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WO2022032144A1 - Inhibiteurs d'adaptateur de substrat de prmt5 et leurs utilisations - Google Patents

Inhibiteurs d'adaptateur de substrat de prmt5 et leurs utilisations Download PDF

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Publication number
WO2022032144A1
WO2022032144A1 PCT/US2021/045016 US2021045016W WO2022032144A1 WO 2022032144 A1 WO2022032144 A1 WO 2022032144A1 US 2021045016 W US2021045016 W US 2021045016W WO 2022032144 A1 WO2022032144 A1 WO 2022032144A1
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Prior art keywords
optionally substituted
compound
prmt5
alkyl
cancer
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Inventor
Dale Porter
William Sellers
Florence Fevrier Wagner
David Mckinney
Alessandra IANARI
Arthur J. Campbell
Martin Drysdale
Robert Hilgraf
Patrick MCCARREN
Brian Mcmillan
Ritu Singh
Kathleen MULVANEY
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Dana Farber Cancer Institute Inc
Broad Institute Inc
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Dana Farber Cancer Institute Inc
Broad Institute Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
    • C07D237/06—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D237/10—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three 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, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D237/14—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • Protein arginine methyltransferase 5 is an arginine methyltransferase and an essential enzyme (Mavrakis, K. J. et al. Science 351, 1208–13 (2016); Tee, W.-W. et al. Genes Dev.24, 2772–7 (2010); Karkhanis, V., Hu, Y. J., Baiocchi, R. A., Imbalzano, A. N. & Sif, S. Trends Biochem. Sci.36, 633–641 (2011)).
  • PRMT5 symmetrically dimethylates substrate proteins by catalyzing the transfer of methyl groups from S-adenosyl methionine (SAM) methyl donor molecules to acceptor arginine residues within a substrate (Gary, J. D. & Clarke, S. Prog. Nucleic Acid Res. Mol. Biol.61, 65–131 (1998); Stopa, N., Krebs, J. E. & Shechter, D. Cell. Mol. Life Sci.72, 2041–2059 (2015)).
  • SAM S-adenosyl methionine
  • Substrate methylation is known to depend upon several co-factors, including WDR77, the requisite heterodimeric binding partner of PRMT5. Together, PRTM5 and WDR77 form a tetramer of heterodimers comprising a stable and functional heterooctamer known as the PRMT5 methylosome (Ho, M. C. et al. PLoS One 8, (2013); Sun, L. et al. Proc. Natl. Acad. Sci.108, 20538–20543 (2011)). In addition to WDR77, the PRMT5 methylosome requires several additional co-factors in order to recognize and methylate substrates (referred to as substrate adaptors herein).
  • PRMT5 substrate adaptors There are three characterized PRMT5 substrate adaptors: pICln/CLNS1A, RIOK1, and COPR5.
  • pICln a chaperone protein that regulates assembly of the splicesome, recruits PRMT5 to three subunits of the splicesome for methylation: SmB/B’, SmD1, and SmD2 (Pesiridis, G. S., Diamond, E. & Van Duyne, G. D. J. Biol. Chem.284, 21347–21359 (2009); Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln. Curr.
  • PRMT5 regulates cellular splicing activity (Bezzi, M. et al. Genes Dev.27, 1903–16 (2013)) and formation of RNA processing bodies.
  • RIOK1 an atypical kinase important for ribosomal biogenesis, recruits the RNA-binding protein nucleolin to the PRMT5 complex for methylation (Guderian, G. et al. J. Biol. Chem.286, 1976–1986 (2011). Through this RIOK1-nucleolin sub-complex, PRMT5 regulates ribosome biogenesis (Ren, J. et al. J. Biol. Chem.285, 12695–705 (2010)). Lastly, COPR5 is a nuclear protein that recruits PRMT5 to nucleosomes to promote methylation of histones, particularly on histone 3R8 and histone 4R3 (Lacroix, M. et al.
  • the PRMT5 complex has been identified as a cancer dependency in MTAP deleted tumors (Kryukov, G. V. et al. Science 351, 1214–8 (2016); Marjon, K. et al. Cell Rep.15, 574–587 (2016)). Specifically, loss of PRMT5 or either of its substrate adaptors RIOK1 and pICLn confers synthetic lethality in MTAP null tumors.
  • the MTAP locus is adjacent to the frequently deleted tumor suppressor, CDKN2A/B (p16), and is thus co-deleted in many tumors.
  • MTAP loss is observed in ⁇ 15% of all tumors and notably in >40% of glioblastomas and 30% of pancreatic tumors, tumor types for which few therapeutic options exist (Cancer Genome Atlas Research Network. Electronic address: andrew_aguirre@dfci.harvard.edu, B. J. et al. Cancer Cell 32, 185-203.e13 (2017); Brennan, C. W. et al. The Somatic Genomic Landscape of Glioblastoma. Cell 155, 462–477 (2013); Cerami, E. et al. Cancer Discov.2, 401–404 (2012)).
  • MTA methylthioadenosine
  • X1 is CH or N
  • R1 is H, halogen, -CN, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -ORO, -N(RN 1 )RN 2 , -C(O)Rb, -C(O)ORO, -C(O)N(RN1)RN 2 , -SRS,
  • X 1 is CH or N
  • R 1 is H, -CN, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -OR O , -N(R N1 )R N2 , -C(O)R b , -C(O)OR O , -C(O)N(R N1 )R N2 ,
  • compositions comprising a compound of Formula (I), or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, and a pharmaceutically acceptable excipient.
  • methods of treating an infectious disease, cardiovascular disease, a proliferative disease, beta-thalassemia, sickle cell disease, a neurological disease, an inflammatory disease, or an autoimmune disease in a subject in need thereof comprising administering a compound of Formula (I), or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, or a pharmaceutical composition comprising a compound of Formula (I), to the subject.
  • the disease is an infectious disease, cardiovascular disease, a proliferative disease, beta- thalassemia, sickle cell disease, a neurological disease, an inflammatory disease, or an autoimmune disease.
  • the disease is cancer.
  • the cancer is a hematological malignancy (e.g., mantle cell lymphoma (MCL)).
  • MCL mantle cell lymphoma
  • methods of inhibiting methylation of a PRMT5 substrate the methods comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent.
  • methods of modulating the transcription of a gene regulated by PRMT5 the methods comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent.
  • methods of modulating PRMT5-dependent mRNA splicing the methods comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent.
  • inhibitors of the binding of PRMT5 with a PRMT5 substrate adaptor wherein the inhibitor disrupts binding of PRMT5 with a PRMT5 substrate adaptor.
  • modulators of PRTM5 activity wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • modulators of PRMT5 methyltransferase activity wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • inhibitors of PRMT5 substrate methylation wherein the inhibitors disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • modulators of the transcription of a gene regulated by PRMT5 wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • modulators of chromatin structure regulation wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • modulators of cellular differentiation wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • modulators of mRNA splicing wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • pharmaceutical compositions comprising a modulator or inhibitor disclosed herein and an excipient.
  • uses of the pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating a disease are provided.
  • the disease is an infectious disease, cardiovascular disease, a proliferative disease, beta- thalassemia, sickle cell disease, a neurological disease, an inflammatory disease, or an autoimmune disease.
  • the disease is cancer.
  • the cancer is a hematological malignancy (e.g., mantle cell lymphoma (MCL)).
  • MCL mantle cell lymphoma
  • FIGs.1A to 1E depict the identification of a conserved PRMT5 substrate adaptor binding motif: GQF[D/E]DA[D/E].
  • FIG.1A Amino acid sequence alignment of three known substrate adaptor proteins of PRMT5 (COPR5, pICLn, and RIOK1) identified an evolutionarily conserved 7mer peptide that lies at the N-terminus of RIOK1 and C-termini of pICLn and COPR5.
  • FIG.1B Peptide distribution of 7 amino acid sequence motifs across all PRMT5 binding proteins (BioGrid) is shown.
  • GQF[D/E]DA[D/E] was enriched in PRMT5 binding proteins compared to other peptide sequences (comparing 7 amino acid length peptides in PRMT5 interactors to 7mer peptides across the human proteome.
  • the BLOSUM Clustered Scoring Matrix was utilized to maximize the flexibility of a potential functional protein pocket (ie allowing for D/E at the same position).
  • FIG.1C Interactions between the PRMT5 complex and PBM peptides were measured by SPR.
  • the PRMT5 complex was immobilized by biotin-neutravidin and the indicated peptides titrated as analyte.13 mer peptides containing the conserved 7mer peptides in (A) derived from each COPR5, pICLn and RIOK1 or a scrambled control. The RU response at equilibrium is shown.
  • FIG.1D Streptavidin affinity purification from cell lysates that were incubated with increasing concentrations of either the RIOK117mer peptide containing the 7mer sequence in (A) or a scrambled control peptide. Western blots show the amount of PRMT5 and WDR77 that were co-purified with the peptides.
  • FIG.1E Streptavidin affinity purification from cell lysates that were incubated with 1ug of either the RIOK117mer peptide containing the 7mer sequence in (A) or a scrambled control peptide. Western blots show the amount of PRMT5, WDR77, PRMT1 and PRMT7 that were co-purified with the peptides. Vinculin served as a loading and affinity purification control.
  • FIGs.2A to 2D depict the structural basis for recognition of the PRMT5-binding motif (PBM).
  • FIG.2A The PRMT5:WDR77 complex bound to a PBM peptide.
  • the heterooctamer complex is shown as a surface with a different color for each protomer unit.
  • the PRMT5 surface for the “front” protomer is colored by amino acid conservation on a sliding scale from cyan (least conserved) to maroon (most conserved).
  • the Riok1 PBM peptide is colored orange and shown as a stick representation (front protomer) or surface (all other protomers).
  • a single protomer unit of the complex is represented in cartoon form with PRMT5 in green and WDR77 in grey.
  • the Riok1 peptide and SAM are shown in orange and purple, respectively.
  • FIG.2B PRMT5 (from the Riok1 bound structure) is represented in cartoon form with a transparent surface colored by electrostatic potential from negative (red) to positive (blue).
  • FIG.2C The PBM binding groove of PRMT5 (green) is shown as a cartoon with transparent surface. Two ordered water molecules are represented as red spheres. The Riok1 peptide is shown in orange.
  • FIG.2D Key hydrogen bonds of the interaction are shown as yellow dotted lines. Hydrogen bonds representing i+3 bonding pattern for ⁇ -turns described in text. [0031]
  • FIGs.3A to 3F depict the results of experiments related to the role of the PBM in PRMT5 binding to substrate adaptor proteins.
  • FIG.3A Interactions between the PRMT5 complex and pICln protein were measured by SPR.
  • the PRMT5 complex was immobilized by biotin-neutravidin and then full-length or mutant pICln protein was titrated as analyte. The RU response at equilibrium is shown.
  • FIG.3B The relative binding affinity of peptides with the wildtype or alanine substituted sequence from pICln were tested via fluorescence polarization. A fluorescent Riok1 peptide sequence was used as probe.
  • FIG.3C The relative interaction between PRMT5 and Riok1 or a deltaPBM Riok1 was measured in cells via Nanobit assay.
  • FIG.3D The Nanobit system was used to test the ability of PBM peptides to compete for PRMT5 binding. Prior to addition of peptides, cells were permeabilized with 0.05% NP-40. Peptides of WT or point mutant PBM sequence were titrated into the permeabilized cells as indicated. Relative luciferase signal was assayed as a measure of functional complementation.
  • FIG. 3D The Nanobit system was used to test the ability of PBM peptides to compete for PRMT5 binding. Prior to addition of peptides, cells were permeabilized with 0.05% NP-40. Peptides of WT or point mutant PBM sequence were titrated into the permeabilized cells as indicated. Relative luciferase signal was assayed as a measure of functional complementation. FIG.
  • FIG.3E Competition between full-length pICln and PBM peptide as measured by SPR.
  • the PRMT5 complex was immobilized by biotin-neutravidin interaction.125 nM pICln was co-injected with increasing concentrations of the indicated peptides. For comparison, the RU response with peptide alone is also shown. The RU response at equilibrium is shown.
  • FIG.3F Co-immunoprecipitation of HA-tagged WT or PBM mutant pICln in the presence or absence of endogenous pICln depletion (dox inducible shRNA). Immunoblot detects amount of PRMT5 and pICln bound. Vinculin serves as a loading control.
  • FIGs.4A to 4E depict the results of experiments related to the role of the PBM groove in PRMT5 binding to substrate adaptor proteins.
  • FIG.4A Co-immunoprecipitation of HA tagged WT or PBM groove mutant PRMT5 in the presence or absence of endogenous PRMT5 depletion (dox. inducible shRNA) was performed. Immunoblot detects amounts of PRMT5, WDR77 and pICln bound. Vinculin serves as a loading control.
  • FIG.4B Co- immunoprecipitation of HA tagged WT, PBM groove mutant (ADA: N239A/K240D/F243A), or catalytically dead (CD: R368A) PRMT5 in the presence or absence of endogenous PRMT5 depletion (CRISPr Cas9 KO). Immunoblot detects amount of PRMT5, WDR77, pICln, RIOK1, and COPR5 bound. Vinculin serves as a loading control.
  • FIG.4C Mutations of the PBM interface on PRMT5 were tested via cell-based NanoBiT as previously described. WT or ADA mutant PRMT5 and RIOK1 were tagged with complementary NanoBiT sequences and co-expressed.
  • FIG.4D Polarization of a fluorescent PBM peptide derived from RIOK1 was measured across the indicated concentrations of either WT or ADA PRMT5.
  • FIG.4E Polarization of a fluorescent PBM peptide derived from RIOK1 was measured across the indicated concentrations of PRMT5 co-incubated with or without SAM analog sinefungin.
  • FIGs.5A to 5E depict the results of experiments related to the role of the PBM- PBM groove interaction in methylation of a subset of PRMT5 substrates.
  • FIG.5A Symmetrical dimethyl arginine (SDMA) detected by western blot in a PRMT5 KO-rescue system utilizing the PBM groove mutant (ADA), catalytically dead (CD) or wildtype (WT) PRMT5 in HCT116 MTAP KO cells.
  • FIG.5B SDMA-modified peptides were immunoprecipitated from PRMT5 WT , PRMT5 ADA and PRMT5 CD rescued MiaPaca2 cells following KO of endogenous PRMT5, and quantified by LC-MS/MS.
  • FIG.5C The mean MS1 peak intensity for all dimethylated peptides of SmD3 that were detected by LC-MS/MS in (b) are shown for each the PRMT5 WT , PRMT5 ADA and PRMT5 CD cell lines. Peptides are ordered 1-5 based on their log fold-change WT/CD ⁇ strength as a PRMT5 substrate.
  • FIG.5D The relative amount of SDMA-modified SmB protein was measured by AlphaLISA proximity assay for each HCT116 MTAP-/-: HcRED, PRMT5 WT , PRMT5 ADA and PRMT5 CD expressing stable cell lines in the presence or absence of 2 PRMT5 targeting sgRNAs.
  • FIG. 5E The amount of total SDMA and H4R3 SDMA was measured by immunoblotting in the HCT116 MTAP-/-; endogenous PRMT5-/-; HA-PRMT5 WT or HA-PRMT5 ADA cell lines following a titration of GSK591 (PRMT5 inhibitor) treatment for 96 hours. Doses:0-5-10-20- 50-100nM.
  • FIG.6A to 6J depict experimental results showing that mutation of the PBM interaction site impair growth in MTAP null cells.
  • FIG.6B Incucyte cell growth plots of PK1 MTAP-/-Cas9 expressing cells infected with AAVS1 control or PRMT5-targeting guide RNA to KO endogenous PRMT5.
  • FIG.6C Incucyte cell growth plots of HCT116 MTAP-/- Cas9 expressing cells infected with AAVS1 control or PRMT5-targeting guide RNA to KO endogenous PRMT5.
  • FIG.6D Immunoblots of the genotypes from (a) MiaPaca2 cells.
  • FIG.6F Quantification of colony formation assays in (e) using acid- solubilization and ABS reading.
  • FIG.6G Immunoblots of KP4 Cas9 expressing cells stably expressing V5-tagged pICln and EV constructs after infection with pICln targeting sgRNA or control (AAVS1) sgRNA. Anti- pICln and anti-V5 measure knockout and overexpression efficiency.
  • FIG.6I Colony formation assay stained with crystal violet of KP4 Cas9 expressing cells infected with AAVS1 control or pICln-targeting guide RNA to KO endogenous pICln and rescue with either Empty Vector, WT pICln or deltaPBM pICln truncation mutant.5 days after KO and 2 days after selection with puromycin, cells are seeded into growth assays.
