WO2023249994A1 - Methods and materials for treating cancer - Google Patents

Methods and materials for treating cancer Download PDF

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Publication number
WO2023249994A1
WO2023249994A1 PCT/US2023/025833 US2023025833W WO2023249994A1 WO 2023249994 A1 WO2023249994 A1 WO 2023249994A1 US 2023025833 W US2023025833 W US 2023025833W WO 2023249994 A1 WO2023249994 A1 WO 2023249994A1
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polypeptide
met
molecule
cancer
met polypeptide
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PCT/US2023/025833
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French (fr)
Inventor
Aaron S. MANSFIELD
Mitesh J. BORAD
Lin Yang
Amarnath Natarajan
Jayapal Reddy MALLAREDDY
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Mayo Foundation For Medical Education And Research
University Of Nebraska Medical Center
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Publication of WO2023249994A1 publication Critical patent/WO2023249994A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/53—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • This document relates to methods and materials involved in treating a mammal (e.g., a human) having cancer.
  • a mammal e.g., a human
  • this document provides bifunctional molecules (e.g., protein targeting chimeras (PROTACs)) that include (a) a targeting moiety that binds to a MET polypeptide, (b) a linker, and (c) an E3 ligase ligand.
  • a targeting moiety that binds to a MET polypeptide
  • linker e.g., a linker
  • E3 ligase ligand e.g., E3 ligase ligand
  • a bifunctional molecule e.g., a PROTAC targeting a MET polypeptide
  • a mammal having cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • Hepatocyte growth factor receptor is a known oncogenic driver in multiple malignancies (Guo etal., Nat. Rev. Clin. Oncol., 17:569-587 (2020)). Mutations that affect the donor or acceptor splice sites oiMEl exon 14 pre-mRNA can lead to skipping of exon 14 during splicing and an mRNA product where exons 13 and 15 are fused (Kong-Beltran et al., Cancer Res., 66:283-289 (2006); Lu et al., Cancer Res., 77:4498-4505 (2017); and Ma e/ al., Cancer Res., 63:6272-6281 (2003)).
  • MET polypeptides have been an elusive target for cancer treatments for decades despite its known oncogenicity.
  • This document provides methods and materials involved in treating a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14).
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14.
  • this document provides bifunctional molecules (e g., PROTACs) that include (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide).
  • a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can recruit an E3 ligase polypeptide to the MET polypeptide resulting in ubiquitination of the MET polypeptide (e.g., to promote degradation of the MET polypeptide).
  • a targeting moiety that binds to a MET polypeptide
  • a linker e.g., a linker
  • an E3 ligase ligand e.g., thalidomide
  • bifunctional molecules e.g., PROTACs
  • bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)).
  • bifunctional molecules e.g., PROTACs
  • bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)
  • a mammal having cancer e.g., to treat the mammal.
  • a PROTAC that includes (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can bind a MET polypeptide and restore ubiquitination of the MET polypeptide to promote degradation of the MET polypeptide. Having the ability to specifically target a MET polypeptide for ubiquitination and degradation provides a unique and unrealized opportunity to treat cancers expressing an oncogenic MET polypeptide.
  • a targeting moiety that binds to a MET polypeptide
  • a linker e.g., a linker
  • an E3 ligase ligand e.g., thalidomide
  • one aspect of this document features molecules comprising capmatinib covalently attached to an E3 ligase ligand.
  • the capmatinib can be directly covalently attached to the E3 ligase ligand.
  • the molecule can be:
  • the capmatinib can be indirectly covalently attached to the E3 ligase ligand via a linker.
  • the linker can be a hydrocarbon linker.
  • the hydrocarbon linker can include from about 3 to about 12 carbon atoms.
  • the hydrocarbon linker can include no more than one oxygen atom.
  • the linker can be:
  • the E3 ligase ligand can be thalidomide, lenalidomide, pomalidomide, VHL032, nutlin 3a, idasanutlin, RG7112, bestatin, MV1, LCL-161, l-[3-(4-bromophenyl)-4,5-dihydro-5-phenyl- 1 h-pyrazol- 1 -yl]-2-chloro-ethanone, or 1 -(3,4-dihydro-6-hydroxy- 1 (2h)-quinolinyl)- 1 - propanone.
  • the E3 ligase ligand can bind to an E3 ligase polypeptide selected from the group consisting of a Von Hippel-Lindau (VHL) polypeptide, a cereblon (CRBN) polypeptide, a MDM2 polypeptide, an inhibitor of apoptosis (IAP) polypeptide, a DDB 1 and CUL4 associated factor 15 (DCAF15) polypeptide, a DDB1 and CUL4 associated factor 16 (DCAF16) polypeptide, a ring finger protein 4 (RNF4) polypeptide, and a ring finger protein 114 (RNF114) polypeptide.
  • VHL Von Hippel-Lindau
  • CRBN cereblon
  • MDM2 MDM2 polypeptide
  • IAP inhibitor of apoptosis
  • DCAF15 DDB 1 and CUL4 associated factor 15
  • DCAF16 DDB1 and CUL4 associated factor 16
  • RRF4 ring finger protein 4
  • RNF114 ring finger
  • the molecule can be:
  • compositions including one or more molecules comprising capmatinib covalently attached to an E3 ligase ligand.
  • compositions including one or more molecules comprising capmatinib covalently attached to an E3 ligase ligand and a pharmaceutically acceptable carrier, excipient, or diluent.
  • this document features methods for treating a mammal having cancer.
  • the methods can include, or consist essentially of, administering, to a mammal having cancer where cancer cells of the cancer express a MET polypeptide, one or more molecules comprising capmatinib covalently attached to an E3 ligase ligand.
  • the mammal can be a human.
  • the MET polypeptide can be a wild-type MET polypeptide.
  • the MET polypeptide can be a mutant MET polypeptide.
  • the mutant MET polypeptide can be a MET polypeptide that lacks the amino acid sequence encoded by at least a portion of exon 14, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TPR polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a KIF5B polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a PTPRZ1 polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a CLIP2 polypeptide, or a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TFG polypeptide.
  • the MET polypeptide can be an oncogenic MET polypeptide.
  • the cancer can be a lung cancer, a gastric cancer, a papillary
  • this document features methods for inducing ubiquitination of a MET polypeptide in a mammal.
  • the methods can include, or consist essentially of, administering, to a mammal, one or more molecules comprising capmatinib covalently attached to an E3 ligase ligand.
  • the mammal can be a human.
  • the MET polypeptide can be a wild-type MET polypeptide.
  • the MET polypeptide can be a mutant MET polypeptide.
  • the mutant MET polypeptide can be a MET polypeptide that lacks the amino acid sequence encoded by at least a portion of exon 14, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TPR polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a KTF5B polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a PTPRZ1 polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a CLIP2 polypeptide, or a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TFG polypeptide.
  • the MET polypeptide can be an oncogenic MET polypeptide.
  • Figures 1 A - IE Live cell imaging screen for MET exon 14 skipping-GFP degradation.
  • Figure 1 A Characterization of HEK293 cells stably transfected with either GFP-vector or GFP-Met-Exon-14 skipping mutant.
  • Figure IB Capmatinib based PROTAC library screened at 1 pM using live cell imaging in HEK293 transfected with GFP-Met- Exon-14 skipping mutant.
  • the bar graph shows the green count values over confluence (phase) normalized to time zero of each well at 0 hours, 8 hours, and 24 hours post addition.
  • the upper and lower broken lines indicate activity relative to capmatinib at 8 and 24 hours respectively.
  • Figure 1C Time course study to assess the effects of the linkers in PROTACs generated with capmatinib and VHL ligand.
  • Figure ID A dose-response and time course study with the most potent capmatinib based PROTAC 48-284 (10000 nM, 5000 nM, 1000 nM, 500 nM, and 100 nM) in HEK293 transfected with GFP-Met-Exon-14 skipping mutant. The images analyzed every 6 hours for 24 hours post-treatment.
  • Figure IE A time course study with the capmatinib based PROTAC 48-284 (1 pM) with HEK293 transfected with GFP-Met-Exon-14 skipping mutant in the presence and absence ofMG132 (10 pM). The images analyzed every 2 hours for 24 hours post-treatment.
  • Figures 2A - 2D Characterization of MET-PROTACs.
  • Figure 2B Volcano plot of the 114 kinases that were quantified in the above study.
  • FIG. 2C and Figure 2D HEK293 transfected with GFP-Met-Exon-14 skipping mutant cells were treated with 48-284 (2 pM, 1 pM ,or 0.5 pM) or SJF-8240 (2 pM, 1 pM, or 0.5 pM) and the cells were imaged every 2 hours for 22 hours using IncuCyte.
  • Figures 3A - 3B Time and dose effects of MET PROTACs.
  • Figure 3A The Hs746T cell line was treated with 48-284 at 1.0 pM and MET was assessed at the indicated timepoints by western blots with P-actin controls. These cells were also treated at the indicated doses for 8 hours. The effects on downstream RAS/AKT and RAS/ERK pathway signaling were also assessed.
  • Figure 3B Similarly, the Hs746T cell line was treated with the foretinib -based PROTAC SJF8240 at 1.0 pM and MET was assessed at the indicated timepoints by western blots with P-actin controls. These cells were also treated at the indicated doses for 8 hours. The effects on downstream RAS/AKT and RAS/ERK pathway signaling were also assessed.
  • Figures 4A - 4C In vivo effects of MET-PROTAC on xenograft.
  • Figure 4A Multiple UW21 xenografts with a MET exon 14 skipping mutation were removed from mouse flanks and stained for MET by immunohistochemistry. In this representative example, intense membranous and cytoplasmic MET expression is seen in almost all the tumor cells.
  • Figure 4B Other UW21 xenografts were treated with to doses of 48-284 through tail vein injection eight hours apart, removed six hours after the second injection and stained for MET by immunohistochemistry. In this representative example, significant reduction in MET expression was observed along with evidence of a treatment effect.
  • Figure 4C Protein lysates were obtained from UW21 xenografts that were treated (T) with 48-284 and controls (Ctrl) and assessed for ubiquitination.
  • Figures 5A - 5D Micro-cancer model with ZM-fusion.
  • Figure 5A A Circos plot summarizing the genomic abnormalities identified in PT425. Breakpoint junctions are presented as lines connecting two breakpoints, including the PTPRZ1MET fusion shown at 7q31.
  • Figure 5B This junction plot visualizes the mate-pair reads supporting the intrachromosomal rearrangement between PTPRZ1 and MET on chromosome 7. Coverage levels across each position are presented in gray-shaded regions.
  • Figures 6A - 6C Figure 6A) Capmatinib docked into MET kinase domain (pdb: 3zbx). Capmatinib is shown in sticks, and the kinase domain of MET is shown as a ribbon structure. The quinoline ring of capmatinib mimics the adenine ring of the ATP, and the quinoline nitrogen is within hydrogen bonding distance of the N-H of Met 1160 (hydrogen bond is shown in black line).
  • Figure 6B Live cell imaging study with a 60 member PROTAC library.
  • HEK293 transfected with GFP-Met-Exon-14 skipping mutant were subjected to 1 pM of capmatinib or the PROTAC library and the cells imaged every 2 hours for 24 hours.
  • Figure 6C HEK293 transfected with GFP-Met-Exonl4-skipping cells were treated with capmatinib (1 pM) or 48-284 (1 pM) for 24 hours, and the resulting lysates were subjected to western blot analyses and probed with a GFP antibody.
  • Figure 7B PCA plot of the proteomics study.
  • Figures 8A - 80 NMR spectra for exemplary PROTACs.
  • Figure 8A PROTAC 48- 269.
  • Figure 8B PROTAC 48-270.
  • Figure 8C PROTAC 48-273.
  • Figure 8D PROTAC 48- 276.
  • Figure 8E PROTAC 48-284.
  • Figure 8F PROTAC 48-285.
  • Figure 8G PROTAC 48- 289.
  • Figure 8H PROTAC 48-295.
  • Figure 8J PROTAC 48- 299.
  • Figure 8K PROTAC 50-210.
  • Figure 8L 50-211.
  • Figure 8M PROTAC 50-212.
  • Figure 8N PROTAC 50-213.
  • Figures 9A - 9B Characterization of PROTAC 48-284.
  • Figure 9A High Performance Liquid Chromatography (HPLC) chromatogram of PROTAC 48-284.
  • Figure 9B High-resolution mass spectrometry (HRMS) of PROTAC 48-284.
  • Figure 11 A picture showing the UW21 xenografts that were removed from the flanks of the mice following treatment with control, PROTAC 48-284 (10 mg/kg or 20 mg/kg,) or capmatinib (5 mg/kg or 10 mg/kg) for two weeks.
  • Figure 14 A graph showing sizes of the UW21 xenografts treated with control or PROTAC 48-284 (10 mg/kg or 20 mg/kg) over two weeks.
  • Figure 15 A graph showing body weight of mice with the Rudin439 xenografts implanted in their flanks and treated with PROTAC 48-284 (10 mg/kg or 20 mg/kg) for two weeks.
  • Figure 17 A graph showing sizes of the Rudin439 xenografts treated with control, PROTAC 48-284 (10 mg/kg or 20 mg/kg), or capmatinib (5 mg/kg or 10 mg/kg) over two weeks.
  • Figure 18 An enlarged picture showing the Rudin439 xenografts that were removed from the flanks of the mice following treatment with control or PROTAC 48-284 (10 mg/kg or 20 mg/kg) for two weeks.
  • Figure 19 A graph showing sizes of the Rudin439 xenografts treated with control or PROTAC 48-284 (10 mg/kg or 20 mg/kg) over two weeks.
  • PROTACs that can bind to a MET polypeptide (e.g., an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) and can be used restore ubiquitination of the MET polypeptide (e.g., to promote degradation of the MET polypeptide), thereby reducing the levels of oncogenic MET polypeptides within a mammal (e.g., a human).
  • a MET polypeptide e.g., an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • restore ubiquitination of the MET polypeptide e.g., to promote degradation of the MET polypeptide
  • a mammal e.g., a human
  • This document provides methods and materials involved in treating a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14).
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14.
  • bifiinctional molecules e.g., PROTACs
  • PROTACs that include (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide).
  • This document also provides methods and materials for making and using bifimctional molecules (e.g., PROTACs) provided herein (e.g., bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)).
  • bifimctional molecules e.g., PROTACs
  • bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)).
  • bifunctional molecules e.g., PROTACs
  • bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)
  • a mammal having cancer e.g., to treat the mammal.
  • Bifunctional molecules e.g., PROTACs
  • bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can be designed to include any appropriate targeting moiety having the ability to bind to a MET polypeptide.
  • a targeting moiety that binds to a MET polypeptide e.g., capmatinib
  • an E3 ligase ligand e.g., thalidomide
  • capmatinib can be used as a targeting moiety that binds to a MET polypeptide to design PROTACs having the ability to (a) degrade a MET polypeptide (e.g., an oncogenic MET polypeptide) and/or (b) inhibit the kinase activity of a MET polypeptide (e.g., catalytically inhibit the kinase activity of a MET polypeptide).
  • a targeting moiety that binds to a MET polypeptide other than capmatinib can be used to design a PROTAC having the ability to target MET polypeptides.
  • tepotinib, savolitinib, crizotinib, amuvatinib, tivantinib, cabozantinib, brigatinib, or fostamatinib can be used as a targeting moiety that binds to a MET polypeptide to design a PROTAC having the ability to target MET polypeptides.
  • a PROTAC provided herein can lack foretinib.
  • a targeting moiety that binds to a MET polypeptide that can be included in a bifunctional molecule can be as shown in Example 2.
  • a targeting moiety that binds to a MET polypeptide that can be included in a bifunctional molecule e.g., a PROTAC
  • a mutant MET polypeptide can lack at least a portion of the amino acid sequence encoded by exon 14. In some cases, a mutant MET polypeptide can have increased kinase activity (e.g., as compared to a MET polypeptide lacking the mutation). In some cases, a mutant MET polypeptide can be a MET fusion polypeptide including a portion of a MET polypeptide and a portion of another polypeptide (e.g., resulting from a genetic translocation creating a fusion gene including a portion of a nucleic acid encoding a MET polypeptide and a portion of another gene).
  • a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a nuclear basket protein (TPR) polypeptide.
  • a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a kinesin family member 5B (KIF5B) polypeptide.
  • KIF5B kinesin family member 5B
  • a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a protein tyrosine phosphatase receptor type ZI (PTPRZ1) polypeptide (e.g., a zm-fiision).
  • PTPRZ1 protein tyrosine phosphatase receptor type ZI
  • a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a CAP-Gly domain containing linker protein 2 (CLIP2) polypeptide.
  • a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a trafficking from ER to golgi regulator (TFG) polypeptide.
  • TGF golgi regulator
  • Examples of MET polypeptides that can be targeted by targeting moiety that binds to a MET polypeptide in a bifunctional molecule (e.g., a PROTAC) provided herein include, without limitation, MET polypeptides having an amino acid sequence set forth in the National Center for Biotechnology Information (NCBI) databases at Accession No.
  • a MET polypeptide that can be targeted by a targeting moiety that binds to a MET polypeptide in a bifunctional molecule can be a MET polypeptide having an altered level of expression.
  • a mutant MET polypeptide can have an increased level of MET expression (e.g., resulting from a genetic translocation allowing a heterologous promoter to drive expression of a nucleic acid encoding a MET polypeptide resulting in an increased level of expression as compared to a level of MET polypeptide when expression of the nucleic acid encoding the MET polypeptide is driven by its endogenous promoter).
  • a MET polypeptide that can be targeted by targeting moiety that binds to a MET polypeptide in a bifunctional molecule (e.g., a PROTAC) provided herein can be as described elsewhere (see, e.g., Stransky et al., Nat.
  • a MET polypeptide that can be targeted by targeting moiety that binds to a MET polypeptide in a bifunctional molecule can be as described in Example 1.
  • Bifunctional molecules e.g., PROTACs
  • bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide)
  • a linker can be used to attach (e.g., covalently attach) a targeting moiety that binds to a MET polypeptide to an E3 ligase ligand.
  • a linker in a bifunctional molecule can be any appropriate type of molecule (e.g., polypeptides, atoms, and polymers). In some cases, a linker can be hydrophobic. Examples of linkers that can be included in a bifunctional molecule (e g., a PROTAC) provided herein include, without limitation, hydrocarbon linkers, polyethylene glycol (PEG) linkers, alkyl chain linkers, and alkyl/ether linkers. A linker can be any appropriate length.
  • the linker when a linker is a hydrocarbon linker, the linker can include from about 3 to about 12 carbon atoms (e.g., from about 3 to about 12, from about 3 to about 10, from about 3 to about 8, from about 3 to about 6, from about 3 to about 5, from about 3 to about 4, from about 4 to about 12, from about 5 to about 12, from about 6 to about 12, from about 8 to about 12, from about 10 to about 12, from about 4 to about 11 , from about 5 to about 10, from about 6 to about 8, from about 4 to about 7, from about 5 to about 8, from about 6 to about 9, from about 7 to about 10, or from about 8 to about 11 carbon atoms).
  • a hydrocarbon linker can have 9 carbon atoms.
  • the hydrocarbon linker does not include more than one oxygen atom.
  • linkers that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein include, without limitation, linkers having one of the following structures.
  • a linker that can be included in a bifunctional molecule e.g., a PROTAC
  • a linker that can be included in a bifunctional molecule e.g., a PROTAC
  • a linker that can be included in a bifunctional molecule e.g., a PROTAC
  • Example 1 see, e.g., Table 1).
  • Bifunctional molecules e.g., PROTACs
  • bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide)
  • An E3 ligase ligand can be any appropriate molecule that can target (e.g., target and bind) to an E3 ligase polypeptide.
  • An E3 ligase ligand can target (e.g., target and bind to) any appropriate E3 ligase polypeptide.
  • E3 ligase polypeptides that can be targeted by an E3 ligase ligand in a bifunctional molecule include, without limitation, E3 ligase polypeptides having an amino acid sequence set forth in the NCBI databases at Accession No. X82654.1, Accession No. NG_008212.3, and Accession No. NC_000003.12.
  • an E3 ligase polypeptide that can be targeted by an E3 ligase ligand in a bifiinctional molecule e.g., a PROTAC
  • E3 ligase ligand can be any appropriate type of molecule (e.g., small molecules, oligonucleotides, and polypeptides).
  • E3 ligase ligands that can be used to design a bifunctional molecule (e.g., a PROTAC) provided herein include, without limitation, thalidomide, lenalidomide, pomalidomide, VE1L032, nutlin 3a, idasanutlin, RG7112, bestatin, MV1, LCL-161, l-[3-(4-bromophenyl)-4,5-dihydro-5-phenyl-lh-pyrazol-l-yl]-2-chloro- ethanone, and l-(3,4-dihydro-6-hydroxy-l(2h)-quinolinyl)-l-propanone.
  • an E3 ligase ligand that can be included in a bifunctional molecule can be as described elsewhere (see, e g., Rana et al., Cancer, 13:5506 (2021) at, for example, Figure 4; and Bricelj et al., Front Chem., 9:707317 (2021) at, for example, Figure 2 and Figure 4).
  • an E3 ligase ligand that can be included in a bifunctional molecule e.g., a PROTAC
  • an E3 ligase ligand that can be included in a bifunctional molecule e.g., a PROTAC
  • a bifunctional molecule e.g., a PROTAC
  • an E3 ligase ligand that can be included in a bifunctional molecule e.g., a PROTAC
  • Example 1 an E3 ligase ligand that can be included in a bifunctional molecule provided herein can be as described in Example 1.
  • a bifunctional molecule e.g., a PROTAC
  • a bifunctional molecule e.g., a PROTAC
  • a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can have the structure:
  • a bifunctional molecule e.g., a PROTAC
  • a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can be as shown in Figure 8.
  • a bifunctional molecule e.g., a PROTAC
  • a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide) can be as described in Example 1.
  • a bifunctional molecule e.g., a PROTAC
  • a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can be made using any appropriate method.
  • a bifunctional molecule e g., a PROTAC
  • a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)
  • a targeting moiety that binds to a MET polypeptide (e.g., capmatinib)
  • a linker e.g., a linker
  • an E3 ligase ligand e.g., thalidomide
  • a bifunctional molecule e.g., a PROTAC
  • a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can be made as described in Example 1.
  • compositions containing one or more bifunctional molecules e.g., PROTACs
  • one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)).
  • a targeting moiety that binds to a MET polypeptide (e.g., capmatinib)
  • a linker e.g., a linker
  • an E3 ligase ligand e.g., thalidomide
  • one or more bifunctional molecules e.g., PROTACs
  • a composition e.g., a pharmaceutically acceptable composition
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14.
  • one or more bifunctional molecules e.g., PROTACs
  • one or more bifunctional molecules e.g., PROTACs
  • one or more bifunctional molecules e.g., PROTACs
  • one or more bifunctional molecules e.g., PROTACs
  • additives e.g., one, two, three, four, or more bifunctional molecules provided herein
  • cyclodextrins e.g., beta- cyclodextrins such as KLEPTOSE®
  • dimethylsulfoxide (DMSO) sucrose
  • lactose starch
  • starch e.g., starch glycolate
  • cellulose e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)
  • HPMC hydroxypropyl cellulose
  • PVP polyvinylpyrrolidone
  • PEG polyethylene glycol
  • crospovidone crosslinked polyvinylpyrrolidone
  • compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules.
  • Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
  • a sterile injectable suspension e.g., a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated using, for example, suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3 -butanediol.
  • acceptable vehicles and solvents include, without limitation, saline, mannitol, water, Ringer’s solution, and isotonic sodium chloride solution.
  • sterile, fixed oils can be used as a solvent or suspending medium. In some cases, a bland fixed oil can be used such as synthetic mono- or di-glycerides.
  • a targeting moiety that binds to a MET polypeptide e.g., capmatinib
  • a linker e.g., an E3 ligase ligand
  • E3 ligase ligand e.g., thalidomide
  • This document also provides methods and materials for using one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)).
  • a targeting moiety that binds to a MET polypeptide (e.g., capmatinib)
  • a linker e.g., a linker
  • an E3 ligase ligand e.g., thalidomide
  • one or more bifunctional molecules e.g., PROTACs
  • a mammal e.g., a human
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14.
  • one or more bifunctional molecules e.g., PROTACs
  • a mammal having cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • administering one or more bifunctional molecules e.g., PROTACs
  • a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide))
  • a mammal e.g., a human
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • one or more bifunctional molecules e.g., PROTACs
  • a mammal e.g., a human
  • a human having cancer e.g., a human having cancer
  • the methods and materials provided herein can be used as described herein to reduce the number of cancer cells in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials provided herein can be used as described herein to reduce the volume of one or more tumors in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • administering one or more bifunctional molecules e.g., PROTACs
  • a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide))
  • a mammal e.g., a human
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • one or more bifunctional molecules e g., PROTACs
  • a mammal e.g., a human
  • a human having cancer e.g., a human having cancer
  • the methods and materials provided herein can be used as described herein to improve the survival of the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials provided herein can be used as described herein to improve the survival of the mammal by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).
  • at least 6 months e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more.
  • administering one or more bifunctional molecules e.g., PROTACs
  • a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide))
  • a mammal e.g., a human
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • a reduced level of a MET polypeptide in a mammal refers to any level that is less than the level of that MET polypeptide observed in that mammal prior to being treated as described herein (e.g., prior to being administered one or more bifunctional molecules provided herein).
