WO2021222147A1 - Modulateurs de gcn2 hétérocycliques - Google Patents

Modulateurs de gcn2 hétérocycliques Download PDF

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Publication number
WO2021222147A1
WO2021222147A1 PCT/US2021/029251 US2021029251W WO2021222147A1 WO 2021222147 A1 WO2021222147 A1 WO 2021222147A1 US 2021029251 W US2021029251 W US 2021029251W WO 2021222147 A1 WO2021222147 A1 WO 2021222147A1
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Prior art keywords
pyrimidin
pyrazol
triazolo
phenyl
amine
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PCT/US2021/029251
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English (en)
Inventor
Rui Wang
Yuxiang Zheng
Parisa ZOLFAGHARI
Loredana PUCA
Anke Klippel-Giese
Edward A. Kesicki
Kannan Karukurichi RAVI
David L. Mcelligott
Lewis C. Cantley
Mark J. Mulvihill
David Surguladze
Paulina WOJNAROWICZ
Feven TAMEIRE
Nandita BOSE
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Hibercell, Inc.
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Publication of WO2021222147A1 publication Critical patent/WO2021222147A1/fr

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    • 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
    • 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
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not 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
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • 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 disclosure is directed to general control nonderepressible 2 kinase (GCN2 kinase, also referred to herein as “GNC2”) modulators useful in the treatment of diseases or disorders associated with GCN2 modulation.
  • GCN2 kinase also referred to herein as “GNC2”
  • the invention is concerned with compounds and compositions that activate GCN2, methods of treating diseases or disorders associated with GCN2, and methods of using GCN2 modulators in combination with other cancer therapies. More specifically, the invention relates to treatment of cancers associated with GCN2 modulation.
  • Background of the Invention [0004] Because protein kinases regulate nearly every cellular process, including metabolism, cell proliferation, cell differentiation, and cell survival, they are attractive targets for therapeutic intervention for various disease states.
  • cell-cycle control, immune modulation, stress response and angiogenesis in which protein kinases play a pivotal role are cellular processes associated with numerous disease conditions such as but not limited to cancer, inflammatory diseases, neurodegenerative diseases, chronic infections, abnormal angiogenesis and diseases related thereto, atherosclerosis, macular degeneration, diabetes, obesity, and pain (Gunther Hoelzemann et al., US 9,617,266).
  • Many strategies of cancer treatment of solid tumors focus on the surgically removal of the tumor mass as far as possible and the subsequent eradication of any residual tumor cells by radiotherapy and chemotherapy with cytotoxic agents or inhibitors that target cancer cell pathways more specifically.
  • the success of such approach is limited and often does not persist.
  • GCN2 general control nonderepressible 2
  • ATF4 activating transcription factor 4
  • the cell By expressing the respective stress response proteins, e.g. enzymes in the in amino acid metabolism, the cell tries to compensate the particular cell stress [Wek et al.2006]. If the stress persists, the same pathway will switch to promoting cell death via transcription of the pro-apoptotic transcription factor, CCAAT/enhancer-binding protein homologous protein (CHOP) [Oyadomari 2004]. It was shown that tryptophan starvation triggers a GCN2-dependent stress signaling pathway. In T cells altering eIF2a phosphorylation and translational initiation leading to a cell growth arrest (Munn et al.2005). Sharma, et al. [2007] published on the direct IDO- induced and GCN2-dependent activation of mature Tregs.
  • CCAAT/enhancer-binding protein homologous protein CCAAT/enhancer-binding protein homologous protein (CHOP) [Oyadomari 2004]. It was shown that tryptophan starvation triggers a G
  • GCN2 pathway to up-regulate ATF4 target genes involved in amino acid synthesis and transport.
  • GCN2 activation/overexpression and increased phospho-eIF2a were observed in human and mouse tumors compared with normal tissues and abrogation of ATF4 or GCN2 expression significantly inhibited tumor growth in vivo. It was concluded that the GCN2- eIF2a-ATF4 pathway is critical for maintaining metabolic homeostasis in tumor cells. [0008]
  • the GCN2/ATF4 pathway has been implicated to play a dual role in cancer progression.
  • GCN2/ATF4 has been shown to promote survival under oncogene (c-MYC) induced stress.
  • c-MYC oncogene
  • N-MYC driven Neuroblastoma ATF4 induced apoptosis through activation of pro-apoptotic PUMA and NOXA halting tumor growth (Qing et al, Cell, 2012).
  • activation of GCN2/ATF4 may result in different outcomes in terms of cell survival depending on the context.
  • GCN2 modulator In cancer cells that rely on GCN2/ATF4 pathway for survival, inhibiting this pathway offers a therapeutic window, whereas activation of the pathway could induce apoptosis in other contexts. Therefore, it is believed that a GCN2 modulator will provide therapeutic benefit in specific cancer types.
  • the pharmacological modulation of GCN2 activity and/or expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions in which cell differentiation, proliferation, and/or motility are compromised, such as cancer or inflammation, and in metabolic disorders.
  • Activation of GCN2 with small molecule inhibitors therefore, has the potential to be a treatment for cancers and other disorders.
  • a first aspect of the disclosure relates to methods for treating cancer. These methods comprise administering to a subject in need thereof, an effective amount of a GCN2 kinase modulator.
  • a second aspect of the disclosure relates to methods of modulating a GCN2 kinase. The methods comprise administering to subject in need thereof a GCN2 kinase modulator that activates said GCN2 kinase.
  • Methods of the present disclosure also find utility in the activation of GCN2 kinase.
  • the present invention is useful in the treatment or prophylaxis against of tumor or cancer growth, invasiveness, intravasation, dissemination, metastasis, and tumor immunotolerance.
  • the instant disclosure relates to methods of treating cancer growth, invasiveness, intravasation, dissemination, metastasis, and immunotolerance using compounds and compositions of Formulae I, II, III, IV, V, VI, VII, and VIII and salts, prodrugs, solvates, hydrates, stereoisomers, and tautomers thereof: (I), wherein R 1 denotes Ar or Het; R 2 denotes furyl, thienyl, pyrrolyl, thiadiazolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, triazolyl or tetrazolyl which is unsubstituted or mono- or disubstituted
  • Ring A is selected from a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur optionally fused to a 5-6 membered aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or
  • each R is independently hydrogen or an optionally substituted group selected from C 1- 6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or two R groups are optionally taken together to form a bivalent C2-4 alkylene chain; two R groups are optionally taken together with their intervening atoms to form an optionally substituted 3-7-membered saturated or partially unsaturated monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur; each R’ is independently hydrogen or a
  • the genus of Formula I is incorporated by reference from Dieter Dorsch et al., US 2014/0378431.
  • the genus of Formula II is incorporated by reference from Jun Fujimoto et al., EP 3498693.
  • the genus of Formula V is incorporated by reference from Ramurthy et al., WO2020/210828.
  • the genus of Formula VI is incorporated by reference from Bui et al., U.S.2019/0375753.
  • the genus of Formula VII is incorporated by reference from Nacro et al., WO2015/108490.
  • the genus of the Formula VIII is incorporated by reference from Dorsch et al., WO2013/110309, Hoelzeman et al., WO2014/135245, Bleich et al., WO2019/148136, Bayly et al., WO2019/148132, and Hoelzeman et al., U.S.9,861,635.
  • a further aspect of the disclosure is related to pharmaceutical compositions comprising a compound of Formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII) or a salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier.
  • the pharmaceutical acceptable carrier may further include an excipient, diluent, or surfactant.
  • the compositions of Formula I also find utility in other cancers wherein GCN2 kinase expression, either in the tumor cell or in the tumor microenvironment, causes tumor progression by mechanisms mediating primary tumor growth, primary tumor invasiveness, tumor intravasation into the blood stream, tumor cell dissemination, tumor metastases to distal tissues, or tumor immunotolerance.
  • the present disclosure further provides compounds and compositions with an improved efficacy and safety profile relative to known GCN2 kinase modulators.
  • the present disclosure also provides agents with novel mechanisms of action toward GCN2 kinase in the treatment of various types of cancer including, but not limited to, hematologic cancer, bladder cancer, colon cancer, rectal cancer, non-small cell lung cancer, head and neck squamous cell cancer, renal cell carcinoma, breast cancer, pancreatic cancer, melanoma, ovarian cancer, hepatocellular carcinoma, neuroendocrine cancer, gastric cancer, and squamous cell carcinoma.
  • FIG.1 depicts tumor cell growth reduction when treated for 6 hrs with known GCN2 inhibitor GCN2iB, also referred to herein as Compound 100, or C-100.
  • Panel (A) is a western blot demonstrating increased ATF4 expression upon treatment with GCN2iB.
  • Panel (B) is a graph depicting change in cell growth (5 relative to treatment with DMSO) upon treatment with GCN2iB.
  • FIGs.2A-2D depict activation of GCN2 by GCN2iB monitored by autophosphorylation of GCN2 and ADP production.
  • FIG 2A and FIG 2B show autophosphorylation of GCN2 by 3 ATP (radiometry).
  • tRNA was used as positive control for activation.
