WO2024097355A1 - Chimères ciblant la désubiquitinase et procédés associés - Google Patents

Chimères ciblant la désubiquitinase et procédés associés Download PDF

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WO2024097355A1
WO2024097355A1 PCT/US2023/036699 US2023036699W WO2024097355A1 WO 2024097355 A1 WO2024097355 A1 WO 2024097355A1 US 2023036699 W US2023036699 W US 2023036699W WO 2024097355 A1 WO2024097355 A1 WO 2024097355A1
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pharmaceutically acceptable
tautomer
stereoisomer
solvate
acceptable salt
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PCT/US2023/036699
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Nathaniel James HENNING
Daniel K. Nomura
Lydia BOIKE
Daniel Marquess
Paul Keitz
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Vicinitas Therapeutics, Inc.
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Publication of WO2024097355A1 publication Critical patent/WO2024097355A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/12—Mucolytics

Definitions

  • UPB Ubiquitin-Proteasome Pathway
  • Ubiquitin and other ubiquitin-like proteins are covalently attached to specific protein substrates, which depending on the specific modification, either ultimately targets these proteins for degradation by the proteasome or affects protein function in other ways.
  • Ubls may be removed through the action of deubiquitinases (DUBs), which hydrolyze the Ubl from a target protein. Removal of a Ubl from a ubiquitinated target protein can modulate the function of the target protein in a number of ways, including improving stability and preventing proteasomal degradation.
  • DABs deubiquitinases
  • the present disclosure features bifunctional compounds, as well as pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers, or tautomers thereof, that are useful for recruiting one or more deubiquitinases to a target protein for modulation (e.g., stabilization) of the target protein, as well as methods of use thereof.
  • a target protein for modulation e.g., stabilization
  • a hallmark of many diseases entails the active ubiquitination and degradation of certain proteins, including misfolded, mutated, or otherwise unstable proteins. As such, targeted stabilization of these key proteins through deliberate deubiquitination may thwart disease progression and impart a therapeutic benefit in a cell or subject.
  • Described herein are a set of bifunctional compounds comprising both a Target Ligand, capable of binding to a target protein (e.g., CFTR), and a DUB recruiter, capable of binding to a deubiquitinase (e.g., OTUB1).
  • a target protein e.g., CFTR
  • a DUB recruiter capable of binding to a deubiquitinase
  • OTUB1 e.g., OTUB1
  • C1-C6 alkyl or ““C1-6 alkyl” is intended to encompass, C1, C2, C 3 , C 4 , C 5 , C 6 , C 1 -C 6 , C 1 -C 5 , C 1 -C 4 , C 1 -C 3 , C 1 -C 2 , C 2 -C 6 , C 2 -C 5 , C 2 -C 4 , C 2 -C 3 , C 3 -C 6 , C 3 -C 5 , C 3 - C4, C4-C6, C4-C5, and C5-C6 alkyl.
  • the following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present invention.
  • alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 6 carbon atoms (“C1–6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C 1–5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C 1–4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C 1–3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1–2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”).
  • an alkyl group has 2 to 6 carbon atoms (“C2–6 alkyl”).
  • C1–6 alkyl groups include methyl (C1), ethyl (C 2 ), propyl (C 3 ) (e.g., n-propyl, isopropyl), butyl (C 4 ) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C6) (e.g., n-hexyl).
  • Alkylene refers to a divalent radical of an alkyl group, e.g., –CH 2 –, –CH 2 CH 2 –, and –CH2CH2CH2–.
  • Heteroalkyl refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • a heteroalkyl group refers to a saturated group having from 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–10 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–9 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1–8 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1–7 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1–6 alkyl”).
  • a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC1–5 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1or 2 heteroatoms within the parent chain (“heteroC 1–4 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC1–3 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC 1–2 alkyl”).
  • a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC1 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC 2–6 alkyl”). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC1–10 alkyl.
  • the heteroalkyl group is a substituted heteroC 1–10 alkyl.
  • Heteroalkylene refers to a divalent radical of a heteroalkyl group.
  • Alkoxy or “alkoxyl” refers to an -O-alkyl radical.
  • the alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n- pentoxy, n-hexoxy, and 1,2-dimethylbutoxy.
  • alkoxy groups are lower alkoxy, i.e., with between 1 and 6 carbon atoms.
  • alkoxy groups have between 1 and 4 carbon atoms.
  • aryl refers to a stable, aromatic, mono- or bicyclic ring radical having the specified number of ring carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, 1-naphthyl, 2-naphthyl, and the like.
  • aryl ring likewise refers to a stable, aromatic, mono- or bicyclic ring having the specified number of ring carbon atoms.
  • heteroaryl refers to a stable, aromatic, mono- or bicyclic ring radical having the specified number of ring atoms and comprising one or more heteroatoms individually selected from nitrogen, oxygen and sulfur.
  • the heteroaryl radical may be bonded via a carbon atom or heteroatom.
  • heteroaryl groups include, but are not limited to, furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazinyl, pyridazinyl, pyrimidyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, indazolyl, oxadiazolyl, benzothiazolyl, quinoxalinyl, and the like.
  • heteroaryl ring likewise refers to a stable, aromatic, mono- or bicyclic ring having the specified number of ring atoms and comprising one or more heteroatoms individually selected from nitrogen, oxygen and sulfur.
  • cycloalkyl refers to a stable, saturated or unsaturated, non- aromatic, mono- or bicyclic (fused, bridged, or spiro) ring radical having the specified number of ring carbon atoms. Examples of cycloalkyl groups include, but are not limited to, the cycloalkyl groups identified above, cyclobutenyl, cyclopentenyl, cyclohexenyl, and the like.
  • the specified number is C 3 –C 12 carbons.
  • the related term “carbocyclic ring” likewise refers to a stable, saturated or unsaturated, non-aromatic, mono- or bicyclic (fused, bridged, or spiro) ring having the specified number of ring carbon atoms.
  • the cycloalkyl can be substituted or unsubstituted.
  • the cycloalkyl can be substituted with 0-4 occurrences of R a , wherein each R a is independently selected from the group consisting of C 1-6 alkyl, C 1-6 alkoxyl, and halogen.
  • heterocyclyl refers to a stable, saturated or unsaturated, non- aromatic, mono- or bicyclic (fused, bridged, or spiro) ring radical having the specified number of ring atoms and comprising one or more heteroatoms individually selected from nitrogen, oxygen and sulfur.
  • the heterocyclyl radical may be bonded via a carbon atom or heteroatom. In an embodiment, the specified number is C3–C12 carbons.
  • heterocyclyl groups include, but are not limited to, azetidinyl, oxetanyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuryl, tetrahydrothienyl, piperidyl, piperazinyl, tetrahydropyranyl, morpholinyl, perhydroazepinyl, tetrahydropyridinyl, tetrahydroazepinyl, octahydropyrrolopyrrolyl, and the like.
  • heterocyclic ring likewise refers to a stable, saturated or unsaturated, non-aromatic, mono- or bicyclic (fused, bridged, or spiro) ring having the specified number of ring atoms and comprising one or more heteroatoms individually selected from nitrogen, oxygen and sulfur.
  • the heterocyclyl can be substituted or unsubstituted.
  • the heterocyclyl can be substituted with 0-4 occurrences of R a , wherein each R a is independently selected from the group consisting of C 1-6 alkyl, C 1-6 alkoxyl, and halogen.
  • spirocycloalkyl or “spirocyclyl” means carbogenic bicyclic ring systems with both rings connected through a single atom.
  • the rings can be different in size and nature, or identical in size and nature. Examples include spiropentane, spriohexane, spiroheptane, spirooctane, spirononane, or spirodecane.
  • One or both of the rings in a spirocycle can be fused to another ring carbocyclic, heterocyclic, aromatic, or heteroaromatic ring.
  • a (C3– C 12 )spirocycloalkyl is a spirocycle containing between 3 and 12 carbon atoms.
  • spiroheterocycloalkyl or “spiroheterocyclyl” means a spirocycle wherein at least one of the rings is a heterocycle wherein one or more of the carbon atoms can be substituted with a heteroatom (e.g., one or more of the carbon atoms can be substituted with a heteroatom in at least one of the rings).
  • One or both of the rings in a spiroheterocycle can be fused to another ring carbocyclic, heterocyclic, aromatic, or heteroaromatic ring.
  • halo or “halogen” refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).
  • haloalkyl means an alkyl group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trichloromethyl.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • each expression e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
  • Various embodiments of the disclosure are described herein. It will be recognized that features specified in each embodiment may be combined with other specified features, including as indicated in the embodiments below, to provide further embodiments of the present disclosure. It is understood that in the following embodiments, combinations of substituents or variables of the depicted formulae are permissible only if such combinations result in stable compounds. Certain compounds described herein may exist in particular geometric or stereoisomeric forms.
  • structures depicted herein are also meant to include geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the disclosed compounds are within the scope of the disclosure. Unless otherwise stated, all tautomeric forms of the compounds described herein are within the scope of the disclosure. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compositions containing 90% of one enantiomer and 10% of the other enantiomer is said to have an enantiomeric excess of 80%.
  • the compounds or compositions described herein may contain an enantiomeric excess of at least 50%, 75%, 90%, 95%, or 99% of one form of the compound, e.g., the S-enantiomer. In other words such compounds or compositions contain an enantiomeric excess of the S enantiomer over the R enantiomer.
  • a particular enantiomer may, in some embodiments be provided substantially free of the corresponding enantiomer, and may also be referred to as “optically enriched.”
  • “Optically enriched,” as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments, the compound is made up of at least about 90% by weight of a preferred enantiomer. In other embodiments, the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer.
  • Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses.
  • HPLC high pressure liquid chromatography
  • Jacques et al. Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ.
  • any resulting racemates of final products or intermediates can be resolved into the optical antipodes by known methods, e.g., by separation of the diastereomeric salts thereof, obtained with an optically active acid or base, and liberating the optically active acidic or basic compound.
  • a basic moiety may thus be employed to resolve the compounds described herein into their optical antipodes, e.g., by fractional crystallization of a salt formed with an optically active acid, e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric acid, di- O,O′-p-toluoyl tartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid.
  • an optically active acid e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric acid, di- O,O′-p-toluoyl tartaric acid, mandelic acid, malic acid or
  • Racemic products can also be resolved by chiral chromatography, e.g., high pressure liquid chromatography (HPLC) using a chiral adsorbent.
  • HPLC high pressure liquid chromatography
  • Other Definitions The following definitions are more general terms used throughout the present disclosure.
  • the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.
  • the term “about” means within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%.
  • “Acquire” or “acquiring” as used herein refer to obtaining possession of a value, e.g., a numerical value, or image, or a physical entity (e.g., a sample), by “directly acquiring” or “indirectly acquiring” the value or physical entity. “Directly acquiring” means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. “Indirectly acquiring” refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value).
  • Directly acquiring a value or physical entity includes performing a process that includes a physical change in a physical substance or the use of a machine or device. Examples of directly acquiring a value include obtaining a sample from a human subject. Directly acquiring a value includes performing a process that uses a machine or device, e.g., mass spectrometer to acquire mass spectrometry data.
  • the terms “administer,” “administering,” or “administration,” as used herein refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing an inventive compound, or a pharmaceutical composition thereof. As used herein, the terms “condition,” “disease,” and “disorder” are used interchangeably.
  • the terms “degrades”, “degrading”, or “degradation” refers to the partial or full breakdown of a target protein by the cellular proteasome system to an extent that reduces or eliminates the biological activity (especially aberrant activity) of target protein.
  • the terms “inhibit”, “inhibition”, or “inhibiting” refer to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
  • the term “modulating a target protein” or “modulating target protein activity” means the alteration of at least one feature of a target protein.
  • modulation may comprise one or more of: (i) modulating the folding of the target protein; (ii) modulating the half-life of the target protein; (iii) modulating trafficking of the target protein to the proteasome; (iv) modulating the level of ubiquitination of the target protein; (v) modulating degradation (e.g., proteasomal degradation) of the target protein; (vi) modulating target protein signaling; (vii) modulating target protein localization; (viii) modulating trafficking of the target protein to the lysosome; (ix) modulating trafficking of the target protein to the ER, Golgi, vesicle, plasma membrane; (x) modulating target protein interactions with another protein (e.g., other proteins in the UPS); (xi) modulating posttranslational modifications of the target protein (e.g., SUMOlyation, phosphorylation, glycosylation).
  • SUMOlyation e.g., SUMOlyation, phosphorylation, glycosy
  • modulating a target protein refers to one or more of: improving the folding of a protein, increasing the half-life of a protein, preventing the trafficking of the target protein to the proteasome, decreasing the level of ubiquitination of the target protein, preventing degradation of the target protein, improving target protein signaling, improving target protein signaling, preventing trafficking of the target protein to the lysosome, and improving target protein interactions with another protein.
  • Modulating a target protein may be achieved by stabilizing the level the target protein in vivo or in vitro.
  • the amount of target protein stabilized can be measured by comparing the amount of target protein remaining after treatment with a bifunctional compound described herein as compared to the initial amount or level of target protein present as measured prior to treatment with a bifunctional compound described herein. In an embodiment, at least about 30% of the target protein is modulated (e.g., stabilized) compared to initial levels. In an embodiment, at least about 40% of the target protein is modulated (e.g., stabilized) compared to initial levels. In an embodiment, at least about 50% of the target protein is modulated (e.g., stabilized) compared to initial levels. In an embodiment, at least about 60% of the target protein is modulated (e.g., stabilized) compared to initial levels.
  • At least about 70% of the target protein is modulated (e.g., stabilized) compared to initial levels. In an embodiment, at least about 80% of the target protein is modulated (e.g., stabilized) compared to initial levels. In an embodiment, at least about 90% of the target protein is modulated (e.g., stabilized) compared to initial levels. In an embodiment, at least about 95% of the target protein is modulated (e.g., stabilized) compared to initial levels. In an embodiment, over 95% of the target protein is modulated (e.g., stabilized) compared to initial levels. In an embodiment, at least about 99% of the target protein is modulated (e.g., stabilized) compared to initial levels.
  • the target protein is modulated (e.g., stabilized) in an amount of from about 30% to about 99% compared to initial levels. In an embodiment, the target protein is modulated (e.g., stabilized) in an amount of from about 40% to about 99% compared to initial levels. In an embodiment, the target protein is modulated (e.g., stabilized) in an amount of from about 50% to about 99% compared to initial levels. In an embodiment, the target protein is modulated (e.g., stabilized) in an amount of from about 60% to about 99% compared to initial levels. In an embodiment, the target protein is modulated (e.g., stabilized) in an amount of from about 70% to about 99% compared to initial levels.
  • the target protein is modulated (e.g., stabilized) in an amount of from about 80% to about 99% compared to initial levels. In an embodiment, the target protein is modulated (e.g., stabilized) in an amount of from about 90% to about 99% compared to initial levels. In an embodiment, the target protein is modulated (e.g., stabilized) in an amount of from about 95% to about 99% compared to initial levels. In an embodiment, the target protein is modulated (e.g., stabilized) in an amount of from about 90% to about 95% compared to initial levels.
  • the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprised therein.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • the term “selectivity for the target protein” means, for example, a bifunctional compound described herein binds to the target protein in preference to, or to a greater extent than, another protein or proteins.
  • the term “subject” refers to an animal. Typically, the animal is a mammal. A subject also refers to, for example, primates (e.g., humans, male or female), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like. In an embodiment, the subject is a primate. In a preferred embodiment, the subject is a human.
  • the term “a therapeutically effective amount” of a compound described herein refers to an amount of the compound described herein that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc.
  • a therapeutically effective amount refers to the amount of the compound described herein that, when administered to a subject, is effective to (1) at least partially alleviate, prevent and/or ameliorate a condition, or a disorder or a disease (i) mediated by a target protein, (ii) associated with activity of a target protein, or (iii) characterized by activity (normal or abnormal) of a target protein; or (2) reduce or inhibit the activity of a target protein; or (3) reduce or inhibit the expression of a target protein.
  • a therapeutically effective amount refers to the amount of the compound described herein that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least prevent or partially prevent reduction of the level of a target protein; or at least maintain or partially increase the activity of a target protein, for example by removing a Ubl covalent bound to the target protein.
  • the terms “treat”, “treating”, or “treatment” of any disease or disorder refer in an embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • “treat”, “treating”, or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • the term “preventing” refers to a reduction in the frequency of, or delay in the onset of, symptoms of the condition or disease.
  • a subject is “in need of” a treatment if such subject would benefit biologically, medically, or in quality of life from such treatment.
  • Bifunctional Compounds The present disclosure describes bifunctional compounds capable of binding to a target protein and a deubiquitinase, e.g., to affect the deubiquitination of the target protein and prevent its degradation by the proteasome.
  • these bifunctional compounds work to bring a deubiquitinase in proximity with a ubiquitinated target protein, such that the deubiquitinase is capable of removing one or more Ubl proteins from the ubiquitinated target protein to modulate (e.g., stabilize and/or prevent degradation of) the target protein.
  • the bifunctional compounds simultaneously bind to a target protein (e.g., CFTR) and a deubiquitinase (e.g., OTUB1).
  • the bifunctional compounds may bind to their respective ligands in any order.
  • the bifunctional compounds first bind to a target protein (e.g., CFTR), then bind to a deubiquitinase (e.g., OTUB1). In another embodiment, the bifunctional compounds first bind to a deubiquitinase (e.g., OTUB1), then a target protein (e.g., CFTR). In still another embodiment, the bifunctional compounds engage with both a target protein (e.g., CFTR) and a deubiquitinase (e.g., OTUB1) at the same time.
  • a target protein e.g., CFTR
  • a deubiquitinase e.g., OTUB1
  • modulating a target protein comprises one or more of: (i) modulating the folding of the target protein; (ii) modulating the half-life of the target protein; (iii) modulating trafficking of the target protein to the proteasome; (iv) modulating the level of ubiquitination of the target protein; (v) modulating degradation (e.g., proteasomal degradation) of the target protein; (vi) modulating target protein signaling; (vii) modulating target protein localization; (viii) modulating trafficking of the target protein to the lysosome; (ix) modulating trafficking of the target protein to the ER, Golgi, vesicle, plasma membrane; (x) modulating target protein interactions with another protein (e.g., other proteins in the UPS); (xi) modulating posttranslational modifications of the target protein (e.g., SUMOlyation, phosphorylation, glycosylation).
  • SUMOlyation e.g., SUMOlyation, phosphorylation,
  • the modulating comprises (i). In an embodiment, the modulating comprises (ii). In an embodiment, the modulating comprises (i). In an embodiment, the modulating comprises (iii). In an embodiment, the modulating comprises (iv). In an embodiment, the modulating comprises (v). In an embodiment, the modulating comprises (vi). In an embodiment, the modulating comprises (vii). In an embodiment, the modulating comprises (viii). In an embodiment, the modulating comprises (ix). In an embodiment, the modulating comprises (x). In an embodiment, the modulating comprises (xi). In an embodiment, the modulating comprises two of (i)-(xi). In an embodiment, the modulating comprises three of (i)-(xi). In an embodiment, the modulating comprises four of (i)-(xi).
  • the modulating comprises five of (i)-(xi). In an embodiment, the modulating comprises six of (i)-(xi). In an embodiment, the modulating comprises seven of (i)-(xi). In an embodiment, the modulating comprises eight of (i)-(xi). In an embodiment, the modulating comprises nine of (i)-(xi). In an embodiment, the modulating comprises ten of (i)-(xi). In an embodiment, the modulating comprises each of (i)-(xi).
  • the bifunctional compound has the structure of Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein (i) the Target Ligand comprises a moiety capable of binding to a target protein; (ii) L1 comprises a linker; and (iii) the DUB recruiter comprises a moiety capable of binding to a deubiquitinase.
  • the bifunctional compound has the structure of Formula (I-a): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the DUB recruiter comprises a moiety capable of binding to a deubiquitinase.
  • Formula (I-a) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the DUB recruiter comprises a moiety capable
  • the bifunctional compound has the structure of Formula (I-b): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein (i) the Target Ligand comprises a moiety capable of binding to a target protein; (ii) L1 comprises a linker; and (iii) the OTUB1 Recruiter comprises a moiety capable of binding to OTUB1.
  • the bifunctional compound has the structure of Formula (I-c): (I-c), or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to OTUB1.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the Target Ligand within the bifunctional compound is a small molecule moiety capable of binding to a target protein or other protein of interest.
  • the Target Ligand binds to a target protein described herein, e.g., an enzyme, receptor, membrane channel, hormone, transcription factor, tumor suppressor, ion channel, apoptotic factor, oncogenic protein, epigenetic regulator, or fragment thereof.
  • the Target Ligand binds to a kinase.
  • the Target Ligand binds to a membrane channel (e.g., CFTR).
  • the Target Ligand binds to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof.
  • the Target Ligand is a CFTR Ligand and comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof.
  • the CFTR Ligand binds to CFTR comprising a sequence mutation (e.g., a Class I, Class II, Class III, Class IV, Class V, or Class VI mutation).
  • the CFTR Ligand binds to a CFTR comprising a Class I mutation, e.g., a nonsense mutation, splice mutation, or deletion (e.g., G542X, W1282X, R553X).
  • the CFTR Ligand binds to a CFTR comprising a Class II mutation, e.g., a processing mutation (e.g., ⁇ F508, N130K, ⁇ I507).
  • the CFTR Ligand binds to a CFTR comprising a Class III mutation, e.g., a gating mutation (e.g., G551D, S549N).
  • the CFTR Ligand binds to a CFTR comprising a Class IV mutation, e.g., a conduction mutation (e.g., D1152H, R347P, R117H). In some embodiments, the CFTR Ligand binds to a CFTR comprising a Class V mutation, e.g., a splice mutation. In some embodiments, the CFTR Ligand binds to CFTR comprising a sequence mutation selected from the group consisting of G551D, R177H, and A445E. In some embodiments, the CFTR Ligand binds to a CFTR comprising a Class VI mutation.
  • a Class IV mutation e.g., a conduction mutation (e.g., D1152H, R347P, R117H).
  • the CFTR Ligand binds to a CFTR comprising a Class V mutation, e.g., a splice mutation.
  • the CFTR binds to CFTR comprising a ⁇ F508 mutation.
  • the CFTR Ligand is a CFTR potentiator.