  • a reduced level of a MET polypeptide can be a level that is at least 5 percent (e.g., at least 10, at least 15, at least 20, at least 25, at least 35, at least 50, at least 75, at least 100, or at least 150 percent) less than the level of that MET polypeptide prior to being treated as described herein.
  • Any appropriate mammal having cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • can be treated as described herein e.g., by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)).
  • a targeting moiety that binds to a MET polypeptide e.g., capmatinib
  • a linker e.g., a linker
  • Examples of mammals that can have cancer and can be treated as described herein include, without limitation, humans, non- human primates such as monkeys, horses, bovine species, porcine species, dogs, cats, mice, and rats.
  • a human having cancer can be treated by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) to the human.
  • a targeting moiety that binds to a MET polypeptide (e.g., capmatinib)
  • a linker e.g., a linker
  • an E3 ligase ligand e.g., thalidomide
  • a mammal e.g., a human having any type of cancer can be treated as described herein (e.g., by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)).
  • a cancer treated as described herein can include one or more solid tumors.
  • a cancer that can be treated as described herein can include one or more cancer cells having an oncogenic MET polypeptide (e.g., a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14).
  • oncogenic MET polypeptide e.g., a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14.
  • cancers that can be treated as described herein include, without limitation, lung cancers (e.g., non-small cell lung cancers), gastric cancers, papillary renal cell carcinomas, brain cancers, and sarcomas.
  • methods described herein also can include identifying a mammal as having cancer.
  • methods for identifying a mammal as having cancer include, without limitation, physical examination, laboratory tests (e.g., blood and/or urine), biopsy, imaging tests (e.g., X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, and/or genetic tests.
  • a mammal can treated as described herein (e.g., by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)).
  • a targeting moiety that binds to a MET polypeptide (e.g., capmatinib)
  • a linker e.g., a linker
  • an E3 ligase ligand e.g., thalidomide
  • One or more bifunctional molecules e.g., PROTACs
  • a mammal e.g., a human
  • any appropriate route e.g, intratumoral injection, intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, oral, intranasal, inhalation, transdermal, and parenteral.
  • one or more bifunctional molecules e.g., PROTACs
  • a mammal e.g., a human
  • one or more bifunctional molecules e.g., PROTACs
  • a mammal e.g., a human
  • a composition containing one or more (e.g., one, two, three, four, or more) bifunctional molecules (e.g., PROTACs) provided herein e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide))
  • a mammal e.g., a human
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • any appropriate dose e.g., any appropriate dose.
  • An effective amount of a composition containing one or more bifunctional molecules can be any amount that can treat a mammal having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) as described herein without producing significant toxicity to the mammal.
  • a mammal having cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • an effective amount of one or more bifunctional molecules can be from about 0.1 milligrams per kilogram body weight (mg/kg) to about 1000 mg/kg (e.g., from about 0.1 mg/kg to about 750 mg/kg, from about 0.1 mg/kg to about 500 mg/kg, from about 0.1 mg/kg to about 400 mg/kg, from about 0.1 mg/kg to about 300 mg/kg, from about 0.1 mg/kg to about 200 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 25 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 1000 mg/kg, from about 10 mg/
  • an effective amount of one or more bifunctional molecules e.g., PROTACs
  • one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)
  • a targeting moiety that binds to a MET polypeptide e.g., capmatinib
  • a linker e.g., a linker
  • an E3 ligase ligand e.g., thalidomide
  • the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment.
  • the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and/or severity of the cancer in the mammal being treated may require an increase or decrease in the actual effective amount administered.
  • a composition containing one or more (e.g., one, two, three, four, or more) bifunctional molecules (e.g., PROTACs) provided herein e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide))
  • a mammal e.g., a human
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and/or route of administration.
  • a composition containing one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide) can include the one or more bifunctional molecules provided herein as the sole active ingredient in the composition that is effective to treat a mammal having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14).
  • a mammal having cancer e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14).
  • a combination therapy used to treat cancer can include administering one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)), and also administering one or more (e.g., one, two, three, four, five or more) agents used to treat cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14).
  • bifunctional molecules e.g., PROTACs
  • an agent that can be administered to a mammal to treat cancer can be a chemotherapeutic agent.
  • an agent that can be administered to a mammal to treat cancer can be an immune checkpoint inhibitor (e.g., PD1 inhibitors, PD-L1 inhibitors, and CTLA4 inhibitors).
  • an agent that can be administered to a mammal to treat cancer can be a cytotoxic agent.
  • agents that can be administered to a mammal to treat cancer include, without limitation, altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, pemetrexed, paclitaxel, docetaxel, vinorelbine, gemcitabine, amivantamab, and any combinations thereof.
  • one or more bifunctional molecules e.g., PROTACs
  • one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)
  • the one or more additional therapies can be performed at the same time or independently of administering the one or more bifunctional molecules (e.g., PROTACs) provided herein.
  • administering one or more bifunctional molecules (e.g., PROTACs) provided herein can be performed before, during, or after the one or more additional therapies are performed.
  • the size of the cancer e.g., the number of cancer cells and/or the volume of one or more tumors
  • the severity of one or more symptoms of the cancer being treated can be monitored. Any appropriate method can be used to determine whether or not the size of the cancer present within a mammal is reduced. For example, imaging techniques can be used to assess the size of the cancer present within a mammal can be used to detect the presence of the cancer present within a mammal.
  • pLenti-MET-GFP plasmid 25 ng of pLenti-MET-GFP plasmid were used as a template, 10 pM forward primer (5’-ATCAGTTTCCTAATTCATTCTAG-3’; SEQ ID NO:1) and 10 pM reverse primer (5’-CTTTAATTTGCTTTCTCTTTTTC-3’; SEQ ID NO:2), 12.5 pL of Q5 Hot Start High-Fidelity 2X master Mix for mutagenesis PCR (30 seconds in 98°C for initial denaturation, 98°C for 10 seconds, 62°C for 30 seconds, and 72°C for 6 minutes, repeated for 25 cycles, plus 2 minutes in 72°C for final extension).
  • the PCR product was processed with KLD reaction and transformation following the protocol provided by the manufacturer (NEB Inc.).
  • the plates were set up in an Incucyte SX3 live cell imager (Sartorius Corporation) to acquire phase contrast and green fluorescence data.
  • the imager was itself housed in a CO2 incubator (5% CO2), and the plates were maintained at 37°C over the course of the experiment. Images of the entire well was captured at 4x magnification, every 2 hours, for the indicated period.
  • the images were analyzed with the included phase contrast cell tracking module software to generate the curves showing % confluence over time.
  • Cell plates were washed twice with cold 1 x PBS, harvested in cold 1 x PBS, and centrifuged at 14,000 g for 10 minutes at 4°C.
  • Cells were lysed in radioimmunoprecipitation assay (RIP A) buffer (Thermo Scientific), sodium orthovandate (NasVCL, Sigma), sodium flouride (NaF, Sigma), P- glycerophosphate (Sigma), and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma).
  • the samples were incubated on ice for 30 minutes, vortexed at 10 minute intervals and centrifuged at 14,000 g for 10 minutes at 4°C.
  • the supernatant was collected, and protein quantified using the BCA Protein Assay kit (23225, Pierce). Samples were stored in -80°C until required for quantitative proteomics analysis.
  • Each sample was analyzed by LC-MS/MS on an RSLCnano system (ThermoFisher Scientific) coupled to an Orbitrap Eclipse Tribrid mass spectrometer (ThermoFisher Scientific).
  • the samples were first injected onto a trap column (Acclaim PepMapTM 100, 75 pm x 2 cm, ThermoFisher Scientific) and desalted for 3.0 minutes at a flow rate of 5 pL/minute, before switching in line with the main column.
  • the UW21 xenograft was removed from the flanks of the mice, fixed in formalin, embedded in paraffin, sectioned at 4 microns, and stained with an antibody directed against MET (clone SP44, Ventana, Arlington, AZ, USA).
  • DNA isolated from fresh-frozen macrodissected primary tumor tissue was sequenced using the whole genome Mate-Pair sequencing (MPseq) protocol and analyzed to detect structural variants.
  • DNA was isolated from macrodissected primary tumor tissue using the Qiagen AllPrep DNA/RNA mini kit (Qiagen, #80204) or Qiagen DNeasy Blood and Tissue Kit (Qiagen, #69504) for peripheral blood mononuclear cell (PBMC) extraction following the manufacture’s protocol.
  • the MPseq protocol was utilized to detect structural variants at the base level resolution through specialized larger 2-5kb fragment tiling of the genome.
  • the binary indexing mapping algorithm maps reads to the GRCh38 reference genome. Structural variants were detected using SVAtools using a minimum of three supporting reads for junction detection.
  • the graph of the PTPZR1-MET fusion was made with the Fusion Editor function in ProteinPaint (pecan, stjude.cloud/proteinpaint).
  • the fusion coordinates detected by sequencing were submitted according to their genomic coordinates to create the graph.
  • Scrbdinger small molecule drug discovery suite 2020-1 was used for molecular modeling.
  • the crystal structure of c-MET kinase (pdb:3zbx) was retrieved from the protein data bank.
  • the protein structure was analyzed using Maestro version 12.3.013 (Scrodinger Inc.) and subjected to docking.
  • the Protein Preparation Wizard was used to add missing hydrogen atoms and side chains and minimized using OPLS3e force field to optimize hydrogen bonding network and converge the heavy atoms to a rmsd of 0.3 A.
  • the capmatinib structure was drawn in Maestro and subjected to Lig Prep to generate conformers, possible protonation at pH of 7 ⁇ 2 that serves as an input for docking process.
  • the receptor grid was generated using Receptor Grid Generation tool in Maestro (Scrodinger Inc.). Docking was performed using GLIDE XP with the van der Waals radii of nonpolar atoms for each of the ligands scaled by a factor of 0.8 and partial charge cutoff 0.15.
  • the binding mode of the ligand in the protein was analyzed using Maestro version 12.3.013 (Scrodinger Inc.).
  • Thalidomide, lenalidomide, and VHL binders were used as E3-targeting ligands to generate a focused set of 37 capmatinib based PROTACs.
  • An additional 23 non-capmatinib based PROTACs with aminopyrazole, palbociclib, staurosporine, futibatinib, BMS345541, YK-4- 279, and APS-2-79 as targeting ligands were added as controls to generate a library of 60 PROTACs (Table 1).
  • composition Compound Head Group E3 Ligand Linker Capmatinib
  • capmatinib based PROTACs did not reduce the GFP signal. Unexpectedly, capmatinib treated cells showed a time-dependent decrease in the GFP signal indicating degradation of METexl4A. This finding suggests that capmatinib could be functioning as a molecular glue.
  • the capmatinib-based PROTACs were binned into 3 groups relative to capmatinib activity (Figure IB). PROTACs with VHL ligand and hydrophobic (all carbon) linkers showed that shorter linkers (3-7) were inactive while the longer linkers (8-11) were active (Figure 1C).
  • a classical feature of a PROTAC is the “hook effect” wherein increasing concentrations of the PROTAC leads to the formation of stable binary complexes between the PROTAC and either the E3-ligase or protein of interest resulting reduced degradation of the target protein.
  • a dose-response study (0 - 10 pM) with one of the most potent degraders, 48-284, monitored every 6 hours showed efficient degradation at ⁇ 0.9 pM with a drop off in efficiency at both lower and higher concentrations (Figure ID).
  • METexl4A-GFP expressing cells were subjected to 1 pM of either capmatinib or 48-284 for 24 hours.
  • the lysates from these samples were subjected to mass spectrometry-based proteomic analyses ( Figures 2A and 2B, and Figure 7). Conjugating the VHL ligand to capmatinib in 48-284 reduced the total number of proteins identified as hits (abundance > 2-fold reduction and p-value ⁇ 0.001) from 64 to 45.
  • bifunctional molecules including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can be used to promote ubiquitination of a MET polypeptide (e.g., to promote degradation of a MET polypeptide), and that such bifunctional molecules can be used to treat cancer.
  • a targeting moiety that binds to a MET polypeptide e.g., capmatinib
  • a linker e.g., a linker
  • an E3 ligase ligand e.g., thalidomide
  • mice that were implanted with the UW21 xenografts were stable over the two- week period that they were treated with control (DMSO and SOLUTOL®), PROTAC 48-284 10 mg/kg, PROTAC 48-284 20 mg/kg, capmatinib 5 mg/kg or capmatinib 10 mg/kg ( Figure 10), suggesting that there was no significant impact on the constitution of the mice.
  • PROTAC 48-284 10 mg/kg, PROTAC 48-284 20 mg/kg, capmatinib 5 mg/kg, or capmatinib 10 mg/kg the UW21 xenografts were removed from the flanks of the mice ( Figure 11).
  • mice that were implanted with the Rudin439 xenografts were stable over the two-week period that they were treated with control, PROTAC 48-284 10 mg/kg, PROTAC 48-284 20 mg/kg, capmatinib 5 mg/kg, or capmatinib 10 mg/kg ( Figure 15), suggesting that there was no significant impact on the constitution of the mice.
  • PROTAC 48-284 10 mg/kg, PROTAC 48-284 20 mg/kg, capmatinib 5 mg/kg, or capmatinib 10 mg/kg were removed from the flanks of the mice ( Figure 16).
  • a human identified as having lung cancer e.g., a lung cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • a composition including one or more bifunctional molecules each including (a) a MET- targeting moiety, (b) a linker, and (c) an E3 ligase ligand.
  • the bifunctional molecule(s) can recruit an E3 ligase to the MET polypeptide, resulting in ubiquitination of the MET polypeptide (e.g., to promote degradation of the MET polypeptide) to treat the human.
  • a human identified as having gastric cancer e.g., a gastric cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14
  • a composition including one or more bifunctional molecules each including (a) a MET- targeting moiety, (b) a linker, and (c) an E3 ligase ligand.
  • the bifunctional molecule(s) can recruit an E3 ligase to the MET polypeptide, resulting in ubiquitination of the MET polypeptide (e.g., to promote degradation of the MET polypeptide) to treat the human.

Abstract

This document relates to methods and materials involved in treating a mammal (e.g., a human) having cancer. For example, bifunctional molecules (e.g., protein targeting chimeras (PROTACs)) that include (a) a targeting moiety that binds to a MET polypeptide, (b) a linker, and (c) an E3 ligase ligand are provided. In some cases, a bifunctional molecule (e.g., a PROTAC targeting a MET polypeptide) provided herein can be administered to a mammal having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) to treat the mammal.

Description

METHODS AND MATERIALS FOR TREATING CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Patent Application Serial No. 63/354,370, filed on June 22, 2022. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.
SEQUENCE LISTING
This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2135W01.xml.” The XML file, created on June 2, 2023, is 3000 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
TECHNICAL FIELD
This document relates to methods and materials involved in treating a mammal (e.g., a human) having cancer. For example, this document provides bifunctional molecules (e.g., protein targeting chimeras (PROTACs)) that include (a) a targeting moiety that binds to a MET polypeptide, (b) a linker, and (c) an E3 ligase ligand. In some cases, a bifunctional molecule (e.g., a PROTAC targeting a MET polypeptide) provided herein can be administered to a mammal having cancer (e g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) to treat the mammal.
BACKGROUND INFORMATION
Hepatocyte growth factor receptor, more commonly referred to as MET, is a known oncogenic driver in multiple malignancies (Guo etal., Nat. Rev. Clin. Oncol., 17:569-587 (2020)). Mutations that affect the donor or acceptor splice sites oiMEl exon 14 pre-mRNA can lead to skipping of exon 14 during splicing and an mRNA product where exons 13 and 15 are fused (Kong-Beltran et al., Cancer Res., 66:283-289 (2006); Lu et al., Cancer Res., 77:4498-4505 (2017); and Ma e/ al., Cancer Res., 63:6272-6281 (2003)). Subsequent translation results in a shortened MET polypeptide without its juxtamembrane domain. This juxtamembrane domain includes a degron that is recognized by the E3 ubiquitin-protein ligase Cbl. In the absence of the MET degron recognized by Cbl, MET polypeptides are not readily ubiquitinated, thus prolonging their half-life by avoiding degradation by the proteasome. In addition, there are a variety of other mutations and genomic alterations that can drive the oncogenicity of MET polypeptides (Guo etal., Nat. Rev. Clin. Oncol., 17:569- 587 (2020)). MET polypeptides have been an elusive target for cancer treatments for decades despite its known oncogenicity.
SUMMARY
This document provides methods and materials involved in treating a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14). For example, this document provides bifunctional molecules (e g., PROTACs) that include (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide). A bifunctional molecule (e.g., a PROTAC) including (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can recruit an E3 ligase polypeptide to the MET polypeptide resulting in ubiquitination of the MET polypeptide (e.g., to promote degradation of the MET polypeptide). This document also provides methods and materials for making and using bifunctional molecules (e.g., PROTACs) provided herein (e.g., bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)). In some cases, bifunctional molecules (e.g., PROTACs) provided herein (e.g., bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal having cancer (e.g., to treat the mammal).
As demonstrated herein, a PROTAC that includes (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can bind a MET polypeptide and restore ubiquitination of the MET polypeptide to promote degradation of the MET polypeptide. Having the ability to specifically target a MET polypeptide for ubiquitination and degradation provides a unique and unrealized opportunity to treat cancers expressing an oncogenic MET polypeptide.
In general, one aspect of this document features molecules comprising capmatinib covalently attached to an E3 ligase ligand. The capmatinib can be directly covalently attached to the E3 ligase ligand. The molecule can be:
Figure imgf000005_0001
The capmatinib can be indirectly covalently attached to the E3 ligase ligand via a linker. The linker can be a hydrocarbon linker. The hydrocarbon linker can include from about 3 to about 12 carbon atoms. The hydrocarbon linker can include no more than one oxygen atom. The linker can be:
Figure imgf000005_0002
Figure imgf000006_0001
Figure imgf000007_0001
The E3 ligase ligand can be thalidomide, lenalidomide, pomalidomide, VHL032, nutlin 3a, idasanutlin, RG7112, bestatin, MV1, LCL-161, l-[3-(4-bromophenyl)-4,5-dihydro-5-phenyl- 1 h-pyrazol- 1 -yl]-2-chloro-ethanone, or 1 -(3,4-dihydro-6-hydroxy- 1 (2h)-quinolinyl)- 1 - propanone. The E3 ligase ligand can bind to an E3 ligase polypeptide selected from the group consisting of a Von Hippel-Lindau (VHL) polypeptide, a cereblon (CRBN) polypeptide, a MDM2 polypeptide, an inhibitor of apoptosis (IAP) polypeptide, a DDB 1 and CUL4 associated factor 15 (DCAF15) polypeptide, a DDB1 and CUL4 associated factor 16 (DCAF16) polypeptide, a ring finger protein 4 (RNF4) polypeptide, and a ring finger protein 114 (RNF114) polypeptide. The molecule can be:
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
5
Figure imgf000011_0001
Figure imgf000012_0001
The molecule can be:
Figure imgf000013_0001
The molecule can promote degradation of a MET polypeptide within cells that express the MET polypeptide. The MET polypeptide can be a wild-type MET polypeptide. The MET polypeptide can be a mutant MET polypeptide. The mutant MET polypeptide can be a MET polypeptide that lacks the amino acid sequence encoded by at least a portion of exon 14, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a nuclear basket protein (TPR) polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a kinesin family member 5B (KIF5B) polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a protein tyrosine phosphatase receptor type Z1 (PTPRZ1) polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a CAP-Gly domain containing linker protein 2 (CL1P2) polypeptide, or a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a trafficking from ER to golgi regulator (TFG) polypeptide. The MET polypeptide can be a human MET polypeptide.
In another aspect, this document features compositions including one or more molecules comprising capmatinib covalently attached to an E3 ligase ligand.
In another aspect, this document features pharmaceutical compositions including one or more molecules comprising capmatinib covalently attached to an E3 ligase ligand and a pharmaceutically acceptable carrier, excipient, or diluent.
In another aspect, this document features methods for treating a mammal having cancer. The methods can include, or consist essentially of, administering, to a mammal having cancer where cancer cells of the cancer express a MET polypeptide, one or more molecules comprising capmatinib covalently attached to an E3 ligase ligand. The mammal can be a human. The MET polypeptide can be a wild-type MET polypeptide. The MET polypeptide can be a mutant MET polypeptide. The mutant MET polypeptide can be a MET polypeptide that lacks the amino acid sequence encoded by at least a portion of exon 14, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TPR polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a KIF5B polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a PTPRZ1 polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a CLIP2 polypeptide, or a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TFG polypeptide. The MET polypeptide can be an oncogenic MET polypeptide. The cancer can be a lung cancer, a gastric cancer, a papillary renal cell carcinoma, a brain cancer, or a sarcoma.
In another aspect, this document features methods for inducing ubiquitination of a MET polypeptide in a mammal. The methods can include, or consist essentially of, administering, to a mammal, one or more molecules comprising capmatinib covalently attached to an E3 ligase ligand. The mammal can be a human. The MET polypeptide can be a wild-type MET polypeptide. The MET polypeptide can be a mutant MET polypeptide. The mutant MET polypeptide can be a MET polypeptide that lacks the amino acid sequence encoded by at least a portion of exon 14, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TPR polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a KTF5B polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a PTPRZ1 polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a CLIP2 polypeptide, or a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TFG polypeptide. The MET polypeptide can be an oncogenic MET polypeptide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF THE DRAWINGS
Figures 1 A - IE: Live cell imaging screen for MET exon 14 skipping-GFP degradation. Figure 1 A) Characterization of HEK293 cells stably transfected with either GFP-vector or GFP-Met-Exon-14 skipping mutant. Figure IB) Capmatinib based PROTAC library screened at 1 pM using live cell imaging in HEK293 transfected with GFP-Met- Exon-14 skipping mutant. The bar graph shows the green count values over confluence (phase) normalized to time zero of each well at 0 hours, 8 hours, and 24 hours post addition. The upper and lower broken lines indicate activity relative to capmatinib at 8 and 24 hours respectively. The bars represent mean ± SD of three independent biological replicates (n = 3). Figure 1C) Time course study to assess the effects of the linkers in PROTACs generated with capmatinib and VHL ligand. The line graph is an average of three independent biological replicates (n = 3), green count values over confluence (phase) normalized to 0 time of each well. The number of linker atoms is indicated in parenthesis. Figure ID) A dose-response and time course study with the most potent capmatinib based PROTAC 48-284 (10000 nM, 5000 nM, 1000 nM, 500 nM, and 100 nM) in HEK293 transfected with GFP-Met-Exon-14 skipping mutant. The images analyzed every 6 hours for 24 hours post-treatment. The line graph is an average of three independent biological replicates (n = 3), green count values over confluence (phase) normalized to time zero of each well. Figure IE) A time course study with the capmatinib based PROTAC 48-284 (1 pM) with HEK293 transfected with GFP-Met-Exon-14 skipping mutant in the presence and absence ofMG132 (10 pM). The images analyzed every 2 hours for 24 hours post-treatment. The line graph is an average of three independent biological replicates (n = 3), green count values over confluence (phase) normalized to 0 time of each well. Figures 2A - 2D: Characterization of MET-PROTACs. Figure 2A) Volcano plot depicting changes of protein abundance in HEK293 transfected with GFP-Met-Exon-14 skipping mutant cells. The cells were treated with 48-284 (1 mM) and incubated for 24 hours. The lysates were subjected to label-free proteomic analyses, and the volcano plot represents 5106 proteins, with the log2 fold change shown on the x-axis and negative log 10 p-values on the y axis. Data are presented as the averages of three independent biological replicates (n = 3). The red circle shows GFP-MET-Exl4-skipping, and the green circle shows MET. Figure 2B) Volcano plot of the 114 kinases that were quantified in the above study. Figure 2C and Figure 2D) HEK293 transfected with GFP-Met-Exon-14 skipping mutant cells were treated with 48-284 (2 pM, 1 pM ,or 0.5 pM) or SJF-8240 (2 pM, 1 pM, or 0.5 pM) and the cells were imaged every 2 hours for 22 hours using IncuCyte.
Figures 3A - 3B: Time and dose effects of MET PROTACs. Figure 3A) The Hs746T cell line was treated with 48-284 at 1.0 pM and MET was assessed at the indicated timepoints by western blots with P-actin controls. These cells were also treated at the indicated doses for 8 hours. The effects on downstream RAS/AKT and RAS/ERK pathway signaling were also assessed. Figure 3B) Similarly, the Hs746T cell line was treated with the foretinib -based PROTAC SJF8240 at 1.0 pM and MET was assessed at the indicated timepoints by western blots with P-actin controls. These cells were also treated at the indicated doses for 8 hours. The effects on downstream RAS/AKT and RAS/ERK pathway signaling were also assessed.
