EP4662202A1 - Pyridine and dihydropyridine compounds and uses thereof - Google Patents

Pyridine and dihydropyridine compounds and uses thereof

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Publication number
EP4662202A1
EP4662202A1 EP24753869.7A EP24753869A EP4662202A1 EP 4662202 A1 EP4662202 A1 EP 4662202A1 EP 24753869 A EP24753869 A EP 24753869A EP 4662202 A1 EP4662202 A1 EP 4662202A1
Authority
EP
European Patent Office
Prior art keywords
optionally substituted
compound
pharmaceutically acceptable
solvate
acceptable salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24753869.7A
Other languages
German (de)
French (fr)
Inventor
Daqing Wu
Frank E. Mcdonald
San PHAM
Zhong-ru XIE
Ruohan CHEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Clark Atlanta University Inc
University of Georgia
Emory University
University of Georgia Research Foundation Inc
Original Assignee
Clark Atlanta University Inc
University of Georgia
Emory University
University of Georgia Research Foundation Inc
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Publication date
Application filed by Clark Atlanta University Inc, University of Georgia, Emory University, University of Georgia Research Foundation Inc filed Critical Clark Atlanta University Inc
Publication of EP4662202A1 publication Critical patent/EP4662202A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/80—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D211/84—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen directly attached to ring carbon atoms
    • C07D211/90—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • C07D213/80—Acids; Esters in position 3

Definitions

  • Cancer remains one of the deadliest threats to human health. In 2023, 1,958,310 new cancer cases and 609,820 cancer deaths are projected to occur in the United States (ACS Cancer Statistics 2023). Globally, nearly 10 million deaths were attributed to cancer in 2020. By 2040, the global cancer burden is expected to grow to 27.5 million new cancer cases and 16.3 million cancer deaths due to the growth and aging of the population. Additionally, cancers can often become multi-drug resistant, further complicating cancer treatment. It is imperative to develop novel therapeutics to overcome this chemoresistance. However, the development of novel therapeutics incurs significant development costs and long timelines.
  • each instance of 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 selected from the group consisting of a bond, -C(O)-, -C(O)2-, -O-, -S-, -S(O)-, and -S(O)2-; or A is selected from the group consisting of a bond, -C(O)-, -C(O)2-, -CH2O-, -O-, - S-, -S(O)-, and -S(O) 2 -;
  • D is optionally substituted Ci-Cs alkylene; such as wherein R 5 and R 6 are each independently selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted cycloalkyl; optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; or wherein R 5 and R 6 and the nitrogen to which each are attached together form a heterocycloalkyl wherein R 7 , R 8 , R 9 , and R 10 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, and Ci-Ce alkyl; or, two of R 7 , R 8 , R 9 , and R 10 or one of R 7 , R 8 , R 9 , and R 10 and one of R 5 and R 6 taken together with the atom to which each is attached form a C3-C7 cycloalkyl or a 4- to 7-membere
  • R 1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and an unsubstituted aryl; or R 1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and optionally substituted aryl;
  • R 2 is selected from the group consisting of 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; wherein R 2A and R 2B are each independently H or Ci-Ce alkyl;
  • R 3 if present, is H or optionally substituted Ci-Ce alkyl
  • R 4 is selected from the group consisting of H, -CN, -OH, halo, -C(O)-(Ci-Ce alkyl), -C(O) 2 -(C1-C 6 alkyl), -S(O)-(Ci-C 6 alkyl), -S-(Ci-C 6 alkyl), -S(O) 2 -(Ci-C 6 alkyl), optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce alkylamino, optionally substituted Ci-Ce alkoxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl.
  • the subject matter described herein is directed to pharmaceutical compositions comprising a therapeutically effective amount of a compound of Formula I, which includes all sub-formulae of Formula I, and a pharmaceutically acceptable carrier.
  • the subject matter described herein is directed to methods for the treatment of disorders associated with histone methyltransferases, the method comprising the step of administering to the subject an effective amount of at least one compound of Formula I, which includes all sub-formulae of Formula I.
  • the subject matter described herein is directed to methods of treatment of disorders associated with histone methyltransferases, the method comprising the step of administering to the subject an effective amount of at least one compound of Formula I, which includes all sub-formulae of Formula I.
  • FIG. 1 ( ⁇ )-Nicardipine (referred to hereinafter as nicardipine) selectively and potently inhibits chemoresistant PCa cells.
  • nicardipine selectively and potently inhibits chemoresistant PCa cells.
  • A In vitro cytotoxicity of nicardipine in the ARCaPE and C4-2B models (72 h).
  • B Flow cytometry results on cell cycle in C4-2B-TaxR cells treated with nicardipine at the indicated concentrations (48 h). *** p ⁇ 0.001 for all pairwise comparisons between the percentages of cells from the control and nicardipine treatment groups in each cell cycle.
  • C Left: flow cytometry analysis on Annexin V staining in C4-2B-TaxR cells treated with nicardipine at the indicated concentrations (72 h). **** p ⁇ 0.0001 for all pairwise comparisons between the control and nicardipine treatment groups; Right: Western blot analysis on the expression of apoptotic markers in C4-2B and C4-2B-TaxR cells treated with nicardipine (2.1 pM) at the indicated time points. P-actin was used as the loading control.
  • D In vitro cytotoxicity of nicardipine in C4-2, PC-3, ARCaP E , and CWR22Rvl cells (72 h).
  • Nicardipine is a putative EED inhibitor.
  • A Left and middle: docking poses of nicardipine and EED; Right: two-dimensional ligand-protein interaction diagram of nicardipine in the binding site of human EED protein (PDB ID: 5WUK). The pink arrow indicates the hydrogen bond; the blue-red line indicates the salt bridge; the red line represents pi-cation.
  • B Left: CETSA analysis of EED expression in C4-2B-TaxR cells treated with DMSO or nicardipine (50 pM, 1 h).
  • FIG. 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 pM; 24 h); Right: The top genes affected by nicardipine treatment in C4-2B-TaxR cells.
  • B IPA analyses of major gene clusters affected by nicardipine treatment in C4-2B-TaxR cells.
  • C GESA of EED-, Gl/S checkpoint-, and EZH2-associated gene signatures 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 score (NES) and false discovery rate (FDR) are indicated for each gene set.
  • Y-axes indicate enrichment scores (top) and ranked list metric (bottom).
  • X-axis bars represent individual genes of the indicated gene sets.
  • FIG. 5 Nicardipine monotherapy inhibits the skeletal growth of chemoresistant PCa in male athymic nude mice.
  • Right two-way ANOVA analysis of the PSA values between different treatment groups. * p ⁇ 0.05.
  • Figure 7 Two-dimensional and three-dimensional interactions of the (R)-enantiomer and (S)-enantiomer of nicardipine and the (S)-enantiomer of compound 27 in the binding site of the human EED protein.
  • Described herein are compounds of Formula I and their uses for the treatment of disorders associated with histone methyltransferases. Also described herein are compounds of Formula I to inhibit dysregulation of poly comb repressive complex 2 (PRC2) and its subunits, including, but not limited to, the embryonic ectoderm development (EED) subunit. Described herein are compounds of Formula I and their uses for the treatment of disorders associated with dysregulation polycomb repressive complex 2 (PRC2) histone methyltransferase. Also described herein are compounds of Formula I and their uses for the treatment of cancers associated with histone methyltransferases.
  • PRC2 poly comb repressive complex 2
  • EED embryonic ectoderm development
  • Described herein are compounds of Formula I and their uses for the treatment of chemoresistant cancers. Also described herein are compounds of Formula I and their uses for the treatment of chemoresistant cancers associated with histone methyltransferases. Described herein are compounds for Formula I and their uses for the treatment of chemoresistant cancers associated with dysregulation of polycomb repressive complex 2 (PRC2), and its subunits, including, but not limited to, the embryonic ectoderm development (EED) subunit.
  • PRC2 polycomb repressive complex 2
  • EED embryonic ectoderm development
  • the compounds described herein exhibit inhibition of dysregulation of histone methyltransferases.
  • the compounds described herein particularly exhibit selective overexpression of various subunits of polycomb repressive complex 2 (PRC2).
  • PRC2 polycomb repressive complex 2
  • the compounds described herein are particularly useful in treating cancers, especially chemoresistant cancers, and other related conditions.
  • EMT Epithelial-to-mesenchymal transition
  • ABSB1 ATP binding cassette B 1 [ABCB1]
  • MDR1 multidrug resistance protein 1 [MDR1], p-glycoprotein
  • survivin anti-apoptotic proteins
  • S-phase kinase-associated protein 2 (SKP2)
  • the substrate recognition component of SCF (SKP1-CUL1-F- box) E3 ubiquitin-protein ligase complex
  • PCa prostate cancer
  • Polycomb repressive complex 2 plays an essential role in transcriptional repression via mono-, di-, and tri-methylation of histone H3 at lysine 27 (H3K27).
  • the core subunits ofPRC2 include enhancer of zeste homolog 1 or 2 (EZH1 orEZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12).
  • EZH2 is a histone methyltransferase (HMT) and acts as the catalytic “writer” subunit of PRC2 in the transcriptional repression of genes. Aberrant overexpression and activation of EZH2 have been associated with clinical progression and poor prognosis of prostate cancer (PCa) and other cancer types. However, the role of EZH2 signaling in chemoresistance remains largely unknown.
  • Enhancer of zeste homolog 2 plays a major role in transcriptional repression via PRC2-dependent histone 3 lysine 27 (H3K27) methylation, as a histone methyltransferase and a subunit of polycomb repressive complex 2 (PRC2).
  • EZH2 also methylates several non-histone protein substrates, such as signal transducer and activator of transcription 3 (Stat3). It acts as a coactivator for androgen receptor (AR), P-catenin and nuclear factor kappa B (NF-KB). This noncanonical function may rely on EZH2 phosphorylation at serine 21 [p-EZH2(S21)].
  • p-EZH2(S21) is significantly increased in mCRPC, a metastatic, castration resistant prostate cancer.
  • EZH2 overexpression and mutation, as well as aberrant EZH2 signaling, have been associated with advanced stages and poor clinical outcomes in various types of cancer.
  • Numerous EZH2 inhibitors have been developed, most of which target the catalytic SET domain via competition with methyl-donating S-adenosylmethionine (SAM).
  • SAM methyl-donating S-adenosylmethionine
  • One of these inhibitors, tazemetostat (EPZ-6438) was approved in 2020 for locally advanced or metastatic epithelioid sarcoma.
  • EZH2 inhibitors have not demonstrated satisfactory clinical outcomes in other solid tumors.
  • EED inhibitors could achieve a general and more efficient blockade of the PRC2 oncogenic signaling in highly heterogeneous cancer cells.
  • An EED inhibitor (MAK683) developed by Novartis has entered Phase I/II trials in patients with advanced malignancies (NCT02900651).
  • a small molecule, LG1980, is an EED inhibitor with promising anticancer activity in chemoresistant PCa cells and xenograft models.
  • MIPDD mechanism-informed phenotypic drug discovery
  • nicardipine acted as a putative EED inhibitor and inhibited noncanonical EED-EZH2 signaling in chemoresistant PCa cells.
  • Nicardipine is an approved drug for treating hypertension, angina, and related cerebrovascular diseases 19,46.
  • CCBs calcium channel blockers
  • nicardipine inhibits the transmembrane influx of calcium into cardiac and smooth muscle without changing serum calcium levels.
  • nicardipine could enhance the in vitro and in vivo effects of certain chemotherapeutics, such as vincristine, carmofur, and nimustine, in experimental models of PCa, esophageal cancer, gastric cancer, glioma, and leukemia; however, it was not clear whether the observed effects of nicardipine in human cancer cells were associated with its function as a CCB. In a recent study, Shi et al.
  • nicardipine could enhance the toxic effect of temozolomide and promote apoptosis in glioma stem cells (GSCs), probably through the upregulation of mTOR and inhibition of autophagy, which is a protective response in glioma cells during chemotherapy.
  • GSCs glioma stem cells
  • nicardipine was found to be capable of increasing the efficacy of vinca alkaloids; in contrast, nicardipine failed to improve adriamycin and vinca alkaloid in seventeen patients with solid tumors or hematologic malignancy.
  • these studies were largely observational in very small patient cohorts, they suggested that there is no straightforward strategy for using nicardipine or other CCBs for cancer treatment in general patient populations.
  • nicardipine The approved schedule of oral administration (i.e., every 8 hours) and a readily measured effect (e.g., change in blood pressure) from nicardipine treatment could allow rapid and convenient adjustments in human trials if any severe adverse effects appear.
  • pharmacological and physiological features of nicardipine suggested that this drug might have a high potential for further clinical development.
  • other approved drugs with high EED-binding affinities i.e., gallopamil, verapamil
  • gallopamil the anti arrhythmic drug gallopamil was withdrawn in 2001 for causing excessive hypotension, bradycardia, or impaired cardiac performance when combined with -adrenoceptor blockers.
  • Verapamil another hypertension drug, was thought to be a functional inhibitor of ABCB 1 (p-glycoprotein) that could re-sensitize cancer cells to chemotherapeutics.
  • ABCB 1 p-glycoprotein
  • verapamil failed to demonstrate clinical benefits in lung cancer patients, mainly due to its poor pharmacokinetics, high dose-limiting toxicity, and low therapeutic window.
  • EED protein is the “reader” component of the PRC2 complex, which binds trimethylated H3K27 (H3K27Me3) through the central pocket formed by its seven WD-40 repeats. The interaction between EED and EZH2 is required for the epigenetic “writer” function of EZH2.
  • EED-EZH2 complex represents an attractive strategy to inhibit EZH2 functions regardless of the mutation status of the EZH2 enzyme.
  • targeting EED-EZH2 interaction often results in the degradation of EZH2 protein and core PRC2 components, EED and SUZ12, which may achieve a general and more efficient blockade of EZH2 functions in highly heterogeneous therapeutic-resistant tumors.
  • EED inhibitors have been developed, most binding to the central pocket and preventing allosteric activation of the catalytic activity of PRC2.
  • Several compounds showed promising in vivo activity in lymphoma xenografts.
  • Nicardipine can be offered to patients with active noncanonical EED-EZH2 signaling as an adjunct therapy in combination with docetaxel and/or ADT, with the expectation of eliminating chemoresistant PCa cells and enhancing the efficacy of standard treatments.
  • the subject matter described herein is directed to methods of treating disorders related to poly comb repressive complex 2 (PRC2) histone methyltransferase and its subunits, the method comprising the step of administering to a subject in need thereof an effective amount of nicardipine, or a compound described in U.S. Patent No. 4,510,310, which is hereby incorporated by reference in its entirety herein.
  • PRC2 poly comb repressive complex 2
  • nicardipine As described herein, the suboptimal nature of nicardipine provided an opportunity to obtain highly specific EED inhibitors with improved anticancer activities and drug-like properties.
  • the compounds described herein represent novel first-in-class EED inhibitors with distinct chemical structures and pharmacological activities.
  • Forma I includes all sub-formulae of Formula I described herein.
  • Alkyl refers to a straight or branched chain hydrocarbon containing from 1 to 12 carbon atoms.
  • Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, 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.
  • alkyl is intended to include both substituted and unsubstituted alkyl unless otherwise indicated and these groups may be substituted with groups selected from halo (e.g., haloalkyl), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl (including spiroalkyl, e.g., C2, C3, or C4 spiroalkyl), cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, amino, carboxy, alkylamino, alkenylamin
  • Cycloalkyl refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in a heterocyclic group as discussed below) and includes spirocyclics.
  • Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may be optionally substituted with additional substituents as described herein such as halo or loweralkyl.
  • the term “cycloalkyl” is generic and intended to include heterocyclic groups as discussed below unless specified otherwise.
  • Alkylene refers to a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of one to eight carbon atoms unless otherwise stated, such as methylene, ethylene, propylene, 1 -methylpropylene, 2- methylpropylene, butylene, pentylene, and the like.
  • Heterocyclic group or “heterocycloalkyl” as used herein alone or as part of another group, refers to an aliphatic (e.g., fully or partially saturated heterocycloalkyl) or aromatic (e.g., heteroaryl) monocyclic- or a 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. The 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds.
  • monocyclic ring systems that are heterocycloalkyls include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3- dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothi azoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophen
  • Bicyclic ring systems are exemplified by any of the above monocyclic ring systems 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, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodi oxine, 1,3 -benzodi oxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline,
  • These rings may be optionally substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, ester,
  • Aryl 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.
  • aryl is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl above.
  • Arylalkyl or “aralkyl” as used herein alone or as part of another group refers to an aryl group, as defined herein, appended 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.
  • Heteroaryl as used herein is as described in connection with heterocycloalkyl above.
  • Heteroaralkyl or “heteroarylalkyl” as used herein alone or as part of another group, refers to a heteroaryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Alkoxy refers to an alkyl group, as defined herein (and thus including substituted versions such as polyalkoxy), appended to the parent molecular moiety through an oxy group, -O-.
  • alkoxy include, but are not limited to, methoxy, ethoxy, propyloxy, 2-propyloxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
  • Halo or “halogen” as used herein refers to any suitable halogen, including F, Cl, Br, and I.
  • Cyano refers to a -CN group.
  • Hydrol refers to an -OH group.