  • the CFTR Ligand comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, or icenticaftor, or a derivative thereof.
  • the CFTR Ligand is a compound disclosed in one or more of. US. Patent No.7,999,113; U.S.
  • the CFTR Ligand has the structure of Formula (II-a): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein X and Z are each independently O, S, or C(R 7a )(R 7b ); Y is C(R 7a )(R 7b ) or NR 7c ; R 1 is H or C1–6 alkyl; R 3a , R 3b , R 4a , R 4b are each independently H, C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, halo, cyano, or -OR A ; each R 5 , R 5’ , and R 6 is independently C1–6 alkyl,
  • X is O. In some embodiments, Z is O. In some embodiments, each of X and Z is independently O. In some embodiments, Y is C(R 7a )(R 7b ). In some embodiments, each of R 7a and R 7b is independently halo (e.g., fluoro). In some embodiments, X is O, Z is O, and Y is C(R 7a )(R 7b ). In some embodiments, X is O, Z is O, and Y is CF 2 . In some embodiments, R 3a and R 3b are each independently H. In some embodiments, R 4a and R 4b are each independently H.
  • each of R 3a , R 3b , R 4a , R 4b is independently H.
  • R 5’ is C1–6 alkyl (e.g., methyl).
  • R 1 is H.
  • p is 0.
  • p’ is 1.
  • q is 0.
  • each of p and q is independently 0.
  • p is 0, q is 0, p’ is 1, and R 5’ is C1–6 alkyl.
  • p is 0, q is 0, p’ is 1, and R 5’ is methyl.
  • the CFTR Ligand has the structure of Formula (II-b): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein X and Z are each independently O, S, or C(R 7a )(R 7b ); Y is C(R 7a )(R 7b ) or NR 7c ; R1 is H or C 1–6 alkyl; R2 is H or C 1–6 alkyl; R3a, R3b, R4a, R4b are each independently H, C 1–6 alkyl, C 1–6 haloalkyl, C 1–6 heteroalkyl, halo, cyano, or -OR A ; each R 5 , R 5’ , and R 6 is independently C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, halo, cyano, -OR A , -C(O), X and
  • X is O. In some embodiments, Z is O. In some embodiments, each of X and Z is independently O. In some embodiments, Y is C(R 7a )(R 7b ). In some embodiments, each of R 7a and R 7b is independently halo (e.g., fluoro). In some embodiments, X is O, Z is O, and Y is C(R 7a )(R 7b ). In some embodiments, X is O, Z is O, and Y is CF2. In some embodiments, R 3a and R 3b are each independently H. In some embodiments, R 4a and R 4b are each independently H.
  • each of R 3a , R 3b , R 4a , R 4b is independently H.
  • R 5’ is C 1–6 alkyl (e.g., methyl).
  • R 1 is H.
  • R 2 is H.
  • each of R 1 and R 2 is independently H.
  • p is 0.
  • p’ is 1.
  • q is 0.
  • each of p and q is independently 0.
  • p is 0, q is 0, p’ is 1, and R 5’ is C1–6 alkyl.
  • p is 0, q is 0, p’ is 1, and R 5’ is methyl.
  • the CFTR Ligand has the structure of Formula (II-c): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein X and Z are each independently O, S, or C(R 7a )(R 7b ); Y is C(R 7a )(R 7b ) or NR 7c ; R 1 is H or C1–6 alkyl; R 3a , R 3b , R 4a , R 4b are each independently H, C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, halo, cyano, or -OR A ; each R 5 , R 5’ , and R 6 is independently C1–6 alkyl, C1–6 haloalkyl, C 1–6 heteroalkyl, halo, cyano, -OR A , -C(O)N(R B )(R C
  • the CFTR Ligand is lumacaftor or a derivative thereof.
  • the CFTR Ligand has the structure of Formula (II-d): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein denotes the point of attachment to L1 in Formula (I).
  • the CFTR Ligand is lumacaftor or a derivative thereof.
  • the CFTR Ligand has the structure of Formula (II-e): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein denotes the point of attachment to L1 in Formula (I).
  • the CFTR Ligand is lumacaftor or a derivative thereof.
  • the CFTR Ligand has the structure of Formula (II-f): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein denotes the point of attachment to L1 in Formula (I).
  • the CFTR Ligand is lumacaftor or a derivative thereof.
  • the CFTR Ligand has the structure of Formula (II-g): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein each of independently denotes a point of attachment to L1 in Formula (I).
  • the CFTR Ligand is lumacaftor or a derivative thereof.
  • the CFTR Ligand has the structure of Formula (II-h): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein denotes the point of attachment to L1 in Formula (I).
  • the CFTR Ligand is lumacaftor or a derivative thereof.
  • the CFTR Ligand has the structure of Formula (II-i): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein denotes the point of attachment to L1 in Formula (I).
  • the CFTR Ligand is lumacaftor or a derivative thereof.
  • the CFTR Ligand has the structure of Formula (II-j): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein denotes the point of attachment to L1 in Formula (I).
  • the CFTR Ligand is lumacaftor or a derivative thereof.
  • the CFTR Ligand has the structure of Formula (II-k): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein denotes the point of attachment to L1 in Formula (I).
  • the bifunctional compound of Formula (I) has the structure (II-l): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein X and Z are each independently O, S, or C(R 7a )(R 7b ); Y is C(R 7a )(R 7b ) or NR 7c ; R 1 is H or C1–6 alkyl; R 3a , R 3b , R 4a , R 4b are each independently H, C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, halo, cyano, or -OR A ; each R 5 , R 5’ , and R 6 is independently C 1–6 alkyl, C 1–6 haloalkyl, C1–6 heteroalkyl, halo, cyano, -OR A , -C(O)N(R B )(R
  • X is O. In some embodiments, Z is O. In some embodiments, each of X and Z is independently O. In some embodiments, Y is C(R 7a )(R 7b ). In some embodiments, R 7a and R 7b are each independently halo (e.g., fluoro). In some embodiments, X is O, Z is O, and Y is C(R 7a )(R 7b ). In some embodiments, X is O, Z is O, and Y is CF2. In some embodiments, R 3a and R 3b are each independently H. In some embodiments, R 4a and R 4b are each independently H.
  • each of R 3a , R 3b , R 4a , R 4b is independently H.
  • R 5’ is C1–6 alkyl (e.g., methyl).
  • R 1 is H.
  • p is 0.
  • p’ is 1.
  • q is 0.
  • each of p and q is independently 0.
  • p is 0, q is 0, p’ is 1, and R 5’ is C1–6 alkyl.
  • p is 0, q is 0, p’ is 1, and R 5’ is methyl.
  • the bifunctional compound of Formula (I) has the structure (II-m): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein X and Z are each independently O, S, or C(R 7a )(R 7b ); Y is C(R 7a )(R 7b ) or NR 7c ; R 1 is H or C 1–6 alkyl; R 2 is H or C 1–6 alkyl; R 3a , R 3b , R 4a , R 4b are each independently H, C 1–6 alkyl, C 1–6 haloalkyl, C 1–6 heteroalkyl, halo, cyano, or -OR A ; each R 5 , R 5’ , and R 6 is independently C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, halo, cyano, -OR A ,
  • X is O. In some embodiments, Z is O. In some embodiments, each of X and Z is independently O. In some embodiments, Y is C(R 7a )(R 7b ). In some embodiments, R 7a and R 7b are each independently halo (e.g., fluoro). In some embodiments, X is O, Z is O, and Y is C(R 7a )(R 7b ). In some embodiments, X is O, Z is O, and Y is CF2. In some embodiments, R 3a and R 3b are each independently H. In some embodiments, R 4a and R 4b are each independently H.
  • each of R 3a , R 3b , R 4a , R 4b is independently H.
  • R 5’ is C1–6 alkyl (e.g., methyl).
  • R 1 is H.
  • R 2 is H.
  • each of R 1 and R 2 is independently H.
  • p is 0.
  • p’ is 1.
  • q is 0.
  • each of p and q is independently 0.
  • p is 0, q is 0, p’ is 1, and R 5’ is C1–6 alkyl.
  • p is 0, q is 0, p’ is 1, and R 5’ is methyl.
  • the bifunctional compound of Formula (I) has the structure (II-n): (II-n) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein X and Z are each independently O, S, or C(R 7a )(R 7b ); Y is C(R 7a )(R 7b ) or NR 7c ; R 1 is H or C 1–6 alkyl; R 3a , R 3b , R 4a , R 4b are each independently H, C 1–6 alkyl, C 1–6 haloalkyl, C 1–6 heteroalkyl, halo, cyano, or -OR A ; each R 5 , R 5’ , and R 6 is independently C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, halo, cyano, -OR A , -C(O)N(
  • X is O. In some embodiments, Z is O. In some embodiments, each of X and Z is independently O. In some embodiments, Y is C(R 7a )(R 7b ). In some embodiments, R 7a and R 7b are each independently halo (e.g., fluoro). In some embodiments, X is O, Z is O, and Y is C(R 7a )(R 7b ). In some embodiments, X is O, Z is O, and Y is CF2. In some embodiments, R 3a and R 3b are each independently H. In some embodiments, R 4a and R 4b are each independently H.
  • each of R 3a , R 3b , R 4a , R 4b is independently H.
  • R 5’ is C1–6 alkyl (e.g., methyl).
  • R 1 is H.
  • R 2 is H.
  • each of R 1 and R 2 is independently H.
  • p is 0.
  • p’ is 1.
  • q is 0.
  • each of p and q is independently 0.
  • p is 0, q is 0, p’ is 1, and R 5’ is C1–6 alkyl.
  • p is 0, q is 0, p’ is 1, and R 5’ is methyl.
  • the bifunctional compound of Formula (I) has the structure (II-o): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L1 and OTUB1 recruiter are as defined for Formula (I).
  • the bifunctional compound of Formula (I) has the structure (II-p): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L1 and OTUB1 recruiter are as defined for Formula (I).
  • the bifunctional compound of Formula (I) has the structure (II-q): (II-q) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L1 and OTUB1 recruiter are as defined for Formula (I).
  • the bifunctional compound of Formula (I) has the structure (II-r): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L1 and OTUB1 recruiter are as defined for Formula (I).
  • the bifunctional compound of Formula (I) has the structure (II-s): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L1 and OTUB1 recruiter are as defined for Formula (I).
  • Linkers The present disclosure features bifunctional compounds comprising a Target Ligand (e.g., CFTR Ligand) and a DUB recruiter (e.g., OTUB1 recruiter), separated by a linker (i.e., L1).
  • the linker is covalently bound to the CFTR Ligand.
  • the linker is covalently bound to the OTUB1 recruiter.
  • the linker is covalently bound to both the CFTR Ligand and the OTUB1 recruiter.
  • the linker may be a cleavable linker or a non-cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is not degraded or hydrolyzed at physiological conditions. In some embodiments, the linker comprises a bond that is not cleavable in a cell (e.g, a cell organelle) or the serum, e.g., of a sample or subject.
  • the linker comprises an alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, ether, amine, alkoxy, aryl, heteroaryl, cycloalkyl, or heterocyclyl. In some embodiments, the linker comprises an alkylene or heteroalkylene.
  • the linker (e.g., L1) has the structure of Formula (III-a): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R 12a , R 12b , R 13a , R 13b , R 14a , and R 14b are each independently H, C 1–6 alkyl, C 1–6 haloalkyl, C1–6 heteroalkyl, halo, cyano, or -OR A ; or each of R 12a and R 12b , R 13a and R 13b , and R 14a and R 14b independently may be taken together with the carbon atom to which they are attached to form an oxo group; W is C(R 15a )(R 15b ), O, N(R 16 ), or S; R 15a and R 15b are each independently H, C 1–6 alkyl, C 1–6 haloalkyl, C 1–6 heteroalkyl
  • each of R 12a , R 12b , R 13a , and R 13b is independently H.
  • each of R 14a and R 14b are taken together with the carbon atom to which they are attached form an oxo group.
  • W is N(R 16 ) (e.g., NH).
  • o is selected from 2, 3, 4, 5, and 6.
  • p is selected from 1, 2, and 3.
  • L1 has the structure of Formula (III-b): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R* is H, C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl; o is an integer between 0 and 10; * denotes the point of attachment to the CFTR Ligand in Formula (I); and denotes the point of attachment to the OTUB1 recruiter in Formula (I).
  • L1 has the structure of Formula (III-c): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R” is H or C1–6 alkyl , and o is an integer between 0 and 10; * denotes the point of attachment to the CFTR Ligand in Formula (I); and denotes the point of attachment to the OTUB1 recruiter in Formula (I). In some embodiments, o is 1. In some embodiments, o is 2. In some embodiments, o is 3.
  • the linker (e.g., L1) is selected from the group consisting of: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,
  • the linker (e.g., L1) is a variant of a linker described herein, e.g., wherein the linker (e.g., L1) comprises an additional C1-C6 alkyl or C1-C6 heteroalkyl moiety at a point of attachment, e.g., to the CFTR Ligand or the OTUB1 recruiter.
  • the linker is a cleavable linker, e.g., a linker that is degraded or hydrolyzed at physiological conditions.
  • the linker comprises a bond cleavable in a cell (e.g, a cell organelle) or the serum, e.g., of a sample or subject.
  • the linker may be pH sensitive (e.g., acid labile or base labile) or cleaved through the action of an enzyme.
  • the rate of hydrolysis of the linker is increased by at least 0.5 times (e.g., at least 1, 1.5, 2, 2.5, 3, 4, 5, 7.5, 10, 12.5, 15, 20, 25, 50, 75, 100, 250, 500, 750, 1000 or more) compared with the rate of hydrolysis of the linker in the absence of an enzyme.
  • the enzyme is an esterase.
  • the linker comprises an ester, disulfide, thiol, hydrazone, ether, or amide.
  • the linker e.g., L1 is selected from the group consisting of: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,
  • the linker (e.g., L1) is a variant of a linker described herein, e.g., wherein the linker (e.g., L1) comprises an additional C 1 -C 6 alkyl or C 1 -C 6 heteroalkyl moiety at a point of attachment, e.g., to the CFTR Ligand or the OTUB1 recruiter.
  • the linker (e.g., L1) is selected from the group consisting of: , , , , , , , , or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein “*” denotes the point of attachment to the CFTR Ligand or the OTUB1 recruiter.
  • the linker (e.g., L1) is a variant of a linker described herein, e.g., wherein the linker (e.g., L1) comprises an additional C 1 -C 6 alkyl or C1-C6 heteroalkyl moiety at a point of attachment, e.g., to the CFTR Ligand or the OTUB1 recruiter.
  • L1 has the structure of Formula (L1-I): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein each of R 7a and R 7b is independently H, C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, cycloalkyl, and halo; G is absent, C1–6 alkyl, C1–6 heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, aryl-(C 1–6 )alkylene, heteroaryl-(C 1–6 )alkylene, aryl-(C 1– 6 )heteroalkylene, heteroaryl-(C 1–6 )heteroalkylene, or -NR’-, wherein R’ is H, C 1–6 alkyl, or – (CH2)1-2-C(O)2H, wherein each alkyl,
  • L1 is selected from the group consisting of: , (L1-8), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein “*” and “**” each independently denote the point of attachment to the Target Ligand or the OTUB1 recruiter.
  • the linker e.g., L1 is a variant of a linker described herein, e.g., wherein the linker (e.g., L1) comprises an additional C 1 -C 6 alkyl or C 1 -C 6 heteroalkyl moiety at a point of attachment, e.g., to the CFTR Ligand or the OTUB1 recruiter.
  • the linker (e.g., L1) has the structure of Formula (III-d): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R 20 and R 22 are each independently H, C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, cycloalkyl, heterocyclyl; R 21a and R 21b are each independently H, C 1–6 alkyl, C 1–6 haloalkyl, C 1–6 heteroalkyl, halo, cyano, or -OR A ; or each of R 12a and R 12b , R 13a and R 13b , and R 14a and R 14b independently may be taken together with the carbon atom to which they are attached to form an oxo group; R A is H, C1–6 alkyl, C2–6 alkenyl, C2–6 alkynyl, C1–6
  • each of R 21a and R 21b is independently H. In some embodiments, each of R 21a and R 21b are taken together with the carbon atom to which they are attached form an oxo group. In some embodiments, m is selected from 2, 3, 4, 5, and 6.
  • DUB recruiter The DUB recruiter within the bifunctional compound is a small molecule moiety capable of binding to a deubiquitinase. In some embodiments, the DUB recruiter is a OTUB1 Recruiter, i.e., binds to the OTUB1 deubiquitinase, or a mutant, fragment, or isoform thereof.
  • the OTUB1 Recruiter binds to a cysteine amino acid residue within OTUB1.
  • the OTUB1 recruiter may bind to OTUB1 covalently or non-covalently.
  • the OTUB1 Recruiter binds to OTUB1 covalently, e.g., through a thiol or thioester bond.
  • the OTUB1 Recruiter binds to OTUB1 non-covalently, e.g., ionically.
  • the OTUB1 recruiter may bind to full-length OTUB1 or a fragment thereof.
  • the OTUB1 recruiter binds to a surface of OTUB1.
  • the OTUB1 Recruiter binds to an internal cavity of OTUB1. In some embodiments, the OTUB1 recruiter binds to a OTUB1 fragment or variant thereof.
  • the deubiquitinase is OTUB1 (Uniprot ID Q96FW1).
  • OTUB1 is a deubiquitinase that cleaves ubiquitin from branched polyubiquitin chains, with limited activity for removing ubiquitin bound directly to ubiquitin substrates.
  • OTUB1 has been shown to remove ubiquitin bound to other ubiquitin molecules through lysine-48 (Lys-48)-linkages, with minimal effect on ubiquitin molecules bound through other linkages.
  • the OTUB recruiter binds to OTUB1 isoform 1. In an embodiment, the OTUB recruiter binds to OTUB1 isoform 2.
  • the OTUB1 Recruiter described herein may bind to (e.g., covalently bind to) any cysteine residue within the OTUB1 sequence, e.g., C23, C91, C204, or C212. In some embodiments, the OTUB1 Recruiter does not bind to a catalytic cysteine amino acid within the OTUB1 sequence. In some embodiments, the OTUB1 recruiter binds to an allosteric cysteine amino acid residue within the OTUB1 sequence.
  • the OTUB1 Recruiter binds to a cysteine residue on a surface of OTUB1. In some embodiments, the OTUB1 recruiter binds to a cysteine residue on or in the interior of OTUB1. In some embodiments, the OTUB1 recruiter binds to C23 within the OTUB1 sequence. In some embodiments, the OTUB1 recruiter binds to C91 within the OTUB1 sequence. In some embodiments, the OTUB1 recruiter binds to C204 within the OTUB1 sequence. In some embodiments, the OTUB1 recruiter binds to C212 within the OTUB1 sequence.
  • the OTUB1 Recruiter binds preferentially to C23 over another cysteine amino acid residue within the OTUB1 sequence (e.g., C91, C204, or C212). In some embodiments, the OTUB1 recruiter binds preferentially to C23 over C91 within the OTUB1 sequence. In some embodiments, the OTUB1 recruiter does not substantially bind to C91 within the OTUB1 sequence.
  • binding of the DUB Recruiter e.g., the OTUB1 Recruiter
  • OTUB1 Recruiter does not modulate the activity of OTUB1 more than 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%, relative to the activity of OTUB1 in the absence of the OTUB1 Recruiter.
  • binding of the DUB Recruiter (e.g., OTUB1 Recruiter) to C23 within the OTUB1 sequence does not modulate the activity of OTUB1 more than 0.1-50%, 1-50%, 1-25%, 1-10%, 0.1-10%, 1-5%, or 0.1-2%, relative to the activity of the deubiquitinase in the absence of the bifunctional compound.
  • the binding of the DUB Recruiter (e.g., OTUB1 Recruiter) to OTUB1 does not substantially modulate (e.g., inhibit) the activity (e.g., deubiquitinase activity) of OTUB1.
  • the binding of the DUB Recruiter (e.g., OTUB1 Recruiter) to C23 within the OTUB1 sequence does not substantially modulate (e.g., inhibit) the activity (e.g., deubiquitinase activity) of OTUB1.
  • the OTUB1 Recruiter binds to a site other than a catalytic site within OTUB1. In some embodiments, the OTUB1 Recruiter binds to an allosteric site within OTUB1.
  • binding of the OTUB1 Recruiter to OTUB1 does not modulate the activity of OTUB1 more than 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%, relative to the activity of OTUB1 in the absence of the OTUB1 Recruiter.
  • binding of the OTUB1 Recruiter to OTUB1 does not modulate the activity of OTUB1 more than 0.1-50%, 1-50%, 1-25%, 1-10%, 0.1-10%, 1-5%, or 0.1-2%, relative to the activity of OTUB1 in the absence of the OTUB1 Recruiter. In some embodiments, the binding of the OTUB1 Recruiter to OTUB1 does not substantially modulate (e.g., inhibit) the activity (e.g., deubiquitinase activity) of OTUB1. In some embodiments, the OTUB1 recruiter binds to a site other than a catalytic site within OTUB1.
  • the OTUB1 Recruiter binds to an allosteric site within OTUB1. In some embodiments, the OTUB1 recruiter binds to a cysteine amino acid residue within OTUB1. In some embodiments, the OTUB1 Recruiter preferentially binds to an allosteric amino acid residue (e.g., an allosteric cysteine amino acid residue) over a catalytic amino acid residue (e.g., a catalytic cysteine amino acid residue). In some embodiments, the OTUB1 recruiter does not substantially bind to a cysteine amino acid residue in the catalytic site of OTUB1 (e.g., a catalytic cysteine).
  • the OTUB1 Recruiter does not substantially bind to cysteine 91 (C91) in OTUB1. In some embodiments, the OTUB1 recruiter binds preferentially to C23 in OTUB1. In some embodiments, the OTUB1 recruiter binds preferentially to C212 in OTUB1. In some embodiments, the OTUB1 Recruiter comprises a functional group selected from the group consisting of an amide, heterocyclyl, cycloalkyl, heterocyclyl, cycloalkyl, carbonyl, ester, alkyl, alkenyl, alkynyl, acyl, or acrylamide.