  • FIG 2C and FIG 2D are dose response plots using transcreener ADP FI assay. C stands for no compound control. Identical concentration range (0- 0.81uM) of GCN2iB was used for both radiometric and transcreener assays.
  • FIGs.3A-3E depict the following related to Compound 100: A) ATF4 activity assay. HEK-293-ATF4-Luc reporter cells treated with increasing C-100 concentrations (0.0001 to 10 ⁇ M); B) GCN2 biochemical assay. Biochemical activity measuring modulation of pGCN2 with C-100 (0.0001 to 10 ⁇ M); C) Cell viability assay. MOLM-16 cells treated with different C-100 concentrations (0.0001 to 10 ⁇ M), DMSO and day 0 were used as CTG assay controls; D) Western blot analysis. Cell lysates obtained from MOLM-16 cells treated with C-100 (0 to 10 ⁇ M) subjected to western blot analysis; and E) In vivo efficacy.
  • FIGs.4A-4E depict the following related to Compound 101: A) ATF4 activity assay. HEK-293-ATF4-Luc reporter cells treated with increasing C-101 concentrations (0.0001 to 10 ⁇ M); B) GCN2 biochemical assay. Biochemical activity measuring modulation of pGCN2 with C-101 (0.0001 to 10 ⁇ M); C) Cell viability assay. MOLM-16 cells treated with different C-101 concentrations (0.0001 to 10 ⁇ M), DMSO and day 0 were used as CTG assay controls; D) Western blot analysis.
  • FIGs.5A-5E depict the following related to Compound 102: A) ATF4 Activity assay. HEK-293-ATF4-Luc reporter cells treated with increasing C-102 concentrations (0.0001 to 10 ⁇ M); B) GCN2 biochemical assay. Biochemical activity measuring modulation of pGCN2 with C-102 (0.0001 to 10 ⁇ M); C) Cell viability assay.
  • FIGs.6A-6G depict the following related to Compound 103: A) ATF4 Activity assay. HEK-293-ATF4-Luc reporter cells treated with increasing C-103 concentrations (0.0001 to 10 ⁇ M); B) GCN2 biochemical assay.
  • Biochemical activity measuring modulation of pGCN2 with C-103 (0.0001 to 10 ⁇ M); C) Cell viability assay. MOLM-16 cells treated with different C-103 concentrations (0.0001 to 10 ⁇ M), DMSO and day 0 were used as CTG assay controls; D) Western blot analysis. Cell lysates obtained from MOLM-16 cells treated with C-103 (0 to 10 ⁇ M) subjected to western blot analysis; E) In vivo efficacy. Effect of C-103 (3, 10 and 30 mg/kg, twice per day, orally) on MOLM-16 tumor growth; F) In vivo efficacy.
  • C-103 (0.3, 1, 3, and 10 mg/kg) on MOLM-16 tumor growth; and G) MOLM-16 PK/PD study.
  • C-103 showed a dose dependent increase in exposure after a single dose administration (Plasma Fu concentrations of C-103 shown in lines, left Y axis) that correlated with ATF4 induction in tumors (PD showed as bar graphs, right Y axis).
  • the grey box indicates the in vitro concentrations (0.04-1.6nM, corrected for protein binding) that induce ATF4 by western blot in MOLM 16 cells.
  • FIGs.7A-7F depict GCN2 dependent, but not other ISR kinase dependent, ATF4 activity in HEK-293T cells according to the following related to Compound 103: (A) si-HRI; (B) si-PKR; (C) Non-Targeted Control (NC); (D) si-PERK; (E) si-GCN2; and (F). HEK-293T-ATF4-Luciferase showing knockdown of GCN2, PERK, PKR, and HRI by western blot.
  • FIGs.8A-8C depict a GCN2 knockdown experiment – ATF4 protein induction western blot analysis) according to the following: (A) ISR kinases, HRI, PKR, PERK, GCN2 were knocked down and the induction of ATF4 was detected as shown by western blot analysis; and the effect of C-103 on ATF4 induction in HT1080 nucleofected cells with siRNAs for the ISR kinases tested at (B) 8h and (C) 24h time points. [00027] FIG.9 depicts a GCN2 knockdown experiment – Cell viability assay for C- 103.
  • FIGs.10A-10D depict the following for Compound 104: A) ATF4 Activity assay. HEK-293-ATF4-Luc reporter cells treated with increasing C-104 concentrations (0.0001 to 10 ⁇ M); B) GCN2 biochemical assay. Biochemical activity measuring modulation of pGCN2 with C-104 (0.0001 to 10 ⁇ M); C) Cell viability assay. MOLM-16 cells treated with different C-104 concentrations (0.0001 to 10 ⁇ M), DMSO and day 0 were used as CTG assay controls; D) Western blot analysis.
  • FIGs.11A-11D depict the following related to Compound 105: A) ATF4 Activity assay. HEK-293-ATF4-Luc reporter cells treated with increasing C-105 concentrations (0.0001 to 10 ⁇ M); B) GCN2 biochemical assay. Biochemical activity measuring modulation of pGCN2 with C-105 (0.0001 to 10 ⁇ M); C) Cell viability assay. MOLM-16 cells treated with different C-105 concentrations (0.0001 to 10 ⁇ M), DMSO and day 0 were used as CTG assay controls; and D) Western blot analysis.
  • FIG.12 depicts the structure of Compound 100, also referred to as C-100.
  • FIG.13 depicts the structure of Compound 101, also referred to as C-101.
  • FIG.14 depicts the structure of Compound 102, also referred to as C-102.
  • FIG.15 depicts the structure of Compound 103, also referred to as C-103.
  • FIG.16 depicts the structure of Compound 104, also referred to as C-104.
  • FIG.17 depicts the structure of Compound 105, also referred to as C-105.
  • FIGs.18-23 depict western blots for MOLM-16 cells treated with GCN2 modulators C-100, C-101, C-102, C-103, C-104 and C-105 respectively in the presence of the prolyl-tRNA synthetase inhibitor and GCN2 agonist, halofuginone.
  • the present disclosure features methods of treating, preventing or ameliorating a disease or disorder in which GCN2 kinase (which may also be referred to herein as “GCN2”) plays a role by administering to a patient in need thereof a therapeutically effective amount of a GCN2 modulator.
  • GCN2 GCN2 kinase
  • the methods of the present invention can be used in the treatment of a variety of GCN2-dependent diseases and disorders by activating the activity of GCN2 enzymes.
  • Activation of GCN2 provides a novel approach to the treatment, prevention, or amelioration of various cancer including, but not limited to, hematologic cancers such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, small lymphocytic leukemia, non-hodgkin lymphoma, or hodgkin lymphoma.
  • GCN2 Activation of GCN2 leads to phosphorylation of eukaryotic initiation factor 2 ⁇ subunit (eIF2 ⁇ ) which in turn leads to active translation of specific mRNAs, including activating transcription factor 4 (ATF4). Therefore, inhibition of pGCN2 leads to inhibition of ATF4 and activation of pGCN2 leads to enhanced translation of ATF4.
  • pGCN2 can be seen to be activated in a biochemical assay (greater than 100% of control) or in a western blot analysis of MOLM- 16 cells at specific non-suppressive concentrations of compounds of Formulas I, II, III, IV, V, VI, VII and VIII.
  • pGCN2 can be seen to be inhibited in a biochemical assay or in a western blot analysis of MOLM-16 cells at specific inhibitory concentrations. Modulation of pGCN2 is therefore compound concentration dependent and can result in either downstream inhibition of translation of ATF4 via inhibition of pGCN2 at higher concentrations or enhanced translation of ATF4 via activation of pGCN2 at lower non-suppressive concentrations.
  • an element means one element or more than one element.
  • the term “and/or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.
  • the term “optionally substituted” is understood to mean that a given chemical moiety (e.g., an alkyl group) can (but is not required to) be bonded other substituents (e.g., heteroatoms).
  • an alkyl group that is optionally substituted can be a fully saturated alkyl chain (i.e., a pure hydrocarbon).
  • the same optionally substituted alkyl group can have one or more substituents different from hydrogen.
  • Suitable substituents used in the optional substitution of the described groups include, without limitation, halogen, oxo, –OH, –CN, –COOH, –CH 2 CN, –O-(C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkoxy, (C 1 -C 6 ) haloalkyl, (C 1 -C 6 ) haloalkoxy, –O-(C2-C6) alkenyl, –O-(C2-C6) alkynyl, (C2-C6) alkenyl, (C2-C6) alkynyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl,–OP(O)(OH) 2 , –OC(O)(C1- C6) alkyl, –C(O)(C 1 -C 6 ) alkyl, –OC(O)O(C 1 -C 6
  • substituents can themselves be optionally substituted.
  • two substituents on the same ring may, together with the atoms to which they are bound, form an aryl, heteroaryl, cycloalkyl, or heterocyclyl, wherein the aryl, heteroaryl, cycloalkyl, or heterocyclyl are optionally substituted.
  • “Optionally substituted” as used herein also refers to substituted or unsubstituted whose meaning is described below. [00043]
  • substituted means that the specified group or moiety bears one or more suitable substituents wherein the substituents may connect to the specified group or moiety at one or more positions.
  • an aryl substituted with a cycloalkyl may indicate that the cycloalkyl connects to one atom of the aryl with a bond or by fusing with the aryl and sharing two or more common atoms.