  • Amino as used herein means the radical -NH2.
  • Alkylamino as used herein alone or as part of another group means the radical -NHR, where R is an alkyl group.
  • “Disubstituted-amino” as used herein alone or as part of another group means the radical -NR a R b , where R a and R b are independently selected from the group alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl.
  • Ester as used herein alone or as part of another group refers to a -C(O)OR radical, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
  • Amide as used herein alone or as part of another group refers to a -C(O)NR a R b radical, where R a and R b are any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
  • the term “residue” or “residue of’ a chemical moiety refers to a chemical moiety that is bound to a molecule, whereby through the binding, at least one covalent bond has replaced at least one atom of the original chemical moiety, resulting in a residue of the chemical moiety in the molecule.
  • the compounds of the present disclosure may have asymmetric centers.
  • Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of materials. All chiral, diastereomeric, all mixtures of chiral or diastereomeric forms and racemic forms are within the scope of this disclosure, unless the specific stereochemistry or isomeric form is specifically indicated.
  • physiological conditions refers to the range of conditions of temperature, pH, and tonicity (or osmolality) normally encountered within tissues in the body of a living human.
  • in vitro refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube).
  • in vivo refers to natural environments (e.g., a cell or organism or body) and to processes or reactions that occur within a natural environment.
  • heterocyclyl group optionally substituted with an alkyl group means that the alkyl may but need not be present, and the description includes situations where the heterocyclyl group is substituted with an alkyl group and situations where the heterocyclyl group is not substituted with alkyl.
  • the phrase “optionally substituted” means that substitution is optional and therefore includes both unsubstituted and substituted atoms and moieties.
  • a “substituted” atom or moiety indicates that any hydrogen on the designated atom or moiety can be replaced with a selection from the indicated substituent groups, provided that the normal valency of the designated atom or moiety is not exceeded, and that the substitution results in a stable compound. For example, if a methyl group is optionally substituted, then 3 hydrogen atoms on the carbon atom can be replaced with substituent groups. Also, double bonds depicted in the structure follow normal valency and can be conjugated or can represent an aromatic system.
  • a “subject” refers to an animal that is the object of treatment, observation or experiment.
  • Animal includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and, in particular, mammals.
  • “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees and apes, and, in particular, humans.
  • the subject can be human.
  • the subject can be a human child and/or a human infant, for example, a child or infant with a fever.
  • the subject can be a human adult.
  • a subject “in need thereof’ is a subject that has been diagnosed with or is believed to be suffering from one or more disorders associated with dysregulation polycomb repressive complex 2 (PRC2) histone methyltransferase.
  • PRC2 dysregulation polycomb repressive complex 2
  • “Treating” or “treatment” of a disease includes:
  • a “therapeutically effective amount” means the amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof that, elicits the biological or medicinal response indicated.
  • the therapeutically effective amount of a compound when administered to a subject for treating a disease, is sufficient to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. This response may occur in a tissue, system, animal or human and includes alleviation of the signs or symptoms of the disease being treated. Determination of an effective amount is well within the capability of those skilled in the art, in view of the disclosure provided herein.
  • the “therapeutically effective amount” of the compounds disclosed herein will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated. Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
  • the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ⁇ 0.5%, 1%, 5%, or 10% from a specified value.
  • compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited.
  • a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients.
  • an antigen or “at least one antigen” can include a plurality of antigens, including mixtures thereof.
  • described herein are compounds that are a novel class of inhibitors of PCR2 and its subunits.
  • the compounds are a novel class of EED inhibitors.
  • the novel class of EED inhibitors have unique chemical structures and display high specificity and potency against disorders associated with EED dysregulation.
  • the disorders associated with EED dysregulation are cancers.
  • the cancers are chemoresistant cancers.
  • the compounds are a novel class of EED inhibitors for treating chemoresistant cancers.
  • the compounds described herein are a novel class of EED inhibitors from treating chemoresistant prostate cancer (PCa).
  • the compounds described herein display high specificity and potency against chemoresistant PCa.
  • chemoresistant cancer is chemoresistance prostate cancer (PCa).
  • the subject matter described herein is directed to compounds of Formula I: or a pharmaceutically acceptable salt or solvate thereof, wherein: each instance of 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 selected from the group consisting of a bond, -C(O)-, -C(O)2-, -O-, -S-, -S(O)-, and -S(O)2-; or A is selected from the group consisting of a bond, -C(O)-, -C(O)2-, -CH2O-, -O-, - S-, -S(O)-, and -S(O) 2 -;
  • D is optionally substituted Ci-Cs alkylene; wherein R 5 and R 6 are each independently selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted cycloalkyl; optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; or wherein R 5 and R 6 and the nitrogen to which each are attached together form a heterocycloalkyl.
  • R 1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and an unsubstituted aryl; or R 1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and an optionally substituted aryl;
  • R 2 is selected from the group consisting of H, -OH, -NR 2A R 2B , -OR 2A , -SR 2A , optionally substituted Ci-Ce alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; wherein R 2A and R 2B are each independently H or Ci-Ce alkyl;
  • R 3 if present, is H or optionally substituted Ci-Ce alkyl
  • R 4 is selected from the group consisting of H, -CN, -OH, halo, -C(O)-(Ci-Ce alkyl), -C(O) 2 -(C1-C 6 alkyl), -S(O)-(Ci-C 6 alkyl), -S-(Ci-C 6 alkyl), -S(O) 2 -(Ci-C 6 alkyl), optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce alkylamino, optionally substituted Ci-Ce alkoxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl.
  • D is selected from the group consisting of :
  • D is wherein R 7 , R 8 , R 9 , and R 10 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, and Ci-Ce alkyl; or, wherein two of R 7 , R 8 , R 9 , and R 10 or one of R 7 , R 8 , R 9 , and R 10 and one of R 5 and R 6 taken together with the atom to which each is attached form a C3-C7 cycloalkyl or a 4- to 7-membered heterocycloalkyl; wherein m, o, q, and r are each independently 0 or 1.
  • D is selected from the group consisting of:
  • D-X is selected from the group consisting of:
  • the subject matter described herein is directed to compounds of Formula la: or a pharmaceutically acceptable salt or solvate thereof.
  • the subject matter described herein is directed to compounds of Formula lb: or a pharmaceutically acceptable salt or solvate thereof.
  • the subject matter described herein is directed to compounds of Formula 1-1 : or a pharmaceutically acceptable salt or solvate thereof.
  • the subject matter described herein is directed to compounds of Formula la- 1 or lb-1 : or a pharmaceutically acceptable salt or solvate thereof.
  • compounds include those wherein A is selected from the group consisting of -C(O)- and -C(O)2-.
  • compounds include those having a structure of Formula la-1, wherein p is 0 and R 1 is selected from the group consisting of H, optionally substituted Ci-Cio alkyl, optionally substituted C1-C10 cycloalkyl, and an optionally substituted aryl.
  • R 1 is phenyl, m-nitrophenyl, cyclobutyl, cyclopentyl, cyclohexyl, methyl, ethyl, propyl, isopropyl, isobutyl, or 3-pentyl.
  • compounds include those having a structure of Formula la-1, wherein p is 1 and R 1 is optionally substituted C1-C10 cycloalkyl. In certain embodiments, p is 1 and R 1 is cyclobutyl, cyclopentyl, or cyclohexyl.
  • compounds include those having a structure of Formula la-1, wherein R 2 is -OR 24 , wherein R 2A is Ci-Ce alkyl. In certain embodiments, R 2 is OCH3.
  • compounds include those having a structure of Formula la-1, wherein R 3 is H.
  • compounds include those having a structure of Formula la-1, wherein R 4 is methyl.
  • compounds include those having a structure of Formula la-1, wherein A is -C(O)2-.
  • compounds include those having a structure of Formula la-1, wherein,
  • compounds include those having a structure of Formula la-1, wherein R 2 is OCH3, R 3 is H, R 4 is CH3, and A is -C(O)2-.
  • compounds include those having a structure of Formula la-1, wherein R 2 is OCH3, R 3 is H, R 4 is CH3, A is -C(O)2-, p is 0, and R 1 is optionally substituted aryl.
  • compounds include those having a structure of Formula la-1 , wherein
  • compounds include those having a structure of Formula la-1, wherein R 2 is OCH3, R 3 is H, R 4 is CH3, A is -C(O)2-, p is 0, R 1 is optionally substituted aryl, and
  • compounds include those wherein X is ve embodiments, compounds include those wherein X is
  • compounds include those wherein D comprises one or more asymmetric centers.
  • compounds include those wherein R 2 is selected from the group consisting of -NR 2A R 2B , -OR 2A , and optionally substituted Ci-Ce alkyl, wherein R 2A and R 2B are each independently H or Ci-Ce alkyl.
  • compounds include those wherein R 2 is -OR 2A , wherein R 2A is Ci-Ce alkyl.
  • compounds include those wherein R 2 is -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH 3 )2, -O(CH 2 )3CH3, -OCH(CH 3 )3, -OCH 2 CH(C H 3 ) 2 , and -OCH(CH 3 )CH2CH3.
  • compounds include those wherein R 2 is -OCH3.
  • compounds include those wherein R 4 is selected from the group consisting of -C(O)-(Ci-Ce alkyl), -C(O)2-(Ci-C6 alkyl), and optionally substituted Ci-Ce alkyl.
  • compounds include those wherein R 4 is optionally substituted Ci-Ce alkyl.
  • compounds include those wherein R 4 is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl or hexyl. [0098] In the above embodiments, compounds include those wherein R 4 is methyl.
  • compounds include those wherein R 4 is -C(O)2-(Ci-C6 alkyl).
  • compounds include those wherein R 1 is selected from the group consisting of optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, and optionally substituted C1-C10 alkoxy.
  • compounds include those wherein R 1 is optionally substituted Ci-Cs alkyl.
  • compounds include those wherein R 1 is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, heptyl, or octyl.
  • compounds include those wherein R 1 is methyl, ethyl, propyl, butyl, isobutyl, or ec-butyl.
  • compounds include those wherein R 1 is optionally substituted C1-C10 cycloalkyl. [0106] In the above embodiments, compounds include those wherein R 1 is selected from the
  • compounds include those wherein R 1 is optional substituted Ci-Cio heterocycloalkyl, wherein the heteroatom is one or more of S, O, or N.
  • compounds include those wherein R 1 is an unsubstituted aryl.
  • compounds include those wherein R 1 is an unsubstituted phenyl.
  • compounds include those wherein R 1 is a substituted aryl.
  • compounds include those wherein R 1 is a substituted phenyl.
  • compounds include those wherein R 1 is a nitro substituted phenyl.
  • compounds include those wherein R 1 is H.
  • compounds include those wherein p is 0.
  • compounds include those wherein p is 1.
  • compounds include those having a structure as shown below in Table A:
  • D-X is selected from the group consisting of:
  • the compounds include those of Formula I, or pharmaceutically acceptable salts thereof, where the compounds inhibit one or more proteins of the poly comb repressive complex 2 (PRC2).
  • PRC2 poly comb repressive complex 2
  • the compounds of Formula I or pharmaceutically acceptable salts or solvates thereof display selectivity for inhibition of embryonic ectoderm development (EED) subunit of PRC2.
  • EED embryonic ectoderm development
  • compositions comprising at least one compound of Formula I as described herein, and a pharmaceutically acceptable excipient or carrier.
  • a “pharmaceutically acceptable excipient” refers to a vehicle for containing a functionalized cell or an acellular extracellular matrix that can be introduced into a subject without significant adverse effects and without having deleterious effects on the functionalized cell or acellular extracellular matrix. That is, “pharmaceutically acceptable” in the context of a formulation refers to any formulation which is safe and provides the appropriate delivery for the desired route of administration of an effective amount of at least one functionalized cell or acellular extracellular matrix for use in the methods disclosed herein. Pharmaceutically acceptable carriers or vehicles or excipients are well known.
  • Such carriers can be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical application).
  • Such pharmaceutical compositions can be buffered, for example, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the functionalized cell or acellular extracellular matrix and route of administration.
  • Suitable pharmaceutically acceptable carriers include, for example, sterile water, salt solutions such as saline, glucose, buffered solutions such as phosphate buffered solutions or bicarbonate buffered solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like.
  • compositions or vaccines may also include auxiliary agents 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, coloring, flavoring, aromatic substances, and the like which do not deleteriously react with a functionalized cell or an acellular extracellular matrix.
  • auxiliary agents 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, coloring, flavoring, aromatic substances, and the like which do not deleteriously react with a functionalized cell or an acellular extracellular matrix.
  • pharmaceutically acceptable carriers may be aqueous
  • 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, including saline and buffered media.
  • 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, a gum, a starch (e.g., corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, or dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • a gum e.g., corn starch, pregeletanized starch
  • a sugar e.g., lactose, mannitol, sucrose, or dextrose
  • a cellulosic material e.g., microcrystalline cellulose
  • an acrylate e.g., polymethylacrylate
  • calcium carbonate e.g., magnesium oxide, talc, or mixtures thereof.
  • sustained or directed release pharmaceutical compositions or vaccines can be formulated. This can be accomplished, for example, through use of liposomes or compositions wherein the active compound is protected with differentially degradable coatings (e.g., by microencapsulation, multiple coatings, and so forth). Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the compositions and use the lyophilisates obtained (e.g., for the preparation of products for injection).
  • the subject matter described herein is directed to methods of treating disorders, the method comprising the step of administering to a subject an effective amount of at least one compound of Formula I.
  • the subject matter described herein is directed to methods of treating disorders related to polycomb repressive complex 2 (PRC2) histone methyltransferase and its subunits, the method comprising the step of administering to a subject in need thereof an effective amount of at least one compound or pharmaceutical salt thereof described in U.S. Patent No. 4,510,310, which is hereby incorporated by reference in its entirety herein.
  • the compound is nicardipine.
  • the disorder is associated with histone methyltransferases.
  • the disorder is associated with polycomb repressive complex 2 (PRC2) histone methyltransferase and its subunits.
  • PRC2 polycomb repressive complex 2
  • EED embryonic ectoderm development
  • the subject matter described herein is directed to methods of treating a cancer, the method comprising the step of administering to a subject an effective amount of at least one compound of Formula I.
  • the cancer is associated with dysregulation of histone methyltransferases.
  • the cancer is associated with dysregulation of polycomb repressive complex 2 (PRC2) and its subunits.
  • PRC2 polycomb repressive complex 2
  • the cancer is associated with the dysregulation of EED subunit of PRC2.
  • the method of treatment further comprises administering an additional active agent, such as an active agent useful in the treatment of cancer.
  • the additional compounds may optionally be administered concurrently.
  • concurrently means sufficiently close in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more events occurring within a short time period before or after each other).
  • the present subject matter is primarily concerned with the treatment of human subjects, but the invention may also be carried out 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.
  • 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 infant, juvenile, adolescent, adult, and geriatric subjects.
  • the subject matter provides pharmaceutical formulations comprising the compounds of Formula I (including the pharmaceutically acceptable salts thereof), in pharmaceutically acceptable carriers for oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, or intravenous, and transdermal administration.
  • the therapeutically effective dosage of any specific compound can vary somewhat from compound to compound, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the active compound, including the cases where a salt is employed. Toxicity concerns at the higher level may restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the active base, including the cases where a salt is employed. A dosage from about 10 mg/kg to about 50 mg/kg may be employed for oral administration.
  • a dosage from about 0.5 mg/kg to 5 mg/kg may be employed for intramuscular injection.
  • dosages are 1 pmol/kg to 50 pmol/kg, and more preferably 22 pmol/kg and 33 pmol/kg of the compound for intravenous or oral administration.
  • the duration of the treatment can be once per day for a period of two to three weeks or until the condition is essentially controlled.
  • 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 (Coming Inc, Corning, NY, USA).
  • C4-2-Luc cells were cultured in the same media as C4-2 with additional G418 (Thermo Fisher Scientific, Waltham, MA) at 400 pg/mL.
  • Human PC-3 cells were routinely maintained in RPMI 1640 medium (Corning Inc) supplemented with 10% FBS and penicillin-streptomycin.
  • the three-dimensional (3D) structures of tested compounds were retrieved from PubChem and built using the Maestro program (Schrodinger, New York, NY, USA), as we described previously 18. All the tested compounds were prepared using Ligprep in Maestro 12.4.
  • the structure of EED protein (PDB ID: 5WUK) was retrieved from RCSB’s Protein Data Bank 22. Using the Protein Preparation Wizard in Maestro, the protein structure was prepared through three steps: preprocessing, optimization, and minimization 23. Preprocessing includes assigning bond orders, adding hydrogens, creating disulfide, and generating het states using Epik 24,25.
  • the process of optimization optimizes hydrogen bonds by using PROPKA 26.
  • the step of minimization is performed by using the OPLS3e force field 27.
  • a receptor grid box was generated based on the five residues (Phe97, Tyrl48, Trp364, Tyr365, Arg367) around the binding site. The size of the receptor grid box was set as default (20 A).
  • Ligand-protein docking was performed in extraprecision (XP) mode using the Ligand Docking panel. After molecular docking, the binding energies were calculated using Prime MM-GBSA (molecular mechanics generalized Bom surface area) in Maestro Program.
  • CETSA Cellular thermal shift assay
  • CETSA was performed following a modified procedure described in 18.