  • the OTUB1 Recruiter comprises a heterocyclyl (e.g., a piperazinonyl). In some embodiments, the OTUB1 recruiter comprises an acrylamide moiety. In some embodiments, the OTUB1 Recruiter comprises a heteroaryl (e.g., a furan moiety).
  • the OTUB1 Recruiter has the structure of Formula (V-a): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein Ring Z is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is substituted with 0-12 R 4 ;
  • L 1 is absent, -O-, C 1–12 alkylene, C 2–12 alkenylene, C 2–12 alkynylene, C 1– 12 heteroalkyl, wherein each alkylene, alkenylene, and heteroalkyl is optionally substituted with one or more R 5 ;
  • each R 1 is independently C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, halo, -OR A , -C(O)R A , -C(O)OR A , -NR B R C , -NR B C(O)R A ,
  • the OTUB1 Recruiter has the structure of Formula (V-b): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein Ring Z is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is substituted with 0-12 R 4 ;
  • L 1 is -O-, C 1–12 alkylene, C 2–12 alkenylene, C 2–12 alkynylene, C 1–12 heteroalkyl, wherein each alkylene, alkenylene, and heteroalkyl is optionally substituted with one or more R 5 ;
  • each R 1 is independently C1–6 alkyl, C1–6 haloalkyl, C1–6 heteroalkyl, halo, -OR A , - C(O)R A , -C(O)OR A , -NR B R C , -NR B C(O)R A , -C
  • the OTUB1 Recruiter has the structure of Formula (V-c): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-c): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-b).
  • the OTUB1 Recruiter has the structure of Formula (V-d): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-e): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-f): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-g): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-):h or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-i): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-j): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-k): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-l): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-m): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-n): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a); each of X and Y is independently C(R 7 ), N, N(R 4a ), or O; R 4a is H, C1–6 alkyl, C1–6 haloalkyl, C1– 6 heteroalkyl, halo, cyano, -C(O)R A ; R 7 is H, C 1–6 alkyl, C 1–6 haloalkyl, C 1–6 heteroalkyl, halo, cyano, -OR A , -C(O)R A , -C(O)OR A , -NR B R C , -NR B C(O)R
  • the OTUB1 Recruiter has the structure of Formula (V-o): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-p): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-a).
  • the OTUB1 Recruiter has the structure of Formula (V-q): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein Ring Z, R 2 , R 3 , R 5 , n, and subvariables thereof are as described for Formula (V- a), Ring W is cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each of cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with 0-12 R 5 ; and p is 0, 1, 2, 3, 4, 5, or 6.
  • the OTUB1 Recruiter has the structure of Formula (V-r): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein Ring Z, L 1 , R 2 , R 3 , n, and subvariables thereof are as described for Formula (V- a).
  • the OTUB1 Recruiter has the structure of Formula (V-s): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein L 1 , R 2 , R 3 , R 4 , n, and subvariables thereof are as described for Formula (V-b).
  • the OTUB1 Recruiter has the structure of Formula (V-t): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein Ring Z, L 1 , R 2 , R 3 , n, and subvariables thereof are as described for Formula (V- a).
  • the OTUB1 recruiter has the structure of Formula (V-u): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein Ring Z, L 1 , R 2 , R 3 , n, and subvariables thereof are as described for Formula (V- a), and m is 0, 1, 2, or 3.
  • Ring Z is heteroaryl (e.g., a monocyclic heteroaryl).
  • Ring Z is a 5-membered heteroaryl (e.g., furanyl).
  • R 2 is an electrophilic moiety.
  • R 2 is H, C 1–6 alkyl, C 2–6 alkenyl, C 2–6 alkynyl, C 1–6 haloalkyl, C1–6 heteroalkyl, halo, cyano, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is substituted with 0-12 R 10 .
  • n is 0.
  • R 2 is an electrophilic moiety.
  • R 2 is a structure selected from one of: or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein: R 16 is H, halogen, -CX 16 3 , -CHX16 2 , -CH 2 X 16 , -CN, -SO n16 R 16A , -SO v16 NR 16A R 16B , - NHNR 16A R 16B , ONR 16A R 16B , - NHC(O)NHNR 16A R 16B , -N(O)m16, -NR 16A R 16B , -C(O)R 16A , -C(O)-OR 16A , -C(O)NR 16A R 16B , -OR 16A , NHC(O)NR 16A R 16B , -NR 16A SO R 16B , -
  • R 2 is selected from the group consisting of: , , , , , , , , , , , , , , , , , , , , , , , , , wherein the electrophilic moiety is bound to the structure of Formula (V-a) at any position.
  • the DUB recruiter is compound is selected from a compound listed in Table 1, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB Recruiter is Compound 100 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 101 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 102 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 103 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 104 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 105 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 106 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 107 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 108 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 109 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 110 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 111 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 112 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 113 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 114 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 115 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 116 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 117 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 118 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 119 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 120 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 121 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 122 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 123 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 124 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 125 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 126 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 127 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 128 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 129 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 130 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 131 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 133 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 134 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 135 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 136 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 137 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 138 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 139 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 140 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 141 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 142 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 143 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 144 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 145 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 146 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 147 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 148 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 149 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 150 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 151 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 152 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 153 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 154 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 155 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 156 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 157 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 158 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 159 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 160 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 161 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 162 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 163 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 164 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 165 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 166 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 168 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 169 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 170 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 171 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 172 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 173 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 174 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 175 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 176 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 177 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 178 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 179 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 180 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 181 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 182 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 183 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 184 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 185 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 186 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 187 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 188 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 189 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 190 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the DUB recruiter is Compound 191 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 192 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 193 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the DUB recruiter is Compound 194 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the disclosure provides a bifunctional compound or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, which is capable of binding to a target protein (e.g., a target protein described herein).
  • the target protein may be any class of protein, for example, any protein found in a cell (e.g., a mammalian cell, a plant cell, a fungal cell, an insect cell, a bacterial cell) or a viral particle.
  • the protein is a soluble protein or a membrane protein.
  • the protein is a soluble protein.
  • the protein is a membrane protein.
  • the target protein may comprise a post-translational modification, e.g., a sugar moiety, acyl moiety, lipid moiety.
  • the target protein is glycosylated, e.g., at an asparagine, serine, threonine, tyrosine, or tryptophan residue.
  • Exemplary target proteins include enzymes (e.g., kinases, hydrolases, phosphatases, ligases, isomerases, oxidoreductases), receptors, membrane channels, hormones, transcription factors, tumor suppressors, ion channels, apoptotic factors, oncogenic proteins, epigenetic regulators, or a fragment thereof.
  • the target protein is an enzyme (e.g., a kinase or phosphatase).
  • the target protein is a kinase (e.g., PKN1, BCR, MAP4K4, TYK2, MAP4K2, EPHB4, MAP4K5, MAP3K2, DDR1, TGFBR1, RIPK2, TNK1, LYN, STK10, PKMYT1, LYN, EGFR, EPHA1, GAK, SIK2, MAP2K2, SLK, PRKACB, EPHA2, WEE1, or glucokinase).
  • the target protein is a tumor suppressor kinase (e.g., WEE1).
  • the target protein is WEE1 or a fragment thereof.
  • the target protein is a ligase (e.g., an E3 ligase, e.g., MDM2).
  • the target protein is a receptor.
  • the target protein is a transcription factor (e.g., MYC).
  • the target protein is a hormone.
  • the target protein is a tumor suppressor (e.g., TP53, AXIN1, BAX, CDKN1A, CKDN1C, PTEN, or SMAD4).
  • the target protein is related to a genetic disorder (e.g., SMN1/2, GLUT1, CFTR, phenylalanine hydroxylase (PAH), fumarylacetoacetate hydrolase (FAH), or acid alpha-glucosidase (GAA)).
  • a genetic disorder e.g., SMN1/2, GLUT1, CFTR, phenylalanine hydroxylase (PAH), fumarylacetoacetate hydrolase (FAH), or acid alpha-glucosidase (GAA)
  • the target protein is a membrane channel (e.g., CFTR).
  • the target protein is CFTR or a fragment thereof.
  • the CFTR comprises a sequence mutation (e.g., a Class I, Class II, Class III, Class IV, or Class V mutation).
  • the CFTR, SMN1/2, GLUT1, PAH, FAH, or GAA comprises a sequence mutation, e.g., an addition mutation, deletion mutation, or substitution mutation (e.g., ⁇ F508-CFTR).
  • the CFTR comprises a sequence mutation selected from the group consisting of G551D, R177H, and A445E.
  • the target protein is BAX or a fragment thereof.
  • the target protein is STING or a fragment thereof
  • the target protein is modified with a ubiquitin or a ubiquitin-like protein (collectively referred to herein as “Ubls”).
  • the Ubl is ubiquitin.
  • the Ubl is SUMO, NEDD8, or Agp12.
  • the target protein is monoubiquitinated or polyubiquitinated.
  • the target protein may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Ubl chains, e.g., on a lysine amino acid residue.
  • the target protein may comprise polyubiquitin chains linked in any manner, for example, K48-linked polyubiquitin chains, K63-linked polyubiquitin linked chains, K29-linked polyubiquitin chains, or K33-linked polyubiquitin chains.
  • the target protein comprises a plurality of polyubiquitin chains.
  • the target protein comprising a Ubl is capable of binding to a protein comprising a Ubl-binding domain (e.g., a ubiquitin binding domain).
  • the target protein may comprise a feature that increases its instability or impairs its activity, e.g., relative to the wild-type target protein.
  • the target protein may be mutated or misfolded.
  • the target protein has a reduced capacity for binding to a binding partner, e.g., by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% relative to the wild type target protein.
  • the target protein is less active than the wild type target protein, e.g., by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%. In some embodiments, the target protein is more active than the wild type target protein, e.g., by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%.
  • Deubiquitinases Described herein are bifunctional compounds comprising a moiety capable of binding to a deubiquitinase (DUB).
  • Deubiquitinases comprise a large family of proteases responsible for hydrolyzing Ubl-Ubl bonds or Ubl-target protein bonds and play a role in numerous cellular processes. Deubiquitinases serve several functions, including generating free ubiquitin monomers from polyubiquitin chains, modulating the size of polyubiquitin chains, and reversing ubiquitin signaling by removal of a from a ubiquitinated target protein. Misregulation of deubiquitinase function is associated with many diseases, including cancer, metabolic diseases, genetic disorders, haploinsufficiency targets, and neurological diseases. Roughly 80 different functional deubiquitinases have been identified in human cells to date.
  • the present disclosure features bifunctional compounds comprising a DUB recruiter capable of binding to a deubiquitinase.
  • the deubiquitinase may be any deubiquitinase, e.g., in a cell, including cysteine protease deubiquitinases and metalloprotease deubiquitinases.
  • the deubiquitinase is a cysteine protease, e.g., comprising a catalytic site cysteine amino acid residue.
  • the deubiquitinase may be a full-length protein or a fragment thereof.
  • the deubiquitinase comprises a single active site.
  • the deubiquitinase is one function of a multifunctional protein.
  • exemplary deubiquitinases include BAP1, CYLD, OTUB1, OTUB2, OTUD3, OTUD5, OTUD7A, OTUD7B, TNFAIP3, UCHL1, UCHL3, UCHL5, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17L1, USP17L2, USP17L24, USP17L3, USP17L5, USP18, USP19, USP2, USP20, USP21, USP22, USP24, USP25, USP26, USP27X, USP28, USP3, USP30, USP31, USP33, USP34, USP35, USP36, USP37, USP38, USP4, USP40, USP41, USP42, USP43, USP44, USP45, USP46, USP47, USP48
  • the deubiquitinase is selected from the group consisting of WDR48, YOD1, OYUD3, OTUB1, USP8, USP5, USP16, UCHL3, UCHL1, and USP14, or a fragment thereof.
  • the deubiquitinase is OTUB1 or a fragment or variant thereof.
  • the bifunctional compounds of the present disclosure may bind to OTUB1 in a covalent or non- covalent manner.
  • the bifunctional compound e.g., the OTUB1 recruiter
  • the bifunctional compound binds to an allosteric site within OTUB1.
  • binding of the bifunctional compound (e.g., the OTUB1 Recruiter) to OTUB1 does not modulate the activity of OTUB1 more than 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%, relative to the activity of OTUB1 in the absence of the bifunctional compound.
  • binding of the bifunctional compound (e.g., the OTUB1 Recruiter) to OTUB1 does not modulate the activity of OTUB1 more than 0.1-50%, 1-50%, 1-25%, 1-10%, 0.1-10%, 1-5%, or 0.1-2%, relative to the activity of OTUB1 in the absence of the bifunctional compound.
  • the binding of the bifunctional compound (e.g., the OTUB1 Recruiter) to OTUB1 does not substantially modulate (e.g., inhibit) the activity (e.g., OTUB1 activity) of OTUB1.
  • the bifunctional compound (e.g., a bifunctional compound described herein) is capable of binding to a cysteine amino acid residue (e.g., a thiol moiety), e.g., within OTUB1.
  • the cysteine amino acid residue is an allosteric cysteine amino acid residue.
  • the cysteine amino acid residue is present on a surface of OTUB1.
  • the cysteine amino acid residue is present on or in the interior of OTUB1.
  • the cysteine amino acid residue is not a catalytic cysteine amino acid residue.
  • the bifunctional compound preferentially binds to an allosteric cysteine amino acid residue over a catalytic cysteine amino acid residue. In some embodiments, the bifunctional compound does not substantially bind to a cysteine amino acid residue in the catalytic site of OTUB1 (e.g., a catalytic cysteine).
  • Pharmaceutically Acceptable Salts Pharmaceutically acceptable salts of the compounds described herein are also contemplated for the uses described herein. As used herein, the terms “salt” or “salts” refer to an acid addition or base addition salt of a compound described herein.
  • Salts include in particular “pharmaceutical acceptable salts.”
  • pharmaceutically acceptable salts refers to salts that retain the biological effectiveness and properties of the compounds disclosed herein and, which typically are not biologically or otherwise undesirable. In many cases, the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table.
  • the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.
  • the bifunctional compound of Formula (I) is provided as an acetate, ascorbate, adipate, aspartate, benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, caprate, chloride/hydrochloride, chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulphate, mucate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate,
  • compositions Another embodiment is a pharmaceutical composition comprising one or more compounds described herein or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and one or more pharmaceutically acceptable carrier(s).
  • pharmaceutically acceptable carrier refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be “acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient.
  • materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;
  • compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • the compositions of the disclosure are administered orally, intraperitoneally or intravenously.
  • Sterile injectable forms of the compositions of this disclosure may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also 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.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di- glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants such as Tween®, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • compositions described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and com starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • the pharmaceutically acceptable compositions of this disclosure may be administered in the form of suppositories for rectal administration.
  • compositions of this disclosure may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.
  • the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.
  • the pharmaceutically acceptable compositions of this disclosure may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • compositions should be formulated so that a dosage of between 0.01–100 mg/kg body weight/day of the inhibitor can be administered to a patient receiving these compositions.
  • Isotopically Labelled Compounds A compound described herein or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds.
  • Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number.
  • isotopes that can be incorporated into compounds described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 2 H, 3H, 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl, 123 I, 124 I, 125 I, respectively.
  • the disclosure includes various isotopically labeled compounds as defined herein, for example, those into which radioactive isotopes, such as 3 H and 14 C, or those into which non-radioactive isotopes, such as 2 H and 13 C are present.
  • isotopically labelled compounds are useful in metabolic studies (with 14 C), reaction kinetic studies (with, for example 2 H or 3 H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • an 18F or labeled compound may be particularly desirable for PET or SPECT studies.
  • Isotopically-labeled compounds described herein or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed. Further, substitution with heavier isotopes, particularly deuterium (i.e., 2 H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index.
  • deuterium i.e., 2 H or D
  • deuterium in this context is regarded as a substituent of a compound described herein or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • concentration of such a heavier isotope, specifically deuterium may be defined by the isotopic enrichment factor.
  • isotopic enrichment factor means the ratio between the isotopic abundance and the natural abundance of a specified isotope.
  • a substituent in a compound described herein is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).
  • Dosages Toxicity and therapeutic efficacy of compounds described herein, including pharmaceutically acceptable salts and deuterated variants, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the LD50 is the dose lethal to 50% of the population.
  • the ED50 is the dose therapeutically effective in 50% of the population.
  • the dose ratio between toxic and therapeutic effects (LD 50 /ED 50 ) is the therapeutic index.
  • Compounds that exhibit large therapeutic indexes are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and thereby reduce side effects. Data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds may lie within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated.
  • the amount of a compound described herein in the composition will also depend upon the particular compound in the composition.
  • the present disclosure features a method of modulating a target protein, e.g., a target protein described herein, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • a target protein e.g., a target protein described herein
  • the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the modulating comprises one or more of: (i) modulating the folding of the target protein; (ii) modulating the half-life of the target protein; (iii) modulating trafficking of the target protein to the proteasome; (iv) modulating the level of ubiquitination of the target protein; (v) modulating degradation (e.g., proteasomal degradation) of the target protein; (vi) modulating target protein signaling; (vii) modulating target protein localization; (viii) modulating trafficking of the target protein to the lysosome; (ix) modulating trafficking of the target protein to the ER, Golgi, vesicle, plasma membrane; (x) modulating target protein interactions with another protein (e.g., other proteins in the UPS); (xi) modulating posttranslational modifications of the target protein (e.g., SUMOlyation, phosphorylation, glycosylation).
  • SUMOlyation e.g., SUMOlyation, phosphorylation, glycosy
  • the present disclosure features a method of stabilizing a target protein, e.g., a target protein described herein, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the stabilizing comprises increasing the half-life of a target protein or removal of a Ubl from a target protein, e.g., compared to a reference standard.
  • the stabilizing improves the function of a target protein.
  • the present disclosure features a method of forming a protein complex comprising a deubiquitinase, e.g., a deubiquitinase described herein, and a target protein, upon administration of a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the protein complex is formed in vitro (e.g., in a sample) or in vivo (e.g., in a cell or tissue, e.g., in a subject).
  • Formulation of the protein complex may be observed and characterized by any method known in the art, e.g., mass spectrometry (native mass spectrometry) or SDS PAGE.
  • forming the protein complex modulates the level of a target protein, e.g., increases the half-life of the target protein, e.g., compared to a reference standard.
  • forming the protein enhances removal of a Ubl from the target protein, e.g., compared to a reference standard.
  • the deubiquitinase is OTUB1.
  • the target protein comprises CFTR.
  • Another embodiment is a method for removing a Ubl (e.g., a ubiquitin or ubiquitin-like protein) from a target protein, e.g., a target protein described herein, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • a Ubl e.g., a ubiquitin or ubiquitin-like protein
  • the present disclosure provides a method of maintaining, improving, or increasing the activity of a target protein, e.g., a target protein described herein, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • a target protein e.g., a target protein described herein
  • the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • maintaining, improving, or increasing the activity of a target protein comprises recruiting a deubiquitinase (e.g., a deubiquitinase of Table 1) with the bifunctional compound described herein (e.g., the DUB recruiter within the bifunctional compound), e.g., a compound of Formula (I), forming a ternary complex of the target protein, the bifunctional compound, and the deubiquitinase, to thereby maintain, improve, or increase the activity of the target protein.
  • a deubiquitinase e.g., a deubiquitinase of Table 1
  • the bifunctional compound described herein e.g., the DUB recruiter within the bifunctional compound
  • a compound of Formula (I) e.g., a compound of Formula (I)
  • the present disclosure features a method of treating or preventing a disease, disorder or condition mediated by a target protein, e.g., a target protein described herein, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • the disease, disorder, or condition is selected from the group consisting of a respiratory disorder, a proliferative disorder, an autoimmune disorder, an autoinflammatory disorder, an inflammatory disorder, a metabolic disorder, a neurological disorder, and an infectious disease.
  • the disease, disorder, or condition is selected from the group consisting of a respiratory disorder, a proliferative disorder, an autoimmune disorder, an autoinflammatory disorder, an inflammatory disorder, a neurological disorder, and an infectious disease.
  • the disease, disorder, or condition comprises a respiratory disorder.
  • the disease, disorder, or condition comprises a proliferative disorder.
  • the disease, disorder, or condition comprises an autoinflammatory disorder.
  • the disease, disorder, or condition comprises an inflammatory disorder.
  • the disease, disorder, or condition comprises a metabolic disorder.
  • the disease, disorder, or condition comprises a neurological disorder.
  • the disease, disorder, or condition comprises an infectious disease.
  • the disease, disorder, or condition is cancer. In some embodiments, the disease, disorder, or condition is cystic fibrosis. In some embodiments, the disease, disorder, or condition is diabetes (e.g., maturity-onset diabetes of the young type 2, MODY2).
  • the disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in inhibiting or modulating a target protein in a subject in need thereof.
  • Another embodiment is a use of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for treating or preventing a respiratory disorder, a proliferative disorder, an autoimmune disorder, an autoinflammatory disorder, an inflammatory disorder, a neurological disorder, and an infectious disease or disorder in a subject in need thereof.
  • a respiratory disorder a proliferative disorder, an autoimmune disorder, an autoinflammatory disorder, an inflammatory disorder, a neurological disorder, and an infectious disease or disorder in a subject in need thereof.
  • an allosteric amino acid residue e.g., an allosteric amino acid residue
  • a catalytic amino acid residue e.g., a catalytic cysteine amino acid residue
  • the CFTR Ligand is capable of modulating CFTR, or a mutant, fragment, or isoform thereof. 17.
  • CFTR Ligand has the structure of Formula (II-a): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein: X and Z are each independently O, S, or C(R 7a )(R 7b ); Y is C(R 7a )(R 7b ) or NR 7c ; R 1 is H or C1–6 alkyl; R 3a , R 3b , R 4a , R 4b are each independently H, C 1–6 alkyl, C 1–6 haloalkyl, C 1–6 heteroalkyl, halo, cyano, or -OR A ; each R 5 , R 5’ , and R 6 is independently C1–6 alkyl, C1–6 haloalkyl, C1
  • bifunctional compound or pharmaceutically acceptable salt hydrate, solvate, stereoisomer, or tautomer thereof according to any one of the preceding embodiments, wherein the bifunctional compound of Formula (I-c) has the structure (II-l): or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein: X and Z are each independently O, S, or C(R 7a )(R 7b ); Y is C(R 7a )(R 7b ) or NR 7c ; R 1 is H or C1–6 alkyl; R 3a , R 3b , R 4a , R 4b are each independently H, C 1–6 alkyl, C 1–6 haloalkyl, C 1–6 heteroalkyl, halo, cyano, or -OR A ; each R 5 , R 5’ , and R 6 is independently C1–6 alkyl, C1–6 haloalkyl
  • OTUB1 Recruiter does not substantially bind to a cysteine amino acid residue in the catalytic site of OTUB1 (e.g., a catalytic cysteine).
