described herein are compounds of formula I and their uses for treating disorders associated with histone methyltransferase. Also described herein are compounds of formula I that inhibit dysregulation of polycomb repressive complex 2 (PRC2) and its subunits (including but not limited to, embryonic ectoderm development (EED) subunits). Described herein are compounds of formula I and their uses for treating disorders associated with dysregulation of polycomb repressive complex 2 (PRC2) histone methyltransferase. Also described herein are compounds of formula I and their uses for treating cancers associated with histone methyltransferase.

[0017] Described herein are compounds of formula I and their use for treating chemotherapy-resistant cancer. Also described herein are compounds of formula I and their use for treating chemotherapy-resistant cancer associated with histone methyltransferase. Described herein are compounds of formula I and their use for treating chemotherapy-resistant cancer associated with dysregulation of polycomb repressive complex 2 (PRC2) and its subunits (including but not limited to, embryonic ectoderm development (EED) subunits).

[0018] The subject matter of the present disclosure will now be described more fully hereinafter. However, numerous variations and other embodiments of the subject matter of the present disclosure described herein will occur to those skilled in the art to which the subject matter of the present disclosure relates, having the benefit of the teachings presented herein. Therefore, it is to be understood that the subject matter of the present disclosure is not limited to the specific embodiments disclosed, but that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein embraces all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including, but not limited to, defined terms, term usage, described techniques, etc., this application shall control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. I. Overview

[0019] The compounds described herein exhibit the inhibition of histone methyltransferase dysregulation.The compounds described herein particularly exhibit the selective overexpression of various subunits of polycomb repressive complex 2 (PRC2).The compounds described herein are particularly useful for the treatment of cancer, particularly chemotherapy-resistant cancer and other related conditions.

[0020] As mentioned above, multiple biological alterations contribute to chemotherapy resistance. Rare subpopulations of cancer cells with stem cell or neuroendocrine characteristics are considered intrinsically resistant, escaping conventional treatments and leading to recurrence and metastasis. Epithelial-mesenchymal transition (EMT) is a major mechanism by which epithelial cancer cells acquire an invasive phenotype, promote self-renewal, and become chemotherapy-resistant. Common mechanisms of chemotherapy resistance include overexpression of membrane-bound drug efflux pumps (e.g., ATP-binding cassette B1 [ABCB1] or multidrug resistance protein 1 [MDR1], p-glycoprotein) and anti-apoptotic proteins (e.g., survivin). For example, S-phase kinase-associated protein 2 (SKP2), a substrate-recognition component of the SCF (SKP1-CUL1-F-box) E3 ubiquitin-protein ligase complex, enhances stem cell and chemotherapy resistance in prostate cancer (PCa) cells.

[0021] Polycomb repressive complex 2 (PRC2) plays an essential role in transcriptional repression through mono-, di-, and trimethylation of histone H3 at lysine 27 (H3K27). Core subunits of PRC2 include enhancer of zeste homolog 1 or 2 (EZH1 or EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12). EZH2 is a histone methyltransferase (HMT) that acts as a catalytic "writer" subunit of PRC2 in transcriptional repression of genes. Aberrant overexpression and activation of EZH2 are associated with clinical progression and poor prognosis in prostate cancer (PCa) and other cancer types. However, the role of EZH2 signaling in chemoresistance remains largely unknown.

[0022] Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and subunit of Polycomb repressive complex 2 (PRC2), plays a major role in transcriptional repression via PRC2-dependent histone 3 lysine 27 (H3K27) methylation. EZH2 also methylates several nonhistone protein substrates, such as signal transducer and activator of transcription 3 (Stat3). EZH2 acts as a coactivator of the androgen receptor (AR), β-catenin, and nuclear factor kappa B (NF-κB). This noncanonical function may depend on EZH2 phosphorylation at serine 21 [p-EZH2(S21)]. Notably, p-EZH2(S21) is significantly increased in metastatic castration-resistant prostate cancer (mCRPC).

[0023] EZH2 overexpression and mutations, as well as aberrant EZH2 signaling, are associated with advanced stage and poor clinical outcomes in various types of cancer. Numerous EZH2 inhibitors have been developed, most of which target the catalytic SET domain through competition with methyl-donating S-adenosylmethionine (SAM). One of these inhibitors, tazemetostat (EPZ-6438), was approved in 2020 for locally advanced or metastatic epithelioid sarcoma. Unfortunately, EZH2 inhibitors have not demonstrated satisfactory clinical outcomes in other solid tumors. The limited success of current EZH2 inhibitors in clinical settings indicates the insufficiency of blocking the catalytic activity of EZH2 alone and highlights the need for novel PRC2-targeting strategies. An alternative approach is to develop small molecules that specifically bind to the H3K27me3-interacting "aromatic cage" in the EED and allosterically affect EZH2 enzymatic activity, thereby resulting in loss of PRC2 function. Interestingly, some allosteric EED inhibitors can also alter the physical interaction between the EED and other core components of the PRC (mainly EZH2 and SUZ12), destabilizing these proteins. Compared with SAM-competitive EZH2 inhibitors, EED inhibitors can achieve a more general and efficient blockade of PRC2 oncogenic signaling in highly heterogeneous cancer cells. An EED inhibitor (MAK683) developed by Novartis is currently in phase I / II trials in patients with advanced malignancies (NCT02900651). Another EED inhibitor, ORIC-944 from ORIC Pharmaceuticals, recently advanced into Phase I trials in metastatic prostate cancer on June 10, 2022 (NCT05413421).

[0024] Recent studies have revealed an essential role for non-canonical EED-EZH2 signaling in chemotherapy-resistant PCa cells. The small molecule LG1980 is an EED inhibitor with promising anticancer activity in chemotherapy-resistant PCa cells and xenograft models. These results indicate that pharmacological targeting of EED may be a promising strategy to overcome chemotherapy resistance and validate our phenotypic screen as a novel "mechanism-informed phenotypic drug discovery" (MIPDD) platform for discovering effective EED modulators. ARCaP E A recent screen using the / C4-2B-based platform identified several potential inhibitors of chemotherapy-resistant PCa, including nicardipine. Experimental evidence that nicardipine is highly specific and effective against chemotherapy-resistant PCa cells in both cellular and animal models is described herein. The mechanism of action by which nicardipine acted as a putative EED inhibitor and inhibited noncanonical EED-EZH2 signaling in chemotherapy-resistant PCa cells is also described herein. These preclinical studies reveal unexpected functions and mechanisms of action of nicardipine in chemotherapy-resistant cancer cells, which may have important translational relevance.

[0025] Nicardipine is a drug approved for the treatment of hypertension, angina pectoris, and related cerebrovascular diseases (19, 46). As a second-generation dihydropyridine class of calcium channel blockers (CCBs), nicardipine inhibits transmembrane calcium influx into cardiac and smooth muscle without altering serum calcium levels. Given the widespread use of CCBs in the management of cardiovascular disease and the well-recognized role of calcium signaling in cancer progression, there has long been interest in the potential effects of CCBs on clinical outcomes in cancer patients. Since the 1980s, several groups have investigated the potential anticancer activity of nicardipine and other CCBs in preclinical and clinical settings. Nicardipine appeared to be able to enhance the in vitro and in vivo effects of certain chemotherapeutic agents, such as vincristine, carmofur, and nimustine, in experimental models of PCa, esophageal cancer, gastric cancer, glioma, and leukemia. However, it was unclear whether the observed effects of nicardipine in human cancer cells were related to its function as a CCB. In a recent study, Shi et al. found that nicardipine could potentiate the toxic effects of temozolomide and promote apoptosis in glioma stem cells (GSCs), possibly by upregulating mTOR and inhibiting autophagy, a protective response in glioma cells during chemotherapy.

[0026] Only a few studies have been published regarding the clinical benefits of nicardipine in cancer patients, and the results are inconclusive and, in part, contradictory. For example, in three patients with recurrent and chemotherapy-resistant non-Hodgkin's lymphoma, nicardipine was found to increase the efficacy of vinca alkaloids. In contrast, nicardipine failed to improve adriamycin and vinca alkaloids in 17 patients with solid tumors or hematologic malignancies. Although these studies were largely based on observations in very small patient cohorts, they suggest that there is no straightforward strategy for using nicardipine or other CCBs for cancer treatment in the general patient population. Supporting this view, a network meta-analysis and trial sequential analysis of 324,168 participants from randomized trials found no significant difference in the risk of cancer or cancer-related death with CCBs or other individual classes of antihypertensive drugs.

[0027] The translational potential of these putative EED inhibitors was the primary measure for selecting nicardipine and evaluating its anticancer activity in preclinical models of chemotherapy-resistant PCa. Nicardipine, an approved drug for treating chronic cardiovascular disease, exhibits an excellent long-term safety profile in humans. This drug also possesses favorable pharmacokinetics and metabolism, with complete absorption and nonlinear accumulation in the circulation after oral administration. The approved schedule of oral administration (i.e., every 8 hours) and easily measurable effects (e.g., changes in blood pressure) from nicardipine treatment may allow for rapid and convenient adjustments in human trials if any severe adverse effects are observed. These pharmacological and physiological characteristics of nicardipine suggested that this drug may have high potential for further clinical development. In comparison, other approved drugs with high EED binding affinity (i.e., gallopamil, verapamil) have limited clinical potential in human subjects, primarily due to their relatively higher toxicity. For example, the antiarrhythmic drug gallopamil was withdrawn in 2001 because it caused excessive hypotension, bradycardia, or impaired cardiac performance when combined with beta-adrenergic receptor blockers. Another hypertension drug, verapamil, was thought to be a functional inhibitor of ABCB1 (p-glycoprotein), which could resensitize cancer cells to chemotherapy. However, verapamil failed to demonstrate clinical benefit in lung cancer patients, primarily due to its poor pharmacokinetics, high dose-limiting toxicity, and narrow therapeutic window.

[0028] The EED protein is a "reader" component of the PRC2 complex that binds to trimethylated H3K27 (H3K27Me3) through a central pocket formed by its seven WD-40 repeats. The interaction between EED and EZH2 is required for EZH2's epigenetic "writer" function. Therefore, disruption of the EED-EZH2 complex represents an attractive strategy for inhibiting EZH2 function regardless of the mutational status of the EZH2 enzyme. Furthermore, compared with SAM-competitive inhibitors, targeting the EED-EZH2 interaction often results in degradation of the EZH2 protein and the core PRC2 components EED and SUZ12, thereby achieving a general and more effective blockade of EZH2 function in highly heterogeneous, treatment-resistant tumors. Several EED inhibitors have been developed, most of which bind to the central pocket and prevent allosteric activation of PRC2 catalytic activity. Some compounds have shown promising in vivo activity in lymphoma xenografts. However, as of February 2023, only two EED inhibitors, MAK683 from Novartis and ORIC-944 from ORIC Pharmaceuticals, have advanced to Phase I trials in advanced cancers, including metastatic prostate cancer (NCT02900651 and NCT05413421). Thus, there is a need for further investigation of EED inhibitors and the development of novel PRC2 targeting strategies. The subject matter described herein addresses this need.

[0029] The studies described herein provide preclinical evidence supporting the promise of nicardipine as a targeted agent for cancer treatment. The translational potential of this disclosure is twofold. First, the discovery of nicardipine as a putative EED inhibitor and a potent compound for chemotherapy-resistant PCa provides a solid basis for designing biomarker-based, subtype-specific studies to test the clinical efficacy of nicardipine in PCa patients. For example, the expression profiles of key components of the noncanonical EED-EZH2 signaling axis, including EED, p-EZH2(S21), SKP2, ABCB1, and survivin, can be assessed in localized tumors from patients with high-volume, high-risk PCa. Nicardipine could be offered as adjuvant therapy in combination with docetaxel and / or ADT to patients with active noncanonical EED-EZH2 signaling, with the hope of eliminating chemotherapy-resistant PCa cells and enhancing the efficacy of standard treatments. Accordingly, in certain embodiments, the subject matter described herein is directed to a method of treating a disorder associated with polycomb repressive complex 2 (PRC2) histone methyltransferase and its subunits, comprising administering to a subject in need thereof an effective amount of nicardipine or a compound described in U.S. Pat. No. 4,510,310, the entirety of which is incorporated herein by reference.