  • C4-2B-TaxR cells were incubated for 1 h in the presence of DMSO or nicardipine (50 pM).
  • a MyCyclerTM thermal cycler system with a gradient option was used to incubate total cell lysates at varying temperatures.
  • RNA samples were collected from C4-2B-TaxR cells treated with DMSO or nicardipine (2.1 pM) for 24 h in triplicates. RNA-seq analyses were performed by Omega Bioservices (Norcross, GA, USA). Data were analyzed by Rosalind® (Rosalind, Inc., San Diego, CA, USA), Ingenuity Pathway Analysis (IP A, Qiagen, Germantown, MD, USA), and Gene Set Enrichment Analysis (GSEA, University of California San Diego and Broad Institute, USA).
  • a total of 2x 106 C4-2B-TaxR or C4-2-Luc cells suspended in 20 pl PBS were injected into the bilateral tibia of male athymic nude mice (5-week-old, Envigo RMS, Inc, Indianapolis, IN). Tumor establishment in mouse bones was confirmed by rising serum levels of human prostatespecific antigen (PSA) using an enzyme-linked immunosorbent assay (ELISA) kit (United Biotech, Inc, Mountain View, CA, USA).
  • PSA prostatespecific antigen
  • ELISA enzyme-linked immunosorbent assay
  • mice For the C4-2B-TaxR xenograft model, tumor-bearing mice were randomly divided into 3 groups and treated with vehicle control (DMSO), docetaxel, or nicardipine, respectively, at the indicated doses and schedule via intraperitoneal (i.p.) injection.
  • vehicle control DMSO
  • docetaxel or nicardipine
  • an additional group of mice was treated with the combination of docetaxel and nicardipine at the indicated doses and schedule via i.p. injection.
  • the vehicle control and docetaxel treatment groups in the C4-2B-TaxR and C4-2-Luc xenografts were the same as those described in a previous study 18. Body weights were monitored twice a week. Intratibial growth of tumors was followed by weekly PSA measurements.
  • the unpaired t-test was performed to examine the significant difference between the means of any two groups.
  • Two-way analysis of variance (ANOVA) was performed to measure the significant difference by comparing the means between groups affected by two independent factors, p ⁇ 0.05 represents statistical significance.
  • a two-tier phenotypic screening system to identify selective inhibitors of chemoresistant PCa cells was established.
  • the primary screening was based on epithelial protein lost in neoplasm (EPLIN) which serves as a molecular regulator of metastasis and chemoresistance.
  • EPLIN epithelial protein lost in neoplasm
  • ARCaPE-shEPLIN cells stably expressing EPLIN shRNA are highly resistant to docetaxel compared with ARCaPE-shCtrl cells, thus representing the characteristics of the intrinsic chemoresistance.
  • Primary screening was performed to identify small-molecule compounds that selectively inhibit ARCaPE-shEPLIN, but not ARCaPE-shCtrl cells.
  • EZH2[S21] phosphorylation of EZH2 at serine 21
  • Stat3 signal transducer and activator of transcription 3
  • S-phase kinase-associated protein 2 S-phase kinase-associated protein 2
  • ABSB1 ATP binding cassette B 1
  • survivin a survival signaling pathway consisting of signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B 1 (ABCB1, p-glycoprotein) and survivin
  • LG1980 effectively interrupts the physical interaction between EED and ZEH2, disassembles PRC2, and promotes the degradation of its core components, thereby inhibiting p-EZH2(S21) and suppressing the expression of its downstream effectors.
  • LG1980 demonstrated high specificity and potent efficacy against the in vitro and in vivo growth of chemoresistant PCa cells.
  • nicardipine acts as a specific and potent inhibitor of chemoresistant PCa in preclinical models. Intriguingly, mechanistic studies demonstrated that nicardipine might function as an EED inhibitor that disrupts the noncanonical EZH2 signaling and confers its anticancer activities in chemoresistant PCa cells.
  • Nicardipine displays high selectivity and potency against chemoresistant PCa
  • nicardipine an anti-hypertensive drug, was identified as a potential inhibitor of chemoresistant PCa cells.
  • SI selectivity index
  • Nicardipine had an IC50 of 29.1 p in ARCaPi -shCtrl cells and 0.5 pM in ARCaPE-shEPLIN cells, with a fold of difference of 58.2 (Fig. 1 A, left).
  • Flow cytometry analyses showed that compared with vehicle control, nicardipine treatment at 2.0 pM and 4.0 pM significantly induced cell cycle arrest at both Gl-S and G2-M checkpoints with an accumulation of a sub-Gl population representing apoptotic cells (Fig. IB).
  • Nicardipine treatment also significantly induced apoptosis dose-dependently, as demonstrated by increased surface staining of Annexin V, a marker of apoptosis (Fig. 1C, left).
  • Western blotting analyses 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 (Fig. 1C, right).
  • nicardipine had high selectivity and potency against chemoresistant PCa cells.
  • Nicardipine s molecular interaction ns with EED
  • EED is the “reader” component of the PRC2 complex that binds trimethylated H3K27 (H3K27me3) and activates the HMT function of EZH2.
  • EED also serves as a scaffolding protein to interact with EZH2 and SUZ12 and maintain the integrity of the PRC2 complex.
  • Current EED inhibitors including EED226, A-395, and BR-001, target the histone-binding central pocket, or the “aromatic cage,” formed by the seven WDRs in EED and prevent allosteric activation of the catalytic activity of PRC2.
  • nicardipine treatment shifted the melting temperature (Tm) of EED protein from 49.8°C to 50.4°C, indicating that nicardipine could specifically bind and stabilize EED protein in live cancer cells (Fig. 2B).
  • Tm melting temperature
  • EED and p-EZH2(S21) were significantly upregulated in C4-2B-TaxR cells compared with parental C4-2B cells.
  • basal levels of EZH2 and SUZ12 were similar between the two cell lines.
  • Treatment with nicardipine at 2.1 pM effectively downregulated EZH2, p- EZH2(S21), EED and SUZ 12 in a time-dependent manner in C4-2B-TaxR cells, but notin parental C4-2B cells (Fig. 3 A).
  • Nicardipine’s affect EZH2 protein stability in chemoresistant PCa cells was ascertained, which would lead to reduced expression of EZH2 and p-EZH2(S21).
  • a CHX chase experiment showed that in the presence of nicardipine, the half-life (Ti,) of EZH2 protein was significantly shortened from > 48 h to 14.8 h (Fig. 3B). This result indicates that nicardipine might facilitate EZH2 degradation via a proteasome-mediated mechanism.
  • Nicardipine s effect on the canonical function of EZH2 on histone methylation was evaluated further. As previously discovered, there was no significant difference in the basal expression of H3K27me3 between C4-2B and C4-2B-TaxR cells, indicating that canonical EZH2 signaling may not play a dominant role in PCa chemoresistance. Treatment with nicardipine at 2.1 pM did not affect the tri-methylation of H3K27 in either C4-2B or C4-2B-TaxR cells during a 72 h period.
  • EZH2 signaling pathway in chemoresistant PCa cells was also determined.
  • p-EZH2(S21), p- Stat3(S727), SKP2, ABCBl 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, ABCBl, and survivin in chemoresistant C4-2B-TaxR cells but not in C4-2B cells (Fig. 3D).
  • nicardipine may effectively target noncanonical EZH2-Stat3-SKP2-ABCBl/survivin signaling and inhibit the proliferation and viability of chemoresistant PCa cells.
  • Nicardipine facilitates cellular uptake of chemotherapeutics in chemoresistant PCa cells
  • Overexpression of ABCB 1 has been recognized as a central molecular mechanism in the multidrug resistance (MDR). Consistently, previous studies have shown that ABCBl depletion effectively increased the intracellular presence of chemotherapeutics in chemoresistant PCa cells. Since nicardipine significantly reduced ABCB1 protein levels in chemoresistant C4-2B-TaxR cells, nicardipine’s facilitation of the uptake of Oregon Green 488- conjugated paclitaxel was explored.
  • Nicardipine pre-treatment resulted in a rapid (within 15 min) accumulation of fluorescent paclitaxel in C4-2B-TaxR cells (Fig. 3E). In comparison, there was no paclitaxel uptake until 30 min in control cells.
  • nicardipine-mediated ABCB1 downregulation could contribute to the increased uptake and retention of chemotherapeutics in chemoresistant PCa cells.
  • nicardipine may bind EED and induce protein degradation of EZH2, thereby reducing p-EZH2 and suppressing Stat3/SKP2/ABCBl/survivin survival signals in chemoresistant PCa cells (Fig. 3F).
  • Nicardipine affects multiple genes implicated in the control of the cell cycle in chemoresistant PCa cells.
  • RNA-seq analyses were performed and gene expression in C4-2B-TaxR cells treated with vehicle control or nicardipine (2.1 pM, 24 h) were compared.
  • p-adj adjusted p-value
  • 336 unique genes were upregulated
  • 259 unique genes were downregulated significantly following nicardipine treatment ( Figure 4A).
  • IPA profiling found that the top canonical pathways affected by nicardipine included cell cycle control and DNA damage responses, which were in line with the known functions of PRC2 in cancer cells (Table 4).
  • Nicardipine inhibits the skeletal growth of chemoresistant C4-2B-TaxR tumors
  • C4-2B-TaxR cells To further elucidate the in vivo efficacy of nicardipine against the skeletal growth of chemoresistant cancers, C4-2B-TaxR cells, a model closely mimicking the clinicopathology of AR-positive, chemoresistant, and bone metastatic PCa, were used.
  • PSA prostate-specific antigen
  • Nicardipine enhances the in vivo efficacy of docetaxel and inhibits the skeletal growth of C4-2 xenografts
  • IC50 15.0 pM
  • IC50 0.72 pM
  • the two drugs demonstrated a synergistic inhibitory effect on the in vitro proliferation of C4-2 cells, which was reflected by the combination indexes (Cis) lower than 1.0 in isobologram analyses using the CompuSyn program ( Figure 6A, right; Table 5).
  • nicardipine may be effective in enhancing the anticancer effect of docetaxel chemotherapy.
  • C4-2 tumors were inoculated into the tibiae of male athymic nude mice. Tumor-bearing mice were treated with vehicle control, docetaxel, nicardipine, and the combination of docetaxel and nicardipine, respectively.
  • the average PSA level of each group was determined as 79.17 ⁇ 17.92 ng/ml (control), 49.04 ⁇ 14.92 ng/ml (docetaxel, 5 mg/kg, once per week), 42.80 ⁇ 9.29 ng/ml (nicardipine, 10 mg/kg, three times per week), and 23.23 ⁇ 6.27 ng/ml (docetaxel and nicardipine).
  • the ratio of change (ROC) in the anticancer potency of (R)-nicardipine or (S)-nicardipine calculated by [ICso of ( ⁇ )- nicardipine/ICso of an analog] in the same sets of cytotoxicity assays (ROC > 1.0 indicating the analog has higher cytotoxicity in chemoresistant PCa cells), was 1.53 and 1.16, respectively.
  • Example A Compound 1, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,6-dimethyl-4- phenyl-l,4-dihydropyridine-3,5-dicarboxylate:
  • Example B Compound 3, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,4,6-trimethyl- l,4-dihydropyridine-3,5-dicarboxylate:
  • Example C and D Compounds 5 and 6 were prepared from a common precursor, 5- (methoxycarbonyl)-2,6-dimethyl-4-phenyl-l,4-dihydropyridine-3-carboxylic acid:
  • the cyanoethyl ester product was eluted using a gradient of 60:40 to 50:50 to 40:60 Hexane/EtOAc, concentrated in vacuo and under high-pressure vacuum overnight to give 3 -(2-cyanoethyl) 5-methyl 2,6-dimethyl-4-phenyl-l,4-dihydropyridine-3,5- dicarboxylate as a thick yellow oil. Yield: 73%.
  • Example C Compound 5, 3-(2-(dimethylamino)ethyl) 5-methyl 2,6-dimethyl-4-phenyl- l,4-dihydropyridine-3,5-dicarboxylate:
  • Example D Compound 6, 3-(6-(benzyl(methyl)amino)hexyl) 5-methyl 2,6-dimethyl-4- phenyl-l,4-dihydropyridine-3,5-dicarboxylate:
  • reaction mixture was stirred on ice for 2.5 hours, then a solution of 6- (benzyl(methyl)amino)hexan-l-ol (1 equiv) in DCM (2 mL) was added to the reaction flask. The reaction was stirred and warmed to room temperature overnight, which became a clear orange mixture. After stirring was stopped, the reaction was diluted with DCM and washed with saturated NaHCCh and brine. The organic layer was dried with anhydrous Na2SC>4 then concentrated in vacuo to give a dark orange oil crude. The reaction mixture was purified via flash column chromatography with 95:5 DCM/MeOH (via wet loading). Fractions were collected (in small quantities when yellow band started eluting) and visualized by UV lamp. The product was identified to be fluorescent under UV light. Yield: 26%, yellow oil.
  • Example E Compound 12, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,6- di m ethy I py ri di n e-3 , 5 -di carb oxy 1 ate :
  • Example G Compound 15, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,6-dimethyl-4- phenylpyridine-3,5-dicarboxylate:
  • Example O Compound 27: [0228] 5-(Methoxycarbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-l,4-dihydropyridine-3-carboxylic acid (100 mg, 0.30 mmol, 1 equiv), 6-(benzyl(methyl)amino)hexan-l-ol (73 mg, 0.33 mmol, 1.1 equiv), and A-methylimidazole (NMI, 74 pL, 0.93 mmol, 3.1 equiv) were added to a 4 mL oven- dried reaction vial charged with stir bar under air.
  • NMI N-methylimidazole
  • DMSO dimethyl sulfoxide
  • the cells were incubated at 37°C, 5% CO2, with 100% humidity.
  • a column of control wells on the same plates contained all materials except cells and compounds (or DMSO). Plates were incubated for 3 days, allowing sufficient time for cell replication and compound-induced cell death.
  • Cells were counted using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MD, USA) following the manufacturer’s instruction.
  • the half-minimal inhibitory concentrations (IC50) of the specified agent were calculated with the SigmaPlot program (Systat Software Inc., San Jose, CA, USA).
  • nicardipine was always included in every in vitro cytotoxicity assay to compare the relative potency of nicardipine analogs.
  • the ratio of change (ROC) in the anticancer potency of a compound was calculated as
  • ROC was obtained from the same sets of cytotoxicity assays used to standardize the assays and as serves as the primary indicator of improvement in terms of anticancer activities, where ROC > 1.0 indicating the analog has higher cytotoxicity than nicardipine in chemoresistant cancer cells.
  • the ROCs for the nicardipine analog compounds disclosed herein is shown in Table 6 below. [0235] Table 6. ROCs of Nicardipine Analogs
  • the (S)-enantiomer of nicardipine has the 1,4-dihydropyridine "flipped": the ammonium cation now has pi-cation interactions only with Phe97 and Tyrl48. Tyr365 is closer to the 1,4-dihydropyridine ring, and the m-nitrophenyl group is exposed to solvent and is not associated with EED.
  • the binding energy between (R)-nicardipine and EED was calculated as -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).

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Abstract

The present disclosure provides compounds of Formula I that are inhibitors of histone methyltransferase polycomb repressive complex 2 (PRC2) and its subunits, including embryonic ectoderm development (BED) protein, and are therefore useful for the treatment of diseases treatable by inhibition of dysregulation of PRC2 and EED such as cancers. Also provided are pharmaceutical compositions containing such compounds and processes for preparing such compounds.

Description

PYRIDINE AND DIHYDROPYRIDINE COMPOUNDS AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/483,480, filed on February 6, 2023.
STATEMEN T REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMEN T
[0002] This invention was made with government support under R01 CA256058 and R42 CA217491 awarded by the National Institute of Health (NIH). The government has certain rights in this invention.
BACKGROUND
[0003] Cancer remains one of the deadliest threats to human health. In 2023, 1,958,310 new cancer cases and 609,820 cancer deaths are projected to occur in the United States (ACS Cancer Statistics 2023). Globally, nearly 10 million deaths were attributed to cancer in 2020. By 2040, the global cancer burden is expected to grow to 27.5 million new cancer cases and 16.3 million cancer deaths due to the growth and aging of the population. Additionally, cancers can often become multi-drug resistant, further complicating cancer treatment. It is imperative to develop novel therapeutics to overcome this chemoresistance. However, the development of novel therapeutics incurs significant development costs and long timelines. Repositioning existing approved or generic drugs for alternate uses in cancer therapy is an attractive approach to generate drugs or drug combinations suitable for new medical indications that shorten clinical development costs and timelines. Effectively repositioning drugs for cancer therapeutics and modifying existing drugs to improve performance as anticancer agents to overcome chemoresistance are highly desired. The subject matter described herein addresses this need.