  • the OTUB1 Recruiter comprises an acrylamide moiety. 47.
  • the bifunctional compound or pharmaceutically acceptable salt hydrate, solvate, stereoisomer, or tautomer thereof according any one of embodiments 48-51, wherein R 2 is C 2–6 alkenyl (e.g., CH CH2). 53.
  • L1 comprises an alkylene or heteroalkylene.
  • bifunctional compound or pharmaceutically acceptable salt hydrate, solvate, stereoisomer, or tautomer thereof according to any one of the preceding embodiments, wherein the bifunctional compound of Formula (I-c) is selected from a bifunctional compound provided in Table 1, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
  • a pharmaceutical composition comprising a bifunctional compound, or pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof according to any one of the preceding embodiments, and one or more pharmaceutically acceptable carriers. 61.
  • compositions for use in providing a compound to a subject comprising a bifunctional compound of Formula (I): (I), or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1. 62.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • a composition for use in treating a disease, disorder, or condition in a subject comprising a bifunctional compound of Formula (I): (I), or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1.
  • compositions for use of claim 62 wherein administering the composition ameliorates a symptom or element of the disease, disorder, or condition.
  • administering the composition ameliorates a symptom or element of the disease, disorder, or condition.
  • 64. The composition for use of any one of embodiments 62-63, wherein the disease, disorder, or condition is cystic fibrosis. 65.
  • a composition for use in treating cystic fibrosis in a subject comprising a bifunctional compound of Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1. 66.
  • a composition for use in modulating a protein in a cell or subject comprising a bifunctional compound of Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1. 67.
  • compositions for use in recruiting a deubiquitinase to a target protein in a cell or subject comprising a bifunctional compound of Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1. 68.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • composition for use of embodiment 67, wherein the deubiquitinase is OTUB1.
  • a composition for use in deubiquitinating a protein comprising a bifunctional compound of Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1. 70.
  • a method of treating a disease, disorder, or condition in a subject comprising administering to the subject a bifunctional compound of Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1, thereby treating disease, disorder, or condition in the subject 71.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the method of embodiment 70 wherein the method comprises ameliorating a symptom or element of the disease, disorder, or condition.
  • 72 The method of any one of embodiments 70-71, wherein the disease, disorder, or condition is cystic fibrosis.
  • 73 A method of treating cystic fibrosis in a subject, the method comprising administering to the subject a bifunctional compound of Formula (I): (I), or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1, thereby treating cystic fibrosis.
  • CFTR cystic fibrosis transmembrane
  • a method of modulating a protein in a cell or subject comprising contacting the cell or administering to the subject a bifunctional compound of Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 Recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1, thereby modulating a protein in a cell or subject.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • a method of recruiting a deubiquitinase to a target protein comprising contacting a mixture (e.g., in a cell or sample) with a bifunctional compound of Formula (I): (I), or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1, thereby recruiting a deubiquitinase to a target protein in a mixture, e.g., a cell or subject.
  • CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant,
  • a method of deubiquitinating a protein comprising contacting a cell or sample with a bifunctional compound of Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein: (i) the CFTR Ligand comprises a moiety capable of binding to the cystic fibrosis transmembrane conductance regulator (CFTR), or a mutant, fragment, or isoform thereof; (ii) L1 comprises a linker; and (iii) the OTUB1 recruiter comprises a moiety capable of binding to the deubiquitinase OTUB1, thereby deubiquitinating a protein in a cell or subject.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • CFBE41o-4.7 ⁇ F508-CFTR Human CF Bronchial Epithelial cells were cultured in MEM (Gibco) containing 10% (v/v) fetal bovine serum (FBS) and maintained at 37 °C with 5% CO2.
  • MEM MEM
  • FBS fetal bovine serum
  • Synthesis of exemplary DUB recruiters and bifunctional compounds Chemical Synthesis and Characterization Starting materials, reagents and solvents were purchased from commercial suppliers and were used without further purification unless otherwise noted. All reactions were monitored by thin layer chromatography. Reaction products were purified by flash column chromatography.1H NMR spectra were recorded on a 400 MHz Bruker Avance spectrometer .
  • Preparative HPLC purification was performed using one of the following HPLC conditions: Condition 1: Column, Phenomenex C1880*40mm*3um; Mobile Phase A: water(NH 4 HCO 3 ); Mobile Phase B: MeCN; Gradient a: 1% B to 30% B in 8 min; Gradient b: 20% B to 55% B in 8 min; Gradient c: 1% B to 20% B in 8 min; Gradient d: 15% B to 45% B in 8 min; Gradient e: 5% B to 35% B in 8 min; Gradient f: 15% B to 50% B in 8 min; Gradient g: 30% B to 60% B in 8 min; Gradient h: 10% B to 35% B in 8 min; Condition 2: Column, Waters Xbridge BEH C18100*30 mm*10 um; Mobile Phase A: water(10 mM NH4HCO3); Mobile Phase B: MeCN; Gradient a: 15% B to 45% B in 8 min; Gradient b: 1% B to
  • Example 1 Synthesis of Compound 100 Step 1: tert-butyl 4-imidazo[1,2-a]pyridin-2-yl-3-oxo-piperazine-1-carboxylate To a solution of 2-bromoimidazo[1,2-a]pyridine (A1, 300 mg, 1.5 mmol, 1 eq) and tert- butyl 3-oxopiperazine-1-carboxylate (A2, 305 mg, 1.5 mmol, 1 eq) in dioxane (4 mL) was added CuI (116 mg, 609 ⁇ mol, 0.4 eq), N,N'-dimethylethane-1,2-diamine (107 mg, 1.2 mmol, 131 uL, 0.8 eq) and K 2 CO 3 (421 mg, 3.05 mmol, 2 eq), and the mixture was stirred at 100 °C for 16 h.
  • 2-bromoimidazo[1,2-a]pyridine A1, 300 mg, 1.5 mmol, 1 eq
  • Step 2 1-(imidazo[1,2-a]pyridin-2-yl)piperazin-2-one
  • DCM dimethylethyl sulfoxide
  • TFA trifluoroacetic acid
  • Step 3 1-imidazo[1,2-a]pyridin-2-yl-4-prop-2-enoyl-piperazin-2-one
  • TEA triethylamine
  • TEA 224.6 mg, 2.2 mmol, 309 uL, 3 eq
  • prop- 2-enoyl chloride 7.3 mg, 814 ⁇ mol, 66.4 uL, 1.1 eq
  • Example 2 Synthesis of Compound 102 Step 1: tert-butyl 3-oxo-4-(2-phenyloxazol-5-yl)piperazine-1-carboxylate To a solution of 5-bromo-2-phenyl-oxazole (A8, 200 mg, 893 ⁇ mol, 1 eq), tert-butyl 3- oxopiperazine-1-carboxylate (A2, 178.7 mg, 893 ⁇ mol, 1 eq) in dioxane (2 mL) was added CuI (25.5 mg, 134 ⁇ mol, 0.15 eq), K 2 CO 3 (370 mg, 2.7 mmol, 3 eq) and N,N'-dimethylethane-1,2- diamine (19.7 mg, 223 ⁇ mol, 24 uL, 0.25 eq), and the mixture was stirred at 100 °C for 16 h.
  • Step 2 1-(2-phenyloxazol-5-yl)piperazin-2-one
  • a solution of tert-butyl 3-oxo-4-(2-phenyloxazol-5-yl)piperazine-1-carboxylate (A9, 100 mg, 291 ⁇ mol, 1 eq) in DCM (1 mL) was added TFA (513.3 mg, 4.5 mmol, 333.3 uL, 15.5 eq) at 0 °C, The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove the solvent. The mixture was used for the next steps without purification.
  • Example 3 Synthesis of Compound 103 Step 1: benzyl 4-(1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydropyridin-4-yl)-3-oxopiperazine-1- carboxylate
  • benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro- 2H-pyridine-1-carboxylate A11, 2 g, 6.47 mmol, 1 eq
  • DMSO 20 mL
  • Step 2 tert-butyl 4-(2-oxopiperazin-1-yl)piperidine-1-carboxylate
  • benzyl 4-(1-tert-butoxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)-3-oxo- piperazine-1-carboxylate (A12, 0.4 g, 963 ⁇ mol, 1 eq) in MeOH (4 mL) and DCM (4 mL), was added Pd/C (0.2 g, 10% purity) under N2 atmosphere.
  • Step 3 tert-butyl 4-(4-acryloyl-2-oxopiperazin-1-yl)piperidine-1-carboxylate
  • a solution of tert-butyl 4-(2-oxopiperazin-1-yl)piperidine-1-carboxylate (A13, 0.1 g, 353 ⁇ mol, 1 eq) in DCM (1 mL) was added TEA (71.4 mg, 706 ⁇ mol, 98.2 uL, 2 eq), then prop- 2-enoyl chloride (32 mg, 353 ⁇ mol, 28.8 uL, 1 eq) in DCM (0.5 mL) was added at 0 °C.
  • Example 4 Synthesis of Compound 104 Step 1: 2-[(3-bromopyrrolo[2,3-b]pyridin-1-yl)methoxy]ethyl-trimethyl-silane To a solution of 3-bromo-1H-pyrrolo[2,3-b]pyridine (A14, 3.0 g, 15.2 mmol, 1 eq) in THF (45 mL) was added portionwise NaH (670 mg, 16.75 mmol, 60% purity, 1.1 eq) at 0°C.
  • Step 2 tert-butyl 3-oxo-4-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-3- yl)piperazine-1-carboxylate
  • 2-[(3-bromopyrrolo[2,3-b]pyridin-1-yl)methoxy]ethyl-trimethyl-silane A15, 900 mg, 2.75 mmol, 1 eq
  • tert-butyl 3-oxopiperazine-1-carboxylate A2, 551 mg, 2.75 mmol, 1 eq
  • CuI 78.6 mg, 412.5 ⁇ mol, 0.15 eq
  • K 2 CO 3 (1.14 g, 8.2 mmol, 3 eq
  • N1,N2- dimethylcyclohexane-1,2-diamine 98 mg, 687.5 ⁇ mol, 0.25 eq) in toluene (13.5 mL
  • Step 3 tert-butyl 3-oxo-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)piperazine-1-carboxylate
  • a solution of tert-butyl 3-oxo-4-[1-(2-trimethylsilylethoxymethyl)pyrrolo[2,3- b]pyridin-3-yl]piperazine-1-carboxylate (A16, 0.5 g, 1.12 mmol, 1 eq) in THF (8 mL) was added TBAF (1 M, 8 mL, 7.15 eq). The mixture was stirred at 60 °C for 16 h. Upon completion, the residue was diluted with water (30 mL).
  • Step 4 1-(1H-pyrrolo[2,3-b]pyridin-3-yl)piperazin-2-one
  • a mixture of tert-butyl 3-oxo-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)piperazine-1- carboxylate (A17, 0.22 g, 695 ⁇ mol, 1 eq) in DCM (5 mL) was added TFA (1.54 g, 13.5 mmol, 1 mL, 19.4 eq), and the mixture was stirred at 25 °C for 1 h.
  • Step 5 4-prop-2-enoyl-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)piperazin-2-one
  • TEA 140.4 mg, 1.4 mmol, 193 uL, 2 eq
  • prop-2-enoyl chloride 62.8 mg, 693.7 ⁇ mol, 56.6 uL, 1 eq
  • Example 5 Synthesis of Compound 105 Step 1: tert-butyl 4-(5-methylfuran-2-yl)-3-oxopiperazine-1-carboxylate A mixture of 2-bromo-5-methyl-furan (A19, 200 mg, 1.24 mmol, 1 eq), tert-butyl 3- oxopiperazine-1-carboxylate (A2, 248.7 mg, 1.24 mmol, 1 eq), CuI (94.6 mg, 497 ⁇ mol, 0.4 eq), DMEDA (87.6 mg, 994 ⁇ mol, 107 uL, 0.8 eq) and K 2 CO 3 (343.4 mg, 2.48 mmol, 2 eq) in dioxane (5 mL) was degassed and purged with N2 (x3), and then the mixture was stirred at 100 °C for 16 h under N2 atmosphere.
  • 2-bromo-5-methyl-furan A19, 200 mg, 1.24 mmol,
  • Step 2 1-(5-methylfuran-2-yl)piperazin-2-one
  • a solution of tert-butyl 4-(5-methyl-2-furyl)-3-oxo-piperazine-1-carboxylate (A20, 270 mg, 963.2 ⁇ mol, 1eq) in DCM (5 mL) was added TFA (109.8 mg, 963.2 ⁇ mol, 71.3 uL, 1 eq). The mixture was stirred at 20 °C for 0.5 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove solvent. The crude product was used to next step without further purification.
  • 1-(5-methyl-2-furyl) piperazin-2-one (A21, 170 mg, crude) was obtained as an oil.
  • Step 3 4-acryloyl-1-(5-methylfuran-2-yl)piperazin-2-one To a mixture of 1-(5-methyl-2-furyl)piperazin-2-one (A21, 85 mg, 471.7 ⁇ mol, 1 eq), TEA (143.2 mg, 1.42 mmol, 197 uL, 3 eq) in DCM (3 mL) and the mixture cooled to 0 °C, then prop- 2-enoyl chloride (47 mg, 519 ⁇ mol, 42.3 uL, 1.1 eq) was added and stirred for 0.5 h.
  • Example 6 Synthesis of Compound 106 Step 1: 4-(2-fluoroprop-2-enoyl)-1-imidazo [1, 2-a] pyridin-2-yl-piperazin-2-one A mixture of 1-imidazo[1,2-a]pyridin-2-ylpiperazin-2-one (A4, 143 mg, 463 ⁇ mol, 1 eq), 2-fluoroprop-2-enoic acid (50 mg, 555.5 ⁇ mol, 1.2 eq), HOBt (68.8 mg, 509 ⁇ mol, 1.1 eq), EDCI (97.6 mg, 509 ⁇ mol, 1.1 eq) and TEA (140.5 mg, 1.39 mmol, 193.3 uL, 3 eq) in DMF (4 mL) was degassed and purged with N2 (x3), and the mixture was stirred at 25°C for 12h under N2 atmosphere.
  • 1-imidazo[1,2-a]pyridin-2-ylpiperazin-2-one
  • Example 7 Synthesis of Compound 107 To a solution of (E)-4-(dimethylamino)but-2-enoic acid (A4, 51.1 mg, 396 ⁇ mol, 1 eq) in DMF (1.2 mL) was added HATU (150.5 mg, 396 ⁇ mol, 1 eq), it was stirred at 25 °C for 0.5 h. Then DIEA (153.4 mg, 1.19 mmol, 206.8 uL, 3 eq) and 1-imidazo[1,2-a]pyridin-2-ylpiperazin-2- one (142.9 mg, 396 ⁇ mol, 70% purity, 1 eq, HCl) were added at 0 °C.
  • Example 8 Synthesis of Compound 108 Step 1: benzyl 4-(1-tert-butoxycarbonylpyrrolidin-3-yl)-3-oxo-piperazine-1-carboxylate
  • benzyl 4-(1-tert-butoxycarbonylpyrrolidin-3-yl)-3-oxo-piperazine-1-carboxylate To a solution of tert-butyl 3-bromopyrrolidine-1-carboxylate (A22, 1 g, 4.0 mmol, 1 eq) and benzyl 3-oxopiperazine-1-carboxylate (936.5 mg, 4.0 mmol, 1 eq) in DMF (40 mL) was added Cs 2 CO 3 (3.9 g, 12 mmol, 3 eq) at 20 °C, and the mixture was stirred at 80 °C for 16 h.
  • Step 2 tert-butyl 3-(2-oxopiperazin-1-yl) pyrrolidine-1-carboxylate
  • Step 3 tert-butyl 3-(2-oxo-4-prop-2-enoyl-piperazin-1-yl) pyrrolidine-1-carboxylate
  • tert-butyl 3-(2-oxopiperazin-1-yl)pyrrolidine-1-carboxylate A24, 100 mg, 371.3 ⁇ mol, 1 eq
  • TEA 112.7 mg, 1.1 mmol, 155 uL, 3 eq
  • DCM 3 mL
  • prop- 2-enoyl chloride 40.3 mg, 445.5 ⁇ mol, 36.3 uL, 1.2 eq
  • Example 9 Synthesis of Compound 109 Step 1: methyl (2-((tert-butoxycarbonyl)amino)ethyl)phenylalaninate
  • tert-butyl N-(2-oxoethyl)carbamate A24, 2 g, 12.6 mmol, 1 eq
  • methyl 2-amino-3-phenyl-propanoate (3.25 g, 15.1 mmol, 1.2 eq, HCl) in MeOH (20 mL)
  • HOAc (1.06 g, 17.6 mmol, 1.01 mL, 1.4 eq
  • NaBH3CN (1.18 g, 18.9 mmol, 1.5 eq
  • Step 2 methyl N-((benzyloxy)carbonyl)-N-(2-((tert-butoxycarbonyl)amino)ethyl)phenylalaninate
  • DCM dimethyl sulfoxide
  • CbzCl 397 mg, 2.33 mmol, 330.7 uL, 1.5 eq
  • DIEA 802 mg, 6.2 mmol, 1.08 mL, 4 eq
  • Step 3 methyl N-(2-aminoethyl)-N-((benzyloxy)carbonyl)phenylalaninate
  • Step 4 benzyl 2-benzyl-3-oxopiperazine-1-carboxylate To a mixture of methyl 2-[2-aminoethyl(benzyloxycarbonyl)amino]-3-phenyl-propanoate (A28, 0.38 g, 1.07 mmol, 1 eq) in DMF (4 mL), was added Cs 2 CO 3 (695 mg, 2.13 mmol, 2 eq). The mixture was stirred at 80 °C for 2 h. Upon completion, water (30 mL) was added, and the aqueous phase was extracted with ethyl acetate (10 mL x3).
  • Step 5 benzy To a solution of 2-bromoimidazo[1,2-a]pyridine (A30, 0.14 g, 710.5 ⁇ mol, 1 eq) and benzyl 2-benzyl-3-oxo-piperazine-1-carboxylate (253.5 mg, 781.6 ⁇ mol, 1.1 eq) in dioxane (3 mL), was added MEDA (15.7 mg, 177.6 ⁇ mol, 19.1 uL, 0.25 eq), CuI (20.3 mg, 106.6 ⁇ mol, 0.15 eq) and K2CO3 (295 mg, 2.13 mmol, 3 eq) under N2. The mixture was stirred at 100 °C for 16 h.
  • Example 10 Synthesis of Compound 110 Step 1: tert-butyl 3-oxo-4-([1,2,4]triazolo[4,3-a]pyridin-3-yl)piperazine-1-carboxylate A mixture of 3-bromo-[1,2,4]triazolo[4,3-a]pyridine (A31, 500 mg, 2.5 mmol, 1 eq), tert- butyl 3-oxopiperazine-1-carboxylate (A2, 607 mg, 3.0 mmol, 1.2 eq), Cs2CO3 (1.65 g, 5.0 mmol, 2 eq), iodocopper;tetrabutylammonium;diiodide (56.5 mg, 50.5 ⁇ mol, 0.02 eq) and 3,4,7,8- tetramethyl-1,10-phenanthroline (11.9 mg, 50.5 ⁇ mol, 0.02 eq) in dioxane (5 mL) was degassed and purged
  • Example 11 Synthesis of Compound 113 Step 1: tert-butyl 4-imidazo[1,2-a]pyridin-7-yl-3-oxo-piperazine-1-carboxylate A mixture of 7-bromoimidazo[1,2-a]pyridine (A40, 300 mg, 1.52 mmol, 1 eq), tert-butyl 3-oxopiperazine-1-carboxylate (305 mg, 1.52 mmol, 1 eq), CuI (43.5 mg, 228 ⁇ mol, 0.15 eq), DMEDA (33.6 mg, 381 ⁇ mol, 41 uL, 0.25 eq) and K 2 CO 3 (421 mg, 3.05 mmol, 2 eq) in dioxane (3 mL) was degassed and purged with N2 (x3), and then the mixture was stirred at 100 °C for 12 h under N2 atmosphere.
  • 7-bromoimidazo[1,2-a]pyridine A40, 300 mg, 1.52 mmol
  • Step 2 1-imidazo[1,2-a]pyridin-7-ylpiperazin-2-one
  • a solution of tert-butyl 4-imidazo[1,2-a]pyridin-7-yl-3-oxo-piperazine-1-carboxylate (A41, 150 mg, 474 ⁇ mol, 1 eq) in DCM (5 mL) was added TFA (1.54 g, 13.5 mmol, 1 mL, 28.5 eq) at 20 °C and stirred at 20 °C for 0.5 h.
  • the reaction mixture was concentrated under reduced pressure to give A42 (1-imidazo[1,2-a]pyridin-7-ylpiperazin-2-one, 156 mg, crude, TFA) as an oil.
  • Step 3 1-imidazo[1,2-a]pyridin-7-yl-4-prop-2-enoyl-piperazin-2-one
  • TEA 143.4 mg, 1.42 mmol, 197.2 uL, 3 eq
  • prop-2- enoyl chloride 47 mg, 519.6 ⁇ mol, 42.4 uL, 1.1 eq
  • Example 12 Synthesis of Compound 114 Step 1: tert-butyl 3-oxo-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)piperazine-1-carboxylate A mixture of 6-bromo-[1,2,4]triazolo[1,5-a]pyridine (A43, 500 mg, 2.52 mmol, 1 eq), tert- butyl 3-oxopiperazine-1-carboxylate (A2, 505.6 mg, 2.52 mmol, 1 eq), CuI (72 mg, 379 ⁇ mol, 0.15 eq), DMEDA (55.7 mg, 631.3 ⁇ mol, 67.9 uL, 0.25 eq) and K2CO3 (697.9 mg, 5.05 mmol, 2 eq) in dioxane (7 mL) was degassed and purged with N 2 (x3), and then the mixture was stirred at 100 °C for 16 h under N 2 atmosphere.