[0030] Given its excellent pharmacological properties and safety profile as a general antihypertensive agent, nicardipine can be readily tested in human trials and integrated into standard treatment for chemotherapy-resistant PCa. Second, while nicardipine exhibits an excellent clinical safety profile, the drug was originally developed as a calcium channel inhibitor, and its antihypertensive activity is a desirable and not "off-target" feature for cancer treatment. Pharmacologically, nicardipine has a short duration of action after oral administration (mean plasma Tmax = 1 hour). This characteristic is ideal for an antihypertensive drug in terms of facilitating dose titration and therefore being safe for patients with hypertension, but is undesirable for cancer treatment. As described herein, the insufficient properties of nicardipine have provided an opportunity to obtain highly specific EED inhibitors with improved anticancer activity and drug-like properties. The compounds described herein are novel, first-in-class EED inhibitors with distinct chemical structures and pharmacological activities. II. Definition

[0031] As used herein, "Formula I" includes all subformulas of Formula I described herein.

[0032] The term "alkyl," as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing 1 to 12 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. The term "alkyl," unless otherwise indicated, is intended to include both substituted and unsubstituted alkyl, and these groups may be substituted with groups selected from halo (e.g., haloalkyl), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl (including spiroalkyl, e.g., C, C or C spiroalkyl), cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby forming polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, ester, amido, sulfonamido, nitro, or cyano.

[0033] "Cycloalkyl," as used herein alone or as part of another group, refers to a saturated or partially unsaturated cyclic hydrocarbon group containing 3, 4, or 5 to 6, 7, or 8 carbons (these carbons may be replaced in heterocyclic groups, as discussed below), including spirocyclic groups. Representative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may be optionally substituted with additional substituents, such as halo or lower alkyl, as described herein. The term "cycloalkyl" is generic and, unless otherwise specified, is intended to include the heterocyclic groups discussed below.

[0034] "Alkylene," as used herein, unless otherwise specified, refers to a linear saturated divalent hydrocarbon radical of 1 to 6 carbon atoms or a branched saturated divalent hydrocarbon radical of 1 to 8 carbon atoms, such as methylene, ethylene, propylene, 1-methylpropylene, 2-methylpropylene, butylene, pentylene, etc.

[0035] The terms "heterocyclic group" or "heterocycloalkyl," as used herein alone or as part of another group, refer to an aliphatic (e.g., fully saturated or partially saturated heterocycloalkyl) or aromatic (e.g., heteroaryl) monocyclic or bicyclic ring system. Monocyclic ring systems are exemplified by any 5- or 6-membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Five-membered rings have 0 to 2 double bonds, and 6-membered rings have 0 to 3 double bonds. Heterocycloalkyls can be monocyclic or polycyclic, which may be fused, bridged, or spiro, and may contain one or more (e.g., 1 to 3) oxo (=O) or N-oxide (NO) groups. -Representative examples of monocyclic ring systems that are heterocycloalkyl include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazolidine, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidinone ... Bicyclic ring systems include azine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, etc. Bicyclic ring systems are exemplified by any of the monocyclic ring systems described above fused to an aryl group, as defined herein, a cycloalkyl group, as defined herein, or another monocyclic ring system, as defined herein. Representative examples of bicyclic ring systems include, but are not limited to, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxin, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like.These groups may be optionally substituted with a group selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, ester, amido, sulfonamide, nitro, or cyano.

[0036] The term "aryl," as used herein alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings. Representative examples of aryl include azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. Unless otherwise specified, the term "aryl" is intended to include both substituted and unsubstituted aryl, and these groups may be substituted with the same groups described above in connection with alkyl.

[0037] The terms "arylalkyl" or "aralkyl," as used herein alone or as part of another group, refer to an aryl group, as defined herein, attached to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.

[0038] "Heteroaryl" as used herein is as described above in connection with heterocycloalkyl.

[0039] The terms "heteroaralkyl" or "heteroarylalkyl" as used herein alone or as part of another group refer to a heteroaryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0040] "Alkoxy," as used herein alone or as part of another group, refers to an alkyl group, as defined herein (and thus includes substituted forms such as polyalkoxy), attached to the parent molecular moiety through an oxy group -O-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propyloxy, 2-propyloxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0041] "Halo" or "halogen" as used herein refers to any suitable halogen, including F, Cl, Br and I.

[0042] "Cyano," as used herein, refers to the group --CN.

[0043] "Hydroxyl," as used herein, refers to an --OH group.

[0044] "Amino" as used herein means the -NH2 radical.

[0045] "Alkylamino" as used herein alone or as part of another group refers to the radical --NHR, where R is an alkyl group.

[0046] "Disubstituted amino" as used herein alone or as part of another group refers to -NR a R b R stands for radical a and R bare independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, and heterocycloalkyl groups.

[0047] "Ester," as used herein alone or as part of another group, refers to the -C(O)OR radical, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0048] "Amido" as used herein alone or as part of another group means -C(O)NR a R b Refers to the radical, R a and R b is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0049] As used herein, the term "residue" or "residue" of a chemical moiety refers to a chemical moiety attached to a molecule through which at least one covalent bond replaces at least one atom of the original chemical moiety, resulting in a residue of the chemical moiety in that molecule.

[0050] The compounds of the present disclosure may have asymmetric centers. Compounds of the present disclosure containing an asymmetrically substituted atom can be isolated in optically active or racemic forms. Methods for preparing optically active forms, such as by resolution of materials, are well known in the art. All chiral, diastereomeric forms, mixtures of chiral or diastereomeric forms, and racemic forms are within the scope of the present disclosure, unless the specific stereochemistry or isomer is specifically indicated. When a bond is drawn from an optically active center, a "flat" bond ( [ka] ) is a "wedge" bond ( [ka] ) and "dashed" bonds ( [ka] It will be well recognized by those skilled in the art that the terms "(R)" and "(S)" refer to and include both (R) and (S) stereoisomers, each representing either the (R) or (S) stereoisomer. It will also be understood by those skilled in the art that when a compound is expressed as the (R) stereoisomer, the compound may contain the corresponding (S) stereoisomer as an impurity, i.e., less than about 5% by weight, preferably less than 2% by weight of the (S) stereoisomer, and will then be expressed as a mixture of R and S isomers, with the amount of R or S isomer in the mixture being greater than about 5% w / w, preferably greater than 2% w / w.

[0051] As used herein, the term "physiological conditions" refers to the range of conditions of temperature, pH and tonicity (or osmolality) normally encountered within the tissues of the living human body.

[0052] The term "in vitro" refers to an artificial environment and to processes or reactions that are carried out within an artificial environment (e.g., a test tube).

[0053] The term "in vivo" refers to the natural environment (eg, a cell or organism or body) and to processes or reactions that occur within a natural environment.

[0054] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not. For example, "a heterocyclyl group optionally substituted with an alkyl group" means that the alkyl may, but need not, be present; the description includes situations in which the heterocyclyl group is substituted with an alkyl group and situations in which the heterocyclyl group is not substituted with an alkyl group. As used herein, the phrase "optionally substituted" means that substitution is optional, and thus includes both unsubstituted atoms and moieties and substituted atoms and moieties. A "substituted" atom or moiety indicates that any hydrogen on the specified atom or moiety may be replaced with a selection from the indicated substituents, provided that the normal valence of the specified atom or moiety is not exceeded and that the substitution results in a stable compound. For example, when a methyl group is optionally substituted, three hydrogen atoms on the carbon atom may be replaced with substituents. Similarly, double bonds depicted in the structures follow normal valences and may be conjugated or represent aromatic systems.

[0055] "Subject" refers to an animal that is the object of treatment, observation, or experiment. "Animal" includes cold-blooded and warm-blooded vertebrates and invertebrates, such as fish, shellfish, reptiles, and especially mammals. "Mammals" include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and especially humans. In some embodiments, the subject can be a human. In some embodiments, the subject can be a human child and / or human infant, such as a child or infant with a fever. In other embodiments, the subject can be a human adult. A subject "in need thereof" is a subject who has been diagnosed with or is believed to be suffering from one or more disorders associated with dysregulation of polycomb repressive complex 2 (PRC2) histone methyltransferase.

[0056] "Treating" or "treatment" of a disease includes: (1) To prevent disease, i.e., to prevent the development of clinical symptoms of disease in subjects who may be exposed to or susceptible to disease but who have not yet experienced or exhibited symptoms of disease; (2) inhibiting the disease, i.e., arresting or reducing the onset of the disease or its clinical symptoms; or (3) To alleviate the disease, i.e., to cause regression of the disease or its clinical symptoms.

[0057] "Therapeutically effective amount" refers to the amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof that elicits the indicated biological or medical response. For example, when administered to a subject to treat a disease, a therapeutically effective amount of a compound is sufficient to prevent the disease, alleviate or ameliorate its symptoms, or prolong the survival of the treated subject. This response may occur in a tissue, system, animal, or human, and includes alleviating the signs or symptoms of the disease being treated. Determining an effective amount is well within the capabilities of one of ordinary skill in the art in light of the disclosure provided herein. The "therapeutically effective amount" of a compound disclosed herein will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. The expression of a range of values ​​includes all integers within or defining this range, and all subranges defined by integers within that range.

[0058] Unless otherwise clear from the context, the term "about" encompasses values ​​within the standard error of measurement (e.g., SEM) of the stated value or within ±0.5%, 1%, 5%, or 10% variation of the specified value.

[0059] A composition or method that "comprising" or "including" one or more recited elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" a protein may contain the protein alone or in combination with other ingredients.

[0060] The singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "antigen" or "at least one antigen" can include multiple antigens, including mixtures thereof.

[0061] Statistically significant means p≦0.05. III. Compound

[0062] In certain embodiments, compounds are described herein that are a novel class of inhibitors of PCR2 and its subunits. In certain embodiments described herein, the compounds are novel class of EED inhibitors. In certain embodiments described herein, the novel class of EED inhibitors has a unique chemical structure and exhibits high specificity and efficacy for disorders associated with EED dysregulation.

[0063] In certain embodiments, the disorder associated with EED dysregulation is cancer.In certain embodiments, the cancer is chemotherapy-resistant cancer.In certain embodiments described herein, the compound is a new class of EED inhibitor for treating chemotherapy-resistant cancer.In certain embodiments, the compound described herein is a new class of EED inhibitor from treating chemotherapy-resistant prostate cancer (PCa).In certain embodiments, the compound described herein shows high specificity and efficacy for chemotherapy-resistant PCa.

[0064] Described herein is a compound that acts as a novel intervention for chemotherapy-resistant cancer.Described herein is a compound that is effective as a targeted treatment for chemotherapy-resistant cancer.In certain embodiments described herein, chemotherapy-resistant cancer is chemotherapy-resistant prostate cancer (PCa).

[0065] In certain embodiments, the subject matter described herein is a compound of formula I: [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein [ka] is independently a single bond or a double bond; n is 0 or 1, p is 0, 1 or 2; -ADX is attached to carbon (a) or (b), A is selected from the group consisting of a bond, —C(O)—, —C(O)—, —O—, —S—, —S(O)— and —S(O)—, or A is selected from the group consisting of a bond, —C(O)—, —C(O)—, —CHO—, —O—, —S—, —S(O)— and —S(O)—; D is an optionally substituted C1-C8 alkylene; X is [ka] and R 5 and R 6 are each independently selected from the group consisting of optionally substituted C1-C6 alkyl, optionally substituted cycloalkyl; optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl, or R 5 and R 6 and the nitrogen to which each is attached, together form a heterocycloalkyl; R 1 H, -CN, -OH, halo, oxo, optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 10 Alkylamino, optionally substituted C1-C 10 Alkoxy, optionally substituted C1-C 10 Cycloalkyl, optionally substituted C1-C 10 selected from the group consisting of heterocycloalkyl and unsubstituted aryl, or R 1 is H, -CN, -OH, halo, oxo, optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 10 Alkylamino, optionally substituted C1-C 10Alkoxy, optionally substituted C1-C 10 Cycloalkyl, optionally substituted C1-C 10 selected from the group consisting of heterocycloalkyl and optionally substituted aryl; R 2 -H, -OH, -NR 2A R 2B , -OR 2A , -SR 2A , optionally substituted C1-C6 alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; R 2A and R 2B are each independently H or C1-C6 alkyl, R 3 is, if present, H or optionally substituted C1-C6 alkyl; R 4 is selected from the group consisting of H, -CN, -OH, halo, -C(O)-(C1-C6 alkyl), -C(O)2-(C1-C6 alkyl), -S(O)-(C1-C6 alkyl), -S-(C1-C6 alkyl), -S(O)2-(C1-C6 alkyl), optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkylamino, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl).