BRIEF SU ARY
[0004] In certain embodiments, the subject matter described herein is directed to compounds of Formula I, which includes all sub-formulae of Formula I:
or a pharmaceutically acceptable salt or solvate thereof, wherein: each instance of 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 selected from the group consisting of a bond, -C(O)-, -C(O)2-, -O-, -S-, -S(O)-, and -S(O)2-; or A is selected from the group consisting of a bond, -C(O)-, -C(O)2-, -CH2O-, -O-, - S-, -S(O)-, and -S(O)2-;
D is optionally substituted Ci-Cs alkylene; such as wherein R5 and R6 are each independently selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted cycloalkyl; optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; or wherein R5 and R6 and the nitrogen to which each are attached together form a heterocycloalkyl wherein R7, R8, R9, and R10 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, and Ci-Ce alkyl; or, two of R7, R8, R9, and R10 or one of R7, R8, R9, and R10 and one of R5 and R6 taken together with the atom to which each is attached form a C3-C7 cycloalkyl or a 4- to 7-membered heterocycloalkyl; wherein m, o, q, and r are each independently 0 or 1 ;
R1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and an unsubstituted aryl; or R1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and optionally substituted aryl;
R2 is selected from the group consisting of H, -OH, -NR2AR2B, -OR2A, -SR2A, optionally substituted C1-C6 alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; wherein R2A and R2B are each independently H or Ci-Ce alkyl;
R3, if present, is H or optionally substituted Ci-Ce alkyl; and
R4 is selected from the group consisting of H, -CN, -OH, halo, -C(O)-(Ci-Ce alkyl), -C(O)2-(C1-C6 alkyl), -S(O)-(Ci-C6 alkyl), -S-(Ci-C6 alkyl), -S(O)2-(Ci-C6 alkyl), optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce alkylamino, optionally substituted Ci-Ce alkoxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl.
[0005] In certain embodiments, the subject matter described herein is directed to pharmaceutical compositions comprising a therapeutically effective amount of a compound of Formula I, which includes all sub-formulae of Formula I, and a pharmaceutically acceptable carrier. [0006] In certain embodiments, the subject matter described herein is directed to methods for the treatment of disorders associated with histone methyltransferases, the method comprising the step of administering to the subject an effective amount of at least one compound of Formula I, which includes all sub-formulae of Formula I.
[0007] In certain embodiments, the subject matter described herein is directed to methods of treatment of disorders associated with histone methyltransferases, the method comprising the step of administering to the subject an effective amount of at least one compound of Formula I, which includes all sub-formulae of Formula I.
BRIEF DESCRIPTION OF THE DRAWINGS
[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.
[0009] Figure 1. (±)-Nicardipine (referred to hereinafter as nicardipine) selectively and potently inhibits chemoresistant PCa cells. (A) In vitro cytotoxicity of nicardipine in the ARCaPE and C4-2B models (72 h). (B) Flow cytometry results on cell cycle in C4-2B-TaxR cells treated with nicardipine at the indicated concentrations (48 h). *** p < 0.001 for all pairwise comparisons between the percentages of cells from the control and nicardipine treatment groups in each cell cycle. (C) Left: flow cytometry analysis on Annexin V staining in C4-2B-TaxR cells treated with nicardipine at the indicated concentrations (72 h). **** p < 0.0001 for all pairwise comparisons between the control and nicardipine treatment groups; Right: Western blot analysis on the expression of apoptotic markers in C4-2B and C4-2B-TaxR cells treated with nicardipine (2.1 pM) at the indicated time points. P-actin was used as the loading control. (D) In vitro cytotoxicity of nicardipine in C4-2, PC-3, ARCaPE, and CWR22Rvl cells (72 h).
[0010] Figure 2. Nicardipine is a putative EED inhibitor. (A) Left and middle: docking poses of nicardipine and EED; Right: two-dimensional ligand-protein interaction diagram of nicardipine in the binding site of human EED protein (PDB ID: 5WUK). The pink arrow indicates the hydrogen bond; the blue-red line indicates the salt bridge; the red line represents pi-cation. (B) Left: CETSA analysis of EED expression in C4-2B-TaxR cells treated with DMSO or nicardipine (50 pM, 1 h). P-actin was used as the loading control; Right: melting temperature curves of EED protein in C4-2B-TaxR cells treated with DMSO or nicardipine. [0011] Figure 3. Nicardipine targets noncanonical EZH2 survival signaling in chemoresistant PCa cells. (A) Western blot analysis on the expression of p-EZH2, EZH2, EED and SUZ12 in C4- 2B and C4-2B-TaxR cells treated with nicardipine (2.1 pM) at the indicated time points. (B) Upper: EZH2 expression in C4-2B-TaxR cells treated with DMSO or nicardipine (2.1 pM) in the presence of CHX (50 pg/ml); Bottom: the calculated half-life of EZH2 protein in C4-2B-TaxR cells treated with DMSO or nicardipine (2.1 pM). (C) Western blot analysis on the expression of H3K27 methylation in C4-2B and C4-2B-TaxR cells treated with nicardipine (2.1 pM) at the indicated time points. (D) Western blot analysis on the expression of p-Stat3, Stat3, SKP2, ABCB1, and survivin in C4-2B and C4-2B-TaxR cells treated with nicardipine (2.1 pM) at the indicated time points. (E) Fluorescence microscopy images of cellular uptake of Oregon Green 488-paclitaxel at the indicated time points in C4-2B-TaxR cells treated with DMSO or nicardipine (2.1 pM) for 72 h before paclitaxel incubation. Scale bar: 50pm. (F) Schematic depiction of the proposed mechanism of action of nicardipine in chemoresistant PCa cells.
[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 pM; 24 h); Right: The top genes affected by nicardipine treatment in C4-2B-TaxR cells. (B) IPA analyses of major gene clusters affected by nicardipine treatment in C4-2B-TaxR cells. (C) GESA of EED-, Gl/S checkpoint-, and EZH2-associated gene signatures 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 score (NES) and false discovery rate (FDR) are indicated for each gene set. Y-axes indicate enrichment scores (top) and ranked list metric (bottom). X-axis bars represent individual genes of the indicated gene sets.
[0013] Figure 5. Nicardipine monotherapy inhibits the skeletal growth of chemoresistant PCa in male athymic nude mice. (A) Left: serum PSA values of C4-2B-TaxR tumor-bearing mice treated with vehicle control (n = 4), docetaxel (5 mg/kg, i.p., once per week; n = 3), or nicardipine (5 mg/kg, i.p., three times per week; n = 5); Right: two-way ANOVA analysis of the PSA values between different treatment groups. * p < 0.05. (B) Left: Average body weights of C4-2B-TaxR tumor-bearing mice in different treatment groups; Middle: pairwise comparison of the body weights between different treatment groups; Right: percentage of body weight change of C4-2B- TaxR tumor-bearing mice in different treatment groups. ** p < 0.01. [0014] Figure 6. Nicardipine synergistically enhances the in vivo efficacy of docetaxel against the skeletal growth of C4-2 tumors in male athymic nude mice. (A) Left: In vitro cytotoxicity of docetaxel in C4-2 cells in varying concentrations of nicardipine (72 h); Right: CompuSyn analyses of the synergistic effect between docetaxel and nicardipine in C4-2 cells. Fa: fraction affected; CI: combination index. (B) Left: serum PSA values of C4-2-Luc tumor-bearing mice treated with vehicle control (n = 5), docetaxel (5 mg/kg, i.p., once per week; n = 5), nicardipine (10 mg/kg, i.p., three times per week; n = 6), or the combination of docetaxel and nicardipine (n = 5); Right: two- way ANOVA analysis of the PSA values between different treatment groups. * p < 0.05, ** p < 0.01, **** p < 0.0001. (C) Left: Average body weights of C4-2-Luc tumor-bearing mice in different treatment groups; Middle: pairwise comparison of the body weights between different treatment groups; Right: percentage of body weight change of C4-2-Luc tumor-bearing mice in different treatment groups. ** p < 0.01.
[0015] Figure 7. Two-dimensional and three-dimensional interactions of the (R)-enantiomer and (S)-enantiomer of nicardipine and the (S)-enantiomer of compound 27 in the binding site of the human EED protein.
DETAILED DESCRIPTION
[0016] Described herein are compounds of Formula I and their uses for the treatment of disorders associated with histone methyltransferases. Also described herein are compounds of Formula I to inhibit dysregulation of poly comb repressive complex 2 (PRC2) and its subunits, including, but not limited to, the embryonic ectoderm development (EED) subunit. Described herein are compounds of Formula I and their uses for the treatment of disorders associated with dysregulation polycomb repressive complex 2 (PRC2) histone methyltransferase. Also described herein are compounds of Formula I and their uses for the treatment of cancers associated with histone methyltransferases.
[0017] Described herein are compounds of Formula I and their uses for the treatment of chemoresistant cancers. Also described herein are compounds of Formula I and their uses for the treatment of chemoresistant cancers associated with histone methyltransferases. Described herein are compounds for Formula I and their uses for the treatment of chemoresistant cancers associated with dysregulation of polycomb repressive complex 2 (PRC2), and its subunits, including, but not limited to, the embryonic ectoderm development (EED) subunit.
[0018] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented herein. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subj ect matter described herein covers 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, or the like, this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
I. Overview
[0019] The compounds described herein exhibit inhibition of dysregulation of histone methyltransferases. The compounds described herein particularly exhibit selective overexpression of various subunits of polycomb repressive complex 2 (PRC2). The compounds described herein are particularly useful in treating cancers, especially chemoresistant cancers, and other related conditions.
[0020] As mentioned above, multiple biological alterations contribute to chemoresistance. The rare subpopulations of cancer cells with sternness or neuroendocrine characteristics are considered intrinsically resistant, evading conventional therapies and resulting in recurrence and metastasis. Epithelial-to-mesenchymal transition (EMT) is a major mechanism by which epithelial cancer cells gain invasive phenotypes, promotes self-renewal capability, and confers chemoresistance. Common mechanisms of chemoresistance include the overexpression of membrane-bound drug efflux pumps (such as ATP binding cassette B 1 [ABCB1), or multidrug resistance protein 1 [MDR1], p-glycoprotein), and anti-apoptotic proteins (such as survivin). For example, S-phase kinase-associated protein 2 (SKP2), the substrate recognition component of SCF (SKP1-CUL1-F- box) E3 ubiquitin-protein ligase complex, increases sternness and chemoresistance in prostate cancer (PCa) cells.
[0021] Polycomb repressive complex 2 (PRC2) plays an essential role in transcriptional repression via mono-, di-, and tri-methylation of histone H3 at lysine 27 (H3K27). The core subunits ofPRC2 include enhancer of zeste homolog 1 or 2 (EZH1 orEZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12). EZH2 is a histone methyltransferase (HMT) and acts as the catalytic “writer” subunit of PRC2 in the transcriptional repression of genes. Aberrant overexpression and activation of EZH2 have been associated with clinical progression and poor prognosis of 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) plays a major role in transcriptional repression via PRC2-dependent histone 3 lysine 27 (H3K27) methylation, as a histone methyltransferase and a subunit of polycomb repressive complex 2 (PRC2). EZH2 also methylates several non-histone protein substrates, such as signal transducer and activator of transcription 3 (Stat3). It acts as a coactivator for androgen receptor (AR), P-catenin and nuclear factor kappa B (NF-KB). This noncanonical function may rely on EZH2 phosphorylation at serine 21 [p-EZH2(S21)]. Particularly, p-EZH2(S21) is significantly increased in mCRPC, a metastatic, castration resistant prostate cancer.
[0023] EZH2 overexpression and mutation, as well as aberrant EZH2 signaling, have been associated with advanced stages and poor clinical outcomes in various types of cancer. Numerous EZH2 inhibitors have been developed, most of which target the catalytic SET domain via 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 indicated that blocking the catalytic activity of EZH2 alone is insufficient and highlighted a need for novel PRC2-targeting strategies. An alternative approach is to develop small-molecule compounds that specifically bind the H3K27me3 -interacting “aromatic cage” in EED and allosterically affect EZH2 enzymatic activity, thereby leading to the loss of PRC2 functions. Interestingly, several allosteric EED inhibitors could also alter physical interactions between EED and other core components of PRCs (mainly EZH2 and SUZ12) and destabilize these proteins. Compared with SAM-competitive EZH2 inhibitors, EED inhibitors could achieve a general and more efficient blockade of the PRC2 oncogenic signaling in highly heterogeneous cancer cells. An EED inhibitor (MAK683) developed by Novartis has entered Phase I/II trials in patients with advanced malignancies (NCT02900651). Another EED inhibitor, ORIC-944 from ORIC Pharmaceuticals, recently entered a Phase I trial in metastatic prostate cancer on June 10, 2022 (NCT05413421). [0024] Recent studies revealed an essential role of noncanonical EED-EZH2 signaling in chemoresistant PCa cells. A small molecule, LG1980, is an EED inhibitor with promising anticancer activity in chemoresistant PCa cells and xenograft models. These results indicated that pharmacological targeting of EED could be a promising strategy to overcome chemoresistance and validated our phenotypic screen as a novel “mechanism-informed phenotypic drug discovery” (MIPDD) platform for discovering effective EED modulators. In a recent screening using the ARCaPE/C4-2B-based platform, several potential inhibitors of chemoresistant PCa were identified, including nicardipine. Described herein is experimental evidence that nicardipine was highly specific and effective against chemoresistant PCa cells in both cellular and animal models. Also described herein is a mechanism of action wherein nicardipine acted as a putative EED inhibitor and inhibited noncanonical EED-EZH2 signaling in chemoresistant PCa cells. These preclinical studies revealed an unexpected function and mechanism of action of nicardipine in chemoresistant cancer cells and could have a significant translational implication.
[0025] Nicardipine is an approved drug for treating hypertension, angina, and related cerebrovascular diseases 19,46. As a second-generation dihydropyridine class of calcium channel blockers (CCBs), nicardipine inhibits the transmembrane influx of calcium into cardiac and smooth muscle without changing serum calcium levels. Given the wide use of CCBs in the management of cardiovascular diseases as well as the well-recognized role of calcium signaling in cancer progression, there has been a longtime interest in the possible effects of CCBs on the clinical outcomes in cancer patients. Since the 1980s, several groups have investigated the potential anticancer activities of nicardipine and other CCBs in preclinical and clinical settings. It appeared that nicardipine could enhance the in vitro and in vivo effects of certain chemotherapeutics, such as vincristine, carmofur, and nimustine, in experimental models of PCa, esophageal cancer, gastric cancer, glioma, and leukemia; however, it was not clear whether the observed effects of nicardipine in human cancer cells were associated with its function as a CCB. In a recent study, Shi et al. found that nicardipine could enhance the toxic effect of temozolomide and promote apoptosis in glioma stem cells (GSCs), probably through the upregulation of mTOR and inhibition of autophagy, which is 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 sometimes conflicting. For example, in three patients with relapsed and chemoresistant non-Hodgkin's lymphoma, nicardipine was found to be capable of increasing the efficacy of vinca alkaloids; in contrast, nicardipine failed to improve adriamycin and vinca alkaloid in seventeen patients with solid tumors or hematologic malignancy. Although these studies were largely observational in very small patient cohorts, they suggested that there is no straightforward strategy for using nicardipine or other CCBs for cancer treatment in general patient populations. Supporting this notion, network meta-analyses and trial sequential analyses of 324,168 participants from randomized trials found no significant differences in the risk of cancer or cancer-related death with CCBs or other individual classes of anti-hypertensive drugs. [0027] The translational potential of these putative EED inhibitors was the primary criterion for selecting nicardipine and evaluating its anticancer activities in preclinical models of chemoresistant PCa. As an approved drug for treating chronic cardiovascular diseases, nicardipine exhibits excellent long-term safety profiles in humans. This drug also has favorable pharmacokinetics and metabolism in terms of its complete absorption and nonlinear accumulation in the circulation following oral administration. The approved schedule of oral administration (i.e., every 8 hours) and a readily measured effect (e.g., change in blood pressure) from nicardipine treatment could allow rapid and convenient adjustments in human trials if any severe adverse effects appear. These pharmacological and physiological features of nicardipine suggested that this drug might have a high potential for further clinical development. In comparison, other approved drugs with high EED-binding affinities (i.e., gallopamil, verapamil) have limited clinical potential, mainly due to their relatively higher toxi cities in human subjects. For example, the anti arrhythmic drug gallopamil was withdrawn in 2001 for causing excessive hypotension, bradycardia, or impaired cardiac performance when combined with -adrenoceptor blockers. Verapamil, another hypertension drug, was thought to be a functional inhibitor of ABCB 1 (p-glycoprotein) that could re-sensitize cancer cells to chemotherapeutics. However, verapamil failed to demonstrate clinical benefits in lung cancer patients, mainly due to its poor pharmacokinetics, high dose-limiting toxicity, and low therapeutic window. [0028] EED protein is the “reader” component of the PRC2 complex, which binds trimethylated H3K27 (H3K27Me3) through the central pocket formed by its seven WD-40 repeats. The interaction between EED and EZH2 is required for the epigenetic “writer” function of EZH2. Therefore, an interruption to the EED-EZH2 complex represents an attractive strategy to inhibit EZH2 functions regardless of the mutation status of the EZH2 enzyme. Furthermore, compared with SAM-competitive inhibitors, targeting EED-EZH2 interaction often results in the degradation of EZH2 protein and core PRC2 components, EED and SUZ12, which may achieve a general and more efficient blockade of EZH2 functions in highly heterogeneous therapeutic-resistant tumors. A few EED inhibitors have been developed, most binding to the central pocket and preventing allosteric activation of the catalytic activity of PRC2. Several compounds showed 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 entered Phase I trials in advanced cancers including metastatic prostate cancer (NCT02900651 and NCT05413421). As such, there is a need for further investigation of EED inhibitors and development of novel PRC2- targeting strategies. The subject matter described herein addresses this need.