  • FIG.7 depicts a graphical model of PRMT5:substrate adaptor site and its cellular consequences. It shows a unified model for substrate recruitment to the PRMT5 enzyme. Substrate adaptor proteins function in an exchangeable, and mutually exclusive, manner via the PBM binding site. This allows for specific methylation and regulation of distinct substrate classes using the same core enzyme.
  • FIGs.8A to 8C depict the electron density of the RIOK1-bound X-ray crystal structure.
  • FIG.8A-8C show 2Fo-Fc density contoured at 1 ⁇ using similar orientation to FIGs.2B-2D.
  • FIG.9 depicts that MTA does not affect substrate adaptor recruitment.
  • FIGs.11A to 11D show hit finding activities.
  • FIG.11A shows PBM peptide from Riok1 (gold) bound to PRMT5 (green) (left panel). Residues involved in covalent compound binding are labeled.
  • FIG. 11B is a HTS flow chart for inhibitors of the PBM-PBM groove interaction.
  • FIG.11C is a chemical structure of compound A1.
  • FIG.11D shows a dose-dependent FP assay of compound A1 displacing a fluorescently-labeled RioK1-derived PBM peptide.
  • FIGs.12A to 12E show the mechanism of binding.
  • FIG.12A is a crystal structure of compound A1 bound to PRMT5. Residues described in the text are labeled. Two alternate conformations of Y286 are shown.
  • FIG.12B is a Cryo-EM structure of compound A6 bound to PRMT5.
  • FIGs.12C and 12D are alternate versions of FIGs.12A and 12B, respectively.
  • FIG.12E is a high resolution (1.9 ⁇ after elliptical truncation of anisotropic data) co-crystal structure of compound A1 with the PRMT5:WDR77 complex (PDB ID: 6V0P).
  • FIGs.13A to 13F illustrate that compound A6 is a covalent binder.
  • FIG.13A shows a Time Course FP Assay of compound A6 displacing the Riok1-KU560 peptide (minutes).
  • FIG.13E presents the same data as FIG.13A with each series represented by a different shape/fill combination, but without trendlines.
  • FIG.13B shows an FP competition assay with compound A6 (squares) or pICln 13-mer control peptide (circles) displacing the fluorescently labeled PBM probe Riok1-KU560 from WT PRMT5 and C278A.
  • FIG.13F presents the same data as FIG.13B with each series represented by a different shape/fill combination, but without trendlines.
  • FIG.13C shows Intact Mass Spec of WT and C278A PRMT5 +/- compound A6.
  • FIG.13D shows a SPR competition assay: PRMT5:WDR77 complex was pre-incubated with compound A6 or DMSO and then immobilized to the chip surface. Full-length pICLn protein was titrated as analyte.
  • “cpmd 6” refers to compound A6.
  • FIGs.14A to 14E show kinetic formation of the covalent adducts between compound A6 and PRMT5.
  • FIG.14A shows dose dependent kinetics of PRMT5 with compound A6 as determined by intact mass spectroscopy.
  • FIG.14D presents the same data as FIG.14A with each series represented by a different shape/fill combination, but without trendlines.
  • FIG 14B shows dose response of observed rates of compound A26 (“compound 26”) compound A25 (“compound 25”) compound A20 (“compound 20”) and compound A6 (“compound 6”) covalent modification determined by intact mass spectroscopy kinetic experiments.
  • FIG.14E presents the same data as FIG.14B with each series represented by a different shape/fill combination, but without trendlines.
  • FIG.14C is a table comparing kinetic parameters for select compounds. [0043]
  • FIGs.15A to 15D show cellular activities of A0639.
  • FIG.15A depicts intact LC/MS time course of PRMT5 adduct formation using A0639.
  • FIG.15B shows a PRMT5-RIOK1 NanoBiT assay in permeabilized cells.293 T cells stably expressing the smBiT-PRMT5 / LgBiT-RIOK1 proteins were first permeabilized with lysis buffer and then treated for 5’ with the indicated compounds. NanoGlo substrate was added and immediately read at the Envision luminometer.
  • FIG.15C shows a NanoBit assay performed in intact cells after 4 hours treatment with A2198 or DMSO.
  • FIG.15D shows WB analysis of total symmetric dimethylation levels in MTAP-/- HCT116 cells, in response to A0639 or A2198 (left panel). WB analysis of total symmetric dimethylation levels in MTAP-/- HCT116 cells overexpressing the PBM groove mutant PRMT5ADA, before and after KO of endogenous PRMT5 (right panel).
  • FIGs.16A to 16C FIG. 16A is a crystal structure of compound A1 bound to PRMT5 (carbon, green). 1 ⁇ 2Fo-Fc density in blue, 3 ⁇ Fo-Fc in red/green. A crystal contact neighbor is in grey.
  • FIG. 16A is a crystal structure of compound A1 bound to PRMT5 (carbon, green). 1 ⁇ 2Fo-Fc density in blue, 3 ⁇ Fo-Fc in red/green. A crystal contact neighbor is in grey.
  • FIG. 16A is a crystal structure
  • FIG. 16B shows crystal contact between two neighboring monomers of PRMT5 (green and orange).
  • Compound A1 is shown in magenta and cyan.
  • FIG.16C is a Cryo-EM structure of compound A6 bound to PRMT5 (carbon, green). Electron density is shown at 6 ⁇ .
  • FIGs.17A to 17D show exemplary mass spectroscopy results of PRMT5 WT DMSO (FIG.17A), PRMT5 WT compound A1 (FIG.17B), PRMT5 C278A DMSO (FIG.17C), and PRMT5 C278A compound A1 (FIG.17D).
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
  • HPLC high pressure liquid chromatography
  • structures depicted herein also include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of 19 F with 18 F, or the replacement of 12 C with 13 C or 14 C are within the scope of the disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.
  • a range of values is listed, it encompasses each value and sub-range within the range.
  • C 1-6 alkyl encompasses C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.
  • aliphatic refers to alkyl, alkenyl, alkynyl, and carbocyclic groups.
  • heteroaliphatic refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.
  • alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C 1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C 1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”).
  • an alkyl group has 1 to 4 carbon atoms (“C 1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C 1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C 1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C 2-6 alkyl”).
  • C 1-6 alkyl groups include methyl (C1), ethyl (C2), propyl (C 3 ) (e.g., n-propyl, isopropyl), butyl (C 4 ) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C6) (e.g., n-hexyl).
  • alkyl groups include n-heptyl (C7), n- octyl (C 8 ), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as F).
  • substituents e.g., halogen, such as F
  • the alkyl group is an unsubstituted C 1-10 alkyl (such as unsubstituted C 1-6 alkyl, e.g., ⁇ CH 3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)).
  • unsubstituted C 1-6 alkyl such as unsubstituted C 1-6 alkyl, e.g., ⁇ CH 3 (Me),
  • the alkyl group is a substituted C 1-10 alkyl (such as substituted C 1-6 alkyl, e.g., ⁇ CF 3 , Bn).
  • haloalkyl is a substituted alkyl group, wherein one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo.
  • the haloalkyl moiety has 1 to 8 carbon atoms (“C1-8 haloalkyl”).
  • the haloalkyl moiety has 1 to 6 carbon atoms (“C 1-6 haloalkyl”).
  • the haloalkyl moiety has 1 to 4 carbon atoms (“C 1-4 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms (“C 1-3 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C 1-2 haloalkyl”). Examples of haloalkyl groups include –CHF 2 , ⁇ CH 2 F, ⁇ CF 3 , ⁇ CH 2 CF 3 , ⁇ CF 2 CF 3 , ⁇ CF 2 CF 2 CF 3 , ⁇ CCl3, ⁇ CFCl 2 , ⁇ CF 2 Cl, and the like.
  • heteroalkyl refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • a heteroalkyl group refers to a saturated group having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-2 0 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 18 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-18 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 16 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-16 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 14 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-14 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 12 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-12 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-10 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-8 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-6 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC 1-4 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC 1-3 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC 1-2 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC1 alkyl”). In some embodiments, the heteroalkyl group defined herein is a partially unsaturated group having 1 or more heteroatoms within the parent chain and at least one unsaturated carbon, such as a carbonyl group. For example, a heteroalkyl group may comprise an amide or ester functionality in its parent chain such that one or more carbon atoms are unsaturated carbonyl groups.
  • each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents.
  • the heteroalkyl group is an unsubstituted heteroC 1-2 0 alkyl.
  • the heteroalkyl group is an unsubstituted heteroC1-10 alkyl.
  • the heteroalkyl group is a substituted heteroC 1-20 alkyl.
  • the heteroalkyl group is an unsubstituted heteroC1-10 alkyl.
  • alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds).
  • an alkenyl group has 2 to 9 carbon atoms (“C2-9 alkenyl”).
  • an alkenyl group has 2 to 8 carbon atoms (“C2-8 alkenyl”).
  • an alkenyl group has 2 to 7 carbon atoms (“C2-7 alkenyl”).
  • an alkenyl group has 2 to 6 carbon atoms (“C 2-6 alkenyl”).
  • an alkenyl group has 2 to 5 carbon atoms (“C 2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C 2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C 2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C 2 alkenyl”).
  • the one or more carbon- carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
  • Examples of C 2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1- butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ), and the like.
  • Examples of C 2-6 alkenyl groups include the aforementioned C 2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C 8 ), octatrienyl (C 8 ), and the like.
  • each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents.
  • the alkenyl group is an unsubstituted C 2-10 alkenyl.
  • the alkenyl group is a substituted C 2-10 alkenyl.
  • heteroalkenyl refers to an alkenyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • a heteroalkenyl group refers to a group having from 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-10 alkenyl”).
  • a heteroalkenyl group has 2 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-9 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-8 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-7 alkenyl”).
  • a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-6 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-5 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-4 alkenyl”).
  • a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC 2-3 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-6 alkenyl”). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC 2-10 alkenyl.
  • the heteroalkenyl group is a substituted heteroC2-10 alkenyl.
  • alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C2-10 alkynyl”).
  • an alkynyl group has 2 to 9 carbon atoms (“C2-9 alkynyl”).
  • an alkynyl group has 2 to 8 carbon atoms (“C 2-8 alkynyl”).
  • an alkynyl group has 2 to 7 carbon atoms (“C 2- 7 alkynyl”).
  • an alkynyl group has 2 to 6 carbon atoms (“C 2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C 2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C 2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C 2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”).
  • the one or more carbon- carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).
  • Examples of C 2-4 alkynyl groups include, without limitation, ethynyl (C 2 ), 1-propynyl (C 3 ), 2- propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like.
  • Examples of C 2-6 alkenyl groups include the aforementioned C 2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C 7 ), octynyl (C 8 ), and the like.
  • each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents.
  • the alkynyl group is an unsubstituted C2-10 alkynyl.
  • the alkynyl group is a substituted C 2-10 alkynyl.
  • heteroalkynyl refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • a heteroalkynyl group refers to a group having from 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-10 alkynyl”).
  • a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-9 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2- 8 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-7 alkynyl”).
  • a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC 2-6 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-5 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-4 alkynyl”).
  • a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC 2-3 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC 2-6 alkynyl”). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents.
  • the heteroalkynyl group is an unsubstituted heteroC 2-10 alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC2-10 alkynyl.
  • the term “carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C 3-14 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (“C3-10 carbocyclyl”).
  • a carbocyclyl group has 3 to 8 ring carbon atoms (“C 3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C 3-7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C 3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C 4-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C 5-6 carbocyclyl”).
  • a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”).
  • Exemplary C 3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C 4 ), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C6), and the like.
  • Exemplary C3-8 carbocyclyl groups include, without limitation, the aforementioned C 3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C 8 ), bicyclo[2.2.1]heptanyl (C 7 ), bicyclo[2.2.2]octanyl (C 8 ), and the like.
  • Exemplary C3-10 carbocyclyl groups include, without limitation, the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C 9 ), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ), and the like.
  • the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • Carbocyclyl also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
  • each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents.
  • the carbocyclyl group is an unsubstituted C3-14 carbocyclyl.
  • the carbocyclyl group is a substituted C3-14 carbocyclyl.
  • “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (“C3-14 cycloalkyl”).
  • a cycloalkyl group has 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”).
  • a cycloalkyl group has 3 to 8 ring carbon atoms (“C 3-8 cycloalkyl”).
  • a cycloalkyl group has 3 to 6 ring carbon atoms (“C 3-6 cycloalkyl”).
  • a cycloalkyl group has 4 to 6 ring carbon atoms (“C 4-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C 5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C 5-10 cycloalkyl”). Examples of C 5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C 3-6 cycloalkyl groups include the aforementioned C 5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C 4 ).
  • C 3-8 cycloalkyl groups include the aforementioned C 3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8).
  • each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.
  • the cycloalkyl group is an unsubstituted C3-14 cycloalkyl.
  • the cycloalkyl group is a substituted C3-14 cycloalkyl.
  • heterocyclyl refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”).
  • heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon- carbon double or triple bonds.
  • Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
  • each instance of heterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents.
  • the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.
  • the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
  • a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”).
  • a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”).
  • a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”).
  • the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, and thiiranyl.
  • Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl.
  • Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione.
  • Exemplary 5- membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.
  • Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl.
  • Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazinyl.
  • Exemplary 7- membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
  • Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8- naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole,
  • aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”).
  • an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl).
  • an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl).
  • an aryl group has 14 ring carbon atoms (“C14 aryl”; e.g., anthracyl).
  • Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
  • the aryl group is an unsubstituted C 6-14 aryl.
  • the aryl group is a substituted C6-14 aryl.
  • “Aralkyl” is a subset of “alkyl” and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety.
  • heteroaryl refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system.
  • a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”).
  • a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”).
  • a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”).
  • the 5- 6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.
  • Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl, and thiophenyl.
  • Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl.
  • Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
  • Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 5,6- bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
  • Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.
  • Heteroaralkyl is a subset of “alkyl” and refers to an alkyl group substituted by a heteroaryl group, wherein the point of attachment is on the alkyl moiety.
  • the term “unsaturated bond” refers to a double or triple bond.
  • the term “unsaturated” or “partially unsaturated” refers to a moiety that includes at least one double or triple bond.
  • the term “saturated” refers to a moiety that does not contain a double or triple bond, i.e., the moiety only contains single bonds.
  • alkylene is the divalent moiety of alkyl
  • alkenylene is the divalent moiety of alkenyl
  • alkynylene is the divalent moiety of alkynyl
  • heteroalkylene is the divalent moiety of heteroalkyl
  • heteroalkenylene is the divalent moiety of heteroalkenyl
  • heteroalkynylene is the divalent moiety of heteroalkynyl
  • carbocyclylene is the divalent moiety of carbocyclyl
  • heterocyclylene is the divalent moiety of heterocyclyl
  • arylene is the divalent moiety of aryl
  • heteroarylene is the divalent moiety of heteroaryl.
  • a group is optionally substituted unless expressly provided otherwise.
  • the term “optionally substituted” refers to being substituted or unsubstituted.
  • alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted.
  • Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group).
  • substituted means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
  • substituted is contemplated to include substitution with all permissible substituents of organic compounds, and includes any of the substituents described herein that results in the formation of a stable compound.
  • the present disclosure contemplates any and all such combinations in order to arrive at a stable compound.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
  • the present disclosure is not limited in any manner by the exemplary substituents described herein.
  • halo or halogen refers to fluorine (fluoro, ⁇ F), chlorine (chloro, ⁇ Cl), bromine (bromo, ⁇ Br), or iodine (iodo, ⁇ I).
  • hydroxyl or “hydroxy” refers to the group ⁇ OH.
  • amino refers to the group ⁇ NH2.
  • substituted amino by extension, refers to a monosubstituted amino, a disubstituted amino, or a trisubstituted amino. In certain embodiments, the “substituted amino” is a monosubstituted amino or a disubstituted amino group.
  • trisubstituted amino refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with three groups, and includes groups selected from ⁇ N(R bb ) 3 and ⁇ N(R bb ) 3 + X ⁇ , wherein R bb and X ⁇ are as defined herein.
  • sulfonyl refers to a group selected from –SO 2 N(R bb )2, –SO 2 R aa , and – SO 2 OR aa , wherein R aa and R bb are as defined herein.
  • acyl groups include aldehydes ( ⁇ CHO), carboxylic acids ( ⁇ CO2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas.
  • Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyl
  • ketones e.g
  • sil refers to the group –Si(R aa )3, wherein R aa is as defined herein.
  • Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms.
  • the substituent present on the nitrogen atom is an nitrogen protecting group (also referred to herein as an “amino protecting group”).
  • Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • Nitrogen protecting groups such as carbamate groups include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD- Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1- methyle
  • Nitrogen protecting groups such as sulfonamide groups include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6- dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanes
  • Ts p-toluenesulfonamide
  • Mtr 2,
  • nitrogen protecting groups include, but are not limited to, phenothiazinyl- (10)-acyl derivative, N′-p-toluenesulfonylaminoacyl derivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3- oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5- dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5- substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5- triazacyclohexan-2-one, 1-substituted 3,5-dinitro
  • a nitrogen protecting group is benzyl (Bn), tert- butyloxycarbonyl (BOC), carbobenzyloxy (Cbz), 9-flurenylmethyloxycarbonyl (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4- dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), 2,2,2-trichloroethyloxycarbonyl (Troc), triphenylmethyl (Tr), tosyl (Ts), brosyl (Bs), nosyl (Ns), mesyl (Ms), triflyl (Tf), or dansyl (Ds).
  • Bn benzyl
  • BOC tert- butyloxycarbonyl
  • Cbz carbobenzyloxy
  • Fmoc 9-flurenylmethyloxycarbony
  • the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an “hydroxyl protecting group”).
  • Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4- methoxytetrahydropyranyl (MT), methyl,
  • an oxygen protecting group is silyl.
  • an oxygen protecting group is t-butyldiphenylsilyl (TBDPS), t- butyldimethylsilyl (TBDMS), triisoproylsilyl (TIPS), triphenylsilyl (TPS), triethylsilyl (TES), trimethylsilyl (TMS), triisopropylsiloxymethyl (TOM), acetyl (Ac), benzoyl (Bz), allyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylsilylethyl carbonate, methoxymethyl (MOM), 1-ethoxyethyl (EE), 2-methyoxy-2-propyl (MOP), 2,2,2- trichloroethoxyethyl, 2-methoxyethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM), methylthiomethyl (MTM), te
  • TDPS t
  • the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a “thiol protecting group”).
  • a sulfur protecting group is acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl.
  • a “counterion” or “anionic counterion” is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality.
  • An anionic counterion may be monovalent (i.e., including one formal negative charge).
  • An anionic counterion may also be multivalent (i.e., including more than one formal negative charge), such as divalent or trivalent.
  • exemplary counterions include halide ions (e.g., F – , Cl – , Br – , I – ), NO 3 – , ClO 4 – , OH – , H 2 PO 4 – , HCO 3 ⁇ , HSO 4 – , sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p–toluenesulfonate, benzenesulfonate, 10–camphor sulfonate, naphthalene–2–sulfonate, naphthalene–1–sulfonic acid–5–sulfonate, ethan–1–sulfonic acid– 2–sulfonate, and the like), carboxylate ions (e.g.,
  • Exemplary counterions which may be multivalent include CO 3 2 ⁇ , HPO 4 2 ⁇ , PO 4 3 ⁇ , B 4 O 7 2 ⁇ , SO 4 2 ⁇ , S 2 O 3 2 ⁇ , carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes.
  • carboxylate anions e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like
  • carboxylate anions e.g., tartrate, citrate, fumarate, maleate,
  • suitable leaving groups include, but are not limited to, halogen (such as F, Cl, Br, or I (iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy, aryloxy, methoxy, N,O- dimethylhydroxylamino, pixyl, and haloformates.
  • halogen such as F, Cl, Br, or I (iodine)
  • alkoxycarbonyloxy such as F, Cl, Br, or I (iodine)
  • alkoxycarbonyloxy such as F, Cl, Br, or I (iodine)
  • alkoxycarbonyloxy such as F, Cl, Br, or I (iodine
  • aryloxycarbonyloxy alkanesulfonyloxy
  • arenesulfonyloxy
  • the leaving group is a brosylate, such as p-bromobenzenesulfonyloxy.
  • the leaving group is a nosylate, such as 2-nitrobenzenesulfonyloxy.
  • the leaving group is a phosphineoxide (e.g., formed during a Mitsunobu reaction) or an internal leaving group such as an epoxide or cyclic sulfate.
  • phosphineoxide e.g., formed during a Mitsunobu reaction
  • an internal leaving group such as an epoxide or cyclic sulfate.
  • Other non-limiting examples of leaving groups are water, ammonia, alcohols, ether moieties, thioether moieties, zinc halides, magnesium moieties, diazonium salts, and copper moieties.
  • non-hydrogen group refers to any group that is defined for a particular variable that is not hydrogen.
  • salt refers to any and all salts, and encompasses pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
  • Pharmaceutically acceptable salts of the compounds of this present disclosure include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N + (C 1-4 alkyl)4 ⁇ salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
  • solvate refers to forms of the compound, or a salt thereof, that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding.
  • solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like.
  • the compounds described herein may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates.
  • the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid.
  • “Solvate” encompasses both solution-phase and isolatable solvates.
  • Representative solvates include hydrates, ethanolates, and methanolates.
  • hydrate refers to a compound that is associated with water. Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R ⁇ x H2O, wherein R is the compound, and x is a number greater than 0.
  • a given compound may form more than one type of hydrate, including, e.g., monohydrates (x is 1), lower hydrates (x is a number greater than 0 and smaller than 1, e.g., hemihydrates (R ⁇ 0.5 H 2 O)), and polyhydrates (x is a number greater than 1, e.g., dihydrates (R ⁇ 2 H 2 O) and hexahydrates (R ⁇ 6 H 2 O)).
  • monohydrates x is 1
  • lower hydrates x is a number greater than 0 and smaller than 1, e.g., hemihydrates (R ⁇ 0.5 H 2 O)
  • polyhydrates x is a number greater than 1, e.g., dihydrates (R ⁇ 2 H 2 O) and hexahydrates (R ⁇ 6 H 2 O)
  • tautomers or “tautomeric” refers to two or more interconvertible compounds resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa).
  • the exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Tautomerizations (i.e., the reaction providing a tautomeric pair) may catalyzed by acid or base.
  • Exemplary tautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim, enamine-to-imine, and enamine-to-(a different enamine) tautomerizations.
  • isomers compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”.
  • stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”.
  • enantiomers When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible.
  • An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or ( ⁇ )-isomers respectively).
  • a chiral compound can exist as either individual enantiomer or as a mixture thereof.
  • a mixture containing equal proportions of the enantiomers is called a “racemic mixture”.
  • the term “polymorph” refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof). All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate.
  • Various polymorphs of a compound can be prepared by crystallization under different conditions.
  • prodrugs refers to compounds that have cleavable groups and become by solvolysis or under physiological conditions the compounds described herein, which are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like. Other derivatives of the compounds described herein have activity in both their acid and acid derivative forms, but in the acid sensitive form often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see Bundgard, H., Design of Prodrugs, pp.7-9, 21-24, Elsevier, Amsterdam 1985).
  • Prodrugs include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides, and anhydrides derived from acidic groups pendant on the compounds described herein are particular prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters.
  • a “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • a human i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)).
  • the non-human animal is a fish, reptile, or amphibian.
  • the non-human animal may be a male or female at any stage of development.
  • the non-human animal may be a transgenic animal or genetically engineered animal.
  • patient refers to a human subject in need of treatment of a disease.
  • the subject may also be a plant.
  • the plant is a land plant. In certain embodiments, the plant is a non- vascular land plant. In certain embodiments, the plant is a vascular land plant. In certain embodiments, the plant is a seed plant. In certain embodiments, the plant is a cultivated plant. In certain embodiments, the plant is a dicot. In certain embodiments, the plant is a monocot. In certain embodiments, the plant is a flowering plant. In some embodiments, the plant is a cereal plant, e.g., maize, corn, wheat, rice, oat, barley, rye, or millet. In some embodiments, the plant is a legume, e.g., a bean plant, e.g., soybean plant.
  • the plant is a tree or shrub.
  • tissue samples such as tissue sections and needle biopsies of a tissue
  • cell samples e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise).
  • tissue refers to any biological tissue of a subject (including a group of cells, a body part, or an organ) or a part thereof, including blood and/or lymph vessels, which is the object to which a compound, particle, and/or composition of the present disclosure is delivered.
  • a tissue may be an abnormal or unhealthy tissue, which may need to be treated.
  • a tissue may also be a normal or healthy tissue that is under a higher than normal risk of becoming abnormal or unhealthy, which may need to be prevented.
  • the tissue is the central nervous system.
  • the tissue is the brain.
  • administer refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject.
  • treatment “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein.
  • treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
  • the terms “condition,” “disease,” and “disorder” are used interchangeably.
  • An “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response.
  • an effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses. [00120] A “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces, or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent.
  • a therapeutically effective amount is an amount sufficient for inhibiting binding of PRMT5 with a PRMT5 substrate adaptor.
  • a therapeutically effective amount is an amount sufficient for treating a cancer.
  • a “prophylactically effective amount” of a compound described herein is an amount sufficient to prevent a condition, or one or more signs or symptoms associated with the condition, or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • a “proliferative disease” refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990).
  • a proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis.
  • Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.
  • angiogenesis refers to the physiological process through which new blood vessels form from pre-existing vessels.
  • Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development.
  • Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue.
  • angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer.
  • Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF).
  • angiogenic proteins such as growth factors (e.g., VEGF).
  • VEGF growth factors
  • neoplasm and tumor are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue.
  • a neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis.
  • a “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin.
  • a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites.
  • Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias.
  • certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor’s neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.”
  • An exemplary pre-malignant neoplasm is a teratoma.
  • a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue.
  • a malignant neoplasm generally has the capacity to metastasize to distant sites.
  • metastasis refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located.
  • a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.
  • cancer refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues.
  • hematological malignancy refers to tumors that affect blood, bone marrow, and/or lymph nodes.
  • Exemplary hematological malignancies include, but are not limited to, leukemia, such as acute lymphocytic leukemia (ALL) (e.g., B- cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma, such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B- cell NHL, such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL, e.g., activated B-cell (AB
  • Additional exemplary cancers include, but are not limited to, lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); kidney cancer (e.g., nephroblastoma, a.k.a.
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung
  • kidney cancer e.g., nephroblastoma, a.k.a.
  • Wilms tumor, renal cell carcinoma); acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary duct cancer; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma);
  • myelofibrosis MF
  • chronic idiopathic myelofibrosis chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)
  • neuroblastoma e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis
  • neuroendocrine cancer e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor
  • osteosarcoma e.g.,bone cancer
  • ovarian cancer e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma
  • papillary adenocarcinoma pancreatic cancer
  • pancreatic cancer e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere
  • leukemia refers to broadly progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • Leukemia diseases include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic
  • sarcoma generally refers to a tumor which arises from transformed cells of mesenchymal origin. Sarcomas are malignant tumors of the connective tissue and are generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma,
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
  • biological refers to a wide range of products such as vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues, nucleic acids, and proteins.
  • Biologics may include sugars, proteins, or nucleic acids, or complex combinations of these substances, or may be living entities, such as cells and tissues. Biologics may be isolated from a variety of natural sources (e.g., human, animal, microorganism) and may be produced by biotechnological methods and other technologies.
  • small molecule or “small molecule therapeutic” refers to molecules, whether naturally occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight.
  • a small molecule is an organic compound (i.e., it contains carbon).
  • the small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.).
  • the molecular weight of a small molecule is not more than about 1,000 g/mol, not more than about 900 g/mol, not more than about 800 g/mol, not more than about 700 g/mol, not more than about 600 g/mol, not more than about 500 g/mol, not more than about 400 g/mol, not more than about 300 g/mol, not more than about 200 g/mol, or not more than about 100 g/mol.
  • the molecular weight of a small molecule is at least about 100 g/mol, at least about 200 g/mol, at least about 300 g/mol, at least about 400 g/mol, at least about 500 g/mol, at least about 600 g/mol, at least about 700 g/mol, at least about 800 g/mol, or at least about 900 g/mol, or at least about 1,000 g/mol. Combinations of the above ranges (e.g., at least about 200 g/mol and not more than about 500 g/mol) are also possible.
  • the small molecule is a therapeutically active agent such as a drug (e.g., a molecule approved by the U.S.
  • the small molecule may also be complexed with one or more metal atoms and/or metal ions.
  • the small molecule is also referred to as a “small organometallic molecule.”
  • Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, more preferably humans. Small molecules include, but are not limited to, radionuclides and imaging agents.
  • the small molecule is a drug.
  • the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body. For example, drugs approved for human use are listed by the FDA under 21 C.F.R.
  • therapeutic agent refers to any substance having therapeutic properties that produce a desired, usually beneficial, effect.
  • therapeutic agents may treat, ameliorate, and/or prevent disease.
  • therapeutic agents, as disclosed herein may be biologics or small molecule therapeutics.
  • the present disclosure relates to the discovery by the inventors of compounds, compositions, and methods for modulating PRMT5 activities and functionalities, including substrate recruitment, including for the treatment of disease such as cancer.
  • This discovery is based, in part, on the identification of a method of PRMT5 substrate recruitment, through the highly conserved PRMT5 binding motif (PBM) in its substrate adaptors.
  • PBM PRMT5 binding motif
  • the inventors posit that the PBM binding site in PRMT5, which is distal to the catalytic site, is useful for substrate adaptor binding and substrate methylation.
  • the PRMT5 substrate recognition motif (RG or GRG) is quite short and does not account for the specificity of PRMT5 substrate methylation observed in the cell. Therefore, the substrate adaptor site (herein referred to as the PBM groove) provides a novel mechanism of selective substrate recruitment to PRMT5.
  • the TIM barrel in which the PBM groove exists, is uniquely present in PRMT5 and lacking in the other PRMTs, suggesting that the PBM- PBM groove interaction is distinct, though the other PRMTs may possess orthogonal binding sites for substrate recruitment.
  • Pathway analysis of PRMT5 substrates described herein reveal PRMT5 substrates largely belonging to the RNA transcription and processing pathways.
  • PRMT5 substrates are modified by a two-step process: 1) recruitment via PBM-adaptor and 2) catalysis of methyl transfer.
  • Molecules MTA, GSK591 and JNJ-64619178 all target the active site, but this novel mechanism provides an alternative or additional route for pathway inhibition. Therefore, therapeutically targeting such a site offers a way to tune the classes of PRMT5 substrates lost and contributes to a more efficacious and less toxic PRMT5 targeting agent.
  • the disclosure posits that the PBM is sufficient and necessary for a stable interaction, and that the peptide alone is sufficient to disrupt interactions between full-length proteins in biophysical and permeabilized cell contexts.
  • the substrates may have some weak intrinsic affinity for PRMT5 through contact sites in and near the catalytic site, as has been shown for the histone 4 tail (Mavrakis, K. J. et al. Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5. Science 351, 1208–13 (2016)), and thereby PBM depleted substrate adaptors are co- complexed weakly with the methylosome indirectly through their binding of substrates in the cell. [00139] Genetic disruption of this novel substrate adaptor binding site may also synergize with other PRMT pathway inhibitors.
  • PRMT5 recruits substrates for methylation.
  • the inventors posit that the newly identified pocket of the methylosome is required for the binding of substrate adaptors pICLn, RIOK1, and COPR5 and for the methylation of select PRMT5 substrates.
  • Modulation of the PRMT5-substrate adaptor binding site leads to hypomorphic effects on cell growth in MTAP null cells and may synergize with existing PRMT pathway inhibitors. Given the effects of perturbing this site on cell growth coupled with previous findings that existing PRMT5 catalytic inhibitors are not selective for MTAP null cells (Mavrakis, K. J. et al.
  • PRMT5 functions as an arginine methyltransferase and is a therapeutic target in MTAP null tumors.
  • PRMT5 utilizes substrate adaptor proteins for the recruitment of substrates.
  • An evolutionarily conserved peptide sequence is shared among the three known substrate adaptors (pICLn/CLNS1A, RIOK1 and COPR5) through which the PRMT5 methylosome recruits substrates for methylation.
  • This novel site is required for binding of pICLn, RIOK1 and COPR5 to PRMT5 and for the methylation of select PRMT5 substrates including SmB and nucleolin.
  • Disruption of the PRMT5-substrate adaptor interaction leads to a hypomorphic decrease in growth of MTAP null tumor cells and is a potential site for modulation of PRMT5 function that is independent of the catalytic site. Disruption of this site may synergize with cells to clinically relevant pathway inhibitors of PRMT1, PRMT5, and MAT2A and thus provide a potential for targeted therapeutic development coupled to existing therapies in the clinic.
  • the compounds may inhibit binding by contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent.
  • the disclosure provides compounds of Formula (I), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co- crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and pharmaceutical compositions thereof.
  • the compounds are useful for the treatment of diseases associated with PRMT5 (e.g., cancer) in a subject in need thereof.
  • Q is CR a or N;
  • X 1 is CH or N;
  • R 1 is H, -CN, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -OR O , -N(R N1 )R N2 , -C(O)R b , -C(O)OR O , -C(O)N(R N1 )R N2 , -SR S , -S(O)R S , -SO 2 R S , -SO 2 OR O , or -SO 2 N(R N1 )R N2 ;
  • R 2 is chloro;
  • R 3 is H, hal
  • Q is CR a or N. In certain embodiments, Q is CR a . In certain embodiments, Q is CH. In certain embodiments, Q is N.
