WO2023039510A1 - Gpx4 inhibitors and uses thereof - Google Patents

Gpx4 inhibitors and uses thereof Download PDF

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Publication number
WO2023039510A1
WO2023039510A1 PCT/US2022/076179 US2022076179W WO2023039510A1 WO 2023039510 A1 WO2023039510 A1 WO 2023039510A1 US 2022076179 W US2022076179 W US 2022076179W WO 2023039510 A1 WO2023039510 A1 WO 2023039510A1
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alkyl
alkenyl
aryl
group
heteroaryl
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PCT/US2022/076179
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French (fr)
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Brent R. Stockwell
Hengrui LIU
Farhad FOROUHAR
Xin XIA
Annie LIN
Qian Wang
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The Trustees Of Columbia University In The City Of New York
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Publication of WO2023039510A1 publication Critical patent/WO2023039510A1/en

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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
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    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
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    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
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    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
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    • C07C2601/14—The ring being saturated

Definitions

  • the present disclosure provides, inter alia, compounds to modulate GPX4 activity. Also provided are pharmaceutical compositions containing the compounds, as well as methods of using such compounds and compositions.
  • Cancer cells are dependent on their lipid composition for establishing and modulating membrane structural integrity, morphology, metabolism, migration, invasiveness, and other functions.
  • PUFA-PLs polyunsaturated-fatty-acid-(PUFA)-containing phospholipids
  • PUFA-PLs are, however, susceptible to peroxidation via iron-catalyzed reaction with molecular oxygen at bis-allylic positions, catalyzed by lipoxygenases and labile iron (Yang et al. 2016).
  • some cancer cells depend on a critical network of proteins to eliminate their PUFA-PL peroxides; a key protein at the center of this defense network is the selenoprotein glutathione peroxidase 4 (GPX4).
  • GPX4 activity is compromised, lipid peroxidation can cause ferroptosis (Stockwell et al. 2017), an oxidative, iron-dependent form of non- apoptotic cell death (Dixon et al. 2012).
  • Ferroptosis acts as a natural tumor suppressive and immune surveillance mechanism, and can be induced by exogenous agents in cells that are addicted to GPX4 (Dixon and Stockwell, 2019). Cancer cells from tissues of diverse origins have been screened for their sensitivity to ferroptosis-inducing compounds (Viswanathan et al. 2017). It has been found that ferroptosis inducers, including GPX4 inhibitors, selectively target cancers with a mesenchymal or otherwise drug-resistant signature (Viswanathan et al. 2017).
  • EMT epithelial- mesenchymal transition
  • GPX4-knockout high-mesenchymal therapy-resistant melanoma regressed after cessation of ferrostatin-1 (a lipophilic antioxidant discovered in the Stockwell Lab that suppresses the loss of GPX4) and did not relapse after ceasing dabrafenib and trametinib treatment, while wt GPX4 xenografts continued to grow in both experiments (Viswanathan et al. 2017).
  • ferrostatin-1 a lipophilic antioxidant discovered in the Stockwell Lab that suppresses the loss of GPX4
  • wt GPX4 xenografts continued to grow in both experiments (Viswanathan et al. 2017).
  • GPX4 inhibitors are selectively lethal to persister and EMT cancer cells, with minimal effects on parental cells and non-transformed cells, suggesting that addiction to GPX4 creates a large therapeutic window.
  • one embodiment of the present disclosure is a compound according to formula (1): wherein:
  • R 1 , R 2 , R 3 , R 4 , R 5 R 6 and R 7 are independently selected from the group consisting of H, D, -OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO 2 , alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C 1-4 alkyl, CF 3 , oxazole
  • Another embodiment of the present disclosure is a compound according to formula (2): wherein: n is a positive integer;
  • R 1 , R 2 , R 3 , and R 4 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO 2 , alkyl, aryl, alkyl-aryl, alkyl- heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C 1-4 alkyl, CF 3 , oxazole, oxadiazole,
  • Rs is selected from the group consisting of no atom, H, D, -OH, halo, -Si(CH 3 ) 3 , - Sn(CH 3 ) 3 , alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl- heteroaryl, wherein the alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, -NO 2 , epoxy, -OH, halo, C 1-4 alkyl, CF 3 , oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N
  • Another embodiment of the present disclosure is a compound according to formula (3): wherein:
  • X is selected from the group consisting of N, S, and SO 2 ;
  • R 1 , R 2 , R 3 , R 4 and R 7 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO 2 , alkyl, aryl, alkyl-aryl, alkylheteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C 1-4 alkyl, CF 3 , oxazole, oxadiazol
  • Another embodiment of the present disclosure is a compound having any one of the following structures:
  • compositions including pharmaceutical compositions, comprising one or more compounds disclosed herein and a pharmaceutically acceptable carrier, adjuvant or vehicle.
  • Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
  • GPX4 glutathione peroxidase 4
  • Another embodiment of the present disclosure is a method for modulating the activity of glutathione peroxidase 4 (GPX4) in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
  • GPX4 glutathione peroxidase 4
  • Another embodiment of the present disclosure is a method for increasing the level of peroxide in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
  • a further embodiment of the present disclosure is a method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
  • Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, which is one or more compounds disclosed herein or one or more compositions disclosed herein, and ii) an effective amount of a second anti-cancer agent.
  • a first anti-cancer agent which is one or more compounds disclosed herein or one or more compositions disclosed herein
  • An additional embodiment of the present disclosure is a kit for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein, packaged with its instructions for use.
  • GPX4 glutathione peroxidase 4
  • FIGS. 1A-1D show MST binding traces of LOC1886 (A) and LOC880 (B) as well as crystal structure of GPX4U46C with LOC1886 (C and D).
  • FIG. 2 is a schematic of assay funnel for candidate validation.
  • FIG. 3 shows observed KdS for representative LOC1886 analogs, measured by MST. Enhancement on binding affinities were reported for all analogs tested.
  • FIG. 4 shows intact LC-MS analysis of GPX4 pre-incubated with LOC1886 analogs.
  • GRL-0496 is a validated commercial LOC1886 analog.
  • FIGS. 5A-5C show characterizations of representative LOC1886 analogs.
  • A MST binding summaries;
  • B LC-MS detection of covalently modified GPX4;
  • C NADPH-coupled GPX4 inhibition assay.
  • FIG. 6 shows observed KdS for representative LOC880 analogs, measured by MST.
  • FIG. 7 shows NADPH-coupled GPX4 inhibition assay for representative LOC880 analogs.
  • FIGS. 8A-8C show SAR (structure-activity relationship) analysis from MST data.
  • A Effect of linker length in region B on binding affinities of LOC880 analogs;
  • B Effect of region A indole ring substitution on binding affinities of LOC880 analogs;
  • C Effect of region B side chain on binding affinities of LOC880 analogs.
  • FIG. 9 shows C11-BODIPY lipid peroxidation flow cytometry assay in HT1080 cells for representative analogs.
  • FIG. 10 shows GI50 of selected analogs.
  • FIGS. 11A-11F show cellular dose response assays of representative LOC1886 analogs with HT1080, HLF, and HepG2, Huh7 and Skhep-1 liver cancer cells.
  • A RSL3 Control - all cell lines undergo ferrostatin-rescuable cell death;
  • B QW-148 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines;
  • C) QW-152 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines;
  • QW-156 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines;
  • E QW-158 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines;
  • QW-162 induces ferrostatin-1 rescuable cell death in Huh7, HLF,
  • FIGS. 13A-13I show crystal structures of GPX4U46C with RSL3, ML162, CDS9, TMT10 and LOC1886.
  • A A cartoon representation of a pseudotrimer of GPX4U46C bound to RSL3.
  • B A close-up view of the GPX4U46C residues that interact with RSL3.
  • RSL3 molecules and the side chain of GPX4 residues are shown with stick models.
  • a 2Fo-Fc omit map at 3. (grey mesh) on one of the RSL3 molecules is also shown.
  • C A cartoon representation of a pseudotrimer of GPX4U46C bound to ML162.
  • FIG. 14 shows MST screening of selected DEL compounds against GPX4.
  • FIG. 15 shows NADPH-coupled GPX4 activity assay for selected DELopen compounds.
  • FIG. 16 shows crystal hit for GPX4 U46C in presence of selected DEL compounds, and LOC880, LOC1886 analogs.
  • one embodiment of the present disclosure is a compound according to formula (1): wherein:
  • R 1 , R 2 , R 3 , R 4 , RS R 6 and R 7 are independently selected from the group consisting of H, D, -OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO 2 , alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C 1-4 alkyl, CF 3 , oxazole
  • the compound has a structure selected from the group consisting of:
  • the compound has the structure of formula (1a): wherein: a dashed line indicates the presence of an optional double bond;
  • R 1 , R 2 , R 3 , and R 4 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO 2 , alkyl, aryl, alkyl-aryl, alkylheteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C 1-4 alkyl, CF 3 , oxazole, oxadiazole,
  • R is selected from the group consisting of H, D, C, 0, N and halo, wherein R is selected from the group consisting of H, D, 0, N, halo, oxazole, oxadiazole, dizaole, triazole, C 1-6 alkyl, C 1-6 alkyl-aryl, C 1-6 alkyl-heteroaryl, C 1-6 alkenyl, C 1-6 alkenyl-aryl, C 1-6 alkenyl-heteroaryl and C 3-12 carbocycle, wherein the C 1-6 alkyl, C 1-6 alkyl-aryl, C 1-6 alkyl-heteroaryl, C 1-6 alkenyl, C 1-6 alkenyl-aryl, C 1-6 alkenyl-heteroaryl and C 3-12 carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N, S, halo, C 1-4 alkyl, CF 3 , -CN
  • the compound has a structure selected from the group consisting of:
  • Another embodiment of the present disclosure is a compound according to formula (2): wherein: n is a positive integer;
  • R 1 , R 2 , R 3 , and R 4 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO 2 , alkyl, aryl, alkyl-aryl, alkylheteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C 1-4 alkyl, CF 3 , oxazole, oxadiazole,
  • Rs is selected from the group consisting of no atom, H, D, -OH, halo, -Si(CH3) 3 , - Sn(CH 3 ) 3 , alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenylheteroaryl, wherein the alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, -NO 2 , epoxy, -OH, halo, C 1-4 alkyl, CF 3 , oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N,
  • the compound has a structure selected from the group consisting of: or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
  • Downstream cellular assays will be performed on the most promising candidates.
  • the structures of all DELopen compounds that are tested positive of GPX4 binding haven been acquired, SAR optimization of the DELopen compounds is currently ongoing.
  • another high-throughput screen was performed using an Enamine Diversity Library of 60,638 compounds. The top ⁇ 180 hits are currently being validated. Further biophysical and biochemical characterizations of the validated hits will follow.
  • Another embodiment of the present disclosure is a compound according to formula (3): wherein:
  • X is selected from the group consisting of N, S, and SO 2 ;
  • R 1 , R 2 , R 3 , R 4 and R 7 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO 2 , alkyl, aryl, alkyl-aryl, alkylheteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C 1-4 alkyl, CF 3 , oxazole, oxadiazol
  • Another embodiment of the present disclosure is a compound having any one of the following structures:
  • compositions including pharmaceutical compositions, comprising one or more compounds disclosed herein and a pharmaceutically acceptable carrier, adjuvant or vehicle.
  • Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
  • GPX4 glutathione peroxidase 4
  • the GPX4-associated disease is selected from the group consisting of a cancer, a neurotic disorder, a neurodegenerative disorder, spondylometaphyseal dysplasia, mixed cerebral palsy, pontocerebellar hypoplasia, and male infertility.
  • the GPX4-associated disease is a cancer.
  • cancer include hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma.
  • the cancer is hepatocellular carcinoma.
  • the subject is a mammal.
  • the mammal is selected from the group consisting of humans, veterinary animals, and agricultural animals.
  • the subject is a human.
  • the cancer is metastatic. In some embodiments, the cancer is under epithelial-to-mesenchymal (EMT) transition. In some embodiments, the cancer is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the cancer is refractory to standard cancer treatment. Nonlimiting examples of standard cancer treatment include chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof.
  • Another embodiment of the present disclosure is a method for modulating the activity of glutathione peroxidase 4 (GPX4) in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
  • the modulation comprises inhibiting GPX4 activity.
  • Another embodiment of the present disclosure is a method for increasing the level of peroxide in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
  • peroxide inclcude hydrogen peroxide organic hydroperoxide, lipid peroxide, and combinations thereof.
  • a further embodiment of the present disclosure is a method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
  • the cell has abberant lipid accumulation.
  • the cell is a cancer cell.
  • the cancer cell is selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma.
  • the cancer is hepatocellular carcinoma.
  • the cell is a human cell. In some embodiments, wherein the cancer cell is metastatic. In some embodiments, the cancer cell is under epithelial-to-mesenchymal (EMT) transition. In some embodiments, the cancer cell is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the hypersensitivity to ferropotosis is identified by NADPH abundance, GCH1 expression, NF2-YAP activity, EMT signature, and GPX4 expression. In some embodiments, the cancer cell is refractory to standard cancer treatment. Non-limiting examples of standard cancer treatment includes chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof.
  • Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, which is one or more compounds disclosed herein or one or more compositions disclosed herein, and ii) an effective amount of a second anti-cancer agent.
  • a first anti-cancer agent which is one or more compounds disclosed herein or one or more compositions disclosed herein
  • the second anti-cancer agent is selected from the group consisting of chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof.
  • the second anticancer agent is an immunotherapy, such as checkpoint inhibitor therapy including PD-1 and CTLA-4 inhibitor therapy.
  • Non-limiting examples of immunotherapy include ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, ofatumumab, blinatumomab, daratumumab, elotuzumab, obinutuzumab, talimogene laherparepvec, necitumumab, lenalidomide, dinutuximab, and combinations thereof.
  • the subject is a human.
  • the cancer is metastatic. In some embodiments, the cancer is under epithelial-to-mesenchymal (EMT) transition. In some embodiments, the cancer is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the cancer is refractory to standard cancer treatment.
  • EMT epithelial-to-mesenchymal
  • the cancer is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the cancer is refractory to standard cancer treatment.
  • the cancer is selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma.
  • the cancer is hepatocellular carcinoma.
  • the first anti-cancer agent is administered to the subject before, concurrently with, or after the second anti-cancer agent.
  • An additional embodiment of the present disclosure is a kit for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein, packaged with its instructions for use.
  • GPX4 glutathione peroxidase 4
  • kits may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each compound of the present disclosure (which, e.g., may be in the form of pharmaceutical compositions) and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the active agents to subjects.
  • suitable storage containers e.g., ampules, vials, tubes, etc.
  • other reagents e.g., buffers, balanced salt solutions, etc.
  • the compounds and/or pharmaceutical compositions of the disclosure and other reagents may be present in the kits in any convenient form, such as, e.g., in a solution or in a powder form.
  • the kits may further include a packaging container, optionally having one or more partitions for housing the compounds and/or pharmaceutical compositions and other optional reagents.
  • the terms "treat,” “treating,” “treatment” and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient.
  • the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development.
  • every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.
  • ameliorate means to decrease the seventy of the symptoms of a disease in a subject.
  • a “subject” is a mammal, preferably, a human.
  • categories of mammals within the scope of the present disclosure include, for example, agricultural animals, veterinary animals, laboratory animals, etc.
  • agricultural animals include cows, pigs, horses, goats, etc.
  • veterinary animals include dogs, cats, etc.
  • laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.
  • the phrase “a subject in need thereof” means a subject in need of treatment for a GPX4-associated disorder, such as, e.g., a cancer.
  • the phrase “a subject in need thereof” menas a subject diagnosed with a GPX4-associated disorder, such as, e.g., a cancer.
  • lipid peroxidation means the oxidative degradation of fats, oils, waxes, sterols, triglycerides, and the like. Lipid peroxidation has been linked with many degenerative diseases, such as atherosclerosis, ischemia- reperfusion, heart failure, Alzheimer’s disease, rheumatic arthritis, cancer, and other immunological disorders. (Ramana et al., 2013).
  • ferropttosis means regulated cell death that is iron- dependent. Ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid reactive oxygen species. (Dixon et al., 2012) Ferroptosis is distinct from apoptosis, necrosis, and autophagy. (Id.) [0072] As used herein, the terms “modulate”, “modulating”, “modulator” and grammatical variations thereof mean to change, such as increasing or decreasing the activity of GPX4. In this embodiment, “contacting” means bringing the compound and optionally one or more additional therapeutic agents into close proximity to the cells in need of such modulation. This may be accomplished using conventional techniques of drug delivery to the subject or in the in vitro situation by, e.g., providing the compound and optionally other therapeutic agents to a culture media in which the cells are located.
  • a "pharmaceutically acceptable salt” means a salt of the compounds of the present disclosure which are pharmaceutically acceptable, as defined herein, and which possess the desired pharmacological activity.
  • Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-
  • Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases.
  • Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide.
  • Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.
  • an "effective amount” or “therapeutically effective amount” of a compound or pharmaceutical composition is an amount of such a compound or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject.
  • Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of the subject, and like factors well known in the arts of, e.g., medicine and veterinary medicine.
  • a suitable dose of a compound or pharmaceutical composition according to the disclosure will be that amount of the compound or composition, which is the lowest dose effective to produce the desired effect with no or minimal side effects.
  • the effective dose of a compound or pharmaceutical composition according to the present disclosure may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
  • a suitable, non-limiting example of a dosage of a compound or pharmaceutical composition according to the present disclosure or a composition comprising such a compound is from about 1 ng/kg to about 1000 mg/kg, such as from about 1 mg/kg to about 100 mg/kg, including from about 5 mg/kg to about 50 mg/kg.
  • Other representative dosages of a compound or a pharmaceutical composition of the present disclosure include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg.
  • a compound, composition, or pharmaceutical composition of the present disclosure may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a compound, composition, or pharmaceutical composition of the present disclosure may be administered in conjunction with other treatments. A compound, composition, or pharmaceutical composition of the present disclosure may be encapsulated or otherwise protected against gastric or other secretions, if desired.
  • compositions or pharmaceutical compositions of the disclosure are pharmaceutically acceptable and comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable carriers or diluents and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the compounds/compositions/pharmaceutical compositions of the present disclosure are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.). More generally, “pharmaceutically acceptable” means that which is useful in preparing a composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
  • Pharmaceutically acceptable carriers and diluents are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and
  • Each pharmaceutically acceptable carrier or diluent used in a composition of the disclosure must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • Carriers or diluents suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers or diluents for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
  • compositions or pharmaceutical compositions of the disclosure may, optionally, contain additional ingredients and/or materials commonly used in such compositions.
  • ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate
  • Compounds, compositions or pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in- water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste.
  • These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.
  • Solid dosage forms for oral administration may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers or diluents and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine.
  • the tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter.
  • compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • the active ingredient can also be in microencapsulated form.
  • Liquid dosage forms for oral administration include pharmaceutically- acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain suitable inert diluents commonly used in the art.
  • the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions may contain suspending agents.
  • compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants.
  • the active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier or diluent.
  • the ointments, pastes, creams and gels may contain excipients.
  • Powders and sprays may contain excipients and propellants.
  • compositions suitable for parenteral administrations comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.
  • Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
  • a drug e.g., pharmaceutical formulation
  • the rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle.
  • injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier or diluent, for example water for injection, immediately prior to use.
  • sterile liquid carrier or diluent for example water for injection
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
  • aliphatic refers to a group composed of carbon and hydrogen that do not contain aromatic rings. Accordingly, aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclyl groups. Additionally, unless otherwise indicated, the term “aliphatic” is intended to include both "unsubstituted aliphatics” and “substituted aliphatics", the latter of which refers to aliphatic moieties having substituents replacing a hydrogen on one or more carbons of the aliphatic group.
  • Such substituents can include, for example, a halogen, a deuterium, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, an aromatic, or heteroaromatic moiety.
  • alkyl refers to the radical of saturated aliphatic groups that does not have a ring structure, including straight-chain alkyl groups, and branched- chain alkyl groups.
  • a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C 1 -C 6 for straight chains, C3-C 6 for branched chains).
  • the “alkyl” may include up to twelve carbon atoms, e.g., C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 or C 12 .
  • substituents include all those contemplated for aliphatic groups, as discussed below, except where stability is prohibitive.
  • alkenyl refers to an aliphatic group containing at least one double bond and unless otherwise indicated, is intended to include both "unsubstituted alkenyls" and “substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group.
  • substituents include all those contemplated for aliphatic groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
  • alkyl as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • all groups recited herein are intended to include both substituted and unsubstituted options.
  • Cx-y when used in conjunction with a chemical moiety, such as, alkyl and cycloalkyl, is meant to include groups that contain from x to y carbons in the chain.
  • C x-y alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-tirf luoroethyl, etc.
  • aryl as used herein includes substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon.
  • the ring is a 3- to 8-membered ring, more preferably a 6-membered ring.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
  • alkyl-aryl refers to an alkyl group substituted with at least one aryl group.
  • alkyl-heteroaryl refers to an alkyl group substituted with at least one heteroaryl group.
  • alkenyl-aryl refers to an alkenyl group substituted with at least one aryl group.
  • alkenyl-heteroaryl refers to an alkenyl group substituted with at least one heteroaryl group.
  • carbocycle refers to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon.
  • a carbocycle ring contains from 3 to 10 atoms, more preferably from 3 to 8 atoms, including 5 to 7 atoms, such as for example, 6 atoms.
  • cabocycle also includes bicycles, tricycles and other multicyclic ring systems, including the adamantyl ring system.
  • halo and halogen are used interchangeably herein and mean halogen and include chloro, fluoro, bromo, and iodo.
  • heteroaryl includes substituted or unsubstituted aromatic single ring structures, preferably 3- to 8-membered rings, more preferably 5- to 7- membered rings, even more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
  • heteroaryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen.
  • Preferred heteroatoms are nitrogen, oxygen, and sulfur; more preferably, nitrogen and oxygen.
