WO2021023609A1 - Combination of a type i protein arginine methyltransferase (type i prmt) inhibitor and a methionine adenosyltransferase ii alpha (mat2a) inhibitor - Google Patents

Combination of a type i protein arginine methyltransferase (type i prmt) inhibitor and a methionine adenosyltransferase ii alpha (mat2a) inhibitor Download PDF

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WO2021023609A1
WO2021023609A1 PCT/EP2020/071460 EP2020071460W WO2021023609A1 WO 2021023609 A1 WO2021023609 A1 WO 2021023609A1 EP 2020071460 W EP2020071460 W EP 2020071460W WO 2021023609 A1 WO2021023609 A1 WO 2021023609A1
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compound
inhibitor
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Ryan G. KRUGER
Jenny LARAIO
Helai MOHAMMAD
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Glaxosmithkline Intellectual Property Development Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a method of treating cancer in a human and to combinations useful in such treatment. Particularly, the present invention relates to combinations of Type I protein arginine methyltransferase (Type I PRMT) inhibitors and methionine adenosyltransferase II alpha (MAT2A) inhibitors.
  • Type I PRMT Type I protein arginine methyltransferase
  • MAT2A methionine adenosyltransferase II alpha
  • cancer results from the deregulation of the normal processes that control cell division, differentiation and apoptotic cell death and is characterized by the proliferation of malignant cells which have the potential for unlimited growth, local expansion and systemic metastasis.
  • Deregulation of normal processes includes abnormalities in signal transduction pathways and response to factors that differ from those found in normal cells.
  • Arginine methylation is an important post-translational modification on proteins involved in a diverse range of cellular processes such as gene regulation, RNA processing, DNA damage response, and signal transduction. Proteins containing methylated arginines are present in both nuclear and cytosolic fractions suggesting that the enzymes that catalyze the transfer of methyl groups on to arginines are also present throughout these subcellular compartments (reviewed in Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013); Lee, Y. H. & Stallcup, M. R. Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.
  • methylated arginine exists in three major forms: w -N G -monomethyl -arginine (MMA), w -N G ,N G -as ym m ct ri c dimethyl arginine (ADMA), or w -N G ,N 'G -symmetric dimethyl arginine (SDMA).
  • MMA monoomethyl -arginine
  • ADMA w -N G ,N G -as ym m ct ri c dimethyl arginine
  • SDMA w -N G ,N 'G -symmetric dimethyl arginine
  • Arginine methylation occurs largely in the context of glycine-, arginine-rich (GAR) motifs through the activity of a family of Protein Arginine Methyltransferases (PRMTs) that transfer the methyl group from S-adenosyl-L-methionine (SAM) to the substrate arginine side chain producing S-adenosyl-homocysteine (SAH) and methylated arginine.
  • PRMTs Protein Arginine Methyltransferases
  • SAM S-adenosyl-L-methionine
  • SAH S-adenosyl-homocysteine
  • This family of proteins is comprised of 10 members, of which 9 have been shown to have enzymatic activity (Bedford, M. T. & Clarke, S. G. Protein arginine methylation in mammals: who, what, and why.
  • the PRMT family is categorized into four sub-types (Type I-IV) depending on the product of the enzymatic reaction.
  • Type IV enzymes methylate the internal guanidino nitrogen and have only been described in yeast (Fisk, J. C. & Read, L. K. Protein arginine methylation in parasitic protozoa. Eukaryot Cell 10, 1013-1022, doi:10.1128/EC.05103-11 (2011)); types T-TTT enzymes generate monomethyl-arginine (MMA, Rmel) through a single methylation event.
  • the MMA intermediate is considered a relatively low abundance intermediate, however, select substrates of the primarily Type III activity of PRMT7 can remain monomethylated, while Types I and II enzymes catalyze progression from MMA to either asymmetric dimethyl-arginine (ADMA, Rme2a) or symmetric dimethyl arginine (SDMA, Rme2s) respectively.
  • ADMA asymmetric dimethyl-arginine
  • SDMA symmetric dimethyl arginine
  • PRMT1 can drive expression of aberrant oncogenic programs through methylation of histone H4 (Takai, H. et al 5- Hydroxymethylcytosine plays a critical role in glioblastomagenesis by recruiting the CHTOP- methylosome complex. Cell Rep 9, 48-60, doi: 10.1016/j.celrep.2014.08.071 (2014); Shia, W. J. et al. PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood 119, 4953-4962, doi: 10.1182/blood-2011-04-347476 (2012); Zhao, X. et al.
  • SAM S-adenosvl methionine
  • MAT1A and MAT2A Two adenosyltransferase genes (MAT1A and MAT2A), which encode distinct catalytic isoforms, as well as a third gene (MAT2B) that encodes a MAT2A regulatory subunit, are known to be differentially impacted in cancer. While MAT1A is specifically expressed in the adult liver, MAT2A activity is widely distributed.
  • hepatocellular carcinoma HCC
  • HCC hepatocellular carcinoma
  • MAT1A-MAT2A switch the downregulation of MAT1A and the upregulation of MAT2A occur, which is known as the MAT1A-MAT2A switch.
  • This switch accompanied with up regulation of MAT2B, results in lower SAM contents and provides a growth advantage to hepatoma cells.
  • MAT2A silencing the via RNAi substantially suppresses growth and induces apoptosis in hepatoma cells. See T. Li, et al, J.
  • the present invention provides a combination of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a methionine adenosyltransferase II alpha (MAT2A) inhibitor.
  • Type I PRMT Type I protein arginine methyltransferase
  • MAT2A methionine adenosyltransferase II alpha
  • a pharmaceutical composition comprising a therapeutically effective amount of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of a methionine adenosyltransferase II alpha (MAT2A) inhibitor are provided.
  • Type I PRMT Type I protein arginine methyltransferase
  • MAT2A methionine adenosyltransferase II alpha
  • methods for treating cancer in a human in need thereof, the methods comprising administering to the human a combination of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a methionine adenosyltransferase II alpha (MAT2A) inhibitor, together with at least one of: a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent, thereby treating the cancer in the human.
  • a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent
  • methods of treating cancer in a human in need thereof comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a pharmaceutical composition comprising of a methionine adenosyltransferase II alpha (MAT2A) inhibitor, thereby treating the cancer in the human.
  • a pharmaceutical composition comprising a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a pharmaceutical composition comprising of a methionine adenosyltransferase II alpha (MAT2A) inhibitor
  • FIG. 1 Methionine salvage pathway, highlighting the function of MAT2A and MTAP.
  • MTAP and MAT2A are both part of the methionine salvage pathway leading to generation of SAM.
  • MTAP deficiency leads to accumulation of MTA.
  • High levels of MTA in MTAP -null tumor cells correlates with decreased levels of SDMA suggesting a pre-existing state of attenuated PRMT5 activity.
  • MTAP deficiency also correlates with increased sensitivity to Type 1 PRMT inhibitors.
  • Targeting MAT2A in this setting further disrupts the pathway leading to increased growth inhibition. From Marjon, K., et al. (2016). MTAP deletions in cancer create vulnerability to targeting of the MAT2A/PRMT5/RIOK1 axis. Cell Reports, 15(3), 574-587.
  • FIG. 2 Schematic of Type 1 PRMT inhibitors and MAT2A inhibitor. Structures of Type I PRMT inhibitors Compound D (left) and Compound A-di-HCl (middle) are shown. The structure of a MAT2A inhibitor (Compound 262) (right) is shown.
  • FIG. 3 Capan-1 (MTAP-null) double titration of Compound D and Compound 262.
  • Drug titrations performed in MTAP-null Capan-1 cells, holding either concentration of (A) Compound 262 or (B) Compound D constant while modulating the other as indicated.
  • CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T 0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves.
  • FIG. 4 Capan-1 (MTAP-null) proliferation assay. Both titrations exhibit greater than 5-fold glC 50 potency shifts starting at 19.5nM Compound 262 in the Compound D titration and at 78.1nM Compound D in the Compound 262 titration. Both titrations exhibit greater than 5-fold glC 100 potency shifts starting at 1250nM Compound 262 in the Compound D titration and at 625nM Compound D in the Compound 262 titration. Shifts from cytostatic to cytotoxic response is observed at 156.3nM Compound 262 in the Compound D titration and at 312.5nM Compound D in the Compound 262 titration.
  • FIG. 5 Capan-1 (MTAP-null) synergistic growth inhibition analyses. Synergy is measured in the Capan-1 cell line at varying concentrations of Compound D and Compound 262 using (A) excess over Bliss and (B) growth index analyses. Growth Index values less than 0 indicate a cytotoxic response. Synergy was observed across most concentration combinations in the double titration in the Capan-1 cell line. Cytotoxicity was also observed when combining these compounds at the higher concentration.
  • FIG. 6 HuP-T4 (MTAP-null) double titration of Compound D and Compound 262.
  • Drug titrations were performed in MTAP-null HuP-T4 cells, holding the either concentration of (A) Compound 262 or (B) Compound D constant while modulating the other as indicated.
  • CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T 0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves.
  • FIG. 7 HuP-T4 (MTAP-null) proliferation assay. Both titrations exhibit greater than 5-fold EC 50 and glC 50 potency shifts starting at 78.1nM Compound 262 in the Compound D titration and at 78. InM Compound D in the Compound 262 titration. Both titrations exhibit greater than 5-fold glC 100 potency shifts starting at 39. InM Compound 262 in the Compound D titration and at 312.5nM Compound D in the Compound 262 titration. Shifts from cytostatic to cytotoxic response is observed at 312.5nM Compound D in the Compound 262 titration.
  • FIG. 8 HuP-T4 (MTAP-null) synergistic growth inhibition analyses. Synergy is measured in the HuP-T4 cell line at varying concentrations of Compound D and Compound 262 using (A) excess over Bliss and (B) growth index analyses. Cytotoxicity and synergy are observed when combining as little as 312.5nM of each compound. Growth Index values less than 0 indicate a cytotoxic response. Synergy is observed across mid-range concentration combinations in the HuP- T4 cell line. Cytotoxicity is also observed when combining these compound at the higher concentrations.
  • FIG. 9 Pane 08.13 double titration of Compound D and Compound 262.
  • FIG. 10 Pane 08.13 proliferation assay. Both titrations exhibit greater than 5-fold glC 50 potency shifts starting at 312.5nM Compound 262 in the Compound D titration and at 78. InM Compound D in the Compound 262 titration. glC 100 potency shifts were not observed. Greater than 20% shifts in growth index were observed with the Compound 262 titration, but a shift from a cytostatic response to a cytotoxic response was only observed at the highest dose tested (10,000nM). If the single agent exhibited a glC 50 or glC 100 value greater than the top concentration tested, >10,000nM was used to calculate fold change.
  • FIG. 11 Pane 08.13 synergistic growth inhibition analyses. Synergy is observed in the Pane 08.13 cell line across concentration combinations above 78. InM of Compound D or Compound 262 using (A) excess over Bliss and (B) growth index analyses. Cytotoxicity is only observed when combining both compounds at the highest dose of 10,000nM. Growth Index values less than 0 indicate a cytotoxic response. Synergy is observed across concentration combinations above 78. InM for each compound in the Pane 08.13 cell line.
  • FIG. 12 Pane 03.27 double titration of Compound D and Compound 262.
  • Drug titrations were performed in Pane 03.27 cells (WT MTAP), holding the either concentration of (A) Compound 262 or (B) Compound D constant while modulating the other as indicated.
  • CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T 0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves.
  • FIG. 13 Pane 03.27 proliferation assay. Greater than 5-fold glC 50 potency shifts were observed above 1250nM Compound 262 in the Compound D titration and above 2500nM Compound D in the Compound 262 titration. Greater than 5-fold glC 100 potency shifts were not observed. Greater than 20% shifts in growth index were observed in both titrations above 156.3nM, but a shift from a cytostatic response to a cytotoxic response was only observed at 5000nM and above. If the single agent exhibited a glC 50 or glC 100 value greater than the top concentration tested, >10,000nM was used to calculate fold change.
  • FIG. 14 Pane 03.27 synergistic growth inhibition analyses.
  • Synergy is measured in the Pane 03.27 cell line at varying concentrations of Compound D and Compound 262 using (A) excess over Bliss and (B) growth index analyses. Synergy and cytotoxicity are only observed at the highest concentration combinations tested (above 5000nM). Growth Index values less than 0 indicate a cytotoxic response.
  • FIG. 15 Pane 03.27 (MTAP KO Clone #31) double titration of Compound D and Compound 262.
  • Drug titrations were performed in Pane 03.27 cells (MTAP KO Clone #31), holding the either concentration of (A) Compound 262 or (B) Compound D constant while modulating the other as indicated.
  • CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T 0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves.
  • FIG. 16 Pane 03.27 (MTAP KO Clone #31) proliferation assay. Knocking out MTAP appears to sensitize the Pane 03.27 cell line to this combination as seen in the shifts in potency for glC 50 and especially glC 100 . Greater than 5-fold glC 50 potency shifts were observed at 78. InM Compound 262 in the Compound D titration and 78. InM Compound D in the Compound 262 titration. Greater than 5-fold glC 100 potency shifts were observed above 156.3nM for each titration. Greater than 20% shifts in growth index were observed above 39.
  • FIG. 17 Pane 03.27 (MTAP KO Clone #31) synergistic growth inhibition analyses. Synergy is measured in the Pane 03.27 cell line at varying concentrations of Compound D and Compound 262 using (A) excess over Bliss and (B) growth index analyses. Synergy and cytotoxicity are observed when each compound was combined at doses as low as 625nM.
  • FIG. 18 Data tables for Capan-1 (MTAP-null) double titration of Compound D and Compound 262. Titrations performed in MTAP-null Capan-1 cells, holding the either concentration of Compound D or Compound 262 constant while modulating the other as indicated. Values in tables reference diagrams in Fig. 3.
  • FIG. 19 Data tables for HuP-T4 (MTAP-null) double titration of Compound D and Compound 262. Titrations performed in MTAP-null HuP-T4 cells, holding the either concentration of Compound D or Compound 262 constant while modulating the other as indicated. Values in tables reference diagrams in Fig. 6.
  • FIG. 20 Data tables for Pane 08.13 double titration of Compound D and Compound 262.
  • FIG. 21 Data tables for Pane 03.27 double titration of Compound D and Compound 262.
  • FIG. 22 Data tables for Pane 03.27 (MTAP KO Clone #31) double titration of Compound D and Compound 262. Titrations performed in Pane 03.27 cells (MTAP KO Clone #31), holding the either concentration of Compound D or Compound 262 constant while modulating the other as indicated. Values in tables reference diagrams in Fig. 15.
  • FIG. 23 Single agent proliferation curves in multiple cell types.
  • A, B MTAP-null Capan-1 and HuP-T4 cell lines or
  • C, D WT MTAP cell lines were treated with the indicated concentration of either Compound A or Compound 262. Proliferation was plotted for each condition in all lines. Note: in FIG. 23, “Compound A” is Compound A-di-HCl.
  • FIG. 24 Combinatorial therapy proliferation assays.
  • A Legend for data table in (B), charting fold change and change in Growth Index for fixed ratio combination of Compound A or Compound 262 in indicated cell lines.
  • C Growth curve for HuP-T4 cells treated with either Compound A, Compound 262, or Compound A + Compound 26. Fold Change and Change in Growth Index for fixed ratio combination were calculated based on the most potent single agent value. Note: in FIG. 24, “Compound A” is Compound A-di-HCl.
  • FIG. 25 Growth indices with combinatorial therapy in multiple cell types (Compound 262 fixed).
  • FIG. 26 Combinatorial therapy titration and growth assays.
  • A Legend for data table in (B), charting fold change and change in Growth Index for combination of Compound A and indicated concentration of Compound 262 in the indicated cell lines.
  • C Growth curve for HuP-T4 cells treated with either Compound A alone or with the indicated concentration of Compound 262.
  • FIG. 27 Growth indices with combinatorial therapy in multiple cell types (Compound 262 fixed).
  • FIG. 28 Combinatorial therapy titration and growth assays.
  • A Legend for data table in (B), charting fold change and change in Growth Index for combination of Compound 262 and indicated concentration of Compound A in the indicated cell lines.
  • C Growth curve for Capan-1 cells treated with either Compound 262 alone or with the indicated concentration of Compound A.
  • FIG. 29 Data table for combinatorial therapy growth assays (fixed ratio). Chart of various indicia of cell growth and proliferation following treatment with Compound A, Compound 262 or both in the indicated cell types. Note: in FIG. 29, “Compound A” is Compound A-di-HCl.
  • FIG. 30 Data table for combinatorial therapy growth assays (fixed Compound A). Chart of various indicia of cell growth and proliferation when following treatment with a fixed concentration of Compound A alone or in combination with the indicated concentration of Compound 262 in the indicated cell types. Note: in FIG. 30, “Compound A” is Compound A-di- HCl.
  • FIG. 31 Data table for combinatorial therapy growth assays (fixed Compound 262). Chart of various indicia of cell growth and proliferation when following treatment with a fixed concentration of Compound 262 alone or in combination with the indicated concentration of Compound A in the indicated cell types. Note: in FIG. 31, “Compound A” is Compound A-di- HC1.
  • FIG. 32 Types of methylation on arginine residues. From Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013).
  • FIG. 33 Functional classes of cancer relevant PRMT1 substrates.
  • Known substrates of PRMT1 and their association to cancer related biology Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013); Shia, W. J. et al. PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential.
  • FIG. 34 Methylscan evaluation of cell lines treated with Compound D. Percent of proteins with methylation changes (independent of directionality of change) are categorized by functional group as indicated.
  • FIG. 35 Mode of inhibition against PRMT1 by Compound A. IC 50 values were determined following a 18 minute PRMT1 reaction and fitting the data to a 3-parameter dose-response equation.
  • B Representative experiment showing IC 50 values plotted as a function of [Peptide]/ K m app .
  • FIG. 36 Potency of Compound A against PRMTl.
  • PRMT1 activity was monitored using a radioactive assay run under balanced conditions (substrate concentrations equal to K m app ) measuring transfer of 3 H from SAM to a H4 1-21 peptide.
  • IC 50 values were determined by fitting the data to a 3-parameter dose-response equation.
  • A IC 50 values plotted as a function of PRMT1: SAM: Compound A-tri-HCl preincubation time. Open and filled circles represent two independent experiments (0.5 nM PRMT1). Inset shows a representative IC 50 curve for Compound A-tri-HCl inhibition of PRMT1 activity following a 60 minute PRMT1: SAM: Compound A-tri-HCl preincubation.
  • B Compound A inhibition of PRMT1 categorized by salt form. IC 50 values were determined following a 60 minute PRMT1: SAM: Compound A preincubation and a 20 minute reaction.
  • FIG. 37 The crystal structure resolved at 2.48 ⁇ for PRMTl in complex with Compound A (orange) and SAH (purple). The inset reveals that the compound is bound in the peptide binding pocket and makes key interactions with PRMT1 sidechains.
  • FIG. 38 Inhibition of PRMT1 orthologs by Compound A.
  • PRMT1 activity was monitored using a radioactive assay run under balanced conditions (substrate concentrations equal to K m app ) measuring transfer of 3 H from SAM to a H4 1-21 peptide.
  • IC 50 values were determined by fitting the data to a 3-parameter dose-response equation.
  • A IC 50 values plotted as a function of PRMT1:SAM:Compound A preincubation time for rat (o) and dog ( ⁇ ) orthologs.
  • B IC 50 values plotted as a function of rat (o), dog ( ⁇ ) or human ( ⁇ ) PRMT1 concentration.
  • FIG. 39 Potency of Compound A against PRMT family members. PRMT activity was monitored using a radioactive assay run under balanced conditions (substrate concentrations at K m app ) following a 60 minute PRMT: SAM: Compound A preincubation.
  • IC 50 values for Compound A were determined by fitting data to a 3-parameter dose-response equation.
  • B IC 50 values plotted as a function of PRMT3 ( ⁇ ), PRMT4 (o), PRMT6 ( ⁇ ) or PRMT8 ( ⁇ ) : SAM: Compound A preincubation time.
  • FIG. 40 MMA in-cell-western. RKO cells were treated with Compound A-tri-HCl, Compound A-mono-HCl, Compound A-free-base, and Compound A-di-HCl for 72 hours.
  • FIG. 41 PRMT1 expression in tumors. mRNA expression levels were obtained from cBioPortal for Cancer Genomics. ACTB levels and TYR are shown to indicate expression of level corresponding to a gene that is ubitiquitously expressed versus one that has restricted expression, respectively.
  • FIG. 42 Antiproliferative activity of Compound A in cell culture.
  • 196 human cancer cell lines were evaluated for sensitivity to Compound A in a 6-day growth assay.
  • glC 50 values for each cell line are shown as bar graphs with predicted human exposure as indicated in (A).
  • Y min -T 0 a measure of cytotoxicity, is plotted as a bar-graph in (B), in which glC 100 values for each cell line are shown as red dots.
  • FIG. 43 Timecourse of Compound A effects on arginine methylation marks in cultured cells.
  • B Representative western blots of arginine methylation marks. Regions quantified are denoted by black bars on the right of the gel.
  • FIG. 44 Dose response of Compound A on arginine methylation.
  • A Representatitve western blot images of MMA and ADMA from the Compound A dose response in the U2932 cell line. Regions quantified for (B) are denoted by black bars to the left of gels.
  • FIG. 45 Durability of arginine methylation marks in response to Compound A in lymphoma cells.
  • B Representative western blots of arginine methylation marks. Regions quantified for (A) are denoted by black bars on the side of the gel.
  • FIG. 47 Anti-proliferative effects of Compound A in lymphoma cell lines at 6 and 10 days.
  • FIG. 48 Anti-proliferative effects of Compound A in lymphoma cell lines as classified by subtype.
  • A glC 50 values for each cell line are shown as bar graphs.
  • Y min-T0 a measure of cytotoxicity, is plotted as a bar-graph in (B), in which glC 100 values for each cell line are shown as red dots.
  • Subtype information was collected from the ATCC or DSMZ cell line repositories.
  • FIG. 50 Caspase-3/7 activation in lymphoma cell lines treated with Compound A. Apoptosis was assessed over a 10-day timecourse in the Toledo (A) and Daudi (B) cell lines. Caspase 3/7 activation is shown as fold-induction relative to DMSO-treated cells. Two independent replicates were performed for each cell line. Representative data are shown for each.
  • FIG. 51 Efficacy of Compound A in mice bearing Toledo xenografts. Mice were treated QD (37.5, 75, 150, 300, 450, or 600 mg/kg) with Compound A orally or BID with 75 mg/kg (B) over a period of 28 (A) or 24 (B) days and tumor volume was measured twice weekly.
  • QD 37.5, 75, 150, 300, 450, or 600 mg/kg
  • BID 75 mg/kg
  • FIG. 52 Effect of Compound A in AML cell lines at 6 and 10 Days.
  • A Average glC 50 values from 6 day (light blue) and 10 day (dark blue) proliferation assays in AML cell lines.
  • B Y min -T 0 at 6 day (light blue) and 10 day (dark blue) with corresponding glC 100 (red points).
  • FIG. 53 In vitro proliferation timecourse of ccRCC cines with Compound A.
  • A Growth relative to control (DMSO) for 2 ccRCC cell lines. Representative curves from a single replicate are shown.
  • FIG. 54 Efficacy of Compound A in ACHN xenografts. Mice were treated daily with Compound A orally over a period of 28 days and tumor volume was measured twice weekly.
  • FIG. 55 Anti-proliferative effects of Compound A in breast cancer cell lines. Bar graphs of glC 50 and growth inhibition (%) (red circles) for breast cancer cell lines profiled with Compound A in the 6-day proliferation assay. Cell lines representing triple negative breast cancer (TNBC) are shown in orange; other subtypes are in blue.
  • TNBC triple negative breast cancer
  • FIG. 56 Effect of Compound A in Breast Cancer Cell Lines at 7 and 12 Days. Average growth inhibition (%) values from 7 day (light blue) and 10 day (dark blue) proliferation assays in breast cancer cell lines with corresponding glC 50 (red points) The increase in potency and percent inhibition observed in long-term proliferation assays with breast cancer, but not lymphoma or AML cell lines, suggest that certain tumor types require a longer exposure to Compound A to fully reveal anti-proliferative activity. DETAILED DESCRIPTION OF THE INVENTION
  • Type I protein arginine methyltransferase (Type I PRMT) and a methionine adenosyltransferase II alpha (MAT2A) for the treatment of cancer.
  • Type I PRMT Type I protein arginine methyltransferase
  • MAT2A methionine adenosyltransferase II alpha
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric 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 are also meant to 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 a carbon by a 13 C- or 14 C-enriched carbon are within the scope of the disclosure.
  • Such compounds are useful, for example, as analytical tools or probes in biological assays.
  • aliphatic includes both saturated and unsaturated, nonaromatic, straight chain (i.e., unbranched), branched, acyclic, and cyclic (i.e., carbocyclic) hydrocarbons.
  • an aliphatic group is optionally substituted with one or more functional groups.
  • aliphatic is intended herein to include alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl moieties.
  • C 1-6 alkyl is intended to encompass, C 1; C 2 , C 3 , C 4 , C 5 , G.
  • Radical refers to a point of attachment on a particular group. Radical includes divalent radicals of a particular group.
  • Alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“C 1-20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C 1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms ("C 1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C 1-8 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”).
  • an alkyl group has 1 to 5 carbon atoms ("C 1-5 alkyl”). In some embodiments, 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 (“C 1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C 2-6 alkyl”).
  • C 1-6 alkyl groups include methyl (C 1 ), ethyl (C 2 ), n- propyl (C 3 ), isopropyl (C 3 ), n-butyl (C 4 ), tert-butyl (C 4 ), sec-butyl (C 4 ), iso-butyl (C 4 ), n-pentyl (C 5 ), 3- pentanyl (C 5 ), amyl (C 5 ), neopentyl (C 5 ), 3-methyl-2-butanyl (C 5 ), tertiary amyl (C 5 ), and n-hexyl (C 6 ).
  • alkyl groups include n-heptyl (C 7 ), n-octyl (C 8 ) and the like.
  • each instance of an alkyl group is independently optionally substituted, e.g. , unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents.
  • the alkyl group is unsubstituted C 1-10 alkyl (e.g., -CH 3 ). In certain embodiments, the alkyl group is substituted C 1-10 alkyl.
  • an alkyl group is substituted with one or more halogens.
  • Perhaloalkyl is a substituted alkyl group as defined herein wherein all of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo.
  • the alkyl moiety has 1 to 8 carbon atoms ("C 1-8 perhaloalkyl”).
  • the alkyl moiety has 1 to 6 carbon atoms ("C 1-6 perhaloalkyl”).
  • the alkyl moiety has 1 to 4 carbon atoms ("C 1-4 perhaloalkyl").
  • the alkyl moiety has 1 to 3 carbon atoms ("C 1-3 perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 2 carbon atoms ("C 1-2 perhaloalkyl”). In some embodiments, all of the hydrogen atoms are replaced with fluoro. In some embodiments, all of the hydrogen atoms are replaced with chloro. Examples of perhaloalkyl groups include - CF 3 , -CF 2 CF 3 , -CF 2 CF 2 CF 3 , -CCI 3 , -CFCI 2 , -CF 2 CI, and the like.
  • alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds), and optionally one or more triple bonds (e.g., 1, 2, 3, or 4 triple bonds) ("C 2-20 alkenyl"). In certain embodiments, alkenyl does not comprise triple bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C 2-10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C 2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C 2-8 alkenyl”).
  • an alkenyl group has 2 to 7 carbon atoms (“C 2-7 alkenyl”) In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C 2-6 alkenyl”). In some embodiments, 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 (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 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 (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ), and the like.
  • alkenyl examples include heptenyl (C 7 ), octenyl (C 8 ), octatrienyl (C 8 ), and the like.
  • each instance of an alkenyl group is independently optionally substituted, e.g. , unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents.
  • the alkenyl group is unsubstituted C 2-10 alkenyl.
  • the alkenyl group is substituted C 2-10 alkenyl.
  • Alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds), and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) ("C 2-20 alkynyl"). In certain embodiments, alkynyl does not comprise double bonds. In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C 2-10 alkynyl "). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C 2-9 alkynyl”) .
  • an alkynyl group has 2 to 8 carbon atoms ("C 2-8 alkynyl”) . In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C 2-7 alkynyl”). In some embodiments, 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”) .
  • an alkynyl group has 2 carbon atoms ("C 2 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 (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ), and the like.
  • Examples of C 2-6 alkenyl groups include the aforementioned C 2-4 alkynyl groups as well as pentynyl (C 5 ), hexynyl (G,). and the like.
  • alkynyl examples include heptynyl (C 7 ), octynyl (C 8 ), and the like.
  • each instance of an alkynyl group is independently optionally substituted, e.g., unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents.
  • the alkynyl group is unsubstituted C 2-10 alkynyl.
  • the alkynyl group is substituted C 2-10 alkynyl.
  • “Fused” or “ortho-fused” are used interchangeably herein, and refer to two rings that have two atoms and one bond in common, e.g.., napthalene "Bridged” refers to a ring system containing (1) a bridgehead atom or group of atoms which connect two or more non-adjacent positions of the same ring; or (2) a bridgehead atom or group of atoms which connect two or more positions of different rings of a ring system and does not thereby form an ortho-fused ring, e.g.,
  • Spiro or “Spiro-fused” refers to a group of atoms which connect to the same atom of a carbocyclic or heterocyclic ring system (geminal attachment), thereby forming a ring, e.g.,
  • Spiro-fusion at a bridgehead atom is also contemplated.
  • 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.
  • a carbocyclyl group refers to a radical of a non aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (C 3-10 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system.
  • a carbocyclyl group has 3 to 8 ring carbon atoms (“C 3-8 carbocyclyl").
  • a carbocyclyl group has 3 to 6 ring carbon atoms ("C 3-6 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 5 to 10 ring carbon atoms ("C 5-10 carbocyclyl”).
  • Exemplary C 3-6 carbocyclyl groups include, without limitation, cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4 ), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C). cyclohexenyl (C). cyclohexadienyl (C). and the like.
  • Exemplary C 3-8 carbocyclyl groups include, without limitation, the aforementioned C 3-6 carbocyclyl groups as well as cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), cyclooctyl (C 8 ). cyclooctenyl (C 8 ). bicyclo[2.2.1]heptanyl (C 7 ), bicyclo[2.2.2]octanyl (C 8 ). and the like.
  • Exemplary C 3-10 carbocyclyl groups include, without limitation, the aforementioned C 3-8 carbocyclyl groups as well as cyclononyl (C 9 ), cyclononenyl (C 9 ), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-lH-indenyl (C 9 ), decahydronaphthalenyl (Cio), spiro[4.5]decanyl (C 10 ), and the like.
  • the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or is a fused, bridged or spiro-fused ring system such as a bicyclic system ("bicyclic carbocyclyl”) and can be saturated or can be partially unsaturated.
  • 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 optionally substituted, e.g., unsubstituted (an "unsubstituted carbocyclyl") or substituted (a "substituted carbocyclyl") with one or more substituents.
  • the carbocyclyl group is unsubstituted C 3-10 carbocyclyl.
  • the carbocyclyl group is a substituted C 3-10 carbocyclyl.