  • X 1 As described herein, Q is CH or N. In certain embodiments, Q is CH. In certain embodiments, Q is N.
  • R 1 is H, halogen, -CN, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -OR O , -N(R N1 )R N2 , -C(O)R b , -C(O)OR O , -C(O)N(R N1 )R N2 , -SR S , -S(O)R S , -SO 2 R S , -SO 2 OR O , or -SO 2 N(R N1 )R N2 .
  • R 1 is H, halogen, optionally substituted alkyl, -alkylamino, or haloalkyl. In certain embodiments, R 1 is H, optionally substituted alkyl, -alkylamino, or haloalkyl. In certain embodiments, R 1 is optionally substituted alkyl substituted with one -OR O . In certain embodiments, R 1 is H, F, Cl, Br, -CH 2 NH2, -CH 2 OH, -CHF 2 , -CF 3 , or -CH 2 F.
  • R 1 is H, -CH 2 NH 2 , -CH 2 OH, -CHF 2 , -CF 3 , or -CH 2 F. In certain embodiments, R 1 is H or halogen. In certain embodiments, R 1 is H or Cl. In certain embodiments, R 1 is halogen. In certain embodiments, R 1 is Cl. In certain embodiments, R 1 is H. R 2 [00149] As described herein, R 2 is a leaving group; or R 1 and R 2 , taken together with the atoms to which they are attached, form an optionally substituted heterocyclic ring.
  • R 2 is chloride, bromide, iodide, fluoride, tosyl, mesyl, triflyl, nonaflyl, nitrate, phosphate, carboxylate, -OR b , -SR S , phenoxide, or -OSO 2 R b .
  • R 2 is chloride, bromide, iodide, fluoride, tosyl, mesyl, triflyl, nonaflyl, carboxylate, -OR b , -SR S , phenoxide, -SO 2 R b , or -OSO 2 R b .
  • R 2 is chloride, bromide, fluoride, -OSO 2 CH3, -OSO 2 CF 3 , -OSO 2 -para-tolyl, or –OSO 2 -para- phenyl-CF 3 .
  • R 2 is chloride, bromide, -OSO 2 CH 3 , -OSO 2 CF 3 , -OSO 2 - para-tolyl, or –OSO 2 -para-phenyl-CF 3 .
  • R 2 is fluoride or chloride.
  • R 2 is fluoride.
  • R 2 is chloride.
  • R 1 and R 2 are both halogen.
  • R 1 and R 2 are both Cl.
  • R 2 is not H or unsubstituted alkyl. In certain embodiments, R 2 is not H or unsubstituted C1-C2 alkyl. In certain embodiments, R 2 is not H. In certain embodiments, R 2 is not unsubstituted alkyl. In certain embodiments, R 2 is not unsubstituted C1-C2 alkyl. [00153] In certain embodiments, R 1 and R 2 , taken together with the atoms to which they are attached, form an optionally substituted heterocyclic ring.
  • R 3 is H, halogen, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -OR O , -N(R N1 )R N2 , -C(O)R b , -C(O)OR O , -C(O)N(R N1 )R N2 , -SR S , -S(O)R S , -SO 2 R S , -SO 2 OR O , or -SO 2 N(R N1 )R N2 .
  • R 3 is H.
  • R 4 and R 5 are independently selected from H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -OR O , -N(R N1 )R N2 , or optionally substituted alkyl substituted with one -OR O ; or R 4 and R 5 , taken together with the atom to which they are attached, form an optionally substituted 4 to 9-membered ring optionally including 1 or 2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1
  • At least one of R 4 and R 5 is optionally substituted alkyl, or halogen. In certain embodiments, at least one of R 4 and R 5 is alkyl optionally substituted with one -OR O . In certain embodiments, at least one of R 4 and R 5 is methyl, ethyl, halogen, or -CH 2 OH. In certain embodiments, one of R 4 and R 5 is H and the other is not H. In certain embodiments, one of R 4 and R 5 is H and the other is methyl. In certain embodiments, R 4 and R 5 are each H. In certain embodiments, R 4 and R 5 are selected so as to provide the (S) absolute configuration at the carbon atom to which they are attached.
  • R 4 and R 5 are selected so as to provide the (R) absolute configuration at the carbon atom to which they are attached. [00158] In certain embodiments, when Q is CH, R 1 is Cl, R 2 is Cl, and one of R 4 and R 5 is not H.
  • R 6 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(O)R b , -C(O)N(R N1 )R N2 , or a nitrogen protecting group; or taken together with one of R 4 and R 5 , forms a 4 to 9-membered ring optionally including 1 or 2 additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1 , and each ring sulfur is optionally substituted 1 to 4 groups each independently selected from R S ; wherein R a and R 6 , taken
  • R 6 is H, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(O)R b , -C(O)N(R N1 )R N2 , or a nitrogen protecting group; or taken together with one of R 4 and R 5 , forms a 4 to 9-membered ring optionally including 1 or 2 additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1 , and each ring sulfur is optionally substituted 1 to 4 groups each independently selected from R S .
  • R 6 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(O)R b , -C(O)N(R N1 )R N2 , or a nitrogen protecting group.
  • R 6 is H, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(O)R b , -C(O)N(R N1 )R N2 , or a nitrogen protecting group.
  • R 6 is H or optionally substituted alkyl.
  • R 6 is optionally substituted alkyl.
  • R 6 is alkyl substituted with a heteroaryl.
  • R 6 is H.
  • R 6 taken together with one of R 4 and R 5 , forms a 4 to 9- membered ring optionally including 1 or 2 additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1 , and each ring sulfur is optionally substituted 1 to 4 groups each independently selected from R S .
  • R a and R 6 taken together with the atoms to which they are attached, optionally form an optionally substituted 5 to 9-membered heterocyclic ring.
  • each R 7 and R 8 is independently selected from H, halogen, - CN, -NO 2 , optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -OR O , -N(R N1 )R N2 , -C(O)R b , -C(O)OR O , -C(O)N(R N1 )R N2 , -SR S , -S(O)R S , -SO 2 R S , -SO 2 OR O , or -SO 2 N(R N1 )R N2 ; or R 7 and R 8 , taken together with
  • each R 7 and R 8 is independently selected from H, halogen, -CN, -NO 2 , optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -OR O , -N(R N1 )R N2 , -C(O)R b , -C(O)OR O , -C(O)N(R N1 )R N2 , -SR S , -S(O)R S , -SO 2 R S , -SO 2 OR O , or -SO 2 N(R N1 )R N2 .
  • R 7 and R 8 taken together with the atoms to which they are attached, form an optionally substituted aryl, optionally substituted carbocyclic, optionally substituted heteroaryl, or optionally substituted heterocyclic ring.
  • at least one of R 7 and R 8 is halogen, optionally substituted alkyl, or optionally substituted heteroalkyl.
  • at least one of R 7 and R 8 is halogen or haloalkyl.
  • each of R 7 and R 8 are H.
  • R 9 is H, halogen, -CN, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -OR O , -N(R N1 )R N2 , -C(O)R b , -C(O)OR O , -C(O)N(R N1 )R N2 , -SR S , -S(O)R S , -SO 2 R S , -SO 2 OR O , or -SO 2 N(R N1 )R N2 ; wherein R 9 and R 8 optionally combine to form an optionally substituted aryl, optionally substituted carbocycloalkyl, optionally substituted al
  • R 9 is H, halogen, -CN, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -OR O , -N(R N1 )R N2 , -C(O)R b , -C(O)OR O , -C(O)N(R N1 )R N2 , -SR S , -S(O)R S , -SO 2 R S , -SO 2 OR O , or -SO 2 N(R N1 )R N2 .
  • R 9 and one of R 10 and R 11 combine to form a 5-8- membered ring optionally including 1 or 2 additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1 , and each ring sulfur is optionally substituted 1 to 4 groups each independently selected from R S .
  • R 9 and R 8 combine to form an optionally substituted aryl, optionally substituted carbocyclic, optionally substituted heteroaryl, or optionally substituted heterocyclic ring.
  • R 9 is optionally substituted alkyl.
  • R 9 is methyl or ethyl. In certain embodiments, R 9 is ethyl. In certain embodiments, R 9 is methyl. In certain embodiments, R 9 is haloalkyl. In certain embodiments, R 9 is -CH 2 F, -CHF 2 , or -CF 3 . In certain embodiments, R 9 is -CH 2 F. In certain embodiments, R 9 is -CHF 2 . In certain embodiments, R 9 is -CF 3 . [00174] In certain embodiments, at least one of R 7 , R 8 , and R 9 is halogen, optionally substituted alkyl, or optionally substituted heteroalkyl.
  • R 10 and R 11 is independently selected from H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted aryl, optionally substituted heteroaryl, or a bond to R 9 to form a 5-, 6-, 7-, or 8-membered ring with the interceding atoms; or R 10 and R 11 , taken together with the atom to which they are attached, form a 4- to 8-membered ring optionally including 1 or 2 additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1 , and each ring sulfur
  • each of R 10 and R 11 is independently selected from H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, or a bond to R 9 to form a 5-, 6-, 7-, or 8-membered ring with the interceding atoms.
  • R 10 and R 11 taken together with the nitrogen atom to which they are attached, form a 5- to 8-membered ring optionally including 1 or 2 additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1 , and each ring sulfur is optionally substituted 1 to 4 groups each independently selected from R S .
  • R a and one of R 10 or R 11 optionally combine to form a 6 to 9-membered ring optionally including 1 or 2 additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1 , and each ring sulfur is optionally substituted 1 to 4 groups each independently selected from R S .
  • one of R 10 and R 11 is of the formula –L-M; wherein L is a bond or optionally substituted alkylene; and M is optionally substituted aryl or optionally substituted heteroaryl. In certain embodiments, M is phenyl.
  • M is pyridyl. In certain embodiments, M is substituted with one or more of a halogen, -SR S , -S(O)R S , -SO 2 R S , -SO 2 OR O , and -SO 2 N(R N1 )R N2 .
  • L is C 1 -C 6 alkylene. In certain embodiments, L is methylene or ethylene. In certain embodiments, L is ethylene. In certain embodiments, L is methylene. [00180] In certain embodiments, one of R 10 and R 11 is of the formula –L-M, and the other of R 10 and R 11 is H.
  • R 10 and R 11 is of the formula: wherein p is 0, 1, 2, 3, 4, or 5; n is 0, 1, 2, 3, 4, 5, or 6; Y is N or CR a ; and R 12 is halogen, alkyl, cycloalkyl, -OR O , -SR S , -S(O)R S , -SO 2 R S , -SO 2 OR O , or -SO 2 N(R N1 )R N2 .
  • p is 0 or 1; and n is 1 or 2.
  • Y is N or CH.
  • R 10 and R 11 is of the formula: [00183] In certain embodiments, one of R 10 and R 11 is alkyl optionally substituted with one, two, or three groups each independently selected from -NHC(O)R b . In certain embodiments, one of R 10 and R 11 is optionally substituted alkyl substituted with one, two, or three groups each independently selected from -NHC(O)R b , and R b is optionally substituted alkyl. [00184] In certain embodiments, R 10 and R 11 are each independently optionally substituted alkyl or optionally substituted cycloalkyl. In certain embodiments, R 10 and R 11 are each methyl.
  • R a is H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(O)R b , -C(O)OR O , -C(O)N(R N3 )R N4 , or a nitrogen protecting group; wherein R a and one of R 10 or R 11 optionally combine to form a 6 to 9-membered ring optionally including 1 or 2 additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1 , and each ring sulfur is optionally substituted 1 to 4 groups each independently selected
  • R a is H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(O)R b , -C(O)OR O , -C(O)N(R N3 )R N4 , or a nitrogen protecting group.
  • R a and one of R 10 and R 11 optionally combine to form a 6 to 9-membered ring optionally including 1 or 2 additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, wherein each ring nitrogen is optionally substituted with 1 or 2 groups each independently selected from R N1 , and each ring sulfur is optionally substituted 1 to 4 groups each independently selected from R S .
  • R a and R 6 taken together with the atoms to which they are attached, optionally form an optionally substituted 5 to 9-membered heterocyclic ring.
  • each R b is independently selected from H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.
  • R b is optionally substituted alkyl. In certain embodiments, R b is unsubstituted alkyl.
  • each R N1 and R N2 is independently selected from H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group.
  • each R N3 and R N4 is independently selected from H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group.
  • each R O is independently selected from H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(O)R b , -C(O)OR b , or an oxygen protecting group.
  • the compound of Formula (I) is a compound of Formula (Ia): , or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • R 2 is not H or unsubstituted alkyl.
  • R 2 is not H or unsubstituted C 1 -C 2 alkyl.
  • Q when Q is CH, R 1 is Cl, and R 2 is Cl, then one of R 4 and R 5 is not H.
  • the compound of Formula (I) is a compound of Formula (Ib): , or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein q is 1, 2, or 3; and Q, X 1 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 11 , and R N1 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ib-1): , (Ib-1) or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein q is 1, 2, or 3; and X 1 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 11 , and R N1 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ib-2): , (Ib-2) or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein q is 1, 2, or 3; and Q, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 11 , and R N1 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ib-3): , (Ib-3) or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein q is 1, 2, or 3; and Q, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , R 11 , and R N1 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ib-4): , (Ib-4) or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein q is 1, 2, or 3; and R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 11 , and R N1 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ib-5): , (Ib-5) or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein q is 1, 2, or 3; and R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , R 11 , and R N1 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic): (Ic), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, X 1 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-1): (Ic-1), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein X 1 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-2): (Ic-2), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-3): (Ic-3), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, X 1 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-4): (Ic-4), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-5): (Ic-5), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, X 1 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-6): (Ic-6), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein X 1 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-7): (Ic-7), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-8): (Ic-8), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, X 1 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-9): (Ic-9), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Ic-10): (Ic-10), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and X 1 , Q, R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Ic-11): (Ic-11), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and X 1 , Q, R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Ic-12): (Ic-12), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 13 is methyl.
  • the compound of Formula (I) is a compound of Formula (Ic-13): (Ic-13), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, X 1 , R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 13 is methyl.
  • the compound of Formula (I) is a compound of Formula (Ic-14): (Ic-14), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Ic-15): (Ic-15), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, X 1 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Ic-16): (Ic-16), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and X 1 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Ic-17): (Ic-17), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Ic-18): (Ic-18), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and X 1 , Q, R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Ic-19): (Ic-19), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id): or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, X 1 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-1): or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein X 1 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-2): or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-3): (Id-3), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, X 1 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-4): (Id-4), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-5): (Id-5), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, X 1 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-6): (Id-6), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-7): (Id-7), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-8): (Id-8), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, X 1 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-9): (Id-9), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein Q, R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein.
  • the compound of Formula (I) is a compound of Formula (Id-10): (Id-10), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, X 1 , R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein. [00244] In certain embodiments of the compound of Formula (Id-10), R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id-11): (Id-11), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and X 1 , R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein. [00246] In certain embodiments of the compound of Formula (Id-11), R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id-12): (Id-12), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id-13): (Id-13), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id-14): (Id-14), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id-15): (Id-15), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, X 1 , R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id-15): (Id-16), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and X 1 , R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein. [00256] In certain embodiments of the compound of Formula (Id-16), R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id-17): (Id-17), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, X 1 , R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id-18): (Id-18), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and Q, X 1 , R 3 , R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of Formula (Id-19): (Id-19), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein R 9 is alkyl; and R 4 , R 6 , R 10 , and R 11 are as defined herein.
  • R 9 is methyl.
  • the compound of Formula (I) is a compound of the formula:
  • the compound of Formula (I) is a compound of the formula:
  • the compound of Formula (I) is a compound of the formula: ,
  • the compound of Formula (I) is a compound of the formula:
  • compounds of Formula (I) are inhibitors of the binding of PRMT5 with a PRMT5 substrate adaptor, wherein the inhibitor disrupts binding of PRMT5 with a PRMT5 substrate adaptor.
  • compounds of Formula (I) are modulators of PRTM5 activity, wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • compounds of Formula (I) are modulators of PRMT5 methyltransferase activity, wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • compounds of Formula (I) are inhibitors of PRMT5 substrate methylation, wherein the inhibitors disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • compounds of Formula (I) are modulators of the transcription of a gene regulated by PRMT5, wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • compounds of Formula (I) are modulators of chromatin structure regulation, wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • compounds of Formula (I) are modulators of cellular differentiation, wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • compounds of Formula (I) are modulators of mRNA splicing, wherein the modulators disrupt binding of PRMT5 with a PRMT5 substrate adaptor.
  • Pharmaceutical Compositions, Kits, and Administration [00276] The present disclosure provides pharmaceutical compositions comprising a compound of Formula (I), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, and optionally a pharmaceutically acceptable excipient.