[0066] In certain embodiments, D is [ka] is selected from the group consisting of:

[0067] In certain embodiments, D is [ka] and R 7 , R 8 , R 9 and R 10 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, and C1-C6 alkyl, or R 7 , R 8 , R 9 and R 10 Two of these, or R 7 , R 8 , R 9 and R 10 One of the R 5 and R 6 together with the atom to which each is attached form a C3-C7 cycloalkyl or a 4-7 membered heterocycloalkyl; m, o, q and r each independently represent 0 or 1.

[0068] In certain embodiments, D is [ka] is selected from the group consisting of:

[0069] In certain embodiments, DX is [ka] is selected from the group consisting of:

[0070] In certain embodiments, the subject matter described herein is a compound of formula 1a: [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0071] In certain embodiments, the subject matter described herein is a compound of formula Ib: [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0072] In certain embodiments, the subject matter described herein is a compound of formula I-1: [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0073] In certain embodiments, the subject matter described herein is a compound of formula Ia-1 or 1b-1: [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0074] In the above embodiment, compounds include those where A is selected from the group consisting of -C(O)- and -C(O)2-.

[0075] In certain embodiments, the compound is 1 H, optionally substituted C1 to C 10 Alkyl, optionally substituted C1-C 10 In certain embodiments, p is 0 and R 1 is phenyl, m-nitrophenyl, cyclobutyl, cyclopentyl, cyclohexyl, methyl, ethyl, propyl, isopropyl, isobutyl, or 3-pentyl.

[0076] In certain embodiments, the compound is selected from the group consisting of: p is 1 and R 1C1 to C are substituted as needed 10 In certain embodiments, p is 1 and R is cycloalkyl, including those having the structure of formula Ia-1. 1 is cyclobutyl, cyclopentyl, or cyclohexyl.

[0077] In certain embodiments, the compound is R 2 -OR 2A and R 2A is C1-C6 alkyl, including those having the structure of formula Ia-1. 2 is CHO3.

[0078] In certain embodiments, the compound is R 3 is H, including those having the structure of formula Ia-1.

[0079] In certain embodiments, the compound is R 4 is methyl, including those having the structure of formula Ia-1.

[0080] In certain embodiments, compounds include those in which A is -C(O)2- and have the structure of formula Ia-1.

[0081] In certain embodiments, the compound is one in which D is [ka] In certain embodiments, D is selected from the group consisting of: [ka] is.

[0082] In certain embodiments, the compound is one in which X is [ka] and includes those having the structure of formula Ia-1.

[0083] In certain embodiments, the compound is R 2 is OCH3 and R 3 is H and R 4 is CH3 and A is -C(O)2-, and has the structure of formula Ia-1.

[0084] In certain embodiments, the compound is R 2 is OCH3 and R 3 is H and R 4 is CH3, A is -C(O)2-, p is 0, and R 1 is optionally substituted aryl, including those having the structure of formula Ia-1.

[0085] In certain embodiments, the compound is R 2 is OCH3 and R 3 is H and R 4 is CH3, A is -C(O)2-, and X is [ka] and includes those having the structure of formula Ia-1.

[0086] In certain embodiments, the compound is R 2 is OCH3 and R 3 is H and R 4 is CH3, A is -C(O)2-, p is 0, and R 1 is optionally substituted aryl, and X is [ka] and includes those having the structure of formula Ia-1.

[0087] In the above embodiment, the compound is one in which X is [ka] Including those that are

[0088] In the above embodiment, the compound is one in which X is [ka] Including those that are

[0089] In the above embodiment, the compound is one in which X is [ka] Including those that are

[0090] In the above embodiment, compounds include those in which D contains one or more asymmetric centers.

[0091] In the above embodiment, the compound is R 2 But, -NR 2A R 2B , -OR 2A and optionally substituted C1-C6 alkyl; R 2A and R 2B are each independently H or C1-C6 alkyl.

[0092] In the above embodiment, the compound is R 2 -OR 2A and R 2A is C1-C6 alkyl.

[0093] In the above embodiment, the compound is R 2 is -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)2, -O(CH2)3CH3, -OCH(CH3)3, -OCH2CH(CH3)2 and -OCH(CH3)CH2CH3.

[0094] In the above embodiment, the compound is R 2 Includes those where is OCH3.

[0095] In the above embodiment, the compound is R 4is selected from the group consisting of -C(O)-(C1-C6 alkyl), -C(O)2-(C1-C6 alkyl) and optionally substituted C1-C6 alkyl.

[0096] In the above embodiment, the compound is R 4 is optionally substituted C1-C6 alkyl.

[0097] In the above embodiment, the compound is R 4 is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl or hexyl.

[0098] In the above embodiment, the compound is R 4 is methyl.

[0099] In the above embodiment, the compound is R 4 is —C(O)2—(C1-C6 alkyl).

[0100] In the above embodiment, the compound is R 4 includes those in which is —C(O)2CH3.

[0101] In the above embodiment, the compound is R 1 C1 to C are substituted as needed 10 Alkyl, optionally substituted C1-C 10 Alkylamino and optionally substituted C1-C 10 Alkoxy is an alkyl group selected from the group consisting of alkoxy.

[0102] In the above embodiment, the compound is R 1 is optionally substituted C1-C8 alkyl.

[0103] In the above embodiment, the compound is R 1is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, heptyl or octyl.

[0104] In the above embodiment, the compound is R 1 is methyl, ethyl, propyl, butyl, isobutyl or sec-butyl.

[0105] In the above embodiment, the compound is R 1 C1 to C are substituted as needed 10 Includes those that are cycloalkyl.

[0106] In the above embodiment, the compound is R 1 but, [ka] The present invention includes those selected from the group consisting of:

[0107] In the above embodiment, the compound is R 1 but, [ka] Including those that are

[0108] In the above embodiment, the compound is R 1 C1 to C are substituted as needed 10 Heterocycloalkyl includes those in which the heteroatom is one or more of S, O, or N.

[0109] In the above embodiment, the compound is R 1 but, [ka] The present invention includes those selected from the group consisting of:

[0110] In the above embodiment, the compound is R 1is unsubstituted aryl.

[0111] In the above embodiment, the compound is R 1 is unsubstituted phenyl.

[0112] In the above embodiment, the compound is R 1 is substituted aryl.

[0113] In the above embodiment, the compound is R 1 is substituted phenyl.

[0114] In the above embodiment, the compound is R 1 is nitro-substituted aryl.

[0115] In the above embodiment, the compound is R 1 is nitro-substituted phenyl.

[0116] In the above embodiment, the compound is R 1 but [ka] Including those that are

[0117] In the above embodiment, the compound is R 1 Includes those where is H.

[0118] In the above embodiment, compounds include those where p is 0.

[0119] In the above embodiment, compounds include those where p is 1.

[0120] In the above embodiments, compounds include those having the structures shown in Table A below. [Table A-1] [Table A-2] [Table A-3]

[0121] In the above embodiment, the compound is one in which D is [ka] The present invention includes compounds having the structure shown in Table A, selected from the group consisting of:

[0122] In the above embodiment, the compound is DX: [ka] The present invention includes compounds having the structure shown in Table A, selected from the group consisting of:

[0123] In the above embodiments, compounds include those shown in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0124] In certain embodiments, the compound comprises a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the compound inhibits one or more proteins of polycomb repressive complex 2 (PRC2).

[0125] In certain embodiments described herein, the compounds of Formula I, or pharmaceutically acceptable salts or solvates thereof, exhibit selectivity for inhibiting the embryonic ectoderm development (EED) subunit of PRC2. IV. Pharmaceutical Compositions

[0126] In certain embodiments, described herein are pharmaceutical compositions comprising at least one compound of Formula I as described herein and a pharmaceutically acceptable excipient or carrier.

[0127] "Pharmaceutically acceptable excipient" refers to a vehicle for containing functionalized cells or acellular extracellular matrices that can be introduced into a subject without significant adverse effects and without adversely affecting the functionalized cells or acellular extracellular matrix. That is, "pharmaceutically acceptable" in the context of a formulation refers to any formulation that is safe and provides for the delivery of an effective amount of at least one functionalized cell or acellular extracellular matrix for use in the methods disclosed herein, suitable for the desired route of administration. Pharmaceutically acceptable carriers or vehicles or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers and the factors involved in their selection can be found in various readily available sources, such as, for example, Remington's Pharmaceutical Sciences, 18th ed., 1990, which is incorporated herein by reference in its entirety for all purposes. Such carriers can be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical application). Such pharmaceutical compositions may be buffered, e.g., the pH is maintained at a particular desired value ranging from pH 4.0 to pH 9.0, depending on the stability of the functionalized cells or acellular extracellular matrix and the route of administration.

[0128] Suitable pharmaceutically acceptable carriers include, for example, sterile water, salt solutions such as saline, glucose, buffer solutions such as phosphate buffer solution or bicarbonate buffer solution, alcohol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (e.g., lactose, amylose, or starch), magnesium stearate, talc, silicic acid, thick paraffin, white paraffin, glycerol, alginate, hyaluronic acid, collagen, flavor oils, fatty acid mono- and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical composition or vaccine may also contain auxiliary substances including, for example, diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffering agents, viscosity-enhancing additives, lubricants, salts for influencing osmotic pressure, buffers, vitamins, colorants, flavorings, aromatic substances, etc., which do not deleteriously react with the functionalized cells or acellular extracellular matrix.

[0129] For liquid preparations, for example, pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, emulsions, or suspensions containing saline and buffered media. Examples of oils include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solid carriers / diluents include, for example, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, or dextrose), cellulose materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0130] If necessary, sustained release or direct release pharmaceutical composition or vaccine can be formulated.This can be done, for example, by using liposome or composition, in which active compound is protected by different degradable coating (for example, by microencapsulation, multiple coating, etc.).Such composition can be formulated for immediate release or slow release.It is also possible to freeze-dry the composition and use the obtained freeze-dried product (for example, for preparing injection products). V. Treatment

[0131] In certain embodiments, the subject matter described herein is directed to a method of treating a disorder, comprising administering to a subject an effective amount of at least one compound of formula I.

[0132] In certain embodiments, the subject matter described herein is directed to a method for treating a disorder associated with polycomb repressive complex 2 (PRC2) histone methyltransferase and its subunits, comprising administering to a subject in need thereof an effective amount of at least one compound described in U.S. Patent No. 4,510,310, herein incorporated by reference in its entirety, or a pharmaceutical salt thereof. In certain aspects, the compound is nicardipine.

[0133] In the embodiments described herein, the disorder is associated with a histone methyltransferase.

[0134] In embodiments described herein, the disorder is associated with polycomb repressive complex 2 (PRC2) histone methyltransferase and its subunits.

[0135] In the embodiments described herein, the disorder is associated with the embryonic ectoderm development (EED) subunit of PRC2.

[0136] In certain embodiments, the subject matter described herein is directed to a method of treating cancer, comprising administering to a subject an effective amount of at least one compound of formula I.

[0137] In certain embodiments, the cancer is associated with dysregulation of histone methyltransferases.

[0138] In the embodiments described herein, the cancer is associated with dysregulation of polycomb repressive complex 2 (PRC2) and its subunits.

[0139] In the embodiments described herein, the cancer is associated with dysregulation of the EED subunit of PRC2.

[0140] In certain embodiments, the method of treatment further comprises administering an additional active agent, such as an active agent useful for the treatment of cancer. The additional compound may be administered simultaneously if necessary. As used herein, the term "simultaneously" means close enough in time to produce a combined effect (i.e., simultaneously may be simultaneous, or may be two or more events that occur within a short period of time before or after each other).

[0141] Although the present subject matter is primarily concerned with the treatment of human subjects, the invention may also be practiced on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, livestock, and horses, for veterinary purposes and for drug screening and drug development purposes. Subjects may be of any age, including infants, juveniles, adolescents, adults, and geriatric subjects.

[0142] As specified above, the present subject matter provides pharmaceutical formulations comprising a compound of Formula I (including pharmaceutically acceptable salts) in a pharmaceutically acceptable carrier for oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, or intravenous and transdermal administration.