  • “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms ("C 3-14 cycloalkyl”). In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms ("C 3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C 3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ("C 3-6 cycloalkyl").
  • 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 (C 5 ) and cyclohexyl (C 5 ). Examples of C 3-6 cycloalkyl groups include the aforementioned C 5-6 cycloalkyl groups as well as cyclopropyl (C 3 ) and cyclobutyl (C 4 ).
  • C 3-8 cycloalkyl groups include the aforementioned C 3-6 cycloalkyl groups as well as cycloheptyl (C 7 ) 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 unsubstituted C 3-10 cycloalkyl.
  • the cycloalkyl group is substituted C 3-10 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 or heterocyclic refers to a radical of a 3-10 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 ("3-10 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 a fused, bridged or spiro-fused ring system such as a bicyclic system ("bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated.
  • Heterocyclyl bicyclic 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 optionally substituted, e.g., unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocyclyl") with one or more substituents.
  • the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 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 independently selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has 1-2 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • Exemplary 3 -membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, and thiorenyl.
  • Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl.
  • Exemplary 5 -membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione.
  • Exemplary 5- membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one.
  • Exemplary 5 -membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl.
  • Exemplary 6- membered heterocyclyl groups containing three heteroatoms include, without limitation, triazinanyl.
  • Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl, and thiocanyl.
  • Exemplary 5-membered heterocyclyl groups fused to a G, aryl ring include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.
  • Exemplary 6- membered heterocyclyl groups fused to an aryl ring include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
  • 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 p electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ("C 1-14 aryl").
  • an aryl group has six ring carbon atoms ("C aryl”; e.g., phenyl).
  • an aryl group has ten ring carbon atoms ("C 10 aryl”; e.g., naphthyl such as 1- naphthyl and 2-naphthyl).
  • an aryl group has fourteen ring carbon atoms ("C 14 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 optionally substituted, e.g.
  • the aryl group is unsubstituted C 6-14 aryl. In certain embodiments, the aryl group is substituted C 6-14 aryl.
  • Heteroaryl refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p 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").
  • heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 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").
  • heteroaryl groups that contain one or more nitrogen atoms the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl bicyclic 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 (aryl/heteroaryl) ring system.
  • Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom e.g., indolyl, quinolinyl, carbazolyl, and the like
  • the point of attachment can be on either ring, e.g., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5- indolyl).
  • a heteroaryl group is a 5-14 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 ("5-14 membered heteroaryl").
  • 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 independently selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1-2 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. In certain embodiments, each instance of a heteroaryl group is independently optionally substituted, e.g., unsubstituted ("unsubstituted heteroaryl") or substituted ("substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is substituted 5-14 membered heteroaryl.
  • Exemplary 5 -membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl.
  • Exemplary 5 -membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl.
  • Exemplary 6- membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
  • Exemplary 7- membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, any one of the following formulae:
  • the point of attachment can be any carbon or nitrogen atom, as valency permits.
  • Partially unsaturated refers to a group that includes at least one double or triple bond.
  • the term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups (e.g., aryl or heteroaryl groups) as herein defined.
  • saturated refers to a group that does not contain a double or triple bond, i.e. , contains all single bonds.
  • aliphatic, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted (e.g., “substituted” or “unsubstituted” aliphatic, "substituted” or “unsubstituted” alkyl, "substituted” or “unsubstituted” alkenyl, "substituted” or “unsubstituted” alkynyl, "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 (e.g., a carbon or nitrogen atom) 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, cycbzation, 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, including 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.
  • a "counterion” or “anionic counterion” is a negatively charged group associated with a cationic quaternary amino group in order to maintain electronic neutrality.
  • exemplary counterions include halide ions (e.g., F-, CI, Br-, G), NO 3 -, CIO 4 - , OH-, H 2 PO 4 -, HSO 4 - , sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5 -sulfonate, ethan-l-sulfonic acid- 2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate
  • Halo or halogen refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).
  • Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quartemary nitrogen atoms.
  • the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to 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.
  • Amide nitrogen protecting groups include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide,
  • Carbamate nitrogen protecting groups include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2.7-di 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), l-(l-adamantyl)-l- methylethyl carbamate (A
  • p-methoxybenzyl carbamate (Moz). p-nitobenzyl carbamate p- bromobenzyl carbamate, p- chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4- methylsulfmylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2- methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfbnyl)ethyl carbamate, [2-(l,3- dithianyl)] methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),
  • p- cyanobenzyl carbamate cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate,p- decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N- dimethylcarboxamido)benzyl carbamate, 1,1 -dimethyl-3 -(N,N- dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2- furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate.
  • p-(/?'-methoxyphenylazo)benzyl carbamate 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1 -methyl- 1 -cyclopropylmethyl carbamate, 1- methyl- 1- (3,5-dimethoxyphenyl)ethyl carbamate, 1 -methyl- 1 -(p-phenylazophenyl)ethyl carbamate, 1 - methyl- 1-phenylethyl carbamate, 1 -methyl- l-(4-pyridyl)ethyl carbamate, phenyl carbamate, p- (phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4- (trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.
  • Sulfonamide nitrogen protecting groups include, but are not limited to, p-toluenesulfonamide (Ts), benzene sulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6- dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5, 6-tetramethyl-4- methoxybenzene sulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2, 2, 5, 7, 8-pentamethylchroman-6-sulfonamide (Pme), methane sulfonamide (Ms),
  • nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, A-p-toluenesulfonylaminoacyl derivative, A-phenylaminothioacyl derivative, N- benzoylphenylalanyl derivative, N-acctylmcthioninc derivative, 4,5-diphenyl- 3-oxazolin-2-one, N-phthalimidc.
  • N-dithiasuccinimidec (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N- 1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl- 1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-mcthylaminc.
  • N- allylamine N-[2-(trimcthylsilyl)cthoxy
  • N-5- dibenzosuberylamine N- triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl] amine (MMTr), N-9- phenylfluorenylamine (PhF), N-2.7-dichloro-9-fluorcnylmcthylcncaminc.
  • the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a 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 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.
  • MOM methoxylmethyl
  • MTM methylthiomethyl
  • t-butylthiomethyl methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl.
  • SMOM phenyldimethylsilylmethoxymethyl
  • BOM benzyloxymethyl
  • PMBM p- methoxybenzyloxymethyl
  • PMBM (4-methoxyphenoxy)methyl
  • GUM guaiacolmethyl
  • POM t-butylthiomethy 4-pentenyloxymethyl
  • siloxymethyl 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetra
  • triphenylsilyl diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate.
  • DPMS diphenylmethylsilyl
  • TMPS t-butylmethoxyphenylsilyl
  • formate benzoylformate
  • acetate chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate.
  • the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group).
  • Sulfur 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.
  • LG is a term understood in the art to refer to a molecular fragment that departs with a pair of electrons upon heterolytic bond cleavage, wherein the molecular fragment is an anion or neutral molecule. See, for example, Smith, March Advanced Organic Chemistry 6th ed. (501-502).
  • Suitable leaving groups include, but are not limited to, halides (such as chloride, bromide, or iodide), alkoxycarbonyloxy, aryloxycarbonyloxy, alkane sulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, haloformates, -N02, trialkylammonium, and aryliodonium salts.
  • the leaving group is a sulfonic acid ester.
  • the sulfonic acid ester comprises the formula -OSO 2 R RG1 wherein R LG1 is selected from the group consisting alkyl optionally, alkenyl optionally substituted, heteroalkyl optionally substituted, aryl optionally substituted, heteroaryl optionally substituted, arylalkyl optionally substituted, and heterarylalkyl optionally substituted.
  • R LG1 is substituted or unsubstituted C 1 -C 6 alkyl.
  • R LG1 is methyl.
  • R LG1 is substituted or unsubstituted aryl.
  • R LG1 is substituted or unsubstitued phenyl.
  • R LG1 is:
  • the leaving group is toluenesulfonate (tosylate, Ts), methanesulfonate (mesylate, Ms), p-bromobenzenesulfonyl (brosylate, Bs), or trifluoromethanesulfonate (triflate, Tf).
  • the leaving group is a brosylate (p-bromobenzenesulfonyl).
  • the leaving group is anosylate (2-nitrobenzenesulfonyl).
  • the leaving group is a sulfonate-containing group.
  • the leaving group is a tosylate group.
  • the leaving group may also be a phosphineoxide (e.g., formed during a Mitsunobu reaction) or an internal leaving group such as an epoxide or cyclic sulfate.
  • “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 other 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.
  • Pharmaceutically acceptable salts of the compounds describe herein 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 used 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 used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzene sulfonate, 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, pec
  • 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, quaternary salts.
  • Type I protein arginine methyltransferase inhibitor or “Type I PRMT inhibitor” means an agent that inhibits any one or more of the following: protein arginine methyltransferase 1 (PRMT1), protein arginine methyltransferase 3 (PRMT3), protein arginine methyltransferase 4 (PRMT4), protein arginine methyltransferase 6 (PRMT6) inhibitor, and protein arginine methyltransferase 8 (PRMT8).
  • the Type I PRMT inhibitor is a small molecule compound.
  • the Type I PRMT inhibitor selectively inhibits any one or more of the following: protein arginine methyltransferase 1 (PRMT1), protein arginine methyltransferase 3 (PRMT3), protein arginine methyltransferase 4 (PRMT4), protein arginine methyltransferase 6 (PRMT6) inhibitor, and protein arginine methyltransferase 8 (PRMT8).
  • the Type I PRMT inhibitor is a selective inhibitor ofPRMT1, PRMT3, PRMT4, PRMT6, and PRMT8.
  • the present invention provides Type I PRMT inhibitors.
  • the Type I PRMT inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein
  • X is N, Z is NR 4 , and Y is CR 5 ; or X is NR 4 , Z is N, and Y is CR 5 ; or X is CR 5 , Z is NR 4 , and Y is N; or X is CR 5 , Z is N, and Y is NR 4 ;
  • R x is optionally substituted C 1-4 alkyl or optionally substituted C3-4 cycloalkyl
  • R w is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; provided that when Li is a bond, R w is not hydrogen, optionally substituted aryl, or optionally substituted heteroaryl;
  • R 3 is hydrogen, C 1-4 alkyl, or C 3-4 cycloalkyl
  • R 4 is hydrogen, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl; or optionally substituted C 1-4 alkyl-Cy;
  • Cy is optionally substituted C 3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
  • R 5 is hydrogen, halo, -CN, optionally substituted C 1-4 alkyl, or optionally substituted C3-4 cycloalkyl.
  • R 3 is a C 1-4 alkyl.
  • R 3 is methyl.
  • R 4 is hydrogen.
  • R 5 is hydrogen.
  • Li is a bond.
  • the Type I PRMT inhibitor is a compound of Formula (I) wherein -L 1 - R w is optionally substituted carbocyclyl.
  • R 3 is a C 1-4 alkyl. In one aspect, R 3 is methyl.
  • R 4 is hydrogen.
  • R 5 is hydrogen.
  • Li is a bond.
  • the Type I PRMT inhibitor is a compound of Formula (V) or a pharmaceutically acceptable salt thereof, wherein Ring A is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In one aspect, Ring A is optionally substituted carbocyclyl.
  • R 3 is a C 1-4 alkyl. In one aspect, R 3 is methyl. In one aspect, R x is unsubstituted C 1-4 alkyl. In one aspect, R x is methyl. In one aspect, Li is a bond.
  • the Type I PRMT inhibitor is a compound of Formula (VI) or a pharmaceutically acceptable salt thereof.
  • Ring A is optionally substituted carbocyclyl.
  • R 3 is a C 1-4 alkyl. In one aspect, R 3 is methyl.
  • R x is unsubstituted C 1-4 alkyl. In one aspect, R x is methyl.
  • the Type I PRMT inhibitor is a compound of Formula (II): or a pharmaceutically acceptable salt thereof.
  • -L 1 -R w is optionally substituted carbocyclyl.
  • R 3 is a C 1-4 alkyl. In one aspect, R 3 is methyl.
  • R x is unsubstituted C 1-4 alkyl. In one aspect, R x is methyl.
  • R 4 is hydrogen.
  • the Type I PRMT inhibitor is Compound A: or a pharmaceutically acceptable salt thereof. Compound A and methods of making Compound A are disclosed in PCT/US2014/029710, in at least page 171 (Compound 158) and page 266, paragraph [00331]
  • the Type I PRMT inhibitor is Compound A-tri-HCl, a tri-HCl salt form of Compound A.
  • the Type I PRMT inhibitor is Compound A-mono- HC1, a mono-HCl salt form of Compound A.
  • the Type I PRMT inhibitor is Compound A-free-base, a free base form of Compound A.
  • the Type I PRMT inhibitor is Compound A-di-HCl, a di-HCl salt form of Compound A.
  • the Type I PRMT inhibitor is Compound D: or a pharmaceutically acceptable salt thereof.
  • Type I PRMT inhibitors are further disclosed in PCT/US2014/029710, which is incorporated herein by reference. Exemplary Type I PRMT inhibitors are disclosed in Table 1A and Table IB of PCT/US2014/029710, and methods of making the Type I PRMT inhibitors are described in at least page 226, paragraph [00274] to page 328, paragraph [00050] of PCT/US2014/029710.
  • MAT2A inhibitor means an agent that inhibits the production of S-adenosylmethionine (SAM) by methionine adenosyltransferase 2A (MAT2A).
  • SAM S-adenosylmethionine
  • MAT2A methionine adenosyltransferase 2A
  • the present invention also provides methionine adenosyltransferase II alpha (MAT2A) inhibitors.
  • the MAT2A inhibitor is a compound of Formula (III):
  • the MAT2A inhibitor binds to its protein target.
  • the MAT2A inhibitor is composed as above (Formula III or pharmaceutically acceptable salt thereof) wherein R D and R E are independently selected from C 3 -C 14 -carbocyclyl, C 6 -C 14 -aryl. and 3- to 14-membered heterocyclyl (wherein 1 to 4 heterocyclyl ring members are heteroatoms selected from N, O, and S.
  • R D and R E of the MAT2A inhibitor are independently selected from C 3 -C 14 - carbocyclyl and C 6 -C 14 -aryl. In some embodiments, R D and R E of the MAT2A inhibitor, as described above (or pharmaceutically acceptable salt thereof), are independently selected from C 5 - C 7 -carbocyclyl, C 6 -C 10 -aryl. In some embodiments, R D and R E of the MAT2A inhibitor, as described above, are independently selected from C 3 -C 14 -carbocyclyl, C 6 -C 14 -aryl.
  • R D and R E of the MAT2A inhibitor are independently selected from cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and phenyl.
  • R D and R E of the MAT2A inhibitor, as described above (Formula III or pharmaceutically acceptable salt thereof) is cyclohexyl or cyclohexenyl and the other is phenyl.
  • R A of the MAT2A inhibitor, as described above (or pharmaceutically acceptable salt thereof) is selected from the group consisting of C 1 -C 6 , -alkyl.
  • R A of the MAT2A inhibitor is selected from the group consisting of C 1 -C 6 -alkyl. - (CH 2 ) 0-6 )NR 1 (CH 2 ) 0-6 )C(O)R 2 , NR' R 2 . and NR 1 C(NR 2 )NR 1 R 2 .
  • R A of the MAT2A inhibitor, as described above (Formula III or pharmaceutically acceptable salt thereof) is C 1 -C 6 , -alkyl or NR'R 2 .
  • R A of the MAT2A inhibitor, as described above (or pharmaceutically acceptable salt thereof) is NR 1 R 2 .
  • R 1 of the MAT2A inhibitor (Formula III or pharmaceutically acceptable salt thereof) is H.
  • the MAT2A inhibitor is a compound of Formula (IV): In an embodiment, the MAT2A inhibitor binds to its protein target. In some embodiments, the MAT2A inhibitor is composed as above (Formula IV or pharmaceutically acceptable salt thereof) R c of the MAT2A inhibitor is C 3 -C 14 carbocyclyl or a 3- to 14-membered heterocyclyl (wherein 1 to 4 heterocyclyl ring members are heteroatoms selected from N, O, and S, each optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, -NH 2 , C 6 -C 14 -aryl.
  • R c of the MAT2A inhibitor is C 3 -C 14 carbocyclyl or a 3- to 14-membered heterocyclyl (wherein 1 to 4 heterocyclyl ring members are heteroatoms selected from N, O, and S, each optionally substituted with one or more substituents selected from the group consisting of hydroxy, hal
  • R D and R E are independently a C 3 -C 14 carbocyclyl or a 3- 14-membered heterocyclyl (with 1 to 4 heteroatoms selected from N, O, and S), each optionally substituted with one or substituents of the following group: hydroxy, halogen, NH 2 , C 6 -C 14 -aryl, ( C 6 -C 14 -aryl)-C 1 -C 6 - alkyl-, carboxy, -CN, oxo, C 1 -C 6 -alkyl.
  • C 3 -C 14 carbocyclyl and a 3- 14-membered heterocyclyl (with 1 to 4 heteroatoms selected from N, O, and S), each optionally substituted with one or more substituents from the following group: hydroxy, halogen, NH2, NO2, -CN, oxo, carboxy, -C(O)OC 1 -C 6 -alkyl.
  • (5- to 7-membered heteroaryl)oxy-, and (5- to 7-membered heteroaryl) ( C 1 -C 6 -alkoxy)- are optionally substituted with one or more of hydroxy, halogen, -NH 2 , (C 1 -C 6 - alkyl)N(H)-, -COOH, -CN and oxo, wherein each heteroaryl in R 1 has 1 to 4 heteroaryl ring members that heteroatoms selected from N, O, and S.
  • the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R D is a C 3 -C 14 carbocyclyl optionally substituted with one or more of the following: hydroxy, halogen, -NH 2 , -C(O)OH, -CN, oxo, alkyl, C 1 -C 6 -alkyl C 1 -C 6 ,-alkoxy and ( C 1 -C 6 -alkyl)N(H)-, wherein C 1 -C 6 -alkyl.
  • C 1 -C 6 -alkoxy and (C 1 -C 6 - alkyl)N(H)- are optionally substituted with one or more of hydroxy, halogen, -NH 2 , -C(O)OH, - CN, and oxo.
  • the MAT2A inhibitor is composed as described above (or pharmaceutically acceptable salt thereof) wherein R D is phenyl. In one embodiment, the MAT2A inhibitor is composed as described above (or pharmaceutically acceptable salt thereof) wherein R D is cyclohex- 1-en-l-yl.
  • the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R E is a C 3 -C 14 carbocyclyl optionally substituted with one or more of the following: hydroxy, halogen, -NH 2 , -C(O)OH, -CN, oxo, alkyl, C 1 -C 6 -alkyl. C 1 -C 6 -alkoxy and (C 1 -C 6 -alkyl)N(H)-. wherein C 1 -C 6 -alkyl.
  • C 1 -C 6 -alkoxy and (C 1 -C 6 - alkyl)N(H)- are optionally substituted with one or more of hydroxy, halogen, -NH 2 , -C(O)OH, - CN, and oxo.
  • the MAT2A inhibitor is composed as described above (or pharmaceutically acceptable salt thereof) wherein R E is selected from a group consisting of the following: cyclohex- 1-en-l-yl, ( 2 H 9 ) cyclohex- 1-en-l-yl, cyclohexan-1,3-dien-l-yl, 4, 4- difluoropiperidin-l-yl, 5,6-dihydro-2H-pyran-3-yl, 3,6-dihydro-2H-pyran-4-yl, lH-pyrrol-3-yl, lH-pyrrol-l-yl, tetrahydrofuran-3-yl, 3,3-difluoropyrrolidin-l-yl, and 3,6-dihydro-2H-pyran-4-yl.
  • the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R E is phenyl. In one embodiment, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R D is cyclohex- 1-en-l-yl and R E is phenyl.
  • the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R c is a C 3 -C 14 carbocyclyl or 3- to 14- membered heterocyclyl (wherein 1 to 4 ring members are heteroatoms selected from N, O, and S) and that optionally substituted with one or more substituents selected from the following: : hydroxy, halogen, -NH 2 , -C(O)OH, -CN, oxo, C 1 -C 6 -alkyl. C 1 -C 6 -alkoxy and (C 1 -C 6 -alkyl)N(H)-. wherein C 1 -C 6 -alkyl.
  • C 1 -C 6 -alkoxy and (C 1 -C 6 -alkyl)N(H)- are optionally substituted with hydroxy, halogen, -NH 2 , -C(O)OH, -CN, and oxo.
  • the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R c is selected from the group consisting of 4- hydroxyphenyl, 4-cholorphenyl, 4-fluorophenyl, 4-methoxyphenyl, 4-ethoxyphenyl, 4- trifluoromethoxyphenyl, 4-hydroxy-2-methylphenyl, 4-hydroxy-2-methoxyphenyl, 3,4- dihydroxyphenyl, 3-fluoro-4-hydroxyphenyl, 3-fluoro-4-methoxyphenyl, 2-chloro-4- hydroxyphenyl, 2-fluoro-4-methoxyphenyl, 3-amino-4-hydroxyphenyl, 3-amino-4-fluorophenyl, 3-(N,N-dimethylaminoethoxy)-4-hydroxyphenyl, 3-chloro-2 -hydroxyphenyl, 3-hydroxyethoxy-4- hydroxyphenyl.
  • R c is selected from the group consisting of 4- hydroxyphenyl
  • the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R c is selected from the group consisting of 6- methoxypyridin-3yl, 2-methoxypyridin-4-yl, lH-pyrazol-4-yl, quinolin-6-yl, 2-methylquinolin-6- yl, 2-methoxyquinolin-6-yl, 4-aminoquinazolin-6-yl, cinnoline-6-yl, quinoxalin-6-yl, chloroquinoxalin-6-yl, 3-chloroquinoxalin-6-yl, 3-aminoquinoxalin-6-yl, 3 -hydroxy quinoxalin-6- yl, 3-methoxyquinoxalin-6-yl, l,8-naphthyridin-3-yl, and imidazo[l,2-a]pyridine-6-yl.
  • R c is selected from the group consisting of 6- methoxypyri
  • the MAT2A inhibitor is composed as described above formula IV or pharmaceutically acceptable salt thereof) wherein R c is 4-methoxyphenyl.
  • the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R 1 is a 3- to 14-membered heterocyclyl optionally substituted with one or more substituents from the following group: hydroxy, halogen, - NH 2 , -NO 2 resort -C(O)0H, )-, -C(O)0C 1 -C 6 -alkyl, -CN, oxo, )-, C 1 -C 6 -alkyl, )-, -C(O)0H, )-, C 1 -C 6 - alkoxy, and (C 1 -C 6 ,-alkyl)N(H)-.
  • R 1 is a 3- to 14-membered heterocyclyl optionally substituted with one or more substituents from the following group: hydroxy, halogen, - NH 2 , -NO 2 contend -C(O)0H, )-, -C(O
  • the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R 1 is selected from the following group: pyridine-2 -yl, pyrazin-2-yl, pyrimidin-2-yl, pyrimidin-4-yl, pyrimidin-5-yl, pyridazine-3-yl, 1,3,5-triazin-2-yl, and 1,2,4- triazin-3-yl, each of which is optionally substituted with one or more of F, Cl, CN, OH, -NO2, -NH 2 , NHMe, -C(O)NH 2 , and methoxy.
  • R 1 is selected from the following group: pyridine-2 -yl, pyrazin-2-yl, pyrimidin-2-yl, pyrimidin-4-yl, pyrimidin-5-yl, pyridazine-3-yl, 1,3,5-triazin-2-yl, and 1,2,4- tri
  • the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R 1 is selected from the following group:
  • the MAT2A inhibitor is a compound of Formula (VII):
  • the MAT2A inhibitor binds to its protein target.
  • the MAT2A inhibitor is composed as above (Formula VII or pharmaceutically acceptable salt thereof) wherein Rings A, B and C are independently a carbocyclyl or heterocyclyl, each optionally substituted with one or more of the following substituents: : hydroxy, halogen, - NH 2 , carboxy, -CN, oxo, alkyl, alkoxy, or alkylamino, wherein said alkyl, alkoxy or alkylamino are optionally substituted with hydroxy, halogen, -NH 2 , carboxy, -CN, or oxo. .
  • R 1 is H, alkyl, carbocyclyl, or hetercyclyl, optionally substituted with hydroxy, halogen, - NH 2 , NO 2 , -CN, oxo, carboxy, alkoxycarbonyl, alkoxyalkly, aminocarbonyl, alkyl, acyl, alkoxy, alkylamino aryl, aralkyl, heteroaryl, heteroaralkyl, aryloxy, aralkoxy, heteroaryloxy and heteroaralkoxy are optionally substituted with hydroxy, halogen, amino, alkylamino, carboxy, -CN or oxo.
  • Type I PRMT inhibitors are further disclosed in PCT/US2014/029710, which is incorporated herein by reference. Exemplary Type I PRMT inhibitors are disclosed in Table 1A and Table IB of PCT US2014/029710, and methods of making the Type I PRMT inhibitors are described in at least page 226, paragraph [00274] to page 328, paragraph [00050] of PCT US2014/029710.
  • MAT2A inhibitors are described in W020180450071 (PCT/US2017/049439). The generic and specific compounds described in these patent applications are incorporated herein by reference and can be used to treats cancer as described herein.
  • combinations of a Type I protein arginine methyltransferase (Type I)
  • the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor.
  • PRMT1 protein arginine methyltransferase 1
  • PRMT3 protein arginine methyltransferase 3
  • PRMT4 protein arginine methyltransferase 4
  • PRMT6 protein arginine methyltransferase 6
  • PRMT8 protein arginine methyltransferase 8
  • the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI. In one aspect, the Type I PRMT inhibitor is Compound A. In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, methods are provided for treating cancer in a human in need thereof, those methods comprising administration to the human a combination of Compound A and Compound 262, together with at least one of: a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent, thereby treating the cancer in the human.
  • a pharmaceutical composition comprising a therapeutically effective amount of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of a methionine adenosyltransferase II alpha (MAT2A) inhibitor are provided.
  • Type I PRMT Type I protein arginine methyltransferase
  • MAT2A methionine adenosyltransferase II alpha
  • the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor.
  • the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI.
  • the Type I PRMT inhibitor is Compound A.
  • the Type I PRMT inhibitor is Compound D.
  • the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a Compound A and a second pharmaceutical composition comprising a therapeutically effective amount of Compound 262 are provided.
  • the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor.
  • PRMT1 protein arginine methyltransferase 1
  • PRMT3 protein arginine methyltransferase 3
  • PRMT4 protein arginine methyltransferase 4
  • PRMT6 protein arginine methyltransferase 6
  • PRMT8 protein arginine methyltransferase 8
  • the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI. In one aspect, the Type I PRMT inhibitor is Compound A. In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, a combination of Compound A and Compound 262 for the manufacture of a medicament is provided. In one embodiment, a product containing a Type I PRMT inhibitor and a MAT2A inhibitor as a combined preparation for simultaneous, separate, or sequential use in medicine is provided.
  • the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor.
  • the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI.
  • the Type I PRMT inhibitor is Compound A.
  • the Type I PRMT inhibitor is Compound D.
  • the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, a product containing Compound A and Compound 262 as a combined preparation for simultaneous, separate, or sequential use in medicine is provided.
  • the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor.
  • the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI.
  • the Type I PRMT inhibitor is Compound A. In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, a product containing Compound A and Compound 262 as a combined preparation for simultaneous, separate, or sequential use in a human subject is provided.
  • a product containing a Type I PRMT inhibitor and a MAT2A inhibitor as a combined preparation for simultaneous, separate, or sequential use in treating cancer in a human subject wherein the cancer is melanoma, breast cancer, lymphoma, triple negative breast cancer (TNBC), bladder cancer or pancreatic cancer.
  • TNBC triple negative breast cancer
  • the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor.
  • the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI.
  • the Type I PRMT inhibitor is Compound A.
  • the Type I PRMT inhibitor is Compound D.
  • the MAT2A inhibitor is a compound of Formula III, IV or VII.
  • the MAT2A inhibitor is Compound 262.
  • a product containing Compound A and Compound 262 as a combined preparation for simultaneous, separate, or sequential use in treating cancer in a human subject is provided, wherein the cancer is melanoma, breast cancer, lymphoma, triple negative breast cancer (TNBC), bladder cancer or pancreatic cancer.
  • the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor.
  • the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI.
  • the Type I PRMT inhibitor is Compound A.
  • the Type I PRMT inhibitor is Compound D.
  • the MAT2A inhibitor is a compound of Formula III, IV or VII.
  • the MAT2A inhibitor is Compound 262.
  • the Type I PRMT inhibitor and the MAT2A inhibitor are administered to the patient in a route selected from: simultaneously, sequentially, in any order, systemically, orally, intravenously, and intratumorally. In one aspect, the Type I PRMT inhibitor and/or the MAT2A inhibitor is administered orally.
  • the cancer is a solid tumor or a haematological cancer. In one aspect, it is melanoma, breast cancer, lymphoma, bladder cancer or pancreatic cancer.
  • the cancer is a solid tumor or a haematological cancer.
  • the tumor is deficient in methylthioadenosine phosphorylase (MTAP).
  • MTAP methylthioadenosine phosphorylase
  • the tumor is normal in its expression of MTAP.
  • the cancer is selected from head and neck cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, prostate cancer, gliomas, glioblastoma, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, medulloblastoma, kidney cancer, liver cancer, melanoma, pancreatic cancer, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid cancer, lymphoblastic T cell leukemia, Chronic myelogenous leukemia, Chronic lymphocytic leukemia, Hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, AML, Chronic neutrophilic leukemia, Acute lymphoblastic T cell leukemia, plasma
  • the methods of the present invention further comprise administering at least one neo-plastic agent to said human.
  • the human has a solid tumor.
  • the tumor is selected from head and neck cancer, gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, non small cell lung carcinoma, prostate cancer, colorectal cancer, ovarian cancer and pancreatic cancer.
  • the human has a liquid tumor such as diffuse large B cell lymphoma (DLBCL), multiple myeloma, chronic lyphomblastic leukemia (CLL), follicular lymphoma, acute myeloid leukemia and chronic myelogenous leukemia.
  • DLBCL diffuse large B cell lymphoma
  • CLL chronic lyphomblastic leukemia
  • follicular lymphoma acute myeloid leukemia and chronic myelogenous leukemia.
  • the present disclosure also relates to a method for treating or lessening the severity of a cancer selected from: brain (gliomas), glioblastomas, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, medulloblastoma, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid, lymphoblastic T-cell leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic neutrophilic leukemia, acute lymphoblastic T-cell leukemia, plasmacytoma, immunoblastic large cell leuk
  • treating means: (1) to ameliorate or prevent the condition of one or more of the biological manifestations of the condition, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition, (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or treatment thereof, or (4) to slow the progression of the condition or one or more of the biological manifestations of the condition.
  • Prophylactic therapy is also contemplated thereby.
  • prevention is not an absolute term.
  • prevention is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof.
  • Prophylactic therapy is appropriate, for example, when a subject is considered at high risk for developing cancer, such as when a subject has a strong family history of cancer or when a subject has been exposed to a carcinogen.
  • cancer As used herein, the terms “cancer,” “neoplasm,” and “tumor” are used interchangeably and, in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination.