  • ether means an organic compound with the structure R-O- R’, wherein neither R nor R' can be hydrogen atoms.
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic mo
  • references to chemical moieties herein are understood to include substituted variants.
  • reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.
  • oxadiazole means any compound or chemical group containing the following structure:
  • oxazole means any compound or chemical group containing the following structure:
  • triazole means any compound or chemical group containing the following structure:
  • indole means any compound or chemical group containg the following structure:
  • stereoisomer refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures which are not interchangeable. The three-dimensional structures are called configurations. Stereoisomers include enantiomers and diastereomers.
  • racemate or “racemic mixture” refer to a mixture of equal parts of enantiomers.
  • chiral center refers to a carbon atom to which four different groups are attached.
  • enantiomeric enrichment refers to the increase in the amount of one enantiomer as compared to the other.
  • Examples of methods to obtain optically active materials include at least the following: i) physical separation of crystals--a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct; ii) simultaneous crystallization--a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state; iii) enzymatic resolutions--a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; iv) enzymatic asymmetric synthesis--a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer;
  • the resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer; vii) first- and second-order asymmetric transformations--a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer.
  • kinetic resolutions--this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions; ix) enantiospecific synthesis from non-racemic precursors--a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis; x) chiral liquid chromatography--a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase.
  • the stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions; xi) chiral gas chromatography--a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase; xii) extraction with chiral solvents--a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; xiii) transport across chiral membranes--a technique whereby a racemate is placed in contact with a thin membrane barrier.
  • the barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane which allows only one enantiomer of the racemate to pass through.
  • the stereoisomers may also be separated by usual techniques known to those skilled in the art including fractional crystallization of the bases or their salts or chromatographic techniques such as LC or flash chromatography.
  • the (+) enantiomer can be separated from the (-) enantiomer using techniques and procedures well known in the art, such as that described by J. Jacques, et al., Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc., 1981.
  • chiral chromatography with a suitable organic solvent, such as ethanol/acetonitrile and Chiralpak AD packing, 20 micron can also be utilized to effect separation of the enantiomers
  • Glutathione peroxidase 4 has been reported to be a promising therapeutic target for metastatic and drug-resistant cancers, based on the elevated dependency of the cancer cells on GPX4 lipid peroxide repair pathway during epithelial-mesenchymal transition (EMT) and the transformation into therapy-tolerant persister states.
  • EMT epithelial-mesenchymal transition
  • LOC1886 and LOC880 are GPX4 allosteric inhibitors identified from the initial biophysical screening of 10,095 Lead Optimized Compounds (LOC).
  • LOC1886 covalently modifies GPX4 Cys66 (allosteric site 1 ) and LOC880 binds to E50-F92-S44 (allosteric site 2) with a d of 8 ⁇ M (FIGS. 1A-1D).
  • the SAR optimizations on LOC1886 and LOC880 were conducted and provided herein.
  • the imidazole group functions as an electrophile for the covalent modification of GPX4, hence remains unmodified.
  • the indole scaffold was chosen to be modified with diverse substitution patterns and electronic effects. The electronic properties of the indole scaffold range from electron withdrawing to electron donating in order to modulate the reactivity of the imidazole.
  • the imidazole was chosen to be linked to the indole via an amide linker because of the rigid structure and poor solubility of LOC1886.
  • the amide linker increases the polarity, when compared to compounds with an all-carbon backbone, which is beneficial for their water solubility, and this prevents aggregation. Since this work focused on the covalent rather than the noncovalent part of the intermolecular interaction between GPX4 and the ligand, relatively small scaffolds were chosen, resulting in fragment-like compounds (Scheme 1).
  • LOC1886 analogs were synthesized and characterized through the assay funnel, and majority of the analogs exhibited enhanced affinity for GPX4 protein (FIG. 3). Specifically, LOC1886 analogs with halogen substitution on the indole ring, such as QW-002 and QW-052, present the most significant improvement on GPX4 binding. Considering that LOC1886 and its analogs are covalent inhibitors, a LCMS-based assay was developed to evaluate and compare the covalent binding potencies of GPX4 covalent inhibitors, in which the doseresponse bound percentage was calculated from the relative intensities of apo protein and protein-inhibitor complex. Accordingly, it was found QW57, QW64, and GRL-0496 featured better or comparable covalent binding potencies as LOC1886 (FIG. 4).
  • Scheme 1a Scaffolds diversed in substitution patterns, and electronic effects
  • LOC880 analogs were synthesized in good to excellent yields by reacting a library of natural/unnatural a-amino acids, /3-amino acids and y/w-amino acids with different phthalic anhydrides, and te/Y-butyl alcohol to furnish the corresponding library. All the substrates furnished the expected LOC880 analogs, almost irrespective of the electronic and steric factors of the substituents present.
  • 2,3-naphthalic anhydride demonstrated good behavior in the methodology.
  • Several analogs from the library were evaluated by thermal denaturation assay and microscale thermophoresis (MST) to validate their bindings to GPX4. All tested compounds showed enhanced binding affinities compared to original LOC880 scaffold, the apparent K d of QW-011 and QW-017 towards GPX4 is in the sub-micromolar range (FIG. 6).
  • LOC1886/QW-044, QW- 078, QW-095, and QW-105 show increased lipid peroxidation that is rescuable by ferrostatin-1 , a ferroptosis inhibitor, potentially suggesting GPX4 inhibition in cells.
  • cellular dose response assays with HT1080, and HepG2, Huh7 and Skhep-1 liver cancer cells have not shown ferrostatin-1 rescue, and Glso values for most compounds have been in the micromolar range (FIG. 10).
  • QW-148, QW-152, QW-156, QW-158 and QW-162 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines (FIGS.
  • FIGS. 13A-13H depict the cartoon representation of the crystal structures of GPX4 U46C with RSL3, ML162, CDS9, and TMT10. As shown in the figure, these four covalent inhibitors are selectively bound to residue Cys-66, as opposed to MAC-5576 and LOC1886, which bound to both Cys-10 and Cys-66 (FIG. 131).
  • Example 2 Large-scale screen for novel compounds that bind in the RSL3-binding site of GPX4
  • DELopen compounds have undergone initial testing in the NADPH- coupled activity assay with many compounds showing promising effects with over 80% inhibition (FIG. 15). Typically, compounds were initially assessed at 1mM, but maximal concentrations were selected for testing the DELopen hits based on solubility limitations in the assay buffer and observed interference with the NADPH absorbance signal at 340nm. Further validation and characterization will be performed via both NADPH-coupled and LC/MS-based GPX4 activity assays. Downstream cellular assays will be performed on the most promising candidates. Meanwhile, more analogs of DEL_B1 , DEL_B2 and DEL_F will be synthesized and tested. Exemplary analogs are listed below:

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Abstract

The present disclosure provides, inter alia, compounds to modulate GPX4 activity. Also provided are pharmaceutical compositions containing same compounds. Further provided are methods for treating or ameliorating the effects of a cancer in a subject, methods of modulating GPX activity in a subject, methods of inducing ferroptosis in a cell, and methods for treating or ameliorating the effects of a cancer in a subject using the compounds or composition in combination with other therapeutic agents.

Description

GPX4 INHIBITORS AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims benefit of U.S. Provisional Patent Application Serial No. 63/242,829, filed on September 10, 2021 , which application is incorporated by reference herein in its entirety.
FIELD OF DISCLOSURE
[0002] The present disclosure provides, inter alia, compounds to modulate GPX4 activity. Also provided are pharmaceutical compositions containing the compounds, as well as methods of using such compounds and compositions.
GOVERNMENT FUNDING
[0003] This disclosure was made with government support under grant no. CA209896, awarded by National Institutes of Health. The government has certain rights in the disclosure.
BACKGROUND OF THE DISCLOSURE
[0004] Cancer cells are dependent on their lipid composition for establishing and modulating membrane structural integrity, morphology, metabolism, migration, invasiveness, and other functions. For example, among the thousands of lipid species that compose eukaryotic cell membranes, the abundance and localization of polyunsaturated-fatty-acid-(PUFA)-containing phospholipids (PUFA-PLs) is a major factor in determining the fluidity of cell membranes (Agmon et al. 2017). Since the cis conformation of double bonds in PUFA-PLs hinders efficient stacking of fatty acyl tails, elevated levels of PUFA-PLs contribute to increasing membrane fluidity and thinning (Agmon et al. 2018). PUFA-PLs are, however, susceptible to peroxidation via iron-catalyzed reaction with molecular oxygen at bis-allylic positions, catalyzed by lipoxygenases and labile iron (Yang et al. 2016). Thus, some cancer cells depend on a critical network of proteins to eliminate their PUFA-PL peroxides; a key protein at the center of this defense network is the selenoprotein glutathione peroxidase 4 (GPX4). When GPX4 activity is compromised, lipid peroxidation can cause ferroptosis (Stockwell et al. 2017), an oxidative, iron-dependent form of non- apoptotic cell death (Dixon et al. 2012). Ferroptosis acts as a natural tumor suppressive and immune surveillance mechanism, and can be induced by exogenous agents in cells that are addicted to GPX4 (Dixon and Stockwell, 2019). Cancer cells from tissues of diverse origins have been screened for their sensitivity to ferroptosis-inducing compounds (Viswanathan et al. 2017). It has been found that ferroptosis inducers, including GPX4 inhibitors, selectively target cancers with a mesenchymal or otherwise drug-resistant signature (Viswanathan et al. 2017). Consistent with the mesenchymal state being associated with drug resistance, an independent study on persister cancer cells, which are proposed to escape from conventional cytotoxic treatment through a dormant state and then revive to cause tumor relapse, revealed a similar selective dependency on GPX4 (Hangauer et al. 2017).
[0005] Examination of persister cells also revealed upregulation of mesenchymal markers and downregulation of epithelial markers (Hangauer et al. 2017). Overexpression of mesenchymal state genes is associated with epithelial- mesenchymal transition (EMT). Since EMT increases motility of tumor cells and enables the invasion of primary tumors to distant sites, EMT is a key step in metastasis. EMT also renders cancer cells resistant to apoptosis and chemotherapy (Viswanathan et al. 2017). EMT requires plasma membrane remodeling to increase fluidity, which is associated with elevated biosynthesis of PUFA-PLs. Given that PUFA-PLs are more susceptible to peroxidation than saturated or mono-unsaturated fatty acid PLs, cells in an EMT state have increased dependency on GPX4 to remove these lipid peroxides (Viswanathan et al. 2017). Therefore, cancer cells undergoing EMT that acquire resistance to apoptosis become vulnerable to lipid peroxidation and ferroptosis induced by GPX4 inhibition (Viswanathan et al. 2017). As cancer cells evolve into a high-mesenchymal drug-resistant state and become resistant to apoptosis, one may selectively target such cells through ferroptosis; the most effective compounds in this context are GPX4 inhibitors (Viswanathan et al. 2017). For example, in-vivo xenografts of GPX4-knockout high-mesenchymal therapy-resistant melanoma regressed after cessation of ferrostatin-1 (a lipophilic antioxidant discovered in the Stockwell Lab that suppresses the loss of GPX4) and did not relapse after ceasing dabrafenib and trametinib treatment, while wt GPX4 xenografts continued to grow in both experiments (Viswanathan et al. 2017). GPX4 inhibitors are selectively lethal to persister and EMT cancer cells, with minimal effects on parental cells and non-transformed cells, suggesting that addiction to GPX4 creates a large therapeutic window.