  • the pharmaceutical composition described herein comprises a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • the compound of Formula (I) is provided in an effective amount in the pharmaceutical composition.
  • the effective amount is a therapeutically effective amount.
  • the effective amount is a prophylactically effective amount.
  • the effective amount is an amount effective for treating an infectious disease, cardiovascular disease, a proliferative disease, beta-thalassemia, sickle cell disease, a neurological disease, an inflammatory disease, or an autoimmune disease in a subject in need thereof.
  • the effective amount is an amount effective for treating cancer in a subject in need thereof.
  • the effective amount is an amount effective for treating cancer in a subject in need thereof.
  • the effective amount is an amount effective for treating a solid tumor or a hematological cancer in a subject in need thereof.
  • the effective amount is an amount effective for treating bladder cancer, breast cancer, biliary duct cancer, a carcinoma, colon cancer, colorectal cancer, gastric cancer, germ cell cancer, glioblastoma multiforme (GBM), glioma, head and neck cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular cancer, or uterine cancer in a subject in need thereof.
  • GBM glioblastoma multiforme
  • the effective amount is an amount effective for treating epithelial ovarian cancer in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating lung adenocarcinoma, lung squamous cell carcinoma, small cell lung carcinoma, or non-small cell lung carcinoma (NSCLC) in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating hepatocellular carcinoma (HCC) in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating osteosarcoma or uterine carcinosarcoma in a subject in need thereof.
  • the effective amount is an amount effective for treating adenoid cystic carcinoma (ACC), chromophobe renal cell carcinoma (chRCC), clear cell renal cell carcinoma (ccRCC), lung adenocarcinoma, or papillary renal cell carcinoma (pRCC) in a subject in need thereof.
  • ACC adenoid cystic carcinoma
  • chRCC chromophobe renal cell carcinoma
  • ccRCC clear cell renal cell carcinoma
  • lung adenocarcinoma or papillary renal cell carcinoma
  • pRCC papillary renal cell carcinoma
  • the effective amount is an amount effective for treating a leukemia, a lymphoma, or multiple myeloma in a subject in need thereof.
  • the effective amount is an amount effective for treating Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), Hodgkin lymphoma, mantle cell lymphoma (MCL), or T-cell lymphoma in a subject in need thereof.
  • the effective amount is an amount effective for treating acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B cell chronic lymphocytic leukemia (B-CLL), chronic myelogenous leukemia (CML), T-cell leukemia, or human t-cell leukemia virus type-1 (HTLV-1) in a subject in need thereof.
  • the effective amount is an amount effective for treating mantle cell lymphoma (MCL) in a subject in need thereof.
  • MCL mantle cell lymphoma
  • the effective amount is an amount effective for treating Epstein-Barr viral (EPV) infection or a retroviral infection in a subject in need thereof.
  • EPV Epstein-Barr viral
  • the effective amount is an amount effective for treating human T lymphotropic viral infection in a subject in need thereof.
  • the effective amount is an amount effective for treating heart disease in a subject in need thereof.
  • compositions comprising a compound that inhibits binding of PRMT5 with a PRMT5 substrate adaptor by contacting a PRMT5 with a PRMT5 substrate adaptor disrupting agent for use in treating an infectious disease, cardiovascular disease, a proliferative disease, beta-thalassemia, sickle cell disease, a neurological disease, an inflammatory disease, or an autoimmune disease in a subject in need thereof.
  • the composition is for use in treating cancer.
  • the composition is for use in treating a solid tumor or a hematological cancer.
  • the composition is for use in treating bladder cancer, breast cancer, biliary duct cancer, a carcinoma, colon cancer, colorectal cancer, gastric cancer, germ cell cancer, glioblastoma multiforme (GBM), glioma, head and neck cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular cancer, or uterine cancer.
  • the composition is for use in treating epithelial ovarian cancer.
  • the composition is for use in treating lung adenocarcinoma, lung squamous cell carcinoma, small cell lung carcinoma, or non-small cell lung carcinoma (NSCLC).
  • NSCLC non-small cell lung carcinoma
  • the composition is for use in treating hepatocellular carcinoma (HCC). In certain embodiments, the composition is for use in treating osteosarcoma or uterine carcinosarcoma. In certain embodiments, the composition is for use in treating adenoid cystic carcinoma (ACC), chromophobe renal cell carcinoma (chRCC), clear cell renal cell carcinoma (ccRCC), lung adenocarcinoma, or papillary renal cell carcinoma (pRCC). In certain embodiments, the composition is for use in treating a leukemia, a lymphoma, or multiple myeloma.
  • HCC hepatocellular carcinoma
  • the composition is for use in treating osteosarcoma or uterine carcinosarcoma.
  • the composition is for use in treating adenoid cystic carcinoma (ACC), chromophobe renal cell carcinoma (chRCC), clear cell renal cell carcinoma (ccRCC), lung adenocarcinoma, or papillary renal cell carcinoma (pRCC).
  • the composition is for use in treating Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), Hodgkin lymphoma, mantle cell lymphoma (MCL), or T-cell lymphoma.
  • the composition is for use in treating acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B cell chronic lymphocytic leukemia (B-CLL), chronic myelogenous leukemia (CML), T-cell leukemia, or human t-cell leukemia virus type- 1 (HTLV-1).
  • the composition is for use in treating mantle cell lymphoma (MCL).
  • the composition is for use in treating Epstein- Barr viral (EPV) infection or a retroviral infection. In certain embodiments, the composition is for use in treating human T lymphotropic viral infection. In certain embodiments, the composition is for use in treating heart disease.
  • the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal.
  • the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat.
  • the subject is a companion animal, such as a dog or cat.
  • the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat.
  • the subject is a zoo animal.
  • the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate.
  • the animal is a genetically engineered animal.
  • the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs).
  • the subject is a fish or reptile.
  • a compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents).
  • the compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, and/or in reducing the risk to develop a disease in a subject in need thereof), improve bioavailability, improve their ability to cross the blood- brain barrier, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell.
  • additional pharmaceutical agents e.g., therapeutically and/or prophylactically active agents.
  • additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in
  • a pharmaceutical composition described herein including a compound described herein and an additional pharmaceutical agent exhibit a synergistic effect that is absent in a pharmaceutical composition including one of the compound and the additional pharmaceutical agent, but not both.
  • the compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies.
  • Pharmaceutical agents include therapeutically active agents.
  • Pharmaceutical agents also include prophylactically active agents.
  • Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S.
  • CFR Code of Federal Regulations
  • proteins proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.
  • CFR Code of Federal Regulations
  • the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g., an infectious disease, cardiovascular disease, a proliferative disease, beta-thalassemia, sickle cell disease, a neurological disease, an inflammatory disease, and/or an autoimmune disease).
  • a disease e.g., an infectious disease, cardiovascular disease, a proliferative disease, beta-thalassemia, sickle cell disease, a neurological disease, an inflammatory disease, and/or an autoimmune disease.
  • Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent.
  • the additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses.
  • the particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved.
  • the compound or pharmaceutical composition is a solid. In certain embodiments, the compound or pharmaceutical composition is a powder. In certain embodiments, the compound or pharmaceutical composition can be dissolved in a liquid to make a solution. In certain embodiments, the compound or pharmaceutical composition is dissolved in water to make an aqueous solution. In certain embodiments, the pharmaceutical composition is a liquid for parental injection. In certain embodiments, the pharmaceutical composition is a liquid for oral administration (e.g., ingestion).
  • the pharmaceutical composition is a liquid (e.g., aqueous solution) for intravenous injection. In certain embodiments, the pharmaceutical composition is a liquid (e.g., aqueous solution) for subcutaneous injection.
  • the pharmaceutical compositions of this present disclosure can be administered to humans and other animals orally, parenterally, intracisternally, intraperitoneally, topically, bucally, or the like, depending on the disease or condition being treated.
  • a pharmaceutical composition comprising a compound of Formula (I) is administered, orally or parenterally, at dosage levels of each pharmaceutical composition sufficient to deliver from about 0.001 mg/kg to about 200 mg/kg in one or more dose administrations for one or several days (depending on the mode of administration).
  • the effective amount per dose varies from about 0.001 mg/kg to about 200 mg/kg, about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic and/or prophylactic effect.
  • the compounds described herein may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 200 mg/kg, from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic and/or prophylactic effect.
  • the desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • the composition described herein is administered at a dose that is below the dose at which the agent causes non-specific effects. [00290] In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.001 mg to about 1000 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 200 mg per unit dose.
  • the pharmaceutical composition is administered at a dose of about 0.01 mg to about 100 mg per unit dose. In certain embodiments, pharmaceutical composition is administered at a dose of about 0.01 mg to about 50 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 10 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.1 mg to about 10 mg per unit dose. [00291] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology.
  • compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition of the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
  • Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.
  • Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross- linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
  • crospovidone cross-linked poly(vinyl-pyrrolidone)
  • sodium carboxymethyl starch sodium starch glycolate
  • Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g.
  • natural emulsifiers e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
  • colloidal clays e.g. bentonite (aluminum silicate) and Veegum (mag
  • stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
  • polyoxyethylene sorbitan monolaurate Tween 20
  • polyoxyethylene sorbitan Tween 60
  • polyoxyethylene sorbitan monooleate Tween 80
  • sorbitan monopalmitate Span 40
  • sorbitan monostearate Span 60
  • sorbitan tristearate Span 65
  • polyoxyethylene esters e.g. polyoxyethylene monostearate (Myrj 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol
  • sucrose fatty acid esters e.g.
  • CremophorTM polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether (Brij 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F-68, Poloxamer-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.
  • Exemplary binding agents include starch (e.g.
  • cornstarch and starch paste examples include gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g.
  • acacia sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.
  • Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
  • the preservative is an antioxidant.
  • the preservative is a chelating agent.
  • Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.
  • EDTA ethylenediaminetetraacetic acid
  • salts and hydrates thereof e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like
  • citric acid and salts and hydrates thereof e.g., citric acid mono
  • antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
  • Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
  • Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
  • Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
  • Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.
  • Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D- gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic s
  • Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
  • Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea
  • Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.
  • Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,
  • oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • agents of the present disclosure are mixed with solubilizing agents such CREMOPHOR EL ® (polyethoxylated castor oil), alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • Sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active agent is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the active agents can also be in micro-encapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the active agent may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, gels, applicants, oil-in-water or water-in-oil emulsions such as creams, ointments, or pastes; or solutions or suspensions such as drops.
  • Formulations for topical administration to the skin surface can be prepared by dispersing the drug with a dermatologically acceptable carrier such as a lotion, cream, ointment, or soap.
  • a dermatologically acceptable carrier such as a lotion, cream, ointment, or soap.
  • Useful carriers are capable of forming a film or layer over the skin to localize application and inhibit removal.
  • the agent can be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to a tissue surface.
  • tissue adhesive or other substance known to enhance adsorption to a tissue surface.
  • tissue-coating solutions such as pectin-containing formulations can be used.
  • Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this present disclosure.
  • transdermal patches which have the added advantage of providing controlled delivery of an agent to the body.
  • dosage forms can be made by dissolving or dispensing the agent in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the agent across the skin.
  • the carrier for a topical formulation can be in the form of a hydroalcoholic system (e.g., quids and gels), an anhydrous oil or silicone based system, or an emulsion system, including, but not limited to, oil-in-water, water-in-oil, water-in-oil-in- water, and oil-in-water-in-silicone emulsions.
  • a hydroalcoholic system e.g., quids and gels
  • an anhydrous oil or silicone based system emulsion system
  • emulsion system including, but not limited to, oil-in-water, water-in-oil, water-in-oil-in- water, and oil-in-water-in-silicone emulsions.
  • the emulsions can cover a broad range of consistencies including thin lotions (which can also be suitable for spray or aerosol delivery), creamy lotions, light creams, heavy creams, and the like.
  • kits e.g., pharmaceutical packs.
  • the kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container).
  • a container e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container.
  • provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein.
  • kits including a first container comprising a compound or pharmaceutical composition described herein.
  • the kits are useful for treating an infectious disease, cardiovascular disease, a proliferative disease, beta-thalassemia, sickle cell disease, a neurological disease, an inflammatory disease, or an autoimmune disease in a subject in need thereof.
  • the kits are useful for treating cancer (e.g., a solid tumor or a hematological cancer) in a subject in need thereof.
  • kits are useful for preventing cancer (e.g., a solid tumor or a hematological cancer) in a subject in need thereof. In certain embodiments, the kits are useful for reducing the risk of developing cancer (e.g., a solid tumor or a hematological cancer) in a subject in need thereof. In certain embodiments, the kits are useful for inhibiting binding of PRMT5 with a PRMT5 substrate adaptor in a subject or cell. [00319] In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information.
  • FDA U.S. Food and Drug Administration
  • a kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.
  • Methods of Use The present disclosure provides methods of inhibiting binding of PRMT5 with a PRMT5 substrate adaptor, the method comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent. In certain embodiments, the method is a method of preventing binding of PRMT5 with a PRMT5 substrate adaptor. [00321] The present disclosure also provides methods of modulating PRTM5 activity, the method comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent. In certain embodiments, the method is a method of inhibiting PRMT5 activity.
  • the present disclosure also provides methods of modulating PRMT5 methyltransferase activity, the method comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent. In certain embodiments, the method is a method of inhibiting PRMT5 methyltransferase activity. [00323] The present disclosure also provides methods of inhibiting methylation of a PRMT5 substrate, the method comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent. In certain embodiments, the method is a method of preventing binding of methylation of a PRMT5 substrate.
  • the PRMT5 substrate is selected from a histone, a spliceosome Sm protein, AR, ASK1, BCL6, CF I m68, CRAF, E2F1, EBNA, EBNA1, EBNA2, EGFR, FCP1, FEN1, FGF-2, G3BP1, GLI1, GM130, HOXA9, KAP1, KLF4, LSM4, MBD2, MBP, MYCN, nucleolin, nucleoplasmin, p53, p65, PAIP, PDCD4, PDGFR ⁇ , Piwi proteins, POLR2A, Rad9, RAF proteins, RPS10, RUVBL1, SERBP, small heterodimer partner (“SHP”), SmB, SPT5, SREBp1, SREBP1a, srGAPs, SUPT5H, TDH, TIP60, and ZNF326.
  • a histone a spliceosome Sm protein
  • AR ASK1, BCL6, CF I m68
  • the spliceosome Sm protein is selected from SmD1, SmD3, SmB, and SmB/B′.
  • the histone is selected from H2A, H2AR3, H4R3, H3R2, and H3R8.
  • the PRMT5 substrate is selected from SmB and nucleolin.
  • the present disclosure also provides methods of modulating regulation of chromatin structure by PRMT5, the method comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent. In certain embodiments, the method is a method of inhibiting regulation of chromatin structure by PRMT5.
  • the present disclosure also provides methods of modulating PRMT5-dependent cellular differentiation, the method comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent. In certain embodiments, the method is a method of inhibiting cellular differentiation by PRMT5.
  • the present disclosure also provides methods of modulating PRMT5-dependent mRNA splicing, the method comprising contacting PRMT5 with a PRMT5 substrate adaptor disrupting agent.
  • the method is a method of inhibiting mRNA splicing mediated by PRMT5.
  • the PRMT5 substrate adaptor disrupting agent is selected from a protein, peptide, a peptide mimetic, a nucleic acid, an aptamer, and a small molecule.
  • the peptide is a competitive peptide inhibitor.
  • the peptide is a PRMT5 Binding Motif (“PBM”) disruption peptide.
  • the protein is an antibody or fragment thereof.
  • the PRMT5 substrate adaptor disrupting agent is an irreversible inhibitor or a suicide inhibitor.
  • the PRMT5 substrate adaptor disrupting agent comprises an amino acid sequence having at least about 57% sequence identity to the sequence GQFXDAX, wherein each X is independently selected from D and E.
  • the sequence is selected from GQFDDAD, GQFDDAE, GQFEDAD, and GQFEDAE.
  • the amino acid sequence has at least about 71% sequence identity to the sequence GQFXDAX.
  • the amino acid sequence has at least about 85% sequence identity to the sequence GQFXDAX.
  • the amino acid sequence has at least about 90% sequence identity to the sequence GQFXDAX.
  • the amino acid sequence has at least about 95% sequence identity to the sequence GQFXDAX. In certain embodiments, the amino acid sequence has at least about 96% sequence identity to the sequence GQFXDAX. In certain embodiments, the amino acid sequence has at least about 97% sequence identity to the sequence GQFXDAX. In certain embodiments, the amino acid sequence has at least about 98% sequence identity to the sequence GQFXDAX. In certain embodiments, the amino acid sequence has at least about 99% sequence identity to the sequence GQFXDAX. In certain embodiments, the amino acid sequence has 100% sequence identity to the sequence GQFXDAX.