  • the definition of a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation on physical examination, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • X-ray X-ray
  • ultrasound or palpation e.g., ultrasound or palpation on physical examination
  • Tumors may be a hematopoietic (or hematologic or hematological or blood-related) cancer, for example, cancers derived from blood cells or immune cells, which may be referred to as “liquid tumors.”
  • liquid tumors include leukemias such as chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies such as multiple myeloma, MGUS and Waldenstrom’s macroglobulinemia; lymphomas such as non-Hodgkin’s lymphoma, Hodgkin’s lymphoma; and the like.
  • the cancer may be any cancer in which an abnormal number of blast cells or unwanted cell proliferation is present or that is diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies.
  • Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or megakaryoblastic) leukemia.
  • leukemias may be referred together as acute myeloid (or myelocytic or myelogenous) leukemia (AML).
  • Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polcythemia vera (PCV).
  • CML chronic myelogenous leukemia
  • CMML chronic myelomonocytic leukemia
  • PCV polcythemia vera
  • Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT); as well as myelofibrosis (MFS) with or without angiogenic myeloid metaplasia.
  • myelodysplasia or myelodysplastic syndrome or MDS
  • MDS myelodysplasia
  • RA refractory anemia
  • RAEB refractory anemia with excess blasts
  • RAEBT refractory anemia with excess blasts in transformation
  • MFS myelofibrosis
  • Hematopoietic cancers also include lymphoid malignancies, which may affect the lymph nodes, spleens, bone marrow, peripheral blood, and/or extranodal sites.
  • Lymphoid cancers include B-cell malignancies, which include, but are not limited to, B-cell non-Hodgkin’s lymphomas (B- NHLs).
  • B-NHLs may be indolent (or low-grade), intermediate-grade (or aggressive) or high-grade (very aggressive).
  • Indolent B-cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated-lymphoid tissue (MALT or extranodal marginal zone) lymphoma.
  • FL follicular lymphoma
  • SLL small lymphocytic lymphoma
  • MZL marginal zone lymphoma
  • LPL lymphoplasmacytic lymphoma
  • MALT mucosa-associated-lymphoid tissue
  • Intermediate-grade B-NHLs include mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML).
  • High-grade B-NHLs include Burkitt’s lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma.
  • B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV associated (or AIDS related) lymphomas, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma.
  • B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom’s macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and Castleman’s disease.
  • NHL may also include T-cell non-Hodgkin’s lymphoma s(T-NHLs), which include, but are not limited to T- cell non-Hodgkin’s lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma/delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides, and Sezary syndrome.
  • T-NHLs T-cell non-Hodgkin’s lymphoma s(T-NHLs)
  • Hematopoietic cancers also include Hodgkin’s lymphoma (or disease) including classical Hodgkin’s lymphoma, nodular sclerosing Hodgkin’s lymphoma, mixed cellularity Hodgkin’s lymphoma, lymphocyte predominant (LP) Hodgkin’s lymphoma, nodular LP Hodgkin’s lymphoma, and lymphocyte depleted Hodgkin’s lymphoma.
  • Hodgkin’s lymphoma or disease
  • classical Hodgkin’s lymphoma including classical Hodgkin’s lymphoma, nodular sclerosing Hodgkin’s lymphoma, mixed cellularity Hodgkin’s lymphoma, lymphocyte predominant (LP) Hodgkin’s lymphoma, nodular LP Hodgkin’s lymphoma, and lymphocyte depleted Hodgkin’s lymphoma.
  • LP lymphocyte predominant
  • Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM) including smoldering MM, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom’s Macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL).
  • MM multiple myeloma
  • MGUS monoclonal gammopathy of undetermined (or unknown or unclear) significance
  • MGUS monoclonal gammopathy of undetermined (or unknown or unclear) significance
  • plasmacytoma bone, extramedullary
  • LPL lymphoplasmacytic lymphoma
  • Waldenstrom’s Macroglobulinemia plasma cell leukemia
  • AL primary amyloidosis
  • Hematopoietic cancers may also include other cancers of additional hematopoietic cells
  • Tissues which include hematopoietic cells referred herein to as "hematopoietic cell tissues” include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa (such as the gut-associated lymphoid tissues), tonsils, Peyer's patches and appendix, and lymphoid tissues associated with other mucosa, for example, the bronchial linings.
  • hematopoietic cell tissues include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa (such as the gut-associated lymphoid tissues), tonsils, Peyer's patches and appendix, and lymphoid tissues associated with other mucosa, for example, the bronchial linings.
  • Compound A 2 means a Type I PRMT inhibitor.
  • Compound A 2 is a compound of Formula I, II, V, or VI.
  • Compound A 2 is Compound A.
  • Compound B 2 means a MAT2A inhibitor.
  • Compound B 2 is a compound of Formula III, IV or VII.
  • Compound B 2 is Compound 262.
  • the combinations of this invention are administered within a “specified period”.
  • specified period and grammatical variations thereof, as used herein, means the interval of time between the administration of one of Compound A 2 and Compound B 2 and the other of Compound A 2 and Compound B 2 . Unless otherwise defined, the specified period can include simultaneous administration. Unless otherwise defined, the specified period refers to administration of Compound A 2 and Compound B 2 during a single day.
  • the specified period will be about 24 hours; suitably they will both be administered within about 12 hours of each other - in this case, the specified period will be about 12 hours; suitably they will both be administered within about 11 hours of each other - in this case, the specified period will be about 11 hours; suitably they will both be administered within about 10 hours of each other - in this case, the specified period will be about 10 hours; suitably they will both be administered within about 9 hours of each other - in this case, the specified period will be about 9 hours; suitably they will both be administered within about 8 hours of each other - in this case, the specified period will be about 8 hours; suitably they will both be administered within about 7 hours of each other - in this case, the specified period will be about 7 hours; suitably they will both be administered within about 6 hours of each other - in this case, the specified period will be about 6 hours; suitably they
  • the compounds when the combination of the invention is administered for a “specified period”, the compounds will be co-administered for a “duration of time”.
  • duration of time and grammatical variations thereof, as used herein means that both compounds of the invention are administered for an indicated number of consecutive days. Unless otherwise defined, the number of consecutive days does not have to commence with the start of treatment or terminate with the end of treatment, it is only required that the number of consecutive days occur at some point during the course of treatment.
  • both compounds will be administered within a specified period for at least one day - in this case, the duration of time will be at least one day; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 3 consecutive days - in this case, the duration of time will be at least 3 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 5 consecutive days - in this case, the duration of time will be at least 5 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 7 consecutive days - in this case, the duration of time will be at least 7 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 14 consecutive days - in this case, the duration of time will be at least 14 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 30 consecutive days - in this case, the duration of time will be at least 30 days.
  • the compounds are not administered during a “specified period”, they are administered sequentially.
  • sequential administration and grammatical derivates thereof, as used herein is meant that one of Compound A 2 and Compound B 2 is administered once a day for two or more consecutive days and the other of Compound A 2 and Compound B 2 is subsequently administered once a day for two or more consecutive days.
  • a drug holiday utilized between the sequential administration of one of Compound A 2 and Compound B 2 and the other of Compound A 2 and Compound B 2 .
  • a drug holiday is a period of days after the sequential administration of one of Compound A 2 and Compound B 2 and before the administration of the other of Compound A 2 and Compound B 2 where neither Compound A 2 nor Compound B 2 is administered.
  • the drug holiday will be a period of days selected from: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,
  • one of Compound A 2 and Compound B 2 is administered for from 1 to 30 consecutive days, followed by an optional drug holiday, followed by administration of the other of Compound A 2 and Compound B 2 for from 1 to 30 consecutive days.
  • one of Compound A 2 and Compound B 2 is administered for from 1 to 21 consecutive days, followed by an optional drug holiday, followed by administration of the other of Compound A 2 and Compound B 2 for from 1 to 21 consecutive days.
  • one of Compound A 2 and Compound B 2 is administered for from 1 to 14 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of the other of Compound A 2 and Compound B 2 for from 1 to 14 consecutive days.
  • one of Compound A 2 and Compound B 2 is administered for from 1 to 7 consecutive days, followed by a drug holiday of from 1 to 10 days, followed by administration of the other of Compound A 2 and Compound B 2 for from 1 to 7 consecutive days.
  • Compound B 2 will be administered first in the sequence, followed by an optional drug holiday, followed by administration of Compound A 2 .
  • Compound B 2 is administered for from 3 to 21 consecutive days, followed by an optional drug holiday, followed by administration of Compound A 2 for from 3 to 21 consecutive days.
  • Compound B 2 is administered for from 3 to 21 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of Compound A 2 for from 3 to 21 consecutive days.
  • Compound B 2 is administered for from 3 to 21 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of Compound A 2 for from 3 to 21 consecutive days.
  • Compound B 2 is administered for 21 consecutive days, followed by an optional drug holiday, followed by administration of Compound A 2 for 14 consecutive days.
  • Compound B 2 is administered for 14 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of Compound A 2 for 14 consecutive days.
  • Compound B 2 is administered for 7 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of Compound A 2 for 7 consecutive days.
  • Compound B 2 is administered for 3 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of Compound A 2 for 7 consecutive days.
  • Compound B 2 is administered for 3 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of Compound A 2 for 3 consecutive days.
  • a “specified period” administration and a “sequential” administration can be followed by repeat dosing or can be followed by an alternate dosing protocol, and a drug holiday may precede the repeat dosing or alternate dosing protocol.
  • the methods of the present invention may also be employed with other therapeutic methods of cancer treatment.
  • Compound A 2 and Compound B 2 may be administered by any appropriate route. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), intratumorally, vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural). It will be appreciated that the preferred route may vary with, for example, the condition of the recipient of the combination and the cancer to be treated. It will also be appreciated that each of the agents administered may be administered by the same or different routes and that Compound A 2 and Compound B 2 may be compounded together in a pharmaceutical composition/formulation.
  • routes include oral, rectal, nasal, topical (including buccal and sublingual), intratumorally, vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural). It will be appreciated that the preferred route may vary with, for example, the condition of the recipient of the combination and the cancer to be treated. It will also be appreciated that each of the agents administered
  • one or more components of a combination of the invention are administered intravenously. In one embodiment, one or more components of a combination of the invention are administered orally. In another embodiment, one or more components of a combination of the invention are administered intratumorally. In another embodiment, one or more components of a combination of the invention are administered systemically, e.g., intravenously, and one or more other components of a combination of the invention are administered intratumorally. In any of the embodiments, e.g., in this paragraph, the components of the invention are administered as one or more pharmaceutical compositions.
  • any anti-neoplastic agent that has activity versus a susceptible tumor being treated may be co-administered in the treatment of cancer in the present invention.
  • examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita, T.S. Lawrence, and S.A. Rosenberg (editors), 10 th edition (December 5, 2014), Lippincott Williams & Wilkins Publishers.
  • a person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved.
  • Typical anti-neoplastic agents useful in the present invention include, but are not limited to, anti-microtubule or anti-mitotic agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such as actinomycins, anthracyclins, and bleomycins; topoisomerase I inhibitors such as camptothecins; topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogues and anti-folate compounds; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; cell cycle signaling inhibitors; proteasome inhibitors; heat shock protein inhibitors; inhibitors of cancer metabolism;
  • anti -neoplastic agents examples include, but are not limited to, chemotherapeutic agents; immuno-modulatory agents; immune-modulators; and immunostimulatory adjuvants.
  • Example 1 Type I PRMT inhibitor and MAT2A inhibitor Combinations
  • a 6-day proliferation screening was performed in 5 pancreatic tumor cell lines: 2 MTAP wild-type, 2 MTAP null, 1 MTAP wt with CRISP knock-out of MTAP.
  • Combinations included double titration of Type 1 PRMT inhibitor Compound D (FIG. 2) with a MAT2A inhibitor Compound 262 (FIG.2).
  • Optimal cell seeding for all cell lines was determined by assessing the growth over a range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 6 days.
  • Cells were then plated at the optimal seeding density 24 hours before treatment (in duplicate) and treated the following day with a 16X16 double titration matrix (two-fold dilution series) of Compound D (Type I PRMT inhibitor) and Compound 262 (MAT2A inhibitor). This double titration was compared to a 16-point two-fold dilution series of each single agent alone or 0.15% DMSO.
  • An untreated plate of cells was harvested at the time of compound addition (T 0 ) to quantify the starting number of cells.
  • Concentrations tested for Compound D and Compound 262 alone or in combination ranged from 0.3 to 10,000nM. Plates were incubated for 6 days at 37 °C in 5% CO 2 . Cells were then lysed with CellTiter-Glo (CTG) (Promega) according to the manufacturer’s protocol and chemiluminescent signal detected on a Cytomat equipped with the Synergy Neo plate reader (ThermoFisher, serial # 140715A). CTG estimates cell number through detection of cellular ATP levels. CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T 0 value, and plotted against compound concentration. Data were fit with a four- parameter equation to generate a concentration response curves.
  • FIGS. 3-22 show that the combination of the Type I PRMT inhibitor (Compound D) with the MAT2A inhibitor (Compound 262) resulted in increased growth inhibition over either single agent alone. Synergistic effects were found in all cell lines tested, though the greatest potency shifts were observed in the MTAP -deficient cell lines. This is most readily observed in the Pane 03.27 wild type line and the matched MTAP knock-out cell line.
  • Example 2 Type I PRMT inhibitor and MAT2A inhibitor Combinations Materials and Methods General Method
  • a 6-day proliferation screening was performed in 4 pancreatic tumor cell lines: 2 MTAP wild type vs 2 MTAP null. Combinations included: 1:1 fixed ratio of Type 1 PRMT inhibitor Compound A-di-HCl (FIG. 2) + MAT2A inhibitor Compound 262 (FIG. 2); titration of the Type 1 PRMT inhibitor Compound A-di-HCl with several fixed concentrations of MAT2A inhibitor Compound 262 (1000 nM, 300 nM, 100 nM); titration of MAT2A inhibitor Compound 262 with several fixed concentrations of the Type I PRMT inhibitor Compound A-di-HCl (1000 nM, 300 nM, 100 nM).
  • Optimal cell seeding for all cell lines was determined by assessing the growth over a range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 6 days. Cells were then plated at the optimal seeding density 24 hours before treatment (in duplicate) and treated the following day with a 20-point two-fold dilution series of Compound A- di-HCl, Compound 262, an equimolar ratio of Compound A-di-HCl: Compound 262, or 0.15% DMSO. An untreated plate of cells was harvested at the time of compound addition (T 0 ) to quantify the starting number of cells. Concentrations tested for Compound A-di-HCl and Compound 262 alone and in combination ranged from 0.00005 to 29.4 mM.
  • CTG CellTiter-Glo
  • Optimal cell seeding was determined for all cell lines by assessing the growth over a range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 6 days. Cells were then plated at the optimal seeding density 24 hours before treatment (in duplicate) and treated the following day with 0.15% DMSO or a 20-point two-fold dilution series of Compound A-di-HCl alone, or with 100, 300, or 1000nM of Compound 262. Additional treatment conditions included a 20-point two-fold dilution series of Compound 262 alone, or with 100, 300, or 1000nM of Compound A-di-HCl. Concentrations tested for each single agent ranged from 0.00005 to 29.4 mM.
  • FIGS. 23-31 show that combining the Type 1 PRMT inhibitor Compound A-di-HCl with the MAT2A inhibitor Compound 262 resulted in increased growth inhibition over either single agent alone.
  • the enhanced effects of the combination were observed in all formats, including: holding Compound A-di-HCl fixed and titrating Compound 262, holding Compound 262 fixed and titrating Compound A and with a 1: 1 fixed ratio of Compound A-di-HCl and Compound 262.
  • a combination effect was observed in both the MTAP -wild-type and MTAP -deficient cell lines.
  • Arginine methylation is an important post-translational modification on proteins involved in a diverse range of cellular processes such as gene regulation, RNA processing, DNA damage response, and signal transduction. Proteins containing methylated arginines are present in both nuclear and cytosolic fractions suggesting that the enzymes that catalyze the transfer of methyl groups on to arginines are also present throughout these subcellular compartments (reviewed in Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013); Lee, Y. H. & Stallcup, M. R. Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.
  • methylated arginine exists in three major forms: w -N G -monomethyl -arginine (MMA), w -N G , N -asymmetric dimethyl arginine (ADMA), or w -N G , N -symmetric dimethyl arginine (SDMA).
  • MMA monoomethyl -arginine
  • ADMA N -asymmetric dimethyl arginine
  • SDMA w -N G , N -symmetric dimethyl arginine
  • Arginine methylation occurs largely in the context of glycine-, arginine-rich (GAR) motifs through the activity of a family of Protein Arginine Methyltransferases (PRMTs) that transfer the methyl group from S-adenosyl-L-methionine (SAM) to the substrate arginine side chain producing S-adenosyl-homocysteine (SAH) and methylated arginine (FIG. 32).
  • PRMTs Protein Arginine Methyltransferases
  • SAM S-adenosyl-L-methionine
  • SAH S-adenosyl-homocysteine
  • FOG. 32 methylated arginine
  • the PRMT family is categorized into four sub-types (Type I-IV) depending on the product of the enzymatic reaction (FIG. 32).
  • Type IV enzymes methylate the internal guanidino nitrogen and have only been described in yeast (Fisk, J. C. & Read, F. K. Protein arginine methylation in parasitic protozoa. Eukaryot Cell 10, 1013-1022, doi:10.1128/EC.05103-11 (2011)); types T-TTT enzymes generate monomethyl-arginine (MMA, Rmel) through a single methylation event.
  • the MMA intermediate is considered a relatively low abundance intermediate, however, select substrates of the primarily Type III activity of PRMT7 can remain monomethylated, while Types I and II enzymes catalyze progression from MMA to either asymmetric dimethyl-arginine (ADMA, Rme2a) or symmetric dimethyl arginine (SDMA, Rme2s) respectively.
  • Type II PRMTs include PRMT5, and PRMT9, however, PRMT5 is the primary enzyme responsible for formation of symmetric dimethylation.
  • Type I enzymes include PRMT1, PRMT3, PRMT4, PRMT6 and PRMT8. PRMT1, PRMT3, PRMT4, and PRMT6 are ubiquitously expressed while PRMT8 is largely restricted to the brain (reviewed in Bedford, M. T. & Clarke, S.
  • PRMT1 is the primary Type 1 enzyme capable of catalyzing the formation of MMA and ADMA on numerous cellular substrates (Bedford, M. T. & Clarke, S. G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 33, 1-13, doi:10.1016/j.molcel.2008.12.013 (2009)).
  • the PRMT1 -dependent ADMA modification is required for the biological activity and trafficking of its substrates (Nicholson, T. B., Chen, T. & Richard, S.
  • Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable. Mol Cell Biol 20, 4859-4869 (2000)). Complete knockout of PRMT1 results in a profound increase in MMA across numerous substrates suggesting that the major biological function for PRMT1 is to convert MMA to ADMA while other PRMTs can establish and maintain MMA (Dhar, S. et al. Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Sci Rep 3, 1311, doi:10.1038/srep01311 (2013)).
  • SDMA levels are increased upon loss of PRMT1, likely a consequence of the loss of ADMA and the corresponding increase of MMA that can serve as the substrate for SDMA-generating Type II PRMTs.
  • Inhibition of Type I PRMTs may lead to altered substrate function through loss of ADMA, increase in MMA, or, alternatively, a switch to the distinct methylation pattern associated with SDMA (Dhar, S. et al Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Sci Rep 3, 1311, doi : 10.1038/srep01311 (2013)).
  • Prmtl locus Disruption of the Prmtl locus in mice results in early embryonic lethality and homozygous embryos fail to develop beyond E6.5 indicating a requirement for PRMT1 in normal development (Pawlak, M. R., Scherer, C. A., Chen, J., Roshon, M. J. & Ruley, H. E. Arginine N- methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable. Mol Cell Biol 20, 4859-4869 (2000); Yu, Z., Chen, T., Hebert, J., Li, E.
  • a mouse PRMT1 null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation. Mol Cell Biol 29, 2982-2996, doi: 10.1128/MCB.00042-09 (2009)).
  • PRMT1 protein and mRNA can be detected in a wide range of embryonic and adult tissues, consistent with its function as the enzyme responsible for the majority of cellular arginine methylation.
  • PRMTs can undergo post-translational modifications themselves and are associated with interacting regulatory proteins, PRMT1 retains basal activity without a requirement for additional modification (reviewed in Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi:10.1038/nrc3409 (2013)).
  • PRMT1 and Cancer Mis-regulation and overexpression of PRMT1 has been associated with a number of solid and hematopoietic cancers (Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013); Yoshimatsu, M. et al. Dysregulation of PRMT1 and PRMT6, Type I arginine methyltransferases, is involved in various types ofhuman cancers. Int J Cancer 128, 562-573, doi:10.1002/ijc.25366 (2011)). The link between PRMT1 and cancer biology has largely been through regulation of methylation of arginine residues found on relevant substrates (FIG. 33).
  • PRMT1 can drive expression of aberrant oncogenic programs through methylation of histone H4 (Takai, H. el al. 5- Hydroxymethylcytosine plays a critical role in glioblastomagenesis by recruiting the CHTOP- methylosome complex. Cell Rep 9, 48-60, doi: 10.1016/j.celrep.2014.08.071 (2014); Shia, W. J. et al. PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood 119, 4953-4962, doi: 10.1182/blood-2011-04-347476 (2012); Zhao,
  • PRMT1 is associated with leukemia development through methylation of key drivers such as MLL and AML1-ETO fusions, leading to activation of oncogenic pathways (Shia, W. J. et al). PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood 119, 4953-4962, doi: 10.1182/blood-2011-04-347476 (2012);
  • PRMT1 is also a component of MLL fusion complexes, promotes aberrant transcriptional activation in association with H4R3 methylation, and knockdown of PRMT1 can suppress MLL-EEN mediated transformation of hematopoietic stem cells (Cheung, N., Chan, L. C., Thompson, A., Cleary, M. L. & So, C. W. Protein arginine- methyltransferase-dependent oncogenesis. Nat Cell Biol 9, 1208-1215, doi: 10.1038/ncbl642 (2007)).
  • PRMT1 has been implicated in the promotion of metastasis and cancer cell invasion (Gao, Y. et al. The dual function of PRMT1 in modulating epithelial-mesenchymal transition and cellular senescence in breast cancer cells through regulation of ZEB1. Sci Rep 6, 19874, doi:10.1038/srepl9874 (2016); Avasarala, S. et al.
  • PRMT1 Is a Novel Regulator of Epithelial-Mesenchymal-Transition in Non-small Cell Lung Cancer. JBiol Chem 290, 13479-13489, doi: 10.1074/jbc.Ml 14.636050 (2015)) and PRMT1 mediated methylation of Estrogen Receptor a (ERa) can potentiate growth-promoting signal transduction pathways. This methylation driven mechanism may provide a growth advantage to breast cancer cells even in the presence of anti-estrogens (Le Romancer, M. et al. Regulation of estrogen rapid signaling through arginine methylation by PRMT1. Mol Cell 31, 212-221, doi:10.1016/j.molcel.2008.05.025 (2008)).
  • PRMT1 promotes genome stability and resistance to DNA damaging agents through regulating both homologous recombination and non- homologous end-joining DNA repair pathways (Boisvert, F. M., Rhie, A., Richard, S. & Doherty, A. J.
  • the GAR motif of 53BP1 is arginine methylated by PRMT1 and is necessary for 53BP1 DNA binding activity. Cell Cycle 4, 1834-1841, doi: 10.4161/cc.4.12.2250 (2005); Boisvert, F. M., Dery, U., Masson, J. Y. & Richard, S. Arginine methylation of MREl 1 by PRMT1 is required for DNA damage checkpoint control.
  • RNA binding proteins and splicing machinery are a major class of PRMT1 substrates and have been implicated in cancer biology through their biological function as well as recurrent mutations in leukemias (Bressan, G. C. et al. Arginine methylation analysis of the splicing- associated SR protein SFRS9/SRP30C. Cell Mol Biol Lett 14, 657-669, doi: 10.2478/s 11658-009- 0024-2 (2009); Sveen, A., K i lpinen, S., Ruusulehto, A., Lothe, R. A. & Skotheim, R. I. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.
  • PRMT1 mediated methylation of RBM15 regulates its expression; consequently, overexpression of PRMT1 in AML cell lines was shown to block differentiation by downregulation of RBM15, thereby preventing its ability to bind pre-mRNA intronic regions of genes important for differentiation.
  • a proteomic approach (Methylscan, Cell Signaling Technology) was utilized to identify proteins with changes in arginine methylation states in response to a tool PRMT1 inhibitor, Compound D. Protein fragments from Compound D- and DSMO-treated cell extracts were immunoprecipitated using methyl arginine specific antibodies (ADMA, MMA, SDMA), and peptides were identified by mass spectrometry. While many proteins undergo changes in arginine methylation, the majority of substrates identified were transcriptional regulators and RNA processing proteins in AML cell lines treated with the tool compound (FIG. 34).
  • PRMT1 anti-tumor activity
  • a PRMT1 inhibitor in hematological and solid tumors including: inhibition of AML-ETO driven oncogenesis in leukemia, inhibition of growth promoting signal transduction in breast cancer, and modulation of splicing through methylation of RNA binding proteins and spliceosome machinery.
  • Inhibition of Type I PRMTs including PRMT1 represents a tractable strategy to suppress aberrant cancer cell proliferation and survival.
  • Compound A was evaluated for time dependent inhibition by measuring IC 50 values following varying SAM :PRMT1: Compound A preincubation time and a 20 minute reaction.
  • An inhibitory mechanism that is uncompetitive with SAM implies that generation of the SAM:PRMT1 complex is required to support binding of Compound A, therefore SAM (held at K m app ) was included during the preincubation.
  • Compound A demonstrated time dependent inhibition of PRMT1 methylation evident by an increase in potency with longer preincubation time (FIG. 36A). Since time dependent inhibition was observed, further IC 50 determinations included a 60 minute SAM: PRMT1: Compound A preincubation and a 40 minute reaction time to provide a better representation of compound potency.
  • affinity selection mass spectrometry was used to examine the binding of Compound A to PRMT1.
  • ASMS affinity selection mass spectrometry
  • a 2 hr preincubation of PRMT 1:SAM with Compound A was used to ensure that the time dependent complex (ESI*) was fully formed based on the profile shown in FIG. 36A) in which maximal potency was observed after 20 minutes of preincubation.
  • ESI* time dependent complex
  • Compound A was detectable using ASMS. This suggests that the primary mechanism is reversible in nature, since ASMS would be unable to detect irreversibly bound Compound A. Definitive reversibility studies including off-rate analysis have not yet been performed and would further validate the mechanism.
  • the co-crystal structure of Compound A bound to PRMT1 and SAH was determined (2.48 ⁇ resolution) (FIG. 37).
  • SAH is the product formed upon removal of the methyl group from SAM by PRMT1; therefore, SAH and SAM should similarly occupy the same pocket of PRMT1.
  • the inhibitor binds in the cleft normally occupied by the substrate peptide directly adjacent to the SAH pocket and its diamine sidechain occupies the putative arginine substrate site.
  • the terminal methylamine forms a hydrogen bond with the Glul62 sidechain residue that is 3.6 A from the thioether of SAH and the SAH binding pocket is bridged to Compound A by Tyr57 and Met66.
  • Compound A binds PRMT1 through the formation of a hydrogen bond between the proton of the pyrazole nitrogen of Compound A and the acidic sidechain of Glu65; the diethoxy branched cyclohexyl moiety lies along the solvent exposed surface in a hydrophobic groove formed by Tyr57, Ile62, Tyrl66 and Tyrl70.
  • the spatial separation between SAH and inhibitor binding, as well as interactions with residues such as Tyr57 could support the SAM uncompetitive mechanism revealed in the enzymatic studies.
  • the finding that Compound A is bound in the substrate peptide pocket and that the diamine sidechain may mimic the amines of the substrate arginine residue implies that inhibitor modality may be competitive with peptide.
  • Biochemical mode of inhibition studies support that Compound A is a mixed inhibitor with respect to peptide (FIG. 36B).
  • the time-dependent behavior of Compound A as well as the potential for exosite binding of the substrate peptide outside of the peptide cleft could both result in a mode of inhibition that is not competitive with peptide, explaining the difference in modality suggested by the structural and biochemical studies.
  • the selectivity of Compound A was assessed across a panel of PRMT family members. IC 50 values were determined against representative Types I (PRMT3, PRMT4, PRMT6 and PRMT8) and II (PRMT5/MEP50 and PRMT9) family members following a 60 minute SAM: Enzyme: Compound A preincubation. Compound A inhibited the activity of all Type I PRMTs tested with varying potencies, but failed to inhibit Type II family members (FIG. 39A). Additional characterization of the Type I PRMTs revealed that Compound A was a time dependent inhibitor of PRMT4, PRMT6 and PRMT8 due to the increase in potency observed following increasing Enzyme: SAM: Compound A preincubation times; whereas, PRMT3 displayed no time dependent behavior (FIG. 39B).
  • Compound A is a potent, reversible, selective inhibitor of Type I PRMT family members showing equivalent biochemical potency against PRMT1, PRMT6 and PRMT8 with IC 50 values ranging between 3-5 nM.
  • the crystal structure of PRMT1 in complex with Compound A reveals that Compound A binds in the peptide pocket and both the crystal structure, as well as enzymatic studies are consistent with a SAM uncompetitive mechanism.
  • PRMT1 Inhibition of PRMT1 is predicted to result in a decrease of ADMA on cellular PRMT1 substrates, including arginine 3 of histone H4 (H4R3me2a), with concomitant increases in MMA and SDMA (Dhar, S. etal. Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Sci Rep 3, 1311, doi: 10.1038/srep01311 (2013)).
  • the dose response associated with increased MMA was evaluated in an in-cell-westem assay using an antibody to detect MMA and the cellular mechanistic EC 50 of 10.1 ⁇ 4.4 nM was determined (FIG. 40).
  • the dose response appeared biphasic, possibly due to differential activity between the Type I PRMTs or differential potency towards a particular subset of substrates.
  • An equation describing a biphasic curve was used to fit the data and since there was no obvious plateau associated with the second inflection over the range of concentrations tested, the first inflection was reported.
  • Various salt forms were tested in this assay format and all demonstrated similar EC 50 values and are, therefore, considered interchangeable for all biology studies (FIG. 40).
  • PRMTs 3, 4, and 6 are also expressed across a range of tumor types while PRMT8 expression appears more restricted as predicted given its tissue specific expression (Fee, T, Sayegh, T, Daniel, T, Clarke, S. & Bedford, M. T. PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family. J Biol Chem 280, 32890-32896, doi:10.1074/jbc.M506944200 (2005)).
  • Compound A was analyzed for its ability to inhibit cultured tumor cell line growth in a 6- day growth-death assay using Cell Titer Glo (Promega) that quantifies ATP as a surrogate of cell number. The growth of all cell lines was evaluated over time across a wide range of seeding densities to identify conditions that permitted proliferation throughout the entire 6-day assay.
  • the glC 50 is the midpoint of the ‘growth window’, the difference between the number of cells at the time of compound addition (T 0 ) and the number of cells after 6 days (DMSO control).