[0006] Accordingly, there is a need for developing GPX4 inhibitors for the treatment of aggressive drug-resistant cancers and other GPX4-associated diseases. This disclosure is directed to meeting these and other needs.
SUMMARY OF THE DISCLOSURE
[0007] One of the most pressing problems in oncology is metastatic, drug- resistant cancers. Indeed, most deaths of cancer patients are caused by aggressive, metastatic, drug-resistant cancers. A surprising finding is that as cancers evolve into aggressive and drug-resistant forms, they acquire an exquisite sensitivity to GPX4 inhibition. These data provide the tantalizing possibility that the most aggressive neoplastic diseases can be treated through the use of GPX4 inhibitors, and that the ideal patients for treatment with these inhibitors are end-stage patients that have exhausted other therapeutic options. In 2012, we reported the existence of a new form of tumor suppressive cell death, ferroptosis (Dixon et al. 2012). In 2014, we discovered that the key negative regulator of ferroptosis was the lipid repair enzyme GPX4, demonstrating that GPX4 functions in ferroptosis in a manner analogous to how the oncogene Bcl-2 functions in apoptosis (Yang et al. 2014). We discovered the first GPX4 inhibitor -- the nanomolar potency small molecule RSL3 (Yang et al. 2014). In 2017, we reported that cancer cells that have undergone epithelial-to- mesenchymal (EMT) transition become hypersensitive to ferroptosis, and to GPX4 inhibitors (Viswanathan et al. 2017). We also discovered how RSL3 inhibits GPX4, obtaining a co-crystal structure of RSL3 bound to GPX4, which revealed a novel drug-binding site on GPX4. We propose, inter alia, to exploit this finding to discover drug-like GPX4 inhibitors with favorable ADMET properties that can be developed as first in class GPX4 inhibitors for drug-resistant cancers having a high EMT gene expression signature.
[0008] Accordingly, one embodiment of the present disclosure is a compound according to formula (1):
Figure imgf000006_0001
wherein:
R1, R2, R3, R4, R5 R6 and R7 are independently selected from the group consisting of H, D, -OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N, halo, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6 alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N,S, halo, C1-4alkyl, CF3, -CN and combinations thereof, with the proviso that the compound is not
Figure imgf000007_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0009] Another embodiment of the present disclosure is a compound according to formula (2):
Figure imgf000007_0002
wherein: n is a positive integer;
R1, R2, R3, and R4 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkyl- heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated Cs-i2carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated Cs-i2carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, R5 and R6 are independently selected from the group consisting of no atom, H, D, - OH, halo, ether, ester, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkylheteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl, wherein the ether, ester, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, epoxy, -OH, halo, C1-4alkyl, CF3, benzyloxy, indole, methyl-indole, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, R7 is selected from the group consisting of 0, N, aryl, C1-6-cycloalkyl, piperazine, and alkyl-aryl, wherein the aryl, C1-6-cycloalkyl, piperazine, and alkyl-aryl may be optionally substituted with an atom or a group selected from the group consisting of N, 0, S, epoxy, -OH, halo, C1-4alkyl, CF3, -(O)C(R), indole, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester, ketone and combinations thereof,
Rs is selected from the group consisting of no atom, H, D, -OH, halo, -Si(CH3)3, - Sn(CH3)3, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl- heteroaryl, wherein the alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, -NO2, epoxy, -OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N, halo, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6 alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N, S, halo, C1-4alkyl, CF3, -CN and combinations thereof, with the proviso that the compound is not
Figure imgf000009_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0010] Another embodiment of the present disclosure is a compound according to formula (3):
Figure imgf000009_0002
wherein:
X is selected from the group consisting of N, S, and SO2;
R1 , R2, R3, R4 and R7 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkylheteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, R5 and R6 are independently selected from the group consisting of no atom, H, H, D, -OH, halo, ether, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, C1-6-cycloalkyl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl, wherein the ether, alkyl, aryl, C1-6-cycloalkyl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl- heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, epoxy, -OH, halo, -(O)C(R), unsubstituted or substituted aryl, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R5 and R6 may together with the N atom attached to form a saturated or unsaturated heterocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N, halo, ether, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6 alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N, S, halo, C1-4alkyl, CF3, -CN, and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0011] Another embodiment of the present disclosure is a compound having any one of the following structures:
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
and
Figure imgf000017_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0012] Another embodiment of the present disclosure is a composition, including pharmaceutical compositions, comprising one or more compounds disclosed herein and a pharmaceutically acceptable carrier, adjuvant or vehicle.
[0013] Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
[0014] Another embodiment of the present disclosure is a method for modulating the activity of glutathione peroxidase 4 (GPX4) in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
[0015] Another embodiment of the present disclosure is a method for increasing the level of peroxide in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
[0016] A further embodiment of the present disclosure is a method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
[0017] Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, which is one or more compounds disclosed herein or one or more compositions disclosed herein, and ii) an effective amount of a second anti-cancer agent.
[0018] An additional embodiment of the present disclosure is a kit for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein, packaged with its instructions for use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] To facilitate further description of the embodiments of this disclosure, the following drawings are provided to illustrate and not to limit the scope of the disclosure.
[0020] FIGS. 1A-1D show MST binding traces of LOC1886 (A) and LOC880 (B) as well as crystal structure of GPX4U46C with LOC1886 (C and D). [0021] FIG. 2 is a schematic of assay funnel for candidate validation.
[0022] FIG. 3 shows observed KdS for representative LOC1886 analogs, measured by MST. Enhancement on binding affinities were reported for all analogs tested.
[0023] FIG. 4 shows intact LC-MS analysis of GPX4 pre-incubated with LOC1886 analogs. GRL-0496 is a validated commercial LOC1886 analog.
[0024] FIGS. 5A-5C show characterizations of representative LOC1886 analogs. (A) MST binding summaries; (B) LC-MS detection of covalently modified GPX4; (C) NADPH-coupled GPX4 inhibition assay.
[0025] FIG. 6 shows observed KdS for representative LOC880 analogs, measured by MST.
[0026] FIG. 7 shows NADPH-coupled GPX4 inhibition assay for representative LOC880 analogs.
[0027] FIGS. 8A-8C show SAR (structure-activity relationship) analysis from MST data. (A) Effect of linker length in region B on binding affinities of LOC880 analogs; (B) Effect of region A indole ring substitution on binding affinities of LOC880 analogs; (C) Effect of region B side chain on binding affinities of LOC880 analogs.
[0028] FIG. 9 shows C11-BODIPY lipid peroxidation flow cytometry assay in HT1080 cells for representative analogs.
[0029] FIG. 10 shows GI50 of selected analogs.
[0030] FIGS. 11A-11F show cellular dose response assays of representative LOC1886 analogs with HT1080, HLF, and HepG2, Huh7 and Skhep-1 liver cancer cells. (A) RSL3 Control - all cell lines undergo ferrostatin-rescuable cell death; (B) QW-148 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines; (C) QW-152 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines; (D) QW-156 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines; (E) QW-158 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines; (F) QW-162 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines. [0031] FIG. 12 shows lipid peroxidation flow cytometry assay of selected LOC1886 analogs.
[0032] FIGS. 13A-13I show crystal structures of GPX4U46C with RSL3, ML162, CDS9, TMT10 and LOC1886. (A) A cartoon representation of a pseudotrimer of GPX4U46C bound to RSL3. (B) A close-up view of the GPX4U46C residues that interact with RSL3. RSL3 molecules and the side chain of GPX4 residues are shown with stick models. A 2Fo-Fc omit map at 3. (grey mesh) on one of the RSL3 molecules is also shown. (C) A cartoon representation of a pseudotrimer of GPX4U46C bound to ML162. (D) A close-up view of the GPX4U46C residues that interact with ML162. (E) A cartoon representation of a pseudo-trimer of the mutant (R1 52H) GPX4U46C bound to CDS9. (F) A close-up view of the mutant GPX4U46C residues that interact with CDS9. (G) A cartoon representation of a pseudo-trimer of the mutant (R152H) GPX4U46C bound to TMT10. (H) A close-up view of the mutant GPX4U46C residues that interact with TMT10. (I) A close-up view of the GPX4U46C residues that interact with LOC1886.
[0033] FIG. 14 shows MST screening of selected DEL compounds against GPX4.
[0034] FIG. 15 shows NADPH-coupled GPX4 activity assay for selected DELopen compounds.
[0035] FIG. 16 shows crystal hit for GPX4U46C in presence of selected DEL compounds, and LOC880, LOC1886 analogs.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0036] In the present disclosure, improved analogs of identified GPX4 binders were created and tested. The output of these studies serves as a starting lead that can be evaluated later for PK/PD, safety, and selectivity. In parallel, the screening assay is optimized, and triage compounds that emerge from the screen are evalutated.
[0037] Structure-Activity Relationship (SAR) optimizations on LOC1886 and LOC880 were conducted. Several analogs of LOC880 and LOC1886 were identified with better binding affinity to GPX4, according to MST tests and their structures provided us with novel pharmacophores for further optimization. MST measurement for the remaining analogs and detailed biophysical and biochemical characterizations are currently tested. GPX4-U46C was crystallized in complex with LOC1886. The crystals are small and diffracted X-ray at APS beam line NE_24ID_C poorly (~4 angstrom).