  • Step 2 1-([1,2,4]triazolo[1,5-a]pyridin-6-yl)piperazin-2-one
  • a solution of tert-butyl 3-oxo-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)piperazine-1- carboxylate (A44, 350 mg, 1.10 mmol, 1 eq) in DCM (4 mL) was added TFA (1.54 g, 13.51 mmol, 1 mL, 12.25 eq). The mixture was stirred at 20 °C for 1 h.
  • Step 3 4-prop-2-enoyl-1-([1,2,4]triazolo[1,5-a]pyridin-7-yl)piperazin-2-one
  • TEA 223.6 mg, 2.21 mmol, 307.6 uL, 4 eq
  • prop- 2-enoyl chloride 60 mg, 662.9 ⁇ mol, 54 uL, 1.2 eq.
  • Example 13 Synthesis of Compound 115 Step 1: tert-butyl 4-(1,3-benzothiazol-2-yl)-3-oxo-piperazine-1-carboxylate A mixture of 2-bromo-1,3-benzothiazole (A45, 500 mg, 2.3 mmol, 1 eq), tert-butyl 3- oxopiperazine-1-carboxylate (468 mg, 2.3 mmol, 1 eq), CuI (66.7 mg, 350.3 ⁇ mol, 0.15 eq), DMEDA (51.5 mg, 584 ⁇ mol, 63 uL, 0.25 eq) and K 2 CO 3 (645.6 mg, 4.7 mmol, 2 eq) in dioxane (8 mL) was degassed and purged with N2 (x3), and then the mixture was stirred at 100 °C for 12 hr under N2 atmosphere.
  • 2-bromo-1,3-benzothiazole A45, 500 mg, 2.3
  • reaction mixture was quenched by water (12 mL), and then extracted with EtOAc (20 mL x3). The combined organic layers were washed with brine (20 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (25-33% EtOAc in PE) to give tert-butyl 4- (1,3-benzothiazol-2-yl)-3-oxo-piperazine-1-carboxylate (A47, 350 mg, 892 umol) as a solid.
  • Step 2 1-(1,3-benzothiazol-2-yl)piperazin-2-one
  • tert-butyl 4-(1,3-benzothiazol-2-yl)-3-oxo-piperazine-1-carboxylate A47, 350 mg, 1.05 mmol, 1 eq
  • TFA 1.54 g, 13.5 mmol, 1 mL, 12.9 eq
  • Example 14 Synthesis of Compound 117 Step 1: tert-butyl 4-imidazo[1, 2-a]pyrimidin-6-yl-3-oxo-piperazine-1-carboxylate A mixture of 6-bromoimidazo[1, 2-a]pyrimidine (A52, 1 g, 5.05 mmol, 1 eq), tert-butyl 3- oxopiperazine-1-carboxylate (1.01 g, 5.05 mmol, 1 eq), CuI (384.71 mg, 2.02 mmol, 0.4 eq), N1, N2-dimethylcyclohexane-1, 2-diamine (574.7 mg, 4.04 mmol, 0.8 eq) and K 2 CO 3 (1.4 g, 10.1 mmol, 2 eq) in toluene (10 mL) was degassed and purged with N2 (x3), and then the mixture was stirred at 120 °C for 16 h under N2 atmosphere.
  • Step 2 1-imidazo[1,2-a]pyrimidin-6-ylpiperazin-2-one
  • a solution of tert-butyl 4-imidazo[1,2-a]pyrimidin-6-yl-3-oxo-piperazine-1-carboxylate (A53, 100 mg, 315 ⁇ mol, 1 eq) in HCl/dioxane (4 M, 3 mL, 38.1 eq) was stirred at 25 °C for 0.5 h.
  • the reaction mixture was concentrated under reduced pressure to give 1- imidazo[1,2-a]pyrimidin-6-ylpiperazin-2-one (A54, 65 mg, crude) as a solid.
  • Step 3 1-imidazo[1, 2-a]pyrimidin-6-yl-4-prop-2-enoyl-piperazin-2-one
  • Example 15 Synthesis of Compound 118 Step 1: benzyl 4-[1-(1-tert-butoxycarbonyl-4-piperidyl)pyrazol-4-yl]-3-oxo-piperazine-1- carboxylate
  • CuI (43.3 mg, 227.1 ⁇ mol, 0.15 eq)
  • DMEDA (33.4 mg, 378.5 ⁇ mol, 40.7 uL, 0.25 eq)
  • K2CO3 48.5 mg, 3.03 mmol, 2 eq) in dioxane (7 mL) was degassed and purged with N 2 (x3), and then the mixture was stirred at 100 °C for 16
  • Step 2 tert-butyl 4-[4-(2-oxopiperazin-1-yl)pyrazol-1-yl]piperidine-1-carboxylate
  • benzyl 4-[1-(1-tert-butoxycarbonyl-4-piperidyl)pyrazol-4-yl]-3-oxo- piperazine-1-carboxylate (A56, 200 mg, 413.6 ⁇ mol, 1 eq) in 75-65-0 (10 mL) was added Pd/C (250 mg), and the mixture was stirred at 20 °C for 1 h.
  • Step 3 tert-butyl 4-[4-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)pyrazol-1-yl]piperidine-1- carboxylate
  • TEA 131.8 mg, 1.3 mmol, 181 uL, 3.5 eq
  • prop-2-enoyl chloride 40.4 mg, 446.5 ⁇ mol, 36.4 uL, 1.2 eq
  • Example 16 Synthesis of Compound 119 Step 1: 2-[(5-bromopyrrolo[2,3-d]pyrimidin-7-yl)methoxy]ethyl-trimethyl-silane To a solution of 5-bromo-7H-pyrrolo[2,3-d]pyrimidine (A58, 5 g, 25.3 mmol, 1 eq) in THF (25 mL) was added dropwise NaH (1.11 g, 27.8 mmol, 60% purity, 1.1 eq) at 0 °C.
  • Step 2 tert-butyl 3-oxo-4-[7-(2-trimethylsilylethoxymethyl)pyrrolo[2,3-d]pyrimidin-5- yl]piperazine-1-carboxylate
  • tert-butyl 3-oxopiperazine-1-carboxylate (2.01 g, 10 mmol, 1 eq)
  • CuI (766 mg, 4 mmol, 0.4 eq
  • DMEDA (709 mg, 8 mmol, 866 uL, 0.8 eq
  • K 2 CO 3 (2.78 g, 20 mmol, 2 eq) in dioxane (35 mL) was degassed and purged with N2 (x3), and then the mixture was stirred at
  • Step 3 tert-butyl 3-oxo-4-(7H-pyrrolo[2,3-d]pyrimidin-5-yl)piperazine-1-carboxylate
  • a solution of tert-butyl 3-oxo-4-[7-(2-trimethylsilylethoxymethyl)pyrrolo[2,3- d]pyrimidin-5-yl]piperazine-1-carboxylate (A60, 400 mg, 894 ⁇ mol, 1 eq) in THF (5 mL) was added TBAF (1 M, 5 mL, 5.60 eq), and the mixture was stirred at 60 °C for 16 h.
  • Step 4 1-(7H-pyrrolo[2,3-d]pyrimidin-5-yl)piperazin-2-one
  • a solution of tert-butyl 3-oxo-4-(7H-pyrrolo[2,3-d]pyrimidin-5-yl)piperazine-1- carboxylate (A61, 160 mg, 504 ⁇ mol, 1 eq) was added HCl/dioxane (4 M, 4.7 mL, 37.3 eq). The mixture was stirred at 25 °C for 1 h.
  • Example 17 Synthesis of Compound 120 Step 1: tert-butyl 4-(5-methylpyrazolo[1,5-a]pyrimidin-3-yl)-3-oxo-piperazine-1-carboxylate A mixture of 3-bromo-5-methyl-pyrazolo[1,5-a]pyrimidine (A63, 400 mg, 1.89 mmol, 1 eq), tert-butyl 3-oxopiperazine-1-carboxylate (377.7 mg, 1.89 mmol, 1 eq), iodocopper (53.9 mg, 283 ⁇ mol, 0.15 eq), N,N'-dimethylethane-1,2-diamine (41.6 mg, 471.6 ⁇ mol, 50.8 uL, 0.25 eq) and dipotassium;carbonate (521.4 mg, 3.8 mmol, 2 eq) in dioxane (6 mL) was degassed and purged with N2 (x3), and then the mixture was stirred at
  • reaction mixture was diluted with H 2 O 10 mL and extracted with EtOAc (5 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (5-100% EtOAc in PE) to give A64 (tert-butyl 4-(5-methylpyrazolo[1,5-a]pyrimidin-3-yl)-3-oxo-piperazine-1- carboxylate (400 mg, 1.21 mmol, 64% yield) as a solid.
  • Step 2 1-(5-methylpyrazolo[1,5-a]pyrimidin-3-yl)piperazin-2-one
  • a solution of tert-butyl 4-(5-methylpyrazolo[1,5-a]pyrimidin-3-yl)-3-oxo-piperazine-1- carboxylate (A64, 200 mg, 603.56 ⁇ mol, 1 eq) in DCM (2 mL) was added TFA (770 mg, 6.75 mmol, 0.5 mL, 11.19 eq). The mixture was stirred at 25 °C for 1 h.
  • Step 3 1-(5-methylpyrazolo[1,5-a]pyrimidin-3-yl)-4-prop-2-enoyl-piperazin-2-one
  • a solution of 1-(5-methylpyrazolo[1,5-a]pyrimidin-3-yl)piperazin-2-one (139 mg, 601.07 ⁇ mol, 1 eq) in DCM (2 mL) was cooled to 0 °C then was added TEA (304.11 mg, 3.01 mmol, 418.31 uL, 5 eq) and prop-2-enoyl chloride (48.96 mg, 540.96 ⁇ mol, 44.11 uL, 0.9 eq).
  • Example 18 Synthesis of Compound 121 Step 1: tert-butyl 4-(5-methylthiazol-2-yl)-3-oxo-piperazine-1-carboxylate A mixture of 2-bromo-5-methyl-thiazole (A66, 500 mg, 2.8 mmol, 1 eq), tert-butyl 3- oxopiperazine-1-carboxylate (562.3 mg, 2.8 mmol, 1 eq), CuI (80.2 mg, 421.2 ⁇ mol, 0.15 eq), DMEDA (61.9 mg, 702 ⁇ mol, 75.6 uL, 0.25 eq) and K2CO3 (776.2 mg, 5.6 mmol, 2 eq) in dioxane (7 mL) was degassed and purged with N 2 (x3), and then the mixture was stirred at 100 °C for 16 h under N 2 atmosphere.
  • 2-bromo-5-methyl-thiazole A66, 500 mg, 2.8 mmol, 1
  • Step 2 1-(5-methylthiazol-2-yl)piperazin-2-one
  • a solution of tert-butyl 4-(5-methylthiazol-2-yl)-3-oxo-piperazine-1-carboxylate (A67, 100 mg, 336.3 ⁇ mol, 1 eq) in DCM (2 mL) was added TFA (550 mg, 4.82 mmol, 357.14 uL, 14.34 eq). The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue that contained A68 (1-(5-methylthiazol-2-yl) piperazin-2-one (66 mg, crude)) as an oil.
  • Step 3 1-(5-methylthiazol-2-yl)-4-prop-2-enoyl-piperazin-2-one
  • TEA 1-(5-methylthiazol-2-yl) piperazin-2-one
  • TEA prop-2-enoyl chloride
  • reaction mixture was diluted with aq. EDTA 10 mL and extracted with EtOAc (10 mL x2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (10% EtOAc in PE) to give A73 (tert-butyl 4-(8-methylimidazo[1,2-a]pyridin-2-yl)-3-oxo-piperazine-1- carboxylate (70 mg, 212 umol)) as a solid.
  • Step 2 1-(8-methylimidazo[1,2-a]pyridin-2-yl)piperazin-2-one
  • a solution of tert-butyl 4-(8-methylimidazo[1,2-a]pyridin-2-yl)-3-oxo-piperazine-1- carboxylate (A73, 70 mg, 212 ⁇ mol, 1 eq) in DCM (2 mL) was added TFA (616 mg, 5.4 mmol, 0.4 mL, 25.5 eq). The mixture was stirred at 25 °C for 1 h.
  • Step 3 1-(8-methylimidazo[1,2-a]pyridin-2-yl)-4-prop-2-enoyl-piperazin-2-one
  • TEA TEA
  • TEA prop-2-enoyl chloride
  • Example 20 Synthesis of Compound 125 Step 1: tert-butyl 4-imidazo[1,2-b]pyridazin-3-yl-3-oxo-piperazine-1-carboxylate To a solution of 3-bromoimidazo[1,2-b]pyridazine (A78, 1 g, 5 mmol, 1 eq) and tert-butyl 3-oxopiperazine-1-carboxylate (1.01 g, 5 mmol, 1 eq) in dioxane (10 mL) was added iodocopper (385 mg, 2 mmol, 0.4 eq), N,N'-dimethylethane-1,2-diamine (356.1 mg, 4 mmol, 435 uL, 0.8 eq) and dipotassium;carbonate (1.4 g, 10 mmol, 2 eq).
  • Step 3 1-imidazo[1,2-b]pyridazin-3-yl-4-prop-2-enoyl-piperazin-2-one
  • A80 180 mg, 829 ⁇ mol, 1 eq
  • prop-2-enoyl chloride 113 mg, 1.24 mmol, 101.4 uL, 1.5 eq
  • TEA 419 mg, 4.14 mmol, 576.7 uL, 5 eq
  • the mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to to give a residue.
  • Example 21 Synthesis of Compound 126 Step 1: tert-butyl 4-imidazo[1,2-a]pyrimidin-3-yl-3-oxo-piperazine-1-carboxylate To a solution of 3-bromoimidazo[1,2-a]pyrimidine (A81, 500 mg, 2.52 mmol, 1 eq), tert- butyl 3-oxopiperazine-1-carboxylate (505.59 mg, 2.52 mmol, 1 eq), DMEDA (178.07 mg, 2.02 mmol, 217.42 uL, 0.8 eq) and K 2 CO 3 (697.94 mg, 5.05 mmol, 2 eq) in dioxane (5 mL) was added CuI (192.35 mg, 1.01 mmol, 0.4 eq) under N2.
  • 3-bromoimidazo[1,2-a]pyrimidine A81, 500 mg, 2.52 mmol, 1 eq
  • Step 2 1-imidazo[1,2-a]pyrimidin-3-ylpiperazin-2-one
  • a mixture of tert-butyl 4-imidazo[1,2-a]pyrimidin-3-yl-3-oxo-piperazine-1-carboxylate (A82, 200 mg, 630.2 ⁇ mol, 1 eq) in HCl/dioxane (4 M, 30 mL, 190.4 eq) was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The crude was used to next step without further purification.
  • Example 22 Synthesis of Compound 127 Step 1: tert-butyl 2-(4-benzyloxycarbonyl-2-oxo-piperazin-1-yl)-6,7-dihydro-4H-thiazolo[4,5- c]pyridine-5-carboxylate A mixture of tert-butyl 2-bromo-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-5-carboxylate (400 mg, 1.25 mmol, 1 eq), benzyl 3-oxopiperazine-1-carboxylate (293.53 mg, 1.25 mmol, 1 eq), CuI (35.8 mg, 188 ⁇ mol, 0.15 eq), DMEDA (27.6 mg, 313.3 ⁇ mol, 33.7 uL, 0.25 eq) and K2CO3 (346.4 mg, 2.5 mmol, 2 eq) in dioxane (6 mL) was degassed and purged with N 2 (
  • Step 2 tert-butyl 2-(2-oxopiperazin-1-yl)-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-5-carboxylate
  • tert-butyl 2-(4-benzyloxycarbonyl-2-oxo-piperazin-1-yl)-6,7-dihydro-4H- thiazolo[4,5-c]pyridine-5-carboxylate 150 mg, 317.42 ⁇ mol, 1 eq
  • Pd/C 200 mg, 10% purity
  • Step 3 tert-butyl 2-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)-6,7-dihydro-4H-thiazolo[4,5- c]pyridine-5-carboxylate
  • a solution of tert-butyl 2-(2-oxopiperazin-1-yl)-6,7-dihydro-4H-thiazolo[4,5- c]pyridine-5-carboxylate (84 mg, 248.21 ⁇ mol, 1 eq) in DCM (2 mL) was added TEA (125.58 mg, 1.24 mmol, 172.74 uL, 5 eq) and prop-2-enoyl chloride (22.46 mg, 248.21 ⁇ mol, 20.24 uL, 1 eq) in DCM (0.25 mL). The mixture was stirred at 0 °C for 1 h.
  • Example 23 Synthesis of Compound 129 Step 1: 2-[(2-bromopyrrolo[2,3-b]pyridin-1-yl)methoxy]ethyl-trimethyl-silane A solution of 2-bromo-1H-pyrrolo[2,3-b]pyridine (2 g, 10.15 mmol, 1 eq) in THF (30 mL) was added NaH (609.04 mg, 15.23 mmol, 60% purity, 1.5 eq) stirred at 0 °C for 0.5 h, then added SEM-Cl (2.54 g, 15.23 mmol, 2.69 mL, 1.5 eq) was drop-wise, the mixture was stirred at 25 °C for 1 h.
  • Step 2 tert-butyl 3-oxo-4-[1-(2-trimethylsilylethoxymethyl)pyrrolo[2,3-b]pyridin-2- yl]piperazine-1-carboxylate
  • tert-butyl 3-oxopiperazine-1-carboxylate (1.54 g, 7.70 mmol, 1.2 eq) in dioxane (30 mL) was added K 2 CO 3 (1.77 g, 12.83 mmol, 2 eq), CuI (488.79 mg, 2.57 mmol, 0.4 eq) and N,N'-dimethylethane-1,2-diamine (452.48 mg, 5.13 mmol, 552.48 uL, 0.8 eq) under N 2 ,
  • Step 3 1-(1H-pyrrolo[2,3-b]pyridin-2-yl)piperazin-2-one
  • Step 4 4-prop-2-enoyl-1-(1H-pyrrolo[2,3-b]pyridin-2-yl)piperazin-2-one
  • TEA 2.3 mg, 924.9 ⁇ mol, 128.7 uL, 2 eq
  • 1-(1H-pyrrolo[2,3-b]pyridin-2- yl)piperazin-2-one 100 mg, 462.5 ⁇ mol, 1 eq
  • DCM 0.2 mL
  • Example 24 Synthesis of Compound 128 Step 1: tert-butyl 3-oxo-4-([1,2,4]triazolo[1,5-a]pyridin-2-yl)piperazine-1-carboxylate
  • 2-bromo-[1,2,4]triazolo[1,5-a]pyridine 90 mg, 454.50 ⁇ mol, 1 eq
  • tert-butyl 3-oxopiperazine-1-carboxylate 91.01 mg, 454.50 ⁇ mol, 1 eq
  • dioxane 3 mL
  • Pd 2 (dba) 3 41.62 mg, 45.45 ⁇ mol, 0.1 eq
  • Xantphos 52.60 mg, 90.90 ⁇ mol, 0.2 eq
  • Step 2 1-([1,2,4]triazolo[1,5-a]pyridin-2-yl)piperazin-2-one
  • a mixture of tert-butyl 3-oxo-4-([1,2,4]triazolo[1,5-a]pyridin-2-yl)piperazine-1- carboxylate (100 mg, 315 ⁇ mol, 1 eq) in DCM (1.5 mL) was added drop-wise TFA (462 mg, 4.05 mmol, 0.3 mL, 12.86 eq), the mixture was stirred at 25 °C for 1 h.
  • Step 3 4-prop-2-enoyl-1-([1,2,4]triazolo[1,5-a]pyridin-2-yl)piperazin-2-one
  • 1-([1,2,4]triazolo[1,5-a]pyridin-2-yl)piperazin-2-one 65 mg, 299.2 ⁇ mol, 1 eq) in THF (1 mL) and H 2 O (1 mL) was added K 2 CO 3 (91 mg, 658 ⁇ mol, 2.2 eq) at 0 °C
  • prop-2-enoyl chloride (27.1 mg, 299.2 ⁇ mol, 24.4 uL, 1 eq) in THF (0.5 mL) was added drop- wise, the mixture was stirred at 0 °C for 0.5 h.
  • Example 25 Synthesis of Compound 133 A solution of (2R)-2-amino-4-phenyl-butanoic acid (5 g, 27.9 mmol, 1 eq) in HCl/MeOH (4 M, 50 mL, 7.17 eq) was stirred at 25 °C for 4 h. Upon completion, the mixture was diluted with 200 mL EtOAc, washed with NaHCO 3 , water, brine, then dried over Na 2 SO 4 . To give methyl (2R)- 2-amino-4-phenyl-butanoate (5.28 g, crude) as an oil. Step 2: methyl (2S)-2-[2-(tert-butoxycarbonylamino)ethylamino]-4-phenyl-butanoate
  • Step 3 methyl (2R)-2-[benzyloxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-4-phenyl- butanoate
  • methyl (2S)-2-[2-(tert-butoxycarbonylamino)ethylamino]-4-phenyl- butanoate (1.45 g, 4.31 mmol, 1 eq) in DCM (15 mL) was added CbzCl (1.10 g, 6.47 mmol, 919 uL, 1.5 eq) and DIEA (2.23 g, 17.2 mmol, 3 mL, 4 eq) at 0 °C, the mxture was stirred at 25 °C for 2 h.
  • Step 6 benzyl (2R)-4-imidazo[1,2-a]pyridin-2-yl-3-oxo-2-(2-phenylethyl)piperazine-1- carboxylate
  • 2-bromoimidazo[1,2-a]pyridine 698.7 mg, 3.55 mmol, 1 eq
  • benzyl (2R)-3- oxo-2-(2-phenylethyl)piperazine-1-carboxylate 1.2 g, 3.55 mmol, 1 eq
  • CuI (101.3 mg, 532 ⁇ mol, 0.15 eq)
  • DMEDA 78.2 mg, 886.5 ⁇ mol, 95.4 uL, 0.25 eq
  • K2CO3 980.2 mg, 7.1 mmol, 2 eq) in dioxane (12 mL) was degassed and purged with N 2 (x3), and then the mixture was stirred at 100 °C for 12 h under N2 atmosphere.