Figures 4A - 4C: In vivo effects of MET-PROTAC on xenograft. Figure 4A) Multiple UW21 xenografts with a MET exon 14 skipping mutation were removed from mouse flanks and stained for MET by immunohistochemistry. In this representative example, intense membranous and cytoplasmic MET expression is seen in almost all the tumor cells. Figure 4B) Other UW21 xenografts were treated with to doses of 48-284 through tail vein injection eight hours apart, removed six hours after the second injection and stained for MET by immunohistochemistry. In this representative example, significant reduction in MET expression was observed along with evidence of a treatment effect. Figure 4C) Protein lysates were obtained from UW21 xenografts that were treated (T) with 48-284 and controls (Ctrl) and assessed for ubiquitination. Figures 5A - 5D: Micro-cancer model with ZM-fusion. Figure 5A) A Circos plot summarizing the genomic abnormalities identified in PT425. Breakpoint junctions are presented as lines connecting two breakpoints, including the PTPRZ1MET fusion shown at 7q31. Figure 5B) This junction plot visualizes the mate-pair reads supporting the intrachromosomal rearrangement between PTPRZ1 and MET on chromosome 7. Coverage levels across each position are presented in gray-shaded regions. Gene regions are labeled with enumeration of exons. There were 112 mate-pair reads supporting the zm fusion and an average bridged coverage of 64X for the specimen. Figure 5C) This graph illustrates the oncogenic zm fusion. The thick solid black horizontal lines mark the involved portions of the genes PTPRZ1 and MET. The unmarked portions are left out. The dotted line joins the breakpoints. The protein domains are shown as stripes inside each protein. Since the MET breakpoint is inside the 5’ UTR, the UTR segment is shown as a grey- filled box on one end of the protein. Figure 5D) The dose response curve of PT425 treated with 48-284 is shown across multiple concentrations. The light grey curve with circle datapoints shows the response when stimulated with 20 ng/mL of HGF and the dark grey with square data pointscurve shows the response without HGF stimulation.
Figures 6A - 6C: Figure 6A) Capmatinib docked into MET kinase domain (pdb: 3zbx). Capmatinib is shown in sticks, and the kinase domain of MET is shown as a ribbon structure. The quinoline ring of capmatinib mimics the adenine ring of the ATP, and the quinoline nitrogen is within hydrogen bonding distance of the N-H of Met1160 (hydrogen bond is shown in black line). Figure 6B) Live cell imaging study with a 60 member PROTAC library. HEK293 transfected with GFP-Met-Exon-14 skipping mutant were subjected to 1 pM of capmatinib or the PROTAC library and the cells imaged every 2 hours for 24 hours. The bar graph shows the green count values over confluence (phase) normalized to 0 time of each well at 0 hours, 8 hours, and 24 hours post addition. The bars represent mean ± SD from three independent biological replicates (n = 3). Figure 6C) HEK293 transfected with GFP-Met-Exonl4-skipping cells were treated with capmatinib (1 pM) or 48-284 (1 pM) for 24 hours, and the resulting lysates were subjected to western blot analyses and probed with a GFP antibody. Figures 7A - 7C: Figure 7A) Volcano plot depicting changes of protein abundance in HEK293 transfected with GFP-Met-Exon-14 skipping mutant cells. The cells were treated with capmatinib (1 pM) and incubated for 24 hours. The lysates were subjected to label-free proteomic analyses, and the volcano plot represents 5100 proteins, with the log2 fold change shown on the x-axis and negative logl O p-values on the y axis. Data presented in the averages of three independent biological replicates (n = 3). The circle shows MET HGF receptor. Figure 7B) PCA plot of the proteomics study. Quantification of MET and MET- Exonl4-skipping-GFP levels using unique peptides from the label free mass spectrometry study. The bar graph represent average ± SD of three independent biological replicates (n = 3). Figure 7C) Quantification of MET-Exonl4-skipping-GFP levels using live cell imaging. The line graph represent average of three independent biological replicates. Degradation time 50 (DT50) is the time it takes to degrade 50% of the protein at the indicated concentration of the drug.
Figures 8A - 80: NMR spectra for exemplary PROTACs. Figure 8A) PROTAC 48- 269. Figure 8B) PROTAC 48-270. Figure 8C) PROTAC 48-273. Figure 8D) PROTAC 48- 276. Figure 8E) PROTAC 48-284. Figure 8F) PROTAC 48-285. Figure 8G) PROTAC 48- 289. Figure 8H) PROTAC 48-295. Figure 81) PROTAC 48-297. Figure 8J) PROTAC 48- 299. Figure 8K) PROTAC 50-210. Figure 8L) 50-211. Figure 8M) PROTAC 50-212. Figure 8N) PROTAC 50-213. Figure 80) PROTAC 50-214.
Figures 9A - 9B: Characterization of PROTAC 48-284. Figure 9A) High Performance Liquid Chromatography (HPLC) chromatogram of PROTAC 48-284. Figure 9B) High-resolution mass spectrometry (HRMS) of PROTAC 48-284.
Figure 10: A graph showing body weight of mice with the UW21 xenografts implanted in their flanks and treated with different compounds for two weeks.
Figure 11 : A picture showing the UW21 xenografts that were removed from the flanks of the mice following treatment with control, PROTAC 48-284 (10 mg/kg or 20 mg/kg,) or capmatinib (5 mg/kg or 10 mg/kg) for two weeks.
Figure 12: A graph showing sizes of the UW21 xenografts treated with control, PROTAC 48-284 (10 mg/kg or 20 mg/kg), or capmatinib (5mg/kg or 10 mg/kg) over two weeks. Figure 13: An enlarged picture showing the UW21 xenografts that were removed from the flanks of the mice following treatment with control or PROTAC 48-284 (10 mg/kg or 20 mg/kg) for two weeks.
Figure 14: A graph showing sizes of the UW21 xenografts treated with control or PROTAC 48-284 (10 mg/kg or 20 mg/kg) over two weeks.
Figure 15: A graph showing body weight of mice with the Rudin439 xenografts implanted in their flanks and treated with PROTAC 48-284 (10 mg/kg or 20 mg/kg) for two weeks.
Figure 16: A picture showing the Rudin439 xenografts that were removed from the flanks of the mice following treatment with control, PROTAC 48-284 (10 mg/kg or 20 mg/kg), or capmatinib (5 mg/kg or 10 mg/kg) for two weeks.
Figure 17: A graph showing sizes of the Rudin439 xenografts treated with control, PROTAC 48-284 (10 mg/kg or 20 mg/kg), or capmatinib (5 mg/kg or 10 mg/kg) over two weeks.
Figure 18: An enlarged picture showing the Rudin439 xenografts that were removed from the flanks of the mice following treatment with control or PROTAC 48-284 (10 mg/kg or 20 mg/kg) for two weeks.
Figure 19: A graph showing sizes of the Rudin439 xenografts treated with control or PROTAC 48-284 (10 mg/kg or 20 mg/kg) over two weeks.
DETAILED DESCRIPTION
This document is based, at least in part, on the discovery of PROTACs that can bind to a MET polypeptide (e.g., an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) and can be used restore ubiquitination of the MET polypeptide (e.g., to promote degradation of the MET polypeptide), thereby reducing the levels of oncogenic MET polypeptides within a mammal (e.g., a human).
This document provides methods and materials involved in treating a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14). For example, this document provides bifiinctional molecules (e.g., PROTACs) that include (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide). This document also provides methods and materials for making and using bifimctional molecules (e.g., PROTACs) provided herein (e.g., bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)). In some cases, bifunctional molecules (e.g., PROTACs) provided herein (e.g., bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal having cancer (e.g., to treat the mammal).
Bifunctional molecules (e.g., PROTACs) provided herein (e.g., bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can be designed to include any appropriate targeting moiety having the ability to bind to a MET polypeptide. As described herein, capmatinib can be used as a targeting moiety that binds to a MET polypeptide to design PROTACs having the ability to (a) degrade a MET polypeptide (e.g., an oncogenic MET polypeptide) and/or (b) inhibit the kinase activity of a MET polypeptide (e.g., catalytically inhibit the kinase activity of a MET polypeptide). In some cases, a targeting moiety that binds to a MET polypeptide other than capmatinib can be used to design a PROTAC having the ability to target MET polypeptides. For example, tepotinib, savolitinib, crizotinib, amuvatinib, tivantinib, cabozantinib, brigatinib, or fostamatinib can be used as a targeting moiety that binds to a MET polypeptide to design a PROTAC having the ability to target MET polypeptides. In some cases, a PROTAC provided herein can lack foretinib.
In some cases, a targeting moiety that binds to a MET polypeptide that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein can have one of the following structures.
Figure imgf000021_0001
In some cases, a targeting moiety that binds to a MET polypeptide that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein can be as shown in Example 2. In some cases, a targeting moiety that binds to a MET polypeptide that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein can be as described in Example 1.
A targeting moiety that binds to a MET polypeptide can target (e.g., target and bind to) any appropriate MET polypeptide. In some cases, a MET polypeptide that can be targeted by a targeting moiety that binds to a MET polypeptide in a bifunctional molecule (e.g., a PROTAC) provided herein can be a mutant MET polypeptide. For example, a MET polypeptide that can be targeted by a targeting moiety that binds to a MET polypeptide in a bifunctional molecule (e.g., a PROTAC) provided herein can include one or more oncogenic mutations. In some cases, a mutant MET polypeptide can lack at least a portion of the amino acid sequence encoded by exon 14. In some cases, a mutant MET polypeptide can have increased kinase activity (e.g., as compared to a MET polypeptide lacking the mutation). In some cases, a mutant MET polypeptide can be a MET fusion polypeptide including a portion of a MET polypeptide and a portion of another polypeptide (e.g., resulting from a genetic translocation creating a fusion gene including a portion of a nucleic acid encoding a MET polypeptide and a portion of another gene). For example, a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a nuclear basket protein (TPR) polypeptide. For example, a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a kinesin family member 5B (KIF5B) polypeptide. For example, a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a protein tyrosine phosphatase receptor type ZI (PTPRZ1) polypeptide (e.g., a zm-fiision). For example, a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a CAP-Gly domain containing linker protein 2 (CLIP2) polypeptide. For example, a MET fusion polypeptide can include a portion of a MET polypeptide and a portion of a trafficking from ER to golgi regulator (TFG) polypeptide. Examples of MET polypeptides that can be targeted by targeting moiety that binds to a MET polypeptide in a bifunctional molecule (e.g., a PROTAC) provided herein include, without limitation, MET polypeptides having an amino acid sequence set forth in the National Center for Biotechnology Information (NCBI) databases at Accession No. AC002080.1, Accession No. AC002543.1, Accession No. M15325.1, Accession No. EU176015.1, Accession No. KY412920.1, Accession No. KY412921.1, Accession No. KY412922.1 and Accession No. KY412923.1. In some cases, a MET polypeptide that can be targeted by a targeting moiety that binds to a MET polypeptide in a bifunctional molecule (e.g., a PROTAC) provided herein can be a MET polypeptide having an altered level of expression. For example, a mutant MET polypeptide can have an increased level of MET expression (e.g., resulting from a genetic translocation allowing a heterologous promoter to drive expression of a nucleic acid encoding a MET polypeptide resulting in an increased level of expression as compared to a level of MET polypeptide when expression of the nucleic acid encoding the MET polypeptide is driven by its endogenous promoter). In some cases, a MET polypeptide that can be targeted by targeting moiety that binds to a MET polypeptide in a bifunctional molecule (e.g., a PROTAC) provided herein can be as described elsewhere (see, e.g., Stransky et al., Nat. Commun.. 5:4846 (2014) at, for example, Figure 2; Suda et al., Transl. Lung Cancer Res., 9(6):2618-2628 (2020) at, for example, page 2621; and You et al., Front. Neurol., Volume 12 | Article 715206 (2021) at, for example, pages 7-8 and Table 2). In some cases, a MET polypeptide that can be targeted by targeting moiety that binds to a MET polypeptide in a bifunctional molecule (e.g., a PROTAC) provided herein can be as described in Example 1.
Bifunctional molecules (e.g., PROTACs) provided herein (e.g., bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide)) can lack a linker or can include any appropriate linker. A linker can be used to attach (e.g., covalently attach) a targeting moiety that binds to a MET polypeptide to an E3 ligase ligand. A linker in a bifunctional molecule (e.g., a PROTAC) provided herein can be any appropriate type of molecule (e.g., polypeptides, atoms, and polymers). In some cases, a linker can be hydrophobic. Examples of linkers that can be included in a bifunctional molecule (e g., a PROTAC) provided herein include, without limitation, hydrocarbon linkers, polyethylene glycol (PEG) linkers, alkyl chain linkers, and alkyl/ether linkers. A linker can be any appropriate length. For example, when a linker is a hydrocarbon linker, the linker can include from about 3 to about 12 carbon atoms (e.g., from about 3 to about 12, from about 3 to about 10, from about 3 to about 8, from about 3 to about 6, from about 3 to about 5, from about 3 to about 4, from about 4 to about 12, from about 5 to about 12, from about 6 to about 12, from about 8 to about 12, from about 10 to about 12, from about 4 to about 11 , from about 5 to about 10, from about 6 to about 8, from about 4 to about 7, from about 5 to about 8, from about 6 to about 9, from about 7 to about 10, or from about 8 to about 11 carbon atoms). In some cases, a hydrocarbon linker can have 9 carbon atoms. In some cases when a linker is a hydrocarbon linker, the hydrocarbon linker does not include more than one oxygen atom.
Examples of linkers that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein include, without limitation, linkers having one of the following structures.
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
In some cases, a linker that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein can be as shown in Example 3. In some cases, a linker that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein can be as described in Example 1 (see, e.g., Table 1).
Bifunctional molecules (e.g., PROTACs) provided herein (e.g., bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide)) can include any appropriate E3 ligase ligand. An E3 ligase ligand can be any appropriate molecule that can target (e.g., target and bind) to an E3 ligase polypeptide. An E3 ligase ligand can target (e.g., target and bind to) any appropriate E3 ligase polypeptide. Examples of E3 ligase polypeptides that can be targeted by an E3 ligase ligand in a bifunctional molecule (e.g., a PROTAC) provided herein include, without limitation, Von Hippel-Lindau (VHL) polypeptides, cereblon (CRBN) polypeptides, MDM2 polypeptides, inhibitor of apoptosis (IAP) polypeptides, DDB1 and CUL4 associated factor 15 (DCAF15) polypeptides, DDB1 and CUL4 associated factor 16 (DCAF16) polypeptides, ring finger protein 4 (RNF4) polypeptides, and ring finger protein 114 (RNF114) polypeptides. In some cases, E3 ligase polypeptides that can be targeted by an E3 ligase ligand in a bifunctional molecule (e.g., a PROTAC) provided herein include, without limitation, E3 ligase polypeptides having an amino acid sequence set forth in the NCBI databases at Accession No. X82654.1, Accession No. NG_008212.3, and Accession No. NC_000003.12. In some cases, an E3 ligase polypeptide that can be targeted by an E3 ligase ligand in a bifiinctional molecule (e.g., a PROTAC) provided herein can be as described in Example 1. An E3 ligase ligand can be any appropriate type of molecule (e.g., small molecules, oligonucleotides, and polypeptides). Examples of E3 ligase ligands that can be used to design a bifunctional molecule (e.g., a PROTAC) provided herein include, without limitation, thalidomide, lenalidomide, pomalidomide, VE1L032, nutlin 3a, idasanutlin, RG7112, bestatin, MV1, LCL-161, l-[3-(4-bromophenyl)-4,5-dihydro-5-phenyl-lh-pyrazol-l-yl]-2-chloro- ethanone, and l-(3,4-dihydro-6-hydroxy-l(2h)-quinolinyl)-l-propanone. In some cases, an E3 ligase ligand that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein can be as described elsewhere (see, e g., Rana et al., Cancer, 13:5506 (2021) at, for example, Figure 4; and Bricelj et al., Front Chem., 9:707317 (2021) at, for example, Figure 2 and Figure 4). In some cases, an E3 ligase ligand that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein can have one of the following structures.
Figure imgf000026_0001
Figure imgf000027_0001
In some cases, an E3 ligase ligand that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein can be as shown in Example 4. In some cases, an E3 ligase ligand that can be included in a bifunctional molecule (e.g., a PROTAC) provided herein can be as described in Example 1.
In some cases, a bifunctional molecule (e.g., a PROTAC) provided herein (e.g., a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide)) can include: (a) a targeting moiety having the structure
Figure imgf000027_0002
° , and (c) an E3 ligase ligand having the structure
Figure imgf000028_0001
For example, a bifunctional molecule (e.g., a PROTAC) provided herein (e.g., a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can have the structure:
Figure imgf000028_0002
In some cases, a bifunctional molecule (e.g., a PROTAC) provided herein (e.g., a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be as shown in Figure 8.
In some cases, a bifunctional molecule (e.g., a PROTAC) provided herein (e.g., a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide)) can be as described in Example 1.
A bifunctional molecule (e.g., a PROTAC) provided herein (e.g., a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be made using any appropriate method. In some cases, a bifunctional molecule (e g., a PROTAC) provided herein (e.g., a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be made using a chemical synthesis. In some cases, a bifunctional molecule (e.g., a PROTAC) provided herein (e.g., a bifunctional molecule including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be made as described in Example 1.
This document also provides compositions containing one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)). In some cases, one or more (e.g., one, two, three, four, or more) bifunctional molecules (e.g., PROTACs) provided herein can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14). For example, one or more bifunctional molecules (e.g., PROTACs) provided herein can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, cyclodextrins (e.g., beta- cyclodextrins such as KLEPTOSE®), dimethylsulfoxide (DMSO), sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene- polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline such as phosphate buffered saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and corn oil.
In some cases, when a composition containing one or more (e.g., one, two, three, four, or more) bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) is administered to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14), the composition can be designed for oral or parenteral (including, without limitation, subcutaneous, intramuscular, intravenous, intradermal, intra-cerebral, intrathecal, intraabdominal, and intraperitoneal injections) administration to the mammal. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
In some cases, a composition containing one or more (e.g., one, two, three, four, or more) bifunctional molecules (e.g., PROTACs) provided herein (e g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be in the form of a sterile injectable suspension (e.g., a sterile injectable aqueous or oleaginous suspension). This suspension may be formulated using, for example, suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3 -butanediol. Examples of acceptable vehicles and solvents that can be used include, without limitation, saline, mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils can be used as a solvent or suspending medium. In some cases, a bland fixed oil can be used such as synthetic mono- or di-glycerides. In some cases, a composition containing one or more (e.g., one, two, three, four, or more) bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
This document also provides methods and materials for using one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)). In some cases, one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be used for treating a mammal (e.g., a human) having cancer (e g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14). For example, one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) to treat the mammal.
In some cases, administering one or more bifunctional molecules (e.g., PROTACs) provided herein (e g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) can be effective to reduce the size of the cancer in the mammal. For example, one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifimctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having cancer) as described herein to reduce the size of the cancer in the mammal. In some cases, the methods and materials provided herein can be used as described herein to reduce the number of cancer cells in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials provided herein can be used as described herein to reduce the volume of one or more tumors in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, administering one or more bifunctional molecules (e.g., PROTACs) provided herein (e g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) can be effective to improve survival of the mammal. For example, one or more bifunctional molecules (e g., PROTACs) provided herein (e g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having cancer) as described herein to improve survival of the mammal. In some cases, the methods and materials provided herein can be used as described herein to improve the survival of the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials provided herein can be used as described herein to improve the survival of the mammal by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more). In some cases, administering one or more bifunctional molecules (e.g., PROTACs) provided herein (e g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) can be effective to reduce a level of one or more MET polypeptides in cells (e.g., cancer cells) within the mammal. The term “reduced level” as used herein with respect to a level of a MET polypeptide in a mammal refers to any level that is less than the level of that MET polypeptide observed in that mammal prior to being treated as described herein (e.g., prior to being administered one or more bifunctional molecules provided herein). In some cases, a reduced level of a MET polypeptide can be a level that is at least 5 percent (e.g., at least 10, at least 15, at least 20, at least 25, at least 35, at least 50, at least 75, at least 100, or at least 150 percent) less than the level of that MET polypeptide prior to being treated as described herein.
Any appropriate mammal having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) can be treated as described herein (e g., by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)). Examples of mammals that can have cancer and can be treated as described herein (e.g., by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) include, without limitation, humans, non- human primates such as monkeys, horses, bovine species, porcine species, dogs, cats, mice, and rats. In some cases, a human having cancer can be treated by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) to the human.
A mammal (e.g., a human) having any type of cancer can be treated as described herein (e.g., by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)). In some cases, a cancer treated as described herein can include one or more solid tumors. In some cases, a cancer that can be treated as described herein can include one or more cancer cells having an oncogenic MET polypeptide (e.g., a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14). Examples of cancers that can be treated as described herein include, without limitation, lung cancers (e.g., non-small cell lung cancers), gastric cancers, papillary renal cell carcinomas, brain cancers, and sarcomas.
In some cases, methods described herein also can include identifying a mammal as having cancer. Examples of methods for identifying a mammal as having cancer include, without limitation, physical examination, laboratory tests (e.g., blood and/or urine), biopsy, imaging tests (e.g., X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, and/or genetic tests. Once identified as having cancer, a mammal can treated as described herein (e.g., by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)).
One or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal (e.g., a human) by any appropriate route (e.g, intratumoral injection, intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, oral, intranasal, inhalation, transdermal, and parenteral). In some cases, one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal (e.g., a human) by intratumoral injection. In some cases, one or more bifunctional molecules (e.g., PROTACs) provided herein (e g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal (e.g., a human) by intravenous injection.
A composition containing one or more (e.g., one, two, three, four, or more) bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) in any appropriate amount (e.g., any appropriate dose). An effective amount of a composition containing one or more bifunctional molecules (e g., PROTACs) provided herein can be any amount that can treat a mammal having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) as described herein without producing significant toxicity to the mammal. In some cases, an effective amount of one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a MET-targeting moiety, (b) a linker, and (c) an E3 ligase ligand) can be from about 0.1 milligrams per kilogram body weight (mg/kg) to about 1000 mg/kg (e.g., from about 0.1 mg/kg to about 750 mg/kg, from about 0.1 mg/kg to about 500 mg/kg, from about 0.1 mg/kg to about 400 mg/kg, from about 0.1 mg/kg to about 300 mg/kg, from about 0.1 mg/kg to about 200 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 25 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 1000 mg/kg, from about 10 mg/kg to about 1000 mg/kg, from about 25 mg/kg to about 1000 mg/kg, from about 50 mg/kg to about 1000 mg/kg, from about 75 mg/kg to about 1000 mg/kg, from about 100 mg/kg to about 1000 mg/kg, from about 250 mg/kg to about 1000 mg/kg, from about 500 mg/kg to about 1000 mg/kg, from about 750 mg/kg to about 1000 mg/kg, from about 1 mg/kg to about 750 mg/kg, from about 10 mg/kg to about 500 mg/kg, from about 25 mg/kg to about 250 mg/kg, from about 50 mg/kg to about 100 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 10 mg/kg to about 25 mg/kg, from about 15 mg/kg to about 30 mg/kg, from about 20 mg/kg to about 50 mg/kg, from about 50 mg/kg to about 75 mg/kg, from about 75 mg/kg to about 100 mg/kg, from about 100 mg/kg to about 200 mg/kg, from about 200 mg/kg to about 300 mg/kg, from about 300 mg/kg to about 400 mg/kg, from about 400 mg/kg to about 500 mg/kg, from about 500 mg/kg to about 600 mg/kg, from about 600 mg/kg to about 700 mg/kg, or from about 700 mg/kg to about 800 mg/kg). For example, an effective amount of one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be from about 15 mg/kg to about 30 mg/kg. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and/or severity of the cancer in the mammal being treated may require an increase or decrease in the actual effective amount administered.
A composition containing one or more (e.g., one, two, three, four, or more) bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide)) can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) in any appropriate frequency. The frequency of administration can be any frequency that can treat a mammal having cancer (e g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) without producing significant toxicity to the mammal. For example, the frequency of administration can be from about twice a day to about one every other day, once a day to about once a week, from about once a week to about once a month, or from about twice a month to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and/or route of administration may require an increase or decrease in administration frequency.
A composition containing one or more (e.g., one, two, three, four, or more) bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) for any appropriate duration. An effective duration for administering or using one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., a composition including one or more bifiinctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide)) can be any duration that can treat a mammal having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) without producing significant toxicity to the mammal. For example, the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and/or route of administration.
In some cases, methods for treating a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) as described herein (e.g., by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) can include administering one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifiinctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide)) as the sole active agent to treat the mammal. For example, a composition containing one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e g., thalidomide) can include the one or more bifunctional molecules provided herein as the sole active ingredient in the composition that is effective to treat a mammal having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14).
In some cases, methods for treating a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) as described herein (e.g., by administering one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) also can include administering to the mammal one or more (e.g, one, two, three, four, five or more) additional agents used to treat cancer to the mammal and/or performing therapies used to treat cancer on the mammal. For example, a combination therapy used to treat cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) can include administering one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)), and also administering one or more (e.g., one, two, three, four, five or more) agents used to treat cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14). In some cases, an agent that can be administered to a mammal to treat cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) can be a chemotherapeutic agent. In some cases, an agent that can be administered to a mammal to treat cancer can be an immune checkpoint inhibitor (e.g., PD1 inhibitors, PD-L1 inhibitors, and CTLA4 inhibitors). In some cases, an agent that can be administered to a mammal to treat cancer can be a cytotoxic agent. Examples of agents that can be administered to a mammal to treat cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) include, without limitation, altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, pemetrexed, paclitaxel, docetaxel, vinorelbine, gemcitabine, amivantamab, and any combinations thereof. In cases where one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) are used in combination with additional agents used to treat cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14), the one or more additional agents can be administered at the same time (e.g., in a single composition containing both one or more bifunctional molecules (e.g., PROTACs) provided herein and the one or more additional agents) or independently. For example, one or more bifunctional molecules (e.g., PROTACs) provided herein can be administered first, and the one or more additional agents administered second, or vice versa.