  • the PRMT5 substrate adaptor disrupting agent comprises an amino acid sequence having 0, 1, 2, or 3 changes from the sequence GQFXDAX, wherein each X is independently selected from D and E. In certain embodiments, the amino acid sequence has 3 changes from the sequence GQFXDAX. In certain embodiments, the amino acid sequence has 2 changes from the sequence GQFXDAX. In certain embodiments, the amino acid sequence has 1 change from the sequence GQFXDAX. In certain embodiments, the amino acid sequence has 0 changes from the sequence GQFXDAX. [00332] In certain embodiments, the PRMT5 substrate adaptor disrupting agent comprises an amino acid sequence having at least about 57% sequence identity to the sequence GKDLYRS.
  • the amino acid sequence has at least about 71% sequence identity to the sequence GKDLYRS. In certain embodiments, the amino acid sequence has at least about 85% sequence identity to the sequence GKDLYRS. In certain embodiments, the amino acid sequence has 100% sequence identity to the sequence GKDLYRS.
  • the PRMT5 substrate adaptor disrupting agent comprises an amino acid sequence having 0, 1, 2, or 3 changes from the sequence GKDLYRS. In certain embodiments, the amino acid sequence has 3 changes from the sequence GKDLYRS. In certain embodiments, the amino acid sequence has 2 changes from the sequence GKDLYRS. In certain embodiments, the amino acid sequence has 1 change from the sequence GKDLYRS.
  • any method disclosed further comprises administering a compound selected from methylthioadenosine (“MTA”), GSK591, JNJ-64619178, EPZ004777, EPZ015666 (also known as GSK3235025), EPZ015866 (also known as GSK3203591), and EPZ015938 (also known as GSK3326595).
  • MTA methylthioadenosine
  • the PRMT5 substrate adaptor is selected from pICLn/CLNS1A, RioK1, COPR5, MEP50, Menin/Men1, hSWI/SNF, a JAK kinase, Blimp1, and AJUBA. In certain embodiments, the PRMT5 substrate adaptor is selected from pICLn/CLNS1A, RioK1, and COPR5. [00336] In certain embodiments, the methods disclosed herein are in vivo methods. In certain embodiments, the methods disclosed herein are in vitro methods. In certain embodiments, the methods are carried out in a cell. In certain embodiments, the methods are carried out in a subject. In certain embodiments, the subject is a mammal.
  • the subject is a human.
  • Methods of Treatment [00337] The present disclosure provides methods for the treatment, prevention, delaying the progression of a disease.
  • Zhu (Zhu, F. et al., Genes Dis.2019, 6, 247-257, incorporated herein by reference) discusses the role of PRMT5 in gene regulation and hematologic malignancies, indicating that modulation of PRMT5 activity has potential utility in the treatment of diseases such as leukemia (e.g., acute myeloid leukemia (AML), human T-cell leukemia virus type-1 (HTLV-1), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), T-cell leukemia), lymphoma (e.g., virus induced lymphomagenesis, Diffuse Large B Cell Lymphoma (DLBCL), mantle cell lymphoma (MCL), Hodgkin’s lymphoma, T- cell lymphoma, and Burkitt lymphoma
  • Stopa discusses PRMT5’s regulation of proliferation and its direct interaction with proteins commonly misregulated or mutated in disease, indicating that modulation of PRMT5 activity has potential utility in the treatment of diseases such as cancer (ovarian, breast, lung, lymphoid, lymphoma, glioblastoma multiforme, melanoma, colon, gastric, testicular, prostate, bladder cancer, and germ cell tumors), infectious disease (Epstein-Barr viral (“EPV”) infection, retroviral infection, human T lymphotropic viral infection), and heart disease.
  • diseases such as cancer (ovarian, breast, lung, lymphoid, lymphoma, glioblastoma multiforme, melanoma, colon, gastric, testicular, prostate, bladder cancer, and germ cell tumors), infectious disease (Epstein-Barr viral (“EPV”) infection, retroviral infection, human T lymphotropic viral infection), and heart disease.
  • the present disclosure provides methods for treating an infectious disease, cardiovascular disease, a proliferative disease, beta-thalassemia, sickle cell disease, a neurological disease, an inflammatory disease, or an autoimmune disease.
  • the proliferative disease is cancer.
  • the cancer is a solid tumor or a hematological cancer.
  • the cancer is in certain embodiments, the cancer is bladder cancer, breast cancer, biliary duct cancer, a carcinoma, colon cancer, colorectal cancer, gastric cancer, germ cell cancer, glioblastoma multiforme (GBM), glioma, head and neck cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular cancer, or uterine cancer.
  • the cancer is epithelial ovarian cancer.
  • the cancer is lung adenocarcinoma, lung squamous cell carcinoma, small cell lung carcinoma, or non-small cell lung carcinoma (NSCLC).
  • NSCLC non-small cell lung carcinoma
  • the cancer is hepatocellular carcinoma (HCC).
  • the cancer is osteosarcoma or uterine carcinosarcoma.
  • the cancer is adenoid cystic carcinoma (ACC), chromophobe renal cell carcinoma (chRCC), clear cell renal cell carcinoma (ccRCC), lung adenocarcinoma, or papillary renal cell carcinoma (pRCC).
  • ACC adenoid cystic carcinoma
  • chRCC chromophobe renal cell carcinoma
  • ccRCC clear cell renal cell carcinoma
  • pRCC papillary renal cell carcinoma
  • the cancer is a leukemia, a lymphoma, or multiple myeloma.
  • the cancer is Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), Hodgkin lymphoma, mantle cell lymphoma (MCL), or T-cell lymphoma.
  • the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B cell chronic lymphocytic leukemia (B-CLL), chronic myelogenous leukemia (CML), T-cell leukemia, or human t-cell leukemia virus type-1 (HTLV-1).
  • the cancer is mantle cell lymphoma (MCL).
  • MCL mantle cell lymphoma
  • the present disclosure provides methods for treating an infectious disease.
  • the infectious disease is Epstein-Barr viral (EPV) infection or a retroviral infection.
  • the infectious disease is a human T lymphotropic viral infection.
  • the present disclosure provides methods for treating a cardiovascular disease.
  • the cardiovascular disease is heart disease.
  • the methods disclosed herein are in vivo methods.
  • the methods disclosed herein are in vitro methods.
  • the methods are carried out in a cell.
  • the methods are carried out in a subject.
  • the subject is a mammal.
  • the subject is a human.
  • the methods comprise administering to a subject in need thereof (e.g., a subject with a cancer) a compound of Formula (I), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof.
  • the method comprises administering a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof, to a subject in need thereof.
  • the methods of the disclosure comprise administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof.
  • the effective amount is a therapeutically effective amount.
  • the effective amount is a prophylactically effective amount.
  • the subject being treated is an animal.
  • the animal may be of either sex and may be at any stage of development.
  • the subject is a mammal.
  • the subject being treated is a human.
  • the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat.
  • the subject is a companion animal, such as a dog or cat.
  • the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat.
  • the subject is a zoo animal.
  • the subject is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate.
  • the animal is a genetically engineered animal.
  • the animal is a transgenic animal.
  • Certain methods described herein may comprise administering one or more additional pharmaceutical agent(s) in combination with the compounds described herein.
  • the additional pharmaceutical agent(s) may be administered at the same time as the compound of Formula (I), or at different times than the compound of Formula (I).
  • the compound of Formula (I) and any additional pharmaceutical agent(s) may be on the same dosing schedule or different dosing schedules.
  • the additional pharmaceutical agent comprises an agent useful in the treatment of cancer.
  • the additional pharmaceutical agent is useful in the treatment of a solid tumor or a hematological cancer.
  • the additional pharmaceutical agent is an anti-cancer agent.
  • the additional pharmaceutical agent is any anti-cancer agent recited herein.
  • the additional pharmaceutical agent is an immunotherapy.
  • CRISPR/Cas9 Cas9 pLXPR023 expressing cells were generated and characterized for Cas9 cutting efficiency (Genetic Perturbation Platform, Broad Institute). All cell lines utilized herein showed greater than 90% Cas9 efficiency in this assay.
  • sgRNAs Guide RNAs
  • sgRNAs were designed using the Genetics Perturbation Platform tool and either guides targeting intron-exon junctions were chosen or codon optimization was performed to allow selective knockout of endogenous genes while allowing expression of ectopic cDNAs.
  • Guide RNAs were cloned into pLXPR023 or pLXPR043 by BsmBI digest.
  • pICLn guide 2 was acquired from Cellecta in vector pRSG17.
  • PRMT5 gRNA 1 GGTTGCTACTCACGTCACCA
  • PRMT5 gRNA 2 ATACAGCTTTATCCGCCGGT
  • pICLn gRNA 1 GTCACACGCTGATTTATCACT
  • pICLn gRNA 2 GCCACACTGGAGAGATTAGA
  • AAVS1 gRNA GGGGCCACTAGGGACAGGAT.
  • Inducible shRNA For PRMT5 cellular binding assays, MiaPaca2 cells were infected with pLKO shRNA lentiviruses containing either a non-targeting control shRNA or PRMT5 targeting shRNAs. The sequences of the PRMT5 shRNAs were previously reported.
  • MiaPaca2 cells were infected with pLKO shRNA lentiviruses containing either a non-targeting control or 1 of 2 pICLn targeting shRNAs. These had the following sequences: pICLn shRNA 1: ccctctgagtaggcctataat ; pICLn shRNA 2: gcctagtgataaatcagcgtt. These and all new constructs in the disclosure have been deposited to AddGene. [00362] Mammalian Expression Constructs: PRMT5 was cloned into pLX305 PGK 3X HA Hygro-2A-GFP vector using Gibson Assembly.
  • Point mutations in PRMT5 were generated using the Q5 site directed mutagenesis kit (New England Biolabs). HcRed was subcloned from pDONR223 by Gateway cloning into pLX305 PGK 3X HA Hygro-2A-GFP. picln was cloned into an N-terminally V5-tagged vector derived from the pLX307 backbone (pSL307). Deletions and point mutations were generated as above for PRMT5.
  • Lysates were cleared by centrifugation, quantified by bicinchoninic assay (BCA; Thermo) and incubated with anti-HA resin (Sigma), washed with lysis buffer and eluted from the beads with a 1:1:1 mixture of 0.5M DTT; NuPAGE 4X SDS loading buffer (Life Technologies); co-IP lysis buffer and boiled for 10 minutes.
  • BCA bicinchoninic assay
  • samples were rotated with SDMA antibody (Cell signaling) followed by rotation with Protein A/G-Sepharose (Roche), then washed 3 times with 0.1% NP40 lysis buffer and eluted with 1:1:1 mixture of 0.5M DTT; 4X SDS loading buffer; co-IP lysis buffer and boiled for 10 minutes.
  • Lysates were resolved on 4-12% SDS-PAGE NuPage gradient gels (Invitrogen), transferred to nitrocellulose membranes, blocked using LiCor PBS blocking buffer and probed using the following antibodies: anti-PRMT5 (Abcam ab31751); anti-SDMA (Cell Signaling 13222); anti-HA (Abcam ab18181); anti-pICLn/CLNS1A (Novus NBP2-33958 or Santa Cruz sc- 271454); anti-SmB/B’/N (Santa Cruz sc-130670); anti-SmB/ SNRPB (Thermo MA513449); anti-SmD1 (Novus NBP2-36427); anti-Vinculin (Sigma V9131); anti-nucleolin (Abcam ab22758); anti-WDR77/MEP50 (Cell Signaling 2828) anti-RIOK1 (Bethyl A302-457A); anti-COPR5 (Novus NBP2-30884); anti-V
  • NanoBit Assays Detection of protein interactions using split luciferase complementation assays were performed as follows. pFC34K LgBiT TK-neo, pFC36K SmBiT TK-neo, pFN33K LgBiT TK-neo and pFN35K SmBiT TK-neo Flexi Vectors were purchased (Promega).
  • Gibson Assembly was performed to clone the cDNA sequence encoding the SmBit peptide sequence to the N-terminus of PRMT5 in pLX304-Blast and the cDNA sequence encoding the LgBit protein to the N-terminus of RIOK1 in pLentiCMV- Puro.
  • HEK293T cells were co-infected with lentiviral pLX304 SmBit-PRMT5 and pLentiCMV LgBit-RIOK1 and selected with puromycin (1ug/mL) and blasticidin (3 ⁇ g/ml). Mutations and truncations of RIOK1 and PRMT5 were introduced using Gibson Assembly.
  • HEK293T cells were plated in 96 well plates at 10,000 cells/well, transfected using TransIT (Mirus Bio.) and 30 ng of each plasmid. Luciferase assays were performed 24h after transient transfection.
  • HEK293T cells stably expressing SmBit-PRMT5 and LgBit-RIOK1 were trypsinized, resuspended in serum-free media and diluted to 100,000-300,000 cells/well.
  • Colony Formation Assays After determining cell counts using a ViaCell counter, cells were seeded in technical triplicates at a limiting density of 500-1,000 cells per well in a 6 well tissue culture coated dish (Corning) to allow single cell colony formation. Cells were allowed to grow for 10-21 days (until colonies were visible in the control wells).
  • Lysates were cleared by centrifugation and proteins quantified by BCA assay.5 ⁇ g of protein was added to each well of a white 96 well plate (Corning). 3 nM 12F5 SmB/B’/N (Santa Cruz) and 0.3 nM anti-SDMA (Cell Signaling) antibody (final concentrations), 10 ⁇ L of Anti-rabbit IgG (AL104C, Perkin Elmer) and AlphaLISA Acceptor Beads (10 ⁇ g/mL final concentration) were added to each well to and plates were incubated 1 hour at RT. Subsequently 25 ⁇ L of the Streptavidin Alpha Donor beads (final concentration: 40 ⁇ g /mL) were added to each well in the dark.
  • the final volume within the wells is 50 ⁇ L. After incubation for 45 minutes at RT (25°C), plates were read on a Perkin Elmer Envision plate reader.
  • Drug Treatments Methylthioadenosine (MTA) and PRMT5 catalytic inhibitor GSK591 were purchased from Sigma.
  • MTA Methylthioadenosine
  • GSK591 PRMT5 catalytic inhibitor
  • Protein production The PRMT5:WDR77 complex was co-expressed in Sf9 cells using the dual promoter pFastBac Dual baculovirus system (Thermo Fisher). Protein purity and molecular mass was confirmed by both SDS-Page Coomassie staining and LC/MS.
  • untagged full-length PRMT5 was co-expressed with a full-length WDR77 protein containing N-terminal (Strep-Tag II, 3C) and C-terminal (ybbR, sortase, His6) tags.
  • untagged full-length PRMT5 was co-expressed with a full-length WDR77 protein containing an N-terminal tag (His8, Strep-Tag II, 3C) only.
  • the complexes were purified by Streptactin XT affinity resin (IBA).
  • the complex was biotin-labeled via incubation with recombinant sortase A5 enzyme (Active Motif) and a custom tri-glycine biotin peptide (Thermo Fisher).
  • the labeled complex was further purified on a Superose 6 size exclusion column (GE Healthcare).
  • HRV 3C protease AG Scientific
  • PRMT5 proteins were purified and stored in 50 mM HEPES 7.4, 10% glycerol, 1mM TCEP, and 150 mM NaCl.
  • pICln protein Full-length (a.a.2-237) or ⁇ C (a.a.2-227) pICln protein was expressed with a N-terminal His6-SUMO tag in BL21(DE3) e. coli cells (Thermo Fisher).
  • pICLn was first affinity purified by HisTrap column (GE Life Science) in 50 mM Tris pH 8, 500 mM NaCl, 10% glycerol, and 0.5 mM TCEP with imidazole elution. Protein was then purified by gel filtration using a Superdex 20026/600 GL column (GE Life Sciences) before cleaving the His6-SUMO tag with ULP1 enzyme. The tag was removed from solution by passage through a HisTrap column.
  • a monoQ column (GE Life Sciences) was then used as a final purification step.
  • pICln protein was diluted tenfold into 25 mM Bis-Tris pH 6.0, 0.5 mM TCEP and passed over a monoQ column followed by elution with a linear gradient to 500 mM NaCl. All proteins were quantified by A280 absorbance. pICLn protein purity and molecular mass was confirmed by both SDS-Page Coomassie staining and LC/MS.
  • Peptides To generate the fluorescent peptide probe, a synthetic peptide acSRVVPGQFDDADSSDCam was reacted with a rigid maleimide conjugate of KU560 fluorophore (KU Dyes) and purified by reverse phase HPLC (Thermo Fisher). All other peptides were synthesized by Genscript and provided at >95% purity. Net peptide content was determined by nitrogen analysis. Peptides were solubilized in DMSO and stored at -80C.