  • the growth-death assay can be used to quantify the net population change, clearly defining cell death (cytotoxicity) as fewer cells compared to the number at the time of compound addition (T 0 ).
  • a negative Y min -T 0 value is indicative of cell death while a glC 100 value represents the concentration of compound required for 100% inhibition of growth.
  • the growth inhibitory effect of Compound A was evaluated using this assay in 196 human cancer cell lines representing solid and hematological malignancies (FIG. 42).
  • Compound A induced near or complete growth inhibition in most cell lines, with a subset showing cytotoxic responses, as indicated by a negative Y min -T 0 value (FIG. 42B). This effect was most pronounced in AML and lymphoma cancer cell lines, where 50 and 54% of cell lines showed cytotoxic responses, respectively.
  • the total AUC or exposure (C ave ) calculated from the rat 14-day MTD (150 mg/kg, C ave 2.1 mM) was used as an estimate of a clinically relevant concentration of Compound A for evaluation of sensitivity.
  • lymphoma cell lines showed cytotoxicity with glC 100 values below 2.1 mM, many cell lines across all tumor types evaluated showed glC 50 values £2.1 mM suggesting that concentrations associated with anti -tumor activity may be achievable in patients.
  • Lymphoma cell lines were highly sensitive to Type I PRMT inhibition, with a median glC 50 of 0.57 mM and cytotoxicity observed in 54%.
  • AML cytotoxicity in 50% of cell lines
  • Renal cell carcinoma glC 50 £ 2.1 mM in 60% of cell lines
  • a human DLBCL cell line (Toledo) was treated with 0.4 mM Compound A or vehicle for up to 120 hours after which protein lysates were evaluated by western analysis using antibodies for various arginine methylation states.
  • ADMA methylation decreased while MMA increased upon compound exposure (PIG. 43).
  • An increase in levels of SDMA was also observed, suggesting that the increase in MMA may have resulted in accumulation in the pool of potential substrates for PRMT5, the major catalyst of SDMA formation.
  • ADMA, SDMA, and MMA levels were assessed in cells treated with Compound A after compound washout (FIG. 45).
  • Toledo cells were cultured with 0.4 mM Compound A for 72 hours to establish robust effects on arginine methylation marks.
  • Cells were then washed, cultured in Compound A-free media, samples were collected daily through 120 hours, and arginine methylation levels were examined by western analysis.
  • MMA levels rapidly decreased, returning to baseline by 24 hours after Compound A washout, while ADMA and SDMA returned to baseline by 24 and 96 hours, respectively.
  • an extended duration growth-death assay was performed in a subset of lymphoma cell lines. Similar to the 6-day proliferation assay described previously, the seeding density was optimized to ensure growth throughout the duration of the assay, and cell number was assessed by CTG at selected timepoints beginning from days 3-10. Growth inhibition was observed as early as 6 days and was maximal by 8 days in Toledo and Daudi lymphoma cell lines (FIG. 46). A larger set of cell lines was evaluated on days 6 and 10 to measure the effects of prolonged exposure to Compound A and determine whether cell lines that displayed a cytostatic response in the 6-day assay might undergo cytotoxicity at later timepoints.
  • the proliferation assay results suggest that the inhibition of PRMT1 induces apparent cytotoxicity in a subset of lymphoma cell lines.
  • the cell cycle distribution in lymphoma cell lines treated with Compound A was evaluated using propidium iodide staining followed by flow cytometry.
  • Cell lines that showed a range of Y min -T 0 and glC 50 values in the 6-day proliferation assay were seeded at low density to allow logarithmic growth over the duration of the assay, and treated with varying concentrations of Compound A.
  • caspase cleavage was performed as an additional measure of apoptosis during a 10-day timecourse. Seeding density was optimized to ensure consistent growth throughout the duration of the assay, and caspase activation was assessed using a luminescent Caspase-Glo 3/7 assay (Promega). Caspase-Glo 3/7 signal was normalized to cell number (assessed by CTG) and shown as fold-induction relative to control (DMSO treated) cells. Caspase 3/7 activity was monitored over a 10-day timecourse in DLBCL cell lines showing cytotoxic (Toledo) and cytostatic (Daudi) responses to Compound A (FIG. 50).
  • the Toledo cell line showed robust caspase activation concurrent with decreases in cell number at all timepoints, while induction of caspase activity in the Daudi cell line was less pronounced and limited to the highest concentrations of Compound A.
  • TGI tumor growth inhibition
  • mice were dosed orally with either vehicle or Compound A (37.5 mg/kg- 150 mg/kg) for 24 days QD or 75 mg/kg BID.
  • BID administration of 75 mg/kg resulted in the same TGI as 150 mg/kg (95% and 96%, respectively) while £ 75 mg/kg QD resulted in partial TGI ( £79%) (FIG. 51, Table 5). No significant body weight loss was observed in any dose group.
  • Compound A had potent, cytotoxic activity in a subset of AML cell lines examined in the 6-day proliferation assay (Table 3). Eight of 10 cell lines had glC 50 values ⁇ 2mM, and Compound A induced cytotoxicity in 5 cell lines.
  • PRMT1 interacts with the AML-ETO fusion characteristic of the M2 AML subtype (Shia, W. J. et al PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential.
  • Renal cell carcinoma cell lines had among the lowest median glC 50 compared with other solid tumor types. Although none of the lines tested showed a cytotoxic response upon treatment with Compound A, all showed complete growth inhibition and 6 of 10 had glC 50 values £ 2 mM (Table 4). 7 of the 10 lines profiled represent clear cell renal carcinoma (ccRCC), the major clinical subtype of renal cancer.
  • mice bearing human renal cell carcinoma xenografts Female SCID mice bearing subcutaneous ACHN cell line tumors were weighed and tumors were measured by callipers and block randomized according to tumor size into treatment groups of 10 mice each. Mice were dosed orally with either vehicle or Compound A (150 mg/kg - 600 mg/kg) for up to 59 days daily. Throughout the study, mice were weighed and tumor measurements were taken twice weekly. Significant tumor growth inhibition was observed at all doses and regressions were observed at doses 3 300 mg/kg. Significant body weight loss was observed in animals treated with 600 mg/kg daily and, therefore, that dosing group was terminated on day 31 (FIG. 54, Table 5).
  • TNBC triple negative breast cancer
  • Compound A had the most potent anti-proliferative effect in melanoma cell lines (FIG. 42). Six of 7 lines assessed had glC 50 values less than 2 mM (Table 6). The effect of Compound A was cytostatic in all melanoma lines, regardless of glC 50 value. Table 6 Summary of Compound A Activity in Melanoma Cell Lines

Abstract

The present invention provides a combination of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a methionine adenosyltransferase II alpha (MAT2A) inhibitor. The present invention also provides methods for treating cancer in a human in need thereof, the methods comprising administering to the human a combination of a Type I PRMT inhibitor and a MAT2A inhibitor, together with at least one of: a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent, thereby treating the cancer in the human. The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a Type I PRMT inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of a MAT2A inhibitor.

Description

COMBINATION OF A TYPE I PROTEIN ARGININE METHYLTRANSFERASE (TYPE I PRMT) INHIBITOR AND A METHIONINE ADENOSYLTRANSFERASE II
ALPHA (MAT2A) INHIBITOR
FIELD OF THE INVENTION
The present invention relates to a method of treating cancer in a human and to combinations useful in such treatment. Particularly, the present invention relates to combinations of Type I protein arginine methyltransferase (Type I PRMT) inhibitors and methionine adenosyltransferase II alpha (MAT2A) inhibitors.
BACKGROUND OF THE INVENTION
Effective treatment of hyperproliferative disorders, such as cancer, is a continuing goal in the oncology field. Generally, cancer results from the deregulation of the normal processes that control cell division, differentiation and apoptotic cell death and is characterized by the proliferation of malignant cells which have the potential for unlimited growth, local expansion and systemic metastasis. Deregulation of normal processes includes abnormalities in signal transduction pathways and response to factors that differ from those found in normal cells.
Arginine methylation is an important post-translational modification on proteins involved in a diverse range of cellular processes such as gene regulation, RNA processing, DNA damage response, and signal transduction. Proteins containing methylated arginines are present in both nuclear and cytosolic fractions suggesting that the enzymes that catalyze the transfer of methyl groups on to arginines are also present throughout these subcellular compartments (reviewed in Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013); Lee, Y. H. & Stallcup, M. R. Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation. Mol Endocrinol 23, 425-433, doi:10.1210/me.2008-0380 (2009)). In mammalian cells, methylated arginine exists in three major forms: w -NG -monomethyl -arginine (MMA), w -NG ,NG -as ym m ct ri c dimethyl arginine (ADMA), or w -NG ,N'G -symmetric dimethyl arginine (SDMA). Each methylation state can affect protein- protein interactions in different ways and therefore has the potential to confer distinct functional consequences for the biological activity of the substrate (Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013)).
Arginine methylation occurs largely in the context of glycine-, arginine-rich (GAR) motifs through the activity of a family of Protein Arginine Methyltransferases (PRMTs) that transfer the methyl group from S-adenosyl-L-methionine (SAM) to the substrate arginine side chain producing S-adenosyl-homocysteine (SAH) and methylated arginine. This family of proteins is comprised of 10 members, of which 9 have been shown to have enzymatic activity (Bedford, M. T. & Clarke, S. G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 33, 1-13, doi:10.1016/j.molcel.2008.12.013 (2009)). The PRMT family is categorized into four sub-types (Type I-IV) depending on the product of the enzymatic reaction. Type IV enzymes methylate the internal guanidino nitrogen and have only been described in yeast (Fisk, J. C. & Read, L. K. Protein arginine methylation in parasitic protozoa. Eukaryot Cell 10, 1013-1022, doi:10.1128/EC.05103-11 (2011)); types T-TTT enzymes generate monomethyl-arginine (MMA, Rmel) through a single methylation event. The MMA intermediate is considered a relatively low abundance intermediate, however, select substrates of the primarily Type III activity of PRMT7 can remain monomethylated, while Types I and II enzymes catalyze progression from MMA to either asymmetric dimethyl-arginine (ADMA, Rme2a) or symmetric dimethyl arginine (SDMA, Rme2s) respectively.
Dysregulation and overexpression of PRMT1 have been associated with a number of solid and hematopoietic cancers (Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013); Yoshimatsu, M. etal. Dysregulation of PRMT1 and PRMT6, Type I arginine methyltransferases, is involved in various types ofhuman cancers. Int J Cancer 128, 562-573, doi:10.1002/ijc.25366 (2011)). The link between PRMT1 and cancer biology has largely been through regulation of methylation of arginine residues found on relevant substrates. In several tumor types, PRMT1 can drive expression of aberrant oncogenic programs through methylation of histone H4 (Takai, H. et al 5- Hydroxymethylcytosine plays a critical role in glioblastomagenesis by recruiting the CHTOP- methylosome complex. Cell Rep 9, 48-60, doi: 10.1016/j.celrep.2014.08.071 (2014); Shia, W. J. et al. PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood 119, 4953-4962, doi: 10.1182/blood-2011-04-347476 (2012); Zhao, X. et al. Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity. Genes Dev 22, 640-653, doi: 10.1101/gad.1632608 (2008), as well as through its activity on non-histone substrates (Wei, H., Mundade, R., Lange, K. C. & Lu, T. Protein arginine methylation of non-histone proteins and its role in diseases. Cell Cycle 13, 32-41, doi: 10.4161/cc.27353 (2014)). In many of these experimental systems, disruption of the PRMT1- dependent ADMA modification of its substrates decreases the proliferative capacity of cancer cells (Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013)).
As the primary methyl donor for arginine methylation in cells, S-adenosvl methionine (SAM) synthesis is crucial to the functionality of this process. The catalysis of methionine and ATP to form SAM by methionine adenosyltransferases is considered the rate- limiting step in this process. Two adenosyltransferase genes (MAT1A and MAT2A), which encode distinct catalytic isoforms, as well as a third gene (MAT2B) that encodes a MAT2A regulatory subunit, are known to be differentially impacted in cancer. While MAT1A is specifically expressed in the adult liver, MAT2A activity is widely distributed. For example, in hepatocellular carcinoma (HCC), the downregulation of MAT1A and the upregulation of MAT2A occur, which is known as the MAT1A-MAT2A switch. This switch, accompanied with up regulation of MAT2B, results in lower SAM contents and provides a growth advantage to hepatoma cells. Because of MAT2A’s critical role in facilitating the growth of hepatoma cells, it is a target for antineoplastic therapy. Recent studies have shown that silencing the via RNAi substantially suppresses growth and induces apoptosis in hepatoma cells. See T. Li, et al, J.
Cancer 7(10) (2016) 1317-27.
Further, Marjon et al ( Cell Reports 15(3), 574-587) report that methylthioadenosine phosphorylase (MTAP) — another component of the methionine cycle — also impacts the epigenetic dysregulation observed in some tumors (FIG. 1). The locus encoding this enzyme that converts methylthioadenosine (MTA) into adenine and 5-melhylthioribose-I-phosphate is deleted in 15% of cancers. Tang et al , Cancer Res. 78(15), 4386-4395. Despite many recent advances in cancer therapies, there remains a need for more effective and/or enhanced treatment for those individuals suffering the effects of cancer.
SUMMARY OF THE INVENTION
In one embodiment the present invention provides a combination of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a methionine adenosyltransferase II alpha (MAT2A) inhibitor.
In one embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of a methionine adenosyltransferase II alpha (MAT2A) inhibitor are provided.
In one embodiment, methods are provided for treating cancer in a human in need thereof, the methods comprising administering to the human a combination of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a methionine adenosyltransferase II alpha (MAT2A) inhibitor, together with at least one of: a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent, thereby treating the cancer in the human.
In one embodiment, methods of treating cancer in a human in need thereof are provided, the methods comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a pharmaceutical composition comprising of a methionine adenosyltransferase II alpha (MAT2A) inhibitor, thereby treating the cancer in the human.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Methionine salvage pathway, highlighting the function of MAT2A and MTAP.
MTAP and MAT2A are both part of the methionine salvage pathway leading to generation of SAM. MTAP deficiency leads to accumulation of MTA. High levels of MTA in MTAP -null tumor cells correlates with decreased levels of SDMA suggesting a pre-existing state of attenuated PRMT5 activity. MTAP deficiency also correlates with increased sensitivity to Type 1 PRMT inhibitors. Targeting MAT2A in this setting further disrupts the pathway leading to increased growth inhibition. From Marjon, K., et al. (2016). MTAP deletions in cancer create vulnerability to targeting of the MAT2A/PRMT5/RIOK1 axis. Cell Reports, 15(3), 574-587.
FIG. 2: Schematic of Type 1 PRMT inhibitors and MAT2A inhibitor. Structures of Type I PRMT inhibitors Compound D (left) and Compound A-di-HCl (middle) are shown. The structure of a MAT2A inhibitor (Compound 262) (right) is shown.
FIG. 3: Capan-1 (MTAP-null) double titration of Compound D and Compound 262. Drug titrations performed in MTAP-null Capan-1 cells, holding either concentration of (A) Compound 262 or (B) Compound D constant while modulating the other as indicated. CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves.
FIG. 4: Capan-1 (MTAP-null) proliferation assay. Both titrations exhibit greater than 5-fold glC50 potency shifts starting at 19.5nM Compound 262 in the Compound D titration and at 78.1nM Compound D in the Compound 262 titration. Both titrations exhibit greater than 5-fold glC100 potency shifts starting at 1250nM Compound 262 in the Compound D titration and at 625nM Compound D in the Compound 262 titration. Shifts from cytostatic to cytotoxic response is observed at 156.3nM Compound 262 in the Compound D titration and at 312.5nM Compound D in the Compound 262 titration. If the single agent exhibited a glC50 or glC100 value greater than the top concentration tested, >10,000nM was used to calculate fold change. FIG. 5: Capan-1 (MTAP-null) synergistic growth inhibition analyses. Synergy is measured in the Capan-1 cell line at varying concentrations of Compound D and Compound 262 using (A) excess over Bliss and (B) growth index analyses. Growth Index values less than 0 indicate a cytotoxic response. Synergy was observed across most concentration combinations in the double titration in the Capan-1 cell line. Cytotoxicity was also observed when combining these compounds at the higher concentration.
FIG. 6: HuP-T4 (MTAP-null) double titration of Compound D and Compound 262. Drug titrations were performed in MTAP-null HuP-T4 cells, holding the either concentration of (A) Compound 262 or (B) Compound D constant while modulating the other as indicated. CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves.
FIG. 7: HuP-T4 (MTAP-null) proliferation assay. Both titrations exhibit greater than 5-fold EC50 and glC50 potency shifts starting at 78.1nM Compound 262 in the Compound D titration and at 78. InM Compound D in the Compound 262 titration. Both titrations exhibit greater than 5-fold glC100 potency shifts starting at 39. InM Compound 262 in the Compound D titration and at 312.5nM Compound D in the Compound 262 titration. Shifts from cytostatic to cytotoxic response is observed at 312.5nM Compound D in the Compound 262 titration. If the single agent exhibited a glC50 or glC100 value greater than the top concentration tested, >10,000nM was used to calculate fold change. If a curve exhibited >20% downward shift in Y-max, EC50 was reported rather than glC50.
FIG. 8: HuP-T4 (MTAP-null) synergistic growth inhibition analyses. Synergy is measured in the HuP-T4 cell line at varying concentrations of Compound D and Compound 262 using (A) excess over Bliss and (B) growth index analyses. Cytotoxicity and synergy are observed when combining as little as 312.5nM of each compound. Growth Index values less than 0 indicate a cytotoxic response. Synergy is observed across mid-range concentration combinations in the HuP- T4 cell line. Cytotoxicity is also observed when combining these compound at the higher concentrations. FIG. 9: Pane 08.13 double titration of Compound D and Compound 262. Drug titrations were performed in Pane 08.13 cells (WT MTAP), holding the either concentration of (A) Compound 262 or (B) Compound D constant while modulating the other as indicated. CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves.
FIG. 10: Pane 08.13 proliferation assay. Both titrations exhibit greater than 5-fold glC50 potency shifts starting at 312.5nM Compound 262 in the Compound D titration and at 78. InM Compound D in the Compound 262 titration. glC100 potency shifts were not observed. Greater than 20% shifts in growth index were observed with the Compound 262 titration, but a shift from a cytostatic response to a cytotoxic response was only observed at the highest dose tested (10,000nM). If the single agent exhibited a glC50 or glC100 value greater than the top concentration tested, >10,000nM was used to calculate fold change.
FIG. 11: Pane 08.13 synergistic growth inhibition analyses. Synergy is observed in the Pane 08.13 cell line across concentration combinations above 78. InM of Compound D or Compound 262 using (A) excess over Bliss and (B) growth index analyses. Cytotoxicity is only observed when combining both compounds at the highest dose of 10,000nM. Growth Index values less than 0 indicate a cytotoxic response. Synergy is observed across concentration combinations above 78. InM for each compound in the Pane 08.13 cell line.
FIG. 12: Pane 03.27 double titration of Compound D and Compound 262. Drug titrations were performed in Pane 03.27 cells (WT MTAP), holding the either concentration of (A) Compound 262 or (B) Compound D constant while modulating the other as indicated. CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves.
FIG. 13: Pane 03.27 proliferation assay. Greater than 5-fold glC50 potency shifts were observed above 1250nM Compound 262 in the Compound D titration and above 2500nM Compound D in the Compound 262 titration. Greater than 5-fold glC100 potency shifts were not observed. Greater than 20% shifts in growth index were observed in both titrations above 156.3nM, but a shift from a cytostatic response to a cytotoxic response was only observed at 5000nM and above. If the single agent exhibited a glC50 or glC100 value greater than the top concentration tested, >10,000nM was used to calculate fold change. FIG. 14: Pane 03.27 synergistic growth inhibition analyses. Synergy is measured in the Pane 03.27 cell line at varying concentrations of Compound D and Compound 262 using (A) excess over Bliss and (B) growth index analyses. Synergy and cytotoxicity are only observed at the highest concentration combinations tested (above 5000nM). Growth Index values less than 0 indicate a cytotoxic response.
FIG. 15: Pane 03.27 (MTAP KO Clone #31) double titration of Compound D and Compound 262. Drug titrations were performed in Pane 03.27 cells (MTAP KO Clone #31), holding the either concentration of (A) Compound 262 or (B) Compound D constant while modulating the other as indicated. CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves.
FIG. 16: Pane 03.27 (MTAP KO Clone #31) proliferation assay. Knocking out MTAP appears to sensitize the Pane 03.27 cell line to this combination as seen in the shifts in potency for glC50 and especially glC100. Greater than 5-fold glC50 potency shifts were observed at 78. InM Compound 262 in the Compound D titration and 78. InM Compound D in the Compound 262 titration. Greater than 5-fold glC100 potency shifts were observed above 156.3nM for each titration. Greater than 20% shifts in growth index were observed above 39. InM Compound 262 in the Compound D titration and a shift from a cytostatic to a cytotoxic response was observed above 39. InM Compound D in the Compound 262 titration. If the single agent exhibited a glC50 or glC100 value greater than the top concentration tested, >10,000nM was used to calculate fold change.
FIG. 17: Pane 03.27 (MTAP KO Clone #31) synergistic growth inhibition analyses. Synergy is measured in the Pane 03.27 cell line at varying concentrations of Compound D and Compound 262 using (A) excess over Bliss and (B) growth index analyses. Synergy and cytotoxicity are observed when each compound was combined at doses as low as 625nM.
FIG. 18: Data tables for Capan-1 (MTAP-null) double titration of Compound D and Compound 262. Titrations performed in MTAP-null Capan-1 cells, holding the either concentration of Compound D or Compound 262 constant while modulating the other as indicated. Values in tables reference diagrams in Fig. 3. FIG. 19: Data tables for HuP-T4 (MTAP-null) double titration of Compound D and Compound 262. Titrations performed in MTAP-null HuP-T4 cells, holding the either concentration of Compound D or Compound 262 constant while modulating the other as indicated. Values in tables reference diagrams in Fig. 6.
FIG. 20: Data tables for Pane 08.13 double titration of Compound D and Compound 262.
Titrations performed in Pane 08.13 cells (WT MTAP), holding the either concentration of Compound D or Compound 262 constant while modulating the other as indicated. Values in tables reference diagrams in Fig. 9.
FIG. 21: Data tables for Pane 03.27 double titration of Compound D and Compound 262.
Titrations performed in Pane 03.27 cells (WT MTAP), holding the either concentration of Compound D or Compound 262 constant while modulating the other as indicated. Values in tables reference diagrams in Fig. 12.
FIG. 22: Data tables for Pane 03.27 (MTAP KO Clone #31) double titration of Compound D and Compound 262. Titrations performed in Pane 03.27 cells (MTAP KO Clone #31), holding the either concentration of Compound D or Compound 262 constant while modulating the other as indicated. Values in tables reference diagrams in Fig. 15.
FIG. 23: Single agent proliferation curves in multiple cell types. (A, B) MTAP-null Capan-1 and HuP-T4 cell lines or (C, D) WT MTAP cell lines were treated with the indicated concentration of either Compound A or Compound 262. Proliferation was plotted for each condition in all lines. Note: in FIG. 23, “Compound A” is Compound A-di-HCl.
FIG. 24: Combinatorial therapy proliferation assays. (A) Legend for data table in (B), charting fold change and change in Growth Index for fixed ratio combination of Compound A or Compound 262 in indicated cell lines. (C) Growth curve for HuP-T4 cells treated with either Compound A, Compound 262, or Compound A + Compound 26. Fold Change and Change in Growth Index for fixed ratio combination were calculated based on the most potent single agent value. Note: in FIG. 24, “Compound A” is Compound A-di-HCl. FIG. 25: Growth indices with combinatorial therapy in multiple cell types (Compound 262 fixed). (A) glC50 or (B) glC100 values for Compound A titrated in the indicated cell lines alone or in combination with the indicated concentration of Compound 262. If Y-max shifted by more than 20% from DMSO, a glC50 value was not calculated. EC50 values were compared if curve exhibited both an upper and lower plateau. Note: in FIG. 25, “Compound A” is Compound A-di-HCl.
FIG. 26: Combinatorial therapy titration and growth assays. (A) Legend for data table in (B), charting fold change and change in Growth Index for combination of Compound A and indicated concentration of Compound 262 in the indicated cell lines. (C) Growth curve for HuP-T4 cells treated with either Compound A alone or with the indicated concentration of Compound 262.
Note: in FIG. 26, “Compound A” is Compound A-di-HCl.
FIG. 27: Growth indices with combinatorial therapy in multiple cell types (Compound 262 fixed). (A) glC50 or (B) glC100 values for Compound 262 in the indicated cell lines alone or in combination with the indicated concentration of Compound A. If Y-max shifted by more than 20% from DMSO, a glC50 value was not calculated. EC50 values were compared if curve exhibited both an upper and lower plateau. Note: in FIG. 27, “Compound A” is Compound A-di-HCl.
FIG. 28: Combinatorial therapy titration and growth assays. (A) Legend for data table in (B), charting fold change and change in Growth Index for combination of Compound 262 and indicated concentration of Compound A in the indicated cell lines. (C) Growth curve for Capan-1 cells treated with either Compound 262 alone or with the indicated concentration of Compound A.
Note: in FIG. 28, “Compound A” is Compound A-di-HCl.
FIG. 29: Data table for combinatorial therapy growth assays (fixed ratio). Chart of various indicia of cell growth and proliferation following treatment with Compound A, Compound 262 or both in the indicated cell types. Note: in FIG. 29, “Compound A” is Compound A-di-HCl.
FIG. 30: Data table for combinatorial therapy growth assays (fixed Compound A). Chart of various indicia of cell growth and proliferation when following treatment with a fixed concentration of Compound A alone or in combination with the indicated concentration of Compound 262 in the indicated cell types. Note: in FIG. 30, “Compound A” is Compound A-di- HCl. FIG. 31: Data table for combinatorial therapy growth assays (fixed Compound 262). Chart of various indicia of cell growth and proliferation when following treatment with a fixed concentration of Compound 262 alone or in combination with the indicated concentration of Compound A in the indicated cell types. Note: in FIG. 31, “Compound A” is Compound A-di- HC1.
FIG. 32: Types of methylation on arginine residues. From Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013).
FIG. 33: Functional classes of cancer relevant PRMT1 substrates. Known substrates of PRMT1 and their association to cancer related biology (Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013); Shia, W. J. et al. PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood 119, 4953-4962, doi: 10.1182/blood-2011-04-347476 (2012); Wei, H., Mundade, R., Lange, K. C. & Lu, T. Protein arginine methylation of non-histone proteins and its role in diseases. Cell Cycle 13, 32-41, doi: 10.4161/cc.27353 (2014); Boisvert, F. M., Rhie, A., Richard, S. & Doherty, A. J. The GAR motif of 53BP1 is arginine methylated by PRMT1 and is necessary for 53BP1 DNA binding activity. Cell Cycle 4, 1834-1841, doi: 10.4161/CC.4.12.2250 (2005); Boisvert, F. M., Dery, U., Masson, J. Y. & Richard, S. Arginine methylation of MRE11 by PRMT1 is required for DNA damage checkpoint control. Genes Dev 19, 671-676, doi: 10.1101/gad.1279805 (2005); Zhang, L. et al. Cross-talk between PRMT1- mediated methylation and ubiquitylation on RBM15 controls RNA splicing. Elife 4, doi: 10.7554/eLife.07938 (2015); Snijders, A. P. et al. Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA. RNA 21, 347-359, doi: 10.1261/ma.045138.114 (2015); Liao, H. W. et al. PRMT1 -mediated methylation of the EGF receptor regulates signaling and cetuximab response. J Clin Invest 125, 4529-4543, doi: 10.1172/JCI82826 (2015); Ng, R. K. et al. Epigenetic dysregulation of leukaemic HOX code in MLL-rearranged leukaemia mouse model. J Pathol 232, 65-74, doi: 10.1002/path.4279 (2014); Bressan, G. C. et al. Arginine methylation analysis of the splicing- associated SR protein SFRS9/SRP30C. Cell Mol Biol Lett 14, 657-669, doi: 10.2478/sl 1658-009- 0024-2 (2009)).
FIG. 34: Methylscan evaluation of cell lines treated with Compound D. Percent of proteins with methylation changes (independent of directionality of change) are categorized by functional group as indicated. FIG. 35: Mode of inhibition against PRMT1 by Compound A. IC50 values were determined following a 18 minute PRMT1 reaction and fitting the data to a 3-parameter dose-response equation. (A) Representative experiment showing Compound A IC50 values plotted as a function of [SAM]/ Km app fit to an equation for uncompetitive inhibition EC50=Ki /(l+(Km/[S])). (B) Representative experiment showing IC50 values plotted as a function of [Peptide]/ Km app. Inset shows data fit to an equation for mixed inhibition to evaluate Compound A inhibition of PRMT1 with respect to peptide H4 1-21 substrate (v = Vmax * [S] / (Km * (1+[I]/Ki) + [S] * (1+[I]/K’))).
An alpha value (a = Ki’/Ki) >0.1 but <10 is indicative of a mixed inhibitor.
FIG. 36: Potency of Compound A against PRMTl. PRMT1 activity was monitored using a radioactive assay run under balanced conditions (substrate concentrations equal to Km app) measuring transfer of 3H from SAM to a H4 1-21 peptide. IC50 values were determined by fitting the data to a 3-parameter dose-response equation. (A) IC50 values plotted as a function of PRMT1: SAM: Compound A-tri-HCl preincubation time. Open and filled circles represent two independent experiments (0.5 nM PRMT1). Inset shows a representative IC50 curve for Compound A-tri-HCl inhibition of PRMT1 activity following a 60 minute PRMT1: SAM: Compound A-tri-HCl preincubation. (B) Compound A inhibition of PRMT1 categorized by salt form. IC50 values were determined following a 60 minute PRMT1: SAM: Compound A preincubation and a 20 minute reaction.
FIG. 37: The crystal structure resolved at 2.48Å for PRMTl in complex with Compound A (orange) and SAH (purple). The inset reveals that the compound is bound in the peptide binding pocket and makes key interactions with PRMT1 sidechains.
FIG. 38: Inhibition of PRMT1 orthologs by Compound A. PRMT1 activity was monitored using a radioactive assay run under balanced conditions (substrate concentrations equal to Km app) measuring transfer of 3H from SAM to a H4 1-21 peptide. IC50 values were determined by fitting the data to a 3-parameter dose-response equation. (A) IC50 values plotted as a function of PRMT1:SAM:Compound A preincubation time for rat (o) and dog (●) orthologs. (B) IC50 values plotted as a function of rat (o), dog (●) or human (□) PRMT1 concentration. (C) IC50 values were determined following a 60 minute PRMT1:SAM:Compound A preincubation and a 20 minute reaction. Data is an average from testing multiple salt forms of Compound A. Ki *app values were calculated based on the equation Ki= IC50/(1+(Km/[S])) for an uncompetitive inhibitor and the assumption that the IC50 determination was representative of the ESI* conformation. FIG. 39: Potency of Compound A against PRMT family members. PRMT activity was monitored using a radioactive assay run under balanced conditions (substrate concentrations at Km app) following a 60 minute PRMT: SAM: Compound A preincubation. IC50 values for Compound A were determined by fitting data to a 3-parameter dose-response equation. (A) Data is an average from testing multiple salt forms of Compound A. Ki *app value were calculated based on the equation Ki=EC50/(l+(Km/[S])) for an uncompetitive inhibitor and the assumption that the IC50 determination was representative of the ESI* conformation. (B) IC50 values plotted as a function of PRMT3 (●), PRMT4 (o), PRMT6 (■) or PRMT8 (□) : SAM: Compound A preincubation time.