In some cases, a combination therapy used to treat cancer (e g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) can include administering one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)), and also can include performing one or more (e g., one, two, three, four, five or more) additional therapies used to treat cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) on the mammal. Examples of therapies used to treat cancer (e.g., a cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) include, without limitation, surgery, radiation therapies, adoptive cell transfer therapies (e.g., CAR-T cell therapies), immunotherapies, radiofrequency ablation, and/or irreversible electroporation. In cases where one or more bifunctional molecules (e.g., PROTACs) provided herein (e.g., one or more bifunctional molecules each including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide)) are used in combination with one or more additional therapies used to treat cancer, the one or more additional therapies can be performed at the same time or independently of administering the one or more bifunctional molecules (e.g., PROTACs) provided herein. For example, administering one or more bifunctional molecules (e.g., PROTACs) provided herein can be performed before, during, or after the one or more additional therapies are performed.
In some cases, the size of the cancer (e.g., the number of cancer cells and/or the volume of one or more tumors) present within a mammal and/or the severity of one or more symptoms of the cancer being treated can be monitored. Any appropriate method can be used to determine whether or not the size of the cancer present within a mammal is reduced. For example, imaging techniques can be used to assess the size of the cancer present within a mammal can be used to detect the presence of the cancer present within a mammal.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1: Restored ubiquitination and degradation of exon 14 skipped hepatocyte growth factor receptor with proteolysis targeting chimeras
This Example demonstrates that MET -targeting PROTACs can restore ubiquitination of MET with exon 14 skipping mutations and promote MET degradation. MATERIALS AND METHODS
Fluorescent models
HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Corning), 10% fetal bovine serum (FBS, Coming)), penicillin/streptomycin (1 :100, Gibco). The plasmid pLenti-MetGFP was purchased from Addgene (#37560) and the exon 14 skipping mutations was introduced using Q5 Site-Directed Mutagenesis Kit (NEB, Cat# E0554S). 25 ng of pLenti-MET-GFP plasmid were used as a template, 10 pM forward primer (5’-ATCAGTTTCCTAATTCATTCTAG-3’; SEQ ID NO:1) and 10 pM reverse primer (5’-CTTTAATTTGCTTTCTCTTTTTC-3’; SEQ ID NO:2), 12.5 pL of Q5 Hot Start High-Fidelity 2X master Mix for mutagenesis PCR (30 seconds in 98°C for initial denaturation, 98°C for 10 seconds, 62°C for 30 seconds, and 72°C for 6 minutes, repeated for 25 cycles, plus 2 minutes in 72°C for final extension). The PCR product was processed with KLD reaction and transformation following the protocol provided by the manufacturer (NEB Inc.).
METexl 4A-GFP screening
HEK293 Affi7exl4A expressing cells (4 x 103 cells / well) were plated in flat bottomed, sterile, 96-well TPP tissue culture plates (Catalog no. 92696 Midwest Scientific), in 90 pL DMEM high media (Catalog no. SH30022 VWR). The cells were allowed to adhere overnight at 37°C in a CO2 incubator (5% CO2). Drug working solutions were prepared from 10 mM DMSO (Catalog no. BP231 Fisher Scientific) stocks. 10 pL of the working solution was added to each well in the plate to yield the indicated final concentration in 0.1% DMSO and a DMSO control was included. The plates were set up in an Incucyte SX3 live cell imager (Sartorius Corporation) to acquire phase contrast and green fluorescence data. The imager was itself housed in a CO2 incubator (5% CO2), and the plates were maintained at 37°C over the course of the experiment. Images of the entire well was captured at 4x magnification, every 2 hours, for the indicated period. The images were analyzed with the included phase contrast cell tracking module software to generate the curves showing % confluence over time. The degradation % was calculated using the following formula: % Degradation = 100 - (100 x (FX/FDMSO)) where F is the normalized green count. Proteomics
A suspension of 5 million cells (HEK293 ATA7'ex l4A-GFP) was seeded into a 15 cm plates in 20 mL ofDMEM high (Catalog # SH30021.FS; Hyclone) with 10% FBS (Catalog # 26140079; Life Technologies) and 1% penicillin-streptomycin (Catalog # 16777-164; Hyclone), and cells were allowed to adhere overnight in a humidified 5% CO2 incubator at 37°C. The following day, cells were treated with 1 pM of capmatinib, 1 pM of 48-284, or DMSO and incubated in a humidified 5% CO2 incubator at 37°C for 24 hours. Cell plates were washed twice with cold 1 x PBS, harvested in cold 1 x PBS, and centrifuged at 14,000 g for 10 minutes at 4°C. Cells were lysed in radioimmunoprecipitation assay (RIP A) buffer (Thermo Scientific), sodium orthovandate (NasVCL, Sigma), sodium flouride (NaF, Sigma), P- glycerophosphate (Sigma), and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma). The samples were incubated on ice for 30 minutes, vortexed at 10 minute intervals and centrifuged at 14,000 g for 10 minutes at 4°C. The supernatant was collected, and protein quantified using the BCA Protein Assay kit (23225, Pierce). Samples were stored in -80°C until required for quantitative proteomics analysis.
Sample Preparation for mass spectrometry experiments
Proteins extracted in RIPA buffer were precipitated using cold acetone and the pellet washed 3 times with 70% ethanol. The proteins were dissolved in 7 M urea, 2 M thiourea, 5 mM DTT, 100 mM tris/HCl, pH 7.8. Protein amounts were quantified using the CB-X™ protein assay (G-Biosciences, St Louis, MO) and a 50 pg aliquot of each sample was alkylated with iodoacetamide (final concentration of 20 mM) for 30 minutes at room temperature (RT) in darkness, then quenched with an equimolar amount of DTT. Samples were diluted to 1 M urea and digested with 1 pg of trypsin to give an enzyme: substrate ratio of 1:50 at 24°C overnight. Digests were acidified to 1% TFA, -pH 3.
LC-MS/MS analysis
Each sample was analyzed by LC-MS/MS on an RSLCnano system (ThermoFisher Scientific) coupled to an Orbitrap Eclipse Tribrid mass spectrometer (ThermoFisher Scientific). The samples were first injected onto a trap column (Acclaim PepMap™ 100, 75 pm x 2 cm, ThermoFisher Scientific) and desalted for 3.0 minutes at a flow rate of 5 pL/minute, before switching in line with the main column. Separation was performed on a C18 nano column (Acquity UPLC® M-class, Peptide CSH™ 130A, 1.7 pm 75 pm x 250 mm, Waters Corp) at 300 nL/minute with a linear gradient from 4-22% over 96 minutes. The LC aqueous mobile phase contained 0.1% (v/v) formic acid in water, and the organic mobile phase contained 0.1% (v/v) formic acid in 100% (v/v) acetonitrile. Mass spectra for the eluted peptides were acquired in the Orbitrap using the data-dependent mode with a mass range of m/z 250-1500, resolution 120,000, AGC target 4 x 106, maximum injection time 50 ms for the MSI peptide measurements. Data-dependent MS2 spectra were acquired by HCD in the ion trap with a normalized collision energy (NCE) set at 30%, AGC target set to 1 x 105, intensity threshold 1 x 105 and a maximum injection time of 35 ms. Dynamic exclusion was set at 45 seconds and the isolation window set to 1.6 m/z.
Proteomics analysis
The identification and quantitation of the proteins was done using Proteome Discoverer (Thermo; version 2.4). All MS/MS samples were searched using Mascot (Matrix Science, London, UK; version 2.6.2). Mascot was set up to search an in-house modified version of the cRAP_20150130.fasta database (124 entries); uniprot-human_20201207 database (75777 entries) assuming the digestion enzyme trypsin and a maximum of 2 missed cleavages. Mascot was searched with a fragment ion mass tolerance of 0.6 Da and a parent ion tolerance of 10.0 ppm. Deamidated of asparagine and glutamine, oxidation of methionine, and protein N-terminal acetylation were specified in Mascot as variable modifications, while carbamidomethyl of cysteine was fixed. Peptides were validated by Percolator with a 0.01 posterior error probability (PEP) threshold. The data were searched using a decoy database to set the false discovery rate to 1% (high confidence). The peptides were quantified using the precursor abundance based on intensity. The peak abundance was normalized using total peptide amount. The peptide group abundances were summed for each sample, and the maximum sum for all files was determined. The normalization factor used was the factor of the sum of the sample and the maximum sum in all files. The protein ratios were calculated using the summed abundance for each replicate separately, and the geometric median of the resulting ratios were used as the protein ratios The significance of differential expression was tested using an ANOVA test, which provided a p-value and an adjusted p- value using the Benjamini-Hochberg method for all the calculated ratios. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031614.
Western blots
The Hs746T cell line were purchased from the American Type Culture Collection (ATCC). Hs746T cells were lysed after the specified treatments in an appropriate volume of NETN lysis buffer (5 M NaCl, 0.5 M EDTA, pH 8.0, 1 M Tris-HCl, pH8.0, NP-40-0.5%) with a protease inhibitor cocktail tablet (complete Mini, EDTA-free, Roche Catalog #11836170001). Protein concentration was measured using Pierce BCA protein assay kit (Thermo Fisher Scientific) and adjusted to 10 to 50 mg per line, using 5X Laemmli Sample Buffer with the addition of 10% 2-mercaptoethanol (Sigma Aldrich). Samples were run on 4 - 20% Mini-Protean TGX pre-cast protein gels and transferred to PVDF membranes using the Trans-Blot Turbo Transfer System (all Bio-Rad). Membranes were blocked for 1 hour at room temperature using 4% milk in TBS-T (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween20), then membranes were incubated in primary antibody at 4°C overnight. After washing three times, membranes were incubated with secondary antibody at room temperature for 1 hour. For imaging, the membranes were incubated in SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo scientific) and developed with HyBlot Cl autoradiography film and X-omat 2000A system (Kodak). The films were scanned by Epson Perfection 2400Photo, and bands were quantified using ImageJ.
Antibodies
The following antibodies were used for the Western blots described above:
1. Anti-MET Cell Signaling Technology (Cat#4060)
2. Anti-Phosphorylation-MET(Y1234/1235): Cell Signaling Technology (Cat#3077S)
3. Anti-Akt: Cell Signaling Technology (Cat#4691P)
4. Anti-Phosphorylation Akt (Ser473) Cell Signaling Technology (Cat#4060)
5. Anti-p44/42 MAPK(Erkl/2): Cell Signaling Technology (Cat#4695) 6. Anti-Phosphorylation p44/42 MAPK(Erkl/2)9T202/Y204): Cell Signaling Technology (Cat#4370S)
7. Anti-Ubiquitin(P37): Cell Signaling Technology (Cat#58395S)
8. Anti-Beta- Actin: Santa Cruz Biotechnology (SC-47778)
9. Anti-GAPDH: Santa Cruz Biotechnology (SC-32233))
10. Anti-GFP Cell Signaling Technology (Cat#2555S)
Xenograft
The UW21 xenograft was derived from the brain metastasis of a patient with non- small cell lung cancer and contains a MET exon 14 skipping mutation as characterized elsewhere (Baschnagel et al., Sei. Rep., 11:2520 (2021)). A short-term treatment experiment was performed on the UW21 model. 48-284 was dissolved in solvent (5% DMSO, 5% Solutol, Kollipgor HS15, Sigma- Aldrich, Cat#42966) at 5 mg/mL concentration, and 15 mg/kg body weight was administered twice through mouse tail vein injection at time point 0 hours and 8 hours. Six hours after the second dose, PDX tumor tissues were harvested, one piece of tumor tissue was snap frozen, and one piece of tumor tissue was fixed in 10% formalin.
MET Immunohistochemistry
Following treatment with 48-284, the UW21 xenograft was removed from the flanks of the mice, fixed in formalin, embedded in paraffin, sectioned at 4 microns, and stained with an antibody directed against MET (clone SP44, Ventana, Tucson, AZ, USA).
Tumor DNA isolation and next-generation sequencing
DNA isolated from fresh-frozen macrodissected primary tumor tissue was sequenced using the whole genome Mate-Pair sequencing (MPseq) protocol and analyzed to detect structural variants. DNA was isolated from macrodissected primary tumor tissue using the Qiagen AllPrep DNA/RNA mini kit (Qiagen, #80204) or Qiagen DNeasy Blood and Tissue Kit (Qiagen, #69504) for peripheral blood mononuclear cell (PBMC) extraction following the manufacture’s protocol. The MPseq protocol was utilized to detect structural variants at the base level resolution through specialized larger 2-5kb fragment tiling of the genome. One microgram of DNA was applied to mate-pair library preparation using the Nextera Mate-Pair Kit (Illumina, &FC-132-1001) following the manufacturer’s protocol. Libraries were sequenced on the Illumina HiSeq4000 platform at a depth of four libraries per lane.
The binary indexing mapping algorithm (BIMA) maps reads to the GRCh38 reference genome. Structural variants were detected using SVAtools using a minimum of three supporting reads for junction detection.
Fusion Diagram
The graph of the PTPZR1-MET fusion was made with the Fusion Editor function in ProteinPaint (pecan, stjude.cloud/proteinpaint). The fusion coordinates detected by sequencing were submitted according to their genomic coordinates to create the graph.
3D micro-cancer models
Fresh surgical tissue was mechanically and enzymatically dissociated into a single cell suspension using the Miltenyi Biotec Tumor Dissociation Kit (Catalog #130-095-929) and the GentleMACSTM Dissociator (Catalog #130-093-235), according to the manufacturer’s instructions. An equal number of cells (5xlO3) was added to each well of a 96-well InspheroTM hanging drop plate, and cells were incubated at 37°C for 6 days in DME/F-12 media containing 10% heat inactivated horse sera, 5 ng/mL insulin, 5 ng/mL hydrocortisone, 10 ng/mL EGF, and pen/ strep. Spheroid cultures representing the cellular diversity of the original tumor (microcancers) were then transferred to the corresponding well of a Corning® 96-Well Clear Round Bottom Ultra Low Attachment 96-well plate and incubated in the presence of the indicated concentrations of drugs or vehicle control at 37°C. Overall cell viability was measured 6 days later using the Cell Titer Glow Luminescent Cell Viability Assay (#G7571). Results were expressed as nM ATP by comparing to a standard curve, transformed into Log 10, and plotted in GraphPad PRISM using a four-parameter dose- response curve. ICso values were calculated using GraphPad PRISM.
Molecular modeling and docking analysis
Scrbdinger small molecule drug discovery suite 2020-1 was used for molecular modeling. The crystal structure of c-MET kinase (pdb:3zbx) was retrieved from the protein data bank. The protein structure was analyzed using Maestro version 12.3.013 (Scrodinger Inc.) and subjected to docking. The Protein Preparation Wizard was used to add missing hydrogen atoms and side chains and minimized using OPLS3e force field to optimize hydrogen bonding network and converge the heavy atoms to a rmsd of 0.3 A.
The capmatinib structure was drawn in Maestro and subjected to Lig Prep to generate conformers, possible protonation at pH of 7 ± 2 that serves as an input for docking process. The receptor grid was generated using Receptor Grid Generation tool in Maestro (Scrodinger Inc.). Docking was performed using GLIDE XP with the van der Waals radii of nonpolar atoms for each of the ligands scaled by a factor of 0.8 and partial charge cutoff 0.15. The binding mode of the ligand in the protein was analyzed using Maestro version 12.3.013 (Scrodinger Inc.).
Chemical synthesis and characterization of PROTACs
General
All reagents were purchased from commercial sources and were used without further purification. ’H NMR and 13C NMR spectra were recorded in DMSO-de on a Varian-500, Varian-600 and Bruker 400 (DMSO-de 2.50 ppm for ’H and 39.00 ppm for 13C). Proton and carbon chemical shifts were reported in ppm relative to the signal from residual solvent proton and carbon. The purity of all final compounds was >96% as determined by analytical HPLC on a reverse-phase column (Phenomenex C18, 150 x 4.6 mm, 5 pm) using a Waters 2695 series system with UV detector, l=254nm. The eluents: solvent A (H2O with 0.1% Formic acid) and solvent B (CH3CN with 0.1% Formic acid), gradient: 10 - 100% B over 15 min with flow rate of 1 .0 mL/minute. HRMS data was generated on an Agilent 6230 LC/TOF system with UV detector (254 nm).
Synthesis of 2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzoic acid (Capmatinib) based PROTACs with amine-containing linkers conjugated to Thalidomide or Pomalidomide or Lenalidomide ligand
Figure imgf000048_0001
Synthesis of 2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzoic acid (Capmatinib) based PROTACs with amine-containing linkers conjugated to VHL ligand
Figure imgf000049_0001
Synthesis of 2-fluoro-4-(7-(quinolin-6-ylmethyl)iniidazo[l,2-b][l,2,4]triazin-2-yl)benzoic acid (1)
Figure imgf000049_0002
The 2-fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2- yl)benzamide (Capmatinib) (1.0 g) was taken in a round bottom flask, added 37% HC1 (8 mL) and heated the reaction mixture at 120°C for 24 hours. The solution was cooled down to room temperature and slowly added water. To this solution was added 5M KOH slowly until bright fluorescent precipitate was observed. The resulting solution was stirred at 0°C for 20 minutes and precipitate was filtered and air dried to yield product as fluorescent solid. Yield: 730 mg (75%). ’l l NMR (400 MHz, DMSO-t76) 3 13.54 (s, 1H), 9.24 (s, 1H), 8.95 (dd, J = 4.4, 1.7 Hz, 1H), 8.51 (dd, J= 8.3, 1.6 Hz, 1H), 8.10 - 8.00 (m, 6H), 7.89 (dd, J= 8.6, 2.0 Hz, 1H), 7.64 (dd, J= 8.3, 4.5 Hz, 1H), 4.67 (s, 2H). 13C NMR (100 MHz, DMSO-d6 ) δ 164.97, 164.94, 163.03, 160.47, 149.35, 144.81, 144.16, 141.74, 141.23, 139.01, 138.92, 138.58, 136.94, 135.30, 133.23, 132.56, 128.51, 127.92, 127.65, 126.65, 123.18, 123.15, 122.26, 121.37, 121.26, 115.97, 115.72, 29.16. HRMS (ESI-MS) calculated for C22HI5FN5O2+ m/z (M+H)+ 400.1204, found: 400.1212.
General procedure for the synthesis of Capmatinib based PROTACs
To a solution of 2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2- yl)benzoic acid (1.0 eq) and amine that was attached to E3 -ligase ligand through linker (1.0 eq) in DMF (1 mL) was added DIPEA (4.0 eq) and stirred for 10 minutes followed by addition of HATU (2.0 eq). The reaction mixture was stirred at room temperature for 16 hours. The crude was purified by reverse phase HPLC (RP-HPLC) with gradient of 10-100% CH3CN in H2O and lyophilized to give product as fluorescent solids. Yields: 40-90%.
N-(5-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)pentyl)-2- fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzamide (48-269)
Figure imgf000050_0001
Yield = 73%. 'H NMR (600 MHz, DMSO-t76) 8 11.08 (s, 1H), 9.18 (s, 1H), 8.97 (dd,
.7= 4.7, 1.6 Hz, 1H), 8.62 (dd, .7 = 8.6, 1.6 Hz, 1H), 8.47 (dt, J= 5.9, 3.0 Hz, 1H), 8.11 - 8.03 (m, 2H), 8.02 - 7.90 (m, 4H), 7.76 - 7.69 (m, 2H), 7.56 (dd, J= 8.6, 7.1 Hz, 1H), 7.09 (d, J = 8.6 Hz, 1H), 7.00 (d, J = 7.0 Hz, 1H), 5.00 (dd, J= 12.9, 5.5 Hz, 1H), 4.68 (s, 2H), 3.32 - 3.24 (m, 4H), 2.89 - 2.78 (m, 1H), 2.62 - 2.60 (m, 2iH), 1.99 (dtd, J= 13.0, 5.5, 2.5 Hz, 1H), 1.59 (dp, J= 26.3, 7.2 Hz, 4H), 1.40 (qd, J= 9.7, 9.0, 6.2 Hz, 2H). HRMS (ESI-MS) calculated for C4oH35FN905+ tn/z (M+H)+ 740.2740, found: 740.2742.
(2S,4R)-l-((S)-15-(tert-butyl)-l-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)phenyl)-l , 13-dioxo-5,8, 1 l-trioxa-2, 14-diazahexadecan- 16-oyl)-4- hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (48-270)
Figure imgf000051_0001
Yield = 60%. 'HNMR (600 MHz, DMS0-d6) 8 9.19 (s, 1H), 9.02 (dd, J= 4.8, 1.6 Hz, 1H), 8.92 (s, 1H), 8.74 - 8.69 (m, 1H), 8.59 (t, 6.1 Hz, 1H), 8.44 (qd, J= 7.5, 5.9, 2.3
Hz, 1H), 8.14 (d, J= 1.8 Hz, 1H), 8.08 (d, J= 8.7 Hz, 1H), 8.03 - 7.94 (m, 4H), 7.81 - 7.73 (m, 2H), 7.42 (d, J= 9.6 Hz, 1H), 7.40 - 7.32 (m, 4H), 4.68 (s, 2H), 4.53 (d, J= 9.6 Hz, 1H), 4.41 (t, J= 8.2 Hz, 1H), 4.41 - 4.31 (m, 2H), 4.23 (td, J= 15.8, 15.1, 5.5 Hz, 1H), 3.94 (s, 2H), 3.65-3.62 (m, 9), 3.58 - 3.48 (m, 4H), 3.41 (p, J= 5.9 Hz, 2H), 2.54 (s, 2H), 2.39 (s, 3H), 2.05 (m 1H), 1.89 (m, 8.9, 4.5 Hz, 1H), 0.90 (s, 9H). HRMS (ESI-MS) calculated for C52H58FNIO08S+ m/z (M+H)+ 1001.4138, found: 1001.4138.
(2S,4R)-l-((S)-18-(tert-butyl)-l-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)phenyl)-l , 16-dioxo-5,8, 11 ,14-tetraoxa-2, 17-diazanonadecan-l 9- oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (48-271)
Figure imgf000052_0001
Yield = 40%. 'H NMR (600 MHz, DMSO-4) 8 9.18 (s, 1H), 9.00 (dd, J= 4.8, 1.6
Hz, 1H), 8.93 (s, 1H), 8.69 (dd, J= 8.5, 1.5 Hz, 1H), 8.60 (t, J= 6.1 Hz, 1H), 8.45 (td, J =
5.6, 2.4 Hz, 1H), 8.12 (d, J= 1.8 Hz, 1H), 8.07 (d, J= 8.7 Hz, 1H), 8.02 - 7.96 (m, 4H), 7.79
- 7.74 (m, 2H), 7.40 (dd, J= 8.9, 4.6 Hz, 1H), 7.36 (q, J= 8.3 Hz, 4H), 4.68 (s, 2H), 4.53 (d, J= 9.6 Hz, 1H), 4.45 - 4.32 (m, 3H), 4.23 (td, J= 16.5, 15.8, 5.6 Hz, 1H), 3.93 (s, 2H), 3.57 (dd, J= 6.2, 3.4 Hz, 1H), 3.-68-3.65 (m, 6H), 3.61 - 3.47 (m, 8H), 3.41 (q, J = 5.8 Hz, 2H), 2.54 (s, 2H), 2.40 (s, 3H), 2.10 - 2.01 (m, 1H), 1.88 (ddd, J= 13.1, 8.9, 4.5 Hz, 1H), 0.90 (s, 9H). HRMS (ESI-MS) calculated for C54H62FNIO09S+ m/z (M+H)+ 1045.4400, found:
1045.4402.
N-(l-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,12- trioxa-3-azatetradecan-14-yl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (48-272)
Figure imgf000052_0002
Yield = 74%. ’H NMR (600 MHz, DMSO-d6 ) δ 11.12 (s, 1H), 9.17 (s, 1H), 8.95 (dd,
.7= 4.6, 1.6 Hz, 1H), 8.63 - 8.54 (m, 1H), 8.43 (td, J = 5.7, 2.4 Hz, 1H), 8.07 (d, J= 1.8 Hz, 1H), 8.03 (d, J= 8.7 Hz, 1H), 8.00 (d, J= 6.7 Hz, 2H), 7.99 - 7.95 (m, 2H), 7.92 (dd, J= 8.8, 2.0 Hz, 1H), 7.76 (q, J= 8.1 Hz, 2H), 7.68 (dd, J= 8.4, 4.6 Hz, 1H), 7.44 (d, .7 = 7.3 Hz, 1H),
7.34 (d, J= 8.5 Hz, 1H), 5.07 (dd, .7 = 12.9, 5.5 Hz, 1H), 4.72 (s, 2H), 4.66 (s, 2H), 3.55 -
3.47 (m, 7H), 3.43 (dt, J= 14.0, 5.8 Hz, 4H), 3.29 (q, J= 5.7 Hz, 2H), 2.87 - 2.81 (m, 1H), 2.59 (dd, .7= 17.3, 3.4 Hz, 1H), 2.54 (s, 2H), 2.02 (dtt, J= 13.0, 5.5, 2.3 Hz, 1H). HRMS (ESI-MS) calculated for C45H43FN9OK/ m/z (M+H)+ 888.3111, found: 888.3111.