[0029] Studies described herein provide preclinical evidence supporting the promise of nicardipine as a targeted agent for cancer treatment. The translational potential of the current disclosure could be two-fold. First, the discovery of nicardipine as a putative EED inhibitor and a potent compound against chemoresistant PCa provided a solid rationale for designing biomarkerbased, subtype-specific trials to test the clinical efficacy of nicardipine in PCa patients. For example, the expression profile of major components of the noncanonical EED-EZH2 signaling axis, including EED, p-EZH2(S21), SKP2, ABCB1 and survivin, can be evaluated in localized tumors from patients with high-volume, high-risk PCa. Nicardipine can be offered to patients with active noncanonical EED-EZH2 signaling as an adjunct therapy in combination with docetaxel and/or ADT, with the expectation of eliminating chemoresistant PCa cells and enhancing the efficacy of standard treatments. Thus, in certain embodiments, the subject matter described herein is directed to methods of treating disorders related to poly comb repressive complex 2 (PRC2) histone methyltransferase and its subunits, the method comprising the step of administering to a subject in need thereof an effective amount of nicardipine, or a compound described in U.S. Patent No. 4,510,310, which is hereby incorporated by reference in its entirety herein. [0030] Given its excellent pharmacological properties and safety profiles as a common antihypertensive, nicardipine could be promptly tested in human trials and integrated with the standard of care for chemoresistant PCa. Second, although nicardipine exhibits excellent clinical safety profiles, the drug was originally developed as an inhibitor of calcium channels and its antihypertensive effect is not a desired and “off-target” feature for cancer treatment. Pharmacologically, nicardipine has a short duration of action (mean I max in plasma =1 h) following oral dosage. Although this feature is ideal for an antihypertensive drug in facilitating dosage titration and therefore is safe for hypertension patients, it is not desired for cancer treatment. As described herein, the suboptimal nature of nicardipine provided an opportunity to obtain highly specific EED inhibitors with improved anticancer activities and drug-like properties. The compounds described herein represent novel first-in-class EED inhibitors with distinct chemical structures and pharmacological activities.
II. Definitions
[0031] As used herein, “Formula I,” includes all sub-formulae of Formula I described herein.
[0032] “Alkyl” as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 12 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, 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” is intended to include both substituted and unsubstituted alkyl unless otherwise indicated and these groups may be substituted with groups selected from halo (e.g., haloalkyl), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl (including spiroalkyl, e.g., C2, C3, or C4 spiroalkyl), cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a 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, amide, sulfonamide, 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 from 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in a heterocyclic group as discussed below) and includes spirocyclics. Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may be optionally substituted with additional substituents as described herein such as halo or loweralkyl. The term “cycloalkyl” is generic and intended to include heterocyclic groups as discussed below unless specified otherwise.
[0034] “Alkylene” as used herein refers to a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of one to eight carbon atoms unless otherwise stated, such as methylene, ethylene, propylene, 1 -methylpropylene, 2- methylpropylene, butylene, pentylene, and the like.
[0035] “Heterocyclic group” or “heterocycloalkyl” as used herein alone or as part of another group, refers to an aliphatic (e.g., fully or partially saturated heterocycloalkyl) or aromatic (e.g., heteroaryl) monocyclic- or a 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. The 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds. A heterocycloalkyl may be a single ring or multiple rings wherein the multiple rings may be fused, bridged or spiro, and may comprise one or more (e.g., 1 to 3) oxo (=0) or N-oxide (N-O ) moieties. Representative examples of monocyclic ring systems that are heterocycloalkyls include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3- dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothi azoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, 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, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems 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, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodi oxine, 1,3 -benzodi oxole, 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 rings may be optionally substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, ester, amide, sulfonamide, nitro or cyano.
[0036] “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. The term “aryl” is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl above.
[0037] “Arylalkyl” or “aralkyl” as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended 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 in connection with heterocycloalkyl above. [0039] “Heteroaralkyl” or “heteroarylalkyl” as used herein alone or as part of another group, refers 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 including substituted versions such as polyalkoxy), appended 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 a -CN group. [0043] “Hydroxyl” as used herein refers to an -OH group.
[0044] “Amino” as used herein means the radical -NH2.
[0045] “Alkylamino” as used herein alone or as part of another group means the radical -NHR, where R is an alkyl group.
[0046] “Disubstituted-amino” as used herein alone or as part of another group means the radical -NRaRb, where Ra and Rb are independently selected from the group alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl.
[0047] ‘Ester” as used herein alone or as part of another group refers to a -C(O)OR radical, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
[0048] “Amide” as used herein alone or as part of another group refers to a -C(O)NRaRb radical, where Ra and Rb are 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 that is bound to a molecule, whereby through the binding, at least one covalent bond has replaced at least one atom of the original chemical moiety, resulting in a residue of the chemical moiety in the molecule.
[0050] The compounds of the present disclosure may have asymmetric centers. Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of materials. All chiral, diastereomeric, all mixtures of chiral or diastereomeric forms and racemic forms are within the scope of this disclosure, unless the specific stereochemistry or isomeric form is specifically indicated. It will also be well recognized by a person skilled in the art that when a bond is drawn from an optically active center, that a “flat” bond ( > - ) represents and encompasses both the “wedge” bond ( and the “dashed” bond each representing the (R) or (S) stereoisomer. It will also be understood by a person of ordinary skill in the art that when a compound is denoted as (R) stereoisomer, it may contain the corresponding (S) stereoisomer as an impurity i.e., the (S) stereoisomer in less than about 5%, preferably 2% by wt. and then it is denoted as a mixture of R and S isomers, the amounts of R or S isomer in the mixture is greater than about 5%, preferably 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 tissues in the body of a living human.
[0052] The term “in vitro” refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube).
[0053] The term “in vivo” refers to natural environments (e.g., 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 where the event or circumstance occurs and instances in which it does not. For example, “heterocyclyl group optionally substituted with an alkyl group” means that the alkyl may but need not be present, and the description includes situations where the heterocyclyl group is substituted with an alkyl group and situations where the heterocyclyl group is not substituted with alkyl. As used herein, the phrase “optionally substituted” means that substitution is optional and therefore includes both unsubstituted and substituted atoms and moieties. A “substituted” atom or moiety indicates that any hydrogen on the designated atom or moiety can be replaced with a selection from the indicated substituent groups, provided that the normal valency of the designated atom or moiety is not exceeded, and that the substitution results in a stable compound. For example, if a methyl group is optionally substituted, then 3 hydrogen atoms on the carbon atom can be replaced with substituent groups. Also, double bonds depicted in the structure follow normal valency and can be conjugated or can represent an aromatic system.
[0055] A “subject” refers to an animal that is the object of treatment, observation or experiment. “Animal” includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and, in particular, mammals. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees and apes, and, in particular, humans. In some embodiments, the subject can be human. In some embodiments, the subject can be a human child and/or a human infant, for example, 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 that has been diagnosed with or is believed to be suffering from one or more disorders associated with dysregulation polycomb repressive complex 2 (PRC2) histone methyltransferase. [0056] “Treating” or “treatment” of a disease includes:
(1) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease;
(2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or
(3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms. [0057] A “therapeutically effective amount” means the amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof that, elicits the biological or medicinal response indicated. For example, when administered to a subject for treating a disease, the therapeutically effective amount of a compound is sufficient to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. This response may occur in a tissue, system, animal or human and includes alleviation of the signs or symptoms of the disease being treated. Determination of an effective amount is well within the capability of those skilled in the art, in view of the disclosure provided herein. The “therapeutically effective amount” of the compounds disclosed herein will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated. Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
[0058] Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ± 0.5%, 1%, 5%, or 10% from a specified value.
[0059] Compositions or methods “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 forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an antigen” or “at least one antigen” can include a plurality of antigens, including mixtures thereof.
[0061] Statistically significant means p <0.05. III. Compounds
[0062] In certain embodiments, described herein are compounds that are a novel class of inhibitors of PCR2 and its subunits. In certain embodiments described herein, the compounds are a novel class of EED inhibitors. In certain embodiments described herein, the novel class of EED inhibitors have unique chemical structures and display high specificity and potency against disorders associated with EED dysregulation.
[0063] In certain embodiments, the disorders associated with EED dysregulation are cancers. In certain embodiments, the cancers are chemoresistant cancers. In certain embodiments described herein, the compounds are a novel class of EED inhibitors for treating chemoresistant cancers. In certain embodiments, the compounds described herein are a novel class of EED inhibitors from treating chemoresistant prostate cancer (PCa). In certain embodiments, the compounds described herein display high specificity and potency against chemoresistant PCa.
[0064] Described herein are compounds that serve as new interventions for chemoresistant cancers. Described herein are compounds that are effective as targeted therapy for chemoresistance cancers. In certain embodiments described herein, the chemoresistant cancer is chemoresistance prostate cancer (PCa).
[0065] In certain embodiments, the subject matter described herein is directed to compounds of Formula I: or a pharmaceutically acceptable salt or solvate thereof, wherein: each instance of 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 selected from the group consisting of a bond, -C(O)-, -C(O)2-, -O-, -S-, -S(O)-, and -S(O)2-; or A is selected from the group consisting of a bond, -C(O)-, -C(O)2-, -CH2O-, -O-, - S-, -S(O)-, and -S(O)2-;
D is optionally substituted Ci-Cs alkylene; wherein R5 and R6 are each independently selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted cycloalkyl; optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; or wherein R5 and R6 and the nitrogen to which each are attached together form a heterocycloalkyl.
R1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and an unsubstituted aryl; or R1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and an optionally substituted aryl;
R2 is selected from the group consisting of H, -OH, -NR2AR2B, -OR2A, -SR2A, optionally substituted Ci-Ce alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; wherein R2A and R2B are each independently H or Ci-Ce alkyl;
R3, if present, is H or optionally substituted Ci-Ce alkyl; and
R4 is selected from the group consisting of H, -CN, -OH, halo, -C(O)-(Ci-Ce alkyl), -C(O)2-(C1-C6 alkyl), -S(O)-(Ci-C6 alkyl), -S-(Ci-C6 alkyl), -S(O)2-(Ci-C6 alkyl), optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce alkylamino, optionally substituted Ci-Ce 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 selected from the group consisting of :
[0067] In certain embodiments, D is wherein R7, R8, R9, and R10 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, and Ci-Ce alkyl; or, wherein two of R7, R8, R9, and R10 or one of R7, R8, R9, and R10 and one of R5 and R6 taken together with the atom to which each is attached form a C3-C7 cycloalkyl or a 4- to 7-membered heterocycloalkyl; wherein m, o, q, and r are each independently 0 or 1.
[0068] In certain embodiments, D is selected from the group consisting of:
[0069] In certain embodiments, D-X is selected from the group consisting of:
[0070] In certain embodiments, the subject matter described herein is directed to compounds of Formula la: or a pharmaceutically acceptable salt or solvate thereof.
[0071] In certain embodiments, the subject matter described herein is directed to compounds of Formula lb: or a pharmaceutically acceptable salt or solvate thereof.
[0072] In certain embodiments, the subject matter described herein is directed to compounds of Formula 1-1 : or a pharmaceutically acceptable salt or solvate thereof.
[0073] In certain embodiments, the subject matter described herein is directed to compounds of Formula la- 1 or lb-1 : or a pharmaceutically acceptable salt or solvate thereof.
[0074] In the above embodiments, compounds include those wherein A is selected from the group consisting of -C(O)- and -C(O)2-.
[0075] In certain embodiments, compounds include those having a structure of Formula la-1, wherein p is 0 and R1 is selected from the group consisting of H, optionally substituted Ci-Cio alkyl, optionally substituted C1-C10 cycloalkyl, and an optionally substituted aryl. In certain embodiments, p is 0 and R1 is phenyl, m-nitrophenyl, cyclobutyl, cyclopentyl, cyclohexyl, methyl, ethyl, propyl, isopropyl, isobutyl, or 3-pentyl.
[0076] In certain embodiments, compounds include those having a structure of Formula la-1, wherein p is 1 and R1 is optionally substituted C1-C10 cycloalkyl. In certain embodiments, p is 1 and R1 is cyclobutyl, cyclopentyl, or cyclohexyl.
[0077] In certain embodiments, compounds include those having a structure of Formula la-1, wherein R2 is -OR24, wherein R2A is Ci-Ce alkyl. In certain embodiments, R2 is OCH3.
[0078] In certain embodiments, compounds include those having a structure of Formula la-1, wherein R3 is H.
[0079] In certain embodiments, compounds include those having a structure of Formula la-1, wherein R4 is methyl.
[0080] In certain embodiments, compounds include those having a structure of Formula la-1, wherein A is -C(O)2-.
[0081] In certain embodiments, compounds include those having a structure of Formula la-1, wherein D is selected from the group consisting of :
[0082] In certain embodiments, compounds include those having a structure of Formula la-1, wherein,
[0083] In certain embodiments, compounds include those having a structure of Formula la-1, wherein R2 is OCH3, R3 is H, R4 is CH3, and A is -C(O)2-.
[0084] In certain embodiments, compounds include those having a structure of Formula la-1, wherein R2 is OCH3, R3 is H, R4 is CH3, A is -C(O)2-, p is 0, and R1 is optionally substituted aryl. [0085] In certain embodiments, compounds include those having a structure of Formula la-1 , wherein
[0086] In certain embodiments, compounds include those having a structure of Formula la-1, wherein R2 is OCH3, R3 is H, R4 is CH3, A is -C(O)2-, p is 0, R1 is optionally substituted aryl, and
[0088] In above embodiments, compounds include those wherein X is ve embodiments, compounds include those wherein X is
[0090] In the above embodiments, compounds include those wherein D comprises one or more asymmetric centers.
[0091] In the above embodiments, compounds include those wherein R2 is selected from the group consisting of -NR2AR2B, -OR2A, and optionally substituted Ci-Ce alkyl, wherein R2A and R2B are each independently H or Ci-Ce alkyl. [0092] In the above embodiments, compounds include those wherein R2 is -OR2A, wherein R2A is Ci-Ce alkyl.
[0093] In the above embodiments, compounds include those wherein R2 is -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)2, -O(CH2)3CH3, -OCH(CH3)3, -OCH2CH(C H3)2, and -OCH(CH3)CH2CH3.
[0094] In the above embodiments, compounds include those wherein R2 is -OCH3.
[0095] In the above embodiments, compounds include those wherein R4 is selected from the group consisting of -C(O)-(Ci-Ce alkyl), -C(O)2-(Ci-C6 alkyl), and optionally substituted Ci-Ce alkyl.
[0096] In the above embodiments, compounds include those wherein R4 is optionally substituted Ci-Ce alkyl.
[0097] In the above embodiments, compounds include those wherein R4 is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl or hexyl. [0098] In the above embodiments, compounds include those wherein R4 is methyl.
[0099] In the above embodiments, compounds include those wherein R4 is -C(O)2-(Ci-C6 alkyl).
[0100] In the above embodiments, compounds include those wherein R4 is -C(O)2CH3.
[0101] In the above embodiments, compounds include those wherein R1 is selected from the group consisting of optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, and optionally substituted C1-C10 alkoxy.
[0102] In the above embodiments, compounds include those wherein R1 is optionally substituted Ci-Cs alkyl.
[0103] In the above embodiments, compounds include those wherein R1 is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, heptyl, or octyl.
[0104] In the above embodiments, compounds include those wherein R1 is methyl, ethyl, propyl, butyl, isobutyl, or ec-butyl.
[0105] In the above embodiments, compounds include those wherein R1 is optionally substituted C1-C10 cycloalkyl. [0106] In the above embodiments, compounds include those wherein R1 is selected from the
[0107] In the above embodiments, compounds include those wherein
[0108] In the above embodiments, compounds include those wherein R1 is optional substituted Ci-Cio heterocycloalkyl, wherein the heteroatom is one or more of S, O, or N.
[0109] In the above embodiments, compounds include those wherein R1 is selected from the
[0110] In the above embodiments, compounds include those wherein R1 is an unsubstituted aryl.
[0U1] In the above embodiments, compounds include those wherein R1 is an unsubstituted phenyl.
[0112] In the above embodiments, compounds include those wherein R1 is a substituted aryl.
[0113] In the above embodiments, compounds include those wherein R1 is a substituted phenyl.
[0114] In the above embodiments, compounds include those wherein R1 is a nitro substituted aryl.
[0115] In the above embodiments, compounds include those wherein R1 is a nitro substituted phenyl.
[0116] In the above embodiments, compounds include those wherein R1 is
[0117] In the above embodiments, compounds include those wherein R1 is H.
[0118] In the above embodiments, compounds include those wherein p is 0.
[0119] In the above embodiments, compounds include those wherein p is 1. [0120] In the above embodiments, compounds include those having a structure as shown below in Table A:
[0121] In the above embodiments, compounds include those having a structure shown in
Table A, wherein D is selected from the group consisting of:
[0122] In the above embodiments, compounds include those having a structure shown in
Table A, wherein D-X is selected from the group consisting of:
[0123] In the above embodiments, compounds include those as shown below in Table 1 :
[0124] In certain embodiments, the compounds include those of Formula I, or pharmaceutically acceptable salts thereof, where the compounds inhibit one or more proteins of the poly comb repressive complex 2 (PRC2).