  • Step 8 (3R)-1-imidazo[1,2-a]pyridin-2-yl-3-(2-phenylethyl)-4-prop-2-enoyl-piperazin-2-one
  • TEA 94.75 mg, 936.37 ⁇ mol, 130.33 uL, 3 eq
  • prop-2-enoyl chloride 33.90 mg, 374.55 ⁇ mol, 30.54 uL, 1.2 eq
  • Step 3 methyl 2-[benzyloxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-5-phenyl- pentanoate
  • DCM methyl 2-[2-(tert-butoxycarbonylamino)ethylamino]-5-phenyl- pentanoate (1.3 g, 3.71 mmol, 1 eq) in DCM (30 mL) was added DIEA (1.92 g, 14.84 mmol, 2.58 mL, 4 eq) and CbzCl (949.22 mg, 5.56 mmol, 791.02 uL, 1.5 eq) at 0 °C under N2.
  • Step 5 benzyl 3-oxo-2-(3-phenylpropyl)piperazine-1-carboxylate
  • DMF methyl 2-[2-aminoethyl(benzyloxycarbonyl)amino]-5-phenyl-pentanoate
  • Cs 2 CO 3 2.75 g, 8.45 mmol, 2.5 eq
  • the reaction mixture was diluted with H 2 O 200 mL and extracted with DCM (100 mL * 3).
  • Example 27 Synthesis of Compound 135 Step1 : tert-butyl 3-oxo-2-(4-phenylbutyl)piperazine-1-carboxylate To a solution of tert-butyl 3-oxopiperazine-1-carboxylate (1 g, 4.99 mmol, 1 eq) in THF (10 mL) was added LDA (2.5 M, 4 mL, 2 eq). The mixture was stirred at -70 °C for 30 min, then a solution of (4-bromobutyl) benzene (745 mg, 3.5 mmol, 0.7 eq) in THF (2 mL) was added to the mixture. The mixture was stirred for 1 h.
  • Step2 tert-butyl 4-imidazo[1 To a solution of tert-butyl 3-oxo-2-(4-phenylbutyl)piperazine-1-carboxylate (350 mg, 1.05 mmol, 1 eq) and 2-bromoimidazo[1,2-a]pyridine (207.4 mg, 1.05 mmol, 1 eq) in dioxane (5 mL) was added K2CO3 (291 mg, 2.11 mmol, 2 eq), DMEDA (23.2 mg, 263.2 ⁇ mol, 28.3 uL, 0.25 eq), then exchanged N 2 (x3), then added CuI (30.1 mg, 157.9 ⁇ mol, 0.15 eq) and exchange N 2 (x3), The mixture was stirred at 100 °C for 12 h.
  • reaction mixture was diluted with H2O (10 mL) and extracted with DCM (10 mL * 3). The combined organic layers were washed with sat. NaCl (15 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (0-50% EtOAc in PE) to give tert-butyl 4-imidazo[1,2-a]pyridin-2-yl-3-oxo-2-(4-phenylbutyl)piperazine-1- carboxylate (210 mg, 468 ⁇ mol, 45% yield) as a solid.
  • Example 28 Synthesis of Compound 139 Step 1: methyl (R)-2-amino-3-(4-fluorophenyl)propanoate To a solution of (2R)-2-amino-3-(4-fluorophenyl)propanoic acid (5 g, 27.3 mmol, 1 eq) in MeOH (60 mL) was added SOCl 2 (13 g, 109 mmol, 7.9 mL, 4 eq) at 0 °C. And then, the reaction mixture was stirred at 60 °C for 2 h.
  • Step 3 methyl (2R)-2-[benzyloxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-3-(4- fluorophenyl)propanoate
  • DCM 50 mL
  • DIEA 7.59 g, 58.8 mmol, 10.2 mL, 4 eq
  • CbzCl 3.8 g, 22 mmol, 3.13 mL, 1.5 eq
  • Step 4 methyl (2R)-2-[2-aminoethyl(benzyloxycarbonyl)amino]-3-(4-fluorophenyl)propanoate
  • a solution of methyl (2R)-2-[benzyloxycarbonyl-[2-(tert-butoxycarbonylamino)- ethyl]amino]-3-(4-fluorophenyl)propanoate (5 g, 10.5 mmol, 1 eq) in HCl/dioxane (4 M, 50 mL, 19 eq) was stirred at 25 °C for 1 h.
  • Step 5 benzyl (2R)-2-[(4-fluorophenyl)methyl]-3-oxo-piperazine-1-carboxylate
  • methyl (2R)-2-[2-aminoethyl(benzyloxycarbonyl)amino]-3-(4- fluorophenyl)propanoate 4.8 g, 11.7 mmol, 1 eq, HCl
  • DBU 1,3-dioethyl
  • Step 6 benzyl (2R)-2-[(4-fluorophenyl)methyl]-4-imidazo[1,2-a]pyridin-2-yl-3-oxo-piperazine- 1-carboxylate
  • benzyl (2R)-2-[(4-fluorophenyl)methyl]-3-oxo-piperazine-1-carboxylate 1.2 g, 3.5 mmol, 1 eq
  • 2-bromoimidazo[1,2-a]pyridine 828.7 mg, 4.2 mmol, 1.2 eq
  • dioxane 15 mL
  • dipotassium;carbonate 968.84 mg, 7.01 mmol, 2 eq
  • Step 8 (3R)-3-[(4-fluorophenyl)methyl]-1-imidazo[1,2-a]pyridin-2-yl-4-prop-2-enoyl-piperazin- 2-one
  • TEA 262 mg, 2.6 mmol, 360.5 uL, 3 eq
  • prop-2-enoyl chloride (78.1 mg, 863.3 ⁇ mol, 70.4 uL, 1 eq) in DCM (1 mL) was added at 0 °C.
  • Step 9 3-[(4-fluorophenyl)methyl]-1-imidazo[1,2-a]pyridin-2-yl-4-prop-2-enoyl-piperazin-2- one (3R)-3-[(4-fluorophenyl)methyl]-1-imidazo[1,2-a]pyridin-2-yl-4-prop-2-enoyl-piperazin- 2-one (Compound 139-(R), 10 mg, 26.4 ⁇ mol, 1 eq) and (3S)-3-[(4-fluorophenyl)methyl]-1- imidazo[1,2-a]pyridin-2-yl-4-prop-2-enoyl-piperazin-2-one (Compound 139-(S), 10 mg, 26.4 ⁇ mol, 1 eq) was dissolved in MeCN (0.5 mL) and H2O (2 mL), The mixture was stirred at 25 °C for 5 min.
  • Example 29 Synthesis of Compound 144 Step 1: methyl (2R)-2-amino-3-(4-nitrophenyl)propanoate A solution of (2R)-2-amino-3-(4-nitrophenyl)propanoic acid (10 g, 47.6 mmol, 1 eq) in MeOH (100 mL) was added SOCl2 (22.6 g, 190.3 mmol, 13.8 mL, 4 eq). The mixture was stirred at 80 °C for 2 hr. Upon completion, the reaction mixture was poured into H2O 300 mL at 20 °C, and then extracted with EtOAc (200 mL * 3).
  • Step 3 methyl (2R)-2-[benzyloxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-3-(4- nitrophenyl)propanoate
  • DCM DCM
  • DIEA 7.74 g, 59.88 mmol, 10.43 mL, 4 eq
  • CbzCl 3.8 g, 22.5 mmol, 3.2 mL, 1.5 eq
  • Step 4 methyl (2R)-2-[2-aminoethyl(benzyloxycarbonyl)amino]-3-(4-nitrophenyl)propanoate
  • methyl (2R)-2-[benzyloxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]- amino]-3-(4-nitrophenyl)propanoate (6.3 g, 12.6 mmol, 1 eq) in HCl/dioxane (4 M, 60 mL) was stirred at 25 °C for 1 hr.
  • Step 5 benzyl (2R)-2-[(4-nitrophenyl)methyl]-3-oxo-piperazine-1-carboxylate
  • a solution of methyl (2R)-2-[2-aminoethyl(benzyloxycarbonyl)amino]-3-(4- nitrophenyl)propanoate (4.6 g, 11.5 mmol, 1 eq) in DMF (60 mL) was added Cs 2 CO 3 (7.47 g, 22.9 mmol, 2 eq). The mixture was stirred at 80 °C for 2 hr. Upon completion, the reaction mixture was quenched by addition H2O 100 mL at 25 °C, and extracted with EtOAc (50 mL * 3).
  • Step 6 benzyl (2R)-4-imidazo[1,2-a]pyridin-2-yl-2-[(4-nitrophenyl)methyl]-3-oxo-piperazine-1- carboxylate
  • 2-bromoimidazo[1,2-a]pyridine (1.25 g, 6.4 mmol, 1.2 eq)
  • K 2 CO 3 (1.47 g, 10.6 mmol, 2 eq)
  • CuI 404.2 mg, 2.12 mmol, 0.4 eq
  • DMEDA 374.2 mg, 4.25 mmol, 456.9 uL, 0.8 eq
  • Step 8 benzyl (2R)-2-[(4-acetamidophenyl)methyl]-4-imidazo[1,2-a]pyridin-2-yl-3-oxo- piperazine-1-carboxylate
  • a solution of benzyl (2R)-2-[(4-aminophenyl)methyl]-4-imidazo[1,2-a]pyridin-2-yl-3- oxo-piperazine-1-carboxylate (500 mg, 1.10 mmol, 1 eq) in DCM (5 mL) was added Py (260.48 mg, 3.29 mmol, 265.79 uL, 3 eq) then Ac2O (123.27 mg, 1.21 mmol, 113.09 uL, 1.1 eq) was added, and was stirred at 20°C for 1 h.
  • Step 9 N-[4-[[(2R)-4-imidazo[1,2-a]pyridin-2-yl-3-oxo-piperazin-2- yl]methyl]phenyl]acetamide
  • Pd/C 660 mg, 1.33 mmol, 10% purity, 1 eq
  • EtOH 5 mL
  • Step 10 N-[4-[[(2R)-4-imidazo[1,2-a]pyridin-2-yl-3-oxo-1-prop-2-enoyl-piperazin-2- yl]methyl]phenyl]acetamide
  • TEA 8.48 mg, 7.98 mmol, 1.11 mL, 5 eq
  • prop-2-enoyl chloride 216.67 mg, 2.39 mmol, 194.50 ⁇ L, 1.5 eq
  • Step 11 N-[4-[(4-imidazo[1,2-a]pyridin-2-yl-3-oxo-1-prop-2-enoyl-piperazin-2- yl)methyl]phenyl]acetamide N-[4-[[(2R)-4-imidazo[1,2-a]pyridin-2-yl-3-oxo-1-prop-2-enoyl-piperazin-2-yl]methyl]- phenyl]acetamide (Compound 152, 6 mg, 14.4 ⁇ mol, 1 eq) and N-[4-[[[(2S)-4-imidazo[1,2- a]pyridin-2-yl-3-oxo-1-prop-2-enoyl-piperazin-2-yl]methyl]phenyl]acetamide (Compound 151, 6 mg, 14.37 ⁇ mol, 1 eq) was dissolved in MeCN 0.5 mL and H2O 2 mL
  • Example 30 Synthesis of Compound 150 Step 1: methyl (2R)-2-[2-(tert-butoxycarbonylamino)ethylamino]-3-(1H-indol-3-yl)propanoate
  • tert-butyl N-(2-oxoethyl)carbamate 1 g, 6.3 mmol, 1 eq
  • methyl (2R)- 2-amino-3-(1H-indol-3-yl)propanoate 1.9 g, 7.5 mmol, 1.2 eq, HCl
  • NaBH 3 CN 592 mg, 9.42 mmol, 1.5 eq
  • AcOH (528.2 mg, 8.79 mmol, 503 uL, 1.4 eq) at 0 °C under N 2 atmosphere, and then the mixture was stirred at 25 °C for 1 h under N 2 atmosphere.
  • reaction mixture was quenched by addition of sat. aq. NaHCO3 (10 mL) at 0 °C, and then diluted with H2O (20 mL) and extracted with DCM (20 mL x3). The combined organic layers were washed with brine (10 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue.
  • Step 2 methyl (2R)-2-[benzyloxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-3-(1H- indol-3-yl)propanoate
  • methyl (2R)-2-[2-(tert-butoxycarbonylamino)ethylamino]-3-(1H-indol-3- yl)propanoate 1.7 g, 4.70 mmol, 1 eq
  • DCM 20 mL
  • CbzCl (1.20 g, 7 mmol, 1 mL, 1.5 eq
  • DIEA 2.43 g, 18.8 mmol, 3.28 mL, 4 eq
  • Step 3 methyl (2R)-2-[2-aminoethyl(benzyloxycarbonyl)amino]-3-(1H-indol-3-yl)propanoate
  • a solution of methyl (2R)-2-[benzyloxycarbonyl-[2-(tert-butoxycarbonylamino)- ethyl]amino]-3-(1H-indol-3-yl)propanoate (1.54 g, 3.11 mmol, 1 eq) and TFA (11.86 g, 104 mmol, 7.70 mL, 33.5 eq) in DCM (24 mL), the mixture was stirred at 25 °C for 1 h.
  • Step 5 tert-butyl 3-[[(2R)-1-benzyloxycarbonyl-3-oxo-piperazin-2-yl]methyl]indole-1- carboxylate
  • benzyl (2R)-2-(1H-indol-3-ylmethyl)-3-oxo-piperazine-1-carboxylate (1 g, 2.75 mmol, 1 eq) and DMAP (33.6 mg, 275.2 ⁇ mol, 0.1 eq) in DCM (10 mL)
  • tert- butoxycarbonyl tert-butyl carbonate 540.5 mg, 2.48 mmol, 568.9 uL, 0.9 eq
  • Step 6 tert-butyl 3-[[(2R)-1-benzyloxycarbonyl-4-imidazo[1,2-a]pyridin-2-yl-3-oxo-piperazin-2- yl]methyl]indole-1-carboxylate
  • tert-butyl 3-[[(2R)-1-benzyloxycarbonyl-3-oxo-piperazin-2- yl]methyl]indole-1-carboxylate 410 mg, 884.5 ⁇ mol, 1 eq
  • 2-bromoimidazo[1,2-a]pyridine (174.3 mg, 884.5 ⁇ mol, 1 eq)
  • CuI (25.3 mg, 132.7 ⁇ mol, 0.15 eq)
  • Example 31 Synthesis of Compound 154 Step 1: methyl 2-(3-oxopiperazin-2-yl)acetate To a solution of dimethyl maleate (10 g, 69.38 mmol, 8.70 mL, 1 eq) in i-PrOH (100 mL) was added ethane-1, 2-diamine (4.57 g, 76.04 mmol, 5.09 mL, 1.10 eq) at 25 °C. The mixture was stirred at 50 °C for 16 h. Upon completion, the reaction mixture filtered and concentrated under reduced pressure to give a residue.
  • Step.2 tert-butyl 2-(2-methoxy-2-oxoethyl)-3-oxopiperazine-1-carboxylate
  • a solution of methyl 2-(3-oxopiperazin-2-yl)acetate (8.8 g, 51.1 mmol, 1 eq) in DCM (150 mL) was added TEA (7.24 g, 71.5 mmol, 9.96 mL, 1.4 eq) and Boc2O (11.15 g, 51.1 mmol, 11.7 mL, 1 eq).
  • TEA 7.24 g, 71.5 mmol, 9.96 mL, 1.4 eq
  • Boc2O 11.15 g, 51.1 mmol, 11.7 mL, 1 eq
  • Step.3 tert-butyl 4-(imidazo[1,2-a]pyridin-2-yl)-2-(2-methoxy-2-oxoethyl)-3-oxopiperazine-1- carboxylate
  • tert-butyl 2-(2-methoxy-2-oxoethyl)-3-oxopiperazine-1-carboxylate 5 g, 18.4 mmol, 1 eq
  • K 2 CO 3 5.1 g, 36.7 mmol, 2 eq
  • N,N'- dimethylethane-1,2-diamine (1.29 g, 14.7 mmol, 1.58 mL, 0.8 eq).
  • Step.4 methyl 2-(4-(imidazo[1,2-a]pyridin-2-yl)-3-oxopiperazin-2-yl)acetate
  • Step 5 methyl 2-(1-acryloyl-4-(imidazo[1,2-a]pyridin-2-yl)-3-oxopiperazin-2-yl)acetate
  • a solution of methyl 2-(4-(imidazo[1,2-a]pyridin-2-yl)-3-oxopiperazin-2-yl)acetate (371 mg, 1.29 mmol, 1 eq) in DCM (2 mL) was added TEA (390.64 mg, 3.86 mmol, 537.34 uL, 3 eq) at 0 °C, then added a solution of prop-2-enoyl chloride (139.76 mg, 1.54 mmol, 125.91 uL, 1.2 eq) in DCM (0.5 mL) to the mixture and was stirred at 25 °C for 1 h.
  • Example 32 Synthesis of Compound 155 Step 1: 2-(1-tert-butoxycarbonyl-4-imidazo[1,2-a]pyridin-2-yl-3-oxo-piperazin-2-yl)acetic acid To a solution of tert-butyl 4-imidazo[1,2-a]pyridin-2-yl-2-(2-methoxy-2-oxo-ethyl)-3- oxo- piperazine-1-carboxylate (1.5 g, 3.86 mmol, 1 eq) in THF (15 mL) and H2O (5 mL) was added LiOH.H2O (486.17 mg, 11.59 mmol, 3 eq).
  • Step 2 2-(4-imidazo[1,2-a]pyridin-2-yl-3-oxo-piperazin-2-yl)acetic acid
  • Step 3 2-(4-imidazo[1,2-a]pyridin-2-yl-3-oxo-1-prop-2-enoyl-piperazin-2-yl)acetic acid
  • TEA 78.18 mg, 772.60 ⁇ mol, 107.54 ⁇ L, 3 eq
  • prop-2-enoyl chloride 13.99 mg, 154.52 ⁇ mol, 12.60 ⁇ L, 0.6 eq
  • Example 33 Synthesis of Compound 156 Step 1: tert-butyl 2-[2-(cyclopropylamino)-2-oxo-ethyl]-4-imidazo[1,2-a]pyridin-2-yl-3-oxo- piperazine-1-carboxylate
  • 2-(1-tert-butoxycarbonyl-4-imidazo[1,2-a]pyridin-2-yl-3-oxo-piperazin- 2-yl)acetic acid 200 mg, 534.2 ⁇ mol, 1 eq
  • DMF 10 mL
  • HATU 203.1 mg, 534.2 ⁇ mol, 1 eq
  • DIEA 69 mg, 534 ⁇ mol, 93 ⁇ L, 1 eq
  • Step 2 N-cyclopropyl-2-(4-imidazo[1,2-a]pyridin-2-yl-3-oxo-piperazin-2-yl)acetamide
  • tert-butyl 2-[2-(cyclopropylamino)-2-oxo-ethyl]-4-imidazo[1,2-a]pyridin- 2-yl-3-oxo-piperazine-1-carboxylate 160 mg, 387 ⁇ mol, 1 eq
  • DCM 3 mL
  • TFA 4 g, 40.4 mmol, 3 mL, 104.4 eq
  • Step 3 N-cyclopropyl-2-(4-imidazo[1,2-a]pyridin-2-yl-3-oxo-1-prop-2-enoyl-piperazin-2- yl)acetamide
  • TEA 161.5 mg, 1.6 mmol, 222 ⁇ L, 5 eq
  • prop-2-enoyl chloride 43.3 mg, 478.7 ⁇ mol, 39 ⁇ L, 1.5 eq
  • Example 34 Synthesis of Compound 160 Step 1: methyl 2-[[3-(nitromethyl)oxetan-3-yl]amino]acetate oxetan-3-one (15 g, 208.2 mmol, 1 eq) nitromethane (19.5 g, 320 mmol, 17.3 mL, 1.54 eq) and TEA (4.21 g, 41.6 mmol, 5.8 mL, 0.2 eq) were stirred at 25 °C for 60 min. DCM (200 mL) was added and the reaction mixture cooled to -70 °C.
  • TEA (42 g, 416.3 mmol, 57.9 mL, 2 eq) was added followed by the dropwise addition of a solution of MsCl (23.16 g, 202.2 mmol, 15.7 mL, 0.97 eq) in DCM (200 mL). The reaction mixture was left to stir at -70 °C for 90 min. Meanwhile, to a solution of methyl 2-aminoacetate; hydrochloride (52.3 g, 416.3 mmol, 2 eq) in DCM (200 mL) was added TEA (42.13 g, 416.3 mmol, 57.9 mL, 2 eq) and stirred at room temperature for 10 min.
  • Step 3 tert-butyl 7-oxo-2-oxa-5,8-diazaspiro[3.5]nonane-5-carboxylate Boc 2 O (12.7 g, 58 mmol, 13.3 mL, 1.5 eq) was added to a suspension of 2-oxa-5,8- diazaspiro[3.5]nonan-7-one (5.5 g, 39 mmol, 1 eq) in DCM (50 mL). The mixture was stirred at 25 °C for 12 h. Upon completion, the solvent was concentrated in vacuum.
  • Step 4 tert-butyl 4-(5-methyl-2-furyl)-3-oxo-piperazine-1-carboxylate
  • tert-butyl 7-oxo-2-oxa-5,8-diazaspiro[3.5]nonane-5-carboxylate 800 mg, 3.30 mmol, 1 eq
  • 2-bromo-5-methyl-furan 797.44 mg, 4.95 mmol, 1.5 eq
  • K2CO3 (1.37 g, 9.91 mmol, 3 eq)
  • CuI (251.55 mg, 1.32 mmol, 0.4 eq) was degassed and purged with N 2 (x3), then N,N'-dimethylethane-1,2-diamine (232.9 mg, 2.6 mmol, 284.3 uL, 0.8 eq) was added under N2, the mixture was stirred at 100 °C for 16 h.
  • Step 5 8-(5-methyl-2-furyl)-2-oxa-5,8-diazaspiro[3.5]nonan-7-one
  • a solution of tert-butyl 8-(5-methyl-2-furyl)-7-oxo-2-oxa-5,8-diazaspiro[3.5]nonane-5- carboxylate (0.5 g, 1.55 mmol, 1 eq) in DCM (3 mL) was added TFA (4.62 g, 40.52 mmol, 3 mL, 26.12 eq), the solution was stirred at 25 °C for 1 h.