  • the term “unsubstituted” means that the specified group bears no substituents.
  • aryl refers to cyclic, aromatic hydrocarbon groups that have 1 to 3 aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl or naphthyl.
  • the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl), or fused (e.g., naphthyl).
  • the aryl group may be optionally substituted by one or more substituents, e.g., 1 to 5 substituents, at any point of attachment.
  • substituents include, but are not limited to, –H, -halogen, –O-(C 1 -C 6 )alkyl, (C 1 -C 6 )alkyl, – O-(C2-C6)alkenyl, –O-(C2-C6) alkynyl, (C2-C6)alkenyl, (C2-C6)alkynyl, –OH, – OP(O)(OH) 2 , –OC(O)(C 1 -C 6 )alkyl, –C(O)(C 1 -C 6 ) alkyl, –OC(O)O(C 1 -C 6 )alkyl, —NH 2 , – NH((C 1 -C 6 )alkyl), –N((C 1 -C 6 )alkyl) 2 , –S(O) 2 -(C 1 -C 6 ) alkyl, –S(O)NH(C 1 -C
  • aryl groups herein defined may have an unsaturated or partially saturated ring fused with a fully saturated ring.
  • Exemplary ring systems of these aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, phenalenyl, phenanthrenyl, indanyl, indenyl, tetrahydronaphthalenyl, tetrahydrobenzoannulenyl, and the like.
  • heteroaryl means a monovalent monocyclic or a polycyclic aromatic radical of 5 to 24 ring atoms, containing one or more ring heteroatoms selected from N, O, S, P, or B, the remaining ring atoms being C.
  • a polycyclic aromatic radical includes two or more fused rings and may further include two or more spiro-fused rings, e.g., bicyclic, tricyclic, tetracyclic, and the like.
  • fused means two rings sharing two ring atoms.
  • spiro-fused means two rings sharing one ring atom.
  • Heteroaryl as herein defined also means a bicyclic heteroaromatic group wherein the heteroatom is selected from N, O, S, P, or B. Heteroaryl as herein defined also means a tricyclic heteroaromatic group containing one or more ring heteroatoms selected from N, O, S, P, or B. Heteroaryl as herein defined also means a tetracyclic heteroaromatic group containing one or more ring heteroatoms selected from N, O, S, P, or B. The aromatic radical is optionally substituted independently with one or more substituents described herein.
  • Examples include, but are not limited to, furyl, thienyl, pyrrolyl, pyridyl, pyrazolyl, pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl, imidazo[1,2-b]pyrazolyl, furo[2,3-c]pyridinyl, imidazo[1,2-a]pyridinyl, indazolyl, pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2-c]pyridinyl, pyrazolo[3,4-c]pyridin
  • the heteroaryl groups defined herein may have one or more saturated or partially unsaturated ring fused with one or more fully unsaturated ring.
  • a saturated or partially unsaturated ring may further be fused with a saturated or partially unsaturated ring described herein.
  • the heteroaryl groups defined herein may have one or more saturated or partially unsaturated ring spiro-fused. Any saturated or partially unsaturated ring described herein is optionally substituted with one or more oxo.
  • Exemplary ring systems of these heteroaryl groups include, for example, indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, 3,4-dihydro-1H--isoquinolinyl, 2,3- dihydrobenzofuranyl, benzofuranonyl, indolinyl, oxindolyl, indolyl, 1,6-dihydro-7H- pyrazolo[3,4-c]pyridin-7-onyl, 7,8-dihydro-6H-pyrido[3,2-b]pyrrolizinyl, 8H-pyrido[3,2- b]pyrrolizinyl, 1,5,6,7-tetrahydrocyclopenta[b]pyrazolo[4,3-e]pyridinyl, 7,8-
  • Halogen or “halo” refers to fluorine, chlorine, bromine, or iodine.
  • Alkyl refers to a straight or branched chain saturated hydrocarbon containing 1-12 carbon atoms. Examples of a (C 1 -C 6 ) alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.
  • Alkoxy refers to a straight or branched chain saturated hydrocarbon containing 1-12 carbon atoms containing a terminal “O” in the chain, i.e., -O(alkyl). Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, butoxy, t-butoxy, or pentoxy groups.
  • Alkenyl refers to a straight or branched chain unsaturated hydrocarbon containing 2-12 carbon atoms. The “alkenyl” group contains at least one double bond in the chain. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group.
  • alkenyl groups examples include ethenyl, propenyl, n- butenyl, iso-butenyl, pentenyl, or hexenyl.
  • An alkenyl group can be unsubstituted or substituted.
  • Alkenyl, as herein defined, may be straight or branched.
  • Alkynyl refers to a straight or branched chain unsaturated hydrocarbon containing 2-12 carbon atoms. The “alkynyl” group contains at least one triple bond in the chain.
  • alkenyl groups include ethynyl, propargyl, n-butynyl, iso-butynyl, pentynyl, or hexynyl.
  • An alkynyl group can be unsubstituted or substituted.
  • alkylene or “alkylenyl” refers to a divalent alkyl radical. Any of the above mentioned monovalent alkyl groups may be an alkylene by abstraction of a second hydrogen atom from the alkyl. As herein defined, alkylene may also be a C 1 -C 6 alkylene. An alkylene may further be a C 1 -C 4 alkylene.
  • Typical alkylene groups include, but are not limited to, -CH 2 -, -CH(CH 3 )-, -C(CH 3 ) 2 -, -CH 2 CH 2 -, -CH 2 CH(CH 3 )-, - CH 2 C(CH 3 ) 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, and the like.
  • Cycloalkyl means monocyclic saturated carbon rings containing 3-18 carbon atoms.
  • cycloalkyl groups include, without limitations, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norbornyl, norborenyl, bicyclo[2.2.2]octanyl, or bicyclo[2.2.2]octenyl.
  • Cycloalkylalkyl means monocyclic saturated carbon rings containing 3-24 carbon atoms further substituted with (C 1 -C 6 ) alkyl groups.
  • m is an integer from 1 to 6 and n is an integer from 1 to 16.
  • the cycloalkyl ring or carbocycle may be optionally substituted by one or more substituents, e.g., 1 to 5 substituents, at any point of attachment.
  • the substituents can themselves be optionally substituted.
  • Examples of cycloalkyl groups include, without limitations, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norbornyl, norborenyl, bicyclo[2.2.2]octanyl, bicyclo[2.2.2]octenyl, decahydronaphthalenyl, octahydro-1H-indenyl, cyclopentenyl, cyclohexenyl, cyclohexa-1,4-dienyl, cyclohexa-1,3-dienyl, 1,2,3,4- tetrahydronaphthalenyl, octahydropen
  • Heterocyclyl or “heterocycloalkyl” monocyclic rings contain carbon and one or more heteroatoms selected from N, O, S, P, or B and wherein the rings are not aromatic.
  • the heterocycloalkyl ring structure may be substituted by one or more substituents.
  • the substituents can themselves be optionally substituted.
  • heterocyclyl rings include, but are not limited to, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, oxazolidinonyl, and homotropanyl.
  • aromatic means a planar ring having 4n + 2 electrons in a conjugated system.
  • conjugated system means a system of connected p- orbitals with delocalized electrons, and the system may include lone electron pairs.
  • hydroxyalkyl means an alkyl group as defined above, where the alkyl group is substituted with one or more OH groups. Examples of hydroxyalkyl groups include HO-CH 2 -, HO-CH 2 -CH 2 - and CH 3 -CH(OH)-.
  • haloalkyl refers to an alkyl group, as defined herein, which is substituted one or more halogen.
  • haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl, etc.
  • haloalkoxy refers to an alkoxy group, as defined herein, which is substituted one or more halogen.
  • haloalkyl groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, pentafluoroethoxy, trichloromethoxy, etc.
  • cyano as used herein means a substituent having a carbon atom joined to a nitrogen atom by a triple bond, i.e., C ⁇ N.
  • amine as used herein refers to primary (R-NH 2 , R ⁇ H), secondary (R2-NH, R2 ⁇ H) and tertiary (R3-N, R ⁇ H) amines.
  • a substituted amine is intended to mean an amine where at least one of the hydrogen atoms has been replaced by the substituent.
  • amino as used herein means a substituent containing at least one nitrogen atom.
  • dialkylamino refers to an amino or -NH 2 group where both of the hydrogens have been replaced with alkyl groups, as defined herein above, i.e., -N(alkyl) 2 .
  • the alkyl groups on the amino group can be the same or different alkyl groups.
  • alkylamino groups include, but are not limited to, dimethylamino (i.e., -N(CH 3 ) 2 ), diethylamino, dipropylamino, diisopropylamino, di-n- butylamino, di-sec-butylamino, di-tert-butylamino, methyl(ethyl)amino, methyl(butylamino), etc.
  • “Spirocycloalkyl” or “spirocyclyl” means carbogenic bicyclic ring systems with both rings connected through a single atom. The ring can be different in size and nature, or identical in size and nature.
  • Examples include spiropentane, spriohexane, spiroheptane, spirooctane, spirononane, or spirodecane.