[0038] Accordingly, one embodiment of the present disclosure is a compound according to formula (1):
Figure imgf000021_0001
wherein:
R1, R2, R3, R4, RS R6 and R7 are independently selected from the group consisting of H, D, -OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated Cs-i2carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated Cs-i2carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, O, N, halo, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6 alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of O, N,S, halo, C1-4alkyl, CF3, -CN and combinations thereof, with the proviso that the compound is not
Figure imgf000022_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0039] In some embodiments, the compound has a structure selected from the group consisting of:
Figure imgf000022_0002
Figure imgf000023_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0040] In some embodiments, the compound has the structure of formula (1a):
Figure imgf000023_0002
wherein: a dashed line indicates the presence of an optional double bond;
R1, R2, R3, and R4 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkylheteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof,
Rs is selected from the group consisting of H, D, C, 0, N and halo, wherein R is selected from the group consisting of H, D, 0, N, halo, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6 alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N, S, halo, C1-4alkyl, CF3, -CN and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0041] In some embodiments, the compound has a structure selected from the group consisting of:
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
and
Figure imgf000029_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0042] Another embodiment of the present disclosure is a compound according to formula (2):
Figure imgf000030_0001
wherein: n is a positive integer;
R1, R2, R3, and R4 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkylheteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated Cs-i2carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated Cs-i2carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, R5 and R6 are independently selected from the group consisting of no atom, H, D, - OH, halo, ether, ester, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkyl- heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl, wherein the ether, ester, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, epoxy, -OH, halo, C1-4alkyl, CF3, benzyloxy, indole, methyl-indole, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, R7 is selected from the group consisting of 0, N, aryl, C1-6-cycloalkyl, piperazine, and alkyl-aryl, wherein the aryl, C1-6-cycloalkyl, and alkyl-aryl may be optionally substituted with an atom or a group selected from the group consisting of N, 0, S, epoxy, -OH, halo, C1-4alkyl, CF3, -(O)C(R), indole, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester, ketone and combinations thereof,
Rs is selected from the group consisting of no atom, H, D, -OH, halo, -Si(CH3)3, - Sn(CH3)3, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenylheteroaryl, wherein the alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, -NO2, epoxy, -OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N, halo, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6 alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N, S, halo, C1-4alkyl, CF3, -CN and combinations thereof, with the proviso that the compound is not
Figure imgf000031_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0043] In some embodiments, the compound has a structure selected from the group consisting of:
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0044] In other aspects of the present disclosure, 4.4 billion compounds from the Wuxi DELopen DNA-encoded library were screened against GPX4. Top hits from the DEL screening, which featured over 10,000-fold enrichment on the immobilized GPX4 target over the negative control, were resynthesized. Preliminary MST tests of selected DELopen compounds further validated the outcomes of the screening, and all selected compounds showed lower micromolar to nanomolar binding affinity. All DELopen compounds have undergone initial testing in the NADPH-coupled activity assay with many compounds showing promising effects with over 80% inhibition. Further validation and characterization will be performed via both NADPH-coupled and LC/MS-based GPX4 activity assays. Downstream cellular assays will be performed on the most promising candidates. The structures of all DELopen compounds that are tested positive of GPX4 binding haven been acquired, SAR optimization of the DELopen compounds is currently ongoing. In addition, another high-throughput screen was performed using an Enamine Diversity Library of 60,638 compounds. The top ~180 hits are currently being validated. Further biophysical and biochemical characterizations of the validated hits will follow.
[0045] Accordingly, another embodiment of the present disclosure is a compound according to formula (3):
Figure imgf000038_0001
wherein:
X is selected from the group consisting of N, S, and SO2;
R1 , R2, R3, R4 and R7 are independently selected from the group consisting of H, D, - OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkylheteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, - OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, R5 and R6 are independently selected from the group consisting of no atom, H, H, D, -OH, halo, ether, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, C1-6-cycloalkyl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl, wherein the ether, alkyl, aryl, C1-6-cycloalkyl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl- heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, epoxy, -OH, halo, -(O)C(R), unsubstituted or substituted aryl, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R5 and R6 may together with the N atom attached to form a saturated or unsaturated heterocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N, halo, ether, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, Ci- ealkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N, S, halo, C1-4alkyl, CF3, -CN, and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0046] In some embodiments, the compound has a structure selected from the group consisting of:
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
and
Figure imgf000047_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0047] Another embodiment of the present disclosure is a compound having any one of the following structures:
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
and
Figure imgf000054_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
[0048] Another embodiment of the present disclosure is a composition, including pharmaceutical compositions, comprising one or more compounds disclosed herein and a pharmaceutically acceptable carrier, adjuvant or vehicle.
[0049] Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
[0050] In some embodiments, the GPX4-associated disease is selected from the group consisting of a cancer, a neurotic disorder, a neurodegenerative disorder, spondylometaphyseal dysplasia, mixed cerebral palsy, pontocerebellar hypoplasia, and male infertility.
[0051] In some embodiments, the GPX4-associated disease is a cancer. Nonlimiting examples of cancer include hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma. In some embodiments, the cancer is hepatocellular carcinoma.
[0052] In some embodiments, the subject is a mammal. In some embodiments, the mammal is selected from the group consisting of humans, veterinary animals, and agricultural animals. In some embodiments, the subject is a human.
[0053] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is under epithelial-to-mesenchymal (EMT) transition. In some embodiments, the cancer is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the cancer is refractory to standard cancer treatment. Nonlimiting examples of standard cancer treatment include chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof.
[0054] Another embodiment of the present disclosure is a method for modulating the activity of glutathione peroxidase 4 (GPX4) in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein. In some embodiments, the modulation comprises inhibiting GPX4 activity.
[0055] Another embodiment of the present disclosure is a method for increasing the level of peroxide in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein. Non-limiting examples of peroxide inclcude hydrogen peroxide, organic hydroperoxide, lipid peroxide, and combinations thereof.
[0056] A further embodiment of the present disclosure is a method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein.
[0057] In some embodiments, the cell has abberant lipid accumulation. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma. In some embodiments, the cancer is hepatocellular carcinoma.
[0058] In some embodiments, the cell is a human cell. In some embodiments, wherein the cancer cell is metastatic. In some embodiments, the cancer cell is under epithelial-to-mesenchymal (EMT) transition. In some embodiments, the cancer cell is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the hypersensitivity to ferropotosis is identified by NADPH abundance, GCH1 expression, NF2-YAP activity, EMT signature, and GPX4 expression. In some embodiments, the cancer cell is refractory to standard cancer treatment. Non-limiting examples of standard cancer treatment includes chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof.
[0059] Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, which is one or more compounds disclosed herein or one or more compositions disclosed herein, and ii) an effective amount of a second anti-cancer agent.
[0060] In some embodiments, the second anti-cancer agent is selected from the group consisting of chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof. In some embodiments, the second anticancer agent is an immunotherapy, such as checkpoint inhibitor therapy including PD-1 and CTLA-4 inhibitor therapy. Non-limiting examples of immunotherapy include ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, ofatumumab, blinatumomab, daratumumab, elotuzumab, obinutuzumab, talimogene laherparepvec, necitumumab, lenalidomide, dinutuximab, and combinations thereof.
[0061] In some embodiments, the subject is a human.
[0062] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is under epithelial-to-mesenchymal (EMT) transition. In some embodiments, the cancer is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the cancer is refractory to standard cancer treatment.
[0063] In some embodiments, the cancer is selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma. In some embodiments, the cancer is hepatocellular carcinoma.
[0064] In some embodiments, the first anti-cancer agent is administered to the subject before, concurrently with, or after the second anti-cancer agent.
[0065] An additional embodiment of the present disclosure is a kit for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising an effective amount of one or more compounds disclosed herein or one or more compositions disclosed herein, packaged with its instructions for use.
[0066] The kits may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each compound of the present disclosure (which, e.g., may be in the form of pharmaceutical compositions) and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the active agents to subjects. The compounds and/or pharmaceutical compositions of the disclosure and other reagents may be present in the kits in any convenient form, such as, e.g., in a solution or in a powder form. The kits may further include a packaging container, optionally having one or more partitions for housing the compounds and/or pharmaceutical compositions and other optional reagents.
[0067] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.
[0068] As used herein, the terms “ameliorate”, "ameliorating" and grammatical variations thereof mean to decrease the seventy of the symptoms of a disease in a subject.
[0069] As used herein, a “subject” is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present disclosure include, for example, agricultural animals, veterinary animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of veterinary animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. in the context of the present disclosure, the phrase “a subject in need thereof” means a subject in need of treatment for a GPX4-associated disorder, such as, e.g., a cancer. Alternatively, the phrase “a subject in need thereof” menas a subject diagnosed with a GPX4-associated disorder, such as, e.g., a cancer.
[0070] As used herein, “lipid peroxidation” means the oxidative degradation of fats, oils, waxes, sterols, triglycerides, and the like. Lipid peroxidation has been linked with many degenerative diseases, such as atherosclerosis, ischemia- reperfusion, heart failure, Alzheimer’s disease, rheumatic arthritis, cancer, and other immunological disorders. (Ramana et al., 2013).
[0071] As used herein, “ferroptosis” means regulated cell death that is iron- dependent. Ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid reactive oxygen species. (Dixon et al., 2012) Ferroptosis is distinct from apoptosis, necrosis, and autophagy. (Id.) [0072] As used herein, the terms “modulate”, “modulating”, “modulator” and grammatical variations thereof mean to change, such as increasing or decreasing the activity of GPX4. In this embodiment, “contacting” means bringing the compound and optionally one or more additional therapeutic agents into close proximity to the cells in need of such modulation. This may be accomplished using conventional techniques of drug delivery to the subject or in the in vitro situation by, e.g., providing the compound and optionally other therapeutic agents to a culture media in which the cells are located.
[0073] As used herein, a "pharmaceutically acceptable salt" means a salt of the compounds of the present disclosure which are pharmaceutically acceptable, as defined herein, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2- hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2- naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4- methylbicyclo[2.2.2]oct-2-ene-1 -carboxylic acid, glucoheptonic acid, 4,4'- methylenebis(3-hydroxy-2-ene-1 -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.
[0074] In the present disclosure, an "effective amount" or “therapeutically effective amount” of a compound or pharmaceutical composition is an amount of such a compound or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of the subject, and like factors well known in the arts of, e.g., medicine and veterinary medicine. In general, a suitable dose of a compound or pharmaceutical composition according to the disclosure will be that amount of the compound or composition, which is the lowest dose effective to produce the desired effect with no or minimal side effects. The effective dose of a compound or pharmaceutical composition according to the present disclosure may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
[0075] A suitable, non-limiting example of a dosage of a compound or pharmaceutical composition according to the present disclosure or a composition comprising such a compound, is from about 1 ng/kg to about 1000 mg/kg, such as from about 1 mg/kg to about 100 mg/kg, including from about 5 mg/kg to about 50 mg/kg. Other representative dosages of a compound or a pharmaceutical composition of the present disclosure include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg.
[0076] A compound, composition, or pharmaceutical composition of the present disclosure may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a compound, composition, or pharmaceutical composition of the present disclosure may be administered in conjunction with other treatments. A compound, composition, or pharmaceutical composition of the present disclosure may be encapsulated or otherwise protected against gastric or other secretions, if desired. [0077] The compositions or pharmaceutical compositions of the disclosure are pharmaceutically acceptable and comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable carriers or diluents and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the compounds/compositions/pharmaceutical compositions of the present disclosure are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.). More generally, “pharmaceutically acceptable” means that which is useful in preparing a composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
[0078] Pharmaceutically acceptable carriers and diluents are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable carrier or diluent used in a composition of the disclosure must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Carriers or diluents suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers or diluents for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
[0079] The compositions or pharmaceutical compositions of the disclosure may, optionally, contain additional ingredients and/or materials commonly used in such compositions. These ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface- active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monosterate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21 ) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.
[0080] Compounds, compositions or pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in- water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.
[0081] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers or diluents and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.
[0082] Liquid dosage forms for oral administration include pharmaceutically- acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.
[0083] Compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.
[0084] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier or diluent. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.
[0085] Compositions suitable for parenteral administrations comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
[0086] In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.
[0087] The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
[0088] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier or diluent, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
[0089] In the foregoing embodiments, the following definitions apply.