FIG. 40: MMA in-cell-western. RKO cells were treated with Compound A-tri-HCl, Compound A-mono-HCl, Compound A-free-base, and Compound A-di-HCl for 72 hours.
Cells were fixed, stained with anti-RmelGGto detect MMA and anti-tubulin to normalize signal, and imaged using the Odyssey imaging system. MMA relative to tubulin was plotted against compound concentration to generate a curve fit (A) in GraphPad using a biphasic curve fit equation. Summary of EC50 (first inflection), standard deviation, and N are shown in (B).
FIG. 41: PRMT1 expression in tumors. mRNA expression levels were obtained from cBioPortal for Cancer Genomics. ACTB levels and TYR are shown to indicate expression of level corresponding to a gene that is ubitiquitously expressed versus one that has restricted expression, respectively.
FIG. 42: Antiproliferative activity of Compound A in cell culture. 196 human cancer cell lines were evaluated for sensitivity to Compound A in a 6-day growth assay. glC50 values for each cell line are shown as bar graphs with predicted human exposure as indicated in (A). Ymin -T0, a measure of cytotoxicity, is plotted as a bar-graph in (B), in which glC100 values for each cell line are shown as red dots. The Cave calculated from the rat 14-day MTD (150 mg/kg, Cave = 2.1 mM ) is indicated as a red dashed line.
FIG. 43: Timecourse of Compound A effects on arginine methylation marks in cultured cells. (A) Changes in ADMA, SDMA, and MMA in Toledo DLBCL cells treated with Compound A. Changes in methylation are shown normalized relative to tubulin + SEM (n=3). (B) Representative western blots of arginine methylation marks. Regions quantified are denoted by black bars on the right of the gel. FIG. 44: Dose response of Compound A on arginine methylation. (A) Representatitve western blot images of MMA and ADMA from the Compound A dose response in the U2932 cell line. Regions quantified for (B) are denoted by black bars to the left of gels. (B) Minimal effective Compound A concentration required for 50% of maximal induction of MMA or 50% maximal reduction ADMA in 5 lymphoma cell lines after 72 hours of exposure ± standard deviation (n=2). Corresponding glC50 values in 6-day growth death assay are as indicated in red.
FIG. 45: Durability of arginine methylation marks in response to Compound A in lymphoma cells. (A) Stability of changes to ADMA, SDMA, and MMA in the Toledo DLBCL cell line cultured with Compound A. Changes in methylation are shown normalized relative to tubulin SEM (n=3). (B) Representative western blots of arginine methylation marks. Regions quantified for (A) are denoted by black bars on the side of the gel.
FIG. 46: Proliferation timecourse of lymphoma cell lines. Cell growth was assessed over a 10- day timecourse in the Toledo (A) and Daudi (B) cell lines (n=2 per cell line). Representative data for a single biological replicate are shown.
FIG. 47: Anti-proliferative effects of Compound A in lymphoma cell lines at 6 and 10 days.
(A) Average glC50 values from 6 day (light) and 10 day (dark) proliferation assays in lymphoma cell lines. (B) Ymin-T0 at 6 day (light) and 10 day (dark) with corresponding glC100 (red points).
FIG. 48: Anti-proliferative effects of Compound A in lymphoma cell lines as classified by subtype. (A) glC50 values for each cell line are shown as bar graphs. Ymin-T0, a measure of cytotoxicity, is plotted as a bar-graph in (B), in which glC100 values for each cell line are shown as red dots. Subtype information was collected from the ATCC or DSMZ cell line repositories.
FIG. 49: Propidium iodide FACS analysis of cell cycle in human lymphoma cell lines. Three lymphoma cell lines, Toledo (A), U2932 (B), and OCI-Lyl (C) were treated with 0, 1, 10, 100, 1000, and 10,000 nM Compound A for 10 days with samples taken on days 3, 5, 7, 10 post treatment. Data represents the average ± SEM of biological replicates, n=2. FIG. 50: Caspase-3/7 activation in lymphoma cell lines treated with Compound A. Apoptosis was assessed over a 10-day timecourse in the Toledo (A) and Daudi (B) cell lines. Caspase 3/7 activation is shown as fold-induction relative to DMSO-treated cells. Two independent replicates were performed for each cell line. Representative data are shown for each.
FIG. 51: Efficacy of Compound A in mice bearing Toledo xenografts. Mice were treated QD (37.5, 75, 150, 300, 450, or 600 mg/kg) with Compound A orally or BID with 75 mg/kg (B) over a period of 28 (A) or 24 (B) days and tumor volume was measured twice weekly.
FIG. 52: Effect of Compound A in AML cell lines at 6 and 10 Days. (A) Average glC50 values from 6 day (light blue) and 10 day (dark blue) proliferation assays in AML cell lines. (B) Ymin-T0 at 6 day (light blue) and 10 day (dark blue) with corresponding glC100 (red points).
FIG. 53: In vitro proliferation timecourse of ccRCC cines with Compound A. (A) Growth relative to control (DMSO) for 2 ccRCC cell lines. Representative curves from a single replicate are shown. (B) Summary of glC50 and % growth inhibition for ccRCC cell lines during the timecourse (Average ± SD; n=2 for each line).
FIG. 54: Efficacy of Compound A in ACHN xenografts. Mice were treated daily with Compound A orally over a period of 28 days and tumor volume was measured twice weekly.
FIG. 55: Anti-proliferative effects of Compound A in breast cancer cell lines. Bar graphs of glC50 and growth inhibition (%) (red circles) for breast cancer cell lines profiled with Compound A in the 6-day proliferation assay. Cell lines representing triple negative breast cancer (TNBC) are shown in orange; other subtypes are in blue.
FIG. 56: Effect of Compound A in Breast Cancer Cell Lines at 7 and 12 Days. Average growth inhibition (%) values from 7 day (light blue) and 10 day (dark blue) proliferation assays in breast cancer cell lines with corresponding glC50 (red points) The increase in potency and percent inhibition observed in long-term proliferation assays with breast cancer, but not lymphoma or AML cell lines, suggest that certain tumor types require a longer exposure to Compound A to fully reveal anti-proliferative activity. DETAILED DESCRIPTION OF THE INVENTION
Described herein is combined inhibition of Type I protein arginine methyltransferase (Type I PRMT) and a methionine adenosyltransferase II alpha (MAT2A) for the treatment of cancer.
DEFINITIONS
Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modem Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, 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. See, for example, Jacques et ah, Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et ah, Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw- Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
It is to be understood that the compounds of the present invention may be depicted as different tautomers. It should also be understood that when compounds have tautomeric forms, all tautomeric forms are intended to be included in the scope of the present invention, and the naming of any compound described herein does not exclude any tautomer form.
Figure imgf000017_0001
Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, 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 a carbon by a 13C- or 14C-enriched carbon are within the scope of the disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.
The term "aliphatic," as used herein, includes both saturated and unsaturated, nonaromatic, straight chain (i.e., unbranched), branched, acyclic, and cyclic (i.e., carbocyclic) hydrocarbons. In some embodiments, an aliphatic group is optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl moieties.
When a range of values is listed, it is intended to encompass each value and subrange within the range. For example "C1-6 alkyl" is intended to encompass, C1; C2, C3, C4, C5, G. C1-6, C1- 5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.
"Radical" refers to a point of attachment on a particular group. Radical includes divalent radicals of a particular group.
"Alkyl" refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms ("C1-20 alkyl"). In some embodiments, an alkyl group has 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 ("C1-7 alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C1-6 alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("C1-5 alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C1-4 alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("C1-3 alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C1-2 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 ("C2-6 alkyl"). Examples of C1-6 alkyl groups include methyl (C1), ethyl (C2), n- propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3- pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. In certain embodiments, each instance of an alkyl group is independently optionally substituted, e.g. , unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents. In certain embodiments, the alkyl group is unsubstituted C1-10 alkyl (e.g., -CH3). In certain embodiments, the alkyl group is substituted C1-10 alkyl.
In some embodiments, an alkyl group is substituted with one or more halogens. "Perhaloalkyl" is a substituted alkyl group as defined herein wherein all of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, the alkyl moiety has 1 to 8 carbon atoms ("C1-8 perhaloalkyl"). In some embodiments, the alkyl moiety has 1 to 6 carbon atoms ("C1-6 perhaloalkyl"). In some embodiments, the alkyl moiety has 1 to 4 carbon atoms ("C1-4 perhaloalkyl"). In some embodiments, the alkyl moiety has 1 to 3 carbon atoms ("C1-3 perhaloalkyl"). In some embodiments, the alkyl moiety has 1 to 2 carbon atoms ("C1-2 perhaloalkyl"). In some embodiments, all of the hydrogen atoms are replaced with fluoro. In some embodiments, all of the hydrogen atoms are replaced with chloro. Examples of perhaloalkyl groups include - CF3, -CF2CF3, -CF2CF2CF3, -CCI3, -CFCI2, -CF2CI, and the like.
"Alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds), and optionally one or more triple bonds (e.g., 1, 2, 3, or 4 triple bonds) ("C2-20 alkenyl"). In certain embodiments, alkenyl does not comprise triple bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C2-10 alkenyl"). In some embodiments, an alkenyl group has 2 to 9 carbon atoms ("C2-9 alkenyl"). In some embodiments, an alkenyl group has 2 to 8 carbon atoms ("C2-8 alkenyl"). In some embodiments, an alkenyl group has 2 to 7 carbon atoms ("C2-7 alkenyl") In some embodiments, an alkenyl group has 2 to 6 carbon atoms ("C2-6 alkenyl"). In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("C2-5 alkenyl"). In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("C2-4 alkenyl"). In some embodiments, an alkenyl group has 2 to 3 carbon atoms ("C2-3 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 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 C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. In certain embodiments, each instance of an alkenyl group is independently optionally substituted, e.g. , unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents. In certain embodiments, the alkenyl group is unsubstituted C2-10 alkenyl. In certain embodiments, the alkenyl group is substituted C2-10 alkenyl.
"Alkynyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds), and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) ("C2-20 alkynyl"). In certain embodiments, alkynyl does not comprise double bonds. In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C2-10 alkynyl "). In some embodiments, an alkynyl group has 2 to 9 carbon atoms ("C2-9 alkynyl") . In some embodiments, an alkynyl group has 2 to 8 carbon atoms ("C2-8 alkynyl") . In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C2-7 alkynyl"). In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("C2-6 alkynyl"). In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("C2-5 alkynyl") . In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C2-4 alkynyl") . In some embodiments, an alkynyl group has 2 to 3 carbon atoms ("C2-3 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 C2-4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (G,). and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. In certain embodiments, each instance of an alkynyl group is independently optionally substituted, e.g., unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents. In certain embodiments, the alkynyl group is unsubstituted C2-10 alkynyl. In certain embodiments, the alkynyl group is substituted C2-10 alkynyl.
"Fused" or "ortho-fused" are used interchangeably herein, and refer to two rings that have two atoms and one bond in common, e.g..,
Figure imgf000019_0001
napthalene "Bridged" refers to a ring system containing (1) a bridgehead atom or group of atoms which connect two or more non-adjacent positions of the same ring; or (2) a bridgehead atom or group of atoms which connect two or more positions of different rings of a ring system and does not thereby form an ortho-fused ring, e.g.,
Figure imgf000020_0001
"Spiro" or "Spiro-fused" refers to a group of atoms which connect to the same atom of a carbocyclic or heterocyclic ring system (geminal attachment), thereby forming a ring, e.g.,
Figure imgf000020_0002
Spiro-fusion at a bridgehead atom is also contemplated.
"Carbocyclyl" or "carbocyclic" refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms ("C3-14 carbocyclyl") and zero heteroatoms in the non-aromatic ring system. In certain embodiments, a carbocyclyl group refers to a radical of a non aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (C3-10 carbocyclyl") and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms ("C3-8 carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C3-6 carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C3-6 carbocyclyl"). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms ("C5-10 carbocyclyl"). Exemplary C3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C). cyclohexenyl (C). cyclohexadienyl (C). and the like. Exemplary C3-8 carbocyclyl groups include, without limitation, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8). cyclooctenyl (C8). bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8). 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 (C10), octahydro-lH-indenyl (C9), decahydronaphthalenyl (Cio), spiro[4.5]decanyl (C10 ), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic ("monocyclic carbocyclyl") or is a fused, bridged or spiro-fused ring system such as a bicyclic system ("bicyclic carbocyclyl") and can be saturated or can be partially unsaturated. "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. In certain embodiments, each instance of a carbocyclyl group is independently optionally substituted, e.g., unsubstituted (an "unsubstituted carbocyclyl") or substituted (a "substituted carbocyclyl") with one or more substituents. In certain embodiments, the carbocyclyl group is unsubstituted C3-10 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3-10 carbocyclyl.
In some embodiments, "carbocyclyl" is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms ("C3-14 cycloalkyl"). In some embodiments, "carbocyclyl" is a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms ("C3-10 cycloalkyl"). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C3-8 cycloalkyl"). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ("C3-6 cycloalkyl"). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms ("C5-6 cycloalkyl"). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms ("C5-10 cycloalkyl"). Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6 cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). In certain embodiments, each instance of a cycloalkyl group is independently unsubstituted (an "unsubstituted cycloalkyl") or substituted (a "substituted cycloalkyl") with one or more substituents. In certain embodiments, the cycloalkyl group is unsubstituted C3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C3-10 cycloalkyl.
"Heterocyclyl" or "heterocyclic" 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"). In certain embodiments, heterocyclyl or heterocyclic refers to a radical of a 3-10 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 ("3-10 membered heterocyclyl"). In 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 a fused, bridged or spiro-fused ring system such as a bicyclic system ("bicyclic heterocyclyl"), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic 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. In certain embodiments, each instance of heterocyclyl is independently optionally substituted, e.g., unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocyclyl") with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.
In some embodiments, 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"). In some embodiments, 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"). In some embodiments, 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"). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.
Exemplary 3 -membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5 -membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5- membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5 -membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6- membered heterocyclyl groups containing three heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl, and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a G, aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6- membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
"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 p electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ("C1-14 aryl"). In some embodiments, an aryl group has six ring carbon atoms ("C aryl"; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen 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. In certain embodiments, each instance of an aryl group is independently optionally substituted, e.g. , unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents. In certain embodiments, the aryl group is unsubstituted C6-14 aryl. In certain embodiments, the aryl group is substituted C6-14 aryl.
"Heteroaryl" refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p 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"). In certain embodiments, heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 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"). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic 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 (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, e.g., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5- indolyl).
In some embodiments, a heteroaryl group is a 5-14 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 ("5-14 membered heteroaryl"). In some embodiments, 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"). In some embodiments, 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"). In some embodiments, 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"). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. In certain embodiments, each instance of a heteroaryl group is independently optionally substituted, e.g., unsubstituted ("unsubstituted heteroaryl") or substituted ("substituted heteroaryl") with one or more substituents. In certain embodiments, the heteroaryl group is unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is substituted 5-14 membered heteroaryl.
Exemplary 5 -membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5 -membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6- membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7- membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, any one of the following formulae:
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
In any of the monocyclic or bicyclic heteroaryl groups, the point of attachment can be any carbon or nitrogen atom, as valency permits.
"Partially unsaturated" refers to a group that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups (e.g., aryl or heteroaryl groups) as herein defined. Likewise, "saturated" refers to a group that does not contain a double or triple bond, i.e. , contains all single bonds.
In some embodiments, aliphatic, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” aliphatic, "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl or "substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted", whether preceded by the term "optionally" or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) 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, cycbzation, elimination, or other reaction. Unless otherwise indicated, 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. The term "substituted" is contemplated to include substitution with all permissible substituents of organic compounds, including 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. For purposes of this disclosure, 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.
Exemplary carbon atom substituents include, but are not limited to, halogen, -CN, -NO2, - N3, -SO2H, -SO3H, -OH, -ORaa, -ON(Rbb)2, -N(Rbb)2, -N(Rbb)3 +X , -N(ORcc)Rbb, -SH, -SRaa, - SSRCC, -C(=O)Raa, -CO2H, -CHO, -C(ORcc)2, -CO2Raa, -OC(=O)Raa, - OCO2Raa, -C(=O)N(Rbb)2, - OC(=O)N(Rbb)2, -NRbbC(=O)Raa, -NRbbCO2Raa, - NRbbC(=O)N(Rbb)2, -C(=NRbb)Raa, - C(=NRbb)ORaa, -OC(=NRbb)Raa, -OC(=NRbb)ORaa, - C(=NRbb)N(Rbb)2, -OC(=NRbb)N(Rbb)2, - NRbbC(=NRbb)N(Rbb)2, -C(=O)NRbbSO2Raa, - NRbbSO2Raa, -SO2N(Rbb)2, -SO2Raa. -SO2ORaa, - OSO2Raa, -S(=O)Raa, -OS(=O)Raa, - Si(Raa)3, -OSi(Raa)3 -C(=S)N(Rbb)2, -C(=O)SRaa, -C(=S)SRaa, - SC(=S)SRaa, -SC(=O)SRaa, -OC(=O)SRaa, -SC(=O)ORaa, -SC(=O)Raa, -P(=O)2Raa, -OP(=O)2Raa, - P(=O)(Raa)2, - OP(=O)(Raa)2, -OP(=O)(ORcc)2, -P(=O)2N(Rbb)2, -OP(=O)2N(Rbb)2, -P(=O)(NRbb)2, - OP(=O)(NRbb)2, -NRbbP(=O)(ORcc)2, -NRbbP(=O)(NRbb)2, -P(RCC)2, -P(RCC)3, -OP(Rcc)2, - OP(Rcc)3, -B(Raa)2, -B(ORcc)2, -BRaa(ORcc), C1-io alkyl, C1-io perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; or two geminal hydrogens on a carbon atom are replaced with the group =O, =S, =NN(Rbb)2, =NNRbbC(=O)Raa, =NNRbbC(=O)ORaa, =NNRbbS(=O)2Raa, =NRbb, or =NORcc; each instance of Raa is, independently, selected from C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Raa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; each instance of Rbb is, independently, selected from hydrogen, -OH, -ORaa, - N(RCC)2, - CN, -C(=O)Raa, -C(=O)N(Rcc)2, -CO2Raa, -S02Raa, -C(=NRcc)ORaa, - C(=NRcc)N(Rcc)2, - SO2N(Rcc)2, -SO2Rcc, -SO2ORcc, -SORaa, -C(=S)N(Rcc)2, -C(=O)SRcc, - C(=S)SRcc, -P(=O)2Raa, - P(=O)(Raa)2, -P(=O)2N(Rcc)2, -P(=O)(NRcc)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rbb groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; each instance of Rcc is, independently, selected from hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; each instance of Rdd is, independently, selected from halogen, -CN, -NO2, -N3, - SO2H, - SO3H, -OH, -ORee, -ON(Rff)2, -N(Rff)2, -N(Rff)3 +X , -N(ORee)Rff, -SH, -SRee, - SSRee, -C(=O)Ree, - CO2H, -CO2Ree, -OC(=O)Ree, -OCO2Ree, -C(=O)N(Rff)2, - OC(=O)N(Rff)2, -NRffC(=O)Ree, - NRffCO2Ree, -NRffC(=O)N(Rff)2, -C(=NRff)ORee, - OC(=NRff)Ree, -OC(=NRff)ORee, - C(=NRff)N(Rff)2, -OC(=NRff)N(Rff)2, - NRffC(=NRff)N(Rff)2,-NRffSO2Ree, -SO2N(Rff)2, -SO2Ree, - S02ORee, -OS02Ree, -S(=O)Ree, -Si(Ree)3, -OSi(Ree)3, -C(=S)N(Rff)2, -C(=O)SRee, -C(=S)SRee, - SC(=S)SRee, -P(=O)2Ree, - P(=O)(Ree)2, -OP(=O)(Ree)2, -OP(=O)(ORee)2, C1-6 alkyl, C1-6 perhaloalkyl, C2-6, alkenyl, C2-6, alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups, or two geminal Rdd substituents can be joined to form =O or =S; each instance of Ree is, independently, selected from C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; each instance of Rff is, independently, selected from hydrogen, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C1-6 aryl and 5-10 membered heteroaryl, or two Rff groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; and each instance of Rgg is, independently, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, - OH, -O1- 6 alkyl, -ON(C1-6 alkyl)2, -N( C1-6 alkyl)2, -N(C1-6 alkyl)3 +X- , -NH(C1-6 alkyl)2 +X- , -NH2(C1-6 alkyl) +X- , -NH3 +X , -N(OC1-6 alkyl)(C1-6 alkyl), -N(OH)(C1-6 alkyl), -NH(OH), -SH, -S1-6 alkyl, - SS(C1-6 alkyl), -C(=O)(C1-6 alkyl), -CO2H, -CO2(C1-6 alkyl), -OC(=O)(C1-6 alkyl), -OCO2(C1-6 alkyl), -C(=O)NH2, -C(=O)N(C1-6 alkyl)2, - OC(=O)NH(C1-6 alkyl), -NHC(=O)( C1-6 alkyl), -N(C1- 6 alkyl)C(=O)( C1-6 alkyl), - NHCO2(C1-6 alkyl), -NHC(=O)N(C1-6 alkyl)2, -NHC(=O)NH(C1-6 alkyl), -NHC(=O)NH2, -C(=NH)0(C1-6 alkyl) ,-OC(=NH)(C1-6 alkyl), -OC(=NH)OC1-6 alkyl, - C(=NH)N( C1-6 alkyl)2, -C(=NH)NH(C1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C1-6 alkyl)2, - OC(NH)NH(C1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C1-6 alkyl)2, -NHC(=NH)NH2, - NHSO2(C1-6 alkyl), -SO2N(C1-6 alkyl)2, -SO2NH(C1-6 alkyl), -SO2NH2,-SO2 C1-6 alkyl, - SO2OC1-6 alkyl, - OSO2C1-6 alkyl, -SOC1-6 alkyl, -Si(C1-6 alkyl)3, -OSi(C1-6 alkyl)3 - C(=S)N(C1-6 alkyl)2, C(=S)NH(C1-6 alkyl), C(=S)NH2, -C(=O)S(C1-6 alkyl), -C(=S)SC1-6 alkyl, -SC(=S)SC1-6 alkyl, - P(=O)2(C1-6 alkyl), -P(=O)(C1-6 alkyl)2, -OP(=O)(C1-6 alkyl)2, - OP(=O)(OC1-6 alkyl)2, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal Rgg substituents can be joined to form =O or =S; wherein X is a counterion.
A "counterion" or "anionic counterion" is a negatively charged group associated with a cationic quaternary amino group in order to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F-, CI, Br-, G), NO3 -, CIO4 - , OH-, H2PO4-, HSO4- , sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5 -sulfonate, ethan-l-sulfonic acid- 2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, and the like).
"Halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).
Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quartemary nitrogen atoms. Exemplary nitrogen atom substitutents include, but are not limited to, hydrogen, -OH, -ORaa, -N(RCC)2, -CN, - C(=O)Raa, - C(=O)N(Rcc)2, -CO2Raa, -SO2Raa, -C(=NRbb)Raa, -C(=NRcc)ORaa, - C(=NRcc)N(Rcc)2, -SO2N(Rcc)2, -SO2Rcc, -SO2ORcc, -SORaa, -C(=S)N(Rcc)2, -C(=O)SRcc, - C(=S)SRcc, -P(=O)2Raa, -P(=O)(Raa)2, - P(=O)2N(Rcc)2, -P(=O)(NRcc)2, C1-10 alkyl, C1-io perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups attached to a nitrogen atom are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined above.
In certain embodiments, the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups include, but are not limited to, -OH, -ORaa, -N(RCC)2, -C(=O)Raa, -C(=O)N(Rcc)2, -CO2Raa, -SO2Raa, -C(=NRcc)Raa, -C(=NRcc)ORaa, -C(=NRCC)N(Rcc)2, -SO2N(Rcc)2, -SO2Rcc, - SO2ORcc, -SORaa, -C(=S)N(Rcc)2, - C(=O)SRcc, -C(=S)SRcc, C1-10 alkyl {e.g., aralkyl, heteroaralkyl), C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R groups, and wherein Raa, Rbb, Rcc, and Rdd are as defined herein. 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.
Amide nitrogen protecting groups (e.g., -C(=O)Raa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide,
3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N- benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-{p- hydroxyphenyl)propanamide, 3- (o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2-methyl-2-(o- phenylazophenoxy)propanamide, 4- chlorobutanamide, 3 -methyl-3 -nitrobutanamide, o- nitrocinnamide, N-acetylmethionine, o- nitrobenzamide, and o-(benzoyloxymethyl)benzamide. Carbamate nitrogen protecting groups (e.g., -C(=O)ORaa) include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2.7-di 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), l-(l-adamantyl)-l- methylethyl carbamate (Adpoc), 1,1 -dimethyl -2 -haloethyl carbamate, 1, 1 -dimethyl -2,2- dibromoethyl carbamate (DB-t-BOC), 1,1 -dimethyl -2,2,2-trichloroethyl carbamate (TCBOC), 1- methyl-l-(4-biphenylyl)ethyl carbamate (Bpoc), 1 -(3.5 -di -t-buty l pheny l)- 1 - methylethyl carbamate (t-Bumeoc). 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-{N.N- dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coe), 4-nitrocinnamyl carbamate (Noe), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz). p-methoxybenzyl carbamate (Moz). p-nitobenzyl carbamate p- bromobenzyl carbamate, p- chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4- methylsulfmylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2- methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfbnyl)ethyl carbamate, [2-(l,3- dithianyl)] methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),
2.4- dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl carbamate (Ppoc), 1,1 -dimethyl -2 -cyanoethyl carbamate, m- chloro- p-acyloxybenzyl carbamate. p-(dihydroxyboryl)benzyl carbamate, 5- benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate,
3.5-dimethoxybenzyl carbamate, o- nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate. p- cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate,p- decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N- dimethylcarboxamido)benzyl carbamate, 1,1 -dimethyl-3 -(N,N- dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2- furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate. p-(/?'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1 -methyl- 1 -cyclopropylmethyl carbamate, 1- methyl- 1- (3,5-dimethoxyphenyl)ethyl carbamate, 1 -methyl- 1 -(p-phenylazophenyl)ethyl carbamate, 1 - methyl- 1-phenylethyl carbamate, 1 -methyl- l-(4-pyridyl)ethyl carbamate, phenyl carbamate, p- (phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4- (trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate. Sulfonamide nitrogen protecting groups (e.g., -S(=O)2Raa) include, but are not limited to, p-toluenesulfonamide (Ts), benzene sulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6- dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5, 6-tetramethyl-4- methoxybenzene sulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2, 2, 5, 7, 8-pentamethylchroman-6-sulfonamide (Pme), methane sulfonamide (Ms), b- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4' ,8'- dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, A-p-toluenesulfonylaminoacyl derivative, A-phenylaminothioacyl derivative, N- benzoylphenylalanyl derivative, N-acctylmcthioninc derivative, 4,5-diphenyl- 3-oxazolin-2-one, N-phthalimidc. N-dithiasuccinimidec (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N- 1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl- 1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-mcthylaminc. N- allylamine, N-[2-(trimcthylsilyl)cthoxy |mcthylaminc (SEM), N-3-acctoxypropylaminc. N- (1 -isopropyl -4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N- benzylamine, N-di(4-methoxyphenyl)methylamine. N-5- dibenzosuberylamine, N- triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl] amine (MMTr), N-9- phenylfluorenylamine (PhF), N-2.7-dichloro-9-fluorcnylmcthylcncaminc. N- ferrocenylmethylamino (Fcm), N-2-picolylamino N' -oxide, N- 1,1- dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N- diphenylmethyleneamine, N-[(2- pyridyl)mesityl]methyleneamine, N-( N' N' - dimethylaminomethylene)amine, N N' - isopropylidenediamine, N- p-nitrobcnzylidcncaminc. N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2- hydroxyphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo- l-cyclohexenyl)amine, N-borane derivative, N- diphenylborinic acid derivative, N- [phenyl(pentaacylchromium- or tungsten)acyl] amine, N- copper chelate, N-zinc chelate, N- nitroamine, N-nitrosoamine, amine A-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).
In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting groups include, but are not limited to, -Raa, -N(Rbb)2, -C(=O)SRaa, -C(=O)Raa, -C O2Raa, - C(=O)N(Rbb)2, -C(=NRbb)Raa, - C(=NRbb)ORaa, -C(=NRbb)N(Rbb)2, -S(=O)Raa, -SO2 Raa, - Si(Raa)3, -P(RCC)2, -P(RCC)3, -P(=O)2Raa, -P(=O)(Raa)2, -P(=O)(ORcc)2, -P(=O)2N(Rbb)2, and - P(=O)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. 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 edition, John Wiley & Sons, 1999, incorporated herein by reference.
Exemplary 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-butylthiomethy 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 (MTHP), 4-methoxytetrahydrothiopyranyl, 4- methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4- yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro- 7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, l-(2-chloroethoxy)ethyl, 1-methyl-l- methoxyethyl, 1 -methyl- 1-benzyloxyethyl, 1- methyl- l-benzyloxy-2-fluoroethyl, 2,2,2- trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, /;-chlorophenyl. p- methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn) p- ethoxybenzyl, 3,4-dimethoxybenzyl, o- nitrobenzyl, p- nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p- yanobenzyl, p- henylbenzyl, 2- picolyl, 4-picolyl, 3- methyl-2 -picolyl N-oxido, diphcnylmcthyl. p.p '-dinitrobenzhydryl, 5- dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p- ethoxyphenyldiphenylmethyl,. di(p- methoxyphenyl)phenylmethyl, tri(p- methoxyphenyl)methyl, 4-(4 '- bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5- dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levubnoyloxyphenyl)methyl, 4, 4', 4"- tris(benzoyloxyphenyl)methyl, 3-(imidazol-l- yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)- 1 -yrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, l,3-benzodisulfuran-2-yl, benzisothiazolyl S,S- dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl. triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate. p-chlorophcnoxyacctatc. 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4- (ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4- methoxycrotonate, benzoate,p- phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), t-butyl carbonate (BOC), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2- trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2- (phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophcnyl carbonate, alkyl benzyl carbonate, alkyl p -methoxybenzyl carbonate, alkyl 3,4- dimethoxybenzyl carbonate, alkyl o- nitrobenzyl carbonate, alkyl p -nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4- ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2- iodobenzoate, 4-azidobutyrate, 4-nitro-4- methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-
(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro- 4-(1,1 ,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(l,l-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2 -methyl-2 -butenoate, o- (methoxyacyl)benzoate, a-naphthoate, nitrate, alkyl N.N.N'.N'- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate. borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methane sulfonate (mesylate), benzylsulfonate, and tosylate (Ts).