  • One or both of the rings in a spirocycle can be fused to another ring carbocyclic, heterocyclic, aromatic, or heteroaromatic ring.
  • One or more of the carbon atoms in the spirocycle can be substituted with a heteroatom (e.g., O, N, S, or P).
  • a (C 3 -C 12 ) spirocycloalkyl is a spirocycle containing between 3 and 12 carbon atoms.
  • One or more of the carbon atoms can be substituted with a heteroatom.
  • spiroheterocycloalkyl or “spiroheterocyclyl” is understood to mean a spirocycle wherein at least one of the rings is a heterocycle (e.g., at least one of the rings is furanyl, morpholinyl, or piperidinyl).
  • solvate refers to a complex of variable stoichiometry formed by a solute and solvent. Such solvents for the purpose of the invention may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, MeOH, EtOH, and AcOH. Solvates wherein water is the solvent molecule are typically referred to as hydrates.
  • Hydrates include compositions containing stoichiometric amounts of water, as well as compositions containing variable amounts of water.
  • the term "isomer” refers to compounds that have the same composition and molecular weight but differ in physical and/or chemical properties. The structural difference may be in constitution (geometric isomers) or in the ability to rotate the plane of polarized light (stereoisomers). With regard to stereoisomers, the compounds of Formula (I), (II), (III), or (IV), may have one or more asymmetric carbon atom and may occur as racemates, racemic mixtures and as individual enantiomers or diastereomers.
  • the present invention also contemplates isotopically-labelled compounds of Formula I (e.g., those labeled with 2 H and 14 C).
  • Deuterated (i.e., 2 H or D) and carbon-14 (i.e., 14 C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances.
  • Isotopically labelled compounds of Formula I can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples herein below, by substituting an appropriate isotopically labelled reagent for a non-isotopically labelled reagent.
  • the disclosure also includes pharmaceutical compositions comprising an effective amount of a disclosed compound and a pharmaceutically acceptable carrier.
  • Representative salts include, e.g., water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, magnesium, malate, maleate, mande
  • a "patient” or “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or rhesus.
  • An "effective amount" when used in connection with a compound is an amount effective for treating or preventing a disease in a subject as described herein.
  • carrier encompasses carriers, excipients, and diluents and means a material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body of a subject.
  • treating refers to improving at least one symptom of the subject's disorder. Treating includes curing, improving, or at least partially ameliorating the disorder.
  • disorder is used in this disclosure to mean, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.
  • administer refers to either directly administering a disclosed compound or salt of the disclosed compound or a composition to a subject, or administering a prodrug derivative or analog of the compound or salt of the compound or composition to the subject, which can form an equivalent amount of active compound within the subject's body.
  • prodrug means a compound which is convertible in vivo by metabolic means (e.g., by hydrolysis) to a disclosed compound
  • salt refers to pharmaceutically acceptable salts
  • pharmaceutically acceptable salt also refers to a salt of the compositions of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base.
  • modulate refers to a biological activity of a compound or substrate that inhibits and/or activates GCN2 enzyme.
  • GCN2 modulators refer to compounds of Formula I and/or compositions comprising a compound of Formulae I, II, III, IV, V, VI, VII and VIII which modulate GCN2.
  • the amount of compound of composition described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose.
  • therapeutic agents e.g. compounds or compositions of Formula I (and/or additional agents) described herein
  • the therapeutic agents are given at a pharmacologically effective dose.
  • a “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease.
  • An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease.
  • administering therapeutic agents to a patient suffering from cancer provides a therapeutic benefit not only when the underlying condition is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the disease, e.g., a decrease in tumor burden, a decrease in circulating tumor cells, an increase in progression free survival.
  • Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • the compound of Formula I is a potent modulator of GCN2, used in the treatment of cancer and/or modulation of GCN2 has the Formula I: wherein R 1 and R 2 are described herein as in Formula I.
  • a composition comprising the compound of Formula I, as described herein, and pharmaceutically acceptable carrier is used in the treatment of cancer and/or modulation of GCN2.
  • the compound or composition of Formula II used in the treatment of cancer and/or modulation of GCN2 activity has the Formula: [00085] wherein A, X 6 , X 7 , X 8 , X 9 , and X 10 are described herein as in Formula II.
  • the compound or composition of Formula III used in the treatment of cancer and/or modulation of GCN2 activity has the Formula: [00087]
  • the compound or composition of Formula IV used in the treatment of cancer and/or modulation of GCN2 activity has the Formula: [00088]
  • the compound or composition of Formula V used in the treatment of cancer and/or modulation of GCN2 activity has the Formula: [00089] wherein A 1 , A 2 , A 3 , X 1 , X 2 , and R 1 are described herein as in Formula V.
  • the compound or composition of Formula VI used in the treatment of cancer and/or modulation of GCN2 activity has the Formula: wherein A, R 1 , R 2 , R 3 , L 1 , L 2 and Z are described herein as in Formula VI.
  • the compound or composition of Formula VII used in the treatment of cancer and/or modulation of GCN2 activity has the Formula: wherein A, B, L, R N1 and R N2 are described herein as in Formula VII.
  • the compound or composition of Formula VIII used in the treatment of cancer and/or modulation of GCN2 activity has the Formula: [00093] wherein A, B, C, R 1 , R 2 , R 3 , R 4 , m, n, p and q are described herein as in Formula VIII.
  • the GCN2 modulator of Formula I is selected from: ”A1” [3-(4-methoxy-phenyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl]- (1-phenyl-1H- pyrazol-4-yl)-amine; “A2” [3-(4-methoxy-phenyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl]- (1- methyl-1H-pyrazol-3-yl)-amine; “A3” [3-(4-methoxy-phenyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl]- (1H- pyrazol-4-yl)-amine; “A4” (3-phenyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)-(1H-pyrazol- 4-
  • the GCN2 modulator of Formula II is selected from: 2,5-dichloro-N-(2,4-difluoro-3-(1H-pyrazolo[3,4-b]pyridin-5-ylethynyl)phenyl)- 3-(hydroxymethyl)benzenesulfonamide; 5-chloro-N-(2,4-difluoro-3-(1H-pyrazolo[3,4-b]pyridin-5-ylethynyl)phenyl)-2- methylpyridine-3-sulfonamide; N-(3-((2-aminopyrimidin-5-yl)ethynyl)-2,4-difluorophenyl)-5-chloro-2- methylpyridine-3-sulfonamide; N-(3-((2-aminopyrimidin-5-yl)ethynyl)-2,4-difluorophenyl)-2,5
  • the GCN2 kinase modulator of Formula I or composition of Formula I is administered in a dosing regime that sufficiently activates GCN2 kinase and blocks and/or reduces tumor cell growth.
  • the dosing regime that sufficiently activates GCN2 kinase and blocks and/or reduces tumor cell growth provides for a compound of Formula I that is between the range of about 10 nM and about 300 nM in concentration within the tumor cell.
  • the dosing regime that sufficiently activates GCN2 kinase and blocks and/or reduces tumor cell growth provides for a compound of Formula I that is between the range of about 30 nM and about 120 nM in concentration within the tumor cell.
  • the dosing regime that sufficiently activates GCN2 kinase and blocks and/or reduces tumor cell growth provides for a compound of Formula I that is between the range of about 114 nM in concentration within a cell.
  • cell growth is reduced to less than 1.0%, less than 0.8%, less than 0.6%, less than 0.4%, less than 0.2%, or about 0%.
  • cell growth is reduced to less than 0.3%. In some embodiments, cell growth is reduced to about 0%.
  • only one diastereomer of the compound of Formulae, I, II, II, IV, V, VI, VII, or VIII is active in GCN2 kinase activation.
  • the dosing regimen of the compound or composition of Formula I is a daily dosing administration.
  • the dosing regimen of the compound or composition of Formula I is a daily dosing administration.
  • the intermittent non-daily dosing regimen may include, without limitation, alternate daily dosing, every third-day dosing, twice weekly dosing, or once weekly dosing.
  • a suitable dosing regimen of the compound or composition of Formula I includes administration twice weekly, once weekly, or alternate weekly.
  • the dosing regimen of the compound or composition of Formula I is twice weekly or once weekly.
  • the dosing regimen of the compound or composition of Formula I is administration twice weekly.
  • the compound of Formula VII is other than C-104.
  • GCN2 modulators of the disclosure may be administered as a single agent or in combination with other therapeutic agents known to treat cancer.
  • Such other therapeutic agents include radiation therapy, anti-tubulin agents, DNA alkylating agents, DNA synthesis-inhibiting agents, DNA intercalating agents, kinase inhibitors, topoisomerase inhibitors, Histone Deacetylase (HDAC) inhibitors, DNA methylation inhibitors, proteasome inhibitors, thalidomide, lenalidomide, antibody-drug-conjugates (ADCs), immunomodulating agents, or cancer vaccines.
  • HDAC Histone Deacetylase
  • GCN2 modulators of the disclosure may be used in combination with chemotherapeutic agents including but not limited to anti-tubulin agents (paclitaxel, paclitaxel protein-bound particles for injectable suspension, eribulin, docetaxel, ixabepilone, vincristine), vinorelbine, DNA-alkylating agents (including cisplatin, carboplatin, oxaliplatin, cyclophosphamide, ifosfamide, temozolomide), DNA intercalating agents (including doxorubicin, pegylated liposomal doxorubicin, daunorubicin, idarubicin, and epirubicin), 5-fluorouracil, capecitabine, cytarabine, decitabine, 5-aza cytadine, gemcitabine and methotrexate.