[0143] The therapeutically effective dosage of any particular compound may vary somewhat from compound to compound and patient to patient, depending on the patient's condition and the route of delivery. As a general rule, dosages of about 0.1 to about 50 mg / kg are therapeutically effective, with all weights calculated based on the weight of the active compound, including when a salt is used. Due to toxicity concerns at higher levels, intravenous dosages may be limited to lower levels, such as up to about 10 mg / kg, with all weights calculated based on the weight of the active base, including when a salt is used. Dosages of about 10 mg / kg to about 50 mg / kg may be used for oral administration. In some embodiments, dosages of about 0.5 mg / kg to 5 mg / kg may be used for intramuscular injection. In some embodiments, dosages range from 1 μmol / kg to 50 μmol / kg, more preferably 22 μmol / kg and 33 μmol / kg, of the compound for intravenous or oral administration. The duration of treatment may be once daily for a period of two to three weeks, or until the condition is essentially controlled.

[0144] The disclosed subject matter is further described in the following non-limiting examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. [Example]

[0145] material and method I. Cell culture and reagents Human PCa ARCaP stably expressing human EPLIN short hairpin RNA (shRNA) E Cells (ARCaP E -shEPLIN) or control shRNA (ARCaP EC4-2 cells (C4-2B-shCtrl) were established and cultured as previously described. C4-2B and its docetaxel-resistant derivative, C4-2B-TaxR, were cultured according to the procedure described in

[21] , with the modification that C4-2B-TaxR cells were maintained in the presence of 100 nM docetaxel (LC Laboratories, Woburn, MA). The final concentration of docetaxel in the culture medium was reduced to 5 nM before experimental assays (18). C4-2 cells were routinely cultured in T-medium (Life Technologies, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA, USA) and penicillin-streptomycin (Corning Inc, Corning, NY, USA). C4-2-Luc cells were cultured in the same medium as C4-2, with the addition of 400 μg / mL of G418 (Thermo Fisher Scientific, Waltham, MA). Human PC-3 cells were routinely maintained in RPMI 1640 medium (Corning Inc.) supplemented with 10% FBS and penicillin-streptomycin. Human CWR22Rv1 cells were maintained in RPMI 1640 medium containing 2% L-glutamine, 10% FBS, penicillin-streptomycin, 1.5 g / L sodium bicarbonate, 10 mmol / L HEPES, 4.5 g / L glucose, and 10 mmol / L sodium pyruvate. Cells were counted using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MD, USA) according to the manufacturer's instructions. The half-minimum inhibitory concentrations (IC50) of the designated drugs were calculated using the SigmaPlot program (Systat Software Inc., San Jose, CA, USA). Cycloheximide (CHX), dimethyl sulfoxide (DMSO), nicardipine hydrochloride, and propidium iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA). II. Molecular docking and binding energy calculations

[0146] Three-dimensional (3D) structures of the test compounds (nicardipine, losartan, EED226, bromocriptine, and metformin) were retrieved from PubChem and constructed using the Maestro program (Schrodinger, New York, NY, USA) as previously described by the inventors. All test compounds were prepared using Ligprep in Maestro 12.4. The structure of the EED protein (PDB ID: 5WUK) was retrieved from the RCSB Protein Data Bank (22). Using the Maestro protein preparation wizard, the protein structures were prepared in three steps: preprocessing, optimization, and minimization (23). Preprocessing included bond order assignment, hydrogen addition, disulfide generation, and heterostate generation using Epik (24, 25). The optimization process optimized hydrogen bonds using PROPKA (26). The minimization step was performed using the OPLS3e force field (27). A receptor grid box was generated based on five residues (Phe97, Tyr148, Trp364, Tyr365, and Arg367) surrounding the binding site. The size of the receptor grid box was set as the default (20 Å). Ligand-protein docking was performed in ultra-precision (XP) mode using the ligand docking panel. After molecular docking, binding energies were calculated using Prime MM-GBSA (molecular mechanics generalized Born surface area) in the Maestro program. III. Cellular Thermal Shift Assay (CETSA)

[0147] CETSA was performed according to a modified procedure described in 18. C4-2B-TaxR cells were incubated for 1 h in the presence of DMSO or nicardipine (50 μM). Whole cell lysates were incubated at various temperatures using a MyCycler™ thermal cycler system with the gradient option (Bio-Rad Laboratories, Hercules, CA, USA). IV. RNA-seq analysis

[0148] RNA samples were collected in triplicate from C4-2B-TaxR cells treated with DMSO or nicardipine (2.1 μM) for 24 hours. RNA-seq analysis was performed by Omega Bioservices (Norcross, GA, USA). Data were analyzed using Rosalind® (Rosalind, Inc., San Diego, CA, USA), Ingenuity Pathway Analysis (IPA, Qiagen, Germantown, MD, USA), and Gene Set Enrichment Analysis (GSEA, University of California, San Diego and Broad Institute, USA). V. In Vivo Efficacy Studies

[0149] A total of 2 × 10 C4-2B-TaxR or C4-2-Luc cells suspended in 20 μl of PBS were injected into the bilateral tibiae of male athymic nude mice (5 weeks old, Envigo RMS, Inc., Indianapolis, IN). Tumor establishment in the mouse bones was confirmed by detecting serum levels of human prostate-specific antigen (PSA) using an enzyme-linked immunosorbent assay (ELISA) kit (United Biotech, Inc., Mountain View, CA, USA). For the C4-2B-TaxR xenograft model, tumor-bearing mice were randomly divided into three groups and treated with vehicle control (DMSO), docetaxel, or nicardipine via intraperitoneal (ip) injection at the indicated doses and schedules. For the C4-2-Luc xenograft model, an additional group of mice was treated with a combination of docetaxel and nicardipine via ip injection at the indicated doses and schedules. Vehicle control and docetaxel-treated groups in C4-2B-TaxR and C4-2-Luc xenografts were the same as those described in a previous study (18). Body weight was monitored twice weekly. Tumor growth within the tibia was followed by weekly PSA measurements. VI. Statistical analysis

[0150] An unpaired t-test was performed to examine significant differences between the means of any two groups. Two-way analysis of variance (ANOVA) was performed to measure significant differences by comparing the means between groups affected by two independent factors. p<0.05 represents statistical significance. Comparative Example 1 - Nicardipine as a Novel EED Inhibitor I. Phenotypic Screening to Identify Selective Inhibitors of Chemoresistance

[0151] We established a two-stage phenotypic screening system to identify selective inhibitors of chemotherapy-resistant PCa cells. In this platform, the primary screen was based on epithelial protein lost in neoplasia (EPLIN), which acts as a molecular regulator of metastasis and chemotherapy resistance. ARCaP stably expressing EPLIN shRNA was used. E -shEPLIN cells are ARCaP E Compared to -shCtrl cells, these cells are highly resistant to docetaxel and therefore exhibit intrinsic chemoresistance characteristics. E -shEPLIN selectively inhibits ARCaP E We identified small molecule compounds that did not inhibit -shCtrl cells. The primary hits were further validated in a second (orthogonal) assay for their high potency against C4-2B-TaxR cells, a cellular model that exhibits acquired chemotherapy resistance, but not in the docetaxel-sensitive parental C4-2B cells.

[0152] These models can closely mimic the complex biology and high heterogeneity of chemotherapy-resistant PCa. Activation of noncanonical EZH2 signaling represents a novel mechanism of chemotherapy resistance in PCa cells. Specifically, phosphorylation of EZH2 at serine 21 (p-EZH2[S21]) activates the survival signaling pathway consisting of signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP-binding cassette B1 (ABCB1, p-glycoprotein), and survivin, thereby conferring chemotherapy resistance. Furthermore, a small molecule compound, namely, LG1980, effectively disrupts the physical interaction between EED and ZEH2, disassembling PRC2 and promoting the degradation of its core components, thereby inhibiting p-EZH2(S21) and suppressing the expression of its downstream effectors. Importantly, LG1980 demonstrated high specificity and potent potency against the growth of chemotherapy-resistant PCa cells in vitro and in vivo. These results suggest that ARCaP E We demonstrated that α-shEPLIN and C4-2B-TaxR cells can be utilized as a mechanism-informed phenotypic drug discovery (MIPDD) platform for the discovery of novel inhibitors of chemotherapy-resistant PCa, a platform introduced by Moffat et al. Using this platform, several FDA-approved non-oncology drugs were identified that selectively and effectively inhibited the growth of chemotherapy-resistant PCa cells in vitro and in vivo. The antihypertensive drug nicardipine acts as a specific and potent inhibitor of chemotherapy-resistant PCa in preclinical models. Interestingly, mechanistic studies demonstrated that nicardipine can function as an EED inhibitor in chemotherapy-resistant PCa cells, blocking noncanonical EZH2 signaling to confer its anticancer activity. II. Nicardipine exhibits high selectivity and efficacy against chemotherapy-resistant PCa

[0153] ARCaP EUsing the / C4-2B screening platform, the antihypertensive drug nicardipine was identified as a potential inhibitor of chemotherapy-resistant PCa cells. When this compound was tested at a single concentration of 12.3 μM, nicardipine exhibited a high selectivity index (SI) of 8.9, where SI is the selectivity index of ARCaP. E Viability and ARCaP in -shEPLIN cells E Chemoresistance ARCaP, defined as the ratio of percentage inhibition with respect to viability in -shCtrl cells E Only 1.1% of -shEPLIN cells survived after this treatment. To confirm the selectivity of nicardipine in chemotherapy-resistant PCa cells, its half-minimal inhibitory concentration (IC 50 ) to ARCaP E -shEPLIN / ARCaP E Nicardipine was measured in the ARCaP E In -shCtrl cells, IC of 29.1 μM 50 ARCaP E -IC of 0.5 μM in shEPLIN cells 50 Consistently, nicardipine significantly inhibited the proliferation of C4-2B cells (IC 50 >51.2 μM) than C4-2B-TaxR cells (IC 50Nicardipine demonstrated high cytotoxicity at concentrations of 2.0 μM and 4.0 μM (=2.1 μM), with a difference of >24.4-fold (Figure 1A, right). Flow cytometry analysis showed that, compared with vehicle control, nicardipine treatment at 2.0 μM and 4.0 μM significantly induced cell cycle arrest at both the G1-S and G2-M checkpoints, with the accumulation of a partial G1 population representing apoptotic cells (Figure 1B). Nicardipine treatment also significantly induced apoptosis in a dose-dependent manner, as demonstrated by increased surface staining of Annexin V, a marker of apoptosis (Figure 1C, left). Western blotting analysis confirmed that nicardipine induced the cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3 in C4-2B-TaxR cells, but not in C4-2B cells (Figure 1C, right).

[0154] Several commonly used PCa systems, namely ARCaP E We further determined the in vitro cytotoxicity of nicardipine in PCa cells, C4-2, CW22Rv1, and PC-3. These cell lines have distinct genetic backgrounds and exhibit various stages of PCa progression, but are relatively sensitive to docetaxel treatment (Table 2). Interestingly, nicardipine was less potent in these chemosensitive PCa cells, and its IC 50 The values ​​ranged from 15.0 μM to >32.0 μM (FIG. 1D). These results demonstrated that nicardipine has high selectivity and potency against chemotherapy-resistant PCa cells.