In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups include, but are not limited to, -Raa, -N(Rbb)2, -C(=O)SRaa, -C(=O)Raa, -CO2Raa, - C(=O)N(Rbb)2, -C(=NRbb)Raa, - C(=NRbb)ORaa, -C(=NRbb)N(Rbb)2, -S(=O)Raa, -SO2 Raa, - Si(Raa)3 -P(RCC)2, -P(RCC)3, -P(=O)2Raa, - P(=O)(Raa)2, -P(=O)(ORcc)2, -P(=O)2N(Rbb)2, and - P(=O)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Sulfur 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, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
As used herein, a "leaving group", or "LG", is a term understood in the art to refer to a molecular fragment that departs with a pair of electrons upon heterolytic bond cleavage, wherein the molecular fragment is an anion or neutral molecule. See, for example, Smith, March Advanced Organic Chemistry 6th ed. (501-502). Examples of suitable leaving groups include, but are not limited to, halides (such as chloride, bromide, or iodide), alkoxycarbonyloxy, aryloxycarbonyloxy, alkane sulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, haloformates, -N02, trialkylammonium, and aryliodonium salts. In some embodiments, the leaving group is a sulfonic acid ester. In some embodiments, the sulfonic acid ester comprises the formula -OSO2RRG1 wherein RLG1 is selected from the group consisting alkyl optionally, alkenyl optionally substituted, heteroalkyl optionally substituted, aryl optionally substituted, heteroaryl optionally substituted, arylalkyl optionally substituted, and heterarylalkyl optionally substituted. In some embodiments, R LG1 is substituted or unsubstituted C1-C6 alkyl. In some embodiments, RLG1 is methyl. I n some embodiments, RLG1 is substituted or unsubstituted aryl. In some embodiments, RLG1 is substituted or unsubstitued phenyl. In some embodiments, RLG1 is:
Figure imgf000036_0001
In some cases, the leaving group is toluenesulfonate (tosylate, Ts), methanesulfonate (mesylate, Ms), p-bromobenzenesulfonyl (brosylate, Bs), or trifluoromethanesulfonate (triflate, Tf). In some cases, the leaving group is a brosylate (p-bromobenzenesulfonyl). In some cases, the leaving group is anosylate (2-nitrobenzenesulfonyl). In some embodiments, the leaving group is a sulfonate-containing group. In some embodiments, the leaving group is a tosylate group. The leaving group may also be a phosphineoxide (e.g., formed during a Mitsunobu reaction) or an internal leaving group such as an epoxide or cyclic sulfate.
These and other exemplary substituents are described in more detail in the Examples. The present disclosure is not intended to be limited in any manner by the above exemplary listing of substituents.
"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 other 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. Pharmaceutically acceptable salts of the compounds describe herein 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 used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzene sulfonate, 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, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)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, quaternary salts.
As used herein “Type I protein arginine methyltransferase inhibitor” or “Type I PRMT inhibitor” means an agent that inhibits any one or more of the following: protein arginine methyltransferase 1 (PRMT1), protein arginine methyltransferase 3 (PRMT3), protein arginine methyltransferase 4 (PRMT4), protein arginine methyltransferase 6 (PRMT6) inhibitor, and protein arginine methyltransferase 8 (PRMT8). In some embodiments, the Type I PRMT inhibitor is a small molecule compound. In some embodiments, the Type I PRMT inhibitor selectively inhibits any one or more of the following: protein arginine methyltransferase 1 (PRMT1), protein arginine methyltransferase 3 (PRMT3), protein arginine methyltransferase 4 (PRMT4), protein arginine methyltransferase 6 (PRMT6) inhibitor, and protein arginine methyltransferase 8 (PRMT8). In some embodiments, the Type I PRMT inhibitor is a selective inhibitor ofPRMT1, PRMT3, PRMT4, PRMT6, and PRMT8.
The present invention provides Type I PRMT inhibitors. In one embodiment, the Type I PRMT inhibitor is a compound of Formula (I):
Figure imgf000037_0001
or a pharmaceutically acceptable salt thereof, wherein
X is N, Z is NR4, and Y is CR5; or X is NR4, Z is N, and Y is CR5; or X is CR5, Z is NR4 , and Y is N; or X is CR5, Z is N, and Y is NR4;
Rx is optionally substituted C1-4 alkyl or optionally substituted C3-4 cycloalkyl;
L1 is a bond, -O-, -N(RB)-, -S-, -C(O)-, -C(O)O-, -C(O)S-, -C(O)N(RB)-, - C(O)N(RB)N(RB)-, -OC(O)-, -OC(O)N(RB)-, -NRBC(O)-, -NRBC(O)N(RB)-, - NRBC(O)N(RB)N(RB)-, -NRBC(O)O-, -SC(O)-, -C(=NRB)-, -C(=NNBb)-, -C(=NORA)-, - C(=NRB)N(RB) -NRBC(=NRB)-, -CCS)-, -C(S)N(RB)-, -NRBC(S)-, -SCO)-, -OS(O)2-, - S(O)2O-, -SO2-, -N(RB)SO2-, -SO2N(RB)-, or an optionally substituted C1-6 saturated or unsaturated hydrocarbon chain, wherein one or more methylene units of the hydrocarbon chain is optionally and independently replaced with -O-, -N(RB)-, -S-, -C(O)-, -C(O)O-, - C(O)S-, -C(O)N(RB) -C(O)N(RB)N(RB)-, -OC(O)-, -OC(O)N(RB)-, -NRBC(O)-, - NRBC(O)N(RB)-, -NRBC(O)N(RB)N(RB)-, -NRBC(O)O-, -SC(O)-, -C(=NRB)-, -C(=NNRB)-, -C(=NORa)-, -C(=NRB)N(RB)-, -NRBC(=NRB)-, -CCS)-, -C(S)N(RB)-, -NRBC(S)-, -S(O)-, - OS(O)2-, -S(O)2O-, -SO2- -N(RB)SO2-, or -SO2N(RB)-; each RA is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, and a sulfur protecting group when attached to a sulfur atom; each RB is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and a nitrogen protecting group, or an RB and Rw on the same nitrogen atom may be taken together with the intervening nitrogen to form an optionally substituted heterocyclic ring;
Rw is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; provided that when Li is a bond, Rw is not hydrogen, optionally substituted aryl, or optionally substituted heteroaryl;
R3 is hydrogen, C1-4 alkyl, or C3-4 cycloalkyl;
R4 is hydrogen, optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl; or optionally substituted C1-4 alkyl-Cy;
Cy is optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and
R5 is hydrogen, halo, -CN, optionally substituted C1-4 alkyl, or optionally substituted C3-4 cycloalkyl. In one aspect, R3 is a C1-4 alkyl. In one aspect, R3 is methyl. In one aspect, R4 is hydrogen. In one aspect, R5 is hydrogen. In one aspect, Li is a bond.
In one embodiment, the Type I PRMT inhibitor is a compound of Formula (I) wherein -L1- Rw is optionally substituted carbocyclyl. In one aspect, R3 is a C1-4 alkyl. In one aspect, R3 is methyl. In another aspect, R4 is hydrogen. In one aspect, R5 is hydrogen. In one aspect, Li is a bond. In one embodiment, the Type I PRMT inhibitor is a compound of Formula (V)
Figure imgf000039_0001
or a pharmaceutically acceptable salt thereof, wherein Ring A is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In one aspect, Ring A is optionally substituted carbocyclyl. In one aspect, R3 is a C1-4 alkyl. In one aspect, R3 is methyl. In one aspect, Rx is unsubstituted C1-4 alkyl. In one aspect, Rx is methyl. In one aspect, Li is a bond.
In one embodiment, the Type I PRMT inhibitor is a compound of Formula (VI)
Figure imgf000039_0002
or a pharmaceutically acceptable salt thereof. In one aspect, Ring A is optionally substituted carbocyclyl. In one aspect, R3 is a C1-4 alkyl. In one aspect, R3 is methyl. In one aspect, Rx is unsubstituted C1-4 alkyl. In one aspect, Rx is methyl.
In one embodiment, the Type I PRMT inhibitor is a compound of Formula (II):
Figure imgf000039_0003
or a pharmaceutically acceptable salt thereof. In one aspect, -L1-Rw is optionally substituted carbocyclyl. In one aspect, R3 is a C1-4 alkyl. In one aspect, R3 is methyl. In one aspect, Rx is unsubstituted C1-4 alkyl. In one aspect, Rx is methyl. In one aspect, R4 is hydrogen. In one embodiment, the Type I PRMT inhibitor is Compound A:
Figure imgf000040_0001
or a pharmaceutically acceptable salt thereof. Compound A and methods of making Compound A are disclosed in PCT/US2014/029710, in at least page 171 (Compound 158) and page 266, paragraph [00331]
In one embodiment, the Type I PRMT inhibitor is Compound A-tri-HCl, a tri-HCl salt form of Compound A. In another embodiment, the Type I PRMT inhibitor is Compound A-mono- HC1, a mono-HCl salt form of Compound A. In yet another embodiment, the Type I PRMT inhibitor is Compound A-free-base, a free base form of Compound A. In still another embodiment, the Type I PRMT inhibitor is Compound A-di-HCl, a di-HCl salt form of Compound A.
In one embodiment, the Type I PRMT inhibitor is Compound D:
Figure imgf000040_0002
or a pharmaceutically acceptable salt thereof.
Type I PRMT inhibitors are further disclosed in PCT/US2014/029710, which is incorporated herein by reference. Exemplary Type I PRMT inhibitors are disclosed in Table 1A and Table IB of PCT/US2014/029710, and methods of making the Type I PRMT inhibitors are described in at least page 226, paragraph [00274] to page 328, paragraph [00050] of PCT/US2014/029710.
As used herein “methionine adenosyltransferase II alpha inhibitor” or “MAT2A inhibitor” means an agent that inhibits the production of S-adenosylmethionine (SAM) by methionine adenosyltransferase 2A (MAT2A). The present invention also provides methionine adenosyltransferase II alpha (MAT2A) inhibitors. In one embodiment, the MAT2A inhibitor is a compound of Formula (III):
Figure imgf000041_0001
In an embodiment, the MAT2A inhibitor binds to its protein target. In some embodiments, the MAT2A inhibitor is composed as above (Formula III or pharmaceutically acceptable salt thereof) wherein RD and RE are independently selected from C3-C14-carbocyclyl, C6-C14-aryl. and 3- to 14-membered heterocyclyl (wherein 1 to 4 heterocyclyl ring members are heteroatoms selected from N, O, and S.
In some embodiments, RD and RE of the MAT2A inhibitor, as described above (Formula III or pharmaceutically acceptable salt thereof), are independently selected from C3-C14- carbocyclyl and C6-C14-aryl. In some embodiments, RD and RE of the MAT2A inhibitor, as described above (or pharmaceutically acceptable salt thereof), are independently selected from C5- C7-carbocyclyl, C6-C10-aryl. In some embodiments, RD and RE of the MAT2A inhibitor, as described above, are independently selected from C3-C14-carbocyclyl, C6-C14-aryl.
In some embodiments, RD and RE of the MAT2A inhibitor, as described above (Formula III or pharmaceutically acceptable salt thereof), are independently selected from cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and phenyl. In some embodiments, RD and RE of the MAT2A inhibitor, as described above (Formula III or pharmaceutically acceptable salt thereof), is cyclohexyl or cyclohexenyl and the other is phenyl. In some embodiments, RA of the MAT2A inhibitor, as described above (or pharmaceutically acceptable salt thereof), is selected from the group consisting of C1-C6, -alkyl. C2-C6 -alkenyl, C1-C6,-alkoxy. C3-C14-carbocyclyl, (C3-C14- carbocyclyl)- C1-C6, -alkyl-. 3-to 14-membered heterocyclyl (C1-C6-alkyl)- wherein 1 to 4 heterocyclyl ring members are heteroatoms selected from N, O, and S), C6-C14-aryl. (C6-C14-aryl)- C1-C6-alkyl-, C6-C14-aryloxy, -(CH2)0-6NR1(CH2)0-6C(O)R2, NR'R2. NR1C(NR2)NR1R2, -CN, and - OH.
In some embodiments, RA of the MAT2A inhibitor, as described above (or pharmaceutically acceptable salt thereof), is selected from the group consisting of C1-C6-alkyl. - (CH2)0-6)NR1(CH2)0-6)C(O)R2, NR' R2. and NR1C(NR2)NR1R2. In some embodiments, RA of the MAT2A inhibitor, as described above (Formula III or pharmaceutically acceptable salt thereof), is C1-C6, -alkyl or NR'R2. In one embodiment, RA of the MAT2A inhibitor, as described above (or pharmaceutically acceptable salt thereof), is NR1 R2. In one embodiment, R1 of the MAT2A inhibitor (Formula III or pharmaceutically acceptable salt thereof) is H.
In one embodiment, the MAT2A inhibitor is a compound of Formula (IV):
Figure imgf000042_0001
In an embodiment, the MAT2A inhibitor binds to its protein target. In some embodiments, the MAT2A inhibitor is composed as above (Formula IV or pharmaceutically acceptable salt thereof) Rc of the MAT2A inhibitor is C3-C14 carbocyclyl or a 3- to 14-membered heterocyclyl (wherein 1 to 4 heterocyclyl ring members are heteroatoms selected from N, O, and S, each optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, -NH2, C6-C14-aryl. (C6-C14-aryl ) - C1-C6, -al k y 1 carboxy, -CN, oxo, C1-C6-alkyl. C1-C6-alkoxy. and -NH(C1-C6-alkyl). wherein the C1-C6-alkyl. C1-C6-alkoxy. and -NH(C1-C6- alkyl) are independently and optionally substituted with hydroxy, halogen, -NH2, carboxy, -CN, and oxo; RD and RE are independently a C3-C14 carbocyclyl or a 3- 14-membered heterocyclyl (with 1 to 4 heteroatoms selected from N, O, and S), each optionally substituted with one or substituents of the following group: hydroxy, halogen, NH2 , C6-C14-aryl, ( C6-C14-aryl)-C1-C6- alkyl-, carboxy, -CN, oxo, C1-C6-alkyl. C1-C6-alkoxy. and -NH(C1-C6-alkyl). wherein the C1-C6- alkyl. C1-C6-alkoxy. and -NH(C1-C6-alkyl) are independently and optionally substituted with hydroxy, halogen, -NH2, carboxy, -CN, and oxo; R1 is selected from the group consisting of H, C1-C6-alkyl. C3-C14 carbocyclyl and a 3- 14-membered heterocyclyl (with 1 to 4 heteroatoms selected from N, O, and S), each optionally substituted with one or more substituents from the following group: hydroxy, halogen, NH2, NO2, -CN, oxo, carboxy, -C(O)OC1-C6-alkyl. (C1-C6- alkyl)O C1-C6-alkyl-, -C(O)NH2, C1-C6-alkyl, -C(O)H, C1-C6-alkoxy, (C1-C6-alkyl)N(H)-aryl-, (C6- C 14-aryl )C 1 -G -alkyl -. 5- to 7-membered heteroaryl, (5- to 7-membered heteroaryl )-C1-C6-alkyl. C6-C14-aryloxy. (C6-C14-aryl)-) C1-C6-alkoxy-. (5- to 7-membered heteroaryl)oxy-, and (5- to 7- membered heteroaryl)(C1-C6-alkoxy)-, wherein the C1-C6-alkyl. C1-C6-alkoxy. C1-C6-alkyl)N(H)-. -C(O)OC1-C6-alkyl, (C1-C6-alkyl)OC1-C6-alkyl-, C(O)NH2, C6-C14-aryl, ( C6-C14-aryl)C1-C6-alkyl-, 5- to 7-membered heteroaryl, (5- to 7-membered heteroaryl)-C1-C6-alkyl. C6-C14-aryloxy, ( C6-C14- aryl)-)C1-C6-alkoxy-. (5- to 7-membered heteroaryl)oxy-, and (5- to 7-membered heteroaryl) ( C1-C6 -alkoxy)- are optionally substituted with one or more of hydroxy, halogen, -NH2, (C1-C6- alkyl)N(H)-, -COOH, -CN and oxo, wherein each heteroaryl in R1 has 1 to 4 heteroaryl ring members that heteroatoms selected from N, O, and S.
In some embodiments, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein RD is a C3-C14 carbocyclyl optionally substituted with one or more of the following: hydroxy, halogen, -NH2, -C(O)OH, -CN, oxo, alkyl, C1-C6 -alkyl C1-C6,-alkoxy and ( C1-C6-alkyl)N(H)-, wherein C1-C6 -alkyl. C1-C6-alkoxy and (C1-C6- alkyl)N(H)- are optionally substituted with one or more of hydroxy, halogen, -NH2, -C(O)OH, - CN, and oxo. In one embodiment, the MAT2A inhibitor is composed as described above (or pharmaceutically acceptable salt thereof) wherein RD is phenyl. In one embodiment, the MAT2A inhibitor is composed as described above (or pharmaceutically acceptable salt thereof) wherein RD is cyclohex- 1-en-l-yl.
In some embodiments, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein RE is a C3-C14 carbocyclyl optionally substituted with one or more of the following: hydroxy, halogen, -NH2, -C(O)OH, -CN, oxo, alkyl, C1-C6-alkyl. C1-C6-alkoxy and (C1-C6-alkyl)N(H)-. wherein C1-C6-alkyl. C1-C6-alkoxy and (C1-C6- alkyl)N(H)- are optionally substituted with one or more of hydroxy, halogen, -NH2, -C(O)OH, - CN, and oxo. In one embodiment, the MAT2A inhibitor is composed as described above (or pharmaceutically acceptable salt thereof) wherein RE is selected from a group consisting of the following: cyclohex- 1-en-l-yl, (2H9) cyclohex- 1-en-l-yl, cyclohexan-1,3-dien-l-yl, 4, 4- difluoropiperidin-l-yl, 5,6-dihydro-2H-pyran-3-yl, 3,6-dihydro-2H-pyran-4-yl, lH-pyrrol-3-yl, lH-pyrrol-l-yl, tetrahydrofuran-3-yl, 3,3-difluoropyrrolidin-l-yl, and 3,6-dihydro-2H-pyran-4-yl. . In one embodiment, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein RE is phenyl. In one embodiment, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein RD is cyclohex- 1-en-l-yl and RE is phenyl.
In some embodiments, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein Rc is a C3-C14 carbocyclyl or 3- to 14- membered heterocyclyl (wherein 1 to 4 ring members are heteroatoms selected from N, O, and S) and that optionally substituted with one or more substituents selected from the following: : hydroxy, halogen, -NH2, -C(O)OH, -CN, oxo, C1-C6-alkyl. C1-C6-alkoxy and (C1-C6-alkyl)N(H)-. wherein C1-C6-alkyl. C1-C6-alkoxy and (C1-C6-alkyl)N(H)- are optionally substituted with hydroxy, halogen, -NH2, -C(O)OH, -CN, and oxo.
In some embodiments, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein Rc is selected from the group consisting of 4- hydroxyphenyl, 4-cholorphenyl, 4-fluorophenyl, 4-methoxyphenyl, 4-ethoxyphenyl, 4- trifluoromethoxyphenyl, 4-hydroxy-2-methylphenyl, 4-hydroxy-2-methoxyphenyl, 3,4- dihydroxyphenyl, 3-fluoro-4-hydroxyphenyl, 3-fluoro-4-methoxyphenyl, 2-chloro-4- hydroxyphenyl, 2-fluoro-4-methoxyphenyl, 3-amino-4-hydroxyphenyl, 3-amino-4-fluorophenyl, 3-(N,N-dimethylaminoethoxy)-4-hydroxyphenyl, 3-chloro-2 -hydroxyphenyl, 3-hydroxyethoxy-4- hydroxyphenyl.
Figure imgf000044_0001
In some embodiments, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein Rc is selected from the group consisting of 6- methoxypyridin-3yl, 2-methoxypyridin-4-yl, lH-pyrazol-4-yl, quinolin-6-yl, 2-methylquinolin-6- yl, 2-methoxyquinolin-6-yl, 4-aminoquinazolin-6-yl, cinnoline-6-yl, quinoxalin-6-yl, chloroquinoxalin-6-yl, 3-chloroquinoxalin-6-yl, 3-aminoquinoxalin-6-yl, 3 -hydroxy quinoxalin-6- yl, 3-methoxyquinoxalin-6-yl, l,8-naphthyridin-3-yl, and imidazo[l,2-a]pyridine-6-yl.
Figure imgf000045_0001
In one embodiment, the MAT2A inhibitor is composed as described above formula IV or pharmaceutically acceptable salt thereof) wherein Rc is 4-methoxyphenyl.
In some embodiments, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R1 is a 3- to 14-membered heterocyclyl optionally substituted with one or more substituents from the following group: hydroxy, halogen, - NH2, -NO2„ -C(O)0H, )-, -C(O)0C1-C6 -alkyl, -CN, oxo, )-, C1-C6-alkyl, )-, -C(O)0H, )-, C1-C6- alkoxy, and (C1-C6,-alkyl)N(H)-. wherein the Ci-G, -alkyl. C1-C6-alkoxy. (C1-C6-alkyl)N(H)-, - C(O)O C1-C6-alkyl. and ( C1-C6 - alkyl) N (H) C (O) - are optionally substituted with one or more of hydroxy, halogen, -NH2, (C1-C6-alkyl)N(H)-. -C(O)0H, -CN, and oxo. In some embodiments, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R1 is selected from the following group: pyridine-2 -yl, pyrazin-2-yl, pyrimidin-2-yl, pyrimidin-4-yl, pyrimidin-5-yl, pyridazine-3-yl, 1,3,5-triazin-2-yl, and 1,2,4- triazin-3-yl, each of which is optionally substituted with one or more of F, Cl, CN, OH, -NO2, -NH2, NHMe, -C(O)NH2, and methoxy.
In some embodiments, the MAT2A inhibitor is composed as described above (Formula IV or pharmaceutically acceptable salt thereof) wherein R1 is selected from the following group:
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
5
In one embodiment, the MAT2A inhibitor is a compound of Formula (VII):
Figure imgf000049_0001
In an embodiment, the MAT2A inhibitor binds to its protein target. In some embodiments, the MAT2A inhibitor is composed as above (Formula VII or pharmaceutically acceptable salt thereof) wherein Rings A, B and C are independently a carbocyclyl or heterocyclyl, each optionally substituted with one or more of the following substituents: : hydroxy, halogen, - NH2, carboxy, -CN, oxo, alkyl, alkoxy, or alkylamino, wherein said alkyl, alkoxy or alkylamino are optionally substituted with hydroxy, halogen, -NH2, carboxy, -CN, or oxo. . In some embodiments, R1 is H, alkyl, carbocyclyl, or hetercyclyl, optionally substituted with hydroxy, halogen, - NH2, NO2, -CN, oxo, carboxy, alkoxycarbonyl, alkoxyalkly, aminocarbonyl, alkyl, acyl, alkoxy, alkylamino aryl, aralkyl, heteroaryl, heteroaralkyl, aryloxy, aralkoxy, heteroaryloxy and heteroaralkoxy are optionally substituted with hydroxy, halogen, amino, alkylamino, carboxy, -CN or oxo.
Some embodiments of Formulae III and VII include the following:
Figure imgf000049_0002
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Type I PRMT inhibitors are further disclosed in PCT/US2014/029710, which is incorporated herein by reference. Exemplary Type I PRMT inhibitors are disclosed in Table 1A and Table IB of PCT US2014/029710, and methods of making the Type I PRMT inhibitors are described in at least page 226, paragraph [00274] to page 328, paragraph [00050] of PCT US2014/029710.
MAT2A inhibitors are described in W020180450071 (PCT/US2017/049439). The generic and specific compounds described in these patent applications are incorporated herein by reference and can be used to treats cancer as described herein. In one embodiment, combinations of a Type I protein arginine methyltransferase (Type I
PRMT) inhibitor and a methionine adenosyltransferase II alpha inhibitor, together with at least one of: a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent, thereby treating the cancer in the human, are provided. In one aspect, the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor. In another aspect, the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI. In one aspect, the Type I PRMT inhibitor is Compound A. In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, methods are provided for treating cancer in a human in need thereof, those methods comprising administration to the human a combination of Compound A and Compound 262, together with at least one of: a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent, thereby treating the cancer in the human.
In a further embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of a methionine adenosyltransferase II alpha (MAT2A) inhibitor are provided. In one aspect, the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor. In another aspect, the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI. In one aspect, the Type I PRMT inhibitor is Compound A. In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a Compound A and a second pharmaceutical composition comprising a therapeutically effective amount of Compound 262 are provided.
In another embodiment, use of a combination of a Type I PRMT inhibitor and a MAT2A inhibitor for the manufacture of a medicament is provided. In one embodiment, use of a combination of a Type I PRMT inhibitor and a MAT2A inhibitor for the treatment of cancer is provided. In one aspect, the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor. In another aspect, the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI. In one aspect, the Type I PRMT inhibitor is Compound A. In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, a combination of Compound A and Compound 262 for the manufacture of a medicament is provided. In one embodiment, a product containing a Type I PRMT inhibitor and a MAT2A inhibitor as a combined preparation for simultaneous, separate, or sequential use in medicine is provided. In one aspect, the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor. In another aspect, the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI. In one aspect, the Type I PRMT inhibitor is Compound A. In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, a product containing Compound A and Compound 262 as a combined preparation for simultaneous, separate, or sequential use in medicine is provided.
In still another embodiment, a product containing a Type I PRMT inhibitor and a MAT2A inhibitor as a combined preparation for simultaneous, separate, or sequential use in treating cancer in a human subject is provided. In one aspect, the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor. In another aspect, the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI. In one aspect, the Type I PRMT inhibitor is Compound A. In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, a product containing Compound A and Compound 262 as a combined preparation for simultaneous, separate, or sequential use in a human subject is provided.
In another embodiment, a product containing a Type I PRMT inhibitor and a MAT2A inhibitor as a combined preparation for simultaneous, separate, or sequential use in treating cancer in a human subject is provided, wherein the cancer is melanoma, breast cancer, lymphoma, triple negative breast cancer (TNBC), bladder cancer or pancreatic cancer. In one aspect, the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor. In another aspect, the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI. In one aspect, the Type I PRMT inhibitor is Compound A. In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262. In one embodiment, a product containing Compound A and Compound 262 as a combined preparation for simultaneous, separate, or sequential use in treating cancer in a human subject is provided, wherein the cancer is melanoma, breast cancer, lymphoma, triple negative breast cancer (TNBC), bladder cancer or pancreatic cancer.
In one embodiment, methods are provided for treating cancer in a human in need thereof, the methods comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising a Type I PRMT inhibitor and a pharmaceutical composition comprising a MAT2A inhibitor, thereby treating the cancer in the human. In one aspect, the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor. In another aspect, the Type I PRMT inhibitor is a compound of Formula I, II, V, or VI. In one aspect, the Type I PRMT inhibitor is Compound A.
In another aspect, the Type I PRMT inhibitor is Compound D. In one aspect, the MAT2A inhibitor is a compound of Formula III, IV or VII. In another aspect, the MAT2A inhibitor is Compound 262.
In any one of the embodiments herein, the Type I PRMT inhibitor and the MAT2A inhibitor are administered to the patient in a route selected from: simultaneously, sequentially, in any order, systemically, orally, intravenously, and intratumorally. In one aspect, the Type I PRMT inhibitor and/or the MAT2A inhibitor is administered orally.
In any one of the embodiments herein, the cancer is a solid tumor or a haematological cancer. In one aspect, it is melanoma, breast cancer, lymphoma, bladder cancer or pancreatic cancer.
In any one of the embodiments herein, the cancer is a solid tumor or a haematological cancer. In one aspect, the tumor is deficient in methylthioadenosine phosphorylase (MTAP). In another aspect, the tumor is normal in its expression of MTAP.
In one aspect the cancer is selected from head and neck cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, prostate cancer, gliomas, glioblastoma, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, medulloblastoma, kidney cancer, liver cancer, melanoma, pancreatic cancer, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid cancer, lymphoblastic T cell leukemia, Chronic myelogenous leukemia, Chronic lymphocytic leukemia, Hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, AML, Chronic neutrophilic leukemia, Acute lymphoblastic T cell leukemia, plasmacytoma, Immunoblastic large cell leukemia, Mantle cell leukemia, Multiple myeloma, Megakaryoblastic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, Erythroleukemia, malignant lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, lymphoblastic T cell lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urothelial cancer, vulval cancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharyngeal cancer, buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal tumor), and testicular cancer.
In one aspect, the methods of the present invention further comprise administering at least one neo-plastic agent to said human.
In one aspect, the human has a solid tumor. In one aspect, the tumor is selected from head and neck cancer, gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, non small cell lung carcinoma, prostate cancer, colorectal cancer, ovarian cancer and pancreatic cancer. In another aspect the human has a liquid tumor such as diffuse large B cell lymphoma (DLBCL), multiple myeloma, chronic lyphomblastic leukemia (CLL), follicular lymphoma, acute myeloid leukemia and chronic myelogenous leukemia.
The present disclosure also relates to a method for treating or lessening the severity of a cancer selected from: brain (gliomas), glioblastomas, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, medulloblastoma, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid, lymphoblastic T-cell leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic neutrophilic leukemia, acute lymphoblastic T-cell leukemia, plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia, multiple myeloma megakaryoblastic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, erythroleukemia, malignant lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, lymphoblastic T cell lymphoma, Burkitt’s lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urothelial cancer, lung cancer, vulval cancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal tumor) and testicular cancer. By the term "treating" and grammatical variations thereof as used herein, is meant therapeutic therapy. In reference to a particular condition, treating means: (1) to ameliorate or prevent the condition of one or more of the biological manifestations of the condition, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition, (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or treatment thereof, or (4) to slow the progression of the condition or one or more of the biological manifestations of the condition. Prophylactic therapy is also contemplated thereby. The skilled artisan will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof. Prophylactic therapy is appropriate, for example, when a subject is considered at high risk for developing cancer, such as when a subject has a strong family history of cancer or when a subject has been exposed to a carcinogen.
As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably and, in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation on physical examination, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient. Tumors may be a hematopoietic (or hematologic or hematological or blood-related) cancer, for example, cancers derived from blood cells or immune cells, which may be referred to as “liquid tumors.” Specific examples of clinical conditions based on hematologic tumors include leukemias such as chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies such as multiple myeloma, MGUS and Waldenstrom’s macroglobulinemia; lymphomas such as non-Hodgkin’s lymphoma, Hodgkin’s lymphoma; and the like.
The cancer may be any cancer in which an abnormal number of blast cells or unwanted cell proliferation is present or that is diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or megakaryoblastic) leukemia. These leukemias may be referred together as acute myeloid (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polcythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT); as well as myelofibrosis (MFS) with or without angiogenic myeloid metaplasia.
Hematopoietic cancers also include lymphoid malignancies, which may affect the lymph nodes, spleens, bone marrow, peripheral blood, and/or extranodal sites. Lymphoid cancers include B-cell malignancies, which include, but are not limited to, B-cell non-Hodgkin’s lymphomas (B- NHLs). B-NHLs may be indolent (or low-grade), intermediate-grade (or aggressive) or high-grade (very aggressive). Indolent B-cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated-lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHLs include mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHLs include Burkitt’s lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV associated (or AIDS related) lymphomas, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom’s macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and Castleman’s disease. NHL may also include T-cell non-Hodgkin’s lymphoma s(T-NHLs), which include, but are not limited to T- cell non-Hodgkin’s lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma/delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides, and Sezary syndrome.