[0125] In certain embodiments described herein, the compounds of Formula I or pharmaceutically acceptable salts or solvates thereof, display selectivity for inhibition of 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] A “pharmaceutically acceptable excipient” refers to a vehicle for containing a functionalized cell or an acellular extracellular matrix that can be introduced into a subject without significant adverse effects and without having deleterious effects on the functionalized cell or acellular extracellular matrix. That is, “pharmaceutically acceptable” in the context of a formulation refers to any formulation which is safe and provides the appropriate delivery for the desired route of administration of an effective amount of at least one functionalized cell or acellular extracellular matrix for use in the methods disclosed herein. Pharmaceutically acceptable carriers or vehicles or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers, and factors involved in their selection, are found in a variety of readily available sources such as, for example, Remington 's Pharmaceutical Sciences, 18th ed., 1990, herein incorporated 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 can be buffered, for example, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the functionalized cell or acellular extracellular matrix and route of administration.
[0128] Suitable pharmaceutically acceptable carriers include, for example, sterile water, salt solutions such as saline, glucose, buffered solutions such as phosphate buffered solutions or bicarbonate buffered solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like. Pharmaceutical compositions or vaccines may also include auxiliary agents 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, coloring, flavoring, aromatic substances, and the like which do not deleteriously react with a functionalized cell or an acellular extracellular matrix. [0129] For liquid formulations, for example, pharmaceutically acceptable carriers may 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, including 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, a gum, a starch (e.g., corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, or dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
[0130] Optionally, sustained or directed release pharmaceutical compositions or vaccines can be formulated. This can be accomplished, for example, through use of liposomes or compositions wherein the active compound is protected with differentially degradable coatings (e.g., by microencapsulation, multiple coatings, and so forth). Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the compositions and use the lyophilisates obtained (e.g., for the preparation of products for injection).
V. Therapeutic Methods
[0131] In certain embodiments, the subject matter described herein is directed to methods of treating disorders, the method comprising the step of 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 methods of treating disorders related to polycomb repressive complex 2 (PRC2) histone methyltransferase and its subunits, the method comprising the step of administering to a subject in need thereof an effective amount of at least one compound or pharmaceutical salt thereof described in U.S. Patent No. 4,510,310, which is hereby incorporated by reference in its entirety herein. In certain aspects, the compound is nicardipine.
[0133] In certain embodiments described herein, the disorder is associated with histone methyltransferases.
[0134] In embodiments described herein, the disorder is associated with polycomb repressive complex 2 (PRC2) histone methyltransferase and its subunits. [0135] In certain embodiments, the disorder is associated with embryonic ectoderm development (EED) subunit of PRC2.
[0136] In certain embodiments, the subject matter described herein is directed to methods of treating a cancer, the method comprising the step of 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 embodiments described herein, the cancer is associated with dysregulation of polycomb repressive complex 2 (PRC2) and its subunits.
[0139] In embodiments described herein, the cancer is associated with the dysregulation of 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 in the treatment of cancer. The additional compounds may optionally be administered concurrently. As used herein, the word “concurrently” means sufficiently close in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more events occurring within a short time period before or after each other).
[0141] The present subject matter is primarily concerned with the treatment of human subjects, but the invention may also be carried out 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 infant, juvenile, adolescent, adult, and geriatric subjects.
[0142] As noted above, the subject matter provides pharmaceutical formulations comprising the compounds of Formula I (including the pharmaceutically acceptable salts thereof), in pharmaceutically acceptable carriers for oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, or intravenous, and transdermal administration.
[0143] The therapeutically effective dosage of any specific compound can vary somewhat from compound to compound, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the active compound, including the cases where a salt is employed. Toxicity concerns at the higher level may restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the active base, including the cases where a salt is employed. A dosage from about 10 mg/kg to about 50 mg/kg may be employed for oral administration. In some embodiments, a dosage from about 0.5 mg/kg to 5 mg/kg may be employed for intramuscular injection. In some embodiments, dosages are 1 pmol/kg to 50 pmol/kg, and more preferably 22 pmol/kg and 33 pmol/kg of the compound for intravenous or oral administration. The duration of the treatment can be once per day 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.
EXAMPLES
Materials and methods
I. Cell culture and reagents
[0145] Human PCa ARCaPi cells stably expressing human EPLIN short hairpin RNA (shRNA) (ARCaPE-shEPLIN) or control shRNA (ARCaPE-shCtrl) were established and cultured as we described in 18,20. C4-2B and its docetaxel-resistant derivative C4-2B-TaxR were cultured following the procedures 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 (Coming Inc, Corning, NY, USA). C4-2-Luc cells were cultured in the same media as C4-2 with additional G418 (Thermo Fisher Scientific, Waltham, MA) at 400 pg/mL. Human PC-3 cells were routinely maintained in RPMI 1640 medium (Corning Inc) supplemented with 10% FBS and penicillin-streptomycin. Human CWR22Rvl 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 Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MD, USA) following the manufacturer’s instruction. The half- minimal inhibitory concentrations (IC50) of the specified agent were calculated with 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 calculation
[0146] The three-dimensional (3D) structures of tested compounds (nicardipine, losartan, EED226, bromocriptine, and metformin) were retrieved from PubChem and built using the Maestro program (Schrodinger, New York, NY, USA), as we described previously 18. All the tested compounds were prepared using Ligprep in Maestro 12.4. The structure of EED protein (PDB ID: 5WUK) was retrieved from RCSB’s Protein Data Bank 22. Using the Protein Preparation Wizard in Maestro, the protein structure was prepared through three steps: preprocessing, optimization, and minimization 23. Preprocessing includes assigning bond orders, adding hydrogens, creating disulfide, and generating het states using Epik 24,25. The process of optimization optimizes hydrogen bonds by using PROPKA 26. The step of minimization is performed by using the OPLS3e force field 27. A receptor grid box was generated based on the five residues (Phe97, Tyrl48, Trp364, Tyr365, Arg367) around the binding site. The size of the receptor grid box was set as default (20 A). Ligand-protein docking was performed in extraprecision (XP) mode using the Ligand Docking panel. After molecular docking, the binding energies were calculated using Prime MM-GBSA (molecular mechanics generalized Bom surface area) in Maestro Program.
III. Cellular thermal shift assay (CETSA)
[0147] CETSA was performed following a modified procedure described in 18. C4-2B-TaxR cells were incubated for 1 h in the presence of DMSO or nicardipine (50 pM). A MyCyclerTM thermal cycler system with a gradient option (Bio-Rad Laboratories, Hercules, CA, USA) was used to incubate total cell lysates at varying temperatures.
IV. RNA-seq analysis
[0148] RNA samples were collected from C4-2B-TaxR cells treated with DMSO or nicardipine (2.1 pM) for 24 h in triplicates. RNA-seq analyses were performed by Omega Bioservices (Norcross, GA, USA). Data were analyzed by Rosalind® (Rosalind, Inc., San Diego, CA, USA), Ingenuity Pathway Analysis (IP A, 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 2x 106 C4-2B-TaxR or C4-2-Luc cells suspended in 20 pl PBS were injected into the bilateral tibia of male athymic nude mice (5-week-old, Envigo RMS, Inc, Indianapolis, IN). Tumor establishment in mouse bones was confirmed by rising serum levels of human prostatespecific 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 3 groups and treated with vehicle control (DMSO), docetaxel, or nicardipine, respectively, at the indicated doses and schedule via intraperitoneal (i.p.) injection. For the C4-2-Luc xenograft model, an additional group of mice was treated with the combination of docetaxel and nicardipine at the indicated doses and schedule via i.p. injection. The vehicle control and docetaxel treatment groups in the C4-2B-TaxR and C4-2-Luc xenografts were the same as those described in a previous study 18. Body weights were monitored twice a week. Intratibial growth of tumors was followed by weekly PSA measurements.
VI. Statistical analysis
[0150] The unpaired t-test was performed to examine the significant difference between the means of any two groups. Two-way analysis of variance (ANOVA) was performed to measure the significant difference 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 screen for identification of selective inhibitors of chemoresistance
[0151] A two-tier phenotypic screening system to identify selective inhibitors of chemoresistant PCa cells was established. In this platform, the primary screening was based on epithelial protein lost in neoplasm (EPLIN) which serves as a molecular regulator of metastasis and chemoresistance. ARCaPE-shEPLIN cells stably expressing EPLIN shRNA are highly resistant to docetaxel compared with ARCaPE-shCtrl cells, thus representing the characteristics of the intrinsic chemoresistance. Primary screening was performed to identify small-molecule compounds that selectively inhibit ARCaPE-shEPLIN, but not ARCaPE-shCtrl cells. Primary hits were further validated in a second (orthogonal) assay for their high potency against C4-2B-TaxR cell s, a cellular model representing acquired chemoresistance, but not in docetaxel-sensitive parental C4-2B cells.
[0152] These models could closely mimic the complex biology and high heterogeneity of chemoresistant PCa. The activation of noncanonical EZH2 signaling represents a novel mechanism of chemoresistance in PCa cells. Specifically, phosphorylation of EZH2 at serine 21 (p-EZH2[S21]) activates a survival signaling pathway consisting of signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B 1 (ABCB1, p-glycoprotein) and survivin, thereby conferring chemoresistance. Furthermore, a smallmolecule compound, namely LG1980, effectively interrupts the physical interaction between EED and ZEH2, disassembles PRC2, and promotes the degradation of its core components, thereby inhibiting p-EZH2(S21) and suppressing the expression of its downstream effectors. Significantly, LG1980 demonstrated high specificity and potent efficacy against the in vitro and in vivo growth of chemoresistant PCa cells. These results indicated that the ARCaPE-shEPLIN and C4-2B-TaxR cells could be exploited as a MIPDD, mechanism-informed phenotypic drug discovery as introduced by Moffat et. al., platform for the discovery of novel inhibitors of chemoresistant PCa. Using this platform, several FDA-approved, non-oncology drugs were identified that selectively and effectively inhibit the in vitro and in vivo growth of chemoresistant PCa cells. An antihypertensive drug, nicardipine, acts as a specific and potent inhibitor of chemoresistant PCa in preclinical models. Intriguingly, mechanistic studies demonstrated that nicardipine might function as an EED inhibitor that disrupts the noncanonical EZH2 signaling and confers its anticancer activities in chemoresistant PCa cells.
II. Nicardipine displays high selectivity and potency against chemoresistant PCa
[0153] Using the ARCaPE/C4-2B screen platform, nicardipine, an anti-hypertensive drug, was identified as a potential inhibitor of chemoresistant PCa cells. When the compound was tested at a single concentration of 12.3 pM, nicardipine exhibited a high selectivity index (SI) of 8.9, where the SI was defined as the ratio of the percentage of inhibition on viability in ARCaPE-shEPLIN cells and that in ARCaPE-shCtrl cells; only 1.1% of chemoresistant ARCaPE-shEPLIN cells survived following the treatment. To confirm the selectivity of nicardipine in chemoresistant PCa cells, its half-minimal inhibitory concentration (IC50) was determined in the ARCaPE- shEPLIN/ARCaPE-shCtrl and C4-2B-TaxR/C4-2B pairs. Nicardipine had an IC50 of 29.1 p in ARCaPi -shCtrl cells and 0.5 pM in ARCaPE-shEPLIN cells, with a fold of difference of 58.2 (Fig. 1 A, left). Consistently, nicardipine demonstrated higher cytotoxicity in C4-2B-TaxR cells (IC50 = 2.1 pM) than in C4-2B cells (IC50 > 51.2 pM), with a fold of difference of > 24.4 (Fig. 1 A, right). Flow cytometry analyses showed that compared with vehicle control, nicardipine treatment at 2.0 pM and 4.0 pM significantly induced cell cycle arrest at both Gl-S and G2-M checkpoints with an accumulation of a sub-Gl population representing apoptotic cells (Fig. IB). Nicardipine treatment also significantly induced apoptosis dose-dependently, as demonstrated by increased surface staining of Annexin V, a marker of apoptosis (Fig. 1C, left). Western blotting analyses 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 (Fig. 1C, right).
[0154] The in vitro cytotoxicity of nicardipine was further determined in several commonly used PCa lines, i.e., ARCaPE, C4-2, CW22Rvl, and PC-3. These cell lines have distinct genetic backgrounds and represent different aspects of PCa progression, but are relatively sensitive to docetaxel treatment (Table 2). Interestingly, nicardipine had low potency in these chemosensitive PCa cells, with its IC50 values ranging from 15.0 pM to > 32.0 pM (Fig. ID). These results indicated that nicardipine had high selectivity and potency against chemoresistant PCa cells.
[0155] Table 2. In vitro cytotoxicity of nicardipine in established PCa cell lines (72h)
III. Nicardipine ’s molecular interaction ns with EED
[0156] EED is the “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 serves as a scaffolding protein to interact with EZH2 and SUZ12 and maintain the integrity of the PRC2 complex. Current EED inhibitors, including EED226, A-395, and BR-001, target the histone-binding central pocket, or the “aromatic cage,” formed by the seven WDRs in EED and prevent allosteric activation of the catalytic activity of PRC2. Previous studies have identified LG1980 as a novel EED inhibitor that effectively blocks noncanonical EZH2 survival signaling and selectively targets chemoresistant PCa cells. To determine whether nicardipine exerts its anticancer effect via a similar mechanism of action, the following experiments were performed: (1) Molecular docking analyses demonstrated that (±)-nicardipine bound the “aromatic cage” of EED, interacting with Phe97 and Tyr365 through Pi-cation interactions, and with Arg414 by forming a salt bridge. Additionally, a hydrogen bond was formed between nicardipine and Arg414 and Trp364, respectively (Fig. 2A). The binding energy between nicardipine and EED was calculated as -65.25 kcal/mol, suggesting that nicardipine had 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 examined calcium channel modulators, losartan, and metformin. As the positive control, LG1980 had the highest binding affinity to EED with the calculated energy of -73.62 kcal/mol (Table 3); (2) Cellular thermal shift assay (CETSA) was performed to determine the intracellular binding of nicardipine and EED protein in live C4-2B-TaxR cells. Compared with vehicle control, nicardipine treatment shifted the melting temperature (Tm) of EED protein from 49.8°C to 50.4°C, indicating that nicardipine could specifically bind and stabilize EED protein in live cancer cells (Fig. 2B). Taken together, these computational and experimental studies indicated that nicardipine could be a novel EED inhibitor.
[0157] Table 3. Predicted binding energies (kcal/mol) between tested compounds/drugs and human EED protein (PDB ID: 5WUK).
IV. Nicardipine inhibiis noncanonical EZH2-Stat3-SKP2-ABCB1 /surviving signaling in chemoresistant prostate cancer (PCa)
[0158] The integrity and function of the PRC2 complex rely 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 the two cell lines. Treatment with nicardipine at 2.1 pM effectively downregulated EZH2, p- EZH2(S21), EED and SUZ 12 in a time-dependent manner in C4-2B-TaxR cells, but notin parental C4-2B cells (Fig. 3 A). Nicardipine’s affect EZH2 protein stability in chemoresistant PCa cells was ascertained, which would lead to reduced expression of EZH2 and p-EZH2(S21). A CHX chase experiment showed that in the presence of nicardipine, the half-life (Ti,) of EZH2 protein was significantly shortened from > 48 h to 14.8 h (Fig. 3B). This result indicates that nicardipine might facilitate EZH2 degradation via a proteasome-mediated mechanism.
[0159] Nicardipine’s effect on the canonical function of EZH2 on histone methylation was evaluated further. As previously discovered, there was no significant difference in the basal expression of H3K27me3 between C4-2B and C4-2B-TaxR cells, indicating that canonical EZH2 signaling may not play a dominant role in PCa chemoresistance. Treatment with nicardipine at 2.1 pM did not affect the tri-methylation of H3K27 in either C4-2B or C4-2B-TaxR cells during a 72 h period. In comparison, nicardipine significantly inhibited the mono-methylation of H3K27 in a time-dependent manner, starting at 24 h, and reduced H3K27 di-methylation after 48 h in C4-2B- TaxR cells but not in C4-2B cells (Fig. 3C). These results indicated that the anticancer activity of nicardipine in chemoresistant PCa cells might be independent of the canonical HMT function of EZH2.
[0160] The effect of nicardipine on the expression of core components of a novel noncanonical
EZH2 signaling pathway in chemoresistant PCa cells was also determined. p-EZH2(S21), p- Stat3(S727), SKP2, ABCBl 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, ABCBl, and survivin in chemoresistant C4-2B-TaxR cells but not in C4-2B cells (Fig. 3D). These results suggested that nicardipine may effectively target noncanonical EZH2-Stat3-SKP2-ABCBl/survivin signaling and inhibit the proliferation and viability of chemoresistant PCa cells.