[0155] [Table 2] III. Molecular Interaction of Nicardipine with EED

[0156] EED is a "reader" component of the PRC2 complex that binds trimethylated H3K27 (H3K27me3) and activates the HMT function of EZH2. As a classical WD40 repeat (WDR)-containing protein, EED also acts as a scaffolding protein, interacting with EZH2 and SUZ12 to maintain the integrity of the PRC2 complex. Current EED inhibitors, including EED226, A-395, and BR-001, target the histone-binding central pocket, or "aromatic cage," formed by seven WDRs in EED, preventing allosteric activation of PRC2 catalytic activity. Previous studies have identified LG1980 as a novel EED inhibitor that effectively blocks noncanonical EZH2 survival signaling and selectively targets chemotherapy-resistant PCa cells. To determine whether nicardipine exerts its anticancer effects through a similar mechanism of action, we performed the following experiments: (1) Molecular docking analysis demonstrated that (±)-nicardipine binds to the "aromatic cage" of EED and interacts with Phe97 and Tyr365 through pi-cation interactions and with Arg414 through the formation of a salt bridge. Furthermore, hydrogen bonds were formed between nicardipine and Arg414 and Trp364, respectively (Figure 2A). The binding energy between nicardipine and EED was calculated to be -65.25 kcal / mol, suggesting that nicardipine has a higher EED affinity than known EED inhibitors such as EED226 (-49.01 kcal / mol) and MAK683 (-56.15 kcal / mol). Nicardipine also had a higher predicted EED affinity than the other tested calcium channel modulators, losartan and metformin. LG1980, as a positive control, had the highest binding affinity to EED, with a calculated energy of −73.62 kcal / mol (Table 3). (2) A cellular thermal shift assay (CETSA) was performed in viable C4-2B-TaxR cells to determine the intracellular binding of nicardipine to EED protein.Compared with the vehicle control, nicardipine treatment shifted the melting temperature (Tm) of EED protein from 49.8 to 50.4 °C, indicating that nicardipine can specifically bind to and stabilize EED protein in viable cancer cells (Figure 2B). Collectively, these computational and experimental studies demonstrated that nicardipine could be a novel EED inhibitor.

[0157] [Table 3] IV. Nicardipine inhibits non-canonical EZH2-Stat3-SKP2-ABCB1 / survival signaling in chemotherapy-resistant prostate cancer (PCa)

[0158] The integrity and function of the PRC2 complex depend on the presence of EED and SUZ12. EED and p-EZH2(S21) were significantly upregulated in C4-2B-TaxR cells compared with parental C4-2B cells. In contrast, the basal levels of EZH2 and SUZ12 were similar between these two cell lines. Treatment with 2.1 μM nicardipine effectively downregulated EZH2, p-EZH2(S21), EED, and SUZ12 in a time-dependent manner in C4-2B-TaxR cells, but not in parental C4-2B cells (Figure 3A). Nicardipine was found to affect EZH2 protein stability in chemotherapy-resistant PCa cells, which likely results in decreased expression of EZH2 and p-EZH2(S21). CHX chase experiments revealed that the half-life (T) of EZH2 protein in the presence of nicardipine was significantly reduced. 1 / 2 ) was significantly shortened from ≥48 h to 14.8 h (Figure 3B). This result indicates that nicardipine can promote EZH2 degradation via a proteasome-mediated mechanism.

[0159] We further evaluated the effect of nicardipine on the canonical function of EZH2 on histone methylation. As previously found, there was no significant difference in basal H3K27me3 expression between C4-2B and C4-2B-TaxR cells, indicating that canonical EZH2 signaling may not play a dominant role in PCa chemoresistance. Treatment with 2.1 μM nicardipine did not affect H3K27 trimethylation in either C4-2B or C4-2B-TaxR cells over a 72-h period. In contrast, nicardipine significantly inhibited H3K27 monomethylation in a time-dependent manner starting at 24 h in C4-2B-TaxR cells, but not in C4-2B cells, and reduced H3K27 dimethylation after 48 h (Figure 3C). These results indicate that the anticancer activity of nicardipine in chemotherapy-resistant PCa cells may be independent of the canonical HMT function of EZH2.

[0160] We also determined the effect of nicardipine on the expression of core components of the novel non-canonical EZH2 signaling pathway in chemotherapy-resistant PCa cells. p-EZH2 (S21), p-Stat3 (S727), SKP2, ABCB1, and survivin were upregulated in C4-2B-TaxR cells compared with parental C4-2B cells. Nicardipine selectively and effectively suppressed the expression of p-EZH2 (S21), p-Stat3 (S727), SKP2, ABCB1, and survivin in chemotherapy-resistant C4-2B-TaxR cells but not in C4-2B cells (Figure 3D). These results suggest that nicardipine can effectively target the non-canonical EZH2-Stat3-SKP2-ABCB1 / survivin signaling pathway and inhibit the proliferation and survival of chemotherapy-resistant PCa cells. V. Nicardipine Facilitates Cellular Uptake of Chemotherapy Agents in Chemoresistant PCa Cells

[0161] Overexpression of ABCB1 (p-glycoprotein) has been recognized as a central molecular mechanism in multidrug resistance (MDR). Consistently, previous studies have shown that depletion of ABCB1 effectively increased the intracellular abundance of chemotherapeutic agents in chemotherapy-resistant PCa cells. Because nicardipine significantly reduced ABCB1 protein levels in chemotherapy-resistant C4-2B-TaxR cells, we explored its potentiation of the uptake of Oregon Green 488-conjugated paclitaxel. Nicardipine pretreatment rapidly (within 15 min) accumulated fluorescent paclitaxel in C4-2B-TaxR cells (Figure 3E). In comparison, paclitaxel uptake was absent in control cells for up to 30 min. These results indicate that nicardipine-mediated downregulation of ABCB1 may contribute to the enhanced uptake and retention of chemotherapy-resistant PCa cells. Based on these molecular and cellular results, nicardipine can bind to EED and induce the proteolysis of EZH2 in chemotherapy-resistant PCa cells, which can reduce p-EZH2 and suppress the survival signal of Stat3 / SKP2 / ABCB1 / survivin ( Figure 3F ). VI. Nicardipine affects multiple genes related to cell cycle control in chemotherapy-resistant PCa cells.

[0162] To gain an unbiased view of the mechanism of action of nicardipine in chemotherapy-resistant PCa cells, we performed RNA-seq analysis to compare gene expression in C4-2B-TaxR cells treated with vehicle control or nicardipine (2.1 μM for 24 hours). Using an adjusted p-value (p-adj) of <0.05, 336 unique genes were upregulated and 259 unique genes were significantly downregulated after nicardipine treatment (Figure 4A). IPA profiling revealed that the top canonical pathways affected by nicardipine included cell cycle control and DNA damage response, consistent with the known functions of PRC2 in cancer cells (Table 4). Among the major signaling nodes affected by nicardipine treatment, CDKN1A (p21), TP53, and RB1-related genes were activated, whereas E2F-regulated genes were repressed (Figure 4B). GSEA studies confirmed the inhibitory effects of nicardipine on EED and cell cycle-related genes (p = 0.026 and 0.015, respectively). Nicardipine appeared to activate genes repressed by EZH2, although the statistical significance was low (p = 0.05) (Figure 4C). These results support the mechanism of action of nicardipine inducing cell cycle arrest and apoptosis through PRC2-mediated signaling in chemotherapy-resistant PCa cells.

[0163] [Table 4] VII. Nicardipine inhibits skeletal growth of chemotherapy-resistant C4-2B-TaxR tumors

[0164] A notable feature of nicardipine is that, as a single agent, it has demonstrated high selectivity and potency in chemotherapy-resistant PCa cells, respectively. E In -shEPLIN cells, IC of 0.5 μM 50 and in C4-2B-TaxR cells, IC 50= 2.1 μM (Figure 1A). To further characterize the in vivo efficacy of nicardipine on skeletal growth of chemotherapy-resistant cancer, we used C4-2B-TaxR cells, a model that closely mimics the clinical pathology of AR-positive, chemotherapy-resistant, bone-metastatic PCa. Serum levels of human prostate-specific antigen (PSA) were measured in mouse bone as a primary indicator of xenograft growth (Figure 5A). At endpoint, mean PSA levels in each group were 43.47 ± 15.62 ng / ml (control), 36.63 ± 19.75 ng / ml (docetaxel, 5 mg / kg, once weekly), and 28.78 ± 11.60 ng / ml (nicardipine, 5 mg / kg, three times weekly). Intraperitoneal (ip) injection of nicardipine significantly inhibited the growth of C4-2B-TaxR tumors in mouse tibias compared with vehicle control (p = 0.020) or docetaxel (p = 0.039). In contrast, docetaxel treatment did not significantly affect the in vivo growth of PCa cells (p = 0.741). Nicardipine treatment was not associated with any obvious in vivo toxicity or reduced mouse body weight (Figure 5B). These results demonstrated that nicardipine, as a monotherapy, can effectively suppress the in vivo growth of chemotherapy-resistant PCa xenografts in mouse bones with a favorable safety profile. VIII. Nicardipine enhances the in vivo efficacy of docetaxel and inhibits skeletal growth of C4-2 xenografts

[0165] IC in the low micromolar range (2.1 μM) in C4-2B-TaxR cells 50 Compared with C4-2 cells (IC), which showed a typical phenotype of docetaxel-responsive PCa, nicardipine 50 = 15.0 μM) had relatively weak cytotoxicity (IC 50= 0.72 μM; Figure 6A). Interestingly, when C4-2 cells were treated with the combination of nicardipine and docetaxel, the two drugs demonstrated a synergistic inhibitory effect on the in vitro proliferation of C4-2 cells, as reflected by a combination index (CI) lower than 1.0 in isobologram analysis using the CompuSyn program (Figure 6A, right; Table 5). These in vitro results suggested that nicardipine may be effective in enhancing the anticancer effect of docetaxel chemotherapy. To test this hypothesis, C4-2 tumors were inoculated into the tibia of male athymic nude mice. Tumor-bearing mice were treated with vehicle control, docetaxel, nicardipine, and the combination of docetaxel and nicardipine, respectively. At endpoint, the mean PSA levels in each group were 79.17±17.92 ng / ml (control), 49.04±14.92 ng / ml (docetaxel, 5 mg / kg, once weekly), 42.80±9.29 ng / ml (nicardipine, 10 mg / kg, three times weekly), and 23.23±6.27 ng / ml (docetaxel and nicardipine). Compared with vehicle control, docetaxel moderately inhibited the in vivo growth of C4-2 tumors (p=0.048), and nicardipine monotherapy was ineffective in inhibiting tumor growth compared with vehicle control (p=0.090) or docetaxel (p=0.706). However, the combination of nicardipine and docetaxel significantly reduced serum PSA levels compared with treatment with vehicle control (p<0.0001), docetaxel (p=0.0025), or nicardipine (p=0.0020) (Figure 6B). Docetaxel treatment significantly reduced body weight compared with vehicle control, while the combination of nicardipine and docetaxel increased body weight compared with the docetaxel group (Figure 6C). These results indicated that nicardipine, as an adjuvant, may be effective in increasing the in vivo efficacy of docetaxel against skeletal growth of C4-2 tumors.

[0166] [Table 5]

[0167] Although nicardipine exhibits an excellent clinical safety profile, the drug was originally developed as a calcium channel inhibitor, and its antihypertensive activity is a desirable and not "off-target" feature for cancer treatment. Pharmacologically, nicardipine has a short duration of action after an oral dose (mean plasma Tmax = 1 hour). This characteristic is ideal for an antihypertensive drug, allowing for easy titration and therefore safety in patients with hypertension, but is undesirable for cancer treatment. Nicardipine's insufficient properties underscore the need for nicardipine analogs to provide highly specific EED inhibitors with improved anticancer activity and drug-like properties. Example 2 Design and synthesis of nicardipine analogues I. Design of Nicardipine Analogues

[0168] Several potential leads with enhanced cytotoxicity were identified in chemotherapy-resistant PCa cells and improved pharmacological properties. Specifically, the (R)-enantiomer of nicardipine (R)-1 had a higher predicted EED binding affinity and was more cytotoxic than the (S)-enantiomer or (±)-nicardipine in chemotherapy-resistant PCa cells. Specifically, in the same set of cytotoxicity assays, the IC of (±)-nicardipine was significantly higher than that of the (S)-enantiomer. 50 / Analog IC 50The ratio of change in anticancer potency (ROC) of (R)-nicardipine or (S)-nicardipine, calculated by [Ratio of Characteristic Curve (ROC) = 1.0, indicating that the analog has higher cytotoxicity in chemotherapy-resistant PCa cells, was 1.53 and 1.16, respectively. This is significant because the (R)-enantiomer had significantly lower antihypertensive activity than the (S)-enantiomer. Therefore, cancer treatment with single-enantiomer compounds can reduce the "off-target" situation caused by racemic nicardipine. (2) The synthetic hexamethylene analog (±)-2 demonstrated excellent cytotoxic activity (ROC = 2.08). Modeling of (R)-2 also demonstrated excellent EED binding affinity. Structural modeling predicted that the hexamethylene linker of 2 would adopt a chiral coil state upon binding to EED, with two (-) synclinal and one (+) anticlinal conformations, and similar pi-cation interactions with the aromatic cage residues Phe97, Tyr148, and Tyr365. Modeling of the enantiomers of 2 revealed distinct ester hydrogen-bonding patterns, favoring the (R)-2 conformation for superior EED binding, with one ester H-bond to the main-chain NH of Trp364 and an ester carbonyl H-bond proximal to the amine to Arg414. (3) Of the family of six alkyl and cycloalkyl analogs prepared and tested to date, the novel 4-cyclopentyl analog (±)-3 was the most promising, exhibiting enhanced cytotoxicity (ROC = 1.47) (Table 6 below).