[0090] The term “aliphatic”, as used herein, refers to a group composed of carbon and hydrogen that do not contain aromatic rings. Accordingly, aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclyl groups. Additionally, unless otherwise indicated, the term “aliphatic” is intended to include both "unsubstituted aliphatics" and "substituted aliphatics", the latter of which refers to aliphatic moieties having substituents replacing a hydrogen on one or more carbons of the aliphatic group. Such substituents can include, for example, a halogen, a deuterium, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, an aromatic, or heteroaromatic moiety.
[0091] The term "alkyl" refers to the radical of saturated aliphatic groups that does not have a ring structure, including straight-chain alkyl groups, and branched- chain alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chains, C3-C6 for branched chains). In other embodiments, the “alkyl” may include up to twelve carbon atoms, e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 or C12. Such substituents include all those contemplated for aliphatic groups, as discussed below, except where stability is prohibitive.
[0092] The term “alkenyl”, as used herein, refers to an aliphatic group containing at least one double bond and unless otherwise indicated, is intended to include both "unsubstituted alkenyls" and "substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents include all those contemplated for aliphatic groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
[0093] Moreover, unless otherwise indicated, the term "alkyl" as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Indeed, unless otherwise indicated, all groups recited herein are intended to include both substituted and unsubstituted options.
[0094] The term “Cx-y” when used in conjunction with a chemical moiety, such as, alkyl and cycloalkyl, is meant to include groups that contain from x to y carbons in the chain. For example, the term “Cx-yalkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-tirf luoroethyl, etc.
[0095] The term “aryl” as used herein includes substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 3- to 8-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
[0096] The term “alkyl-aryl” refers to an alkyl group substituted with at least one aryl group.
[0097] The term “alkyl-heteroaryl” refers to an alkyl group substituted with at least one heteroaryl group.
[0098] The term “alkenyl-aryl” refers to an alkenyl group substituted with at least one aryl group.
[0099] The term “alkenyl-heteroaryl” refers to an alkenyl group substituted with at least one heteroaryl group.
[0100] The terms “carbocycle”, “carbocyclyl”, and “carbocyclic”, as used herein, refer to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. Preferably a carbocycle ring contains from 3 to 10 atoms, more preferably from 3 to 8 atoms, including 5 to 7 atoms, such as for example, 6 atoms. The term “cabocycle” also includes bicycles, tricycles and other multicyclic ring systems, including the adamantyl ring system.
[0101] The terms “halo” and “halogen” are used interchangeably herein and mean halogen and include chloro, fluoro, bromo, and iodo.
[0102] The term “heteroaryl” includes substituted or unsubstituted aromatic single ring structures, preferably 3- to 8-membered rings, more preferably 5- to 7- membered rings, even more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term “heteroaryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
[0103] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur; more preferably, nitrogen and oxygen.
[0104] The term “ketone” means an organic compound with the structure RC(=O)R', wherein neither R nor R' can be hydrogen atoms.
[0105] The term “ether” means an organic compound with the structure R-O- R’, wherein neither R nor R' can be hydrogen atoms.
[0106] The term “ester” means an organic compound with the structure RC(=O)OR’, wherein neither R nor R' can be hydrogen atoms.
[0107] The term “polyyne” means is an organic compound with alternating single and triple bonds; that is, a series of consecutive alkynes, (-C=C-) n with n greater than 1 .
[0108] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
[0109] As set forth previously, unless specifically stated as “unsubstituted,” references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.
[0110] As used herein, the term “oxadiazole” means any compound or chemical group containing the following structure:
Figure imgf000069_0001
[0111] As used herein, the term “oxazole” means any compound or chemical group containing the following structure:
Figure imgf000069_0002
[0112] As used herein, the term “triazole” means any compound or chemical group containing the following structure:
Figure imgf000069_0003
[0113] As used herein, the term “indole” means any compound or chemical group containg the following structure:
Figure imgf000070_0001
[0114] It is understood that the disclosure of a compound herein encompasses all stereoisomers of that compound. As used herein, the term "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures which are not interchangeable. The three-dimensional structures are called configurations. Stereoisomers include enantiomers and diastereomers.
[0115] The terms "racemate" or "racemic mixture" refer to a mixture of equal parts of enantiomers. The term "chiral center" refers to a carbon atom to which four different groups are attached. The term "enantiomeric enrichment" as used herein refers to the increase in the amount of one enantiomer as compared to the other.
[0116] It is appreciated that to the extent compounds of the present disclosure have a chiral center, they may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present disclosure encompasses any racemic, optically-active, diastereomeric, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the disclosure, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).
[0117] Examples of methods to obtain optically active materials are known in the art, and include at least the following: i) physical separation of crystals--a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct; ii) simultaneous crystallization--a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state; iii) enzymatic resolutions--a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; iv) enzymatic asymmetric synthesis--a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v) chemical asymmetric synthesis--a synthetic technique whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry (i.e., chirality) in the product, which may be achieved using chiral catalysts as disclosed in more detail herein or chiral auxiliaries; vi) diastereomer separations--a technique whereby a racemic compound is reacted with an enantiomerically pure reagent (the chiral auxiliary) that converts the individual enantiomers to diastereomers. The resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer; vii) first- and second-order asymmetric transformations--a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer. The desired enantiomer is then released from the diastereomer; viii) kinetic resolutions--this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions; ix) enantiospecific synthesis from non-racemic precursors--a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis; x) chiral liquid chromatography--a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase. The stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions; xi) chiral gas chromatography--a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase; xii) extraction with chiral solvents--a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; xiii) transport across chiral membranes--a technique whereby a racemate is placed in contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane which allows only one enantiomer of the racemate to pass through.
[0118] The stereoisomers may also be separated by usual techniques known to those skilled in the art including fractional crystallization of the bases or their salts or chromatographic techniques such as LC or flash chromatography. The (+) enantiomer can be separated from the (-) enantiomer using techniques and procedures well known in the art, such as that described by J. Jacques, et al., Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc., 1981. For example, chiral chromatography with a suitable organic solvent, such as ethanol/acetonitrile and Chiralpak AD packing, 20 micron can also be utilized to effect separation of the enantiomers [0119] The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.
EXAMPLES
Example 1
Improved analogs of fragments and leads that bind to GPX4
[0120] Glutathione peroxidase 4 (GPX4) has been reported to be a promising therapeutic target for metastatic and drug-resistant cancers, based on the elevated dependency of the cancer cells on GPX4 lipid peroxide repair pathway during epithelial-mesenchymal transition (EMT) and the transformation into therapy-tolerant persister states. We proposed to exploit this finding to discover drug-like GPX4 inhibitors with favorable ADMET properties that can be developed as a first in class GPX4 inhibitor for drug-resistant cancers having a high EMT gene expression signature.
[0121] LOC1886 and LOC880 are GPX4 allosteric inhibitors identified from the initial biophysical screening of 10,095 Lead Optimized Compounds (LOC). LOC1886 covalently modifies GPX4 Cys66 (allosteric site 1 ) and LOC880 binds to E50-F92-S44 (allosteric site 2) with a d of 8 μM (FIGS. 1A-1D). By employing the established assay funnel designed for quick identification of potential lead compounds (FIG. 2), the SAR optimizations on LOC1886 and LOC880 were conducted and provided herein.
SAR of LOC1886
[0122] For SAR optimizations based on LOC1886 scaffold, the imidazole group functions as an electrophile for the covalent modification of GPX4, hence remains unmodified. The indole scaffold was chosen to be modified with diverse substitution patterns and electronic effects. The electronic properties of the indole scaffold range from electron withdrawing to electron donating in order to modulate the reactivity of the imidazole. In addition, the imidazole was chosen to be linked to the indole via an amide linker because of the rigid structure and poor solubility of LOC1886. The amide linker increases the polarity, when compared to compounds with an all-carbon backbone, which is beneficial for their water solubility, and this prevents aggregation. Since this work focused on the covalent rather than the noncovalent part of the intermolecular interaction between GPX4 and the ligand, relatively small scaffolds were chosen, resulting in fragment-like compounds (Scheme 1).
[0123] In total, nine LOC1886 analogs were synthesized and characterized through the assay funnel, and majority of the analogs exhibited enhanced affinity for GPX4 protein (FIG. 3). Specifically, LOC1886 analogs with halogen substitution on the indole ring, such as QW-002 and QW-052, present the most significant improvement on GPX4 binding. Considering that LOC1886 and its analogs are covalent inhibitors, a LCMS-based assay was developed to evaluate and compare the covalent binding potencies of GPX4 covalent inhibitors, in which the doseresponse bound percentage was calculated from the relative intensities of apo protein and protein-inhibitor complex. Accordingly, it was found QW57, QW64, and GRL-0496 featured better or comparable covalent binding potencies as LOC1886 (FIG. 4).
Figure imgf000074_0001
Scheme 1a: Scaffolds diversed in substitution patterns, and electronic effects
Figure imgf000074_0002
Scheme 1b: Am ide-linked scaffolds
Figure imgf000075_0001
Molecular Weight: 256.22 Molecular weight: 290.12
QW-049 QW-052
Figure imgf000076_0001
Molecular Weight 225,25 Molecular Weight: 226.24
QW-057 QW-065
Scheme 1. LOC1886 synthesis design and synthesized analogs.
[0124] Using newly available crystal structures as guidance, several LOC1886 analogs bearing a similar warhead as RSL3 were also designed and synthesized. (Scheme 2) Specifically, the imidazole warhead was replaced from the original LOC1886 with the chloroacetyl group from RSL3 for enhanced reactivity. The indole scaffold was connected to the warhead via a 2-methylpropyl linker that extends further into the long hydrophobic cavity, bringing the indole ring closer to W136 and allowing for improved π -π stacking. The indole ring was also modified with diverse substitution patterns and electronic effects to modulate the stacking strength.
Additionally, we chose to link the indole to the 2-methylpropyl linker via an amide bond to enhance the solubility and prevent aggregation.
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Exact Mass: 316.44 Exact Mass; 364.04
QW-168 QW-169
Scheme 2. More on LOC1886 SAR. [0125] In total, six LOC1886 analogs within this new category of SAR have been synthesized and characterized through the assay funnel, and the majority of the analogs exhibited enhanced affinity for GPX4 protein (Scheme 2, FIG. 5A). Specifically, QW-152, QW-156 and QW-158 showed 400-fold, 100-fold and 1000- fold binding enhancement, respectively, compared to the original LOC1886. The LC- MS binding assay also confirmed the total exhaustion of apo-GPX4 when incubated with 8-fold excess of inhibitors (FIG. 5B). The in vitro inhibitory efficiencies of those analogs on GPX4 are further validated using the NADPH-coupled biochemical assay. Among the 6 analogs tested, QW-152, QW-156 and QW-158 showed significant improvement on GPX4 inhibition, with 200 μM of each analog achieving comparable inhibitory output as 100 μM of RSL3 (FIG. 5C).
SAR O LOC880
[0126] For SAR optimizations based on LQC880 scaffold, 3 regions of the molecule were proposed to be individually targeted: (1) modifications on the phthalimide ring in region A by adding substituted groups to rapidly prepare a variety of analogues in a parallel chemistry format, (2) substitutions with various amino acids in region with the goal of optimizing the physicochemical properties (pKa, tPSA, MW, cLogP, and hydrogen bond donors) for good oral bioavailability and brain penetration, (3) replacement of the Tin in region C with a carbon or silicon atom while keeping the methyl groups fixed in the terminus to reduce potential toxicity (Scheme 3).