N-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)oxy)acetamido)butyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo|l,2-b]|l,2,4]triazin-
2-yl)benzamide (48-273)
Figure imgf000053_0001
Yield = 72%. ’H NMR (600 MHz, DMSO-d6 ) δ 11. 11 (s, 1H), 9. 18 (s, 1H), 8.95 (dd, .7= 4.6, 1.6 Hz, 1H), 8.61 - 8.56 (m, 1H), 8.48 (td, J= 5.7, 1.7 Hz, 1H), 8.10 - 7.90 (m, 8H), 7.79 (dd, J= 8.5, 7.3 Hz, 1H), 7.74 (t, J= 7.7 Hz, 1H), 7.69 (dd, J = 8.4, 4.6 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 5.08 (dd, J = 12.9, 5.5 Hz, 1H), 4.76 (s, 2H), 4.67 (s, 2H), 3.26 (q, J= 6.3 Hz, 2H), 3.19 (q, J= 6.1, 5.7 Hz, 2H), 2.89 - 2.79 (m, 1H), 2.54 (s, 2H), 2.09 - 1.98 (m, 1H), 1.51 (dtt, J= 11.5, 8.3, 4.8 Hz, 4H). HRMS (ESI-MS) calculated for CYFFsFNyO?- m/z (M+H)+ 784.2638, found: 7842636.
N-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (48-274)
Figure imgf000053_0002
Yield = 58%. 'HNMR (499 MHz, DMSO-dfc) 5 11.10 (s, 1H), 9.22 (s, 1H), 8.98 (dd,
.7= 4.5, 1.7 Hz, 1H), 8.55 (d, J= 8.5 Hz, 1H), 8.39 (dt, .7= 5.7, 3.8 Hz, 1H), 8.10 - 7.98 (m, 5H), 7.92 (dd, J= 8.7, 2.0 Hz, 1H), 7.79 (t, J= 7.8 Hz, 1H), 7.67 (dd, J= 8.4, 4.5 Hz, 1H), 7.52 (dd, J= 8.5, 7.0 Hz, 1H), 7.10 (d, ./~ 8.6 Hz, 1H), 6.97 (d, J = 7.0 Hz, 1H), 6.59 (s, 1H), 5.05 (dd, J= 12.7, 5.4 Hz, 1H), 4.68 (s, 2H), 3.67 - 3.54 (m, 8H), 3.45 (q, J= 5.8 Hz, 4H), 2.93 - 2.82 (m, 1H), 2.63 - 2.52 (m, 2H), 2.02 (ddd, J = 12.0, 6.5, 4.0 Hz, 1H). HRMS (ESI- MS) calculated for C4iH37FN9O7+ m/z (M+H)+ 786.2794, found: 786.2798.
N-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)ethyl)-2-fluoro-
4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzamide (48-275)
Figure imgf000054_0001
9.25 (s, 1H), 8.97 (dd,
.7 = 4.5, 1.7 Hz, 1H), 8.69 (q, J= 5.0 Hz, 1H), 8.52 (d, ./ - 8.5 Hz, 1H), 8.14 - 7.98 (m, 5H), 7.91 (dd, J= 8.8, 2.0 Hz, 1H), 7.80 (t, J= 7.7 Hz, 1H), 7.71 - 7.56 (m, 2H), 7.27 (d, J= 8.7 Hz, 1H), 7.06 (d, J= 7.0 Hz, 1H), 6.89 - 6.79 (m, 1H), 5.07 (dd, J= 12.7, 5.5 Hz, 1H), 4.69 (s, 2H), 3.59 - 3.53 (m, 2H), 3.50 (q, J= 7.2, 6.2 Hz, 2H), 2.89 (ddd, J= 16.7, 13.4, 5.4 Hz, 1H), 2.66 - 2.56 (m, 2H), 2.03 (dp, J= 11.2, 3.6 Hz, 1H). HRMS (ESI-MS) calculated for C37H29FN9O5+ m/z (M+H)+ 698.2270, found: 698.2266.
N-(6-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)oxy)hexyl)-2-fluoro-4-
(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzamide (48-276)
Figure imgf000054_0002
Yield = 69%. 'HNMR (499 MHz, DMSO-r/r,) 8 11.11 (s, 1H), 9.24 (s, 1H), 8.96 (dd,
J= 4.6, 1.6 Hz, 1H), 8.61 - 8.41 (m, 2H), 8.12 - 7.99 (m, 4H), 7.91 (dd, J= 8.8, 2.0 Hz, 1H), 7.84 - 7.74 (m, 2H), 7.66 (dd, J= 8.3, 4.5 Hz, 1H), 7.53 (d, J= 8.5 Hz, 1H), 7.44 (d, J= 7.3 Hz, 1H), 5.08 (dd, J= 12.8, 5.5 Hz, 1H), 4.68 (s, 2H), 4.23 (t, J= 6.4 Hz, 2H), 3.29 (q, J = 6.6 Hz, 2H), 2.94 - 2.84 (m, 1H), 2.58 (d, J= 30.4 Hz, 2H), 2.03 (dp, J= 11.1, 3.5 Hz, 1H), 1.79 (p, J= 6.7 Hz, 2H), 1.55 (dp, J= 22.7, 7.1 Hz, 4H), 1.43 (q, J= 7.8 Hz, 2H). HRMS (ESI-MS) calculated for C4IH36FNSO6+ m/z (M+H)+ 755.2736, found: 755.2739.
N-(4-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)oxy)butyl)-2-fluoro-4- (7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzamide (48-277)
Figure imgf000055_0001
Yield = 77%. 'HNMR (499 MHz, DMSO-rA) 8 11.10 (s, 1H), 9.24 (s, 1H), 8.96 (dd, .7 = 4.5, 1.6 Hz, 1H), 8.53 (t, ./ ~ 5.6 Hz, 2H), 8.12 - 7.99 (m, 4H), 7.91 (dd, J = 8.7, 2.0 Hz, 1H), 7.86 - 7.71 (m, 2H), 7.65 (dd, J= 8.4, 4.5 Hz, 1H), 7.55 (d, J= 8.5 Hz, 1H), 7.46 (d, J = 7.2 Hz, 1H), 5.08 (dd, J= 12.7, 5.4 Hz, 1H), 4.68 (s, 2H), 4.28 (t, J= 6.3 Hz, 2H), 3.37 (q, J = 6.6 Hz, 2H), 2.92 - 2.83 (m, 1H), 2.60 (d, J= 3.4 Hz, 2H), 2.03 (ddd, J= 11.4, 6.3, 3.8 Hz, 1H), 1.86 (dq, J= 11.7, 6.4 Hz, 2H), 1.75 (q, J= 13 Hz, 2H). HRMS (ESI-MS) calculated for C39H32FN8O6+ m/z (M+H)+ 727.2423, found: 727.2421.
N-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)oxy)acetamido)ethyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin- 2-yl)benzamide (48-278)
Figure imgf000055_0002
Yield = 78%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.12 (s, 1H), 9.24 (s, 1H), 8.94 (dd, .7 = 4.5, 1.7 Hz, 1H), 8.49 (s, 2H), 8.16 (t, J= 5.7 Hz, 1H), 8.10 - 7.94 (m, 5H), 7.89 (dd, J = 8.8, 2.0 Hz, 1H), 7.83 - 7.72 (m, 2H), 7.63 (dd, J= 8.4, 4.4 Hz, 1H), 7.46 (d, J= 7.2 Hz, 1H), 7.40 (d, J= 8.6 Hz, 1H), 5.10 (dd, J= 12.7, 5.4 Hz, 1H), 4.80 (s, 2H), 4.68 (s, 2H), 3.38 (dt, J= 10.6, 5.7 Hz, 3H), 2.94 - 2.82 (m, 2H), 2.31 - 2.58 (m, 2H), 2.01 (ddd, J= 10.9, 6.0, 3.3 Hz, 1H). HRMS (ESI-MS) calculated for C39H3IFN9O7 + m/z (M+H)+ 756.2325, found: 756.2327.
(2S,4R)-l-((S)-2-(5-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b][l,2,4]triazin-2-yl)benzamido)pentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-
(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (48-279)
Figure imgf000056_0001
Yield = 61%. ’H NMR (499 MHz, DMSO-d6 ) δ 9.24 (s, 1H), 8.98 (d, J= 3.5 Hz, 2H), 8.57 (t, J= 6.2 Hz, 2H), 8.46 (t, J= 5.2 Hz, 1H), 8.11 - 8.01 (m, 5H), 7.95 - 7.86 (m, 2H), 7.77 (t, J= 7.7 Hz, 1H), 7.69 (dd, J= 8.4, 4.5 Hz, 1H), 7.46 - 7.34 (m, 4H), 4.68 (s, 2H), 4.56 (d, J= 9.4 Hz, 1H), 4.47 - 4.41 (m, 2H), 4.38 - 4.34 (m, 2H), 4.22 (dd, J= 15.9, 5.4 Hz, 2H), 3.27 (q, J= 6.2 Hz, 2H), 2.40 - 2.26 (m, 2H), 2.17 (dd, J= 14.5, 7.6 Hz, 2H), 2.09 - 1.98 (m, 2H), 1.91 (ddd, J= 14.4, 8.6, 4.6 Hz, 2H), 1.62 - 1.50 (m, 4H), 0.95 (s, 9H) HRMS (ESI-MS) calculated for C49H52FNIO05S+ m/z (M+H)+ 911.3821, found: 911.3816.
(2S,4R)-l-((S)-2-(3-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b][l,2,4]triazin-2-yl)benzamido)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4- (4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxainide (48-280)
Figure imgf000057_0001
Yield = 74%. 'HNMR (499 MHz, DMSO-d6 ) δ 9.24 (s, 1H), 9.02 - 8.95 (m, 2H),
8.58 (t, J= 6.3 Hz, 2H), 8.43 (q, J= 5.3 Hz, 1H), 8.12 - 8.01 (m, 6H), 7.93 (dd, J= 8.7, 2.0
Hz, 1H), 7.81 (t, J= 7.8 Hz, 1H), 7.69 (dd, J= 8.3, 4.5 Hz, 1H), 7.45 - 7.34 (m, 4H), 4.69 (s, 2H), 4.59 (s, 1H), 4.48 - 4.42 (m, 2H), 4.39 - 4.35 (m, 2H), 4.24 - 4.20 (m, 2H), 3.70 - 3.64
(m, 3H), 3.49 (p, J= 6.7 Hz, 3H), 2.59 (dt, J= 15.0, 7.5 Hz, 2H), 2.05 (t, J= 10.5 Hz, 1H), 1.92 (ddd, J = 11.0, 8.5, 4.6 Hz, 1H), 0.95 (s, 9H). HRMS (ESI-MS) calculated for C47H48FNIO05S+ m/z (M+H)+ 883.3508, found: 883.3503. (2S,4R)-l-((S)-2-(7-(2-fhioro-4-(7-(qiiinolin-6-ylmethyl)imidazo[l,2- b][l,2,4]triazin-2-yl)benzamido)heptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4- (4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (48-281)
Figure imgf000057_0002
Yield = 75%. 'HNMR (499 MHz, DMSO-d6 ) δ 9.24 (s, 1H), 8.98 (d, J= 3.6 Hz, 2H), 8.56 (t, J = 6.1 Hz, 2H), 8.48 - 8.40 (m, 1H), 8.09 - 8.01 (m, 5H), 7.92 (dd, J = 8.8, 2.0
Hz, 1H), 7.86 (d, J= 9.4 Hz, 1H), 7.77 (t, J= 7.7 Hz, 1H), 7.68 (dd, J= 8.4, 4.5 Hz, 1H), 7.45 - 7.35 (m, 4H), 4.68 (s, 2H), 4.56 (d, J= 9.4 Hz, 1H), 4.43 (dd, J= 9.5, 6.6 Hz, 2H), 4.37 - 4.33 (m, 2H), 4.24 (d, J= 5.6 Hz, 2H), 3 67 (d, J= 8.4 Hz, 2H), 3.26 (q, J= 6.7 Hz, 2H), 2.28 (dt, J= 14.6, 7.5 Hz, 2H), 2.16 - 2.12 (m, 1H), 2.03 (d, J= 8.6 Hz, 1H), 1.93 - 1.88 (m, 1H), 1.52 (t, J= 7.9 Hz, 4H), 1.30 (d, J= 12.6 Hz, 5H), 0.95 (s, 9H). HRMS (ESI- MS) calculated for CsiHseFNioOsS4 m/z (M+H)+ 939.4134, found: 939.4134.
(2S,4R)-l-((S)-2-(ll-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b]ll,2,4]triazin-2-yl)benzamido)undecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4- (4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (48-282)
Figure imgf000058_0001
Yield = 68%. 'HNMR (499 MHz, DMSO-d6 ) δ 9.24 (s, 1H), 8.98 (d, J= 4.3 Hz, 2H), 8.56 (t, J = 5.9 Hz, 2H), 8.44 (t, J = 5.4 Hz, 1H), 8.11 - 8.00 (m, 5H), 7.92 (dd, J = 8.1, 2.0 Hz, 1H), 7.84 (d, J= 9.4 Hz, 1H), 7.76 (t, J= 7.7 Hz, 1H), 7.68 (dd, J= 8.4, 4.5 Hz, 1H), 7.47 - 7.35 (m, 4H), 4.68 (s, 2H), 4.55 (d, J= 9.4 Hz, 1H), 4.47 - 4.40 (m, 2H), 4.37 - 4.33 (m, 2H), 4.22 (dd, J= 15.9, 5.4 Hz, 2H), 3.71 - 3.64 (m, 5H), 3.26 (q, J= 6.7 Hz, 2H), 2.44 (s, 3H), 2.27 (dt, J= 14.7, 7.6 Hz, 1H), 2.12 (dd, J= 8.0, 6.1 Hz, 1H), 2.03 (t, J= 10.2 Hz, 1H), 1.90 (ddd, J= 12.8, 8.6, 4.6 Hz, 1H), 1.58 - 1.43 (m, 4H), 1.29 (d, J= 24.4 Hz, 8H), 0.93 (s, 9H). HRMS (ESI-MS) calculated for CSSHMFNIOOSS4 m/z (M+H)+ 995.4760, found: 995.4762.
(2S,4R)-l-((S)-12-(tert-butyl)-l-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)phenyl)-l , 10-dioxo-5,8-dioxa-2, 11-diazatridecan- 13-oyl)-4-hydroxy- N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (48-283)
Figure imgf000059_0001
Yield = 56%. ’H NMR (499 MHz, DMSO-d6 ) δ 9.24 (s, 1H), 8.99 - 8.93 (m, 2H), 8.57 (t, J= 6.0 Hz, 1H), 8.53 (d, J= 8.3 Hz, 1H), 8.47 (q, J= 52 Hz, 1H), 8.12 - 7.99 (m, 5H), 7.91 (dd, J= 8.7, 2.0 Hz, 1H), 7.79 (t, J= 7.8 Hz, 1H), 7.66 (dd, J= 8.3, 4.5 Hz, 1H), 7.45 (d, ./= 9.6 Hz, 1H), 7.39 (s, 4H), 4.68 (s, 2H), 4.58 (d, J= 9.6 Hz, 1H), 4.48 - 4.33 (m, 4H), 4.26 (dd, .7 = 15.7, 5.6 Hz, 2H), 4.00 (s, 2H), 3.66 - 3.59 (m, 7H), 3.47 (q, J = 5.9 Hz, 2H), 2.43 (s, 3H), 2.09 - 2.01 (m, 1H), 1.90 (ddd, J= 13.0, 8.5, 4.5 Hz, 1H), 0.94 (s, 9H). HRMS (ESI-MS) calculated for C5OH54FNIO07S+ m/z (M+H)+ 957.3876, found: 957.3875.
(2S,4R)-l-((S)-2-(9-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b][l,2,4]triazin-2-yl)benzamido)nonanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4- (4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (48-284)
Figure imgf000059_0002
Yield = 53%. 'HNMR (600 MHz, DMSO-d6 ) δ 9.26 (s, 1H), 9.06 (dd, J= 4.7, 1.6 Hz, 1H), 8.99 (s, 1H), 8.70 (d, J= 8.3 Hz, 1H), 8.57 (t, J= 6.1 Hz, 1H), 8.44 (td, J= 5.6, 1.6 Hz, 1H), 8.14 (d, J = 1.9 Hz, 1H), 8.10 (d, J= 8.7 Hz, 1H), 8.06 - 8.02 (m, 3H), 7.99 (dd, J = 8.8, 1.9 Hz, 1H), 7.86 (d, J= 9.3 Hz, 1H), 7.77 (dt, J= 9.0, 6.2 Hz, 2H), 7.44 - 7.36 (m, 4H), 4.70 (s,2H), 4.55 (d, J= 9.4 Hz, 1H), 4.46 - 4.40 (m, 2H), 4.35 (tt, J= 4.6, 2.6 Hz, 1H), 4.22 (dd, J= 15.8, 5.5 Hz, 1H), 3.66 (qd, J= 9.8, 9.0, 2.9 Hz, 2H), 3.26 (q, J= 6.6 Hz, 2H), 2.44 (s, 3H), 2.31 - 2.24 (m, 1H), 2.12 (ddd, J = 14.2, 8.2, 6.2 Hz, 1H), 2.06 - 2.01 (m, 1H), 1.91 (ddd, ./- 12.9, 8.6, 4.6 Hz, 1H), 1.56 - 1.43 (m, 4H), 1.37 - 1.21 (m, 9H), 0.94 (s, 9H). 13C NMR (151 MHz, DMSO) 8 172.05, 171.91, 169.67, 162.88, 160.06, 158.56, 158.41, 158.31, 158.07, 157.83, 151.44, 147.62, 144.04, 141.23, 140.98, 139.48, 137.29, 136.13, 134.45, 133.19, 131.14, 130.86, 129.56, 128.59, 127.60, 127.38, 125.93, 122.81, 121.93, 116.49, 114.64, 114.56, 114.47, 68.83, 58.66, 56.32, 56.23, 41.61, 39.90, 39.77, 39.63, 39.49, 39.35, 39.21, 39.07, 37.94, 35.19, 34.84, 28.87, 28.72, 28.64, 28.62, 26.35, 25.42, 15.90. HRMS (ESI-MS) calculated for CssHeoFNioOsS4 m/z (M+H)+ 967.4447, found: 967.4449.
(2S,4R)-l-((S)-2-acetamido-3,3-dimethylbutanoyl)-N-(2-((6-(2-fluoro-4-(7- (quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzamido)hexyl)oxy)-4-(4- methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (48-285)
Figure imgf000060_0001
Yield = 74%. 'H NMR (499 MHz, DMSO-d6 ) δ 9.24 (s, 1H), 8.98 (d, J = 7.3 Hz, 2H), 8.56 (d, J= 8.3 Hz, 1H), 8.46 (t, J= 5.9 Hz, 2H), 8.12 - 7.99 (m, 5H), 7.99 - 7.88 (m, 2H), 7.77 (t, J= 7.9 Hz, 1H), 7.68 (dd, J= 8.4, 4.5 Hz, 1H), 7.47 (d, J= 7.8 Hz, 1H), 7.00 (d,
J= 1.7 Hz, 1H), 6.90 (dd, J= 7.8, 1.6 Hz, 1H), 4.69 (s, 2H), 4.54 (d, J= 9.4 Hz, 1H), 4.47 (t,
7 = 8.0 Hz, 1H), 4.37 - 4.32 (m, 2H), 4.16 (dd, 7 = 16.6, 5.5 Hz, 2H), 4.06 (d, 7 = 6.4 Hz, 2H), 3.67 - 3.63 (m, 3H), 3.29 (q, 7= 6.6 Hz, 2H), 2.46 (s, 3H), 2.06 - 2.02 (m, 1H), 1.89 (s,
3H), 1.78 (q, 7= 6.7, 6.2 Hz, 2H), 1.63 - 1.54 (m , 2H) 1.58 (p, 7= 7.1 Hz, 2H), 1.51 (q, 7 =
7.5, 7.0 Hz, 2H), 1.43 (q, 7= 7.9 Hz, 2H), 0 92 (s, 9H). HRMS (ESI-MS) calculated for C52H58FNIO06S+ m/z (M+H)+ 969.4240, found: 969.4240.
(2S,4R)-l-((S)-2-(2-(2-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b][l,2,4]triazin-2-yl)benzamido)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N- (4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (48-286)
Figure imgf000061_0001
Yield = 63%. 'HNMR (499 MHz, DMSO-d6 ) δ 9.22 (s, 1H), 9.01 - 8.96 (m, 1H),
8.94 (s, 1H), 8.58 (dt, J= 13.5, 7.0 Hz, 3H), 8.10 - 7.99 (m, 5H), 7.92 (dd, J= 8.7, 2.1 Hz, 1H), 7.84 (t, J= 7.8 Hz, 1H), 7.69 (dd, J= 8.3, 4.5 Hz, 1H), 7.49 (d, J= 9.5 Hz, 1H), 7.37 (q,
J= 8.4 Hz, 4H), 4.66 (s, 2H), 4.58 (d, J= 9.5 Hz, 1H), 4.50 - 4.33 (m, 3H), 4.25 (dd, J = 15.8, 5.6 Hz, 3H), 4.03 (d, J= 3.7 Hz, 3H), 3.51 - 3.44 (m, 2H), 2.40 (s, 3H), 2.11 - 2.00 (m, 2H), 1.91 (ddd, J= 13.1, 8.8, 4.6 Hz, 2H), 0.94 (s, 9H). HRMS (ESI-MS) calculated for C48H5OFNIO06S+ m/z (M+H)+ 913.3614, found: 913.3610.
(2S,4S)-l-((S)-2-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-
2-yl)benzamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide (48-287)
Figure imgf000061_0002
Yield = 68%. 'HNMR (499 MHz, DMSO-d6 ) δ 9.25 (s, 1H), 9.05 - 8.91 (m, 2H), 8.69 (t, J = 6.1 Hz, 1H), 8.56 (d, J= 8.5 Hz, 1H), 8.36 (dd, J= 8.6, 3.6 Hz, 1H), 8.16 - 7.97 (m, 5H), 7.92 (dd, J= 8.7, 2.0 Hz, 1H), 7.78 (t, J= 7.8 Hz, 1H), 7.68 (dd, J= 8.3, 4.5 Hz, 1H), 7.51 - 7.32 (m, 4H), 4.69 (d, J= 5.2 Hz, 2H), 4.49 - 4.40 (m, 2H), 4.34 - 4.29 (m, 2H), 4.02 (d, J= 5.7 Hz, 2H), 3.53 (dd, J= 10.1, 5.4 Hz, 2H), 2.45 (s, 3H), 2.39 (dd, J= 6.4, 2.3 Hz, 1H), 1.78 (dt, J= 12.3, 6.1 Hz, 1H), 1.06 (s, 9H). HRMS (ESI-MS) calculated for C44H43FN9O4S+ m/z (M+H)+ 812.3137, found: 812.3141. N-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (48-288)
Figure imgf000062_0001
Yield = 70%. 'HNMR (499 MHz, DMSO-rA) 8 11.11 (s, 1H), 9.24 (s, 1H), 8.99 (dd, J= 4.5, 1.7 Hz, 1H), 8.57 (d, J= 8.3 Hz, 1H), 8.44 (td, J= 5.8, 2.4 Hz, 1H), 8.10 - 8.00 (m, 5H), 7.92 (dd, J= 8.7, 2.0 Hz, 1H), 7.83 - 7.73 (m, 2H), 7.69 (dd, J= 8.3, 4.6 Hz, 1H), 7.50 (d, J= 8.5 Hz, 1H), 7.42 (d, J= 7.2 Hz, 1H), 5.08 (dd, J= 12.8, 5.4 Hz, 1H), 4.68 (s, 2H), 4.32 (dd, J= 5.6, 3.6 Hz, 2H), 3.82 - 3.77 (m, 2H), 3.65 (dd, J= 5.9, 3.7 Hz, 2H), 3.56 (d, J = 6.3 Hz, 8H), 3.44 (q, J= 5.8 Hz, 2H), 2.94 - 2.83 (m, 1H), 2.59 (d, J= 19.7 Hz, 2H), 2.03 (ddd, J= 11.4, 5.9, 3.5 Hz, 1H). HRMS (ESI-MS) calculated for C43H40FN8O9+ m/z (M+H)+ 831.2897, found: 831.2899.