  • Fluorescence polarization The Fluoresence polarization assay buffer (FP buffer) was composed of 10 nM peptide probe, 50 mM HEPES 7.4, 100 mM NaCl, 0.5 mM TCEP, and 0.01% v/v Tween 20.
  • FP buffer Fluoresence polarization assay buffer
  • the PRMT5:WDR77 concentration was fixed at 200 nM protomer. Samples were incubated at room temperature for 30 minutes before reading. Data were collected in black, 384 well non-binding surface plates (Corning) using a Spectramax Paradigm with Rhodamine FP filter set or a Perkin-Elmer Envision with Bodipy TMR FP filter set.
  • the assay buffer was 25 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% v/v Tween 20, and 1% DMSO. All reported points were measured at binding equilibrium. Data were solvent corrected using a DMSO standard curve and fit with Prism software using a one-site saturation with linear non-specific binding model.
  • PBM 7-amino acid sequence
  • BLOSUM Clustered Scoring Matrix BLOSUM100 from R package Biostrings was taken into consideration to obtain extended 7-mers when counting the occurrence of a specific 7- mer seed to maximize the flexibility of a potential functional protein pocket.
  • 2e6 cells stably expressing 3X HA tagged WT, ADA or CD mutants were infected with lentiviral sgRNA targeting PRMT5 in 6 well dishes containing 10 ⁇ g/mL polybrene (Santa Cruz).48 hours after infections, cells were split into 10 cm dishes and selected with puromycin (1 ⁇ g/mL) for 48 hours. Following selection, cells were expanded to 3 x 150mm dishes and grown for an additional 5 days. Cells were harvested by scraping and washing 3 times in sterile DPBS.5% of the cell pellets were set aside to confirm equivalent expression of the PRMT5 constructs and efficient and equivalent knockout of the endogenous PRMT5 across samples.
  • PTMScan® Urea Lysis Buffer (20 mM HEPES (pH 8.0), 9.0 M urea, 1 mM sodium orthovanadate (activated), 2.5 mM sodium pyrophosphate, and 1 mM ß-glycerol-phosphate). Protein concentration was determined by BCA and 2.8 mg of cell lysate was used from each sample for subsequent analyses.
  • Mass spectrometry samples were analyzed using the PTMScan method. Briefly, cellular extracts were prepared in urea lysis buffer, sonicated, centrifuged, reduced with DTT, and alkylated with iodoacetamide.
  • Enzyme specificity was limited to trypsin, with at least one tryptic (K- or R-containing) terminus required per peptide and up to four mis-cleavages allowed.
  • Cysteine carboxamidomethylation was specified as a static modification, oxidation of methionine and mono- or di-methylation on arginine residues were allowed as variable modifications.
  • Reverse decoy databases were included for all searches to estimate false discovery rates, and filtered using a 2.5% FDR in the Linear Discriminant module of Core. Peptides were also manually filtered using a -/+ 5ppm mass error range and presence of a di-methyl arginine residue. All quantitative results were generated using Skyline 41 to extract the integrated peak area of the corresponding peptide assignments.
  • Mass Spectrometry Data Processing and Analysis Normalized abundance for 408 peptides containing di-methylated arginines was obtained from mass spectrometry (as described above) in three different PRMT5 variants (WT, ADA and CD), each with two replicates. The missing data in each condition replicate was filled by the minimum values of the replicate MS run. For 3*2 replicates, median normalization was performed after log2 transformation of the abundance values. Further, the measurements in WT condition were averaged across two replicates.
  • the up- and down-regulated methylation activity profiling of ADA and CD relative to WT PRMT5 was quantified by the difference between the average log2 values in WT and log2 transformed values in four replicates. More Negative values (blue) in the Heatmap represent more enriched methylation activity in the condition replicate. On the other hand, depleted methylation was observed for peptides in each condition replicate when they are annotated with positive values (red). Column hierarchical clustering was performed for four condition replicates while the rows of Heatmap was organized in the order of ascending order of values in ADA replicate 1. Identification of a PRMT5 Binding Motif [00381] PRMT5 recruits many well characterized substrates through substrate adaptor proteins.
  • GQFDDAD was found as the most enriched peptide among PRMT5 interacting proteins ( Figure 1B) and the GQF[D/E]DA[D/E] peptide sequence is unique to pICln, COPR5, and RIOK1 across the proteome ( Figure 1B). Given the conservation of the peptide among adaptor proteins and the uniqueness within the proteome, we hypothesized that this site might mediate substrate adaptor interaction with PRMT5.
  • the PRMT5 Binding Motif is sufficient to bind the PRMT5 methylosome [00383]
  • SPR surface plasmon resonance
  • the PBM binding site is conserved across PRMT5 homologs in comparison to the protein as a whole ( Figure 2A), albeit to a lesser degree than the nearly universally conserved catalytic pocket residues of the Rossmann fold.
  • the interaction between the PBM peptide and PRMT5 occurs within a shallow groove of the TIM barrel, that is created by a protruding “finger” of two short antiparallel ⁇ - strands (Figure 2B). At the tip of the finger are two projecting lysine residues, K240 and K241. At the base of the finger F243 forms the back wall of the groove, and N239, which serves several intermolecular hydrogen bonds (Figure 2B-2D).
  • RIOK1 For a more detailed view of the interaction the higher resolution RIOK1 structure is shown (Fig.8).
  • the three main elements of PBM interaction are electrostatic attraction (Figure 2B), hydrophobic interaction of the peptide phenylalanine ( Figure 2C), and a complex hydrogen-bonding network (Figure 2D).
  • Both RIOK1 and pICln peptides have a strong overall negative charge with 3 ordered acidic side-chains in proximity to and complemented by three conserved lysine residues in the PRMT5 binding site ( Figure 2B).
  • the PRMT5 binding site has a shallow, hydrophobic “pocket” the floor of which is composed of Y283 and Y286 while the back wall is formed by F243 of the ⁇ -strand finger (Figure 2C).
  • the phenyl ring and the ⁇ / ⁇ carbons of the peptide Phe and Gln side- chains, respectively ( Figure 2C).
  • the positioning of these side-chains is enforced by an unusual peptide conformation discussed below.
  • Also contained within the pocket are two ordered waters.
  • the PBM-PRMT5 interaction is promoted by several intermolecular and intrapeptide hydrogen bonds (Figure 2D).
  • PRMT5 N239 has a prominent role in the interaction network and bonds with the main-chain carbonyl groups of 3 peptide residues.
  • Two water molecules also bridge the main-chain of PRMT5 via hydrogen bonds to the peptide Gln.
  • the positioning of the peptide Gln and Phe side-chains is enabled by an unusual peptide conformation requiring two consecutive ⁇ -turn motifs. The first turn is facilitated by the RIOK1 Pro-Gly sequence (Ala-Gly in pICln or Thr-Gly in COPR5) with typical i+3 bonding pattern (“1” in Figure 2D), but with the addition of a second stabilizing main-chain (valine amide) to side-chain (glutamine ⁇ -carbonyl) bond.
  • the second ⁇ -turn (“2” in Figure 2D) forces the Gln and Phe residues into the “stacked” conformation required for interaction with the shallow pocket described above.
  • the PRMT5 Binding Motif is necessary for substrate adaptor binding to the methylosome [00390]
  • SPR was performed using the PRMT5:WDR77 complex and pICln or pICln lacking the PBM (pICln ⁇ PBM : deletion of residues 228-237). Concentration-dependent binding was observed for full length pICln, but not pICln ⁇ PBM (Fig.3A).
  • HEK293T cells were stably infected with a PRMT5 WT Small-BiT fusion protein and a Large-BiT fusion to either RIOK1 WT or a ⁇ PBM mutant lacking residues 2-23 (RIOK1 ⁇ PBM ).
  • Productive interaction between PRMT5-SmBiT and RIOK1-LgBiT is expected to reconstitute luciferase activity (Fig.3C).
  • Deletion of the PBM sequence led to a ⁇ 98% decrease in luciferase activity relative to WT RIOK1, suggesting that the PBM sequence is required for PRMT5:RIOK1 interactions in cells (Fig 3C).
  • HA-tagged constructs were expressed with and without shRNA knockdown of endogenous PRMT5 as endogenous and exogenous molecules might co-assemble in the same hetero- octamer.
  • Immunoprecipitates of HA-tagged PRMT5 proteins were assessed for co- purification of pICln by immunoblot ( Figure 4A).
  • Figure 4A In the absence of endogenous PRMT5 knockdown, when compared to wild-type PRMT5 the K240D and F243A single mutants showed partial loss, while the ADA triple mutant and N239A mutant showed nearly complete loss of pICln binding (Figure 4A). With knockdown of endogenous PRMT5 we did not detect coprecipitated pICln with any of the mutants ( Figure 4A).
  • RIOK1 and COPR5 substrate adaptors were examined by expressing HA-tagged PRMT5 as either WT, ADA, or catalytically dead (“CD”, R368A 24 ) forms and using CRISPR-Cas9 to deplete endogenous PRMT5.
  • HA-PRMT5 proteins were then immunoprecipitated and probed for co-purification of pICln, RIOK1 or COPR5 by immunoblot. All three substrate adaptors were detected in immunopecipitates of the WT or CD forms of PRMT5. While PRMT5 ADA exhibited 85-95% loss of substrate adaptor binding compared to control ( Figure 4B).
  • PRMT5:RIOK1 NanoBiT complementation assay was utilized where RIOK1-LgBiT was co-expressed with either WT or ADA mutant forms of PRMT5-SmBiT.
  • RIOK1-LgBiT was co-expressed with either WT or ADA mutant forms of PRMT5-SmBiT.
  • a rapamycin-inducible complex between FKBP-SmBiT and FRB-LgBiT was included.
  • PRMT5 ADA showed significantly reduced binding compared to WT, suggesting that the PRMT5 groove is required for full RIOK1 adaptor binding (Figure 4C: 92% reduction with PRMT ADA versus PRMT5 WT ).
  • PRMT5 binding Motif-PBM groove interaction is necessary for substrate methylation
  • PRMT5 WT and binding deficient mutants were expressed in the context of inactivation of endogenous PRMT5. Specifically, MiaPaca2 or HCT116 cells stably expressing Cas9 were infected with lentiviruses bearing sgRNA resistant cDNAs of HA-tagged PRMT5 WT or PRMT5 mutants and then infected with sgRNA targeting endogenous PRMT5. PRMT5 knockout efficiency was ⁇ 85% ( Figure 5A).
  • the PBM-dependent substrates including the known RIOK1-dependent substrates nucleolin and RPS10, and known pICln-dependent substrates SmB, SmD3, SMN and LSM4 were all markedly decreased in cells expressing the PRMT5 ADA mutant, consistent with a requirement for the PBM interaction for methylation (Figure 5B-C).
  • a number of substrates were also identified as PBM groove-dependent for which no substrate adaptor has been identified, such as SERBP ( Figure 5B).
  • Pathway enrichment analysis demonstrated that both PRMT5 ADA and PRMT5 WT substrates are primarily from RNA binding and RNA processing pathways.
  • KP4 pancreatic cancer cells with CDKN2A, MTAP deletion stably expressing CAS9 were infected with lentiviruses encoding either sgRNA-resistant pICln WT or pICln ⁇ PBM or an empty vector.
  • the three resulting stable cell lines were then stably infected with lentivirus directing the expression of sgRNAs targeting either the AAVS1 safe-harbor site or pICln.
  • the cells expressing pICln ⁇ PBM showed a hypomorphic growth rescue when compared to the empty vector ( ⁇ 65% versus 35% rescued growth; Fig.6H-J).
  • a hypomorphic decrease in MTAP -/- cell viability following genetic perturbation of the pICln PBM suggest a hypomorphic decrease in MTAP -/- cell viability following genetic perturbation of the pICln PBM.
  • untagged full-length PRMT5 was co-expressed with a full-length WDR77 protein containing N-terminal (Strep-Tag II, 3C) and C-terminal (ybbR, sortase, His6) tags.
  • untagged full-length PRMT5 was co-expressed with a full-length WDR77 protein containing an N-terminal tag (His8, Strep-Tag II, 3C) only.
  • the complexes were purified by Streptactin XT affinity resin (IBA).
  • the complex was biotin-labeled via incubation with recombinant sortase A5 enzyme (Active Motif) and a custom tri-glycine biotin peptide (Thermo Fisher).
  • the labeled complex was further purified on a Superose 6 size exclusion column (GE Healthcare).
  • HRV 3C protease AG Scientific
  • PRMT5 proteins were purified and stored in 50 mM HEPES 7.4, 10% glycerol, 1mM TCEP, and 150 mM NaCl.
  • pICln protein Full-length (a.a.2-237) or ⁇ C (a.a.2-227) pICln protein was expressed with a N-terminal His6-SUMO tag in BL21(DE3) e. coli cells (Thermo Fisher).
  • pICLn was first affinity purified by HisTrap column (GE Life Science) in 50 mM Tris pH 8, 500 mM NaCl, 10% glycerol, and 0.5 mM TCEP with imidazole elution. Protein was then purified by gel filtration using a Superdex 20026/600 GL column (GE Life Sciences) before cleaving the His6-SUMO tag with ULP1 enzyme. The tag was removed from solution by passage through a HisTrap column.
  • a monoQ column (GE Life Sciences) was then used as a final purification step.
  • pICln protein was diluted tenfold into 25 mM Bis- Tris pH 6.0, 0.5 mM TCEP and passed over a monoQ column followed by elution with a linear gradient to 500 mM NaCl. All proteins were quantified by A280 absorbance.
  • Peptides [00410] To generate the custom fluorescent probe, synthetic peptide of sequence ac SRVVPGQFDDADSSDC am was reacted with a rigid maleimide conjugate of KU560 fluorophore (KU Dyes) and purified by reverse phase HPLC (Thermo Fisher). All other peptides were synthesized by Genscript and provided at >95% purity.
  • Glutathione (GSH) Stability [00411] The assay volume in each well was 200 ⁇ l, consisting of 190 ⁇ l of glutathione in phosphate buffered saline and 10 ⁇ l of compound or control working solution. The compounds at a final concentration of 0.1, 1 and 10 ⁇ M were incubated at 37°C with glutathione (5 mM) in phosphate buffered saline (100 mM PBS, pH 7.4) for 0, 15, 30, 60, 120 and 1440 min, in duplicate.
  • Samples with compound concentration at 0.1, 1 and 10 ⁇ M were diluted with 100 ⁇ l, 300 ⁇ l and 600 ⁇ l water, respectively. Samples were analyzed by LC-MS/MS (Sciex API 4000). The percent of parent remaining at each time-point was determined based on peak area ratios at the 0 min time-point and half-life was calculated using the first order kinetics equation. In addition, the samples were analyzed for formation of the predicted GSH adduct at each time-point.
  • the experimental assay was composed of 200 nM PRMT5:WDR77 protomer, 10 nM peptide probe, 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.5 mM TCEP, and 0.01% v/v Tween 20.5 microliters of the above experimental assay mixture was added to assay ready plates containing 50 nanoliters of DMSO containing either test compounds, a positive control, or a DMSO blank. Experiments were mixed immediately prior to incubation resulting in a 10-point, 2-fold dilution series with top final concentration of compounds at 100 mM in the assay.
  • the plates were incubated for 30 minutes at room temperature, and the final endpoint read at 40 minutes. Data were collected in triplicate in black, 384-well non- binding surface plates (Corning) using a Spectramax Paradigm with Rhodamine FP filter set or a Perkin-Elmer Envision with Bodipy TMR FP filter set.
  • Peptide Probe was Riok1 KU560 labeled peptide (custom peptide sequence [ace]SRVVPGQFDDADSSD[C ⁇ KU560][amide]) Positive control was Riok1 peptide LMSRVVPGQFDDADSSD at 20 micromolar test concentration.
  • Method 1 (2 min UPLC): Instrument: Waters Acquity; Column: Waters Acquity BEH C1850 mm x 2.1 mm x 1.7 ⁇ m; eluent A: Water (MilliQ) + 0.01 vol % formic acid, eluent B: acetonitrile + 0.01 vol % formic acid; gradient: 0-0.3 min 3-4% B, 0.3-1.5 min 4- 95% B, 1.5-1.9 min 95% B; 1.9-2.0 min 5% B; flow: 0.65 mL/min; temperature: 50°C; DAD scan: 200-500 nm.
  • Method 2 (2.5 min UPLC): Instrument: Waters Acquity; Column: Waters Acquity BEH C1850 mm x 2.1 mm x 1.7 ⁇ m; eluent A: Water (MilliQ) + 0.01 vol % formic acid, eluent B: acetonitrile + 0.01 vol % formic acid; gradient: 0-0.5 min 2% B, 0.5-1.5 min 2-95% B, 1.5-1.9 min 95% B; 1.9-2.0 min 95-2% B, 2.0-2.5 min, 2% B; flow: 0.65 mL/min; temperature: 45°C; DAD scan: 200-500 nm.