V. Nicardipine facilitates cellular uptake of chemotherapeutics in chemoresistant PCa cells [0161] Overexpression of ABCB 1 (p-glycoprotein) has been recognized as a central molecular mechanism in the multidrug resistance (MDR). Consistently, previous studies have shown that ABCBl depletion effectively increased the intracellular presence of chemotherapeutics in chemoresistant PCa cells. Since nicardipine significantly reduced ABCB1 protein levels in chemoresistant C4-2B-TaxR cells, nicardipine’s facilitation of the uptake of Oregon Green 488- conjugated paclitaxel was explored. Nicardipine pre-treatment resulted in a rapid (within 15 min) accumulation of fluorescent paclitaxel in C4-2B-TaxR cells (Fig. 3E). In comparison, there was no paclitaxel uptake until 30 min in control cells. These results indicated that nicardipine-mediated ABCB1 downregulation could contribute to the increased uptake and retention of chemotherapeutics in chemoresistant PCa cells. Based on these molecular and cellular results, nicardipine may bind EED and induce protein degradation of EZH2, thereby reducing p-EZH2 and suppressing Stat3/SKP2/ABCBl/survivin survival signals in chemoresistant PCa cells (Fig. 3F).
VI. Nicardipine affects multiple genes implicated in the control of the cell cycle in chemoresistant PCa cells.
[0162] To obtain an unbiased view of the mechanism of action of nicardipine in chemoresistant PCa cells, RNA-seq analyses were performed and gene expression in C4-2B-TaxR cells treated with vehicle control or nicardipine (2.1 pM, 24 h) were compared. When an adjusted p-value (p-adj) < 0.05 was used, 336 unique genes were upregulated, and 259 unique genes were downregulated significantly following nicardipine treatment (Figure 4A). IPA profiling found that the top canonical pathways affected by nicardipine included cell cycle control and DNA damage responses, which were in line with the known functions of PRC2 in cancer cells (Table 4). Among the significant signaling nodes affected by nicardipine treatment, CDKN1A (p21)-, TP53- and RBI-related genes were activated, whereas E2F-regulated genes were suppressed (Figure 4B). GSEA studies confirmed the inhibitory effect of nicardipine on EED- and cell cycle-related genes (p = 0.026 and 0.015, respectively). Nicardipine appeared to activate EZH2-repressed genes, although with low statistical significance (p = 0.05) (Figure 4C). These results supported a mechanism of action that nicardipine induces cell cycle arrest and apoptosis in chemoresistant PCa cells via PRC2-mediated signaling.
[0163] Table 4. Top canonical pathways affected by nicardipine in C4-2B-TaxR cells
VII. Nicardipine inhibits the skeletal growth of chemoresistant C4-2B-TaxR tumors
[0164] A notable feature of nicardipine was that as a single agent, it demonstrated high selectivity and potency in chemoresistant PCa cells, with an IC50 of 0.5 pM in ARCaPE-shEPLIN cells and IC50 = 2.1 pM in C4-2B-TaxR cells, respectively (Figure 1A). To further elucidate the in vivo efficacy of nicardipine against the skeletal growth of chemoresistant cancers, C4-2B-TaxR cells, a model closely mimicking the clinicopathology of AR-positive, chemoresistant, and bone metastatic PCa, were used. Serum levels of human prostate-specific antigen (PSA) were measured as the primary indicator of xenograft growth in mouse bones (Figure 5A). At the endpoint, the average PSA level of each group was determined as 43.47±15.62 ng/ml (control), 36.63±19.75 ng/ml (docetaxel, 5 mg/kg, once per week) and 28.78±11.60 ng/ml (nicardipine, 5 mg/kg, three times per week). Compared with vehicle control (p = 0.020) or docetaxel (p = 0.039), intraperitoneal (i.p.) injection of nicardipine significantly inhibited the growth of C4-2B-TaxR tumors in mouse tibias. On the other hand, docetaxel treatment did not significantly affect the in vivo growth of PCa cells (p = 0.741). Nicardipine treatment was not associated with obvious in vivo toxicity or reduced body weights of mice (Figure 5B). These results indicated that as a monotherapy, nicardipine could effectively suppress the in vivo growth of chemoresistant PCa xenografts in mouse bones with a good safety profile.
VIII. Nicardipine enhances the in vivo efficacy of docetaxel and inhibits the skeletal growth of C4-2 xenografts
[0165] Compared with an IC50 at the low-micromolar range (2.1 pM) in C4-2B-TaxR cells, nicardipine had relatively weak cytotoxicity in C4-2 cells (IC50 = 15.0 pM) that exhibited typical phenotypes of docetaxel-responsive PCa (IC50 = 0.72 pM; Figure 6A). Interestingly, when C4-2 cells were treated with a combination of nicardipine and docetaxel, the two drugs demonstrated a synergistic inhibitory effect on the in vitro proliferation of C4-2 cells, which was reflected by the combination indexes (Cis) lower than 1.0 in isobologram analyses 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 tibiae of male athymic nude mice. Tumor-bearing mice were treated with vehicle control, docetaxel, nicardipine, and the combination of docetaxel and nicardipine, respectively. At the endpoint, the average PSA level of each group was determined as 79.17±17.92 ng/ml (control), 49.04±14.92 ng/ml (docetaxel, 5 mg/kg, once per week), 42.80±9.29 ng/ml (nicardipine, 10 mg/kg, three times per week), and 23.23±6.27 ng/ml (docetaxel and nicardipine). Compared with the vehicle control, docetaxel could moderately retard the in vivo growth of C4-2 tumors (p = 0.048), and nicardipine monotherapy was ineffective in suppressing tumor growth compared with the vehicle control (p = 0.090) or docetaxel (p = 0.706). However, the combination of nicardipine and docetaxel significantly decreased serum PSA levels compared to the treatment with vehicle control (p < 0.0001), docetaxel (p = 0.0025), or nicardipine (p = 0.0020) (Figure 6B). Compared with vehicle control, docetaxel treatment significantly decreased the body weight, whereas the combination of nicardipine and docetaxel increased the body weight compared to the docetaxel group (Figure 6C). These results indicated that as an adjunct agent, nicardipine could be effective in enhancing the in vivo efficacy of docetaxel against the skeletal growth of C4-2 tumors. [0166] Table 5. Combination index (CI) data for the combinational treatment with docetaxel and nicardipine
[0167] Although nicardipine exhibits excellent clinical safety profiles, the drug was originally developed as an inhibitor of calcium channels and its antihypertensive effect is not a desired and “off-target” feature for cancer treatment. Pharmacologically, nicardipine has a short duration of action (mean A max in plasma =1 h) following oral dosage. Although this feature is ideal for an antihypertensive drug in facilitating titration and therefore is safe for hypertension patients, it is not desired for cancer treatment. The suboptimal nature of nicardipine underscores the need for nicardipine analogs to obtain highly specific EED inhibitors with improved anticancer activities and drug-like properties.
Example 2 - Design and Synthesis of Nicardipine Analogs
I. Designing Nicardipine Analogs
[0168] Several potential leads with enhanced cytotoxicity were identified in chemoresistant PCa cells and improved pharmacological properties. Specifically, the (R)-enantiomer of nicardipine (R)-l had a higher predicted EED binding affinity and was more cytotoxic than the (S)-enantiomer or (±)-nicardipine in chemoresistant PCa cells. Specifically, the ratio of change (ROC) in the anticancer potency of (R)-nicardipine or (S)-nicardipine, calculated by [ICso of (±)- nicardipine/ICso of an analog] in the same sets of cytotoxicity assays (ROC > 1.0 indicating the analog has higher cytotoxicity in chemoresistant PCa cells), was 1.53 and 1.16, respectively. This is significant, because the (R)-enantiomer had markedly less anti-hypertensive activity than the (S)-enantiomer, therefore cancer treatment with single enantiomer compounds may diminish "off- target" aspects arising from racemic nicardipine; (2) The synthetic hexamethylene analog (±)-2 demonstrated superior cytotoxic activity (ROC = 2.08). Modeling of (R)-2 also showed better EED binding affinity. The structural model predicted that the hexamethylene linker of 2 adopts a chiral coil upon binding with EED, with two (-)-synclinal and one (+)-anticlinal conformations and with similar pi-cation interactions with the aromatic cage residues Phe97, Tyrl48, and Tyr365. Modeling the enantiomers of 2 revealed different hydrogen bonding patterns of the esters, favoring the (R)-2 structure for superior EED binding, with one ester H-bonding to the main-chain NH of Trp364 and the ester carbonyl proximal to the amine H-bonding to Arg414; (3) Among a family of six alkyl and cycloalkyl analogs prepared and tested to date, the novel 4-cyclopentyl analog (±)- 3 was the most promising, with increased cytotoxicity (ROC=1.47) (Table 6, below).
[0169] Compounds less cytotoxic than nicardipine also informed the pharmacophore model for EED inhibitors. For example, the pyridine metabolite of nicardipine exhibited ROC of 0.89. 4- Phenyl analogs were slightly less cytotoxic than 4-(m-nitrophenyl) compounds, but in N,N- dimethylamine analog (±)-5, removing the benzyl group of the tertiary amine substantially diminished cytotoxic activity (ROC=0.05), demonstrating the importance of the tertiary benzylic amine. Modeling revealed that the small N,N-dimethylammonium cation bound weakly and more deeply into the central pocket so that the tertiary ammonium cation was no longer within the Phe97-Tyrl48-Tyr365 aromatic cage. Small and linear alkyl analogs, such as 4-methyl- substituted-(±)-6, were less cytotoxic (ROC=0.65) than branched or cyclic alkyl analogs, i.e. (±)- 3 (ROC=1.47). [0170] Materials and reagents: All chemicals were purchased from sources 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 and argon-charged glassware unless otherwise specified. Argon was passed through a drying column containing drying agents. Reaction vials and stir bars were treated with concentrated nitric acid, washed with water and acetone thoroughly, dried in oven before usage. Thin layer chromatography (TLC) was performed on precoated aluminum-backed plates purchased from Whatman (silica gel 60F254), visualized by p-anisaldehyde stain and UV lamp. Flash column chromatography was carried out with silica gel 60 (230-400 mesh ASTM) from Silicycle.
[0172] Analysis: Proton and carbon NMR spectra were recorded on INOVA 400 (400 MHz), BROKER 400 (400 MHz), INOVA-500 (500 MHz), INOVA-600 (600 MHz), or a BROKER 600 (600 MHz) instrument equipped with cryogen probe. NMR spectra were recorded in solutions of deuterated chloroform (CDCI3) with the residual chloroform (7.26 ppm for 'H NMR and 77.23 ppm for 13C NMR) taken as the internal standard and were reported in parts per million (ppm). Abbreviations for signal coupling 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 protocols
[0174] Example A: Compound 1, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,6-dimethyl-4- phenyl-l,4-dihydropyridine-3,5-dicarboxylate:
[0175] To a 4 mL or 8 mL reaction vial, methyl (£)-3-aminobut-2-enoate (1 equiv) and 2- (benzyl(methyl)amino)ethyl 3-oxobutanoate (1 equiv) were added and placed under argon atmosphere via purging, which was followed by addition of benzaldehyde (1 equiv) via syringe. The reaction mixture was dissolved with isopropanol (1 .4 M), and was bubbled with argon for 10- 15 minutes. The reaction vial was capped with solid cap, and placed in sand bath or heat block to reflux overnight. After the reaction was cooled to room temperature, the mixture was transferred to a round bottom flask for concentrating in vacuo to give a thick yellow oil. The crude mixture (wet loaded) was purified via flash column chromatography in silica gel (30 cm in height in the 1- inch diameter column) using 60:40 EtOAc/Hexane mixture (-500-700 mL). Fractions were visualized by TLC and p-anisaldehyde stain, combined appropriately, and concentrated in vacuo and under high-pressure vacuum overnight. The pure product is a yellow residue. Yield: 32%, >95% purity (estimated by !H NMR).
[0176] 'H NMR (400 MHz, CDCh) 8 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] 13C NMR (101 MHZ, CDCh) 8 168.16, 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 calc, for C26H3oN204 + [M+H]+: 435.2278, found 435.2276.
[0179] Example B: Compound 3, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,4,6-trimethyl- l,4-dihydropyridine-3,5-dicarboxylate:
[0180] A mixture of methyl (£)-3-aminobut-2-enoate (1 equiv), 2-(benzyl(methyl)amino)ethyl 3- oxobutanoate (1 equiv), acetaldehyde (2 equiv), and tetrabutylammonium hydrogensulfate (0.12 equiv) was dissolved in ethylene glycol (2.4 M) in a 4 mL reaction vial. The mixture was bubbled with argon inlet for 10 minutes before being capped with solid cap, and was stirred at 80 - 85 °C overnight. The reaction mixture was cooled to room temperature, then diluted with EtOAc. The solution was then poured over brine (-3-5 mL), then extracted with EtOAc (x5). The combined organic layer was dried with anhydrous MgSO4, filtered and concentrated in vacuo. The crude mixture was subjected (wet loading) to flash column chromatography (silica gel) in 50:50 Hexane/EtOAc. Appropriate fractions were collected and concentrated to give a yellow oily residue (17% yield).
[0181] 1HNMR (400 MHz, CDCh) 5 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] 13C NMR (101 MHz, CDCh) 6 168.30, 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 calc, for C21H28N2OC [M+H]+: 373.2122, found 373.2119.
[0184] Example C and D: Compounds 5 and 6 were prepared from a common precursor, 5- (methoxycarbonyl)-2,6-dimethyl-4-phenyl-l,4-dihydropyridine-3-carboxylic acid:
[0185] In the first step, following the protocol for preparing compound (±)-l, a mixture of methyl (£)-3-aminobut-2-enoate (1 equiv), 2-cyanoethyl 3-oxobutanoate (1 equiv), and benzaldehyde (1.2 eq), in isopropanol (4 mL, 1.6 M) was heated overnight at reflux under argon atmosphere. The crude mixture was dry-loaded onto celite before subjecting to a silica gel flash column chromatography in 60:40 Hexane/EtOAc. The cyanoethyl ester product was eluted using a gradient of 60:40 to 50:50 to 40:60 Hexane/EtOAc, concentrated in vacuo and under high-pressure vacuum overnight to give 3 -(2-cyanoethyl) 5-methyl 2,6-dimethyl-4-phenyl-l,4-dihydropyridine-3,5- dicarboxylate as a thick yellow oil. Yield: 73%.
[0186] 'H NMR (400 MHz, CDCh) 5 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-Cy anoethyl) 5-methyl 2,6-dimethyl-4-phenyl-l ,4-dihydropyridine-3,5-dicarboxylate (4.7 mmol, 1 eq) was dissolved in acetone (1.87 mL/mmol of cyanoethyl ester) under air, then a solution of 1 M NaOH (3.74 mL/mmol of cyanoethyl ester) was poured into the reaction flask (producing smoky vapor). The reaction was stirred at 28-30 °C over 1 hour under an argon atmosphere. The clear yellow mixture was diluted with 20 mL of DI water, then transferred into a separatory funnel. DCM was used to wash the reaction flask and then poured into the separatory funnel, which caused the organic layer to become cloudy light yellow after mixing. The aqueous layer was washed three times with DCM and collected into an Erlenmeyer flask, which was then acidified slowly by adding drops of concentrated HC1 until pH 1-2 (monitored closely 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 via fritted funnel and collected after air-drying and drying on the high-pressure vacuum overnight to give 5-(methoxycarbonyl)-2,6-dimethyl-4-phenyl-l,4- dihydropyridine-3 -carboxylic acid as a white powder. Yield: 88%.
[0188] rH NMR (400 MHz, DMSO) 8 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- l,4-dihydropyridine-3,5-dicarboxylate:
[0190] Compound 5 was prepared following the same protocol as the synthesis of compound 6, using 2-(dimethylamino)ethan-l-ol. Yield: 11%, yellow oil.
[0191] 1 H NMR (400 MHz, CDCh) 5 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] 13C NMR (101 MHZ, CDCh) 8 168.04, 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 calc, for C2oH26N204+ [M+H]+: 359.1965, found 359.1963. [0194] Example D: Compound 6, 3-(6-(benzyl(methyl)amino)hexyl) 5-methyl 2,6-dimethyl-4- phenyl-l,4-dihydropyridine-3,5-dicarboxylate:
[0195] 5-(Methoxycarbonyl)-2,6-dimethyl-4-phenyl-l,4-dihydropyridine-3-carboxylic acid (0.7 mmol, 1 equiv) was dissolved with dry DMF (1.2 mL, 0.6 M) and DCM (12 mL) under argon atmosphere, and the reaction was cooled to 0 °C on an ice bath. Next, SOCh (0.98 mmol, 1.4 equiv) was added via microsyringe, which turned the white suspension into a clear yellow mixture over time. The reaction mixture was stirred on ice for 2.5 hours, then a solution of 6- (benzyl(methyl)amino)hexan-l-ol (1 equiv) in DCM (2 mL) was added to the reaction flask. The reaction was stirred and warmed to room temperature overnight, which became a clear orange mixture. After stirring was stopped, the reaction was diluted with DCM and washed with saturated NaHCCh and brine. The organic layer was dried with anhydrous Na2SC>4 then concentrated in vacuo to give a dark orange oil crude. The reaction mixture was purified via flash column chromatography with 95:5 DCM/MeOH (via wet loading). Fractions were collected (in small quantities when yellow band started eluting) and visualized by UV lamp. The product was identified to be fluorescent under UV light. Yield: 26%, yellow oil.
[0196] 'H NMR (400 MHz, CDCh) 5 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, CDCI3) 8 167.87, 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 (APC1): m z calc, for C30H39N2OU [M+H]+: 491.2904, found 491.2903.