N-(l-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,12,15- tetraoxa-3-azaheptadecan-17-yl)-2-fluoro-4-(7-(qiiinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (48-289)
Figure imgf000062_0002
Yield = 59%. 'HNMR (499 MHz, DMSO-r/r,) 8 11.12 (s, 1H), 9.24 (s, 1H), 8.98 (dd, J= 4.5, 1.7 Hz, 1H), 8.55 (d, J= 8.2 Hz, 1H), 8.45 (dt, J= 5.6, 3.7 Hz, 1H), 8.10 - 7.96 (m, 6H), 7.92 (dd, J= 8.8, 2.0 Hz, 1H), 7.80 (dd, J= 8.4, 7.2 Hz, 2H), 7.68 (dd, J= 8.3, 4.5 Hz, 1H), 7.48 (d, J= 7.2 Hz, 1H), 7.39 (d, J= 8.6 Hz, 1H), 5.11 (dd, 12.8, 5.4 Hz, 1H), 4.78 (s, 2H), 4.68 (s, 2H), 3.59 - 3.49 (m, 14H), 3.49 - 3.40 (m, 4H), 3.31 (q, J= 5.7 Hz, 2H), 2.90 (ddd, ./- 17.4, 13.9, 5.5 Hz, 1H), 2.67 - 2.56 (m, 2H), 2.06 - 2.01 (m, 1H). HRMS (ESI-MS) calculated for C47H47FN90II+ m/z (M+H)+ 932.3374, found: 932.3373.
N-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)oxy)ethyl)-2-fluoro-4- (7-(quinolin-6-ylmethyl)imidazo|l,2-b]|l,2,4]triazin-2-yl)benzamide (48-290)
Figure imgf000063_0001
11.13 (s, 1H), 9.25 (s, 1H), 8.99 (dd, J= 4.6, 1.7 Hz, 1H), 8.65 (td, J= 5.5, 2.4 Hz, 1H), 8.56 (d, J= 8.4 Hz, 1H), 8.11 - 8.01 (m, 5H), 7.93 (dd, J = 8.1, 2.0 Hz, 1H), 7.89 - 7.80 (m, 2H), 7.68 (dd, J= 8.4, 4.5 Hz, 1H), 7.62 (d, J= 8.6 Hz, 1H), 7.50 (d, J= 7.2 Hz, 1H), 5.10 (dd, J= 12.7, 5.4 Hz, 1H), 4.69 (s, 2H), 4.42 (t, J= 6.0 Hz, 2H), 3.71 (q, J= 5.8 Hz, 2H), 2.89 (ddd, J= 17.0, 13.9, 5.4 Hz, 1H), 2.55 (s, 2H), 2.04 (ddq, J= 10.5, 5.5, 3.2, 2.6 Hz, 1H). HRMS (ESI-MS) calculated for C37H2SFNSO6+ m/z (M+H)+ 699.2110, found: 699.2110.
N-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)oxy)acetamido)ethoxy)ethyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (48-292)
Figure imgf000063_0002
Yield = 66%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.12 (s, 1H), 9.21 (s, 1H), 8.97 (dd, J= 4.6, 1.7 Hz, 1H), 8.54 (d, J= 8.4 Hz, 1H), 8.38 (dt, J= 5.4, 3.6 Hz, 1H), 8.09 - 8.02 (m, 2H), 8.05 - 7.96 (m, 4H), 7.91 (dd, J= 8.7, 2.0 Hz, 1H), 7.82 - 7.71 (m, 2H), 7.67 (dd, J = 8.4, 4.5 Hz, 1H), 7.39 (dd, J= 23.2, 7.9 Hz, 2H), 5.12 (dd, J= 12.8, 5.4 Hz, 1H), 4.78 (s, 2H), 4.68 (s, 2H), 3.55 (dt, J= 24.0, 5.8 Hz, 4H), 3.47 (q, 5.8 Hz, 2H), 3.36 (q, J= 5.8
Hz, 2H), 2.90 (ddd, J= 16.7, 13.7, 5.4 Hz, 1H), 2.56 - 2.56 (m, 2H), 2.08 - 2.01 (m, 1H). HRMS (ESLMS) calculated for C41H35FN9OY m/z (M+H)+ 800.2587, found: 800.2587.
N-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)- 1 ,3-dioxoisoindolin-4- yl)oxy)acetamido)ethoxy)ethoxy)ethyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (48-293)
Figure imgf000064_0001
Yield = 90%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.12 (s, 1H), 9.24 (s, 1H), 8.97 (dd, .7= 4.5, 1.7 Hz, 1H), 8.54 (d, J= 8.4 Hz, 1H), 8.45 (dt, J = 1.5, 3.7 Hz, 1H), 8.09 - 7.98 (m, 6H), 7.91 (dd, J = 8.7, 2.0 Hz, 1H), 7.83 - 7.75 (m, 2H), 7.67 (dd, J= 8.4, 4.5 Hz, 1H), 7.47 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 5.11 (dd, J = 12.8, 5.4 Hz, 1H), 4.78 (s, 2H), 4.68 (s, 2H), 3.57 (qd, J = 5.5, 2.5 Hz, 6H), 3.47 (dt, J= 21.8, 5.8 Hz, 4H), 3.33 (q, J = 5.7 Hz, 2H), 2.90 (ddd, J = 17.3, 13.8, 5.4 Hz, 1H), 2.64 - 2.53 (m, 2H), 2.04 (dp, J = 10.6, 3.4 Hz, 1H). HRMS (ESLMS) calculated for C43H39FN9O9+ m/z (M+H)+ 844.2849, found: 844.2847.
N-(6-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)oxy)acetamido)hexyl)-2-fluoro-4-(7-(quiiiolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (48-294)
Figure imgf000064_0002
Yield = 87%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.12 (s, 1H), 9.24 (s, 1H), 8.95 (dd,
J= 4.5, 1.7 Hz, 1H), 8.51 (d, J= 8.4 Hz, 1H), 8.47 - 8.41 (m, 1H), 8.04 (dd, J= 12.3, 3.7 Hz, 5H), 7.96 (t, J= 5.7 Hz, 1H), 7.90 (dd, J = 8.7, 2.0 Hz, 1H), 7.85 - 7.73 (m, 2H), 7.65 (dd, J = 8.3, 4.4 Hz, 1H), 7.49 (d, J= 7.2 Hz, 1H), 7.40 (d, J= 8.5 Hz, 1H), 5.13 (dd, J= 12.8, 5.4 Hz, 1H), 4.78 (s, 2H), 4.68 (s, 2H), 3.26 (q, J= 6.6 Hz, 2H), 3.21 - 3.13 (m, 2H), 2.90 (ddd, J= 16.9, 13.8, 5.4 Hz, 1H), 2.64 - 2.52 (m, 2H), 2.07 - 2.00 (m, 1H), 1.49 (dq, J = 21 A, 6.4, 6.0 Hz, 4H), 1.37 - 1.30 (m, 4H). HRMS (ESI-MS) calculated for C43H39FN9O7+ m/z (M+H)+ 812.2951, found: 812.2953.
N-(3-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)propyl)-2- fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzainide (48-295)
Figure imgf000065_0001
Yield = 65%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.10 (s, 1H), 9.25 (s, 1H), 8.98 (dd, J = 4.6, 1.7 Hz, 1H), 8.61 - 8.53 (m, 2H), 8.10 - 8.01 (m, 5H), 7.92 (dd, J = 8.8, 2.0 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.68 (dd, J= 8.3, 4.5 Hz, 1H), 7.60 (dd, J= 8.6, 7.1 Hz, 1H), 7.14 (d, J= 8.7 Hz, 1H), 7.04 (d, J= 7.0 Hz, 1H), 6.80 - 6.75 (m, 1H), 5.06 (dd, J= 12.7, 5.4 Hz, 1H), 4.69 (s, 2H), 3.39 (dt, J= 19.3, 6.3 Hz, 4H), 2.88 (ddd, J= 16.6, 13.6, 5.3 Hz, 1H), 2.63 - 2.54 (m, 2H), 2.03 (ddt, J= 12.9, 5.5, 3.0 Hz, 1H), 1.83 (p, J= 6.7 Hz, 2H). HRMS (ESI- MS) calculated for C38H3iFN9O5+ m/z (M+H)+ 712.2427, found: 712.2425.
N-(8-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)octyl)-2-fluoro-
4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzamide (48-296)
Figure imgf000065_0002
Yield = 60%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.09 (s, 1H), 9.24 (s, 1H), 8.98 (dd, J= 4.6, 1.6 Hz, 1H), 8.56 (d, J= 7.9 Hz, 1H), 8.47 - 8.40 (m, 1H), 8.05 (dd, J= 16.8, 8.9 Hz, 5H), 7.92 (dd, J= 8.7, 2.0 Hz, 1H), 7.76 (t, J= 7.7 Hz, 1H), 7.68 (dd, J= 8.4, 4.5 Hz, 1H), 7.57 (dd, J= 8.6, 7.0 Hz, 1H), 7.09 (d, J= 8.6 Hz, 1H), 7.01 (d, J= 7.0 Hz, 1H), 6.55 - 6.51 (m, 1H), 5.05 (dd, J= 12.8, 5.5 Hz, 1H), 4.69 (s, 2H), 3.27 (dt, J= 13.1, 7.0 Hz, 4H), 2.94 - 2.83 (m, 1H), 2.67 - 2.50 (m, 2H), 2.03 (tt, J= 7.4, 4.1 Hz, 1H), 1.56 (dt, J= 28.3, 7.2 Hz, 4H), 1.34 (s, 7H). HRMS (ESI-MS) calculated for C43H4IFN9O5 + m/z (M+H)+ 782.3209, found: 782.3211.
N-(14-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)-3,6,9,12- tetraoxatetradecyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2- yl)benzamide (48-297)
Figure imgf000066_0001
Yield = 76%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.10 (s, 1H), 9.24 (s, 1H), 8.99 (dd, .7= 4.5, 1.7 Hz, 1H), 8.57 (d, J= 8.4 Hz, 1H), 8.44 (td, J= 5.6, 2.3 Hz, 1H), 8.10 - 8.00 (m, 5H), 7.92 (dd, J= 8.7, 2.0 Hz, 1H), 7.80 (t, J= 7.9 Hz, 1H), 7.69 (dd, J= 8.3, 4.5 Hz, 1H), 7.55 (dd, J = 8.6, 7.0 Hz, 1H), 7.11 (d, J= 8.6 Hz, 1H), 7.01 (d, J= 1A Hz, 1H), 6.60 - 6.55 (m, 1H), 5.05 (dd, J= 12.7, 5.4 Hz, 1H), 4.68 (s, 2H), 3.60 (t, J= 5.5 Hz, 2H), 3.58 - 3.47 (m, 14H), 3.44 (q, J= 5.8 Hz, 4H), 2.89 (ddd, J= 13.9, 11.7, 7.0 Hz, 1H), 2.63 - 2.52 (m, 2H), 2.07 - 1.98 (m, 1H). HRMS (ESI-MS) calculated for C45H45FN9O/ m/z (M+H)+ 874.3314, found: 874.3319.
N-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (48-298)
Figure imgf000067_0001
Yield = 61%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.10 (s, 1H), 9.24 (s, 1H), 8.99 (dd, J= 4.6, 1.6 Hz, 1H), 8.57 (d, J = 8.3 Hz, 1H), 8.46 - 8.39 (m, 1H), 8.10 - 8.00 (m, 5H), 7.93 (dd, J= 8.8, 2.0 Hz, 1H), 7.80 (t, J= 7.8 Hz, 1H), 7.69 (dd, J= 8.4, 4.6 Hz, 1H), 7.54 (dd, J = 8.6, 7.1 Hz, 1H), 7. 10 (d, ./- 8.6 Hz. 1H), 7.00 (d, 7.0 Hz, 1H), 6.60 - 6.55 (m, 1H),
5.05 (dd, J= n.1, 5.4 Hz, 1H), 4.68 (s, 2H), 3.61 (t, J= 5.5 Hz, 2H), 3.56 (d, J= 5.1 Hz, 10H), 3.44 (q, J= 5.8 Hz, 4H), 2.89 (ddd, J= 14.0, 11.5, 7.1 Hz, 1H), 2.63 - 2.54 (m, 2H), 2.07 - 1.98 (m, 1H). HRMS (ESI-MS) calculated for C43H4IFN9O8+ m/z (M+H)+ 830.3057, found: 830.3056.
N-(7-((2-(2,6-dioxopiperidiii-3-yl)-l,3-dioxoisoindolin-4-yl)ainino)heptyl)-2- fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzamide (48-299)
Figure imgf000067_0002
Yield = 89%. 'HNMR (499 MHz, DMSO-fife) 8 11.09 (s, 1H), 9.24 (s, 1H), 8.96 (dd, J= 4.5, 1.6 Hz, 1H), 8.52 (d, J= 8.4 Hz, 1H), 8.44 (t, J= 5.6 Hz, 1H), 8.08 - 8.00 (m, 5H), 7.90 (dd, J= 8.8, 2.0 Hz, 1H), 7.76 (t, J= 7.8 Hz, 1H), 7.65 (dd, J= 8.4, 4.5 Hz, 1H), 7.58 (dd, J= 8.6, 7.0 Hz, 1H), 7.10 (d, J= 8.6 Hz, 1H), 7.01 (d, J= 7.1 Hz, 1H), 6.60 - 6.54 (m, 1H), 5.05 (dd, J= 12.7, 5.4 Hz, 1H), 4.68 (s, 2H), 3.33 - 3.23 (m, 4H), 2.88 (ddd, J= 16.7, 13.7, 5.4 Hz, 1H), 2.68 - 2.47 (m, 2H), 2.02 (dp, J= 10.9, 3.5 Hz, 1H), 1.60 (t, J= 7.0 Hz, 2H), 1.54 (t, 7= 6.9 Hz, 2H), 1.36 (d, 7= 4.3 Hz, 6H). HRMS (ESI-MS) calculated for C42H39FN9O5+ m/z (M+H)+ 768.3053, found: 768.3055.
(2S,4R)-l-((S)-2-(6-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b]ll,2,4]triazin-2-yl)benzamido)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (50-207)
Figure imgf000068_0001
Yield = 68%. 'HNMR (499 MHz, DMSO-d6 ) δ 9.25 (s, 1H), 9.04 - 8.95 (m, 2H), 8.64 - 8.53 (m, 2H), 8.47 - 8.40 (m, 1H), 8.12 - 8.00 (m, 5H), 7.95 (dd, J = 8.7, 2.0 Hz, 1H), 7.87 (d, J= 9.3 Hz, 1H), 7.77 (t, J= 7.7 Hz, 1H), 7.72 (dd, J= 8.3, 4.6 Hz, 1H), 7.46 - 7.36 (m, 4H), 4.69 (s, 2H), 4.56 (d, J= 9.4 Hz, 1H), 4.48 - 4.40 (m, 2H), 4.38 - 4.32 (m, 1H), 4.22 (dd, J= 15.8, 5.5 Hz, 1H), 3.73 - 3.62 (m, 2H), 3.26 (q, J= 6.8 Hz, 2H), 2.44 (s, 3H), 2.29 (dt, 7 = 14.7, 7.6 Hz, 1H), 2.15 (dq, J = 14.3, 7.5, 6.8 Hz, 1H), 2.08 - 2.00 (m, 1H), 1.91 (ddd, J= 12.8, 8.5, 4.6 Hz, 1H), 1.53 (qt, J= 11.9, 6.8 Hz, 4H), 1.37 - 1.20 (m, 2H), 0.94 (s, 9H). HRMS (ESI-MS) calculated for Cso^FNioOsS4 m/z (M+H)+ 925.3978, found: 925.3977.
N-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-4-yl)amino)ethoxy)ethyl)-2- fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-2-yl)benzamide (50-208)
Figure imgf000068_0002
Yield = 78%. 'HNMR (499 MHz, DMSO-d6 ) δ 10.99 (s, 1H), 9.24 (s, 1H), 8.99 (dd, .7 = 4.6, 1.6 Hz, 1H), 8.58 (d, J= 8.4 Hz, 1H), 8.47 (dt, J= 5.8, 3.7 Hz, 1H), 8.11 - 8.03 (m, 2H), 8.02 (dd, J= 7.8, 2.1 Hz, 3H), 7.94 (dd, J= 8.8, 2.0 Hz, 1H), 7.77 (t, J= 7.7 Hz, 1H), 7.69 (dd, J= 8.4, 4.6 Hz, 1H), 7.28 (t, J= 7.7 Hz, 1H), 6.93 (d, J= 1A Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 5.09 (dd, J= 13.3, 5.1 Hz, 1H), 4.70 (s, 2H), 4.22 (d, J = 17.1 Hz, 1H), 4.12 (d, J= 17.1 Hz, 1H), 3.66 (t, J= 5.8 Hz, 2H), 3.60 (t, J= 5.8 Hz, 2H), 3.47 (q, J= 5.8 Hz, 2H), 3.34 (t, J= 5.8 Hz, 2H), 2.89 (ddd, J= 17.2, 13.6, 5.5 Hz, 1H), 2.55 (s, 1H), 2.24 (qd, J= 13.2, 4.5 Hz, 1H), 2.00 (dtd, J= 11.8, 6.7, 6.0, 3.3 Hz, 1H). HRMS (ESI-MS) calculated for C39H35FN9O5+ m/z (M+H)+ 728.2740, found: 728.2741.
(2S,4R)-l-((S)-2-(8-(2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b][l,2,4]triazin-2-yl)benzamido)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (50-209)
Figure imgf000069_0001
Yield = 71%. ‘HNMR (499 MHz, DMSO-d6 ) δ 9.25 (s, 1H), 9.03 - 8.95 (m, 2H), 8.63 - 8.53 (m, 2H), 8.47 - 8.41 (m, 1H), 8.12 - 8.01 (m, 5H), 7.94 (dd, J = 8.8, 2.0 Hz, 1H), 7.85 (d, J= 9.3 Hz, 1H), 7.76 (t, J= 7.7 Hz, 1H), 7.71 (dd, J= 8.3, 4.6 Hz, 1H), 7.46 - 7.35 (m, 4H), 4.69 (s, 2H), 4.55 (d, J= 9.4 Hz, 1H), 4.48 - 4.39 (m, 2H), 4.35 (s, 1H), 4.22 (dd, J = 15.9, 5.4 Hz, 1H), 3.71 - 3.61 (m, 4H), 3.26 (q, J= 6.7 Hz, 2H), 2.44 (s, 3H), 2.28 (dt, J =
14.8, 7.7 Hz, 1H), 2.13 (dt, J= 14.2, 7.1 Hz, 1H), 2.03 (t, J= 10.7 Hz, 1H), 1.91 (ddd, J =
12.9, 8.6, 4.6 Hz, 1H), 1.50 (d, J= 15.9 Hz, 4H), 1.31 (d, J= 6.9 Hz, 5H), 0.94 (s, 9H). HRMS (ESI-MS) calculated for C52H58FNIO05S+ m/z (M+H)+ 953.4291, found: 953.4293. N-(8-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)oxy)acetamido)octyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin-
2-yl)benzamide (50-210)
Figure imgf000070_0001
Yield = 70%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.12 (s, 1H), 9.24 (s, 1H), 8.98 (dd, J= 4.6, 1.7 Hz, 1H), 8.56 (d, J= 8.5 Hz, 1H), 8.43 (dt, J= 5.7, 3.3 Hz, 1H), 8.08 - 8.01 (m, 5H), 7.97 - 7.89 (m, 2H), 7.84 - 7.73 (m, 2H), 7.68 (dd, J= 8.4, 4.5 Hz, 1H), 7.49 (d, J= 7.3 Hz, 1H), 7.39 (d, J= 8.6 Hz, 1H), 5.12 (dd, J= 12.8, 5.5 Hz, 1H), 4.77 (s, 2H), 4.69 (s, 2H), 3.26 (q, J= 6.7 Hz, 2H), 3.15 (q, J= 6.7 Hz, 2H), 2.90 (ddd, J= 16.0, 13.6, 5.4 Hz, 1H), 2.58 (d, J= 35.4 Hz, 2H), 2.04 (dp, J= 11.6, 4.2, 3.8 Hz, 1H), 1.49 (dt, J= 36.4, 7.0 Hz, 5H), 1.29 (s, 7H). HRMS (ESI-MS) calculated for C45H43FN9O7+ m/z (M+H)+ 840.3264, found:
840.3263.
N-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)ethoxy)ethyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2-b][l,2,4]triazin- 2-yl)benzamide (50-212)
Figure imgf000070_0002
(s, 1H), 9.24 (s, 1H), 8.98 (dd, J= 4.6, 1.7 Hz, 1H), 8.56 (d, J= 8.4 Hz, 1H), 8.43 (td, J= 5.7, 2.3 Hz, 1H), 8.10 - 7.99 (m, 5H), 7.92 (dd, .7 = 8.7, 2.0 Hz, 1H), 7.78 (t, 7.8 Hz, 1H), 7.68 (dd, J= 8.4, 4.6 Hz, 1H),
7.57 (dd, J= 8.5, 7.1 Hz, 1H), 7.17 (d, J= 8.6 Hz, 1H), 7.02 (d, J= 7.0 Hz, 1H), 6.64 (s, 1H), 5.03 (dd, J= 12.8, 5.4 Hz, 1H), 4.69 (s, 2H), 3.64 (dt, J = 30.4, 5.6 Hz, 4H), 3.49 (dq, J = 17.5, 5.6 Hz, 4H), 2.92 - 2.80 (m, 1H), 2.56 - 2.52 (m, 2H), 2.00 (ddd, ./~ 11.0, 6.0, 3.4 Hz, 1H). HRMS (ESI-MS) calculated for C39H33FN9O6+ m/z (M+H)+ 742.2532, found: 742.2532.
N-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (50-213)
Figure imgf000071_0001
Yield = 42%. ‘HNMR (499 MHz, DMSO-d6 ) δ 11.00 (s, 1H), 9.23 (s, 1H), 9.00 (dd, J= 4.7, 1.6 Hz, 1H), 8.59 (d, J= 8.4 Hz, 1H), 8.45 (td, J= 5.6, 2.3 Hz, 1H), 8.13 - 7.99 (m, 6H), 7.94 (dd, .7 = 8.7, 2.0 Hz, 1H), 7.79 (t, 7.9 Hz, 1H), 7.70 (dd, J= 8.4, 4.6 Hz, 1H),
7.24 (t, J= 7.7 Hz, 1H), 6.90 (d, J= 1A Hz, 1H), 6.77 (d, J= 8.1 Hz, 1H), 5.11 (dd, J= 13.3, 5.1 Hz, 1H), 4.69 (s, 2H), 4.21 (d, J= 17.1 Hz, 1H), 4.12 (d, J = 17.1 Hz, 1H), 3.65 - 3.54 (m, 7H), 3.45 (p, J= 6.1, 5.5 Hz, 2H), 3.31 (t, J= 5.9 Hz, 2H), 2.98 - 2.86 (m, 1H), 2.69 - 2.52 (m, 2H), 2.30 (qd, J= 13.2, 4.5 Hz, 1H), 2.03 (dtd, J= 11.4, 6.4, 5.8, 3.1 Hz, 1H). HRMS (ESI-MS) calculated for C41H39FN9O6+ m/z (M+H)+ 772.3002, found: 772.3000.
N-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-2-fluoro-4-(7-(quinolin-6-ylmethyl)imidazo[l,2- b] [ 1 ,2,4] triazin-2-yl)benzamide (50-214)
Figure imgf000071_0002
Yield = 62%. 'HNMR (499 MHz, DMSO-d6 ) δ 11.00 (s, 1H), 9.24 (s, 1H), 8.99 (dd, .7 = 4.6, 1.6 Hz, 1H), 8.57 (d, J = 8.4 Hz, 1H), 8.45 (td, J = 5.6, 2.3 Hz, 1H), 8.10 - 8.00 (m, 6H), 7.93 (dd, J = 8.8, 2.0 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.69 (dd, J = 8.4, 4.6 Hz, 1H), 7.24 (t, J= 7.7 Hz, 1H), 6.91 (d, J= 7.4 Hz, 1H), 6.77 (d, J= 8.1 Hz, 1H), 5.11 (dd, J= 13.3, 5.1 Hz, 1H), 4.68 (s, 2H), 4.21 (d, J = 17.1 Hz, 1H), 4.11 (d, J = 17.1 Hz, 1H), 3.62 - 3.52
(m, 4H), 3.55 (s, 8H), 3.44 (q, J= 5.9 Hz, 2H), 3.30 (t, J = 6.0 Hz, 2H), 2.92 (ddd, J= 17.5, 13.7, 5.5 Hz, 1H), 2.65 - 2.58 (m, 1H), 2.30 (qd, J= 13.2, 4.5 Hz, 1H), 2.03 (dtd, J= 11.4, 6.3, 5.8, 3.1 Hz, 1H). HRMS (ESI-MS) calculated for C43H43FN9O7+ m/z (M+H)+ 816.3264, found: 816.3265. Synthesis of 6-methoxy-4-((2-methyl-4-phenoxyphenyl)amino)quinazolin-7-ol based
PROTACs with amine-containing linkers conjugated to Pomalidomide
Figure imgf000072_0001
Figure imgf000073_0001
2-methyl-l-nitro-4-phenoxybenzene (3) ne (4)
Figure imgf000073_0002
benzyl 6-((4-chloro-6-methoxyquinazolin-7-yl)oxy)hexanoate (5)
Figure imgf000073_0003
To a solution of 4-chloro-6-methoxyquinazolin-7-ol (150 mg, 1.0 eq) and 2 (243 mg, 1.2 eq) in DMF (5 mL) was added NaHCCh (90 mg, 1.5 eq) and refluxed for 24 hours. To this reaction mixture was added water extracted with EtOAC and extracted, washed with brine, dried on MgSCU. The solvent was evaporated, and the crude was purified using silica gel column chromatography with hexane/ethyl acetate to yield off-white solid (0.2 g, 68%).