[0169] Compounds less cytotoxic than nicardipine also informed the pharmacore modeling of EED inhibitors. For example, the pyridine metabolite of nicardipine showed an ROC of 0.89. The 4-phenyl analog was slightly less cytotoxic than the 4-(m-nitrophenyl) compound, but removal of the tertiary benzyl group in the N,N-dimethylamine analog (±)-5 significantly reduced cytotoxic activity (ROC = 0.05), demonstrating the importance of the tertiary benzylic amine. Modeling revealed that the small N,N-dimethylammonium cation binds weaker and more deeply in the central pocket, such that the tertiary ammonium cation no longer resides within the Phe97-Tyr148-Tyr365 aromatic cage. Small linear alkyl analogs, such as the 4-methyl-substituted (±)-6, were less cytotoxic (ROC = 0.65) than branched or cyclic alkyl analogs, i.e., (±)-3 (ROC = 1.47). II. General synthetic scheme [ka] General information

[0170] Materials and Reagents: All chemicals were purchased from suppliers such as Sigma Aldrich, Oakwood Chemical, TCI Chemicals, Ambeed, Synthonix and Combi-blocks.

[0171] Experimental Protocol: All reactions were carried out in oven-dried or flame-dried, argon-filled glassware unless otherwise specified. Argon was passed through a drying column containing a desiccant. Reaction vials and stir bars were treated with concentrated nitric acid, washed thoroughly with water and acetone, and dried in an oven before use. Thin-layer chromatography (TLC) was performed on pre-coated aluminum-backed plates (silica gel 60F254) purchased from Whatman and visualized with p-anisaldehyde staining and a UV lamp. Flash column chromatography was performed on silica gel 60 (230-400 mesh ASTM) from Silicycle.

[0172] Analysis: Proton and carbon NMR spectra were recorded on an INOVA 400 (400 MHz), BRUKER 400 (400 MHz), INOVA-500 (500 MHz), INOVA-600 (600 MHz) or BRUKER 600 (600 MHz) instrument equipped with a cryogen probe. NMR spectra were recorded in solutions of deuterated chloroform (CDCl3) and residual chloroform ( 1 For H NMR, it is 7.26 ppm, 13 For C NMR, C NMR was recorded using 77.23 ppm as the internal standard and reported in parts per million (ppm). Abbreviations for signal couplings are as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; ddd, doublet of doublet of doublet; dt, doublet of triplet; m, multiplet. Mass spectra (high-resolution ESI and APCI) were recorded on a Thermo LTQ (linear quadrupole ion trap) FTMS (Fourier transform mass spectrometer) based on ion cyclotron resonance mass spectrometry (ICR-MS).

[0173] Representative Experimental Protocol

[0174] Example A: Compound 1, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate: [ka]

[0175] Methyl (E)-3-aminobut-2-enoate (1 equivalent) and 2-(benzyl(methyl)amino)ethyl 3-oxobutanoate (1 equivalent) were added to a 4 mL or 8 mL reaction vial and purged under argon, after which benzaldehyde (1 equivalent) was added via syringe. The reaction mixture was dissolved in isopropanol (1.4 M) and purged with argon for 10–15 minutes. The reaction vial was capped with a heavy-duty stopper and refluxed overnight in a sand bath or heating block. After cooling to room temperature, the mixture was transferred to a round-bottom flask and concentrated under vacuum to a thick yellow oil. The crude mixture (wet load) was purified by flash column chromatography on silica gel (30 cm height, 1 inch column diameter) using a 60:40 EtOAc / hexane mixture (approximately 500–700 mL). Fractions were visualized by TLC and p-anisaldehyde staining, appropriately combined, and concentrated under vacuum and high vacuum overnight. The pure product is a yellow residue. Yield: 32%, >95% purity ( 1 (Estimated by H NMR).

[0176] 1 H NMR (400 MHz, CDCl3) δ 7.33 - 7.22 (m, 6H), 7.18 (tq, J = 8.2, 1.2 Hz, 2H), 7.14 - 7.09 (m, 1H), 5.70 (s, 1H), 5.02 (s, 1H), 4.19 (t, J = 6.0 Hz, 2H), 3.63 (s, 3H), 3.52 (s, 2H), 2.66 (td, J = 6.1, 3.3 Hz, 2H), 2.33 (d, J = 1.6 Hz, 6H), 2.21 (s, 3H).

[0177] 13 C NMR (101 MHz, CDCl3) δ 168.16, 167.63, 147.54, 144.42, 144.26, 138.95, 129.11, 128.35, 128.10, 127.89, 127.13, 126.30, 104.06, 62.59, 61.75, 55.67, 51.11, 42.37, 39.42, 19.81, 19.73.

[0178] HRMS (APCI): m / z C 26 H 30 N2O4 + [M+H] + Calculated value: 435.2278, measured value: 435.2276.

[0179] Example B: Compound 3, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,4,6-trimethyl-1,4-dihydropyridine-3,5-dicarboxylate: [ka]

[0180] In a 4 mL reaction vial, a mixture of methyl (E)-3-aminobut-2-enoate (1 equiv.), 2-(benzyl(methyl)amino)ethyl 3-oxobutanoate (1 equiv.), acetaldehyde (2 equiv.), and tetrabutylammonium hydrogen sulfate (0.12 equiv.) was dissolved in ethylene glycol (2.4 M). The mixture was purged with argon for 10 min, then capped with a heavy-duty stopper and stirred at 80–85 °C overnight. The reaction mixture was cooled to room temperature and then diluted with EtOAc. The solution was then poured onto brine (approximately 3–5 mL) and extracted with EtOAc (×5). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude mixture was subjected to flash column chromatography (silica gel) with 50:50 hexanes / EtOAc (wet load). The appropriate fractions were collected and concentrated to give a yellow oily residue (17% yield).

[0181] 1 H NMR (400 MHz, CDCl3) δ 7.36 - 7.24 (m, 4H), 7.27 - 7.19 (m, 1H), 5.72 (s, 1H), 4.35 - 4.19 (m, 2H), 3.84 (q, J = 6.5 Hz, 1H), 3.68 (s, 3H), 3.57 (s, 2H), 2.73 (t, J = 6.0 Hz, 2H), 2.30 - 2.20 (m, 9H), 0.96 (d, J = 6.5 Hz, 3H).

[0182] 13 C NMR (101 MHz, CDCl3) δ 168.30, 167.75, 144.77, 144.73, 139.05, 128.99, 128.92, 128.29, 128.25, 127.17, 126.99, 104.48, 104.41, 62.57, 62.51, 61.66, 55.72, 54.47, 50.99, 42.43, 28.43, 22.86, 22.36, 19.54, 19.42.

[0183] HRMS (APCI): m / z C 21 H 28 N2O4 + [M+H] + Calculated value: 373.2122, measured value: 373.2119.

[0184] Examples C and D: Compounds 5 and 6 were prepared from the common precursor 5-(methoxycarbonyl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3-carboxylic acid: [ka]

[0185] In the first step, following the protocol for preparing compound (±)-1, a mixture of methyl (E)-3-aminobut-2-enoate (1 equiv.), 2-cyanoethyl 3-oxobutanoate (1 equiv.), and benzaldehyde (1.2 equiv.) in isopropanol (4 mL, 1.6 M) was heated to reflux overnight under an argon atmosphere. The crude mixture was dry-loaded onto Celite and then subjected to silica gel flash column chromatography with 60:40 hexanes / EtOAc. The cyanoester product was eluted using a gradient of 60:40 to 50:50 to 40:60 hexanes / EtOAc and concentrated under vacuum and high pressure overnight to afford 3-(2-cyanoethyl) 5-methyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate as a thick yellow oil. Yield: 73%.

[0186] 1 H NMR (400 MHz, CDCl3) δ 7.32 - 7.18 (m, 4H), 7.19 - 7.10 (m, 1H), 5.68 (s, 1H), 4.97 (s, 1H), 4.33 - 4.16 (m, 2H), 3.64 (s, 3H), 2.66 - 2.56 (m, 2H), 2.35 (d, J = 7.3 Hz, 6H).

[0187] 3-(2-Cyanoethyl) 5-methyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (4.7 mmol, 1 equiv.) was dissolved in acetone (1.87 mL per mmol of cyanoethyl ester) under air, and then a solution of 1 M NaOH (3.74 mL per mmol of cyanoethyl ester) was poured into the reaction flask (smoky vapors formed). The reaction was stirred at 28–30°C under an argon atmosphere for 1 hour. The clear yellow mixture was diluted with 20 mL of DI water and then transferred to a separatory funnel. DCM was used to wash the reaction flask and then poured into the separatory funnel, whereupon the organic layer became cloudy and light yellow after mixing. The aqueous layer was washed three times with DCM and collected in an Erlenmeyer flask, which was then slowly acidified by adding concentrated HCl dropwise until the pH reached 1–2 (closely monitored by pH paper). At this point, a white solid precipitated, and the mixture was placed on ice and stirred for 2 hours. The solid was filtered through a fritted funnel, air-dried, collected, and dried under high vacuum overnight to give 5-(methoxycarbonyl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3-carboxylic acid as a white powder. Yield: 88%.

[0188] 1 H NMR (400 MHz, DMSO) δ 11.67 (s, 1H), 8.75 (s, 1H), 7.15 (tt, J = 20.9, 7.3 Hz, 5H), 4.88 (s, 1H), 3.54 (s, 3H), 2.25 (d, J = 3.1 Hz, 6H).

[0189] Example C: Compound 5, 3-(2-(dimethylamino)ethyl) 5-methyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate: [ka]

[0190] Compound 5 was prepared using 2-(dimethylamino)ethan-1-ol following the same protocol as the synthesis of compound 6. Yield: 11%, yellow oil.

[0191] 1 H NMR (400 MHz, CDCl3) δ 7.30 - 7.08 (m, 4H), 5.69 (s, 1H), 5.00 (s, 1H), 4.21 - 4.07 (m, 2H), 3.64 (s, 3H), 2.62 - 2.47 (m, 2H), 2.33 (d, J = 5.1 Hz, 6H), 2.24 (s, 6H), 1.30 - 1.22 (m, 1H).

[0192] 13 C NMR (101 MHz, CDCl3) δ 168.04, 167.49, 147.42, 144.34, 144.07, 128.00, 127.79, 126.21, 103.99, 103.87, 61.87, 57.70, 51.01, 45.75, 39.35, 19.69, 19.64.

[0193] HRMS (APCI): m / z C 20 H 26 N2O4 + [M+H] + Calculated value: 359.1965, measured value: 359.1963.

[0194] Example D: Compound 6, 3-(6-(benzyl(methyl)amino)hexyl) 5-methyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate: [ka]

[0195] 5-(Methoxycarbonyl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3-carboxylic acid (0.7 mmol, 1 equiv.) was dissolved in dry DMF (1.2 mL, 0.6 M) and DCM (12 mL) under an argon atmosphere, and the reaction was cooled to 0 °C in an ice bath. SOCl (0.98 mmol, 1.4 equiv.) was then added via microsyringe, and the white suspension turned into a clear, yellow mixture over time. The reaction mixture was stirred on ice for 2.5 h, and then a solution of 6-(benzyl(methyl)amino)hexan-1-ol (1 equiv.) in DCM (2 mL) was added to the reaction flask. The reaction was stirred and warmed to room temperature overnight, turning into a clear, orange mixture. After stirring was stopped, the reaction was diluted with DCM and washed with saturated NaHCO and brine. The organic layer was dried over anhydrous Na2SO4 and then concentrated in vacuo to give a crude dark orange oil. The reaction mixture was purified by flash column chromatography with 95:5 DCM / MeOH (wet load). Fractions were collected (a small amount when the yellow band began to elute) and visualized by UV lamp. The product was identified as fluorescent under UV light. Yield: 26%, yellow oil.