  • chemotherapeutic agents including but not limited to anti-tubulin agents (paclitaxel, paclitaxel protein
  • GCN2 modulators of the disclosure may be used in combination with kinase inhibitors including but not limited to erlotinib, gefitinib, lapatanib, everolimus, temsirolimus, LY2835219, LEE011, PD 0332991, crizotinib, cabozantinib, sunitinib, pazopanib, sorafenib, regorafenib, axitinib, dasatinib, imatinib, nilotinib, vemurafenib, dabrafenib, trametinib, idelalisib, osimertinib, alpelisib, and quizartinib.
  • kinase inhibitors including but not limited to erlotinib, gefitinib, lapatanib, everolimus, temsirolimus, LY2835219, LEE011,
  • GCN2 modulators of the disclosure may be used in combination with topoisomerase I inhibitors including but not limited to irinotecan, camptothecin, and topotecan.
  • the compositions of Formula I may be used in combination with topoisomerase II inhibitors including but not limited to etoposide, etoposide phosphate, and mitoxantrone.
  • GCN2 modulators of the disclosure may be used in combination with Histone Deacetylase (HDAC) inhibitors including but not limited to vorinostat, romidepsin, panobinostat, valproic acid, and belinostat.
  • HDAC Histone Deacetylase
  • GCN2 modulators of the disclosure may be used in combination with DNA methylation inhibitors including but not limited to DZNep and 5-aza-2'-deoxycytidine.
  • GCN2 modulators of the disclosure may be used in combination with proteasome inhibitors including but not limited to bortezomib and carfilzomib.
  • GCN2 modulators of the disclosure may be used in combination with thalidomide, lenalidomide and pomalidomide.
  • GCN2 modulators of the disclosure may be used in a method of treating various types of cancer, including hematologic cancer, bladder cancer, colon cancer, rectal cancer, non-small cell lung cancer, head and neck squamous cell cancer, renal cell carcinoma, breast cancer, pancreatic cancer, melanoma, ovarian cancer, hepatocellular carcinoma, neuroendocrine cancer, gastric cancer, and squamous cell carcinoma.
  • Types of hematologic cancers include may include acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, myelodysplastic syndrome, non-Hodgkin lymphoma.
  • Such non-Hodgkin lymphomas include anaplastic large cell lymphoma, B-cell lymphoma, Burkitt’s lymphoma, Cutaneous T-cell lymphoma, Diffuse large B-cell lymphoma, follicular lymphoma, Histiocytic lymphoma, Mantle cell lymphoma, Mixed-cell type lymphoma, Peripheral T-cell lymphoma, Primary mediastinal large B-cell lymphoma, T-cell lymphoblastic lymphoma, and small-cell lymphocytic lymphoma.
  • the hematologic cancer is selected from acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, small-cell lymphocytic lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, and myelodysplastic syndrome.
  • the hematologic cancer is acute myeloid leukemia.
  • the hematologic cancer is chronic myeloid leukemia.
  • the hematologic cancer is acute lymphoblastic leukemia.
  • the hematologic cancer is chronic lymphocytic leukemia.
  • the hematologic cancer is Hodgkin lymphoma. In some embodiments, the hematologic cancer is myelodysplastic syndrome. In some embodiments, the hematologic cancer is small-cell lymphocytic lymphoma. In some embodiments, the hematologic cancer is non-Hodgkin lymphoma. In some embodiments, the hematologic cancer is anaplastic large cell lymphoma. In some embodiments, the hematologic cancer is B-cell lymphoma. In some embodiments, the hematologic cancer is Burkitt’s lymphoma. In some embodiments, the hematologic cancer is Cutaneous T-cell lymphoma.
  • the hematologic cancer is Diffuse large B-cell lymphoma. In some embodiments, the hematologic cancer is follicular lymphoma. In some embodiments, the hematologic cancer is Histiocytic lymphoma. In some embodiments, the hematologic cancer is Mantle cell lymphoma. In some embodiments, the hematologic cancer is Mixed-cell type lymphoma. In some embodiments, the hematologic cancer is Peripheral T-cell lymphoma. In some embodiments, the hematologic cancer is Primary mediastinal large B-cell lymphoma. In some embodiments, the hematologic cancer is T-cell lymphoblastic lymphoma.
  • the hematologic cancer is small lymphocytic lymphoma. In some embodiments, the hematological cancer is myelodysplastic syndrome/myeloproliferative neoplasm overlap syndromes.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with an agent selected from an immune checkpoint inhibitor, an immunosuppressive targeting agent, a BCL2 inhibitor, a FLT3 inhibitor, an amino acid biosynthesis modulator, an anti-vascular agent, an ER stress modulator, a kinase inhibitor, a T-cell costimulatory agent, tumor-associated macrophage targeting agent, an innate immune checkpoint inhibitor, an innate immune activator, and an adoptive cell therapy.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with an immune checkpoint inhibitor, including, but not limited to, an anti-CTLA4 inhibitor, an anti-PD1 inhibitor, an anti- PDL1 inhibitor, an anti-LAG3 inhibitor, and anti-TIM3 inhibitor, and an anti-TIGIT inhibitor.
  • the immune checkpoint inhibitor may be selected from ipilimumab, pembrolizumab, and nivolumab.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with an innate immune checkpoint inhibitor, selected from, but not limited to, an anti-CD47 agent, an anti-SIRPa agent, and an A 2a receptor antagonist.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with T-cell costimulatory agents, selected from, but not limited to, an anti-OX40 agent, an anti-ICOS agent, an anti-GITR, and an anti-41BB agent.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with an agent that targets immunosuppressive metabolic pathways, including, but not limited to an arginase-1 inhibitor, an IDO inhibitor, a TDO inhibitor, and an IDO/TDO dual inhibitor.
  • the immunosuppressive targeting agent may be selected from Imprime PGG.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with a BCL2 inhibitor, including, but not limited to, venetoclax.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with an FLT3 inhibitor, including, but not limited to, gilteritinib.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with an ER stress modulator, including, but not limited to, a PERK inhibitor.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with an amino acid biosynthesis modulator, including, but not limited to, L-asparaginase.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with an anti-vascular agent, selected from, but not limited to, bevacizumab, ramucirumab, cabozantinob, and axitinib.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with innate immune activators, selected from, but not limited to, a TLR agonist, a STING agonist, and a dectin agonist.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with a tumor-associated macrophage targeting agent, selected from, but not limited to, an anti-CSF1R inhibitor and an anti- CSF1R antibody.
  • a tumor-associated macrophage targeting agent selected from, but not limited to, an anti-CSF1R inhibitor and an anti- CSF1R antibody.
  • the methods of treating cancer comprise administering GCN2 modulators of the disclosure in combination with an adoptive cell therapy, selected from, but not limited to, CAR T cell therapy and TCR engineered autologous T cell therapy.
  • the methods of treating cancer comprise administering GCN2 inhibitors of the disclosure in combination with an agent selected from wherein the agent is selected from epacadostat, 680C91, CMG017, HC5404-FU, GSK-157, GSK 2606414, AMG PERK 44, AMG510, PF-04518600, KY1044, BMS-986156, PF- 05082566, Pexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, emactuzumab, AMG820, IMC-CS4, MCS110, AO-176, CC-95251, istradefylline, CPI-444, CPI-006, imiquimod, MEDI9197, CMP-001, ODN2395, Pam2CSK4, DMXAA, MSA 2, MEDI9197, CMP-001, imprime PGG, UCART123, CTL119, MB-102
  • the methods of treating hematologic cancer comprise administering GCN2 modulators of the disclosure in combination with an agent effective for treatment of acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the agent effective for treating AML may be selected from Cyclophosphamide, Cytarabine, Daunorubicin Hydrochloride, Glasdegib Maleate, Dexamethasone, Doxorubicin Hydrochloride, Enasidenib Mesylate, Gemtuzumab Ozogamicin, Gilteritinib Fumarate, Glasdegib Maleate, Idarubicin Hydrochloride, Enasidenib Mesylate, Ivosidenib, Mitoxantrone Hydrochloride, Gemtuzumab, Ozogamicin, Midostaurin, Thioguanine, Ivosidenib, Arsenic Trioxide, Venetoclax, Vincristine Sulfate, Gilteritinib Fumarate, Az
  • the methods of treating hematologic cancer comprise administering GCN2 modulators of the disclosure in combination with an agent effective for treatment of chronic myeloid leukemia (CML).
  • CML chronic myeloid leukemia
  • the agent effective for treating CML may be selected from Bosutinib, Busulfan, Cyclophosphamide, Cytarabine, Dasatinib, Dexamethasone, Hydroxyurea, Imatinib Mesylate, Mechlorethamine Hydrochloride, Nilotinib, Omacetaxine Mepesuccinate, Ponatinib Hydrochloride, Lenalidomide, or Pomalidomide.