  • Step 6 8-(5-methyl-2-furyl)-5-prop-2-enoyl-2-oxa-5,8-diazaspiro[3.5]nonan-7-one
  • TEA 682.98 mg, 6.75 mmol, 939.45 ⁇ L, 5 eq
  • prop-2-enoyl chloride 183.26 mg, 2.02 mmol, 165.10 ⁇ L, 1.5 eq
  • Example 35 Synthesis of Compound 163 Step 1: 5-methylpiperazin-2-one and 6-methylpiperazin-2-one A solution of ethyl 2-chloroacetate (16.5 g, 134.9 mmol, 14.4 mL, 0.2 eq) in EtOH (600 mL) was added the solution of propane-1,2-diamine (50 g, 674.5 mmol, 57.6 mL, 1 eq) in EtOH (100 mL) drop-wise at 20 °C over 1.5 h, after 2 h K2CO3 (18.7 g, 134.9 mmol, 0.2 eq) was added. And the mixture was stirred at 20 °C for another 2 h.
  • ethyl 2-chloroacetate 16.5 g, 134.9 mmol, 14.4 mL, 0.2 eq
  • EtOH 600 mL
  • propane-1,2-diamine 50 g, 674.5 mmol, 57.6 mL, 1 e
  • Step 2 tert-butyl 2-methyl-5-oxo-piperazine-1-carboxylate and tert-butyl 3-methyl-5-oxo- piperazine-1-carboxylate
  • 6-methylpiperazin-2-one 37.5 g, 328.53 mmol, 1 eq
  • 5-methylpiperazin- 2-one (12.5 g, 109.51 mmol, 3.33e-1 eq) in DCM (1000 mL)
  • Boc)2O 93.21 g, 427.09 mmol, 98.12 mL, 1.3 eq
  • Step 4 5-methyl-1-(5-methyl-2-furyl)piperazin-2-one
  • TFA 200 mg, 1.75 mmol, 130 ⁇ L, 5.15 eq
  • the reaction mixture was concentrated under reduced pressure to give 5-methyl-1-(5-methyl-2-furyl)piperazin-2-one (60 mg, crude) as an oil.
  • MS (ESI) m/z 195.3 [M+H] + .
  • Step 5 5-methyl-1-(5-methyl-2-furyl)-4-prop-2-enoyl-piperazin-2-one
  • TEA 3-methyl-1-(5-methyl-2-furyl)piperazin-2-one
  • TEA 1.3 mg, 308.9 ⁇ mol, 43 ⁇ L, 1 eq
  • prop-2-enoyl chloride 28 mg, 308.9 ⁇ mol, 25.1 ⁇ L, 1 eq
  • Example 36 Synthesis of Compound 167 Step 1: tert-butyl 4-(2,1,3-benzoxadiazol-5-yl)-3-oxo-piperazine-1-carboxylate
  • tert-butyl 3-oxopiperazine-1-carboxylate 528.2 mg, 2.6 mmol, 1.5 eq
  • dioxane 14 mL
  • Pd 2 (dba) 3 161 mg, 176 ⁇ mol, 0.1 eq
  • Xantphos (203.5 mg, 352 ⁇ mol, 0.2 eq) and then K 2 CO 3 (729 mg, 5.3 mmol, 3 eq)
  • Step 2 1-(2,1,3-benzoxadiazol-5-yl)piperazin-2-one
  • a solution of tert-butyl 4-(2,1,3-benzoxadiazol-5-yl)-3-oxo-piperazine-1-carboxylate (159 mg, 499 ⁇ mol, 1 eq) in HCl/dioxane (3 mL) was stirred for 1 h at 20 °C. Upon completion, the reaction was concentrated to give 1-(2,1,3-benzoxadiazol-5-yl)piperazin-2-one (108 mg, 495 ⁇ mol, 99% yield) as a solid.
  • MS (ESI) m/z 219.2 [M+H] + .
  • Step 3 1-(2,1,3-benzoxadiazol-5-yl)-4-prop-2-enoyl-piperazin-2-one
  • TEA 150 mg, 1.5 mmol, 207 uL, 3 eq
  • Prop-2-enoyl chloride 58 mg, 643 ⁇ mol, 52.5 uL, 1.3 eq
  • the mixture was stirred for 1 h at 0 °C. Upon completion, the reaction was concentrated to give crude.
  • Example 37 Synthesis of Compound 169 Step 1: 6-bromo-2-[(4-methoxyphenyl)methyl]-[1,2,4]triazolo[4,3-a]pyridin-3-one
  • 6-bromo-2H-[1,2,4]triazolo[4,3-a]pyridin-3-one 800 mg, 3.74 mmol, 1 eq
  • 1-(chloromethyl)-4-methoxy-benzene 643.9 mg, 4.11 mmol, 560 ⁇ L, 1.1 eq
  • DMF (12 mL) was added K2CO3 (1.55 g, 11.2 mmol, 3 eq). The mixture was stirred for 5 h at 50 °C.
  • Step 2 tert-butyl 4-[2-[(4-methoxyphenyl)methyl]-3-oxo-[1,2,4]triazolo[4,3-a]pyridin-6-yl]-3- oxo-piperazine-1-carboxylate
  • 6-bromo-2-[(4-methoxyphenyl)methyl]-[1,2,4]triazolo[4,3-a]pyridin-3- one 230 mg, 688.28 ⁇ mol, 1 eq
  • tert-butyl 3-oxopiperazine-1-carboxylate 551.27 mg, 2.75 mmol, 4 eq
  • Xantphos 79.65 mg, 137.66 ⁇ mol, 0.2 eq
  • Pd 2 (dba) 3 63.03 mg, 68.83 ⁇ mol, 0.1 eq
  • Step 3 2-[(4-methoxyphenyl)methyl]-6-(2-oxopiperazin-1-yl)-[1,2,4]triazolo[4,3-a]pyridin-3- one
  • Step 4 2-[(4-methoxyphenyl)methyl]-6-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)- [1,2,4]triazolo[4,3-a]pyridin-3-one
  • 2-[(4-methoxyphenyl)methyl]-6-(2-oxopiperazin-1-yl)- [1,2,4]triazolo[4,3-a]pyridin-3-one 311 mg, 880 ⁇ mol, 1 eq
  • prop-2-enoyl chloride 95.6 mg, 1.06 mmol, 86.1 ⁇ L, 1.2 eq
  • DCM 30 mL
  • Step 5 6-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)-2H-[1,2,4]triazolo[4,3-a]pyridin-3-one
  • a solution of 2-[(4-methoxyphenyl)methyl]-6-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)- [1,2,4]triazolo[4,3-a]pyridin-3-one (110 mg, 269.99 ⁇ mol, 1 eq) in methanesulfonic acid (3 mL) was stirred for 1 h at 70°C. Upon completion, the solution was filtered to give crude.
  • Example 38 Synthesis of Compound 170 Step 1: tert-butyl 4-[5-ethoxycarbonyl-4-methyl-1-(2-trimethylsilylethoxymethyl)imidazol-2-yl]- 3-oxo-piperazine-1-carboxylate
  • Step 2 ethyl 4-methyl-2-(2-oxopiperazin-1-yl)-1H-imidazole-5-carboxylate
  • tert-butyl 4-[5-ethoxycarbonyl-4-methyl-1-(2-trimethylsilylethoxymethyl)- imidazol-2-yl]-3-oxo-piperazine-1-carboxylate (100 mg, 207 ⁇ mol, 1 eq) in DCM (5 mL) was added diethyloxonio(trifluoro)boranuide (117.6 mg, 829 ⁇ mol, 102 ⁇ L, 4 eq) and the mixture was stirred at 25 °C for 1 h.
  • Example 39 Synthesis of Compound 171 Step 1: methyl 2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)imidazo[1,2-a]pyridine-6- carboxylate
  • a solution of methyl 2-bromoimidazo[1,2-a]pyridine-6-carboxylate (5 g, 19.6 mmol, 1 eq) , tert-butyl 3-oxopiperazine-1-carboxylate (4.7 g, 23.5 mmol, 1.2 eq) in dioxane (100 mL) was added K2CO3 (5.4 g, 39.2 mmol, 2 eq) , CuI (1.49 g, 7.8 mmol, 0.4 eq) and N,N'-dimethylethane- 1,2-diamine (1.38 g, 15.7 mmol, 1.7 mL, 0.8 eq) under N2, the mixture was stirred at 110 °C for 16 h.
  • Step 2 2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)imidazo[1,2-a]pyridine-6-carboxylic acid
  • a solution of methyl 2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)imidazo[1,2- a]pyridine-6-carboxylate (2.15 g, 4.6 mmol, 80% purity, 1 eq) in MeOH (40 mL) and H2O (13 mL) was added K 2 CO 3 (1.9 g, 13.8 mmol, 3 eq), the mixture was stirred at 50 °C for 2 h.
  • Step 3 tert-butyl 4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl]-3-oxo-piperazine-1- carboxylate
  • 2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)imidazo[1,2-a]pyridine-6- carboxylic acid 200 mg, 554.99 ⁇ mol, 1 eq
  • HATU 316.5 mg, 832.5 ⁇ mol, 1.5 eq
  • DIEA 143.5 mg, 1.1 mmol, 193.3 uL, 2 eq
  • DCM 3 mL
  • Step 4 N-methyl-2-(2-oxopiperazin-1-yl)imidazo[1,2-a]pyridine-6-carboxamide
  • Step 5 N-methyl-2-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)imidazo[1,2-a]pyridine-6- carboxamide
  • a solution of N-methyl-2-(2-oxopiperazin-1-yl)imidazo[1,2-a]pyridine-6-carboxamide (80 mg, 293 ⁇ mol, 1 eq) in DCM (7 mL) was added TEA (89 mg, 878 ⁇ mol, 122.2 uL, 3 eq), then prop-2-enoyl chloride (26.5 mg, 293 ⁇ mol, 24 uL, 1 eq) in DCM (1 mL) was added drop-wise at 0 °C, the mixture was stirred at 0 °C for 1 h.
  • Step 2 methyl 2-[4-tert-butoxycarbonyl-2-oxo-3-(2-phenoxyethyl)piperazin-1-yl]imidazo[1,2- a]pyridine-6-carboxylate
  • a mixture of tert-butyl 3-oxo-2-(2-phenoxyethyl)piperazine-1- carboxylate 300 mg, 936 ⁇ mol, 1 eq
  • methyl 2-bromoimidazo[1,2-a]pyridine-6-carboxylate (239 mg, 936 ⁇ mol, 1 eq)
  • CuI 27 mg, 140.5 ⁇ mol, 0.15 eq
  • N,N'-dimethylethane-1,2-diamine 20.6 mg, 234 ⁇ mol, 25 ⁇ L, 0.25 eq
  • K 2 CO 3 259 mg, 1.87 mmol, 2 eq) in dioxane (10 mL) was degassed and purged with N 2 (x3), and then the mixture was
  • Step 3 tert-butyl 4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl]-3-oxo-2-(2-
  • methyl 2-[4-tert-butoxycarbonyl-2-oxo-3-(2-phenoxyethyl)piperazin-1- yl]imidazo[1,2-a]pyridine-6-carboxylate 100 mg, 202.2 ⁇ mol, 1 eq
  • EtOH mL
  • MeNH 2 (1 g, 9.66 mmol, 30% purity, 47.8 eq
  • Step 5 N-methyl-2-[2-oxo-3-(2-phenoxyethyl)-4-prop-2-enoyl-piperazin-1-yl]imidazo[1,2-
  • TEA 99 mg, 978.6 ⁇ mol, 391.4 ⁇ L, 5 eq
  • Step 2 tert-butyl 2-(2-hydroxyethyl)-4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl]-3-oxo- piperazine-1-carboxylate
  • tert-butyl 2-(2-hydroxyethyl)-3-oxo-piperazine-1-carboxylate 900 mg, 3.7 mmol, 1.1 eq
  • 2-bromo-N-methyl-imidazo[1,2-a]pyridine-6-carboxamide 851 mg, 3.35 mmol, 1 eq) in dioxane (30 mL) was added K2CO3 (926 mg, 6.7 mmol, 2 eq) and DMEDA (236.2 mg, 2.7 mmol, 288 ⁇ L, 0.8 eq) then CuI (255.2 mg, 1.34 mmol, 0.4 eq) was added and stirred under N 2 at 100 °C for 16 h.
  • Step 3 tert-butyl 4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl]-3-oxo-2-[2-(p- tolylsulfonyloxy)ethyl]piperazine-1-carboxylate
  • Step 4 tert-butyl 2-[2-(3-methoxycarbonylphenoxy)ethyl]-4-[6-(methylcarbamoyl)imidazo[1,2- a]pyridin-2-yl]-3-oxo-piperazine-1-carboxylate
  • methyl 3-hydroxybenzoate 95.8 mg, 630 ⁇ mol, 1.2 eq
  • t-BuOK 88.3 mg, 787 ⁇ mol, 1.5 eq
  • DMF 3 mL
  • tert-butyl 4-[6-(methylcarbamoyl)imidazo[1,2- a]pyridin-2-yl]-3-oxo-2-[2-(p-tolylsulfonyloxy)ethyl]piperazine-1-carboxylate 300 mg x3, 524.8 ⁇ mol, 1 eq
  • Example 42 Synthesis of Compound 174 Step 1 : dimethyl (2-((tert-butoxycarbonyl)amino)ethyl)glutamate
  • tert-butyl N-(2-oxoethyl)carbamate 7 g, 43.97 mmol, 1 eq
  • MeOH 100 mL
  • dimethyl 2-aminopentanedioate;hydrochloride (10.9 g, 44 mmol, 1 eq, HCl)
  • NaBH 3 CN 4.15 g, 66 mmol, 1.5 eq
  • AcOH 3.7 g, 61.6 mmol, 3.5 mL, 1.4 eq
  • Step 2 dimethyl N-((benzyloxy)carbonyl)-N-(2-((tert-butoxycarbonyl)amino)ethyl)glutamate
  • reaction mixture was diluted with H 2 O (150 mL), extracted with DCM (100 mL*2) and washed with sat. NaCl (100 mL*2). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (0-50% EtOAc in PE) to give dimethyl 2-[benzyloxycarbonyl-[2-(tert- butoxycarbonylamino)ethyl]amino]pentanedioate (11.6 g, 26 mmol, 58% yield) as an oil.
  • Step3 dimethyl N-(2-aminoethyl)-N-((benzyloxy)carbonyl)glutamate
  • a mixture of dimethyl 2-[benzyloxycarbonyl-[2-(tert-butoxycarbonylamino) ethyl]amino]pentanedioate (11.6 g, 25.64 mmol, 1 eq) and HCl/dioxane (4 M, 116 mL, 18.1 eq) was stirred at 25 °C for 1 h. Upon completion, the reaction was concentrated in vacuum to give dimethyl 2-[2-aminoethyl (benzyloxycarbonyl) amino] pentanedioate (9 g, crude) as an oil.
  • Step4 benzyl 2-(3-methoxy-3-oxopropyl)-3-oxopiperazine-1-carboxylate
  • DMF dimethyl 2-[2-aminoethyl(benzyloxycarbonyl)amino]pentanedioate (9 g, 25.5 mmol, 1 eq) in DMF (90 mL) was added Cs2CO3 (20.8 g, 64 mmol, 2.5 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with H 2 O (100 mL) and extracted with DCM (100 mL*3).
  • Step5 3-(1-((benzyloxy)carbonyl)-3-oxopiperazin-2-yl)propanoic acid
  • benzyl 2-(3-methoxy-3-oxo-propyl)-3-oxo-piperazine-1-carboxylate (1 g, 3.1 mmol, 1 eq) in H 2 O (2.3 mL) and MeOH (7 mL) was added K 2 CO 3 (1.3 g, 9.4 mmol, 3 eq).
  • the mixture was stirred at 50 °C for 1 h.
  • the reaction mixture was added 1M HCl adjust to pH ⁇ 2, then extracted with EtOAc (15 mL*3).
  • Step6 3-(1-((benzyloxy)carbonyl)-4-(6-(methoxycarbonyl)imidazo[1,2-a]pyridin-2-yl)-3- oxopiperazin-2-yl)propanoic acid
  • 3-(1-benzyloxycarbonyl-3-oxo-piperazin-2-yl)propanoic acid 100 mg, 327 ⁇ mol, 1 eq
  • methyl 2-bromoimidazo[1,2-a]pyridine-6-carboxylate 83.3 mg, 326.5 ⁇ mol, 1 eq) in dioxane (20 mL) was added K 2 CO 3 (90.2 mg, 653 ⁇ mol, 2 eq) and DMEDA (7.2 mg, 81.6 ⁇ mol, 8.8 ⁇ L, 0.25 eq), then exchange N2 (x3), then added CuI (9.33 mg, 49 ⁇ mol, 0.15 eq) and exchange N2 (x3), the mixture was stirred at 100
  • Step7 3-(1-((benzyloxy)carbonyl)-4-(6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl)-3- oxopiperazin-2-yl)propanoic acid
  • Step8 3-[4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl]-3-oxo-piperazin-2-yl]propanoic acid
  • 3-[1-benzyloxycarbonyl-4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2- yl]-3-oxo-piperazin-2-yl]propanoic acid 105 mg, 219 ⁇ mol, 1 eq
  • EtOH 2 mL
  • H2(15 psi) and Pd/C 105 mg, 219 ⁇ mol, 10% purity
  • Step 9 3-(1-acryloyl-4-(6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl)-3-oxopiperazin-2- yl)propanoic acid
  • 3-[4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl]-3-oxo-piperazin- 2-yl]propanoic acid 70 mg, 202.7 ⁇ mol, 1 eq
  • KOH 17 mg, 304 ⁇ mol, 1.5 eq
  • H2O 1 mL
  • prop-2-enoyl chloride (18.4 mg, 202.7 ⁇ mol, 16.5 ⁇ L, 1 eq) at 0 °C.
  • Example 43 Synthesis of Compound 175 Step 1: benzyl 2-(3-hydroxypropyl)-3-oxo-piperazine-1-carboxylate To a solution of benzyl 2-(3-methoxy-3-oxo-propyl)-3-oxo-piperazine-1-carboxylate (1.2 g, 3.75 mmol, 1 eq) in THF (15 mL) was added LiAlH 4 (284.32 mg, 7.49 mmol, 2 eq) at 0 °C, the mixture was stirred at 0 °C for 1 h.
  • reaction mixture was quenched by addition H2O (0.3 mL) and 15% NaOH (0.3 mL) and H2O (0.9 mL) at 20 °C, and then diluted with EtOAc (20 mL) and extracted with EtOAc (20 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (0-9% MeOH in EtOAc) to obtain benzyl 2-(3-hydroxypropyl)-3-oxo-piperazine-1-carboxylate (600 mg, 1.3 mmol, 36% yield, 65% purity) as an oil.
  • Step 3 benzyl 2-(3-hydroxypropyl)-4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl]-3-oxo- piperazine-1-carboxylate
  • MeNH 2 8 mL, 30% purity in EtOH
  • Step 4 benzyl 4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2-yl]-3-oxo-2-[3-(p- tolylsulfonyloxy)propyl]piperazine-1-carboxylate
  • benzyl 2-(3-hydroxypropyl)-4-[6-(methylcarbamoyl)imidazo[1,2- a]pyridin-2-yl]-3-oxo-piperazine-1-carboxylate 210 mg, 451.1 ⁇ mol, 1 eq
  • DMAP 5.51 mg, 45.1 ⁇ mol, 0.1 eq
  • Et 3 N 68.5 mg, 676.7 ⁇ mol, 94.2 ⁇ L, 1.5 eq
  • TosCl 129 mg, 676.7 ⁇ mol, 1.5 eq
  • Step 5 benzyl 2-[3-(dimethylamino)propyl]-4-[6-(methylcarbamoyl)imidazo[1,2-a]pyridin-2- yl]-3-oxo-piperazine-1-carboxylate
  • Step 6 2-[3-[3-(dimethylamino)propyl]-2-oxo-piperazin-1-yl]-N-methyl-imidazo[1,2-a]pyridine- 6-carboxamide
  • benzyl 2-[3-(dimethylamino)propyl]-4-[6-(methylcarbamoyl)imidazo[1,2- a]pyridin-2-yl]-3-oxo-piperazine-1-carboxylate 100 mg, 203 ⁇ mol, 1 eq
  • EtOH 6 mL
  • Pd/C 108 mg, 101.5 ⁇ mol, 10% purity, 0.5 eq
  • Step 7 2-[3-[3-(dimethylamino)propyl]-2-oxo-4-prop-2-enoyl-piperazin-1-yl]-N-methyl- i
  • TEA 0.8 mg, 502.2 ⁇ mol, 69.9 ⁇ L, 3 eq
  • prop-2-enoyl chloride 18.18 mg, 200.87 ⁇ mol, 16.32 ⁇ L, 1.2 eq
  • Example 44 Synthesis of Compound 176 Step 1 : 4-bromo-1-methyl-imidazole-2-carbaldehyde LDA (2 M, 10.25 mL, 1.1 eq) was added to a solution of 4-bromo-1-methyl-imidazole (3 g, 18.6 mmol, 1 eq) in THF (90 mL) at -10 °C, after 1 h, DMF (2.04 g, 28 mmol, 2.15 mL, 1.5 eq) was added, then the mixture was stirred for another 1 h at 0 °C.
  • reaction mixture was quenched by saturated aqueous citric acid solution (30 Ml), and then extracted with ethyl acetate (30 mL x2). The combined organic layers were washed with aqueous NaCl (30 mL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to 4-bromo-1-methyl- imidazole-2-carbaldehyde (2.7 g, crude) as a solid.