Figure imgf000081_0002
Synthesis of LOC880 analogues.
Figure imgf000081_0003
“Reagents and conditions: (i) Et3N, PhMe, reflux, 6 h; (ii) H2SO4, MgSO4, CH2CI2, overnight, it
Figure imgf000081_0001
Molecular Weight: 317.39 Molecular Weight: 351.40
QW-003 QW-005
Figure imgf000082_0002
Molecular Weight: 335.42 Molecular Weight: 261.28
QW-007 QW-010
Figure imgf000082_0003
Molecular Weight: 275.30 Molecular Weight: 289.33
QW-011 QW-017
Figure imgf000082_0001
Molecular Weight: 289.33 Molecular Weight: 275.30
QW-020 QW-023
Figure imgf000083_0001
Molecular Weight: 347.37 Molecular Weight: 404.47
QW-024 QW-026
Figure imgf000083_0002
Molecular Weight: 317.39 Molecular Weight: 334.33
QW-029 QW-033
Figure imgf000083_0003
Molecular Weight: 317.39 Molecular Weight: 337.38
QW-034 QW 036
Figure imgf000084_0001
Molecular Weight: 319.36 Molecular Weight: 457.53
QW-038 QW-043
Figure imgf000084_0002
Molecular Weight: 323.77 Molecular Weight: 317.39
QW-046 QW-050
Figure imgf000085_0001
Molecular Weight: 405.45 Molecular Weight: 351.83
QW-061 QW-063
Scheme 3. LOC880 synthesis design and synthesized analogs.
[0127] A series of LOC880 analogs were synthesized in good to excellent yields by reacting a library of natural/unnatural a-amino acids, /3-amino acids and y/w-amino acids with different phthalic anhydrides, and te/Y-butyl alcohol to furnish the corresponding library. All the substrates furnished the expected LOC880 analogs, almost irrespective of the electronic and steric factors of the substituents present. As shown in Scheme 2, various amino acids including glycine, alanine, phenylalanine, leucine, isoleucine, methionine, 2-phenylglycine, 2-methylalanine, te/t-leucine, O-benzyl-tyrosine, 1-methyl-tryptophan, glutamic acid 5-methyl ester, aspartic acid-4-te/t-butyl ester, /3-alanine, y-aminobutyric acid, and w-amino valeric acid proceeded well in this protocol. Meanwhile, phthalic anhydrides bearing electron-withdrawing groups such as 5-NO2 and 5-CI and an electron-donating group 4-OMe was compatible in this process to deliver the products. 2,3-naphthalic anhydride demonstrated good behavior in the methodology. Several analogs from the library were evaluated by thermal denaturation assay and microscale thermophoresis (MST) to validate their bindings to GPX4. All tested compounds showed enhanced binding affinities compared to original LOC880 scaffold, the apparent Kd of QW-011 and QW-017 towards GPX4 is in the sub-micromolar range (FIG. 6). Specifically, 1) replacement of tin with carbon, QW-017, has no significant effects on binding affinity; 2) chloride substitution on region A of the LOC880 scaffold improves binding affinity, while other substitutions render less efficient GPX4 binding, which indicates small electrophiles can be further explored, and 3) there is an optimal cut-off regarding the length of the carbon chain in region B, increase the length from CH2 to (CH2)3 yields 8-fold improvement on the observed binding affinity, while further increase in the length to (CH2)3 negatively impacts the binding affinity.
[0128] Following the same strategy outlined in Scheme 3, more LOC88O analogs were synthesized:
Figure imgf000087_0001
Exact Mass: 303.15 Exact Mass: 285.10
QW-069 QW-070
Figure imgf000087_0002
Exact Mass: 351.12
QW-078 QW-079
Figure imgf000087_0003
Exact Mass: 382.12 Exact Mass: 274.13
QW-087 QW-089
Figure imgf000088_0001
Exact Mass: 288.15 Exact Mass: 274.13
QW-094
QW-091
Figure imgf000089_0001
Exact Mass: 395.07 Exact Mass: 350.14
QW-095 QW-097
Figure imgf000089_0002
Exact Mass: 394.09 Exact Mass: 362.15
QW-100 QW-102
Figure imgf000089_0003
Exact Mass: 366.19
Exact Mass: 381.13
QW-106 QW-108
Figure imgf000090_0002
Figure imgf000090_0001
Exact Mass: 334.17
Exact Mass: 335.15
QW-099
QW-098
Figure imgf000090_0003
Exact Mass: 367.18
Exact Mass: 361.16
QW-105
QW-103
Figure imgf000090_0004
Exact Mass: 333.13
Exact Mass: 380.15
QW-109 QW-110
Figure imgf000091_0001
Exact Mass: 347.17 Exact Mass: 346.19
QW-114 QW-115
Figure imgf000091_0002
Exact Mass: 350.14 Exact Mass: 322.11
QW-116 QW-117
[0129] Similarly, the scope of amino acids in the above newly synthesized LOC880 analogs was explored. The reaction of amino acids, such as alanine, valine, 5-aminovaleric acid, O- tert-Butyl-L-serine under the conditions resulted in the formation of QW-089, QW-082, QW-079, QW-108 in good yields. The transformation also proceeded well for several alcohols, such as 3-methyl-1 -butanol, 3-butynol, 2- ethoxyethanol, and 4-nitrophenol, providing the desired products QW-069, QW-070, QW-074, QW-087 smoothly. Furthermore, reacting with tert-Leucine and tert-butyl alcohol, phthalic anhydrides bearing various substituted groups successfully participated in the reaction to form the desired products. Lastly, parallel synthesis of amides was conducted by replacing alcohols with amines and the primary amines underwent the reaction with phthalic anhydrides and amino acids to generate 13 corresponding products, whereas secondary amines gave 2 desired products.
[0130] More LOC880 analogs were synthesized and tested (Scheme 4):
Figure imgf000092_0001
Scheme 4. LOC880 analogs installed with a warhead via piperazine link.
[0131] Analogs of LOC880 and LOC1886 have also been screened in the NADPH-coupled GPX4 activity assay (FIGS. 5C and 7). LOC880 analogs were assessed at 1 mM, while LOC1886 analogs were assessed at 200 uM due to absorbance interference constraints. Compounds with poor solubility in the assay buffer were assessed at lower concentrations. Many analogs show improved inhibition of GPX4 activity over the parent compound. A few compounds show greater than 50% inhibition of GPX4 activity. Further characterization and validation of these hits are currently in progress, and results will be used to inform further inhibitor optimization.
[0132] In parallel, MST analysis of the synthesized LOC880 analogs have provided insights into future SAR optimizations. The full list of measured KdS of LOC880 and LOC1886 analogs can be found in Table 1. Specifically, linker length has played a major role in binding affinity, with 3- and 4-atom linker length ideal for tighter binding. (FIG. 8A) Additionally, halogen substitution on the indole ring has shown improvements in both solubility and binding affinity (FIG. 8B), and the long hydrophobic side chain in region B also enhances the observed binding affinity (FIG.
8C)
Table 1. Summary of Kd values from MST measurements.
Figure imgf000093_0002
Figure imgf000093_0001
Figure imgf000093_0003
[0133] Some of the best performing compounds thus far have been tested for induction of lipid peroxidation using flow cytometry with C11-BODIPY in HT1080 fibrosarcoma cells, an established ferroptosis model system (FIG. 9). 2.5 x 105 HT1080 cells per well were seeded in 6-well plates and cells were treated with drug the next day for 2 hours, then stained with 1.5uM C11-BODIPY for 20 min. Cells were then washed with HBSS and harvested for analysis. LOC1886/QW-044, QW- 078, QW-095, and QW-105 show increased lipid peroxidation that is rescuable by ferrostatin-1 , a ferroptosis inhibitor, potentially suggesting GPX4 inhibition in cells. However, cellular dose response assays with HT1080, and HepG2, Huh7 and Skhep-1 liver cancer cells have not shown ferrostatin-1 rescue, and Glso values for most compounds have been in the micromolar range (FIG. 10). In another set of test, QW-148, QW-152, QW-156, QW-158 and QW-162 induces ferrostatin-1 rescuable cell death in Huh7, HLF, and SKHEP-1 liver cancer cell lines (FIGS. 11A-11 F), and show increased lipid peroxidation that is rescuable by ferrostatin-1 (FIG. 12), and. Further optimization of these compounds may be required to improve potency and specificity in the cellular context. We will use the GPX4 crystal structures that we have obtained to inform the further design and development of improved molecules for synthesis.
[0134] So far, we have solved and refined six crystal structures of GPX4U46C with six different inhibitors (RSL3, ML162, CDS9, TMT10, MAC_5576, and LOC1886) covalently bound to Cys-66 of GPX4. FIGS. 13A-13H depict the cartoon representation of the crystal structures of GPX4U46C with RSL3, ML162, CDS9, and TMT10. As shown in the figure, these four covalent inhibitors are selectively bound to residue Cys-66, as opposed to MAC-5576 and LOC1886, which bound to both Cys-10 and Cys-66 (FIG. 131).
[0135] Taken together, these data suggested that we have identified several analogs of LOC880 and LOC1886 with better binding affinity to GPX4 according to MST tests, and their structures provided us with novel pharmacophores for further optimization. MST measurement for the remaining analogs and detailed biophysical and biochemical characterizations are currently underway via our established test funnels.
Example 2 Large-scale screen for novel compounds that bind in the RSL3-binding site of GPX4
[0136] Other than LOC880 and LOC1886 analogs described above, 2.8 billion compounds wer screened from the Wuxi DELopen DNA-encoded library against GPX4. HisTag-GPX4 was immobilized onto affinity matrix and incubated with pooled DEL molecules, the bound and unbound molecules were separated, and the isolated binders were decoded through DNA sequencing. Top hits from the DEL screening, which featured over 10,000-fold enrichment on the immobilized GPX4 target over the negative control, were re-synthesized without DNA tag for subsequent bioactivity confirmation (see below, DEL_A to DEL_G). Preliminary MST tests of selected DELopen compounds validated the outcomes of the screening, and all selected compounds showed lower micromolar to nanomolar binding affinity (FIG. 14).
Figure imgf000095_0001
Figure imgf000096_0001
[0137] All DELopen compounds have undergone initial testing in the NADPH- coupled activity assay with many compounds showing promising effects with over 80% inhibition (FIG. 15). Typically, compounds were initially assessed at 1mM, but maximal concentrations were selected for testing the DELopen hits based on solubility limitations in the assay buffer and observed interference with the NADPH absorbance signal at 340nm. Further validation and characterization will be performed via both NADPH-coupled and LC/MS-based GPX4 activity assays. Downstream cellular assays will be performed on the most promising candidates. Meanwhile, more analogs of DEL_B1 , DEL_B2 and DEL_F will be synthesized and tested. Exemplary analogs are listed below:
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
[0138] Structural studies of GPX4U46C with DELopen compounds and LOC880, LOC1886 analogs were also conducted. GPX4 with each of these molecules were robotically screened for crystallization conditions, from which several promising crystal hits were obtained. Images of representative hit for each GPX4- inihibitor complex is shown in FIG. 16.