  • the methods of treating hematologic cancer comprise administering GCN2 modulators of the disclosure in combination with an agent effective for treatment of acute lymphoblastic leukemia (ALL).
  • ALL acute lymphoblastic leukemia
  • the agent effective for treating ALL may be selected from Asparaginase Erwinia chrysanthemi, Blinatumomab, Calaspargase Pegol-mknl, Clofarabine, Cyclophosphamide, Cytarabine, Dasatinib, Daunorubicin Hydrochloride, Dexamethasone, Doxorubicin Hydrochloride, Inotuzumab Ozogamicin, Imatinib Mesylate, Mercaptopurine, Methotrexate, Nelarabine, Pegaspargase, Ponatinib Hydrochloride, Prednisone, Tisagenlecleucel, Vincristine Sulfate, Vincristine Sulfate Liposome, or Venetoclax.
  • the methods of treating hematologic cancer comprise administering GCN2 modulators of the disclosure in combination with an agent effective for treatment of chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • the agent effective for treating CLL may be selected from Alemtuzumab, Bendamustine Hydrochloride, Chlorambucil, Cyclophosphamide, Dexamethasone, Duvelisib, Fludarabine Phosphate, Ibrutinib, Idelalisib, Mechlorethamine Hydrochloride, Obinutuzumab, Ofatumumab, Prednisone, Rituximab, Rituximab and Hyaluronidase Human, or Venetoclax.
  • the methods of treating hematologic cancer comprise administering GCN2 modulators of the disclosure in combination with an agent effective for treatment of a non-Hodgkin lymphoma (NHL).
  • NHL non-Hodgkin lymphoma
  • the agent effective for treating NHL may be selected from Acalabrutinib, Axicabtagene Ciloleucel, Belinostat, Bendamustine Hydrochloride, Bleomycin Sulfate, Bortezomib, Brentuximab Vedotin, Carmustine, Chlorambucil, Copanlisib Hydrochloride, Cyclophosphamide, Denileukin Diftitox, Dexamethasone, Doxorubicin Hydrochloride, Duvelisib, Ibritumomab Tiuxetan, Ibrutinib, Idelalisib, Lenalidomide, Mechlorethamine Hydrochloride, Methotrexate, Mogamuli
  • the methods of treating hematologic cancer comprise administering GCN2 modulators of the disclosure in combination with an agent effective for treatment of a Hodgkin lymphoma (HL).
  • the agent effective for treating HL may be selected from Bleomycin Sulfate, Brentuximab Vedotin, Carmustine, Chlorambucil, Cyclophosphamide, dacarbazine, Dexamethasone, Doxorubicin Hydrochloride, Lomustine, Mechlorethamine Hydrochloride, Nivolumab, Pembrolizumab, Prednisone, Procarbazine Hydrochloride, Vinblastine Sulfate, or Vincristine Sulfate.
  • the methods of treating hematologic cancer comprise administering GCN2 modulators of the disclosure in combination with an agent effective for treatment of a myelodysplastic syndrome.
  • the agent effective for treating myelodysplastic syndrome may be selected from Cytarabine, Hydroxyurea, Azacitidine, Decitabine, or Lenalidomide.
  • patients with tumors showing expression of wt DNMT3B, DNMT3A-R882, SMAD1, alone or as part of a defined gene signature such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, small-cell lymphocytic lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, or myelodysplastic syndrome, are preferentially treated.
  • the subject has tumor cells showing tumor growth control after ex-vivo treatment.
  • patients with tumor cells showing tumor growth control after ex-vivo treatment such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, small- cell lymphocytic lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, or myelodysplastic syndrome, are preferentially treated.
  • the subject has tumor cells showing induction of ATF4 or other integrated stress response genes, either alone or as part of a gene signature after ex-vivo treatment.
  • patients with tumor cells showing induction of ATF4 or other integrated stress response genes are preferentially treated.
  • acute myeloid leukemia chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, small-cell lymphocytic lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, or myelodysplastic syndrome
  • induction of ATF4, phospho-eIF2a or CHOP either alone or as part of a multicolor flowcytometry panel or a gene signature after ex-vivo treatment allows dose and schedule optimization.
  • Formulations, Administration, Dosing, and Treatment Regimens [000143] The present disclosure includes the described GCN2 modulators of Formulae I, II, III, IV, V, VI, VII, VIII (and/or additional agents) in various formulations.
  • compositions (and/or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155).
  • suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447- 1676 (Alfonso R. Gennaro eds., 19th ed.1995), incorporated herein by reference.
  • the compounds and/or compositions herein described can also include a solubilizing agent.
  • the agents can be delivered with a suitable vehicle or delivery device as known in the art.
  • Combination therapies outlined herein can be co- delivered in a single delivery vehicle or delivery device.
  • Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.
  • a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.
  • the compounds and/or compositions of Formulae I, II, III, IV, V, VI, VII or VIII (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration.
  • routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • the administering is effected orally or by parenteral injection.
  • the mode of administration can be left to the discretion of the practitioner, and depend in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream.
  • compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • compositions can be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time.
  • Selectively permeable membranes surrounding an osmotically active driving the salt of Formula I (and/or additional agents) described herein are also suitable for orally administered compositions.
  • fluid from the environment surrounding the capsule is imbibed by the driving composition, which swells to displace the agent or agent composition through an aperture.
  • delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations.
  • a time-delay material such as glycerol monostearate or glycerol stearate can also be useful.
  • Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.
  • the excipients are of pharmaceutical grade.
  • Suspensions in addition to the active compositions, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, etc., and mixtures thereof.
  • suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, etc., and mixtures thereof.
  • intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
  • the dosage of the compounds and/or composition of Formulae I, II, III, IV, V, VI, VII, or VIII (and/or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject’s general health, and the administering physician’s discretion.
  • Any agent described herein can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional therapeutic agent, to a subject in need thereof.
  • any agent described herein is administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, or 11 hours to 12 hours apart.
  • the dosage of the compound and/or composition of Formulae I, II, III, IV, V, VI, VII, or VIII (and/or additional agents) described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated.
  • pharmacogenomic the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic
  • dosage used may affect dosage used.
  • the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.
  • the dosage of any composition of Formula I (and/or additional agents) described herein may be 0.001 mg/kg/day to 100 mg/kg/day, 0.01 mg/kg/day to 50 mg/kg/day, or 0.1 mg/kg/day to 10 mg/kg/day.
  • the dosage of any agent described herein is normally 0.001 mg to 1500 mg per day, 1 mg to 600 mg per day, or 5 mg to 30 mg per day.
  • the dosage of the salt (or agent) ranges from 57 mg to 1200 mg per day.
  • the dosage of the agents or salt ranges from 100 mg to 200 mg per day.
  • the dosage is normally 0.1 mg to 250 mg per day, 1 mg to 20 mg per day, or 3 mg to 5 mg per day. Injections may be given up to four times daily.
  • the dosage of any agent described herein is normally 0.1 mg to 1500 mg per day, or 0.5 mg to 10 mg per day, or 0.5 mg to 5 mg per day. A dosage of up to 3000 mg per day can be administered.
  • Administration of the compounds and/or compositions (and/or additional agents) described herein can, independently, be one to four times daily.
  • administration of the salt can be once a day at a dosing regimen of the salt is from about 50 mg to 1500 mg. Suitable daily dosage for the prophylactic effects sought is 57-1200 mg/day. If administered twice daily, a suitable dosage is 100 mg to 200mg of the salt. Administration of the salt may also be intermittently non-daily. In particular, administration of the compound and/or composition may be done one to four times per month or one to six times per year or once every two, three, four or five years. In some embodiments administration of the compound and/or composition is done weekly or bi- weekly. When administered weekly or bi-weekly, a suitable compound or composition dosing regimen ranges from 50-200 mg/ per administration.
  • dosage is 200-400 mg/ per administration.
  • Yet other mode of weekly or bi-weekly administration include 400-500 mg/ per administration, 500-600 mg/ per administration, 600-700 mg/ per administration, 700-800 mg/ per administration, 800- 900 mg/ per administration, 900-1000 mg/ per administration, 1000-1100 mg/ per administration, or 1100-1200 mg/ per administration.
  • Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject. Chronic, long-term administration will be indicated in many cases.
  • the dosage may be administered as a single dose or divided into multiple doses.
  • the desired dosage should be administered at set intervals for a prolonged period, usually at least over several weeks or months, although longer periods of administration of several months or years or more may be needed.
  • the compound is administered in a dosing regimen sufficient to activate ATF4.
  • the compound is administered in a dosing regimen sufficient to enhance translation of ATF4.
  • the compound is administered in a dosing regimen insufficient to inhibit ATF4.
  • the compound is administered in a dosing regimen insufficient to inhibit GCN2 kinase.
  • the compound is administered in a dosing regimen sufficient to activate GCN2 kinase, wherein the compound is effective to enhance translation of ATF4.
  • the compound is administered in a dosing regimen, which achieves concentrations in the subject non-suppressive of GCN2 kinase.
  • EXAMPLES Example 1: Biochemical Assay General overview of GCN2 kinase activity assessment. [000161] GCN2 is purchased as an active kinase enzyme. It is active because it has become autophosphorylated during protein preparation and purification. In order to accurately assess whether compounds can activate GCN2 (via autophosphorylation), the commercial GCN2 must be dephosphorylated to a less active form first.