I I NMR (400 MHz, CDCh) 5 8.91 (s, 1H), 7.45 (s, 1H), 7.41 (s, 1H), 7.38 (d, J= 3.8 Hz, 5H), 5.15 (s, 2H), 4.23 (t, J= 6.6 Hz, 2H), 4.07 (s, 3H), 2.44 (t, J= 7.4 Hz, 2H), 2.03 - 1.93 (m, 2H), 1.79 (p, J= 7.5 Hz, 2H), 1.63 - 1.55 (m, 2H). HRMS (ESI-MS) calculated for C22H24C1N2O4+ m/z (M+H)+ 415.1419, found: 415.1422. benzyl 6-((6-methoxy-4-((2-methyl-4-phenoxyphenyl)amino)quinazolin-7- yl)oxy)hexanoate (6)
Figure imgf000074_0001
The solution of 5 (25 mg, 1.0 eq) and 4 (14 mg, 1.2 eq) in isopropanol (1 mL) heated to 80 °C for 16 hours. The reaction mixture was cooled to room temperature. The formed crystals were fdtered and washed with 2-propanol and hexanes. The compound was air dried to yield light pink color solid (19 mg, 56%). !H NMR (400 MHz, CDCh) 8 11.44 (s, 1H), 8.63 (s, 1H), 8.02 (s, 1H), 7.46 (s, 1H), 7.38 - 7.26 (m, 6H), 7.18 - 7.06 (m, 2H), 7.01 - 6.97 (m, 2H), 6.65 (d, J= 2.7 Hz, 1H), 6.59 (dd, J= 8.6, 2.7 Hz, 1H), 5.12 (s, 2H), 4.14 (s, 3H), 4.09 (t, J= 6.4 Hz, 2H), 2.39 (t, J= 7.5 Hz, 2H), 2.17 (s, 3H), 1.89 (p, J= 6.5 Hz, 2H), 1.72 (q, J= 7.6 Hz, 2H), 1.58 - 1.45 (m, 2H). nC NMR (101 MHz, CDCh) 8 173.24, 159.59, 157.16, 156.49, 156.01, 151.39, 146.74, 137.19, 136.02, 134.91, 129.88, 129.65, 128.70, 128.56, 128.22, 128.20, 123.99, 119.88, 119.55, 115.60, 107.23, 104.84, 99.93, 69.60, 66.18, 58.02, 34.05, 28.37, 25.51, 24.59, 18.64. HRMS (ESI-MS) calculated for CvHvMCK m/z (M+H)+ 578.2649, found: 578.2646.
6-((6-methoxy-4-((2-methyl-4-phenoxyphenyl)amino)quinazolin-7- yl)oxy)hexanoic acid (7)
Figure imgf000074_0002
benzyl 6-((6-methoxy-4-((2-methyl-4-phenoxyphenyl)amino)quinazolin-7- yl)oxy)hexanoate (50 mg, 0.08 mmol) was dissolved in EtOH followed by the addition of Pd/C (10% w/w) under inert conditions. The reaction mixture was stirred under H2 atmosphere for 2 days at room temperature. After completion of reaction, the mixture was filtered through Celite and solvent was evaporated under reduced pressure to obtain green color solid (30 mg, 71%). ’H NMR (400 MHz, DMSO) 8 9.35 (s, 1H), 8.28 (s, 1H), 7.83 (s, 1H), 7.42 (t, J= 7.8 Hz, 2H), 7.30 (d, J= 8.5 Hz, 1H), 7.19 - 7.11 (m, 2H), 7.06 (d, J= 7.9 Hz, 2H), 6.99 (d, J= 2.9 Hz, 1H), 6.89 (dd, J= 8.4, 2.8 Hz, 1H), 4.11 (t, J= 6.6 Hz, 2H), 3.94 (s, 3H), 2.16 (s, 3H), 2.01 (t, J= 7.2 Hz, 2H), 1.82 - 1.76 (m, 2H), 1.53 (q, J= 7.4 Hz, 2H), 1.44 (d, J= 7.9 Hz, 2H). HRMS (ESI-MS) calculated for C28H3oN305+ m/z (M+H)+ 488.2180, found: 488.2184.
General procedure for the synthesis of 6-methoxy-4-((2-methyl-4- phenoxyphenyl)amino)quinazolin-7-ol based PROTACs
To a solution of 6-((6-methoxy-4-((2-methyl-4-phenoxyphenyl)amino)quinazolin-7- yl)oxy) hexanoic acid (1.0 eq) and amine that was attached to pomalidomide through linker (1.0 eq) in DMF (1 mL) was added DIPEA (4.0 eq) and stirred for 10 minutes followed by addition of HATU (2.0 eq). The reaction mixture was stirred at room temperature for 16h. The crude was purified by reverse phase HPLC (RP-HPLC) with gradient of 10-100% CH3CN in H2O and lyophilized to give product as fluorescent solids. Yields: 25-35%.
N-(5-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)pentyl)-6-((6- methoxy-4-((2-methyl-4-phenoxyphenyl)amino)quinazolin-7-yl)oxy)hexanamide (48- 039)
Figure imgf000075_0001
Yield: 35%. ’H NMR (400 MHz, DMSO) 8 11.08 (s, 1H), 10.89 (s, 1H), 8.72 (s, 1H), 8.03 (s, 1H), 7.77 (t, J= 5.7 Hz, 1H), 7.56 (dd, J= 8.6, 7.1 Hz, 1H), 7.49 - 7.41 (m, 2H), 7.35 (d, J= 8.6 Hz, 1H), 7.25 - 7.16 (m, 2H), 7.12 - 7.04 (m, 4H), 7.01 (d, J= 7.0 Hz, 1H), 6.95 (dd, J= 8.6, 2.7 Hz, 1H), 6.50 (d, J= 6.3 Hz, 1H), 5.05 (dd, J = 12.9, 5.4 Hz, 1H), 4.18 (t, J= 6.6 Hz, 2H), 3.98 (s, 3H), 3.27 (q, J= 6.7 Hz, 2H), 3.05 (q, J= 6.5 Hz, 2H), 2.88 (ddd, J= 17.6, 14.0, 5.4 Hz, 1H), 2.63 - 2.52 (m, 1H), 2.18 (s, 3H), 2.10 (t, J= 7.2 Hz, 2H), 2.07 - 1.99 (m, 1H), 1.83 (p, J= 6.7 Hz, 2H), 1.58 (q, J= 7.5 Hz, 4H), 1.45 (s, 4H), 1.45 - 1.35 (m, 1H), 1.34 (t, J = 7.7 Hz, 2H). 13C NMR (151 MHz, DMSO) 5 172.80, 171.76, 170.08, 168.93, 167.26, 156.34, 155.96, 155.60, 150.23, 146.36, 137.16, 136.23, 132.14, 130.17, 129.17, 123.80, 120.25, 118.89, 117.12, 116.36, 110.36, 108.97, 106.54, 103.27, 69.08, 56.53, 48.52, 41.80, 38.22, 35.28, 30.96, 28.86, 28.35, 27.95, 25.09, 24.98, 23.72, 22.13, 17.84. HRMS (ESI-MS) calculated for C46H5oN708 + tn/z (M+H)+ 828.3715, found: 828.3716.
N-(14-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)-3,6,9,12- tetraoxatetradecyl)-6-((6-methoxy-4-((2-methyl-4-phenoxyphenyl)amino)quinazolin-7- yl)oxy)hexanamide (48-041)
Figure imgf000076_0001
Yield: 25%. *H NMR (600 MHz, DMSO) 6 11.08 (s, 1H), 10.93 (s, 1H), 8.72 (s, 1H), 8.03 (s, 1H), 7.84 (t, J = 5.7 Hz, 1H), 7.56 (dd, J= 8.6, 7.1 Hz, 1H), 7.46 - 7.40 (m, 2H), 7.33 (d, J= 8.6 Hz, 1H), 7.23 - 7.10 (m, 3H), 7.09 - 6.98 (m, 5H), 6.94 (dd, J= 8.6, 2.8 Hz, 1H), 6.58 (t, J= 5.9 Hz, 1H), 5.04 (dd, J= 12 9, 5.4 Hz, 1H), 4.17 (t, J= 6.5 Hz, 2H), 3.97 (s, 3H), 3.60 (t, J = 5.4 Hz, 2H), 3.57 - 3.43 (m, 8H), 3.37 (d, J= 11.9 Hz, 1H), 3.17 (q, J = 5.8 Hz, 2H), 2.91 - 2.82 (m, 1H), 2.60 - 2.50 (m, 2H), 2.16 (s, 3H), 2.10 (t, J= 7.3 Hz, 2H), 2.01 (ddd, J= 12.8, 6.6, 4.2 Hz, 1H), 1.81 (p, J= 6.8 Hz, 2H), 1.56 (p, J= 7.5 Hz, 2H), 1.45 - 1.37 (m, 2H). HRMS (ESI-MS) calculated for C5IH6ON70I2 + m/z (M+H)+ 962.4294, found: 962.4297. N-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethyl)-6-((6-methoxy-4-((2-methyl-4- phenoxyphenyl)amino)quinazolin-7-yl)oxy)hexanamide (48-042)
Figure imgf000077_0001
Yield: 31%. 'H NMR (600 MHz, DMSO) 8 11.08 (s, 1H), 10.94 (s, 1H), 8.72 (s, 1H),
8.02 (s, 1H), 7.82 (t, J= 5.7 Hz, 1H), 7.56 (dd, J= 8.6, 7.0 Hz, 1H), 7.46 - 7.40 (m, 2H), 7.33 (d, J= 8.6 Hz, 1H), 7.21 (s, 1H), 7.20 - 7.15 (m, 1H), 7.11 (d, J= 8.6 Hz, 1H), 7.09 - 7.03 (m, 3H), 7.03 - 6.98 (m, 1H), 6.94 (dd, J= 8.6, 2.8 Hz, 1H), 6.58 (t, J= 5.8 Hz, 1H), 5.04 (dd, J= 12.9, 5.4 Hz, 1H), 4.16 (t, J= 6.5 Hz, 2H), 3.96 (s, 2H), 3.60 (t, J= 5.4 Hz,
3H), 3.57 - 3.53 (m, 4H), 3.53 - 3.49 (m, 7H), 3.45 (d, J= 5.7 Hz, 15H), 3.39 (t, J= 5.9 Hz, 7H), 3.18 (q, J= 5.8 Hz, 2H), 2.91 - 2.82 (m, 1H), 2.60 - 2.51 (m, 2H), 2.16 (s, 2H), 2.09 (t, J= 7.3 Hz, 2H), 2.01 (ddt, J= 15.0, 7.8, 3.7 Hz, 1H), 1.81 (p, J= 6.8 Hz, 2H), 1.56 (p, J = 7.4 Hz, 2H), 1.41 (tt, J= 10.0, 6.3 Hz, 2H). HRMS (ESI-MS) calculated for C47H52N70IO+ m/z (M+H)+ 874.3770, found: 874.3770.
General procedure for the synthesis of Nl-(l,8-dimethylimidazo[l,2-a]quinoxalin-4- yl)ethane-l,2-diamine based PROTACs
To a solution of Nl-(l,8-dimethylimidazo[l,2-a]quinoxalin-4-yl)ethane-l,2-diamine hydrogen chloride (1.0 eq) and acid linker conjugated with pomalidomide (1.0 eq) in DMF (1 mL) was added DIPEA (4.0 eq) and stirred for 10 minutes followed by addition of HATU (2.0 eq). The reaction mixture was stirred at room temperature for 16 hours. The crude was purified by reverse phase HPLC (RP-HPLC) with gradient of 10-100% CH3CN in H2O and lyophilized to give product as fluorescent solids. Yields: 30-52%.
Figure imgf000078_0001
N-(2-((l,8-dimethylimidazo[l,2-a]quinoxalin-4-yl)amino)ethyl)-2-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)acetamide (48-069)
Figure imgf000078_0002
Yield: 52%. ’ll NMR. (600 MHz, DMSO-c/6) 5 11.11 (s, 1H), 8.25 (t, J= 5.7 Hz, 1H),
8.00 (s, 1H), 7.61 - 7.57 (m, 1H), 7.45 (s, 1H), 7.31 - 7.25 (m, 2H), 6.96 (t, J= 5.8 Hz, 1H),
6.88 (d, J= 7.1 Hz, 1H), 6.71 (d, J = 8.5 Hz, 1H), 5.06 (dd, J= 12.9, 5.5 Hz, 1H), 3.90 (d, J = 4.9 Hz, 2H), 3.69-3.64 (m, 2H), 3.48 (m, 2H), 2.89 (s, 3H), 2.64 - 2.52 (m, 3H), 2.46 (s, 3H), 2.07 - 2.00 (m, 1H). HRMS (ESI-MS) calculated
Figure imgf000078_0003
569.2255, found: 569.2253.
N1-(2-((l,8-dimethylimidazo[l,2-a]quinoxalin-4-yl)amino)ethyl)-N4-(3-(2-(2-(3-
((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)propoxy)ethoxy)ethoxy)propyl)succinimide (48-074)
Figure imgf000079_0001
Yield: 30%. ’H NMR (400 MHz, DMSO-c/e) 5 11.08 (s, 1H), 8.13 (s, 1H), 8.02 (s, 1H), 7.77 (t, J= 5.6 Hz, 1H), 7.64 (s, 1H), 7.57 (dd, J= 8.5, 7.1 Hz, 1H), 7.47 (s, 1H), 7.32 (d, J= 8.3 Hz, 1H), 7.08 (d, J= 8.6 Hz, 1H), 7.01 (d, J= 7.0 Hz, 1H), 6.65 (t, J= 6.2 Hz, 1H), 5.05 (dd, J= 12.9, 5.4 Hz, 1H), 3.64 (d, J= 6.1 Hz, 2H), 3.57 - 3.42 (m, 10H), 3.46 - 3.34 (m, 5H), 3.34 (s, 1H), 3.04 (q, J= 6.6 Hz, 2H), 2.89 (s, 3H), 2.58 (dd, J= 18.9, 8.1 Hz, 2H), 2.48 (s, 3H), 2.31 (s, 4H), 2.08 - 1.98 (m, 1H), 1.80 (p, J = 6.4 Hz, 2H), 1.58 (p, J= 6.7 Hz, 2H). HRMS (ESI-MS) calculated for C41H52N9O9WZ (M+H)+ 814.3883, found: 814.3883.
N-(2-((l,8-dimethylimidazo[l,2-a]quinoxalin-4-yl)amino)ethyl)-3-(2-(2-(2-((2-
(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethoxy)propenamide (48-084)
Figure imgf000079_0002
Yield: 48%. ’H NMR (600 MHz, DMSO-c/e) 5 11.09 (s, 1H), 8.12 (s, 1H), 8.00 (s, 1H), 7.65 (s, 1H), 7.55 (dd, J= 8.6, 7.1 Hz, 1H), 7.48 (s, 1H), 7.33 (s, 1H), 7.01 (dd, J= 7.8, 2.6 Hz, 2H), 6.45 (t, J~ 5.5 Hz, 1H), 5.04 (dd, J- 12.8, 5.5 Hz, 1H), 3.64 (m, 7H), 3.40 (m, 4H), 3.19 (q, J = 6.5 Hz, 2H), 2.88 (s, 3H), 2.57 (d, J= 34.0 Hz, 2H), 2.45 (s, 3H), 2.12 - 2.06 (m, 2H), 2.02 (did, J= 13.0, 5.4, 2.3 Hz, 1H), 1.50 (dp, J= 10.5, 7.5 Hz, 4H), 1.32 - 1.22 (m, 2H). HRMS (ESI-MS) calculated for C36H43N8O8 +in/z (M+H)+ 715.3198, found: 715.3197 N-(2-((l,8-dimethylimidazo[l,2-a]quinoxalin-4-yl)amino)ethyl)-6-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)hexanamide (48-085)
Figure imgf000080_0001
11.09 (s, 1H), 8.16 (s, 1H), 8.01 (s, 1H), 7.65 (s, 1H), 7.54 (dd, J= 8.5, 7.1 Hz, 1H), 7.48 (s, 1H), 7.33 (s, 1H), 7.09 (d, J= 8.6
Hz, 1H), 7.01 (d, ./= 7,0 Hz, 1H), 6.56 (t, J = 6.0 Hz, 1H), 5.04 (dd, J= 12.9, 5.5 Hz, 1H), 3.61 - 3.54 (m, 6H), 3.52 (dd, J= 5.9, 3.4 Hz, 2H), 3.49 - 3.46 (m, 2H), 3.44 - 3.39 (m, 3H),
2.88 (s, 3H), 2.61 - 2.51 (m, 2H), 2.46 (s, 3H), 2.32 (t, J= 6.4 Hz, 2H), 2.02 (dtd, J= 13.0,
5.4, 2.3 Hz, 1H). HRMS (ESI-MS) calculated for CYH^NsO.E m/z (M+H)+ 625.2881, found: 625.2882.
Synthesis of (S)-l-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)pyrrolidin-l-yl)prop-2-en-l-one (Futibatinib) based PROTACs
To a solution of Nl-(l,8-dimethylimidazo[l,2-a]quinoxalin-4-yl)ethane-l,2-diamine hydrogen chloride (1.0 eq) and acid linker conjugated with pomalidomide (1.0 eq) in DMF (1 mL) was added DIPEA (4.0 eq) and stirred for 10 minutes followed by addition of HATU (2.0 eq). The reaction mixture was stirred at room temperature for 16 hours. The crude was purified by reverse phase HPLC (RP-HPLC) with gradient of 10-100% CH3CN in H2O and lyophilized to give product as fluorescent solids. Yields: 30-52%.
Figure imgf000081_0001
N-(2-((3-((S)-3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)pyrrolidin-l-yl)-3-oxopropyl)thio)ethyl)-2-((2-(2,6-dioxopiperidin-3- yl)-l,3-dioxoisoindolin-4-yl)amino)acetamide (48-134)
Figure imgf000081_0002
Yield: 37%. ’H NMR (499 MHz, DMSO-c/e) 5 11.10 (s, 1H), 8.28 (d, J= 1.8 Hz, 1H), 8.24 (dt, J= 10.8, 5.8 Hz, 1H), 7.62 - 7.55 (m, 1H), 7.07 (dd, J= 7.1, 5.2 Hz, 1H), 7.02 - 6.89 (m, 3H), 6.87 (t, J= 7.9 Hz, 1H), 6.62 (t, J= 2.4 Hz, 1H), 5.56 - 5.38 (m, 2H), 5.07 (dd, J= 12.8, 5.4 Hz, 1H), 4.02 (dd, J= 11.0, 7.0 Hz, 1H), 3.93 (dd, J= 8.1, 4.6 Hz, 2H), 3.84 (dt, J= 12.5, 6.3 Hz, 2H), 3.79 (s, 6H), 3.73 (dd, J= 13.9, 9.7 Hz, 2H), 3.32 - 3.21 (m, 3H), 2.89 (ddd, J= 16.7, 13.5, 5.2 Hz, 2H), 2.72 (dt, J= 13.3, 7.1 Hz, 3H), 2.65 - 2.53 (m, 6H). HRMS (ESI-MS) calculated for C39H4INIO08S+ m/z (M+H)+ 809.2824, found: 809.2824.
N-(2-((3-((S)-3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)pyrrolidin-l-yl)-3-oxopropyl)thio)ethyl)-3-(2-(2-(2-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propenamide (48-138)
Figure imgf000082_0001
Yield: 38%. ’H NMR (499 MHz, DMSO-c/()) 5 11.09 (s, 1H), 8.29 (d, J = 2.5 Hz,
1H), 8.07 - 7.91 (m, 2H), 7.60 - 7.50 (m, 1H), 7.14 (dd, J= 8.6, 3.9 Hz, 1H), 7.04 (dd, J =
7.0, 2.8 Hz, 1H), 6.92 (d, J = 2.3 Hz, 1H), 6.62 (t, J= 2.4 Hz, 2H), 5.47 (dt, J = 37.6, 6.0 Hz, 2H), 5.06 (dd, J= 12.6, 5.4 Hz, 1H), 4.02 (dd, J= 11.0, 7.1 Hz, 1H), 3.84 (td, J= 11.5, 10.1, 5.9 Hz, 1H), 3.79 (s, 6H), 3.66 - 3.49 (m, 19H), 3.32 (q, J= 6.5 Hz, 2H), 3.21 (dq, J= 12.9, 6.4 Hz, 2H), 2.89 (ddd, J= 17.9, 13.5, 5.4 Hz, 2H), 2.79 - 2.66 (m, 2H), 2.58 (dd, J= 19.2, 12.7 Hz, 4H), 2.33 - 2.25 (m, 2H), 2.07 - 1.98 (m, 1H). HRMS (ESI-MS) calculated for C46H55NIOOIIS+ m/z (M+H)+ 955.3767, found: 955.3766.
N-(2-((3-((S)-3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)pyrrolidin-l-yl)-3-oxopropyl)thio)ethyl)-6-((2-(2,6-dioxopiperidin-3- yl)-l,3-dioxoisoindolin-4-yl)amino)hexanamide (48-139)
Figure imgf000082_0002
Yield: 22%. ’H NMR (499 MHz, DMSO-c4,) 5 11.09 (s, 1H), 8.28 (d, J= 2.4 Hz, 1H), 7.92 (dt, J= 11.5, 5.8 Hz, 1H), 7.57 (ddd, J= 8.8, 7.0, 2.1 Hz, 1H), 7.07 (dd, J= 8.6, 4.7 Hz, 1H), 7.01 (dd, J= 6.9, 1.5 Hz, 1H), 6.91 (d, J= 2.4 Hz, 1H), 6.62 (t, J= 2.3 Hz, 1H), 6.51 (m, 2H), 5.54 - 5.38 (m, 2H), 5.05 (dd, J= 12.7, 5.4 Hz, 1H), 3.74 - 3.51 (m, 3H), 3.25 - 3.15 (m, 2H), 4.02 (dd, J= 10.9, 7.1 Hz, 2H), 3.79 (s, 6H), 2.71 (dt, J= 14.0, 7.2 Hz, 3H), 2.66 - 2.54 (m, 4H), 2.15 - 1.96 (m, 5H), 1.53 (dp, J= 21.1, 7.5 Hz, 5H), 1.42 - 1.24 (m, 4H). HRMS (ESI-MS) calculated for C43H49NIO08S+ m/z (M+H)+ 865.3450, found: 865.3451.
Synthesis of Amino pyrazole based PROTACs
The synthesis of amino pyrazole based PROTACs was as described elsewhere (King et al., Bioorg Med Chem Lett., 43: 128061 (2021)).
N-(5-cyclobutyl-lH-pyrazol-3-yl)-2-(4-((16-((2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-4-yl)amino)-2-oxo-7,10,13-trioxa-3-azahexadecyl)oxy)phenyl)acetamide
Figure imgf000083_0001
N-(5-cyclobutyl-lH-pyrazol-3-yl)-2-(4-(2-((8-((2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-4-yl)amino)octyl)amino)-2-oxoethoxy)phenyl)acetamide (43-108)
Figure imgf000083_0002
N-(5-cyclobutyl-lH-pyrazol-3-yl)-2-(4-(2-((4-((2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-4-yl)amino)butyl)amino)-2-oxoethoxy)phenyl)acetamide (43-115)
Figure imgf000083_0003
N-(5-cyclobutyl-lH-pyrazol-3-yl)-2-(4-(2-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)- l,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)amino)-2- oxoethoxy)phenyl)acetamide (43- 116)
Figure imgf000084_0001
4-(4-(2-((5-cyclobutyl-lH-pyrazol-3-yl)amino)-2-oxoethyl)phenoxy)-N-(4-((2-
(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)butyl)butanamide (43-117)
Figure imgf000084_0002
4-(4-(2-((5-cyclobutyl-lH-pyrazol-3-yl)amino)-2-oxoethyl)phenoxy)-N-(2-(2-(2-
((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethyl)butanamide (43-118)
Figure imgf000084_0003
4-(4-(2-((5-cyclobutyl-lH-pyrazol-3-yl)amino)-2-oxoethyl)phenoxy)-N-(3-(2-(2-
(3-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)propoxy)ethoxy)ethoxy)propyl)butanamide (43-119)
Figure imgf000085_0001
N-(5-cyclobutyl-lH-pyrazol-3-yl)-2-(4-((16-((2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-4-yl)amino)-ll-oxo-3,6,9-trioxa-12-azahexadecyl)oxy)phenyl)acetamide
Figure imgf000085_0002
N-(5-cyclobutyl-lH-pyrazol-3-yl)-2-(4-((l-((2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-4-yl)amino)-10-oxo-3,6,12,15,18-pentaoxa-9-azaicosan-20- yl)oxy)phenyl)acetamide (43-127)
Figure imgf000085_0003
N-(5-cyclobutyl-lH-pyrazol-3-yl)-2-(4-((25-((2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-4-yl)amino)-ll-oxo-3,6,9,16,19,22-hexaoxa-12- azapentacosyl)oxy)phenyl)acetamide (43-128)
Figure imgf000085_0004
Synthesis of (2S,4R)-l-((S)-20-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-yl)ainino)pyridin-3-yl)piperazin-l-yl)-2-(tert-butyl)-
4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide (48-124)
Figure imgf000086_0001
tert-butyl 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-l-yl)acetate
Figure imgf000086_0002
(2S,4R)-l-((S)-20-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-yl)ainino)pyridin-3-yl)piperazin-l-yl)-2-(tert-butyl)- 4,19-dioxo-6,9,l 2, 15-tetraoxa-3, 18-diazaicosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide (48-124)
Figure imgf000087_0001
To a solution of tert-butyl 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyL7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-l-yl)acetate (5.5. mg, 0.01 mmol) in DCM: TFA (1 : 1) (2 mL) and stirred for 0.5 hours at room temperature. The solvent was evaporated under reduced pressure to give an acid intermediate. The acid was then dissolved in 1.0 mL ofDMF, followed by addition of (2S,4R)-l-((S)-17-amino-2-(tert- butyl)-4-oxo-6,9, 12, 15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (7.2 mg, 0.01 mmol) and DIPEA (0.007 mL, 0.04 mmol) and stirred for 10 minutes followed by addition of HATU (7.5 mg, 0.02 eq). The reaction mixture was stirred at room temperature for 16 hours. The crude was purified by reverse phase HPLC (RP-HPLC) with gradient of 10-100% CH3CN in H2O and lyophilized to give product as fluorescent solid (4.0 mg, 36%).