Example 3
Other high-throughput screen for novel compounds that inhibit GPX4
[0139] In addition, we have completed another high-throughput screen using an Enamine Diversity Library of 60,638 compounds. Library compounds were first screened for decreased cell viability in HT1080 fibrosarcoma cells with empty vector pBP, then counter screened in HT1080 cells containing a GPX4 overexpression vector. Hits were defined as compounds for which GPX4 overexpression provided at least 20% rescue in cell viability. Some exemplary compounds are listed below:
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
DOCUMENTS CITED
1. Agmon E, Solon J, Bassereau P, Stockwell BR. Modeling the effects of lipid peroxidation during ferroptosis on membrane properties. Sci Rep. 2018;8(1 ):5155. Epub 2018/03/28. doi: 10.1038/s41598-018-23408-0. PubMed PMID: 29581451 ; PMCID: PMC5979948.
2. Agmon, E. & Stockwell, B. R. Lipid homeostasis and regulated cell death. Curr Opin Chem Biol 39, 83-89, doi: 10.1016/j.cbpa.2O17.06.002 (2017).
3. Dixon SJ, Stockwell BR. The Hallmarks of Ferroptosis. Annual Review of Cancer Biology. 2019;3:35-54.
4. Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Ce// 149, 1060-1072, doi:10.1016/j.cell.2012.03.042 (2012).
5. Hangauer, M. J. et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature 551, 247-250, doi:10.1038/nature24297 (2017).
6. Stockwell BR, Friedmann Angeli JP, Bayir H, Bush Al, Conrad M, Dixon SJ, Fulda S, Gascon S, Hatzios SK, Kagan VE, Noel K, Jiang X, Linkermann A, Murphy ME, Overholtzer M, Oyagi A, Pagnussat GC, Park J, Ran Q, Rosenfeld CS, Salnikow K, Tang D, Torti FM, Torti SV, Toyokuni S, Woerpel KA, Zhang DD. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell. 2017;171 (2):273-85. Epub 2017/10/07. doi: 10.1016/j.celL2017.09.021. PubMed PMID: 28985560; PMCID: PMC5685180.
7. Viswanathan, V. S. et al. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway. Nature 547, 453-457, doi:10.1038/nature23007 (2017).
8. Yang, W. S. et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc Natl Acad Sci U S A 113, E4966-4975, doi: 10.1073/pnas.1603244113 (2016).
9. Yang, W. S. et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317-331 , doi: 10.1016/j. cell.2013.12.010 (2014). [0140] All documents cited in this application are hereby incorporated by reference as if recited in full herein.
[0141] Although illustrative embodiments of the present disclosure have been described herein, it should be understood that the disclosure is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the disclosure.

Claims

What is claimed is:
1. A compound according to formula (1 ):
Figure imgf000109_0001
wherein:
R1, R2, R3, R4, RS R6 and R7 are independently selected from the group consisting of H, D, -OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkylaryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, -OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or
R1 and R2 may together form a saturated or unsaturated Cs-i2carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1- 4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N, halo, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6 alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of O, N,S, halo, C1- 4alkyl, CF3, -CN and combinations thereof, with the proviso that the compound is not
Figure imgf000110_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. The compound of claim 1 having the structure of formula (1a):
Figure imgf000110_0002
wherein: a dashed line indicates the presence of an optional double bond;
R1, R2, R3, and R4 are independently selected from the group consisting of H, D, -OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl- aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl- heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, -OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1- 4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof,
Rs is selected from the group consisting of H, D, C, 0, N and halo, wherein R is selected from the group consisting of H, D, 0, N, halo, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1- 6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N, S, halo, C1- 4alkyl, CF3, -CN and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. A compound according to formula (2):
Figure imgf000111_0001
wherein: n is a positive integer;
R1, R2, R3, and R4 are independently selected from the group consisting of H, D, -OH, halo, ether, ester, amide, -(O)C(R), -C(0)0R, -NO2, alkyl, aryl, alkyl- aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl- heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, -OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1- 4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, R5 and R6 are independently selected from the group consisting of no atom, H, D, -OH, halo, ether, ester, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl, wherein the ether, ester, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, epoxy, -OH, halo, C1-4alkyl, CF3, benzyloxy, indole, methyl-indole, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, R7 is selected from the group consisting of 0, N, aryl, C1-6-cycloalkyl, piperazine, and alkyl-aryl, wherein the aryl, C1-6-cycloalkyl, piperazine, and alkyl-aryl may be optionally substituted with an atom or a group selected from the group consisting of N, 0, S, epoxy, -OH, halo, C1-4alkyl, CF3, -(O)C(R), indole, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester, ketone and combinations thereof,
R8 is selected from the group consisting of no atom, H, D, -OH, halo, - Si(CH3)3, -Sn(CH3)3, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl- aryl, and alkenyl-heteroaryl, wherein the alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, -NO2, epoxy, - OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N, halo, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1- 6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N, S, halo, C1- 4alkyl, CF3, -CN and combinations thereof, with the proviso that the compound is not
Figure imgf000113_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. A compound according to formula (3):
Figure imgf000113_0002
wherein:
X is selected from the group consisting of N, S, and SO2;
R1, R2, R3, R4 and R7 are independently selected from the group consisting of H, D, -OH, halo, ether, ester, amide, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide, wherein the ether, ester, amide, alkyl, aryl, alkyl-aryl, alkyl- heteroaryl, alkenyl, alkenyl-aryl, alkenyl-heteroaryl, peptide, and polypeptide may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, -OH, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R1 and R2 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R2 and R3 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1- 4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R3 and R4 may together form a saturated or unsaturated C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, R5 and R6 are independently selected from the group consisting of no atom, H, H, D, -OH, halo, ether, -(O)C(R), -C(O)OR, -NO2, alkyl, aryl, C1-6-cycloalkyl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl, wherein the ether, alkyl, aryl, C1-6-cycloalkyl, alkyl-aryl, alkyl-heteroaryl, alkenyl, alkenyl-aryl, and alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, S, epoxy, -OH, halo, -(O)C(R), unsubstituted or substituted aryl, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof; or R5 and R6 may together with the N atom attached to form a saturated or unsaturated heterocycle that may be optionally substituted with an atom or a group selected from the group consisting of 0, N, halo, C1-4alkyl, CF3, oxazole, oxadiazole, dizaole, triazole, amide, ether, ester and combinations thereof, wherein R is selected from the group consisting of H, D, 0, N, halo, ether, oxazole, oxadiazole, dizaole, triazole, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl- heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3- 12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1- 6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of 0, N, S, halo, C1-4alkyl, CF3, -CN, and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. The compound according to claim 1 having a structure selected from the
Figure imgf000115_0001
Figure imgf000116_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. The compound according to claim 2 having a structure selected from the group consisting of:
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. The compound according to claim 3 having a structure selected from the group consisting of:
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. The compound according to claim 4 having a structure selected from the group consisting of:
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
and
Figure imgf000139_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. A compound having any one of the following structures:
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
and
Figure imgf000147_0001
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. A composition comprising one or more compounds of any one of claims 1-9 and a pharmaceutically acceptable carrier, adjuvant or vehicle. A method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds of any one of claims 1 -9 or one or more compositions of claim 10. The method of claim 11 , wherein the GPX4-associated disease is selected from the group consisting of a cancer, a neurotic disorder, a neurodegenerative disorder, spondylometaphyseal dysplasia, mixed cerebral palsy, pontocerebellar hypoplasia, and male infertility. The method of 12, wherein the GPX4-associated disease is a cancer. The method of claim 13, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma. The method of claim 13, wherein the cancer is hepatocellular carcinoma. The method of claim 11 , wherein the subject is a mammal. The method of claim 16, wherein the mammal is selected from the group consisting of humans, veterinary animals, and agricultural animals. The method of claim 11 , wherein the subject is a human. The method of claim 13, wherein the cancer is metastatic. The method of claim 13, wherein the cancer is under epithelial-to- mesenchymal (EMT) transition. The method of claim 13, wherein the cancer is hypersensitive to ferroptosis. The method of claim 13, wherein the cancer is refractory to standard cancer treatment. The method of claim 22 wherein the standard cancer treatment comprises chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof. A method for modulating the activity of glutathione peroxidase 4 (GPX4) in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds of any one of claims 1-9 or one or more compositions of claim 10. The method of claim 24, wherein the modulation comprises inhibiting GPX4 activity. A method for increasing the level of peroxide in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds of any one of claims 1-9 or one or more compositions of claim 10. The method of claim 26, wherein the peroxide is selected from the group consisting of hydrogen peroxide, organic hydroperoxide, lipid peroxide, and combinations thereof. A method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds of any one of claims 1-9 or one or more compositions of claim 10. The method of claim 28, wherein the cell has abberant lipid accumulation. The method of claim 28, wherein the cell is a cancer cell. The method of claim 30, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma. The method of claim 30, wherein the cancer is hepatocellular carcinoma. The method of claim 28, wherein the cell is a human cell. The method of claim 30, wherein the cancer cell is metastatic. The method of claim 30, wherein the cancer cell is under epithelial-to- mesenchymal (EMT) transition. The method of claim 30, wherein the cancer cell is hypersensitive to ferroptosis. The method of claim 30, wherein the cancer cell is refractory to standard cancer treatment. The method of claim 37, wherein the standard cancer treatment comprises chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof. A method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, which is one or more compounds of any one of claims 1-9 or one or more compositions of claim 10, and ii) an effective amount of a second anti-cancer agent. The method of claim 39, wherein the second anti-cancer agent is selected from the group consisting of chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof. The method of claim 39, wherein the second anti-cancer agent is an immunotherapy. The method of claim 41 , wherein the immunotherapy is selected from ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, ofatumumab, blinatumomab, daratumumab, elotuzumab, obinutuzumab, talimogene laherparepvec, necitumumab, lenalidomide, dinutuximab, and combinations thereof. The method of claim 39, wherein the subject is a human. The method of claim 39, wherein the cancer is metastatic. The method of claim 39, wherein the cancer is under epithelial-to- mesenchymal (EMT) transition. The method of claim 39, wherein the cancer is hypersensitive to ferroptosis. The method of claim 46, wherein the hypersensitivity to ferropotosis is identified by NADPH abundance, GCH1 expression, NF2-YAP activity, EMT signature, and GPX4 expression. The method of claim 39, wherein the cancer is refractory to standard cancer treatment. The method of claim 39, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma. The method of claim 39, wherein the cancer is hepatocellular carcinoma. The method of claim 39, wherein the first anti-cancer agent is administered to the subject before, concurrently with, or after the second anti-cancer agent. A kit for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising an effective amount of one or more compounds of any one of claims 1-9 or one or more compositions of claim 10, packaged with its instructions for use.
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Citations (1)

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