  • lambda phosphatase (Uniprot P49593) from EMD Millipore (Cat. No.14- 405, expressed with C-term histag, 25 kDa) was used.
  • the dephosphorylation mixture (25uL) contained 50 mM HEPES buffer, pH 7.4, 2mM MnCl2, 2mM DTT, 2uL of lambda phosphatase, and 4 ug full length GCN2. This mixture was incubated at 30°C for 3 hrs. A parallel control was done without adding lambda phosphatase.
  • a 23 mer peptide G-A-G-A-G-A-R-K-L-E-L-S-F-Y-P-I-R-G-A-G-A-G-A (SEQ ID NO: 1) was synthesized from Anaspec and used as the substrate for GCN2.
  • the peptide was dissolved in milliQ water to 2.5 mM and pH was adjusted to 7 with conc.
  • NaOH.10mM stocks of compounds were serially diluted (3X) in neat DMSO.5X compound stocks were prepared in 25% DMSO in water from neat DMSO stocks.
  • full length GCN2 along with buffer components, was incubated with or without compound at 27°C for 1h.
  • reaction was initiated by the addition of 5uL of 125uM ATP.
  • a typical assay mixture (25 uL) contained 40mM HEPES buffer, pH 7.4, 25 mM MgCl2, 0.01% v/v triton-X-100, 5% v/v DMSO, 20 mM NaCl, 20-25 nM GCN2, 25 uM ATP, and 100 uM substrate peptide.
  • the reaction was allowed to proceed for 45 min and 1.5h after which time, 10 uL of reaction mixture was quenched with 25uL of transcreener reagent. The contents were incubated at RT for 1h and fluorescence was measured using a plate reader (Paradigm, Molecular Devices).
  • a positive control for activation was run using crude bovine liver tRNA (type XI, Sigma, Cat. No. R4752). Lyophilized tRNA powder was directly dissolved in RNase free water and assayed at 2 uM. A calibration curve was generated under identical buffer conditions with varying ADP amounts. Using that, the observed fluorescence was converted to [ADP] in uM. A plot between [ADP] and log[I] yielded dose-response curves.
  • ⁇ 70 uCi (70uL) of hot ATP was mixed with 192 uM (130 uL) cold ATP to yield 125 uM ATP stock.
  • the assay procedure was identical to the activation/inhibition assay except the reaction was initiated with 5 uL of 125 uM hot ATP. After 30 min and 1 h, 10 uL of the reaction was quenched with 5 uL of 6X loading buffer. From this mixture, 12uL was loaded per well in a 4-12% bis-tris, 15 well, 1.5mm NuPAGE gel (Cat. No. NP0336BOX). After electrophoretic separation of proteins, the gels were washed multiple times in distilled water and exposed to storage phosphor screen.
  • FIG.1 depicts tumor cell growth reduction when treated for 6 hrs with known GCN2 inhibitor GCN2iB, also referred to herein as Compound 100 or C-100.
  • FIG 1A is a western blot demonstrating increased ATF4 expression upon treatment with GCN2iB.
  • FIG 1B is a graph depicting change in cell growth (5 relative to treatment with DMSO) upon treatment with GCN2iB.
  • FIG 2 depicts activation of GCN2 by GCN2iB monitored by autophosphorylation of GCN2 and ADP production.
  • FIG 2A and FIG 2B show autophosphorylation of GCN2 by 32 P ATP (radiometry). tRNA was used as positive control for activation.
  • FIG 2C and FIG 2D are dose response plots using transcreener ADP FI assay. C stands for no compound control. Identical concentration range (0- 0.81uM) of GCN2iB was used for both radiometric and transcreener assays.
  • Example 2 Biochemical assays [000167] GCN2 protein was obtained from Carna Biosciences (GCN2 cat#: 05-153).
  • the protein was diluted in assay buffer (ThermoFisher Scientific, #PV6135), 2 mM dithiothreitol (DTT), to obtain a final concentration of 2 nM and 5 ⁇ L was plated in a 384-well white assay plate.
  • Test compounds were serially diluted to 11 concentrations by 3-fold dilution in DMSO and 10 nL of stock was plated into 384 well white assay plate.
  • DMSO was used as a vehicle control.
  • GFP-eIF2 ⁇ protein was obtained from ThermoFisher (cat# PV4809).
  • the protein was diluted in assay buffer to a 2x concentration of 200 nM along with 300 mM ATP (final concentration of 100 nM GFP- eIF2 ⁇ and 150 mM ATP) in the presence of 2 mM DTT and a 5 ⁇ L aliquot was added to each well containing the GCN2 protein and test compound.
  • the plate was incubated in the dark at 25°C for 1.5 hours, shaking at 1250 rpm.
  • Tb-anti P-eIF2 ⁇ (ThermoFisher cat# PV4810) were diluted to a concentration of 4 nM with 20 mM EDTA (final concentration of 2 nM Tb-anti P-eIF2 ⁇ and 10 nM EDTA) in TR-FRET Dilution Buffer (ThermoFisher cat# PV3574).10 ⁇ L of the Tb-anti P-eIF2 ⁇ solution was added to the TR-FRET reaction. The plate was incubated in the dark for 2 h at 25°C shaking at 600 rpm.
  • the FRET signal from the plate was read on a Envision (PerkinElmer) plate reader: Label 1: Excitation: 340 nm, bandwidth 30 nm; Emission: 495 nm, bandwidth 10 nm. Lag time: 100 ⁇ sec. Integration time: 400 ⁇ sec. Flashes: 30. Label 2: Excitation: 340 nm, bandwidth 30 nm; Emission: 520 nm, bandwidth 25 nm. Lag time: 100 ⁇ sec. Integration time: 400 ⁇ sec. Flashes: 30 [000168] The data were analyzed using GraphPad Prism employing a 4-parameter sigmoidal curve fit.
  • FIGs.3B, 4B, 5B, 6B, 10B, and 11B depict the experimental results for C-100, C-101, C-102, C-103, C-104 and C-105, respectively, in the GCN2 activity assay described above.
  • Example 3 Cell based assays ATF4 Activity Assay [000170] HEK293-ATF4-Luc cells were with culture medium 1.5e 5 cells/mL and 25 ⁇ L of cell suspension was added into each well of 384-well cell culture plate (Corning, CLS3570-50EA) as designated and transferred into 37°C - 5% CO2 incubator (Thermo Scientific) overnight for cell attachment.
  • Test compounds were serially diluted to 11 concentrations by 3-fold dilution in DMSO and 25 nL of stock was plated into cell plate by Echo550(Labcyte, Echo550), then incubated for 30 minutes at 37oC. After compound treatment for 6 hours, add 25 ⁇ L of One-Glo reagent (Promega, cat#: E6120) into each well to be detected (at 1:1 to culture medium). Then the plates were placed at room temperature for 10 min followed by luciferase luminescence reading on EnVision (PerkinElmer). The data were analyzed by Xlfit (v5.3.1.3), equation 201.
  • FIGs.3A, 4A, 5A, 6A, 10A, and 11A depict the experimental results for C- 100, C-101, C-102, C-103, C-104 and C-105, respectively, in the ATF4 activity assay described above.
  • MOLM-16 cells were obtained from DSMZ (#ACC 555) cells were cultivated using RPMI 1640 (Gibco, #11875119) with 20% FBS (Invitrogen, #10099141C) in T-225 flasks in a cell culture incubator (Thermo Scientific) set at 37°C - 5% CO 2 , 95% relative humidity.30 ⁇ L Cell suspension (2k cells/well) was added to each well in 384-well cell culture plates (Corning, #CLS3764-100EA). The plates were then incubated overnight.
  • Test compounds were diluted in 10 concentrations by 3-fold dilution in 384pp-plate using TECAN EVO200.40 nL of the compounds stock were transferred to 384-well cell culture plate using Echo550.10 uL ASNase (Sigma, #A3809-100UN) dilution was added to each well in one plate (final concentration was 0.1U/mL). For another plate, 10 uL culture media was added to each well. The plates were incubated for 72hr in incubator at 37°C, 5% CO 2 .30 uL CellTiter-Glo® 2.0 Assay (Promega, G9243) was added to each well. The plates were then incubated at RT for 30 min.
  • FIGs.3C, 4C, 5C, 6C, 10C, and 11C depict the experimental results for C-100, C-101, C-102, C-103, C-104 and C-105, respectively, in the cell viability assay described above.
  • Knockdown Assay - MOLM-16 Nucleofection Protocol ⁇ MOLM-16 cells were harvested from flasks into cell culture medium and then counted. The required number of cells were obtained by centrifugation at 90xg for 10 minutes at room temperature and the supernatant removed.
  • the cell pellet ( ⁇ 3e 6 ) was carefully resuspended at room temperature in 20 ⁇ L 4D-Nucleofector Solution (LONZA, #V4XC-2032.1.2 ⁇ L 100 uM siRNA were added to each aliquot.
  • the master mixes were transferred into the Nucleocuvette Vessels.