[0196] 1 H NMR (400 MHz, CDCl3) δ 7.35 - 7.16 (m, 9H), 7.16 - 7.04 (m, 1H), 5.62 (s, 1H), 4.98 (s, 1H), 4.02 (ddt, J = 33.2, 10.8, 6.6 Hz, 2H), 3.64 (s, 3H), 3.51 (s, 2H), 2.33 (d, J = 5.8 Hz, 8H), 2.20 (s, 3H), 1.63 - 1.44 (m, 3H), 1.29 - 1.24 (m, 5H).

[0197] 13C NMR (101 MHz, CDCl3) δ 167.87, 167.43, 147.37, 143.84, 143.80, 129.00, 128.07, 127.76, 127.60, 126.88, 125.97, 103.96, 103.80, 63.70, 61.98, 57.11, 50.80, 39.24, 28.50, 26.88, 25.77, 24.60, 19.50, 19.48.

[0198] HRMS (APCI): m / z C 30 H 39 N2O4 + [M+H] + Calculated value: 491.2904, measured value: 491.2903.

[0199] Example E: Compound 12, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,6-dimethylpyridine-3,5-dicarboxylate [ka]

[0200] Compound 12 was synthesized following a similar protocol to that for substrate (±)-1, using formaldehyde instead of benzaldehyde. The crude mixture was an orange oil, which was subjected to flash column chromatography (silica gel) with 50:50 hexane / EtOAc (dry load). The appropriate fractions were collected and concentrated to give a yellow powder (65% yield). The 1,4-dihydropyridine product 4 was obtained as follows: 1 Oxidative decomposition to the pyridine product 12 was confirmed by 1 H NMR, which appeared as spots in the NMR sample.

[0201] 1H NMR (400 MHz, CDCl3) δ 6.90 - 6.70 (m, 5H), 4.76 (s, 1H), 3.76 (t, J = 6.0 Hz, 2H), 3.20 (s, 3H), 3.09 (s, 2H), 2.80 (t, J = 1.1 Hz, 2H), 2.22 (t, J = 5.9 Hz, 2H), 1.80 (s, 3H), 1.71 (d, J = 5.2 Hz, 6H).

[0202] The 1,4-dihydropyridine intermediate 4 (130 mg, 0.36 mmol) was transferred to a 4 mL reaction vial and 2 M HNO3 (1.5 mL) was added. The reaction mixture was stirred under air at 55–60 °C for 20 min, during which time the yellow mixture turned into a clear solution. The reaction was neutralized with saturated NaHCO3 and extracted with DCM. The combined organic layers were washed with DI water and brine and dried over anhydrous Na2SO4. The mixture was purified on a pipette column containing silica gel using 1:1 hexane / EtOAc as the eluent. Fractions (1–2 mL each) were collected and concentrated in vacuo. Note: The oxidized pyridine product 12 is only UV-active and cannot be visualized by p-anisaldehyde staining. The product is a white residue. Yield: 36%.

[0203] 1 H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.34 - 7.18 (m, 6H), 4.44 (t, J = 5.9 Hz, 2H), 3.91 (s, 3H), 3.59 (s, 2H), 2.84 (d, J = 8.9 Hz, 6H), 2.79 (t, J = 5.8 Hz, 2H), 2.33 (s, 3H).

[0204] 13C NMR (101 MHz, CDCl3) δ 166.28, 165.82, 162.62, 162.56, 141.09, 138.79, 128.88, 128.29, 127.11, 122.89, 122.62, 63.05, 62.66, 55.33, 52.33, 42.62, 29.73, 25.05, 24.99.

[0205] HRMS (APCI): m / z C 20 H 25 N2O4 + [M+H] + Calculated value: 357.1809, measured value: 357.1807.

[0206] Example F: Compound 13, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,4,6-trimethylpyridine-3,5-dicarboxylate: [ka]

[0207] Compound (±)-3 (26 mg, 0.07 mmol) was transferred to a 4 mL reaction vial, and 2 M HNO (0.2 mL) was added. The reaction mixture was stirred under air at 55–60° C. overnight. The reaction mixture was neutralized with saturated NaHCO and extracted with DCM. The combined organic layers were washed with DI water and brine and dried over anhydrous NaSO. The mixture was purified using a pipette column containing silica gel packed with 1:1 hexane / EtOAc (wet loaded). Fractions (1–2 mL each) were collected and concentrated in vacuo. The product was a pale yellow residue. Yield: 17%.

[0208] 1H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 1.2 Hz, 2H), 7.25 - 7.17 (m, 3H), 4.46 (t, J = 5.7 Hz, 2H), 3.91 (s, 3H), 3.55 (s, 2H), 2.75 (d, J = 6.0 Hz, 2H), 2.48 (d, J = 2.5 Hz, 6H), 2.25 (s, 3H), 2.22 (s, 3H).

[0209] Example G: Compound 15, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,6-dimethyl-4-phenylpyridine-3,5-dicarboxylate: [ka]

[0210] Compound (±)-1 (58 mg, 0.13 mmol) was transferred to a 4 mL reaction vial and 2 M HNO3 (0.8 mL) was added. The reaction mixture was stirred under air at 55-60 °C, turning from a cloudy yellow mixture to a slightly more opaque solution. The reaction was neutralized with saturated NaHCO3 and extracted with DCM. The combined organic layers were washed with DI water and brine and dried over anhydrous Na2SO4. The mixture was purified by flash column chromatography using silica gel packed with 5:1 hexane / EtOAc (wet loaded). The compound was eluted using a gradient of 5:1 to 3:1 hexane / EtOAc as the eluent. Fractions were collected and concentrated in vacuo. The product is a yellow residue. Yield: 32%.

[0211] 1 H NMR (400 MHz, CDCl3) δ 7.36 - 7.27 (m, 5H), 7.25 - 7.19 (m, 5H), 4.05 (t, J = 6.1 Hz, 2H), 3.52 (s, 3H), 3.39 (s, 2H), 2.60 (d, J = 5.0 Hz, 6H), 2.29 (t, J = 6.1 Hz, 2H), 2.09 (s, 3H).

[0212] 13 C NMR (101 MHz, CDCl3) δ 168.47, 167.96, 155.74, 155.54, 146.16, 138.49, 136.54, 128.95, 128.54, 128.26, 128.24, 127.89, 127.12, 126.77, 126.74, 63.11, 62.36, 54.79, 52.22, 42.23, 23.06, 23.02.

[0213] HRMS (APCI): m / z C 26 H 28 N2O4 + [M+H] + Calculated value: 433.2127, measured value: 433.2120.

[0214] Examples HM: Compounds 2, 7, 8, 9, 10 and 11:

[0215] Compounds 2, 7, 8, 9, 10 and 11 were synthesized by the same method as described in Example A above, using instead the following aldehydes:

[0216] Compound 2, cyclohexanecarbaldehyde

[0217] Compound 7, cyclopropanecarbaldehyde

[0218] Compound 8, cyclopentanecarbaldehyde

[0219] Compound 9, isobutyraldehyde

[0220] Compound 10, butyraldehyde

[0221] Compound 11, 3-Methylbutanaldehyde

[0222] Compound 30, 2-ethylbutanal

[0223] Compound 31, cyclobutanecarbaldehyde

[0224] Compound 32, 2-cyclopentylacetaldehyde

[0225] Example N: Compound 16:

[0226] Compound 16 was synthesized starting from dihydropyridine compound 7 according to Examples F and G above.

[0227] Example O: Compound 27: [ka]

[0228] To a 4 mL oven-dried reaction vial containing a stir bar, 5-(methoxycarbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid (100 mg, 0.30 mmol, 1 equiv.), 6-(benzyl(methyl)amino)hexan-1-ol (73 mg, 0.33 mmol, 1.1 equiv.), and N-methylimidazole (NMI, 74 μL, 0.93 mmol, 3.1 equiv.) were added under air. The mixture was then purged with argon and dissolved in acetonitrile (MeCN, 2 mL, 20 mL / g limit reagent), followed by the addition of N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH, 101 mg, 0.36 mmol, 1.2 equiv.). The reaction mixture was placed under argon, sealed with a heavy-duty screw cap and electrical tape, placed on a heating block, and stirred at 40 °C for 16 hours. The reaction mixture rapidly became a clear, yellow homogeneous material over time. After cooling to room temperature, deionized water was added to quench the reaction, and the organic material was extracted three times with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation in vacuo. The crude mixture was purified by column chromatography on silica gel using a gradient elution with hexane / acetone mixtures from 70:30 to 65:35 to 60:40 to 50:50. Appropriate fractions were collected and concentrated by rotary evaporation (40 °C, trapped solvent removed) to give the pure product as a viscous, yellow oil (139 mg, 84% yield).

[0229] 1H NMR (400 MHz, CDCl3) δ 8.10 (t, J = 2.0 Hz, 1H), 7.99 (ddd, J = 8.2, 2.3, 1.1 Hz, 1H), 7.63 (dt, J = 7.9, 1.3 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 7.32 - 7.21 (m, 5H), 5.77 (s, 1H), 5.08 (s, 1H), 4.06 (dt, J = 10.8, 6.7 Hz, 1H), 3.98 (dt, J = 10.9, 6.7 Hz, 1H), 3.64 (s, 3H), 3.49 (s, 2H), 2.37 (s, 3H), 2.36 (s, 3H), 2.35 (m, 2H), 2.19 (s, 3H), 1.58 (width quintet, J = 6.7 Hz, 2H), 1.48 (width quintet, J = 7.4 Hz, 2H), 1.30 - 1.19 (m, 4H).

[0230] 13 C NMR (101 MHz, CDCl3) δ 167.7, 167.2, 149.9, 148.4, 145.0, 144.9, 139.2, 134.4, 129.2, 128.8, 128.3, 127.0, 123.0, 121.5, 103.5, 103.3, 64.3, 62.4, 57.5, 51.3, 42.3, 39.9, 28.8, 27.4, 27.2, 26.1, 19.79, 19.76.

[0231] HRMS (APCI): m / z C 30 H 38 N3O6 + [M+H] + Calculated value of についての: 536.27551, measured value 536.27548. (Example 3) Biotechnology I. In vitro cytotoxicity of anti-がん compounds

[0232] The human prostate cancer cell line C4-2B and its docetaxel-resistant derivative C4-2B-TaxR were cultured according to the procedure described in Zhu et al., Mol. Cancer Ther. 2013, with modifications such that C4-2B-TaxR cells were maintained in the presence of 100 nM docetaxel (LC Laboratories, Woburn, MA). The final concentration of docetaxel in the culture medium was reduced to 5 nM prior to the experimental assay (Li et al., Theranostics 2021). Approximately 1,500–3,000 cancer cells were plated in 100 μL of growth medium in a 96-well flat-bottom microtiter plate. Cells were incubated overnight and allowed to recover. Compounds to be tested were added to the cells in sextuplicate at a range of concentrations using dimethyl sulfoxide (DMSO) as a vehicle. The cells were incubated at 37°C, 5% CO2, and 100% humidity. A row of control wells on the same plate contained cells and all substances except the compound (or DMSO). The plates were incubated for 3 days to allow sufficient time for cell replication and compound-induced cell death. Cells were counted using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MD, USA) according to the manufacturer's instructions. The half-minimum inhibitory concentration (IC) of the indicated drug was determined. 50 ) was calculated using the SigmaPlot program (Systat Software Inc., San Jose, CA, USA).

[0233] To minimize potential variability due to cancer cell passage number and culture conditions, nicardipine was always included in each in vitro cytotoxicity assay to compare the relative potency of nicardipine analogs. The ratio of change in anticancer potency (ROC) of the compounds was calculated as follows: ROC = IC of (±)-nicardipine 50 / Compound IC 50

[0234] The ROC was obtained from the same set of cytotoxicity assays used to standardize the assays and serves as a primary indicator of improvement in anticancer activity, where an ROC > 1.0 indicates that the analog has greater cytotoxicity than nicardipine in chemotherapy-resistant cancer cells. The ROCs of the nicardipine analog compounds disclosed herein are shown in Table 6 below.