Hematopoietic cancers also include Hodgkin’s lymphoma (or disease) including classical Hodgkin’s lymphoma, nodular sclerosing Hodgkin’s lymphoma, mixed cellularity Hodgkin’s lymphoma, lymphocyte predominant (LP) Hodgkin’s lymphoma, nodular LP Hodgkin’s lymphoma, and lymphocyte depleted Hodgkin’s lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM) including smoldering MM, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom’s Macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers may also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes and natural killer cells. Tissues which include hematopoietic cells referred herein to as "hematopoietic cell tissues" include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa (such as the gut-associated lymphoid tissues), tonsils, Peyer's patches and appendix, and lymphoid tissues associated with other mucosa, for example, the bronchial linings.
As used herein the term “Compound A2” means a Type I PRMT inhibitor. In some embodiments, Compound A2 is a compound of Formula I, II, V, or VI. Suitably Compound A2 is Compound A.
As used herein the term “Compound B2” means a MAT2A inhibitor. In some embodiments, Compound B2 is a compound of Formula III, IV or VII. Suitably Compound B2 is Compound 262.
Suitably, the combinations of this invention are administered within a “specified period”.
The term “specified period” and grammatical variations thereof, as used herein, means the interval of time between the administration of one of Compound A2 and Compound B2 and the other of Compound A2 and Compound B2. Unless otherwise defined, the specified period can include simultaneous administration. Unless otherwise defined, the specified period refers to administration of Compound A2 and Compound B2 during a single day.
Suitably, if the compounds are administered within a “specified period” and not administered simultaneously, they are both administered within about 24 hours of each other - in this case, the specified period will be about 24 hours; suitably they will both be administered within about 12 hours of each other - in this case, the specified period will be about 12 hours; suitably they will both be administered within about 11 hours of each other - in this case, the specified period will be about 11 hours; suitably they will both be administered within about 10 hours of each other - in this case, the specified period will be about 10 hours; suitably they will both be administered within about 9 hours of each other - in this case, the specified period will be about 9 hours; suitably they will both be administered within about 8 hours of each other - in this case, the specified period will be about 8 hours; suitably they will both be administered within about 7 hours of each other - in this case, the specified period will be about 7 hours; suitably they will both be administered within about 6 hours of each other - in this case, the specified period will be about 6 hours; suitably they will both be administered within about 5 hours of each other - in this case, the specified period will be about 5 hours; suitably they will both be administered within about 4 hours of each other - in this case, the specified period will be about 4 hours; suitably they will both be administered within about 3 hours of each other - in this case, the specified period will be about 3 hours; suitably they will be administered within about 2 hours of each other - in this case, the specified period will be about 2 hours; suitably they will both be administered within about 1 hour of each other - in this case, the specified period will be about 1 hour. As used herein, the administration of Compound A2 and Compound B2 in less than about 45 minutes apart is considered simultaneous administration.
Suitably, when the combination of the invention is administered for a “specified period”, the compounds will be co-administered for a “duration of time”.
The term “duration of time” and grammatical variations thereof, as used herein means that both compounds of the invention are administered for an indicated number of consecutive days. Unless otherwise defined, the number of consecutive days does not have to commence with the start of treatment or terminate with the end of treatment, it is only required that the number of consecutive days occur at some point during the course of treatment.
Regarding “specified period” administration:
Suitably, both compounds will be administered within a specified period for at least one day - in this case, the duration of time will be at least one day; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 3 consecutive days - in this case, the duration of time will be at least 3 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 5 consecutive days - in this case, the duration of time will be at least 5 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 7 consecutive days - in this case, the duration of time will be at least 7 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 14 consecutive days - in this case, the duration of time will be at least 14 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 30 consecutive days - in this case, the duration of time will be at least 30 days.
Suitably, if the compounds are not administered during a “specified period”, they are administered sequentially. By the term “sequential administration”, and grammatical derivates thereof, as used herein is meant that one of Compound A2 and Compound B2 is administered once a day for two or more consecutive days and the other of Compound A2 and Compound B2 is subsequently administered once a day for two or more consecutive days. Also, contemplated herein is a drug holiday utilized between the sequential administration of one of Compound A2 and Compound B2 and the other of Compound A2 and Compound B2. As used herein, a drug holiday is a period of days after the sequential administration of one of Compound A2 and Compound B2 and before the administration of the other of Compound A2 and Compound B2 where neither Compound A2 nor Compound B2 is administered. Suitably the drug holiday will be a period of days selected from: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,
11 days, 12 days, 13 days and 14 days.
Regarding sequential administration:
Suitably, one of Compound A2 and Compound B2 is administered for from 1 to 30 consecutive days, followed by an optional drug holiday, followed by administration of the other of Compound A2 and Compound B2 for from 1 to 30 consecutive days. Suitably, one of Compound A2 and Compound B2 is administered for from 1 to 21 consecutive days, followed by an optional drug holiday, followed by administration of the other of Compound A2 and Compound B2 for from 1 to 21 consecutive days. Suitably, one of Compound A2 and Compound B2 is administered for from 1 to 14 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of the other of Compound A2 and Compound B2 for from 1 to 14 consecutive days. Suitably, one of Compound A2 and Compound B2 is administered for from 1 to 7 consecutive days, followed by a drug holiday of from 1 to 10 days, followed by administration of the other of Compound A2 and Compound B2 for from 1 to 7 consecutive days.
Suitably, Compound B2 will be administered first in the sequence, followed by an optional drug holiday, followed by administration of Compound A2. Suitably, Compound B2 is administered for from 3 to 21 consecutive days, followed by an optional drug holiday, followed by administration of Compound A2 for from 3 to 21 consecutive days. Suitably, Compound B2 is administered for from 3 to 21 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of Compound A2 for from 3 to 21 consecutive days. Suitably, Compound B2 is administered for from 3 to 21 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of Compound A2 for from 3 to 21 consecutive days. Suitably, Compound B2 is administered for 21 consecutive days, followed by an optional drug holiday, followed by administration of Compound A2 for 14 consecutive days. Suitably, Compound B2 is administered for 14 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of Compound A2 for 14 consecutive days. Suitably, Compound B2 is administered for 7 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of Compound A2 for 7 consecutive days. Suitably, Compound B2 is administered for 3 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of Compound A2 for 7 consecutive days. Suitably, Compound B2 is administered for 3 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of Compound A2 for 3 consecutive days.
It is understood that a “specified period” administration and a “sequential” administration can be followed by repeat dosing or can be followed by an alternate dosing protocol, and a drug holiday may precede the repeat dosing or alternate dosing protocol.
The methods of the present invention may also be employed with other therapeutic methods of cancer treatment.
Compound A2 and Compound B2 may be administered by any appropriate route. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), intratumorally, vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural). It will be appreciated that the preferred route may vary with, for example, the condition of the recipient of the combination and the cancer to be treated. It will also be appreciated that each of the agents administered may be administered by the same or different routes and that Compound A2 and Compound B2 may be compounded together in a pharmaceutical composition/formulation.
In one embodiment, one or more components of a combination of the invention are administered intravenously. In one embodiment, one or more components of a combination of the invention are administered orally. In another embodiment, one or more components of a combination of the invention are administered intratumorally. In another embodiment, one or more components of a combination of the invention are administered systemically, e.g., intravenously, and one or more other components of a combination of the invention are administered intratumorally. In any of the embodiments, e.g., in this paragraph, the components of the invention are administered as one or more pharmaceutical compositions.
Typically, any anti-neoplastic agent that has activity versus a susceptible tumor being treated may be co-administered in the treatment of cancer in the present invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita, T.S. Lawrence, and S.A. Rosenberg (editors), 10th edition (December 5, 2014), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Typical anti-neoplastic agents useful in the present invention include, but are not limited to, anti-microtubule or anti-mitotic agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such as actinomycins, anthracyclins, and bleomycins; topoisomerase I inhibitors such as camptothecins; topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogues and anti-folate compounds; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; cell cycle signaling inhibitors; proteasome inhibitors; heat shock protein inhibitors; inhibitors of cancer metabolism; and cancer gene therapy agents such as genetically modified T cells.
Examples of a further active ingredient or ingredients for use in combination or co-administered with the present methods or combinations are anti -neoplastic agents. Examples of anti -neoplastic agents include, but are not limited to, chemotherapeutic agents; immuno-modulatory agents; immune-modulators; and immunostimulatory adjuvants.
EXAMPLES
The following examples illustrate various non-limiting aspects of this invention.
Example 1 : Type I PRMT inhibitor and MAT2A inhibitor Combinations
Materials and Methods
A 6-day proliferation screening was performed in 5 pancreatic tumor cell lines: 2 MTAP wild-type, 2 MTAP null, 1 MTAP wt with CRISP knock-out of MTAP. Combinations included double titration of Type 1 PRMT inhibitor Compound D (FIG. 2) with a MAT2A inhibitor Compound 262 (FIG.2).
Optimal cell seeding for all cell lines was determined by assessing the growth over a range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 6 days. Cells were then plated at the optimal seeding density 24 hours before treatment (in duplicate) and treated the following day with a 16X16 double titration matrix (two-fold dilution series) of Compound D (Type I PRMT inhibitor) and Compound 262 (MAT2A inhibitor). This double titration was compared to a 16-point two-fold dilution series of each single agent alone or 0.15% DMSO. An untreated plate of cells was harvested at the time of compound addition (T0) to quantify the starting number of cells.
Concentrations tested for Compound D and Compound 262 alone or in combination ranged from 0.3 to 10,000nM. Plates were incubated for 6 days at 37 °C in 5% CO2. Cells were then lysed with CellTiter-Glo (CTG) (Promega) according to the manufacturer’s protocol and chemiluminescent signal detected on a Cytomat equipped with the Synergy Neo plate reader (ThermoFisher, serial # 140715A). CTG estimates cell number through detection of cellular ATP levels. CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T0 value, and plotted against compound concentration. Data were fit with a four- parameter equation to generate a concentration response curves.
Results
FIGS. 3-22 show that the combination of the Type I PRMT inhibitor (Compound D) with the MAT2A inhibitor (Compound 262) resulted in increased growth inhibition over either single agent alone. Synergistic effects were found in all cell lines tested, though the greatest potency shifts were observed in the MTAP -deficient cell lines. This is most readily observed in the Pane 03.27 wild type line and the matched MTAP knock-out cell line.
Example 2: Type I PRMT inhibitor and MAT2A inhibitor Combinations Materials and Methods General Method
A 6-day proliferation screening was performed in 4 pancreatic tumor cell lines: 2 MTAP wild type vs 2 MTAP null. Combinations included: 1:1 fixed ratio of Type 1 PRMT inhibitor Compound A-di-HCl (FIG. 2) + MAT2A inhibitor Compound 262 (FIG. 2); titration of the Type 1 PRMT inhibitor Compound A-di-HCl with several fixed concentrations of MAT2A inhibitor Compound 262 (1000 nM, 300 nM, 100 nM); titration of MAT2A inhibitor Compound 262 with several fixed concentrations of the Type I PRMT inhibitor Compound A-di-HCl (1000 nM, 300 nM, 100 nM).
Fixed Ratio Combination
Optimal cell seeding for all cell lines was determined by assessing the growth over a range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 6 days. Cells were then plated at the optimal seeding density 24 hours before treatment (in duplicate) and treated the following day with a 20-point two-fold dilution series of Compound A- di-HCl, Compound 262, an equimolar ratio of Compound A-di-HCl: Compound 262, or 0.15% DMSO. An untreated plate of cells was harvested at the time of compound addition (T0) to quantify the starting number of cells. Concentrations tested for Compound A-di-HCl and Compound 262 alone and in combination ranged from 0.00005 to 29.4 mM. Plates were incubated for 6 days at 37°C in 5% CO2. Cells were then lysed with CellTiter-Glo (CTG) (Promega) according to the manufacturer’s protocol and chemiluminescent signal detected on a Cytomat equipped with the Synergy Neo plate reader (ThermoFisher, serial # 140715A). CTG estimates cell number through detection of cellular ATP levels. CTG values obtained after the 6-day treatment were background subtracted, expressed as a percent of the T0 value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate a concentration response curve.
Fixed Concentration Combinations Optimal cell seeding was determined for all cell lines by assessing the growth over a range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 6 days. Cells were then plated at the optimal seeding density 24 hours before treatment (in duplicate) and treated the following day with 0.15% DMSO or a 20-point two-fold dilution series of Compound A-di-HCl alone, or with 100, 300, or 1000nM of Compound 262. Additional treatment conditions included a 20-point two-fold dilution series of Compound 262 alone, or with 100, 300, or 1000nM of Compound A-di-HCl. Concentrations tested for each single agent ranged from 0.00005 to 29.4 mM. An untreated plate of cells was harvested at the time of compound addition (TO) to quantify the starting number of cells. Plates were incubated for 6 days at 37°C in 5% CO2. Cells were then lysed with CellTiter-Glo (CTG) (Promega) according to the manufacturer’s protocol and chemiluminescent signal detected on a Cytomat equipped with the Synergy Neo plate reader (ThermoFisher, serial # 140715A). CTG estimates cell number through detection of cellular ATP levels. CTG values obtained after the 6 day treatment were background subtracted, expressed as a percent of the TO value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate a concentration response curve.
Results
FIGS. 23-31 show that combining the Type 1 PRMT inhibitor Compound A-di-HCl with the MAT2A inhibitor Compound 262 resulted in increased growth inhibition over either single agent alone. The enhanced effects of the combination were observed in all formats, including: holding Compound A-di-HCl fixed and titrating Compound 262, holding Compound 262 fixed and titrating Compound A and with a 1: 1 fixed ratio of Compound A-di-HCl and Compound 262. A combination effect was observed in both the MTAP -wild-type and MTAP -deficient cell lines.
Example 3 : Type I PRMT inhibition
Arginine Methylation and PRMTs
Arginine methylation is an important post-translational modification on proteins involved in a diverse range of cellular processes such as gene regulation, RNA processing, DNA damage response, and signal transduction. Proteins containing methylated arginines are present in both nuclear and cytosolic fractions suggesting that the enzymes that catalyze the transfer of methyl groups on to arginines are also present throughout these subcellular compartments (reviewed in Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013); Lee, Y. H. & Stallcup, M. R. Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation. Mol Endocrinol 23, 425-433, doi:10.1210/me.2008-0380 (2009)). In mammalian cells, methylated arginine exists in three major forms: w -NG -monomethyl -arginine (MMA), w -NG, N -asymmetric dimethyl arginine (ADMA), or w -NG, N -symmetric dimethyl arginine (SDMA). Each methylation state can affect protein- protein interactions in different ways and therefore has the potential to confer distinct functional consequences for the biological activity of the substrate (Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013)).
Arginine methylation occurs largely in the context of glycine-, arginine-rich (GAR) motifs through the activity of a family of Protein Arginine Methyltransferases (PRMTs) that transfer the methyl group from S-adenosyl-L-methionine (SAM) to the substrate arginine side chain producing S-adenosyl-homocysteine (SAH) and methylated arginine (FIG. 32). This family of proteins is comprised of 10 members of which 9 have been shown to have enzymatic activity (Bedford, M. T. & Clarke, S. G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 33, 1-13, doi:10.1016/j.molcel.2008.12.013 (2009)). The PRMT family is categorized into four sub-types (Type I-IV) depending on the product of the enzymatic reaction (FIG. 32). Type IV enzymes methylate the internal guanidino nitrogen and have only been described in yeast (Fisk, J. C. & Read, F. K. Protein arginine methylation in parasitic protozoa. Eukaryot Cell 10, 1013-1022, doi:10.1128/EC.05103-11 (2011)); types T-TTT enzymes generate monomethyl-arginine (MMA, Rmel) through a single methylation event. The MMA intermediate is considered a relatively low abundance intermediate, however, select substrates of the primarily Type III activity of PRMT7 can remain monomethylated, while Types I and II enzymes catalyze progression from MMA to either asymmetric dimethyl-arginine (ADMA, Rme2a) or symmetric dimethyl arginine (SDMA, Rme2s) respectively. Type II PRMTs include PRMT5, and PRMT9, however, PRMT5 is the primary enzyme responsible for formation of symmetric dimethylation. Type I enzymes include PRMT1, PRMT3, PRMT4, PRMT6 and PRMT8. PRMT1, PRMT3, PRMT4, and PRMT6 are ubiquitously expressed while PRMT8 is largely restricted to the brain (reviewed in Bedford, M. T. & Clarke, S. G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 33, 1-13, doi:10.1016/j.molcel.2008.12.013 (2009)). PRMT1 is the primary Type 1 enzyme capable of catalyzing the formation of MMA and ADMA on numerous cellular substrates (Bedford, M. T. & Clarke, S. G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 33, 1-13, doi:10.1016/j.molcel.2008.12.013 (2009)). In many instances, the PRMT1 -dependent ADMA modification is required for the biological activity and trafficking of its substrates (Nicholson, T. B., Chen, T. & Richard, S. The physiological and pathophysiological role of PRMT1 -mediated protein arginine methylation. Pharmacol Res 60, 466-474, doi: 10.1016/j.phrs.2009.07.006 (2009)), and the activity of PRMT1 accounts for -85% of cellular ADMA levels (Dhar, S. et al. Foss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Sci Rep 3, 1311, doi:10.1038/srep01311 (2013); Pawlak, M. R., Scherer, C. A., Chen, J., Roshon, M. J. & Ruley, H. E. Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable. Mol Cell Biol 20, 4859-4869 (2000)). Complete knockout of PRMT1 results in a profound increase in MMA across numerous substrates suggesting that the major biological function for PRMT1 is to convert MMA to ADMA while other PRMTs can establish and maintain MMA (Dhar, S. et al. Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Sci Rep 3, 1311, doi:10.1038/srep01311 (2013)). In addition, SDMA levels are increased upon loss of PRMT1, likely a consequence of the loss of ADMA and the corresponding increase of MMA that can serve as the substrate for SDMA-generating Type II PRMTs. Inhibition of Type I PRMTs may lead to altered substrate function through loss of ADMA, increase in MMA, or, alternatively, a switch to the distinct methylation pattern associated with SDMA (Dhar, S. et al Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Sci Rep 3, 1311, doi : 10.1038/srep01311 (2013)).
Disruption of the Prmtl locus in mice results in early embryonic lethality and homozygous embryos fail to develop beyond E6.5 indicating a requirement for PRMT1 in normal development (Pawlak, M. R., Scherer, C. A., Chen, J., Roshon, M. J. & Ruley, H. E. Arginine N- methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable. Mol Cell Biol 20, 4859-4869 (2000); Yu, Z., Chen, T., Hebert, J., Li, E.
& Richard, S. A mouse PRMT1 null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation. Mol Cell Biol 29, 2982-2996, doi: 10.1128/MCB.00042-09 (2009)). Conditional or tissue specific knockout will be required to better understand the role for PRMT1 in the adult. Mouse embryonic fibroblasts derived from Prmtl null mice undergo growth arrest, polyploidy, chromosomal instability, and spontaneous DNA damage in association with hypomethylation of the DNA damage response protein MREl 1, suggesting a role for PRMT1 in genome maintenance and cell proliferation (Y u, Z., Chen, T., Hebert, J., Li, E. & Richard, S. A mouse PRMT1 null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation. Mol Cell Biol 29, 2982-2996, doi: 10.1128/MCB.00042-09 (2009)). PRMT1 protein and mRNA can be detected in a wide range of embryonic and adult tissues, consistent with its function as the enzyme responsible for the majority of cellular arginine methylation. Although PRMTs can undergo post-translational modifications themselves and are associated with interacting regulatory proteins, PRMT1 retains basal activity without a requirement for additional modification (reviewed in Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi:10.1038/nrc3409 (2013)).
PRMT1 and Cancer Mis-regulation and overexpression of PRMT1 has been associated with a number of solid and hematopoietic cancers (Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013); Yoshimatsu, M. et al. Dysregulation of PRMT1 and PRMT6, Type I arginine methyltransferases, is involved in various types ofhuman cancers. Int J Cancer 128, 562-573, doi:10.1002/ijc.25366 (2011)). The link between PRMT1 and cancer biology has largely been through regulation of methylation of arginine residues found on relevant substrates (FIG. 33). In several tumor types, PRMT1 can drive expression of aberrant oncogenic programs through methylation of histone H4 (Takai, H. el al. 5- Hydroxymethylcytosine plays a critical role in glioblastomagenesis by recruiting the CHTOP- methylosome complex. Cell Rep 9, 48-60, doi: 10.1016/j.celrep.2014.08.071 (2014); Shia, W. J. et al. PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood 119, 4953-4962, doi: 10.1182/blood-2011-04-347476 (2012); Zhao,
X. et al. Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity. Genes Dev 22, 640-653, doi:10.1101/gad.1632608 (2008)), as well as through its activity on non-histone substrates (Wei, H., Mundade, R., Lange, K. C. & Lu, T.
Protein arginine methylation of non-histone proteins and its role in diseases. Cell Cycle 13, 32-41, doi: 10.4161/cc.27353 (2014)). In many of these experimental systems, disruption of the PRMT1- dependent ADMA modification of its substrates decreases the proliferative capacity of cancer cells (Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat Rev Cancer 13, 37-50, doi: 10.1038/nrc3409 (2013)).
Several studies have linked PRMT1 to the development of hematological and solid tumors. PRMT1 is associated with leukemia development through methylation of key drivers such as MLL and AML1-ETO fusions, leading to activation of oncogenic pathways (Shia, W. J. et al). PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood 119, 4953-4962, doi: 10.1182/blood-2011-04-347476 (2012);
Cheung, N. et al. Targeting Aberrant Epigenetic Networks Mediated by PRMT1 and KDM4C in Acute Myeloid Leukemia. Cancer Cell 29, 32-48, doi:10.1016/j.ccell.2015.12.007 (2016)). Knockdown of PRMT1 in bone marrow cells derived from AML1-ETO expressing mice suppressed clonogenicity, demonstrating a critical requirement for PRMT1 in maintaining the leukemic phenotype of this model (Shia, W. J. et al. PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood 119, 4953- 4962, doi: 10.1182/blood-2011-04-347476 (2012)). PRMT1 is also a component of MLL fusion complexes, promotes aberrant transcriptional activation in association with H4R3 methylation, and knockdown of PRMT1 can suppress MLL-EEN mediated transformation of hematopoietic stem cells (Cheung, N., Chan, L. C., Thompson, A., Cleary, M. L. & So, C. W. Protein arginine- methyltransferase-dependent oncogenesis. Nat Cell Biol 9, 1208-1215, doi: 10.1038/ncbl642 (2007)). In breast cancer patients, high expression of PRMT1 was found to correlate with shorter disease free survival and with tumors of advanced histological grade (Mathioudaki, K. et al. Clinical evaluation of PRMT1 gene expression in breast cancer. Tumour Biol 32, 575-582, doi:10.1007/sl3277-010-0153-2 (2011)). To this end, PRMT1 has been implicated in the promotion of metastasis and cancer cell invasion (Gao, Y. et al. The dual function of PRMT1 in modulating epithelial-mesenchymal transition and cellular senescence in breast cancer cells through regulation of ZEB1. Sci Rep 6, 19874, doi:10.1038/srepl9874 (2016); Avasarala, S. et al. PRMT1 Is a Novel Regulator of Epithelial-Mesenchymal-Transition in Non-small Cell Lung Cancer. JBiol Chem 290, 13479-13489, doi: 10.1074/jbc.Ml 14.636050 (2015)) and PRMT1 mediated methylation of Estrogen Receptor a (ERa) can potentiate growth-promoting signal transduction pathways. This methylation driven mechanism may provide a growth advantage to breast cancer cells even in the presence of anti-estrogens (Le Romancer, M. et al. Regulation of estrogen rapid signaling through arginine methylation by PRMT1. Mol Cell 31, 212-221, doi:10.1016/j.molcel.2008.05.025 (2008)). In addition, PRMT1 promotes genome stability and resistance to DNA damaging agents through regulating both homologous recombination and non- homologous end-joining DNA repair pathways (Boisvert, F. M., Rhie, A., Richard, S. & Doherty, A. J. The GAR motif of 53BP1 is arginine methylated by PRMT1 and is necessary for 53BP1 DNA binding activity. Cell Cycle 4, 1834-1841, doi: 10.4161/cc.4.12.2250 (2005); Boisvert, F. M., Dery, U., Masson, J. Y. & Richard, S. Arginine methylation of MREl 1 by PRMT1 is required for DNA damage checkpoint control. Genes Dev 19, 671-676, doi: 10.1101/gad.1279805 (2005)). Therefore, inhibition of PRMT1 may sensitize cancers to DNA damaging agents, particularly in tumors where DNA repair machinery may be compromised by mutations (such as BRCA1 in breast cancers) (O'Donovan, P. J. & Livingston, D. M. BRCA1 and BRCA2: breast/ovarian cancer susceptibility gene products and participants in DNA double-strand break repair. Carcinogenesis 31, 961-967, doi: 10.1093/carcin/bgq069 (2010)). Together, these observations demonstrate key roles for PRMT1 in clinically-relevant aspects of tumor biology, and suggest a rationale for exploring combinations with therapies such as those that promote DNA damage.
RNA binding proteins and splicing machinery are a major class of PRMT1 substrates and have been implicated in cancer biology through their biological function as well as recurrent mutations in leukemias (Bressan, G. C. et al. Arginine methylation analysis of the splicing- associated SR protein SFRS9/SRP30C. Cell Mol Biol Lett 14, 657-669, doi: 10.2478/s 11658-009- 0024-2 (2009); Sveen, A., Kilpinen, S., Ruusulehto, A., Lothe, R. A. & Skotheim, R. I. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes. Oncogene 35, 2413-2427, doi:10.1038/onc.2015.318 (2016); Hsu, T. Y. et al. The spliceosome is a therapeutic vulnerability in MYC-driven cancer. Nature 525, 384-388, doi: 10.1038/naturel4985 (2015)). In a recent study, PRMT1 was shown to methylate the RNA binding protein, RBM15, in acute megakaryocytic leukemia (Zhang, L. etal. Cross-talk between PRMT1 -mediated methylation and ubiquitylation on RBM15 controls RNA splicing. Elife 4, doi: 10.7554/eLife.07938 (2015)). PRMT1 mediated methylation of RBM15 regulates its expression; consequently, overexpression of PRMT1 in AML cell lines was shown to block differentiation by downregulation of RBM15, thereby preventing its ability to bind pre-mRNA intronic regions of genes important for differentiation. To identify putative PRMT1 substrates, a proteomic approach (Methylscan, Cell Signaling Technology) was utilized to identify proteins with changes in arginine methylation states in response to a tool PRMT1 inhibitor, Compound D. Protein fragments from Compound D- and DSMO-treated cell extracts were immunoprecipitated using methyl arginine specific antibodies (ADMA, MMA, SDMA), and peptides were identified by mass spectrometry. While many proteins undergo changes in arginine methylation, the majority of substrates identified were transcriptional regulators and RNA processing proteins in AML cell lines treated with the tool compound (FIG. 34).
In summary, the impact of PRMT1 on cancer relevant pathways suggests inhibition may lead to anti -tumor activity, providing a novel therapeutic mechanism for the treatment of AML, lymphoma, and solid tumor indications. As described in the emerging literature, several mechanisms support a rationale for the use of a PRMT1 inhibitor in hematological and solid tumors including: inhibition of AML-ETO driven oncogenesis in leukemia, inhibition of growth promoting signal transduction in breast cancer, and modulation of splicing through methylation of RNA binding proteins and spliceosome machinery. Inhibition of Type I PRMTs including PRMT1 represents a tractable strategy to suppress aberrant cancer cell proliferation and survival.
BIOCHEMISTRY
Detailed in vitro biochemical studies were conducted with Compound A to characterize the potency and mechanism of inhibition against Type I PRMTs.
Mechanism of Inhibition
The inhibitory mechanism of Compound A for PRMT1 was explored through substrate competition experiments. Inhibitor modality was examined by plotting Compound A IC50 values as a function of substrate concentration divided by its Km app and comparing the resulting plots to the Cheng-Prusoff relationship for competitive, non-competitive, and uncompetitive inhibition (Copeland, R. A. Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem Anal 46, 1-265 (2005)). Compound A IC50 values decreased with increasing SAM concentration indicating that inhibition of PRMT1 by Compound A was uncompetitive with respect to SAM with a Ki app value of 15 nM when fit to an equation for uncompetitive inhibition (FIG. 35A). No clear modality trend was observed when Compound A IC50 values were plotted as a function of H4 1-21 peptide (FIG. 35B) suggesting mixed type inhibition. Further analysis was performed using a global analysis resulting in an a value of 3.7 confirming the peptide mechanism as mixed and yielding a Ki app value of 19 nM (FIG. 35B, inset).
Time Dependence and Reversibility
Compound A was evaluated for time dependent inhibition by measuring IC50 values following varying SAM :PRMT1: Compound A preincubation time and a 20 minute reaction. An inhibitory mechanism that is uncompetitive with SAM implies that generation of the SAM:PRMT1 complex is required to support binding of Compound A, therefore SAM (held at Km app ) was included during the preincubation. Compound A demonstrated time dependent inhibition of PRMT1 methylation evident by an increase in potency with longer preincubation time (FIG. 36A). Since time dependent inhibition was observed, further IC50 determinations included a 60 minute SAM: PRMT1: Compound A preincubation and a 40 minute reaction time to provide a better representation of compound potency. These conditions yield an IC50 of 3.1 ± 0.4 nM (n=29) that is > 10-fold above the theoretical tight-binding limit (0.25 nM) of the assay. Examining IC50 values at varying PRMT1 concentrations revealed that the actual tight binding limit would be significantly lower than 0.25 nM potentially due to a low active fraction (FIG. 36B). The salt form of Compound A did not significantly affect the IC50 value determined against PRMT1 (FIG. 36B).
Two explanations for time dependent inhibition are slow-binding reversible inhibition and irreversible inhibition. To distinguish between these two mechanisms, affinity selection mass spectrometry (ASMS) was used to examine the binding of Compound A to PRMT1. ASMS first separates bound from unbound ligand, and then detects reversibly bound ligand by MS. A 2 hr preincubation of PRMT 1:SAM with Compound A was used to ensure that the time dependent complex (ESI*) was fully formed based on the profile shown in FIG. 36A) in which maximal potency was observed after 20 minutes of preincubation. Under these conditions, Compound A was detectable using ASMS. This suggests that the primary mechanism is reversible in nature, since ASMS would be unable to detect irreversibly bound Compound A. Definitive reversibility studies including off-rate analysis have not yet been performed and would further validate the mechanism.