  • the NucleocuvetteTM Vessels were gently tapped to make sure the sample covered the bottom of the cuvette and then placed with the lid closed into the retainer of the 4D-NucleofectorTM Core Unit (4D-NucleofectorTM Core Unit, AAF-1002B).
  • the nucleofection was started using Lonza’s 4D Core Unit EP- 100 program.
  • FIG.9 depicts the results of the above-described knockdown assay for C-103.
  • siRNA Knock Down Procedure for HT1080 or HEK293-ATF4-Luc cells [000176] Non-targeting control siRNA and siRNAs against GCN2, PERK, HRI and PKR were dissolved at the concentration of 10 ⁇ M in DNAse/RNAase free water. Cells were plated in 6-well plates and incubated at 37 ⁇ , 5% CO2 overnight.
  • FIGs.7A-7F and 8A depict GCN2 dependent, but not other ISR kinase dependent, ATF4 activity in HEK-293T cells or HT1080 cells. Specifically, FIGs.7A-7E illustrate an ATF4 activity assay that measures transcriptional activity of ATF4.
  • non-targeting control transfected cells show an induction of ATF4 activity at select concentrations in response to GCN2 modulator, C-103, treatment. Knocking down GCN2 abolished this response. However, ATF4 activity was still present in cells with knock down other ISR kinases (PERK, HRI and PKR) indicating that ATF4 activation was dependent on GCN2 and not other ISR kinases.
  • FIG.7F shows that knockdown of GCN2, PERK, HRI and PKR was achieved by siRNA compared to non-targeting control siRNA transfected cells.
  • FIG.8A-8C demonstrate that treating HT1080 cells with C-103 induced ATF4 in non-targeting control transfected cells as well as in cells transfected with siRNAs against PERK, HRI and PKR.
  • induction of ATF4 protein was absent in cells transfected with GCN2 siRNA suggesting that GCN2 is required for activation of ATF4 during treatment with GCN2 modulator, C-103.
  • FIG.8B and FIG.8C show quantification of the ATF4 western band for 8hr and 24hr, respectively.
  • Example 4 Western blot analysis [000179] Cell lysates obtained from MOLM-16 cells treated with C-100, C-101, C-102, C-103, C-104 and C-105 (0 to 10 ⁇ M) and subjected to western blot analysis.
  • FIGs.3D, 4D, 5D, 6D, 10D, and 11D depict the experimental results for C- 100, C-101, C-102, C-103, C-104 and C-105, respectively, in the western blot analysis described above.
  • Example 5 In vivo TGI studies [000183] Effect of C-100, C-101, C-102 and C-103 (1, 3 and 10 mg/kg, twice per day, orally) on MOLM-16 tumor growth was investigated. 1.
  • Animals were housed in polycarbonate cage, which is in the size of 300 ⁇ 180 ⁇ 150 mm 3 and in an environmentally monitored, well-ventilated room maintained at a temperature of (22 ⁇ 3°C) and a relative humidity of 40%-80%. Fluorescent lighting was provided illumination approximately 12 hours per day. The bedding material is soft wood, which was changed once per week. [000186] 1.2.3 Animal ID: Each animal was assigned an identification number; the following identification method was applied. Each cage card was labeled with such information as study number, group, sex, dose, animal number, initiation date, study director and telephone number. Individual animal was identified by ear coding.
  • mice were assigned to respective groups such that the average starting tumor size is the same for each treatment group.
  • Vehicle-1 for test compounds 10% Tween 80 in water 2.
  • Experimental method and measurement parameters 2.1 Method for Tumor Inoculation [000191] Each mouse was inoculated subcutaneously on the right flank with MOLM-16 tumor cells (1 x 10 7 ) in 0.1 ml of RPMI 1640 medium without serum for tumor development. The treatment was started when the mean tumor size reached approximately 200 mm 3 . Animals were randomized based on tumor volume in the groups of 8-10 animals, typically including a vehicle control and treatment groups at various dose levels.
  • FIGs.3E, 4E, 5E, and 6E depict the experimental results for C-100, C-101, C- 102, and C-103 respectively, in the TGI study described above.
  • FIG.6F depicts experimental results for C-103 in the TGI study described above except using doses at 0.3, 1, 3, and 10 mg/kg.
  • Example 6 Western blot analysis Samples [000198] MOLM-16 cells were treated with GCN2 modulators C-100, C-101, C-102, C- 103, C-104 and C-105 in the presence of the prolyl-tRNA synthetase inhibitor and GCN2 agonist, halofuginone (Keller TL, et al.2012, Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase. Nat Chem Biol 8(3):311–317).
  • FIGs.18-23 depict results of the above-described western analysis for MOLM-16 cells treated with GCN2 modulators C-100, C-101, C-102, C-103, C-104 and C-105, respectively.
  • halofuginone acts as a GCN2 agonist by inhibiting prolyl-tRNA synthetase.
  • Cells treated with halofuginone showed activation of GCN2 and ATF4.
  • GCN2 modulator, C-103 inhibited halofuginone induced p-GCN2 and ATF4 in a dose dependent manner.
  • This result demonstrates the inhibitory nature of GCN2 modulator, C-103, where it acts as an inhibitor of pGCN2 and ATF4 at certain concentrations, those higher than lower non-suppressive concentrations, which result in activation of pGCN2 and ATF4.
  • Example 7 KINOMEscanTM screening at concentrations of 1.0 mM versus EIF2AK1 and EIF2AK4 kinases
  • the KINOMEscanTM screening platform employs a novel and proprietary active site-directed competition binding assay to quantitatively measure interactions between test compounds and more than 450 human kinases and disease relevant mutant variants.
  • This robust and reliable assay technology affords investigators the ability to extensively annotate compounds with accurate, precise and reproducible data.
  • KINOMEscanTM assays do not require ATP and thereby report true thermodynamic interaction affinities, as opposed to IC50 values, which can depend on the ATP concentration. Nat. Biotechnol.23, 329-336 (2005). Nat.
  • Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays.
  • the liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1 % BSA, 0.05 % Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non- specific phage binding.
  • Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1x binding buffer (20 % SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT).
  • Test compounds were prepared as 40x stocks in 100% DMSO and directly diluted into the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.02 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1x PBS, 0.05 % Tween 20). The beads were then re-suspended in elution buffer (1x PBS, 0.05 % Tween 20, 0.5 ⁇ M non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
  • LC-MS/MS tandem mass spectrometry
  • FIG.6G depicts the results of a PK/PD study for C-103 in MOLM-16 cells according to the above procedure.
  • a dose dependent increase in exposure after a single dose administration of C-103 correlates with ATF4 induction in tumors (PD showed as bar graphs, right Y axis).
  • the grey box indicates the in vitro concentrations (0.04-1.6nM, corrected for protein binding) that induce ATF4 by western blot in MOLM 16 cells.

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Abstract

L'invention concerne des procédés d'utilisation de modulateurs de GCN2 et de compositions associées, dans le traitement de cancers et d'autres maladies et affections associées à l'activation de GCN2, lesdits modulateurs ayant les formules I, II, III, IV, V, VI, VII et/ou VIII.
PCT/US2021/029251 2020-04-27 2021-04-26 Modulateurs de gcn2 hétérocycliques WO2021222147A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023230567A1 (fr) * 2022-05-25 2023-11-30 Hibercell, Inc. Modulateur de gcn2 pour le traitement du cancer
US11945799B2 (en) 2020-06-09 2024-04-02 Ip2Ipo Innovations Limited 4-ethynylpyridine derivatives useful as GCN2 inhibitors

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013110309A1 (fr) * 2012-01-28 2013-08-01 Merck Patent Gmbh Dérivés de triazolo[4,5-d]pyrimidine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013110309A1 (fr) * 2012-01-28 2013-08-01 Merck Patent Gmbh Dérivés de triazolo[4,5-d]pyrimidine

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIANGBIN YE ET AL: "The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation", THE EMBO JOURNAL / EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, vol. 29, no. 12, 16 June 2010 (2010-06-16), Oxford, pages 2082 - 2096, XP055429392, ISSN: 0261-4189, DOI: 10.1038/emboj.2010.81 *
JUN FUJIMOTO ET AL: "Identification of Novel, Potent, and Orally Available GCN2 Inhibitors with Type I Half Binding Mode", ACS MEDICINAL CHEMISTRY LETTERS, vol. 10, no. 10, 19 September 2019 (2019-09-19), US, pages 1498 - 1503, XP055663172, ISSN: 1948-5875, DOI: 10.1021/acsmedchemlett.9b00400 *
LOUGH LEA ET AL: "Triazolo[4,5-d]pyrimidines as Validated General Control Nonderepressible 2 (GCN2) Protein Kinase Inhibitors Reduce Growth of Leukemia Cells", COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL, vol. 16, 1 January 2018 (2018-01-01), Sweden, pages 350 - 360, XP055821879, ISSN: 2001-0370, DOI: 10.1016/j.csbj.2018.09.003 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11945799B2 (en) 2020-06-09 2024-04-02 Ip2Ipo Innovations Limited 4-ethynylpyridine derivatives useful as GCN2 inhibitors
WO2023230567A1 (fr) * 2022-05-25 2023-11-30 Hibercell, Inc. Modulateur de gcn2 pour le traitement du cancer

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