Figure imgf000087_0002
(600 MHz, DMSO-de) 8 10.28 (s, 1H), 10.11 (s, 1H), 8.98 (d, 11.3 Hz, 2H), 8.66 (s, 1H), 8.59 (t, J= 6.0 Hz, 1H), 8.11
(d, J= 3.0 Hz, 1H), 7.90 (d, J= 9.0 Hz, 1H), 7.57 (dd, J= 9.1, 3.0 Hz, 1H), 7.45 - 7.39 (m, 4H), 5.83 (p, J= 8.9 Hz, 1H), 4.57 (d, J= 9.6 Hz, 1H), 4.43 (t, J= 8.2 Hz, 1H), 4.42 - 4.33 (m, 2H), 4.31 - 4.22 (m, 1H), 4.01 (s, 2H), 3.97 (s, 2H), 3.82 (m, 4H), 3.67 (dd, J= 10.7, 4.0 Hz, 1H), 3.65 - 3.50 (m, 6H), 3.46 (t, J= 5.5 Hz, 2H), 3.32 (q, J= 5.6 Hz, 2H), 3.15 (s, 2H), 2.54 (s, 1H), 2.62 - 2.59 (m, 4H), 2.40 - 2.37 (m, 4H), 2.44 (s, 3H), 2.42 (s, 3H), 2.32 (s, 3H), 2.25 (s, 2H), 2.28 - 2.20 (m, 1H), 2.06 (dd, J= 12.8, 7.9 Hz, 1H), 1.90 (tq, J= 9.2, 4.9 Hz, 3H), 1.78 (p, J= 9.2 Hz, 2H), 1.63 - 1.55 (m, 2H), 0.95 (s, 9H). HRMS (ESI-MS) calculated for C58H79Ni20iiS+ m/z (M+H)+ 1151.5706, found: 1151.5702. Synthesis of N-(4-(2-(4,7-dichloro-3-hydroxy-2-oxoindolin-3-yl)acetyl)phenyl)-6-(2-((2-
(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)acetamido)-N- methylhexanamide (45-164) N-(4-(2-(4,7-dichloro-3-hydroxy-2-oxoindolin-3-yl)acetyl)phenyl)-6-(2-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)acetamido)-N-methylhexananiide
Figure imgf000088_0001
Synthesis of 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropy rido [2,3- d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin- l-yl)-N-(3-(2-(2-(3-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)propoxy)ethoxy)ethoxy)propyl)acetamide (45-053)
2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin- l-yl)-N-(3-(2-(2-(3-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)propoxy)ethoxy)ethoxy)propyl)acetamide (45-053)
Figure imgf000088_0002
RESULTS
Synthesis and screening of a PROTAC library to identify MET-Exon 14 skipping mutant degraders
To develop MET-targeting PROTACs, the tyrosine kinase inhibitor capmatinib was selected, as it had a lower molecular weight, logP, polar surface area and greater potency than tepotinib. Analysis of the Schrodinger GLIDE docked capmatinib into MET structure (Figure 6A) and the co-crystal structure of a close analog (PDB: 3ZBX) indicated that N- methyl amide was solvent exposed. Hydrolysis of the N-methyl amide in capmatinib yielded the corresponding acid. Since linker length and composition are involved in PROTAC performance, the above acid was conjugated to an array of linkers through amide chemistry. Thalidomide, lenalidomide, and VHL binders were used as E3-targeting ligands to generate a focused set of 37 capmatinib based PROTACs. An additional 23 non-capmatinib based PROTACs with aminopyrazole, palbociclib, staurosporine, futibatinib, BMS345541, YK-4- 279, and APS-2-79 as targeting ligands were added as controls to generate a library of 60 PROTACs (Table 1).
Table 1. PROTAC library with different MET -targeting moiety head groups (HGs) and E3 ligands.
Figure imgf000090_0001
Composition Compound Head Group E3 Ligand Linker Capmatinib
Figure imgf000090_0002
Figure imgf000091_0001
Figure imgf000092_0002
Figure imgf000092_0001
Figure imgf000093_0002
Figure imgf000093_0003
Figure imgf000093_0001
Figure imgf000094_0001
*A direct carboxamide bond between HG1 and E4.
HG 2-7 were used as controls to demonstrate selectivity
Figure imgf000094_0002
To screen the PROTAC library of MET-Exon-14 skipping degradation, cells that express METexl4A-GFP were generated (Figure 1A). The METexl4A-GFP HEK293 cells were used to screen the PROTAC library along with capmatinib as a control in a live cell imaging study. The treated cells were imaged every 2 hours for GFP signal and confluence (phase). METexl4A degraders induced a time-dependent loss of GFP signal with minimal effect on the confluence of the cells. The bar graph represents the effect of the PROTACs on GFP signal normalized to confluence at 8 hours and 24 hours post treatment (Figure 6B). The
23 non-capmatinib based PROTACs did not reduce the GFP signal. Unexpectedly, capmatinib treated cells showed a time-dependent decrease in the GFP signal indicating degradation of METexl4A. This finding suggests that capmatinib could be functioning as a molecular glue. The capmatinib-based PROTACs were binned into 3 groups relative to capmatinib activity (Figure IB). PROTACs with VHL ligand and hydrophobic (all carbon) linkers showed that shorter linkers (3-7) were inactive while the longer linkers (8-11) were active (Figure 1C). The activity progressively improved with increasing linker lengths, with the most potent PROTAC (48-284) having a 9-carbon linker, and then dropped off slightly with a 11 -carbon linker (48-282). Replacing 2 carbon atoms with oxygen atoms in the 8 atom linker resulted in a complete loss of activity (50-209 vs. 48-283). All the PROTACs with VHL ligand and hydrophilic linkers (5-14) were inactive. Although no clear trends were observed among PROTACs with CRBN ligand, the 4 active PROTACs with CRBN ligand had longer hydrophilic linkers (9-14). Consistent with the live cell imaging study, lysates from METexl4A-GFP HEK293 cells treated with capmatinib (1 pM) and 48-284 (1 pM) for
24 hours when probed with anti-GFP antibody showed 48-284 was more potent than capmatinib (Figure 6C).
A classical feature of a PROTAC is the “hook effect” wherein increasing concentrations of the PROTAC leads to the formation of stable binary complexes between the PROTAC and either the E3-ligase or protein of interest resulting reduced degradation of the target protein. A dose-response study (0 - 10 pM) with one of the most potent degraders, 48-284, monitored every 6 hours showed efficient degradation at ~0.9 pM with a drop off in efficiency at both lower and higher concentrations (Figure ID). To assess if the degradation of METexl4A-GFP is induced by 48-284 is mediated by the proteasome, MET-Exon-14 skipping-GFP expressing cells were subjected to 48-284 in the presence and absence of a proteasome inhibitor (MG132). The loss of GFP signal induced by 48-284 was blocked by MG132 demonstrating 48-284 induces proteasomal degradation of MET-Exon-14 skipping- GFP (Figure IE).
To confirm degradation of METexl4A-GFP and proteome wide selectivity of capmatinib and PROTAC 48-284, METexl4A-GFP expressing cells were subjected to 1 pM of either capmatinib or 48-284 for 24 hours. The lysates from these samples were subjected to mass spectrometry-based proteomic analyses (Figures 2A and 2B, and Figure 7). Conjugating the VHL ligand to capmatinib in 48-284 reduced the total number of proteins identified as hits (abundance > 2-fold reduction and p-value < 0.001) from 64 to 45. Under the criteria described above among the 124 kinases quantified, eight kinases were identified as hits in capmatinib treated samples while only 4 kinases (MET, PDPK1, EPHA2 and PIK3R4) were identified as hits in the 48-484 treated samples. Quantification of unique peptides associated with MET and METexl4A-GFP in capmatinib and 48-284 treated samples show that 48-284 more potently degrades both proteins (Figure 7B). Previously, foretinib was used as a MET-recruiting ligand to create a MET PROTAC degrader (SJF- 8240), which reduced the half-life of exon- 14 deleted MET by > 50% upon treatment with SJF-8420 (Burslem et al., Cell Chem. Biol., 25:67-77 e63 (2018); and Bondeson et al., Cell Chem. Biol., 25:78-87 e75 (2018)). In a dose-response head-to-head comparison study, 48- 284 was able to degrade METexl4A-GFP at lower concentrations than SJF-8420 (Figures 2C and 2D). In a live cell imaging study, the rate of METexl4A-GFP degradation in the presence of capmatinib (1 pM) and SJF-8240 (2 pM) was comparable. In the same study, 48- 284 (1 pM) was twice as efficient in degrading METexl4A-GFP (Figure 7C). Together, the synthesis and screening effort led to the identification of a lead METexl4A degrader that was more potent than the reported compounds.
In vitro degradation and diminished downstream activity in native model
To confirm the results obtained with 48-284 in the METexl4A-GFP models, cell lines with native A7ETexl4 A mutations or MET amplification were used. The Hs746T cell line that was derived from a gastric cancer was selected since this model contains MET amplification with aAffiTexl4A mutation. Treatment ofHs746T cells showed a time dependent decrease in MET when treated with 48-284 at 1.0 pM as assessed by western blot (Figure 3 A). There was also a dose-dependent decrease in the levels of MET protein as assessed by western blot, except at levels above 1.0 pM consistent with a hook effect (Figure 3 A) that was also seen in the HEK-293T METexl4A-GFP models (Figure ID) at similar concentrations. The degradation of MET with the foretinib-based PROTAC (SFF-8240) treated Hs746T cells was modest (Figure 3B). The effects of 48-284 and SJF-8240 RAS/AKT and RAS/ERK pathway signaling was assessed. Significant reduction of phosphorylation of AKT and MAPK was observed in 48-284 treated cells when compared to that using SJF8240 (Figures 3A-3B). Together, these studies show that 48-284 is a potent degrader of MET and disrupts downstream signaling in a cell line with a native AffiTexl4A mutation.
In vivo degradation in short perturbation xenograft
To assess the potential in vivo efficacy of 48-284, a short perturbation study was performed. A7A7exl4A-mutant UW21 xenografts were implanted into the flanks of mice and were allowed to grow for 2-weeks. Half of the mice were treated with 48-284 through tail vein injection twice, eight hours apart, and the tumors were removed six hours after the second injection. Compared to the untreated controls (Figure 4A), the treated tumors showed significant reduction in MET expression by immunohistochemistry (Figure 4B). Protein lysates of the treated UW21 xenografts had evidence for higher molecular weight species of MET, which were absent in the untreated controls (Figure 4C), suggesting that treatment with 48-284 resulted in target ubiquitination and degradation.
Activity against zm-fusion spheroid
It was hypothesized that 48-284 may have activity against other MET abnormalities without A£E7exl4A. 48-284 was tested against PT425, a 3D cancer spheroid with an oncogenic PTPRZ1-MET rearrangement, also known as a zm-fusion that occurs in gliomas. PT425 was derived from a resected glioma and sequencing identified variant 2 of the zm fusion where exon 2 of PTPRZ1 joins exon 2 oiMET which is still in the 5’UTR region of MET (Figure 5A-B). This fusion was predicted to result in an in-frame fusion with five novel amino acids between exon 2 of PTPRZ1 and the start of the coding region oiMET (Figure 5C). In zm fusions, the PTPRZ1 promoter can drive transcription of MET, which retains its dimerization and phosphorylation properties (Chen et al., FEBS Lett., 589: 1437-1443 (2015)). Since the ligand for MET, HGF or scatter factor, is often required for MET activation in an autocrine or paracrine manner, 48-284 was tested with and without HGF in the PT425 spheroid. Cytotoxicity with an IC50 of 0.1 pM was observed in the presence of stimulation with HGF and a less dramatic effect in the absence of HGF (Figure 5D).
Together, these results demonstrate that bifunctional molecules including (a) a targeting moiety that binds to a MET polypeptide (e.g., capmatinib), (b) a linker, and (c) an E3 ligase ligand (e.g., thalidomide) can be used to promote ubiquitination of a MET polypeptide (e.g., to promote degradation of a MET polypeptide), and that such bifunctional molecules can be used to treat cancer.
Example 2: Exemplary MET-Targeting Moieties
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Example 4: Exemplary E3 Ligase Ligands
Figure imgf000101_0001
Example 5: Treating Cancer
This Example demonstrates that MET-targeting PROTACs can reduce the size of the cancer (e.g., the number of cancer cells and/or the volume of one or more tumors) present in a mammal.
Mice were implanted with patient-derived xenografts with MET exon 14 skipping mutations in their flanks. UW21 and Rudin439 model were used. Treatments mentioned below were administered every 12 hours.
The body weights of mice that were implanted with the UW21 xenografts were stable over the two- week period that they were treated with control (DMSO and SOLUTOL®), PROTAC 48-284 10 mg/kg, PROTAC 48-284 20 mg/kg, capmatinib 5 mg/kg or capmatinib 10 mg/kg (Figure 10), suggesting that there was no significant impact on the constitution of the mice. After two weeks of treatment with control, PROTAC 48-284 10 mg/kg, PROTAC 48-284 20 mg/kg, capmatinib 5 mg/kg, or capmatinib 10 mg/kg, the UW21 xenografts were removed from the flanks of the mice (Figure 11). The sizes of the UW21 xenografts were measured over the two weeks, suggesting that treatment with PROTAC 48-284 was more effective than treatment with control, but treatment with capmatinib was most effective (Figure 12). A picture of the removed UW21 xenografts treated with control or PROTAC 48- 284 over two weeks is shown in Figure 13. A graph of the tumor sizes over two weeks of the UW21 xenografts treated with control or PROTAC 48-284 is shown in Figure 14.
The body weights of mice that were implanted with the Rudin439 xenografts were stable over the two-week period that they were treated with control, PROTAC 48-284 10 mg/kg, PROTAC 48-284 20 mg/kg, capmatinib 5 mg/kg, or capmatinib 10 mg/kg (Figure 15), suggesting that there was no significant impact on the constitution of the mice. After two weeks of treatment with control, PROTAC 48-284 10 mg/kg, PROTAC 48-284 20 mg/kg, capmatinib 5 mg/kg, or capmatinib 10 mg/kg, the Rudin439 xenografts were removed from the flanks of the mice (Figure 16). The sizes of the Rudin439 xenografts were measured over the two weeks, suggesting that treatment with PROTAC 48-284 was more effective than treatment with control, but treatment with capmatinib was most effective (Figure 17). A picture of the removed Rudin439 xenografts treated with control or PROTAC 48-284 over two weeks is shown (Figure 18). A graph of the tumor sizes over two weeks of the Rudin439 xenografts treated with control or PROTAC 48-284 is shown (Figure 19).
Example 6: Treating Lung Cancer
A human identified as having lung cancer (e.g., a lung cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) is administered a composition including one or more bifunctional molecules each including (a) a MET- targeting moiety, (b) a linker, and (c) an E3 ligase ligand. The bifunctional molecule(s) can recruit an E3 ligase to the MET polypeptide, resulting in ubiquitination of the MET polypeptide (e.g., to promote degradation of the MET polypeptide) to treat the human.
Example 7: Treating Gastric Cancer
A human identified as having gastric cancer (e.g., a gastric cancer including one or more cancer cells having an oncogenic MET polypeptide such as a MET polypeptide lacking at least a portion of the amino acid sequence encoded by exon 14) is administered a composition including one or more bifunctional molecules each including (a) a MET- targeting moiety, (b) a linker, and (c) an E3 ligase ligand. The bifunctional molecule(s) can recruit an E3 ligase to the MET polypeptide, resulting in ubiquitination of the MET polypeptide (e.g., to promote degradation of the MET polypeptide) to treat the human.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A molecule comprising capmatinib covalently attached to an E3 ligase ligand.
2. The molecule of claim 1, wherein said capmatinib is directly covalently attached to said E3 ligase ligand.
3. The molecule of claim 2, wherein said molecule is:
Figure imgf000104_0001
4. The molecule of claim 1, wherein said capmatinib is indirectly covalently attached to said E3 ligase ligand via a linker.
5. The molecule of claim 4, wherein said linker is a hydrocarbon linker.
6. The molecule of claim 5, wherein said hydrocarbon linker comprises from about 3 to about 12 carbon atoms.
7. The molecule of any one of claims 5-6, wherein said hydrocarbon linker does not include more than one oxygen atom.
8. The molecule of claim 4, wherein said linker is selected from the group consisting of:
Figure imgf000104_0002
Figure imgf000105_0001
Figure imgf000106_0001
9. The molecule of claim 5, wherein said linker is:
Figure imgf000106_0002
10. The molecule of any one of claims 1-9, wherein said E3 ligase ligand is selected from the group consisting of thalidomide, lenalidomide, pomalidomide, VHL032, nutlin 3 a, idasanutlin, RG7112, bestatin, MV1, LCL-161, l-[3-(4-bromophenyl)-4,5-dihydro-5-phenyl- 1 h-pyrazol- 1 -yl]-2-chloro-ethanone, and 1 -(3 ,4-dihydro-6-hydroxy- 1 (2h)-quinolinyl)- 1 - propanone.
11. The molecule of any one of claims 1-10, wherein said E3 ligase ligand binds to an E3 ligase polypeptide selected from the group consisting of a Von Hippel-Lindau (VHL) polypeptide, a cereblon (CRBN) polypeptide, a MDM2 polypeptide, an inhibitor of apoptosis (IAP) polypeptide, a DDB1 and CUL4 associated factor 15 (DCAF15) polypeptide, a DDB1 and CUL4 associated factor 16 (DCAF16) polypeptide, a ring finger protein 4 (RNE4) polypeptide, and a ring finger protein 114 (RNF114) polypeptide.
12. The molecule of claim 1, wherein said molecule is selected from the group consisting of:
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
(48-276);
Figure imgf000110_0001
Figure imgf000111_0001
13. The molecule of claim 1, wherein said molecule is:
Figure imgf000112_0001
14. The molecule of any one of claims 1-13, wherein said molecule promotes the degradation of a MET polypeptide within cells that express said MET polypeptide.
15. The molecule of claim 14, wherein said MET polypeptide is a wild-type MET polypeptide.
16. The molecule of claim 14, wherein said MET polypeptide is a mutant MET polypeptide.
17. The molecule of claim 16, wherein said mutant MET polypeptide is selected from the group consisting of a MET polypeptide that lacks the amino acid sequence encoded by at least a portion of exon 14, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a nuclear basket protein (TPR) polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a kinesin family member 5B (KIF5B) polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a protein tyrosine phosphatase receptor type Z1 (PTPRZ1) polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a CAP-Gly domain containing linker protein 2 (CLIP2) polypeptide, and a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a trafficking from ER to golgi regulator (TFG) polypeptide.
18. The molecule of any one of claims 14-17, wherein said MET polypeptide is a human
MET polypeptide.
19. Acomposition comprising the molecule of any one of claims 1-18.
20. A pharmaceutical composition comprising the molecule of any one of claims 1-18 and a pharmaceutically acceptable carrier, excipient, or diluent.
21. A method for treating a mammal having cancer, wherein cancer cells of said cancer express a MET polypeptide, wherein said method comprises administering, to said mammal, the molecule of any one of claims 1-18.
22. The method of claim 21, wherein said mammal is a human.
23. The method of any one of claims 21-22, wherein said MET polypeptide is a wild-type MET polypeptide.
24. The method of any one of claims 21-22, wherein said MET polypeptide is a mutant MET polypeptide.
25. The method of claim 24, wherein said mutant MET polypeptide is selected from the group consisting of a MET polypeptide that lacks the amino acid sequence encoded by at least a portion of exon 14, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TPR polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a KIF5B polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a PTPRZ1 polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a CLIP2 polypeptide, and a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TFG polypeptide.
I l l
26. The method of any one of claims 24-25, wherein said MET polypeptide is an oncogenic MET polypeptide.
27. The method of any one of claims 21-27, wherein said cancer is selected from the group consisting of a lung cancer, a gastric cancer, a papillary renal cell carcinoma, a brain cancer, and a sarcoma.
28. A method for ubiquitination of a MET polypeptide in a mammal, wherein said method comprises administering, to said mammal, the molecule of any one of claims 1-18.
29. The method of claim 28, wherein said mammal is a human.
30. The method of any one of claims 28-29, wherein said MET polypeptide is a wild-type MET polypeptide.
31. The method of any one of claims 28-29, wherein said MET polypeptide is a mutant MET polypeptide.
32. The method of claim 31 , wherein said mutant MET polypeptide is selected from the group consisting of a MET polypeptide that lacks the amino acid sequence encoded by at least a portion of exon 14, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TPR polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a KIF5B polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a PTPRZ1 polypeptide, a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a CLIP2 polypeptide, and a MET fusion polypeptide including a portion of a MET polypeptide and a portion of a TFG polypeptide.
33. The method of any one of claims 31-32, wherein said MET polypeptide is an oncogenic MET polypeptide.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8461330B2 (en) * 2006-11-22 2013-06-11 Incyte Corporation Imidazotriazines and imidazopyrimidines as kinase inhibitors
US8901123B2 (en) * 2008-05-21 2014-12-02 Incyte Corporation Salts of 2-fluoro-N-methyl-4-[7-(quinolin-6-yl-methyl)-imidazo[1,2-B][1,2,4]triazin-2-yl]benzamide and processes related to preparing the same
WO2020038415A1 (en) * 2018-08-22 2020-02-27 Cullgen (Shanghai), Inc. Tropomyosin receptor kinase (trk) degradation compounds and methods of use
US10596178B2 (en) * 2014-07-25 2020-03-24 Novartis Ag Tablet formulation of 2-fluoro-N-methyl-4-[7-(quinolin-6-ylmethyl) imidazo [1,2-B] [1,2,4] triazin-2-yl] benzamide

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8461330B2 (en) * 2006-11-22 2013-06-11 Incyte Corporation Imidazotriazines and imidazopyrimidines as kinase inhibitors
US8901123B2 (en) * 2008-05-21 2014-12-02 Incyte Corporation Salts of 2-fluoro-N-methyl-4-[7-(quinolin-6-yl-methyl)-imidazo[1,2-B][1,2,4]triazin-2-yl]benzamide and processes related to preparing the same
US10596178B2 (en) * 2014-07-25 2020-03-24 Novartis Ag Tablet formulation of 2-fluoro-N-methyl-4-[7-(quinolin-6-ylmethyl) imidazo [1,2-B] [1,2,4] triazin-2-yl] benzamide
WO2020038415A1 (en) * 2018-08-22 2020-02-27 Cullgen (Shanghai), Inc. Tropomyosin receptor kinase (trk) degradation compounds and methods of use

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CARLISLE JENNIFER W., HARVEY R. DONALD: "Tyrosine Kinase Inhibitors, Antibody-Drug Conjugates, and Proteolysis-Targeting Chimeras: The Pharmacology of Cutting-Edge Lung Cancer Therapies", EDUCATIONAL BOOK, AMERICAN SOCIETY OF CLINICAL ONCOLOGY, US, no. 41, 1 June 2021 (2021-06-01), US , pages e286 - e293, XP093125271, ISSN: 1548-8748, DOI: 10.1200/EDBK_320667 *
DUPONT CAMILLE AMANDINE, RIEGEL KRISTINA, POMPAIAH MALVIKA, JUHL HARTMUT, RAJALINGAM KRISHNARAJ: "Druggable genome and precision medicine in cancer: current challenges", THE FEBS JOURNAL, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 288, no. 21, 1 November 2021 (2021-11-01), GB , pages 6142 - 6158, XP093125273, ISSN: 1742-464X, DOI: 10.1111/febs.15788 *
SINGH, S ET AL.: "Pyrrolo[2; 1-f][1,2,4]triazine: a promising fused heterocycle to target kinases in cancer therapy", MEDICINAL CHEMISTRY RESEARCH, vol. 31, no. 1, 2022 - 13 November 2021 (2021-11-13), pages 1 - 25, XP037660890, DOI: 10.1007/s00044-021-02819-1 *
WANG, B ET AL.: "An overview of kinase downregulators and recent advances in discovery approaches", SIGNAL TRANSDUCTION AND TARGETED THERAPY, vol. 6, no. 1, 20 December 2021 (2021-12-20), pages 1 - 19, XP055940125, DOI: 10.1038/s41392-021-00826-7 *

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