[0235] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] Example 4 modeling

[0236] Molecular docking analysis using Schrodinger's Maestro program demonstrated that (R)-nicardipine binds to the "aromatic cage" of the EED, interacting with Phe97, Tyr148, and Tyr365 through pi-cation interactions, with the benzylammonium cation extending relatively deep into the central pocket. Furthermore, the carbonyl of the ester distal to the tertiary amine is hydrogen-bonded to Arg414, and the m-nitrophenyl group is located near Trp364. The 1,4-dihydropyridine core is less strongly associated with the EED and is partially exposed to the solvent. In contrast, the (S)-enantiomer of nicardipine has a "flipped" 1,4-dihydropyridine: the ammonium cation now has pi-cation interactions only with Phe97 and Tyr148. Tyr365 is close to the 1,4-dihydropyridine ring, and the m-nitrophenyl group is exposed to the solvent and does not associate with the EED. The binding energy between (R)-nicardipine and EED was calculated to be -59.32 kcal / mol, suggesting a higher EED affinity than known EED inhibitors such as EED226 (-49.01 kcal / mol) and MAK683 (-56.15 kcal / mol). The binding energy of (S)-nicardipine with EED was calculated to be -57.09 kcal / mol.

[0237] Two analogs of compound 6 bearing an additional meta-nitro group (±) exhibited nanomolar cytotoxicity (IC) in chemotherapy-resistant C4-2B-TaxR cells. 50 =0.53-0.65 μM) and has a higher ROC value (2.99 and 2.54, respectively) compared to (±)-nicardipine.

[0238] Modeling of the analogs using an induced fit algorithm indicated that compounds 27 and 28 have potentially high EED binding affinity, as shown in Table 7. For example, as shown in Figure 7, there are stabilizing interactions between (S)-compound 27 and residues in the binding pocket of EED, including π-π stacking between the aromatic ring and Trp364, a π-cation interaction between the ammonium group and Tyr148, and two H-bonds between the carbonyl group and Arg414.

[0239] [Table 7]

[0240] Because the linkers of nicardipine and analogs assume a chiral coil state upon binding to EED, analogs with additional substituents and rings on the linker may have increased binding affinity to EED. Modeling results using achiral linkers suggest that the introduction of substituents and chiral centers reduces the conformational freedom of the linker by strengthening the dihedral angles within the chiral protein environment.

[0241] Many modifications and other embodiments of the inventions described herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the accompanying drawings. It is to be understood, therefore, that the invention is not to be limited to the particular embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. Compounds of Formula I 【Chemistry 43】 or a pharmaceutically acceptable salt or solvate thereof, wherein: 【Chemistry 44】 is independently a single bond or a double bond; n is 0 or 1; p is 0, 1 or 2; -A-D-X is attached to carbon (a) or (b), A is a bond, —C(O)—, —C(O) ２ -, -CH ２ O-, -O-, -S-, -S(O)- and -S(O) ２ - selected from the group consisting of D is an optionally substituted C １ ~C ８ alkylene; or 【Chemistry 45】 and X is 【Chemistry 46】 and R ５ and R ６ is an optionally substituted C １ ~C ６ optionally substituted heterocycloalkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted aralkyl; and optionally substituted heteroaralkyl; or R ５ and R ６ and the nitrogen to which each is attached, together form a heterocycloalkyl; R ７ , R ８ , R ９ and R １０ is hydrogen, halogen, hydroxy, C １ ~C ６ alkyl, or R ７ , R ８ , R ９ and R １０ Two of these, or R ７ , R ８ , R ９ and R １０ One of the R ５ and R ６ One of the groups, together with the atom to which each of them is attached, is C ３ ~C ７ forming a cycloalkyl or a 4- to 7-membered heterocycloalkyl, m, o, q, and r each independently represent 0 or 1; R １ is H, —CN, —OH, halo, oxo, optionally substituted C １ ~C １０ Alkyl, optionally substituted C １ ~C １０ alkylamino, optionally substituted C １ ~C １０ Alkoxy, optionally substituted C １ ~C １０ Cycloalkyl, optionally substituted C １ ~C １０ selected from the group consisting of heterocycloalkyl and optionally substituted aryl; R ２ is H, —OH, —NR ２Ａ R ２Ｂ , -OR ２Ａ , -SR ２Ａ , optionally substituted C １ ~C ６ selected from the group consisting of alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; R ２Ａ and R ２Ｂ are each independently H or C １ ~C ６ is alkyl, R ３ When present, is H or optionally substituted C １ ~C ６ is alkyl, R ４ is H, —CN, —OH, halo, —C(O)—(C １ ~C ６ alkyl), —C(O) ２ -(C １ ~C ６ alkyl), -S(O)-(C １ ~C ６ alkyl), -S-(C １ ~C ６ alkyl), -S(O) ２ -(C １ ~C ６ alkyl), optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ alkylamino, optionally substituted C １ ~C ６ alkoxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl and optionally substituted heteroaralkyl.

2. A is -C(O) ２ - and -CH ２ 10. The compound of claim 1, or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of:

3. X is, 【Chemistry 47】 3. The compound of claim 1 or claim 2, wherein:

4. R １ The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein is nitro-substituted aryl.

5. R １ but, 【Chemistry 48】 5. The compound of claim 4, wherein:

6. below Table 8-1 Table 8-2 Table 8-3 Table 8-4 Table 8-5 Table 8-6 Table 8-7 Table 8-8 Table 8-9 wherein D is 【Chemistry 49】 or is selected from the group consisting of D-X is, [Transformation 50] selected from the group consisting of 10. The compound of claim 1 selected from the group consisting of: or a pharmaceutically acceptable salt or solvate thereof.

7. A is a bond, —C(O)—, —C(O) ２ -, -O-, -S-, -S(O)- and -S(O) ２ - selected from the group consisting of R ５ and R ６ is optionally substituted C １ ~C ６ optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; R １ is H, —CN, —OH, halo, oxo, optionally substituted C １ ~C １０ Alkyl, optionally substituted C １ ~C １０ alkylamino, optionally substituted C １ ~C １０ Alkoxy, optionally substituted C １ ~C １０ Cycloalkyl, optionally substituted C １ ~C １０ selected from the group consisting of heterocycloalkyl and unsubstituted aryl; The compound of claim 1.

8. A is —C(O)— and —C(O) ２ 8. The compound of claim 7, selected from the group consisting of: - or a pharmaceutically acceptable salt or solvate thereof.

9. X is, 【Chemistry 51】 9. The compound of claim 7 or claim 8, wherein:

10. X is, 【Chemistry 52】 10. The compound of claim 9, wherein:

11. R ２ But, -NR ２Ａ R ２Ｂ , -OR ２Ａ and optionally substituted C １ ~C ６ is selected from the group consisting of alkyl, R ２Ａ and R ２Ｂ are each independently H or C １ ~C ６ is alkyl, 11. The compound according to any one of claims 7 to 10, or a pharmaceutically acceptable salt or solvate thereof.

12. R ２ Ga-OR ２Ａ and R ２Ａ is C １ ~C ６ 12. The compound of claim 11, or a pharmaceutically acceptable salt or solvate thereof, wherein: R is alkyl;

13. R ２ But, -OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ CH ３ , -OCH(CH ３ ) ２ , -O(CH ２ ) ３ CH ３ , -OCH(CH ３ ) ３ , -OCH ２ CH (CH ３ ) ２ and -OCH(CH ３ ) CH ２ CH ３ 13. The compound of claim 12, wherein:

14. R ２ Ga-OCH ３ 14. The compound of claim 13, wherein:

15. R ４ is -C(O)-(C １ ~C ６ alkyl), —C(O) ２ -(C １ ~C ６ alkyl) and optionally substituted C １ ~C ６ 15. The compound of any one of claims 1 to 14, or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of alkyl.

16. R ４ is optionally substituted C １ ~C ６ 16. The compound of claim 15, or a pharmaceutically acceptable salt or solvate thereof, wherein: R is alkyl;

17. R ４ 17. The compound of claim 16, or a pharmaceutically acceptable salt or solvate thereof, wherein is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, or hexyl.

18. R ４ 18. The compound of claim 17, or a pharmaceutically acceptable salt or solvate thereof, wherein is methyl.

19. R ４ But -C(O) ２ -(C １ ~C ６ 16. The compound of claim 15, or a pharmaceutically acceptable salt or solvate thereof, wherein:

20. R ４ -C(O) ２ CH ３ 20. The compound of claim 19, wherein:

21. R １ is optionally substituted C １ ~C １０ Alkyl, optionally substituted C １ ~C １０ Alkylamino and optionally substituted C １ ~C １０ 21. The compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of alkoxy.

22. R １ is optionally substituted C １ ~C ８ 22. The compound of claim 21, or a pharmaceutically acceptable salt or solvate thereof, wherein:

23. R １ is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, heptyl, or octyl, or a pharmaceutically acceptable salt or solvate thereof.

24. R １ 24. The compound of claim 23, or a pharmaceutically acceptable salt or solvate thereof, wherein is methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, or sec-butyl.

25. R １ is optionally substituted C １ ~C １０ 21. The compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt or solvate thereof, which is cycloalkyl.

26. R １ but 【Chemistry 53】 26. The compound of claim 25, selected from the group consisting of: or a pharmaceutically acceptable salt or solvate thereof.

27. R １ is optionally substituted C １ ~C １０ 11. The compound of any one of claims 7 to 10, or a pharmaceutically acceptable salt or solvate thereof, which is heterocycloalkyl, wherein the heteroatom is one or more of S, O, or N.

28. R １ but 【Chemistry 54】 28. The compound of claim 27, selected from the group consisting of: or a pharmaceutically acceptable salt or solvate thereof.

29. R １ The compound according to any one of claims 1 to 20, or a pharmaceutically acceptable salt or solvate thereof, wherein is unsubstituted aryl.

30. R １ 30. The compound of claim 29, or a pharmaceutically acceptable salt or solvate thereof, wherein is unsubstituted phenyl.

31. R １ 21. The compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt or solvate thereof, wherein

32. Structure of Formula 1a: 【Transformation 55】 32. The compound of any one of claims 1 to 31, or a pharmaceutically acceptable salt or solvate thereof, having the formula:

33. Structure of Formula 1b: 【Transformation 56】 32. The compound of any one of claims 1 to 31, or a pharmaceutically acceptable salt or solvate thereof, having the formula:

34. Structure of Formula I-1: 【Chemistry 57】 34. The compound of any one of claims 1 to 33, or a pharmaceutically acceptable salt or solvate thereof, having the formula:

35. Structure of Formula Ia-1 or 1b-1: 【Transformation 58】 35. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt or solvate thereof, having the formula:

36. 36. The compound of any one of claims 1 to 35, or a pharmaceutically acceptable salt or solvate thereof, wherein p is 0.

37. 36. The compound of any one of claims 1 to 35, or a pharmaceutically acceptable salt or solvate thereof, wherein p is 1.

38. below Table 9-1 Table 9-2 Table 9-3 Table 9-4 Table 9-5 8. The compound of claim 7, selected from the group consisting of: or a pharmaceutically acceptable salt or solvate thereof.

39. A pharmaceutical composition comprising the compound according to any one of claims 1 to 38 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.

40. 37. A method of treating a disorder in a subject in need thereof, comprising administering to said subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 38, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 33.

41. 41. The method of claim 40, wherein the disorder is associated with a histone methyltransferase.

42. 42. The method of claim 41, wherein the disorder is associated with polycomb repressive complex 2 (PRC2) histone methyltransferase and its subunits.

43. 37. The method of claim 36, wherein the disorder is associated with the embryonic ectoderm development (EED) subunit of PRC2.

44. 44. The method of any one of claims 40 to 43, wherein the disorder is cancer.

45. 45. The method of claim 44, wherein the cancer is associated with dysregulation of histone methyltransferase.

46. 46. ​​The method of claim 45, wherein the cancer is associated with dysregulation of polycomb repressive complex 2 (PRC2) histone methyltransferase and its subunits.

47. 47. The method of claim 46, wherein the cancer is associated with dysregulation of the embryonic ectoderm development (EED) subunit of PRC2.

48. 48. The method of any one of claims 44 to 47, wherein the cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, colon cancer, head and neck cancer, pancreatic cancer, brain cancer, bladder cancer, osteosarcoma, and lymphoma.

49. 49. The method of any one of claims 40-48, wherein said treatment of said disorder further comprises a second therapeutic agent or therapy.

50. 41. The method of claim 40, wherein the disorder is selected from the group consisting of sickle cell disease, acute kidney injury, and an autoimmune or inflammatory disease.

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