  • Method 3 (5 min UPLC): Instrument: Waters Acquity; Column: Waters Acquity BEH C1850 mm x 2.1 mm x 1.7 ⁇ m; eluent A: Water (MilliQ) + 0.01 vol % formic acid, eluent B: acetonitrile + 0.01 vol % formic acid; gradient: 0-0.5 min 2-5% B, 0.5-4.0 min 5- 95% B, 4.0-4.5 min 95% B, 4.5-5.0 min 95-2% B; flow: 0.65 mL/min; temperature: 45°C; DAD scan: 200-500 nm.
  • Method 4 (5-95AB_R_220&254.M): Instrument: Agilent 1200 ⁇ G1956A; Column: Kinetex EVO C1830*2.1mm,5um; eluent A: 0.0375% TFA in water (v/v); eluent B: 0.01875% TFA in Acetonitrile (v/v); gradient 0-0.8 min5-95% B, 0.8-1.2 min 95% B, 1.2-1.5 5%B; flow 1.5 mL/min; temperature 50°C; DAD scan: 200-500 nm.
  • Method 5 (0-60CD_R_220&254_POS): Instrument: SHIMADZU LCMS-2020; Column: Kinetex EVO C182.1X30mm,5um; eluent A: 0.025% NH3 ⁇ H2O in Water(v/v); eluent B: Acetonitrile; gradient 0-0.8 min 0-60% B, 0.8-1.2 min 60% B, 1.2-1.150%B; flow 1.5 mL/min; temperature 40°C; DAD scan: 200-500 nm.
  • Method 6 (5-95CD_R_220&254_POS): Instrument: SHIMADZU LCMS-2020; Column: Kinetex EVO C182.1X30mm,5um; eluent A: 0.025% NH3 ⁇ H2O in water(v/v); eluent B: Acetonitrile; gradient 0-0.8 min 5-95% B, 0.8-1.2 min 95% B, 1.2-1.155%B; flow 1.5 mL/min; temperature 40°C; DAD scan: 200-500 nm.
  • Method 7 (0-60AB_0_R_220&254.M): Instrument: Agilent 1200 ⁇ G1956A; Column: Kinetex EVO C1830*2.1mm,5um; eluent A: 0.0375% TFA in water (v/v); eluent B: 0.01875% TFA in Acetonitrile (v/v); gradient 0-0.8 min 0-60% B, 0.8-1.2 min 60% B, 1.2-1.50%B; flow 1.5 mL/min; temperature 50°C; DAD scan: 200-500 nm.
  • Method 8 (5-95AB_R_220&254): Instrument: SHIMADZU LCMS-2020; Column: Kinetex EVO C1830*2.1mm,5um; eluent A: 0.0375% TFA in water (v/v); eluent B: 0.01875% TFA in Acetonitrile (v/v); gradient 0-0.8 min 5-95% B, 0.8-1.2 min 95% B, 1.2-1.55%B; flow 1.5 mL/min; temperature 50°C; DAD scan: 200-500 nm.
  • Method 9 (0-60AB_R_220&254): Instrument: SHIMADZU LCMS-2020; Column: Kinetex EVO C1830*2.1mm,5um; eluent A: 0.0375% TFA in water (v/v); eluent B: 0.01875% TFA in Acetonitrile (v/v); gradient 0-0.8 min 0-60% B, 0.8-1.2 min 60% B, 1.2-1.50%B; flow 1.5 mL/min; temperature 50°C; DAD scan: 200-500 nm.
  • Preparatory HPLC conditions [00425] Prep-HPLC has been done on different columns; Instrument: Gilson GX-281or SHIMADZU LC-20AD, please see below detail information: [00426] Method A: prep-HPLC (Instrument: GX-P)column: Phenomenex Synergi C18 150*25*10um;mobile phase: [water(0.225%FA)-ACN];B%: 33%-63%,10min gradient) Flow/Rate (ml/min): 25 [00427] Method B: prep-HPLC (Instrument: ACSWH-GX-B, column: Phenomenex luna C18150*40mm* 15um;mobile phase: [water(0.225%FA)-ACN];B%: 2%-32%,9min gradient)Flow/Rate (ml/min): 50 [00428] Method C: prep-HPLC (Instrument:ASCWH-GX-Q; column: Shim-pack C18 150*25
  • N-(3-(azepan-1-ylsulfonyl)-4-methylphenyl)-2-(4,5-dichloro-6-oxopyridazin- 1(6H)-yl)acetamide (A1).
  • a mixture of 2-(4,5-dichloro-6-oxopyridazin-1(6H)-yl)acetic acid (32, 997.17 mg, 4.47 mmol) and DMF (27.24 mg, 372.61 umol, 28.67 uL) and thionyl chloride (664.95 mg, 5.59 mmol) in THF (20 mL) at 0 °C was heated at 60 °C for 30 minutes.
  • reaction mixture was diluted with saturated aqueous ammonium chloride, extracted twice with ethyl acetate, combined organic layers were dried over sodium sulfate, insoluble materials were removed by filtration, volatiles were removed under reduced pressure and the residue was purified by prep-HPLC (column: Kromasil 250*50 mm*10 um; mobile phase: [water (0.225% FA)-ACN]; B%: 37ACN%-67ACN%, 26 min, 85% min) to give the title compound as a white solid (1.4 g, 79% yield).
  • Compounds A2, A4-A14 were made analogously to A1.
  • Compound A3 was purchased from Enamine (Catalog ID: Z19024493) and used without further purification.
  • N-(6-(benzylthio)-5-methylpyridin-2-yl)acetamide 38.
  • N-(6- chloro-5-methylpyridin-2-yl)acetamide 37, 10.1 g, 54.71 mmol
  • DMF 100 mL
  • potassium carbonate 30.24 g, 218.82 mmol
  • phenylmethanethiol 20.38 g, 164.12 mmol, 19.23 mL
  • tert-butyl N-[4-(dimethylsulfamoyl)-5-methyl-2-pyridyl]carbamate 48.
  • reaction mixture was diluted with water and extracted 4 times with ethyl acetate, combined organic phases were washed three times with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials were removed by filtration, volatiles removed under reduced pressure and the residue purified by column chromatography on silica gel eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as an off-white solid (565 mg, 59% yield).
  • ethyl 2-(4-fluoro-6-oxo-pyridazin-1-yl)acetate 63.
  • a mixture of ethyl 2-(5- chloro-4-fluoro-6-oxopyridazin-1(6H)-yl)acetate (59, 300 mg, 1.21 mmol), palladium on carbon (60 mg, 5% w/w) and triethylamine (366.28 mg, 3.62 mmol, 503.82 uL) in EtOAc (6 mL) was stirred under an atmosphere of hydrogen at 25 °C for 12 hr.
  • reaction mixture was cooled to room temperature, volatiles removed under reduced pressure, and the residue purified by column chromatography on silica gel, eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a light-yellow solid (1.8 g).
  • reaction mixture was filtered through a pad of Celite, volatiles removed under reduced pressure, and the resulting residue was dissolved in 110 o C toluene (10 ml), filtered to remove insoluble material, cooled to room temperature to afford a white solid, which was isolated by filtration as the title compound (1.2g).
  • reaction mixture slowly warmed to 20°C and stirred for 2 hr. Water was added and the mixture was extracted three times with dichloromethane, combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, and volatiles removed under reduced pressure to afford the title compound as a yellow oil (11.6 g).
  • reaction mixture was filtered through Celite and the filter cake was washed with methanol, volatiles were removed from the combined filtrate and washes under reduced pressure to give the title compound as a yellow oil (460 mg), which was used without further manipulation.
  • reaction mixture was diluted with saturated aqueous sodium hydrogen carbonate, extracted twice with ethyl acetate, combined organic layers were washed with saturated aqueous sodium chloride, dried over Na 2 SO 4 , insoluble materials removed by filtration, volatiles removed under reduced pressure to afford the title compound as a yellow oil (0.647 g), used without further manipulation.
  • the mixture was diluted with water, and washed with ethyl acetate, the aqueous phase was then acidified to pH 5 by the slow addition of cold hydrochloric acid (1M in water), and extracted three times with dichloromethane.
  • the combined DCM layers were dried over sodium sulfate, insoluble materials removed by filtration, and volatiles removed under reduced pressure to afford the title compound as a white solid (540 mg).
  • the mixture was stirred at 90 °C for 12 hours, cooled to room temperature, and poured onto crushed ice.
  • the mixture was extracted twice with dichloromethane, the combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure, and the residue was purified by column chromatography on silica gel, eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a brown solid (940 mg).
  • the mixture was diluted with saturated aqueous sodium hydrogen carbonate, and extracted with ethyl acetate, the organic phase was washed with saturated aqueous sodium chloride, dried over sodium hydrogen sulfate, insoluble materials were removed by filtration and volatiels removed under reduced pressure to afford the title compound as a yellow solid (485 mg), which was used without further manipulation.
  • the mixture was diluted with hydrochloric acid (1M in water), and extracted with ethyl acetate, the organic layer was washed with saturated aqueous sodium hydrogen carbonate, saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration and volatiles removed under reduced pressure to afford the title compound as a yellow oil (2.95 g), which was used without further manipulation.
  • the mixture was diluted with saturated aqueous ammonium chloride and ethyl acetate, the layers were separated and the organic phase was further extracted with ethyl acetate, combined organic phases were washed with aqueous sodium thiosulfate (10% w/v), then with saturated aqueous sodium chloride, dried over magnesium sulfate, insoluble materials removed by filtration, and the crude material was adsorbed onto silica gel and purified by column chromatography on silica gel eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a yellow solid (1.5 g).
  • the mixture was heated to 60°C for 12 hours.
  • the reaction mixture was diluted with water and extracted 4 times with ethyl acetate, combined organic phases were washed three times with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials were removed by filtration, volatiles removed under reduced pressure and the residue purified by column chromatography on silica gel eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as an off-white solid (565 mg).
  • reaction mixture was stirred at 0 °C for 15 minute. Then TEA (750.68 mg, 7.42 mmol, 1.03 mL) was added. The reaction mixture was stirred at 0 °C for 30 minutes. The reaction mixture was diluted with ethyl acetate, washed with water twice and then with saturated aqueous sodium chloride, organic phase was dried over sodum sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure and the residue purified by column chromatography on silica gel eluting with a gradient of ethyl acetate in petroleum ether to give the title compound as a white solid (1.2 g).
  • the reaction mixture was diluted with ethyl acetate and insoluble materials removed by filtration, the filtrate was washed twice with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure and the residue purified by chromatography on silica gel eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a light-yellow solid (120 mg).
  • reaction mixture was diluted with water, extracted with ethyl acetate.
  • organic layer was washed successively with an aqueous solution of hydrochloric acid (1M), three times with saturated aqueous sodium hydrogen carbonate, and finally saturated aqueous sodium chloride, then dried over sodium sulfate, insoluble materials removed by filtration, and volatiles removed under reduced pressure to afford the title compound as a yellow solid (710 mg), which was used without further manipulation.
  • the mixture was stirred at 20°C for 12 hr.
  • the mixture was diluted with ice water and extracted three times with ethyl acetate, combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration and volatiles removed under reduced pressure to afford the title compound as a yellow oil (15 g), which was used without further manipulation.
  • the mixture was diluted with water, acidified to pH 7 with hydrochloric acid (1M in water), and extracted twice with ethyl acetate, combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure, and the residue purified by column chromatography on silica gel eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a white solid (9.28 g).
  • the mixture was diluted with aqueous potassium fluoride, insoluble materials removed by filtration, the mixture was extracted twice with ethyl acetate, combined organic phases were washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtrations, volatiles removed under reduced pressure and the residue purified by column chromatography on silica gel eluting with a gradient of ethyl acetate in petroleum ether to give the title compound as a yellow oil (500 mg).
  • the mixture was cooled to 0 °C, treated with 3-(azepan-1-ylsulfonyl)-4-methylaniline (Intermediate 20, 370 mg, 1.38 mmol) and TEA (279.02 mg, 2.76 mmol, 383.79 uL), and the reaction mixture was warmed to 25 °C and stirred for 1 hour.
  • the reaction mixture was diluted with saturated aqueous ammonium chloride and saturated aqueous sodium hydrogen carbonate, then extracted twice with ethyl acetate.
  • the mixture was stirred at 25°C for 12h.
  • the mixture was diluted with saturated aqueous ammonium chloride, then saturated aqueous sodium hydrogen carbonate, and extracted twice with ethyl acetate.
  • the combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure, and the residue was purified by column chromatography on silica gel, eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a white solid (400 mg).
  • the mixture was stirred at 25°C for 12 hr.
  • the mixture was diluted with saturated aqueous ammonium chloride and extracted three times with dichloromethane, the combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure and the residue purified by column chromatography on silica gel eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a yellow oil (0.9 g).
  • the mixture was diluted with ethyl acetate and water, the organic layer was separated, washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure and the residue purified by column chromatography on silica gel, eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a white solid (900 mg).
  • the mixture was diluted with water, extracted twice wit hethyl acetate, combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure and the residue purified by column chromatography on silica gel, eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a white solid (150 mg).
  • the mixture was diluted with saturated aqueous sodium bicarbonate, extracted with dichloromethane, combined organic phases were washed with saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure and the residue purified by column chromatography on silica gel, eluting with a gradient of ethyl acetate in petroleum ether to afford the title compound as a light-yellow solid (120 mg).
  • the mixture was diluted with ethyl acetate and water, layers were separated and the aqueous phase was extracted three times with ethyl acetate, combined organics were washed sequentially with water, saturated aqueous sodium chloride, dried over sodium sulfate, insoluble materials removed by filtration, volatiles removed under reduced pressure, and the residue was purified by column chromatography on silica gel eluting with ethyl acetate in petroleum ether to afford the title compound as a yellow solid (2.8 g).

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Abstract

L'invention concerne des composés qui modulent l'activité de PRMT5. Les composés peuvent inhiber la liaison de PRMT5 à un adaptateur de substrat de PRMT5. Les composés peuvent moduler l'activité de la PRMT5 méthyltransférase, moduler la transcription d'un gène régulé par PRMT5, moduler la régulation de la structure de la chromatine, moduler la différentiation cellulaire et/ou moduler l'épissage de l'ARNm, par exemple par rupture de la liaison de PRMT5 avec un adaptateur de substrat de PRMT5. L'invention concerne également des compositions pharmaceutiques comprenant les composés, des procédés de modulation de l'activité de PRMT5 et des procédés pour le traitement d'une maladie (par exemple, un cancer) chez un sujet par administration d'un composé ou d'une composition décrit dans l'invention.
PCT/US2021/045016 2020-08-07 2021-08-06 Inhibiteurs d'adaptateur de substrat de prmt5 et leurs utilisations Ceased WO2022032144A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
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CN115684428A (zh) * 2022-11-17 2023-02-03 暨南大学 一种基于丹磺酰氯衍生结合质谱源内裂解非靶向定性检测双酚类物质的方法
CN116199605A (zh) * 2023-02-27 2023-06-02 上海睿腾医药科技有限公司 一种5-氯-2-羟基-二甲基苯磺酰胺的合成方法
CN116283831A (zh) * 2023-03-13 2023-06-23 中国医学科学院医药生物技术研究所 一种对硝基苯衍生物及其制备方法和应用
CN117903024A (zh) * 2024-01-12 2024-04-19 浙江扬帆新材料股份有限公司 一种芳基硫酚衍生物的合成方法
CN119242606A (zh) * 2024-08-30 2025-01-03 中国农业大学 疫霉属植物病原卵菌精氨酸甲基转移酶蛋白及其编码基因与应用

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115684428A (zh) * 2022-11-17 2023-02-03 暨南大学 一种基于丹磺酰氯衍生结合质谱源内裂解非靶向定性检测双酚类物质的方法
CN115684428B (zh) * 2022-11-17 2024-06-07 暨南大学 一种基于丹磺酰氯衍生结合质谱源内裂解非靶向定性检测双酚类物质的方法
CN116199605A (zh) * 2023-02-27 2023-06-02 上海睿腾医药科技有限公司 一种5-氯-2-羟基-二甲基苯磺酰胺的合成方法
CN116283831A (zh) * 2023-03-13 2023-06-23 中国医学科学院医药生物技术研究所 一种对硝基苯衍生物及其制备方法和应用
CN117903024A (zh) * 2024-01-12 2024-04-19 浙江扬帆新材料股份有限公司 一种芳基硫酚衍生物的合成方法
CN119242606A (zh) * 2024-08-30 2025-01-03 中国农业大学 疫霉属植物病原卵菌精氨酸甲基转移酶蛋白及其编码基因与应用

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