[0199] Example E: Compound 12, 3-(2-(benzyl(methyl)amino)ethyl) 5-methyl 2,6- di m ethy I py ri di n e-3 , 5 -di carb oxy 1 ate :
[0200] Compound 12 was synthesized according to similar protocol as substrate (±)-l, using formaldehyde instead of benzaldehyde. The crude mixture was an orange oil, which was subjected (dry loading) to flash column chromatography (silica gel) in 50:50 Hexane/EtOAc. Appropriate fractions were collected and concentrated to give a yellow powder (65% yield). The 1 ,4- dihydropyridine product 4 was confirmed by rH NMR, however, oxidative decomposition to the pyridine product 12 was spotted in the NMR sample.
[0201] LH NMR (400 MHz, CDCh) 6 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 2M HNO3 (1.5 mL) was added. The reaction mixture was stirred at 55-60 °C under air for 20 minutes, which turned from a yellow mixture to a clear solution. The reaction was neutralized with saturated NaHCCh, extracted with DCM, and the combined organic layer was washed with DI water and brine, and was dried with anhydrous Na2SO4. The mixture was purified on a pipette column with silica gel with 1 : 1 Hexane/EtOAc as eluent. Fractions (1-2 mL/each) was collected and concentrated in vacuo. Sidenote-, the oxidized pyridine product 12 is only UV-active and cannot be visualized by p-anisaldehyde staining. Product is a white residue. Yield: 36%.
[0203] rH NMR (400 MHz, CDCh) 8 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, CDCh) 6 166.28, 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 calc, for C20H25N2OC [M+H]+: 357.1809, found 357.1807. [0206] Example F: Compound 13, 3-(2-(benzyl(methyl)amino)ethyl) 5 -methyl 2,4,6- tri methyl pyri di ne-3 , 5 -di carb oxy 1 ate :
[0207] Compound (±)-3 (26 mg, 0.07 mmol) was transferred to a 4 mL reaction vial, and 2M HNO3 (0.2 mL) was added. The reaction mixture was stirred at 55-60 °C under air overnight. The reaction was neutralized with saturated NaHCOi, extracted with DCM, and the combined organic layer was washed with DI water and brine, and was dried with anhydrous Na2SC>4. The mixture was purified via pipette column with silica gel packed in 1 : 1 Hexane/EtOAc (via wet loading). Fractions (1-2 mL/each) was collected and concentrated in vacuo. Product is a pale-yellow residue. Yield: 17%.
[0208] 'H NMR (400 MHz, CDCh) 5 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:
[0210] Compound (±)-l (58 mg, 0.13 mmol) was transferred to a 4 mL reaction vial, and 2M HNO3 (0.8 mL) was added. The reaction mixture was stirred at 55-60 °C under air overnight, which turned from a cloudy yellow mixture to a slightly clearer solution. The reaction was neutralized with saturated NaHCCh, extracted with DCM, and the combined organic layer was washed with DI water and brine, and was dried with anhydrous NaiSCU. The mixture was purified via flash column chromatography with silica gel packed in 5: 1 Hexane/EtOAc (via wet loading). The compound was eluted using a gradient of 5: 1 to 3: 1 Hexane/EtOAc as eluent. Fractions were collected and concentrated in vacuo. Product is a yellow residue. Yield: 32%.
[0211] 'H NMR (400 MHz, CDCh) 8 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] 13C NMR (101 MHZ, CDCh) 6 168.47, 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 calc, for C26H2sN2O4+ [M+H]+: 433.2127, found 433.2120.
[0214] Examples H-M: Compounds 2, 7, 8, 9, 10, and 11:
[0215] Compounds 2, 7, 8, 9, 10, and 11 were synthesized by the same methods as described above in Example A, using the following aldehydes instead:
[0216] Compound 2, cyclohexanecarbaldehyde
[0217] Compound 7, cyclopropanecabaldehyde
[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 according to examples F and G described above, beginning with the dihydropyridine compound 7.
[0227] Example O: Compound 27: [0228] 5-(Methoxycarbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-l,4-dihydropyridine-3-carboxylic acid (100 mg, 0.30 mmol, 1 equiv), 6-(benzyl(methyl)amino)hexan-l-ol (73 mg, 0.33 mmol, 1.1 equiv), and A-methylimidazole (NMI, 74 pL, 0.93 mmol, 3.1 equiv) were added to a 4 mL oven- dried reaction vial charged with stir bar under air. The mixture was then purged with argon and dissolved with acetonitrile (MeCN, 2 mL, 20 mL/g of limiting reagent), followed by addition of '.A.A', A"-tetramethylchloro-formamidinium hexafluorophosphate (TCFH, 101 mg, 0.36 mmol, 1.2 equiv). The reaction mixture was placed under argon, sealed with solid screw cap and electrical tape, placed on heat block, and stirred at 40 °C over 16 h. The reaction mixture quickly became clear yellow homogeneous over time. After the reaction was cooled to room temperature, deionized water was added to quench the reaction, and the organic material was extracted with ethyl acetate three times. The organic layer was washed with brine, dried with anhydrous sodium sulfate, and concentrated in vacuo via rotary evaporation. The crude mixture was purified by column chromatography on silica gel using gradient elution with a 70:30 - 65:35 - 60:40 - 50:50 Hexane/acetone mixture. The appropriate fractions were collected and concentrated by rotary evaporation (at 40 °C to remove trapped solvent) to afford the pure product as a viscous yellow oil (139 mg, 84% yield).
[0229] 1 H NMR (400 MHz, CDCk) 5 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 overlapping), 2.19 (s, 3H), 1.58 (broad pentet, J = 6.7 Hz, 2H), 1.48 (broad pentet, J = 7.4 Hz, 2H), 1.30 - 1.19 (m, 4H).
[0230] 13C NMR (101 MHZ, CDCh) 5 167.7, 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 calc, for CsolLsNsC [M+H]+: 536.27551, found 536.27548.
Example 3 - Biological Assays
I. In vitro cytotoxicity assay of anticancer compounds
[0232] Human prostate cancer cell line C4-2B and its docetaxel-resistant derivative C4-2B- TaxR were cultured following the procedures described in Zhu et. al., Mol. Cancer Ther. 2013, 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 (Li et. al., Theranostics 2021). Approximately 1,500- 3,000 cancer cells were plated in 100 pL of growth medium in 96-well flat-bottomed microtiter plates. Cells were incubated overnight to allow recovery. Compounds to be tested were added to the cells in six replicates in a range of concentrations, using dimethyl sulfoxide (DMSO) as the vehicle. The cells were incubated at 37°C, 5% CO2, with 100% humidity. A column of control wells on the same plates contained all materials except cells and compounds (or DMSO). Plates were incubated for 3 days, allowing sufficient time for cell replication and compound-induced cell death. Cells were counted using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MD, USA) following the manufacturer’s instruction. The half-minimal inhibitory concentrations (IC50) of the specified agent were calculated with the SigmaPlot program (Systat Software Inc., San Jose, CA, USA).
[0233] To minimize potential variations due to the passage numbers and culture conditions of cancer cells, nicardipine was always included in every in vitro cytotoxicity assay to compare the relative potency of nicardipine analogs. The ratio of change (ROC) in the anticancer potency of a compound was calculated as
ROC = IC50 of (±)-nicardipine/ICso of the compound
[0234] ROC was obtained from the same sets of cytotoxicity assays used to standardize the assays and as serves as the primary indicator of improvement in terms of anticancer activities, where ROC > 1.0 indicating the analog has higher cytotoxicity than nicardipine in chemoresistant cancer cells. The ROCs for the nicardipine analog compounds disclosed herein is shown in Table 6 below. [0235] Table 6. ROCs of Nicardipine Analogs
Example 4 - Modeling
[0236] ‘ ‘Molecular docking analyses using Schrodinger’s Maestro program demonstrated that (R)-nicardipine bound the “aromatic cage” of EED, interacting with Phe97, Tyrl48 and Tyr365 through Pi-cation interactions, with the benzyl ammonium cation extending relatively deeply into the central pocket. In addition, the ester carbonyl distal to the tertiary amine hydrogen bonds with Arg414, and the m-nitrophenyl group is near Trp364. The 1,4-dihydropyridine core is less strongly associated with EED and is partially exposed to solvent. In contrast, the (S)-enantiomer of nicardipine has the 1,4-dihydropyridine "flipped": the ammonium cation now has pi-cation interactions only with Phe97 and Tyrl48. Tyr365 is closer to the 1,4-dihydropyridine ring, and the m-nitrophenyl group is exposed to solvent and is not associated with EED. The binding energy between (R)-nicardipine and EED was calculated as -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). (S)-nicardipine binding energy with EED was calculated as -57.09 kcal/mol. [0237] Two analogs of (±) compound 6 with an additional meto-nitro group, exhibit nanomolar cytotoxicity (ICso = 0.53-0.65 pM) in chemoresistant C4-2B-TaxR cells and have higher ROC values (2.99 and 2.54, respectively) when compared with (±)-nicardipine.
[0238] Modeling the analogs using an induced-fit algorithm showed that compounds 27 and 28 have potentially high EED binding affinity, as shown in Table 7. For example, there are stable interactions between (S)-compound 27 and the residues in the binding pocket of EED, including the it- Ti stacking between the aromatic ring and Trp364, the 7t-cation interaction between ammonium group and Tyrl48, and two H-bonds between the carbonyl group and Arg414 as shown in Figure 7. [0239] Table 7: Predicted binding affinity of enantiomers of compounds 27 and 28
[0240] As the linker of nicardipine and analogs adopts a chiral coil upon binding with EED, analogs that add substituents and rings onto the linker may have enhanced binding affinity for EED. The introduction of substituents and chiral centers diminishes the conformational freedom of the linker by enforcing dihedral angles within the chiral protein environment, in accordance with modeling results with achiral linkers.
[0241] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which the inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific 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

WHAT IS CLAIMED IS:
1. A compound of Formula I or a pharmaceutically acceptable salt or solvate thereof, wherein: each instance of ~~~~~~ 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 selected from the group consisting of a bond, -C(O)-, -C(O)2-, -CH2O-, -O-, -S-, - S(O)-, and -S(O)2-;
D is optionally substituted Ci-Cs alkylene; or wherein R5 and R6 are each independently selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted cycloalkyl; optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; or wherein R3 and R6 and the nitrogen to which each are attached together form a heterocycloalkyl; wherein R7, R8, R9, and R10 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, Ci-Ce alkyl; or two of R7, R8, R9, and R10 or one of R7, R8, R9, and R10 and one of R5 and R6 taken together with the atom to which each is attached form a C3-C7 cycloalkyl or a 4- to 7-membered heterocycloalkyl; wherein m, o, q, and r are each independently 0 or 1 ;
R1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and an optionally substituted aryl;
R2 is selected from the group consisting of H, -OH, -NR2AR2B, -OR2A, -SR2A, optionally substituted Ci-Ce alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; wherein R2A and R2B are each independently H or Ci-Ce alkyl;
R3, if present, is H or optionally substituted Ci-Ce alkyl; and
R4 is selected from the group consisting of H, -CN, -OH, halo, -C(O)-(Ci-Ce alkyl), -C(O)2-(Ci-C6 alkyl), -S(O)-(Ci-C6 alkyl), -S-(Ci-C6 alkyl), -S(O)2-(Ci-C6 alkyl), optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce alkylamino, optionally substituted Ci-Ce alkoxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl.
2. The compound of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein A is selected from the group consisting of -C(O)2- and -CH2O-.
3. The compound of claim 1 or claim 2, or a pharmaceutically acceptable salt or solvate thereof, wherein
4. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is a nitro- substituted aryl.
5. The compound of claim 4, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is
6. The compound of claim 1 , or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of: wherein D is selected from the group consisting of: wherein D-X is selected from the group consisting of:
7. The compound of claim 1, wherein
A is selected from the group consisting of a bond, -C(O)-, -C(O)2-, -O-, -S-, -S(O)-, and -S(O)2-; wherein R3 and R6 are each independently selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted cycloalkyl; optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted heteroaralkyl; and
R1 is selected from the group consisting of H, -CN, -OH, halo, oxo, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkylamino, optionally substituted C1-C10 alkoxy, optionally substituted C1-C10 cycloalkyl, optionally substituted C1-C10 heterocycloalkyl, and an unsubstituted aryl;
8. The compound of claim 7, or a pharmaceutically acceptable salt or solvate thereof, wherein A is selected from the group consisting of -C(O)- and -C(O)2-.
9. The compound of claim 7 or claim 8, or a pharmaceutically acceptable salt or solvate thereof, wherein
10. The compound of claim 9, or a pharmaceutically acceptable salt or solvate thereof, wherein
11. The compound of any one of claims 7-10, or a pharmaceutically acceptable salt or solvate thereof, wherein R2 is selected from the group consisting of -NR2AR2B, -OR2A, and optionally substituted Ci-Ce alkyl; wherein R2A and R2B are each independently H or Ci-Ce alkyl.
12. The compound of claim 11, or a pharmaceutically acceptable salt or solvate thereof, wherein R2 is -OR2A, wherein R2A is Ci-Ce alkyl.
13. The compound of claim 12, or a pharmaceutically acceptable salt or solvate thereof, wherein R2 is -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)2, -O(CH2)3CH3, -OCH(CH3)3, -OCH2CH(C H3)2, and -OCH(CH3)CH2CH3.
14. The compound of claim 13, or a pharmaceutically acceptable salt or solvate thereof, wherein R2 is -OCH3.
15. The compound of any one of claims 1-14, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is selected from the group consisting of -C(O)-(Ci-C6 alkyl), -C(O)2-(Ci-Ce alkyl), and optionally substituted Ci-Ce alkyl.
16. The compound of claim 15, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is optionally substituted Ci-Ce alkyl.
17. The compound of claim 16, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, terf-butyl, ec-butyl, pentyl or hexyl.
18. The compound of claim 17, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is methyl.
19. The compound of claim 15, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is -C(O)2-(Ci-Ce alkyl).
20. The compound of claim 19, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is -C(O)2CHs.
21. The compound of any one of claims 1-20, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is selected from the group consisting of optionally substituted Ci-Cio alkyl, optionally substituted Ci-Cio alkylamino, and optionally substituted Ci-Cio alkoxy.
22. The compound of claim 21, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is optionally substituted Ci-Cs alkyl.
23. The compound of claim 22, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, heptyl, or octyl.
24. The compound of claim 23, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, or sec-butyl.
25. The compound of any one of claims 1-20, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is optionally substituted Ci-Cio cycloalkyl.
26. The compound of claim 25, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is selected from the group consisting
27. The compound of any one of claims 7-10, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is optional substituted Ci-Cio heterocycloalkyl, wherein the heteroatom is one or more of S, O, or N.
28. The compound of claim 27, or a pharmaceutically acceptable salt or solvate thereof,
29. The compound of any one of claims 1-20, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is an unsubstituted aryl.
30. The compound of claim 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is an unsubstituted phenyl.
31. The compound of any one of claims 1-20, or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is H.
32. The compound of any one of claims 1-31, having a structure of Formula la: or a pharmaceutically acceptable salt or solvate thereof.
33. The compound of any one of claims 1-31, having a structure of Formula lb: or a pharmaceutically acceptable salt or solvate thereof.
34. The compound of any one of claims 1-33, having a structure of Formula 1-1:
or a pharmaceutically acceptable salt or solvate thereof.
35. The compound of any one of claims 1-34, having a structure of Formula la-1 or lb-1 : or a pharmaceutically acceptable salt or solvate thereof.
36. The compound of any one of claims 1-35, or a pharmaceutically acceptable salt or solvate thereof, wherein p is 0.
37. The compound of any one of claims 1-35, or a pharmaceutically acceptable salt or solvate thereof, wherein p is 1.
38. The compound of claim 7 selected from the group consisting of: or a pharmaceutically acceptable salt or solvate thereof.
39. A pharmaceutical composition comprising a compound of any one of claims 1-38, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.
40. A method of treating a disorder in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-38, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 33.
41. The method of claim 40, wherein the disorder is associated with histone methyltransferases.
42. The method of claim 41, wherein the disorder is associated with polycomb repressive complex 2 (PRC2) histone methyltransferase, and its subunits.
43. The method of claim 36, wherein the disorder is associated with embryonic ectoderm development (EED) subunit of PRC2.
44. The method of any one of claims 40-43, wherein the disorder is a cancer.
45. The method of claim 44, wherein the cancer is associated with dysregulation of histone methyltransferases.
46. The method of claim 45, wherein the cancer is associated with dysregulation of polycomp repressive complex 2 (PRC2) histone methyltransferase, and its subunits.
47. The method of claim 46, wherein the cancer is associated with dysregulation of embryonic ectoderm development (EED) subunit of PRC2.
48. The method of any one of claims 44-47, wherein the cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, colorectal cancer, head and neck cancer, pancreatic cancer, brain cancer, bladder cancer, osteosarcoma, and lymphoma.
49. The method of any one of claims 40-48, wherein the treatment of the disorder further comprises a second therapeutic agent or therapeutic method.
50. 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.
EP24753869.7A 2023-02-06 2024-02-05 Pyridine and dihydropyridine compounds and uses thereof Pending EP4662202A1 (en)

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