Crystallography
To determine inhibitor binding mode, the co-crystal structure of Compound A bound to PRMT1 and SAH was determined (2.48 Å resolution) (FIG. 37). SAH is the product formed upon removal of the methyl group from SAM by PRMT1; therefore, SAH and SAM should similarly occupy the same pocket of PRMT1. The inhibitor binds in the cleft normally occupied by the substrate peptide directly adjacent to the SAH pocket and its diamine sidechain occupies the putative arginine substrate site. The terminal methylamine forms a hydrogen bond with the Glul62 sidechain residue that is 3.6 A from the thioether of SAH and the SAH binding pocket is bridged to Compound A by Tyr57 and Met66. Compound A binds PRMT1 through the formation of a hydrogen bond between the proton of the pyrazole nitrogen of Compound A and the acidic sidechain of Glu65; the diethoxy branched cyclohexyl moiety lies along the solvent exposed surface in a hydrophobic groove formed by Tyr57, Ile62, Tyrl66 and Tyrl70. The spatial separation between SAH and inhibitor binding, as well as interactions with residues such as Tyr57 could support the SAM uncompetitive mechanism revealed in the enzymatic studies. The finding that Compound A is bound in the substrate peptide pocket and that the diamine sidechain may mimic the amines of the substrate arginine residue implies that inhibitor modality may be competitive with peptide. Biochemical mode of inhibition studies support that Compound A is a mixed inhibitor with respect to peptide (FIG. 36B). The time-dependent behavior of Compound A as well as the potential for exosite binding of the substrate peptide outside of the peptide cleft could both result in a mode of inhibition that is not competitive with peptide, explaining the difference in modality suggested by the structural and biochemical studies.
Orthologs
To facilitate interpretation of toxicology studies, the potency of Compound A was evaluated against the rat and dog orthologs of PRMT1. As with human PRMT1, Compound A revealed time dependent inhibition against rat and dog PRMT1 with IC50 values decreasing with increasing preincubation (FIG. 38A). Additionally, no shift in Compound A potency was observed across a range of enzyme concentrations (0.25- 32 nM) suggesting the IC50 values measured did not approach the tight-binding limit of the assay for human, rat or dog (FIG. 38B). IC50 values were determined using conditions equivalent to those used to assess human PRMT1 and revealed that Compound A potency varied < 2-fold across all species (FIG. 38C).
Selectivity
The selectivity of Compound A was assessed across a panel of PRMT family members. IC50 values were determined against representative Types I (PRMT3, PRMT4, PRMT6 and PRMT8) and II (PRMT5/MEP50 and PRMT9) family members following a 60 minute SAM: Enzyme: Compound A preincubation. Compound A inhibited the activity of all Type I PRMTs tested with varying potencies, but failed to inhibit Type II family members (FIG. 39A). Additional characterization of the Type I PRMTs revealed that Compound A was a time dependent inhibitor of PRMT4, PRMT6 and PRMT8 due to the increase in potency observed following increasing Enzyme: SAM: Compound A preincubation times; whereas, PRMT3 displayed no time dependent behavior (FIG. 39B).
To further characterize selectivity of Compound A, the inhibition of twenty-one methyltransferases was evaluated at a single concentration of Compound A (10 mM, Reaction Biology). The highest degree of inhibition, 18%, was observed against PRDM9. Overall, Compound A showed minimal inhibition of the methyltransferases tested suggesting it is a selective inhibitor of Type I PRMTs (Table 1). Additional selectivity assays are described in the Safety sections.
Table 1 Methyltransferases tested for inhibition by Compound A. Enzymes were assayed at a fixed concentration of SAM (1 mM) independent of the SAM Km value.
Figure imgf000092_0001
In summary, Compound A is a potent, reversible, selective inhibitor of Type I PRMT family members showing equivalent biochemical potency against PRMT1, PRMT6 and PRMT8 with IC50 values ranging between 3-5 nM. The crystal structure of PRMT1 in complex with Compound A reveals that Compound A binds in the peptide pocket and both the crystal structure, as well as enzymatic studies are consistent with a SAM uncompetitive mechanism. BIOLOGY
Cellular Mechanistic Effects
Inhibition of PRMT1 is predicted to result in a decrease of ADMA on cellular PRMT1 substrates, including arginine 3 of histone H4 (H4R3me2a), with concomitant increases in MMA and SDMA (Dhar, S. etal. Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Sci Rep 3, 1311, doi: 10.1038/srep01311 (2013)). To evaluate the effect of Compound A on arginine methylation the dose response associated with increased MMA was evaluated in an in-cell-westem assay using an antibody to detect MMA and the cellular mechanistic EC50 of 10.1 ± 4.4 nM was determined (FIG. 40). The dose response appeared biphasic, possibly due to differential activity between the Type I PRMTs or differential potency towards a particular subset of substrates. An equation describing a biphasic curve was used to fit the data and since there was no obvious plateau associated with the second inflection over the range of concentrations tested, the first inflection was reported. Various salt forms were tested in this assay format and all demonstrated similar EC50 values and are, therefore, considered interchangeable for all biology studies (FIG. 40). Additional studies were performed to examine the timing, durability, and impact on other methylation states in select tumor types as indicated below. The potency of Compound A on induction of MMA indicates that Compound A can be used to investigate the biological mechanism associated with inhibition of Type 1 PRMTs in cells.
Type I PRMT Expression in Cancer
Analysis of gene expression data from multiple tumor types collected from > 100 cancer studies through The Cancer Genome Atlas (TCGA) and other primary tumor databases represented in cBioPortal indicates that PRMT1 is highly expressed g in cancer, with highest levels in lymphoma (diffuse large B-cell lymphoma, DLBCL) relative to other solid and hematological malignancies (FIG. 41). Expression of ACTB, a common housekeeping gene and TYR, a gene selectively expressed in skin, were also surveyed to characterize the range associated with high ubiquitous expression or tissue restricted expression, respectively. High expression in lymphoma among other cancers provides additional confidence that the target of Compound A inhibition is present in primary tumors that correspond to cell lines evaluated in preclinical studies. PRMTs 3, 4, and 6 are also expressed across a range of tumor types while PRMT8 expression appears more restricted as predicted given its tissue specific expression (Fee, T, Sayegh, T, Daniel, T, Clarke, S. & Bedford, M. T. PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family. J Biol Chem 280, 32890-32896, doi:10.1074/jbc.M506944200 (2005)).
Cellular Phenotypic Effects Compound A was analyzed for its ability to inhibit cultured tumor cell line growth in a 6- day growth-death assay using Cell Titer Glo (Promega) that quantifies ATP as a surrogate of cell number. The growth of all cell lines was evaluated over time across a wide range of seeding densities to identify conditions that permitted proliferation throughout the entire 6-day assay.
Cells were plated at the optimal seeding density and after overnight incubation, a 20-point 2-fold titration of compound was added and plates were incubated for 6 days. A replicate plate of cells was harvested at the time of compound addition to quantify the starting number of cells (T0). Values obtained after the 6 day treatment were expressed as a function of the T0 value and plotted against compound concentration. The T0 value was normalized to 100% and represents the number of cells at the time of compound addition. The data were fit with a 4 parameter equation to generate a concentration response curve and the growth IC50 (glC50) was determined. The glC50 is the midpoint of the ‘growth window’, the difference between the number of cells at the time of compound addition (T0) and the number of cells after 6 days (DMSO control). The growth-death assay can be used to quantify the net population change, clearly defining cell death (cytotoxicity) as fewer cells compared to the number at the time of compound addition (T0). A negative Ymin-T0 value is indicative of cell death while a glC100 value represents the concentration of compound required for 100% inhibition of growth. The growth inhibitory effect of Compound A was evaluated using this assay in 196 human cancer cell lines representing solid and hematological malignancies (FIG. 42).
Compound A induced near or complete growth inhibition in most cell lines, with a subset showing cytotoxic responses, as indicated by a negative Ymin-T0 value (FIG. 42B). This effect was most pronounced in AML and lymphoma cancer cell lines, where 50 and 54% of cell lines showed cytotoxic responses, respectively. The total AUC or exposure (Cave) calculated from the rat 14-day MTD (150 mg/kg, Cave=2.1 mM) was used as an estimate of a clinically relevant concentration of Compound A for evaluation of sensitivity. While lymphoma cell lines showed cytotoxicity with glC100 values below 2.1 mM, many cell lines across all tumor types evaluated showed glC50 values £2.1 mM suggesting that concentrations associated with anti -tumor activity may be achievable in patients. The dog 21-day MTD was slightly higher (25 mg/kg; total AUC or Cave= 3.2 mM), therefore the lower concentration from the rat provides a more conservative target for appreciating cell line sensitivity. Lymphoma cell lines were highly sensitive to Type I PRMT inhibition, with a median glC50 of 0.57 mM and cytotoxicity observed in 54%. Among solid tumor types, potent anti-proliferative activity of Compound A was observed in melanoma and kidney cancer cell lines (primarily representing clear cell renal carcinoma), however, the responses were predominantly cytostatic in this assay format (FIG. 42, Table 2). Table 1 Compound A 6-day proliferation summary. glC50 £2.1 mM was used as target based on concentration achieved in the rat 14-day MTD (150 mg/kg, Cave=2.1 mM).
Figure imgf000095_0001
Evaluation of the anti-proliferative effects of Compound A indicates that inhibition of PRMT1 results in potent anti-tumor activity across cell lines representing a range of solid and hematological malignancies. Together, these data suggest that clinical development in solid and hematological malignancies is warranted. Prioritized indications include:
• Lymphoma: cytotoxicity in 54% of cell lines
• AML: cytotoxicity in 50% of cell lines · Renal cell carcinoma: glC50 £ 2.1 mM in 60% of cell lines
• Melanoma: g IC50 £ 2.1 mM in 71% of cell lines
• Breast cancer including TNBC: glC50 £ 2.1 mM in 41% of cell lines
Lymphoma Biology Cell Mechanistic Effects
To evaluate the effect of Compound A on arginine methylation in lymphoma, a human DLBCL cell line (Toledo) was treated with 0.4 mM Compound A or vehicle for up to 120 hours after which protein lysates were evaluated by western analysis using antibodies for various arginine methylation states. As predicted, ADMA methylation decreased while MMA increased upon compound exposure (PIG. 43). An increase in levels of SDMA was also observed, suggesting that the increase in MMA may have resulted in accumulation in the pool of potential substrates for PRMT5, the major catalyst of SDMA formation. Given the detection of numerous substrates with varying kinetics, and variability of ADMA levels among DMSO-treated samples, both the full lane and a prominent 45kDa band were characterized to assess ADMA. Increases in MMA were apparent by 24 hours and near maximal by 48 hours while decreases in the 45 kDa ADMA band required 72-96 hours to achieve maximal effect. Increases in SDMA were apparent after 48 hours of compound exposure and continued to increase through 120 hours, consistent with the potential switch from conversion of MMA to ADMA by Type I PRMTs to SDMA by Type II PRMTs (FIG. 43).
The dose response associated with Compound A effects on arginine methylation (MMA, ADMA, SDMA) was determined in a panel of lymphoma cell lines (FIG. 44). ADMA decreases were measured across the full lane and the single 45 kDa band that decreased to undetectable levels across all cell lines evaluated. Overall, concentrations required to achieve 50% of the maximal effect were similar across cell lines and did not correspond to the glC50 in the 6-day growth death assay, suggesting that the lack of sensitivity is not explained by poor target engagement.
To determine the durability of global changes in arginine methylation in response to Compound A, ADMA, SDMA, and MMA levels were assessed in cells treated with Compound A after compound washout (FIG. 45). Toledo cells were cultured with 0.4 mM Compound A for 72 hours to establish robust effects on arginine methylation marks. Cells were then washed, cultured in Compound A-free media, samples were collected daily through 120 hours, and arginine methylation levels were examined by western analysis. MMA levels rapidly decreased, returning to baseline by 24 hours after Compound A washout, while ADMA and SDMA returned to baseline by 24 and 96 hours, respectively. Notably, recovery of the 45kDa ADMA band appeared delayed relative to most other species in the ADMA western blots, suggesting the durability of arginine methylation changes by Compound A may vary by substrate. SDMA appeared to continue to increase even after 6 hours of washout. This is consistent with the continued increase observed through 120 hours without any obvious plateau (FIG. 43) coupled with the durable increase in MMA that has not yet returned to baseline after washout. Durability of each modification generally reflected the kinetics of arginine methylation changes brought about by Compound A, with MMA being the most rapid.
Cell Phenotypic Effects
To assess the time course associated with inhibition of growth by Compound A, an extended duration growth-death assay was performed in a subset of lymphoma cell lines. Similar to the 6-day proliferation assay described previously, the seeding density was optimized to ensure growth throughout the duration of the assay, and cell number was assessed by CTG at selected timepoints beginning from days 3-10. Growth inhibition was observed as early as 6 days and was maximal by 8 days in Toledo and Daudi lymphoma cell lines (FIG. 46). A larger set of cell lines was evaluated on days 6 and 10 to measure the effects of prolonged exposure to Compound A and determine whether cell lines that displayed a cytostatic response in the 6-day assay might undergo cytotoxicity at later timepoints. The extended time of exposure to Compound A had minimal effects on potency (glC50) or cytotoxicity ( Ymin-T0) across lymphoma cell lines evaluated (FIG. 47) indicating that 6-day proliferation evaluation could be utilized for assessment of sensitivity.
Given that growth inhibition was apparent at day 6 and prolonged exposure had minimal impact on potency or percent inhibition, a broad panel of lymphoma cell lines representing Hodgkin’s and non-Hodgkin’s subtypes was evaluated in the 6-day growth-death assay format (FIG. 48). All subtypes appeared equally sensitive in this format and many cell lines underwent cytotoxicity (as indicated by negative Ymin-T0) independent of classification, suggesting that Compound A has anti -tumor effects in all subtypes of lymphoma evaluated.
The proliferation assay results suggest that the inhibition of PRMT1 induces apparent cytotoxicity in a subset of lymphoma cell lines. To further elucidate this effect, the cell cycle distribution in lymphoma cell lines treated with Compound A was evaluated using propidium iodide staining followed by flow cytometry. Cell lines that showed a range of Ymin-T0 and glC50 values in the 6-day proliferation assay were seeded at low density to allow logarithmic growth over the duration of the assay, and treated with varying concentrations of Compound A.
Consistent with the growth-death assay results, an accumulation of cells in sub-G1 (<G1), indicative of cell death, was observed in Toledo cells in a time and dose dependent manner beginning after 3 days of treatment with Compound A concentrations ³ 1000 nM (FIG. 49). By day 7, an increase in the sub-G1 population was apparent at concentrations ³ 100 nM. In U2932 and OCI-Lyl, cell lines that underwent apparent cytostatic growth inhibition in the 6-day proliferation assay, this effect was only evident at 10 mM Compound A. No profound effect in any other cell cycle phase was revealed in this assay format.
To confirm the FACS analysis of cell cycle, evaluation of caspase cleavage was performed as an additional measure of apoptosis during a 10-day timecourse. Seeding density was optimized to ensure consistent growth throughout the duration of the assay, and caspase activation was assessed using a luminescent Caspase-Glo 3/7 assay (Promega). Caspase-Glo 3/7 signal was normalized to cell number (assessed by CTG) and shown as fold-induction relative to control (DMSO treated) cells. Caspase 3/7 activity was monitored over a 10-day timecourse in DLBCL cell lines showing cytotoxic (Toledo) and cytostatic (Daudi) responses to Compound A (FIG. 50). Consistent with the profile observed in the growth-death assay, the Toledo cell line showed robust caspase activation concurrent with decreases in cell number at all timepoints, while induction of caspase activity in the Daudi cell line was less pronounced and limited to the highest concentrations of Compound A.
Together with the cell cycle profiles, these data indicate that Compound A induces caspase-mediated apoptosis in the Toledo DLBCL cell line, suggesting the cytotoxicity observed in other lymphoma cell lines may reflect activation of apoptotic pathways by Compound A.
Anti-tumor Effects in Mouse Xenografts
The effect of Compound A on tumor growth was assessed in a Toledo (human DLBCL) xenograft model. Female SCID mice bearing subcutaneous Toledo tumors were weighed, tumors were measured with callipers, and mice were block randomized according to tumor size into treatment groups of 10 mice each. Mice were dosed orally with either vehicle or Compound A (150 mg/kg- 600 mg/kg) for 28 days daily. Throughout the study, mice were weighed and tumor measurements were taken twice weekly. Significant tumor growth inhibition (TGI) was observed at all doses and regressions were observed at doses ³ 300 mg/kg (FIG. 51, Table 5). There was no significant body weight loss in any dose group.
Given that complete TGI was observed at all doses evaluated, a second study was performed to test the anti-tumor effect of Compound A at lower doses as well as to compare twice daily (BID) dosing relative to daily (QD). In this second study, mice were dosed orally with either vehicle or Compound A (37.5 mg/kg- 150 mg/kg) for 24 days QD or 75 mg/kg BID. In this study, BID administration of 75 mg/kg resulted in the same TGI as 150 mg/kg (95% and 96%, respectively) while £ 75 mg/kg QD resulted in partial TGI ( £79%) (FIG. 51, Table 5). No significant body weight loss was observed in any dose group. These data suggest that either BID or QD dosing with the same total daily dose should result in similar efficacy.
Additional Tumor Types
AML
In addition to lymphoma cell lines, Compound A had potent, cytotoxic activity in a subset of AML cell lines examined in the 6-day proliferation assay (Table 3). Eight of 10 cell lines had glC50 values < 2mM, and Compound A induced cytotoxicity in 5 cell lines. Although PRMT1 interacts with the AML-ETO fusion characteristic of the M2 AML subtype (Shia, W. J. et al PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood 119, 4953-4962, doi: 10.1182/blood-2011-04-347476 (2012)), cell lines carrying this fusion protein (Kasumi-1 and SKNO-1) were not the only lines showing sensitivity to Compound A as measured by glC50 or that underwent cytotoxicity (Table 3, FIG.
52), therefore, the presence of this oncogenic fusion protein does not exclusively predict sensitivity of AML cell lines to Compound A. Table 3 Summary of Compound A activity in AML cell lines
Figure imgf000099_0001
Similar to studies in lymphoma, a set of cell lines was evaluated on days 6 and 10 to measure the effects of prolonged exposure to Compound A and determine whether AML cell lines that displayed a cytostatic response in the 6-day assay might undergo cytotoxicity at later timepoints. Consistent with the lymphoma result, extending time of exposure to Compound A had minimal effects on potency ( glC50) or cytotoxicity (Ymin-T0) across AML cell lines evaluated (FIG. 52). Renal Cell Carcinoma
Renal cell carcinoma cell lines had among the lowest median glC50 compared with other solid tumor types. Although none of the lines tested showed a cytotoxic response upon treatment with Compound A, all showed complete growth inhibition and 6 of 10 had glC50 values £ 2 mM (Table 4). 7 of the 10 lines profiled represent clear cell renal carcinoma (ccRCC), the major clinical subtype of renal cancer.
Table 4 Summary of Compound A anti-proliferative effects in renal cell carcinoma cells
Figure imgf000099_0002
Figure imgf000100_0001
To assess the time course of growth inhibition in renal carcinoma cell lines by Compound A, cell growth was assessed by CTG in a panel of 4 ccRCC cell lines at days 3,4,5, and 6 (FIG. 53). The largest shift in activity occurred between days 3 and 4, where all cell lines showed decreases glC50 values and increases growth inhibition. Potency of Compound A (assessed by glC50) was maximal by 4 days in 3 of 4 lines and did further not change through the 6 day assay duration. Additionally, percent growth inhibition reached 100% in all cell lines evaluated. Therefore, maximal growth inhibition in ccRCC cell lines was apparent within the 6-day growth window utilized in the cell line screening strategy.
Caspase activation was evaluated during the proliferation timecourse and, consistent with the lack of overt cytotoxicity as indicated by the Ymin-T0 values, caspase cleavage only occurred at the highest concentration (30 mM) indicating that apopotosis may have a minimal contribution to the overall growth inhibitory effect induced by Compound A in ccRCC cell lines.
The effect of Compound A on tumor growth was assessed in mice bearing human renal cell carcinoma xenografts (ACHN). Female SCID mice bearing subcutaneous ACHN cell line tumors were weighed and tumors were measured by callipers and block randomized according to tumor size into treatment groups of 10 mice each. Mice were dosed orally with either vehicle or Compound A (150 mg/kg - 600 mg/kg) for up to 59 days daily. Throughout the study, mice were weighed and tumor measurements were taken twice weekly. Significant tumor growth inhibition was observed at all doses and regressions were observed at doses ³ 300 mg/kg. Significant body weight loss was observed in animals treated with 600 mg/kg daily and, therefore, that dosing group was terminated on day 31 (FIG. 54, Table 5).
Table 5 Efficacy of Compound A in vivo
Figure imgf000101_0001
Together, these data suggest that 100% TGI can be achieved at similar doses in subcutaneous xenografts of human solid and hematologic tumors.
Breast Cancer
Breast cancer cell lines displayed a range of sensitivities to Compound A and in many cases, showed partial growth inhibition in the 6-day proliferation assay (FIG. 55). Cell lines representing triple negative breast cancer (TNBC) had slightly lower median glC50 values compared with non-TNBC cell lines (3.6 mM and 6.8 mM for TNBC and non-TNBC, respectively).
Since the effect on proliferation by Compound A was cytostatic and did not result in complete growth inhibition in the majority of breast cancer cell lines, an extended duration growth-death assay was performed to determine whether the sensitivity to Compound A would increase with prolonged exposure. In 7/17 cell lines tested there was an increase in percent maximal inhibition by ³ 10% and a ³ 2-fold decrease in glC50 (FIG. 56). In the prolonged exposure assay, 11/17 cell lines had glC50 £ 2 mM (65%) while 7/17 (41%) met these criteria in the 7 day assay format. Melanoma
Among solid tumor types, Compound A had the most potent anti-proliferative effect in melanoma cell lines (FIG. 42). Six of 7 lines assessed had glC50 values less than 2 mM (Table 6). The effect of Compound A was cytostatic in all melanoma lines, regardless of glC50 value. Table 6 Summary of Compound A Activity in Melanoma Cell Lines
Figure imgf000102_0001

Claims

What is claimed is:
1. A method of treating cancer in a human in need thereof, the method comprising administering to the human an effective amount of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and administering to the human an effective amount of a methionine adenosyltransferase II alpha (MAT2A) inhibitor.
2. The method of claim 1, wherein the Type I PRMT inhibitor is a protein arginine methyltransferase 1 (PRMT1) inhibitor, a protein arginine methyltransferase 3 (PRMT3) inhibitor, a protein arginine methyltransferase 4 (PRMT4) inhibitor, a protein arginine methyltransferase 6 (PRMT6) inhibitor, or a protein arginine methyltransferase 8 (PRMT8) inhibitor.
3. The method of claim 1 or 2, wherein the Type I PRMT inhibitor is a compound of
Formula (I):
Figure imgf000103_0001
or a pharmaceutically acceptable salt thereof, wherein
X is N, Z is NR4, and Y is CR5; or X is NR4, Z is N, and Y is CR5; or X is CR5, Z is NR4 , and Y is N; or X is CR5, Z is N, and Y is NR4;
Rx is optionally substituted C1-4 alkyl or optionally substituted C3-4 cycloalkyl;
Li is a bond, -O-, -N(RB)-, -S-, -C(O)-, -C(O)O-, -C(O)S-, -C(O)N(RB)-, - C(O)N(RB)N(RB)-, -OC(O)-, -OC(O)N(RB)-, -NRBC(O)-, -NRBC(O)N(RB)-, - NRBC(O)N(RB)N(RB)-, -NRBC(O)O-, -SC(O)-, -C(=NRB)-, -C(=NNRB)-, -C(=NORA)-, - C(=NRB)N(RB)-, -NRBC(=NRB)-, -CCS)-, -C(S)N(RB)-, -NRBC(S)-, -SCO)-, -OS(O)2-, - S(O)2O-, -SO2-, -N(RB)SO2-, -SO2N(RB)-, or an optionally substituted C1-6 saturated or unsaturated hydrocarbon chain, wherein one or more methylene units of the hydrocarbon chain is optionally and independently replaced with -O-, -N(RB)-, -S-, -C(O)-, -C(O)O-, - C(O)S-, -C(O)N(RB) -C(O)N(RB)N(RB)-, -OC(O)-, -OC(O)N(RB)-, -NRBC(O)-, - NRBC(O)N(RB) -NRBC(O)N(RB)N(RB)-, -NRBC(O)O-, -SC(O)-, -C(=NRB)-, -C(=N RB)-, -C(=NORa)-, -C(=NRB)N(RB)-, -NRBC(=NRB)-, -CCS)-, -C(S)N(RB)-, -NRBC(S)-, -S(O)-, - OS(O)2-, -S(O)2O-, -SO2- -N(RB)SO2-, or -SO2N(RB)-; each RA is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, and a sulfur protecting group when attached to a sulfur atom; each RB is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and a nitrogen protecting group, or an RB and Rw on the same nitrogen atom may be taken together with the intervening nitrogen to form an optionally substituted heterocyclic ring;
Rw is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; provided that when Li is a bond, Rw is not hydrogen, optionally substituted aryl, or optionally substituted heteroaryl;
R3 is hydrogen, C1-4 alkyl, or C3-4 cycloalkyl;
R4 is hydrogen, optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl; or optionally substituted C1-4 alkyl-Cy;
Cy is optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and
R5 is hydrogen, halo, -CN, optionally substituted C1-4 alkyl, or optionally substituted C3-4 cycloalkyl.
4. The method of any one of claims 1-3, wherein the Type I PRMT inhibitor is a compound of Formula (II):
Figure imgf000105_0001
or a pharmaceutically acceptable salt thereof.
5. The method of claim 3 or 4, wherein the Type I PRMT inhibitor is a compound of Formula (I) or Formula (II) wherein -L1-Rw is optionally substituted carbocyclyl.
6. The method of any one of claims 1-5, wherein the Type I PRMT inhibitor is Compound A:
Figure imgf000105_0002
or a pharmaceutically acceptable salt thereof.
7. The method of any one of claims 1-6, wherein the MAT2A inhibitor is a compound of
Formula (III):
Figure imgf000105_0003
III or a pharmaceutically acceptable salt thereof, wherein RA is selected from the group consisting of H, C1-C -alkyl C2 6 -alkenyl. C1-6 -alkoxy. C3-C14- carbocyclyl, ( C3-C14-carbocyclo)- C1-C6-alkyl-, 3- to 14-membered heterocyclyl or heterocyclyl- C1-C6-alkyl- (wherein 1 to 4 heterocyclyl ring members are heteroatoms selected from N, O, and S), (3- to 14-membered heterocyclyl)oxy-, C6-C14-aryl. ( C6-C14- aryl)- C1-C6-alkyl-, C6-C14-aryloxy-, -(CH2)0-6NR1(CH2)0-6(O)R2, NR'R2. C(O)NR1R2, NR1C(NR2)NR1R2, NR1C(NR2)(=NR1), SR1, -CN, and -OH; wherein each alkyl, alkenyl, alkoxy, aryl, and heterocyclyl is optionally substituted with one or more substituents selected from the group consisting of R1, OR1, halo, -N=N-R'. NR1R2. -( C1-C6-alkyl) NR1R2, -C(O)OR1, -C(O)NR1R2, -OC(O)R1, -CN, -OR(O)(OR1)1-2, and oxo;
RB is selected from the group consisting of H, C1-C6-alkenyl. and C1-C6-alkyl. wherein RB is optionally substituted by one or more R1;
Rc, RD, and RE are independently selected from the group consisting of C3-C - carbocyclyl, C6-C14-aryl. and 3- to 14-membered heterocyclyl (wherein 1 to 4 heterocycle ring members are heteroatoms selected from N, O, and S), wherein Rc, RD, and RE are optionally substituted with one or more substituents selected from the group consisting of R1, -OR1, halo, -NR'R2. -(C1-C6-alkyl)-NR' R2. - C(O)OR'. - C(O)NR1R2, -NO2, -CN, and oxo; and
R1 and R2 are independently selected from the group consisting of H, D (2H), -CN, -OH, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-alkenyl, C1-C6-alkynyl, NH2, -S(O)0-2-( C1-C6- alkyl), - S(O)0-2-( C6-C14-aryl), -C(O)(C1-C6-alkyl), -C(O)( C3-C14-carbocyclyl), - C3-C14- carbocyclyl, C6-C14-aryl. 3- to 14-membered heterocyclyl (C1-C6-alkyl)- (wherein 1 to 4 heterocyclyl ring members are heteroatoms selected from N, O, and S), wherein each alkyl, alkoxy, alkenyl, alkynyl, aryl, carbocyclyl, and heterocyclyl moiety of R1 and R2 is optionally substituted with one or more substituents selected from the group consisting of hydroxy, halo, -NH2, -NHC(O)(OC1-C6, alkyl), -NO2, -CN, oxo, -C(O)OH, -C(O)O(C1-C6- alkyl), -C1-C6-alkyl(C1-C6- alkoxy), -C(O)NH2, C1-C6-alkyl, -C(O)C1-C6-alkyl, -OC1-C6- alkyl, -Si(C1-C6-alkyl)’,. C6-C14-aryl. -( C1-C6-alkyl )( C6-C14-aryl) 3- to 14-membered heterocyclyl (C1-C6-alkyl)- (wherein 1 to 4 heterocyclyl ring members are heteroatoms selected from N, O, and S), and -O(C6-C14-aryl) wherein each alkyl, aryl, and heterocyclyl in R1 and R2 is optionally substituted with one or more substituents selected from the group consisting of hydroxy, -O C1-C6-alkyl. halo, -NH2, -(C1-C6-alkyl) NH2, -C(O)OH, CN, and oxo.
8. The method of any one of claims 1-7, wherein the MAT2A inhibitor is Compound 262:
Figure imgf000107_0001
or a pharmaceutically acceptable salt thereof.
9. The method of any one of claims 1-8, wherein the Type I PRMT inhibitor and the MAT2A inhibitor are administered simultaneously or sequentially.
10. The method of any one of claims 1-9, wherein the Type I PRMT inhibitor and/or the MAT2A inhibitor is administered orally, intravenously, or intratumorally.
11. The method of any one of claims 1-10, wherein the cancer is a solid tumor or a haematological cancer.
12. The method of any one of claims 1-11, wherein the cancer is MTAP null.
13. The method of any one of claims 1-12, wherein the cancer is pancreatic cancer.
14. A pharmaceutical product comprising a first pharmaceutical composition comprising a therapeutically effective amount of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of a methionine adenosyltransferase II alpha (MAT2A) inhibitor.
15. A Type I protein arginine methyltransferase (Type I PRMT) inhibitor for use in treating cancer, wherein the Type I PRMT inhibitor is to be administered simultaneously or sequentially with a methionine adenosyltransferase II alpha (MAT2A) inhibitor.
16. A methionine adenosyltransferase II alpha (MAT2A) inhibitor for use in treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with a Type I protein arginine methyltransferase (Type I PRMT) inhibitor.
17. Use of a Type I protein arginine methyltransferase (Type I PRMT) inhibitor in the manufacture of a medicament for treating cancer, wherein the Type I PRMT inhibitor is to be administered simultaneously or sequentially with a methionine adenosyltransferase II alpha (MAT2A) inhibitor.
18. Use of a methionine adenosyltransferase II alpha (MAT2A) inhibitor in the manufacture of a medicament for treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with a Type I protein arginine methyltransferase (Type I PRMT) inhibitor.
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