  • Step 2 methyl (E)-3-(4-bromo-1-methyl-imidazol-2-yl)prop-2-enoate
  • methyl 2-diethoxyphosphorylacetate 3.56 g, 16.93 mmol, 1 eq
  • NaH 1.22 g, 30.60 mmol, 60 % purity, 1.81 eq
  • a solution of 4-bromo-1-methyl-imidazole-2-carbaldehyde 3.2 g, 16.93 mmol, 1 eq
  • Step 3 tert-butyl 4-[2-[(E)-3-methoxy-3-oxo-prop-1-enyl]-1-methyl-imidazol-4-yl]-3-oxo- piperazine-1-carboxylate
  • tert-butyl 3-oxopiperazine-1-carboxylate (2.54 g, 12.7 mmol, 1 eq)
  • CuI 362.5 mg, 1.9 mmol, 0.15 eq
  • DMEDA (279.7 mg, 3.17 mmol, 341.5 ⁇ L, 0.25 eq
  • K 2 CO 3 (3.51 g, 25.4 mmol, 2 eq) in dioxane (50 mL) was degassed and purged with N 2 (x3), and then the
  • Step 4 tert-butyl 4-[2-(3-methoxy-3-oxo-propyl)-1-methyl-imidazol-4-yl]-3-oxo-piperazine-1- carboxylate
  • tert-butyl 4-[2-[(E)-3-methoxy-3-oxo-prop-1-enyl]-1-methyl-imidazol-4- yl]-3-oxo-piperazine-1-carboxylate (500 mg, 1.37 mmol, 1 eq) in EtOH (10 mL) was added Pd/C (500 mg, 10% purity) under N 2 atmosphere. The suspension was degassed and purged with H 2 (x3).
  • Step5 tert-butyl 4-[1-methyl-2-[3-(methylamino)-3-oxo-propyl]imidazol-4-yl]-3-oxo- piperazine-1-carboxylate tert-butyl 4-[2-(3-methoxy-3-oxo-propyl)-1-methyl-imidazol-4-yl]-3-oxo-piperazine-1- carboxylate (500 mg, 1.4 mmol, 1 eq) was added to methanamine (12.4 g, 120 mmol, 30% purity, 88 eq) (methylamine ethanol solution), and then the temperature was raised to 50 °C for 16 h.
  • Step6 N-methyl-3-[1-methyl-4-(2-oxopiperazin-1-yl)imidazol-2-yl]propanamide
  • tert-butyl 4-[1-methyl-2-[3-(methylamino)-3-oxo-propyl]imidazol-4-yl]- 3-oxo-piperazine-1-carboxylate 500 mg, 1.37 mmol, 1 eq
  • TFA 2 mL
  • DCM 6-mL
  • Step 7 N-methyl-3-[1-methyl-4-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)imidazol-2- yl]propanamide
  • TEA 610 mg, 6 mmol, 839 ⁇ L, 4 eq
  • prop-2-enoyl chloride 164 mg, 1.8 mmol, 147.5 ⁇ L, 1.2 eq
  • Example 45 Synthesis of Compound 177 Step 1: 2-[(2-bromoimidazol-1-yl)methoxy]ethyl-trimethyl-silane To a solution of 2-bromo-1H-imidazole (10.5 g, 71.44 mmol, 1 eq) in THF (100 mL) was added dropwise NaH (3.43 g, 85.73 mmol, 60% purity, 1.2 eq) at 0 °C. After addition, the mixture was stirred at this temperature for 0.5 h and then was added dropwise SEMCl (15.48 g, 92.87 mmol, 16.44 mL, 1.3 eq). The mixture was stirred at 25 °C for 1.5 h.
  • Step 2 2-bromo-3-(2-trimethylsilylethoxymethyl)imidazole-4-carbaldehyde
  • 2-[(2-bromoimidazol-1-yl)methoxy]ethyl-trimethyl-silane (12 g, 43.28 mmol, 1 eq) in THF (250 mL) was added LDA (2 M, 23.81 mL, 1.1 eq).
  • LDA 2 M, 23.81 mL, 1.1 eq
  • the mixture was stirred at -65 °C for 1 h.
  • DMF (6.33 g, 86.57 mmol, 6.66 mL, 2 eq).
  • the mixture was stirred at -65 °C for 0.5 h.
  • Step 3 methyl (E)-3-[2-bromo-3-(2-trimethylsilylethoxymethyl)imidazol-4-yl]prop-2-enoate
  • DBU dimethyl 2-diethoxyphosphorylacetate
  • the mixture was stirred at 0 °C for 0.5 h.
  • 2-bromo-3-(2-trimethylsilylethoxymethyl)imidazole-4-carbaldehyde 6.5 g, 21.29 mmol, 1 eq).
  • Step 5 methyl (E)-3-(2-bromo-3-methyl-imidazol-4-yl)prop-2-enoate
  • DMF 25 mL
  • NaOH sodium sulfate
  • CH3I CH3I
  • reaction mixture was quenched by aqueous H 2 O 5 mL and then extracted with EtOAc (10 mL * 2). The combined organic ayers were washed with aqueous NaCl (15 mL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue.
  • Step 6 tert-butyl 4-[5-[(E)-3-methoxy-3-oxo-prop-1-enyl]-1-methyl-imidazol-2-yl]-3-oxo- piperazine-1-carboxylate
  • methyl (E)-3-(2-bromo-3-methyl-imidazol-4-yl)prop-2-enoate 700 mg, 2.9 mmol, 1 eq
  • tert-butyl 3-oxopiperazine-1-carboxylate 1.7 g, 8.6 mmol, 3 eq
  • CuI 272 mg, 1.4 mmol, 0.5 eq) 1,10-phenanthroline (360.3 mg, 2 mmol, 0.7 eq)
  • K2CO3 395 mg, 2.9 mmol, 1 eq).
  • Step 7 tert-butyl 4-[5-(3-methoxy-3-oxo-propyl)-1-methyl-imidazol-2-yl]-3-oxo-piperazine-1- carboxylate
  • tert-butyl 4-[5-[(E)-3-methoxy-3-oxo-prop-1-enyl]-1-methyl-imidazol-2- yl]-3-oxo-piperazine-1-carboxylate 500 mg, 1.37 mmol, 1 eq
  • EtOH 10 mL
  • Pd/C 706.6 mg, 664 ⁇ mol, 10% purity
  • Step 8 tert-butyl 4-[1-methyl-5-[3-(methylamino)-3-oxo-propyl]imidazol-2-yl]-3-oxo- piperazine-1-carboxylate
  • tert-butyl 4-[5-(3-methoxy-3-oxo-propyl)-1-methyl-imidazol-2-yl]-3-oxo- piperazine-1-carboxylate 500 mg, 1.36 mmol, 1 eq
  • MeNH2/EtOH 15 mL
  • Step 9 N-methyl-3-[3-methyl-2-(2-oxopiperazin-1-yl)imidazol-4-yl]propanamide
  • tert-butyl 4-[1-methyl-5-[3-(methylamino)-3-oxo-propyl]imidazol-2-yl]- 3-oxo-piperazine-1-carboxylate 300 mg, 821 ⁇ mol, 1 eq
  • DCM 7 mL
  • TFA 2 mL
  • Step 10 N-methyl-3-[3-methyl-2-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)imidazol-4- yl]propanamide
  • TEA 240.3 mg, 2.4 mmol, 330.5 ⁇ L, 3 eq
  • prop-2-enoyl chloride 85.97 mg, 949.8 ⁇ mol, 77.2 ⁇ L, 1.2 eq
  • Example 46 Synthesis of Compound 178 Step 1: 2-chlorooxazole-4-carbaldehyde To a solution of ethyl 2-chlorooxazole-4-carboxylate (3 g, 17.09 mmol, 1 eq) in THF (30 mL), was added DIBAL-H (1 M, 68.35 mL, 4 eq), the mixture was stirred at -78 °C for 2 h. Upon completion, the reaction mixture was quenched by addition sshapedte salt 150 mL at -78 °C, and then diluted with EtOAc 200 mL and extracted with EtOAc (200 mL * 3). The combined organic layers were washed with NaCl aq.
  • Step 2 benzyl (E)-3-(2-chlorooxazol-4-yl)prop-2-enoate
  • benzyl 2-diethoxyphosphorylacetate 652.98 mg, 2.28 mmol, 1 eq
  • NaH 165.13 mg, 4.13 mmol, 60% purity, 1.81 eq
  • 2-chlorooxazole-4-carbaldehyde 300 mg, 2.28 mmol, 1 eq
  • Step 3 tert-butyl 4-[4-[(E)-3-benzyloxy-3-oxo-prop-1-enyl]oxazol-2-yl]-3-oxo-piperazine-1- carboxylate
  • benzyl (E)-3-(2-chlorooxazol-4-yl)prop-2-enoate 600 mg, 2.28 mmol, 1 eq
  • tert-butyl 3-oxopiperazine-1-carboxylate 546.8 mg, 2.7 mmol, 1.2 eq
  • Cs2CO3 (1.48 g, 4.55 mmol, 2 eq)
  • tBuXPhos Pd G3 (180.8 mg, 227.6 ⁇ mol, 0.1 eq).
  • Step 4 3-[2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)oxazol-4-yl]propanoic acid
  • tert-butyl 4-[4-[(E)-3-benzyloxy-3-oxo-prop-1-enyl]oxazol-2-yl]-3-oxo- piperazine-1-carboxylate (698 mg, 1.63 mmol, 1 eq) in EtOH (15 mL) was added Pd/C (608.72 mg, 572 ⁇ mol, 10% purity, 0.35 eq) under N2.
  • Pd/C 608.72 mg, 572 ⁇ mol, 10% purity, 0.35 eq
  • Step 5 tert-butyl 4-[4-[3-(methylamino)-3-oxo-propyl]oxazol-2-yl]-3-oxo-piperazine-1- carboxylate
  • HATU 3-[2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)oxazol-4-yl]propanoic acid (310 mg, 913.5 ⁇ mol, 1 eq) in DMF (1 mL)
  • HATU 416.8 mg, 1.1 mmol, 1.2 eq
  • DIPEA 354.2 mg, 2.74 mmol, 477.4 ⁇ L, 3 eq
  • the mixture was stirred at 25 °C for 30 min, methanamine; hydrochloride (95 mg, 913.5 ⁇ mol, 1 eq, HCl) was added, the mixture was stirred at 25 °C for 30 min.
  • Step 6 N-methyl-3-[2-(2-oxopiperazin-1-yl)oxazol-4-yl]propanamide
  • TFA 1,3-(methylamino)-3-oxo-propyl]oxazol-2-yl]-3-oxo- piperazine-1-carboxylate
  • Step 7 N-methyl-3-[2-(2-oxopiperazin-1-yl)oxazol-4-yl]propanamide (150 mg, crude) as an oil.
  • Step 7 N-methyl-3-[2-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)oxazol-4-yl]propanamide
  • K 2 CO 3 (197.2 mg, 1.43 mmol, 3 eq)
  • prop-2- enoyl chloride (30 mg, 333 ⁇ mol, 27 ⁇ L, 0.7 eq) was added, the mixture was stirred at 0 °C for 1 h.
  • Example 47 Synthesis of Compound 181 Step 1: benzyl 4-hydroxy-3-nitro-benzoate To a solution of 4-hydroxy-3-nitro-benzoic acid (10 g, 54.6 mmol, 1 eq) in DMF (500 mL) was added NaHCO3 (6.88 g, 81.9 mmol, 3.2 mL, 1.5 eq) and bromomethylbenzene (14 g, 81.9 mmol, 9.73 mL, 1.5 eq). The mixture was stirred at 50 °C for 16 h. Upon completion, the reaction mixture was quenched by addition H 2 O 800 mL, and then extracted with EtOAc 2.4 L (800 mL * 3).
  • Step 2 benzyl 3-amino-4-hydroxy-benzoate
  • a mixture of benzyl 4-hydroxy-3-nitro-benzoate (2 g, 7.32 mmol, 1 eq) and NH4Cl (3.9 g, 73.2 mmol, 10 eq) in EtOH (20 mL) and H2O (4 mL) was degassed and purged with N2 (x3), then added Fe (2 g, 36.6 mmol, 5 eq) at 60 °C. Then the mixture was stirred at 80 °C for 1 h under N 2 atmosphere.
  • Step 4 benzyl 2-bromo-1,3-benzoxazole-5-carboxylate
  • benzyl 1,3-benzoxazole-5-carboxylate 600 mg, 2.37 mmol, 1 eq
  • LiHMDS 1 M, 2.84 mL, 1.2 eq
  • NBS 632.5 mg, 3.6 mmol, 1.5 eq
  • the mixture was stirred at 20 °C for 1 h.
  • the reaction mixture was quenched by addition NH4CI 15 mL, and then extracted with EtOAc (20 mL * 3).
  • Step 5 benzyl 2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)-1,3-benzoxazole-5-carboxylate
  • benzyl 2-bromo-1,3-benzoxazole-5-carboxylate 200 mg, 602.14 ⁇ mol, 1 eq
  • ACN 14 mL
  • K 2 CO 3 249.67 mg, 1.81 mmol, 3 eq
  • tert-butyl 3- oxopiperazine-1-carboxylate 241.14 mg, 1.20 mmol, 2 eq.
  • the mixture was stirred at 80 °C for 16 h.
  • Step 6 2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)-1,3-benzoxazole-5-carboxylic acid
  • Pd/C 420 mg, 394.7 ⁇ mol, 10% purity, 0.4 eq
  • benzyl 2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)-1,3-benzoxazole-5-carboxylate 420 mg, 930.3 ⁇ mol, 1 eq
  • Step 7 tert-butyl 4-[5-(methylcarbamoyl)-1,3-benzoxazol-2-yl]-3-oxo-piperazine-1-carboxylate
  • 2-(4-tert-butoxycarbonyl-2-oxo-piperazin-1-yl)-1,3-benzoxazole-5- carboxylic acid 280 mg, 774.9 ⁇ mol, 1 eq
  • HATU 353.6 mg, 929.9 ⁇ mol, 1.2 eq
  • DIEA 300.4 mg, 2.3 mmol, 404.9 ⁇ L, 3 eq
  • Step 9 N-methyl-2-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)-1,3-benzoxazole-5-carboxamide
  • TEA 270.2 mg, 2.7 mmol, 371.7 ⁇ L, 5 eq
  • prop-2-enoyl chloride 43.5 mg, 481 ⁇ mol, 39 ⁇ L, 0.9 eq
  • Example 48 Synthesis of Compound 183 Step 1: methyl 3-(3-iodopyrazol-1-yl)propanoate To a solution of 3-iodo-1H-pyrazole (3 g, 15.47 mmol, 1 eq) in ACN (45 mL) was added DBU (1.18 g, 7.73 mmol, 1.17 mL, 0.5 eq) at 0 °C, followed by methyl prop-2-enoate (2.66 g, 30.93 mmol, 2.79 mL, 2 eq) then was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched with 1 M HCl (30 mL) and extracted with EtOAc (30 mL*2).
  • Step 2 tert-butyl 4-[1-(3-methoxy-3-oxo-propyl)pyrazol-3-yl]-3-oxo-piperazine-1-carboxylate
  • reaction mixture was diluted with aq. EDTA 30 mL and extracted with EtOAc (20 mL*2). The combined organic layers were dried over sat.Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (10-30% EtOAc in PE) to give tert-butyl 4-[1-(3-methoxy-3-oxo-propyl)pyrazol-3-yl]-3-oxo-piperazine-1- carboxylate (1.9 g, 5.4 mmol, 76% yield) as an oil.
  • Step 3 tert-butyl 4-[1-[3-(methylamino)-3-oxo-propyl]pyrazol-3-yl]-3-oxo-piperazine-1- carboxylate tert-butyl 4-[1-(3-methoxy-3-oxo-propyl)pyrazol-3-yl]-3-oxo-piperazine-1-carboxylate (1 g, 2.84 mmol, 1 eq) was added to methanamine (25.860 g, 249.80 mmol, 30% purity, 88.03 eq) (methylamine ethanol solution), and then the temperature was raised to 50 °C for 16 h.
  • Step 4 N-methyl-3-[3-(2-oxopiperazin-1-yl)pyrazol-1-yl]propanamide
  • tert-butyl 4-[1-[3-(methylamino)-3-oxo-propyl]pyrazol-3-yl]-3-oxo- piperazine-1-carboxylate 300 mg, 854 ⁇ mol, 1 eq
  • TFA 2.30 g, 20.19 mmol, 1.5 mL, 23.65 eq
  • the mixture was stirred at 25 °C for 1 h.
  • Step 5 N-methyl-3-[3-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)pyrazol-1-yl]propanamide
  • TEA 422.8 mg, 4.2 mmol, 581.6 ⁇ L, 5 eq
  • prop- 2-enoyl chloride 83.2 mg, 919.3 ⁇ mol, 74.7 ⁇ L, 1.1 eq
  • Example 49 Synthesis of Compound 185 Step 1: 1-benzyl-3,5-dibromo-1H-pyrazole To a solution of 3,5-dibromo-1H-pyrazole (5 g, 22.1 mmol, 1 eq) in DMF (30 mL) was added NaH (2.2 g, 55.3 mmol, 60% purity, 2.5 eq) and BnBr (4.54 g, 26.6 mmol, 3.16 mL, 1.2 eq) at 0 °C. The mixture was then allowed to 25 °C and stirred for 12 h.
  • Step 2 2-benzyl-5-bromo-pyrazole-3-carbaldehyde
  • THF 2-butyl-3,5-dibromo-pyrazole
  • chloro(isopropyl)magnesium 2 M, 2.85 mL, 1.2 eq
  • DMF 2.43 g, 33.23 mmol, 2.56 mL, 7 eq
  • Step 3 methyl (E)-3-(2-benzyl-5-bromo-pyrazol-3-yl)prop-2-enoate
  • DBU methyl 2-diethoxyphosphorylacetate
  • 2- benzyl-5-bromo-pyrazole-3-carbaldehyde 870 mg, 3.3 mmol, 1.2 eq
  • Step 4 tert-butyl 4-[1-benzyl-5-[(E)-3-methoxy-3-oxo-prop-1-enyl]pyrazol-3-yl]-3-oxo- piperazine-1-carboxylate
  • tert-butyl 3-oxopiperazine-1-carboxylate 613.5 mg, 3.06 mmol, 1.2 eq
  • K 2 CO 3 705.7 mg, 5.1 mmol, 2 eq
  • DMEDA 180 mg, 2.04 mmol, 219.8 ⁇ L, 0.8 eq
  • CuI (194.5 mg, 1 mmol, 0.4 eq) in dioxane (41 mL) was degassed and purged with N2 (x3), and then the mixture was stirred at 100
  • Step 6 tert-butyl 4-[5-[3-(methylamino)-3-oxo-propyl]-1H-pyrazol-3-yl]-3-oxo-piperazine-1- carboxylate
  • a solution tert-butyl 4-[5-(3-methoxy-3-oxo-propyl)-1H-pyrazol-3-yl]-3-oxo-piperazine- 1-carboxylate (285 mg, 808.8 ⁇ mol, 1 eq) in MeNH 2 (7.37 g, 71.17 mmol, 30% purity, 88 eq) (ethanol solution) was stirred at 50 °C for 2 h. Upon completion, the residue was evaporated to dryness. The crude was used directly.
  • Step 7 N-methyl-3-[3-(2-oxopiperazin-1-yl)-1H-pyrazol-5-yl]propanamide
  • tert-butyl 4-[5-[3-(methylamino)-3-oxo-propyl]-1H-pyrazol-3-yl]-3-oxo- piperazine-1-carboxylate 280 mg, 796.8 ⁇ mol, 1 eq
  • TFA 4 g, 40.4 mmol, 3 mL, 50.7 eq
  • the mixture was stirred at 25 °C for 0.5 h. Upon completion, the residue was evaporated to dryness.
  • Step 8 N-methyl-3-[3-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)-1H-pyrazol-5-yl]propanamide
  • TEA 403 mg, 3.98 mmol, 554 ⁇ L, 4 eq
  • prop- 2-enoyl chloride 45 mg, 497.4 ⁇ mol, 40.4 ⁇ L, 0.5 eq
  • Example 50 Synthesis of Compound 186 Step 1: methyl 2-[allyl(tert-butoxycarbonyl)amino]acetate To a solution of methyl 2-(tert-butoxycarbonylamino)acetate (3.7 g, 19.6 mmol, 2.9 mL, 1 eq) in DMF (37 mL) was cooled to 0 °C then NaH (938.6 mg, 23.5 mmol, 60% purity, 1.2 eq) was added and stirred at 0 °C for 0.5 h. Then 3-bromoprop-1-ene (2.84 g, 23.5 mmol, 1.2 eq) was added and the mixture was stirred at 0 °C for 1.5 h.
  • Step 2 methyl 2-[tert-butoxycarbonyl(2-oxoethyl)amino]acetate
  • MeOH MeOH
  • Step 3 amino 2,4,6-trimethylbenzenesulfonate To a solution of (tert-butoxycarbonylamino) 2,4,6-trimethylbenzenesulfonate (60 g, 190.2 mmol, 1 eq) in TFA (460.5 g, 4.04 mol, 300 mL, 21.23 eq) was stirred at 0 °C for 1 h.
  • reaction mixture was quenched by ice and water (400 mL) and maintained inter- temperature below 5 °C. The mixture was stirred for 15 min to form a slurry. The slurry was filtered and the filter cake was washed with water (10 mL x3) and air dried to afford amino 2,4,6- trimethylbenzenesulfonate (46 g, crude) as a solid.
  • Step 4 2-(4-bromo-2-pyridyl)acetonitrile
  • a solution of 4-bromo-2-fluoro-pyridine (15 g, 85.2 mmol, 1 eq) and acetonitrile (7 g, 170.5 mmol, 8.97 mL, 2 eq) in THF (200 mL) was cooled to -60 °C, then added LiHMDS (1 M, 29.8 mL, 0.35 eq) dropwise for 1 h. The mixture was stirred at 20 °C for 1 h.
  • Step 5 2-(1-amino-4-bromo-pyridin-1-ium-2-yl)acetonitrile
  • 2-(4-bromo-2-pyridyl)acetonitrile 23 g, 116.73 mmol, 1 eq
  • DCM 100 mL
  • amino 2,4,6-trimethylbenzenesulfonate 46 g, 213.7 mmol, 1.83 eq
  • DCM 150 mL
  • Step 7 methyl 2-aminopyrazolo[1,5-a]pyridine-5-carboxylate
  • Step 8 2-amino-N-methyl-pyrazolo[1,5-a]pyridine-5-carboxamide To a solution of methyl 2-aminopyrazolo[1,5-a]pyridine-5-carboxylate (0.3 g, 1.6 mmol, 1 eq) in EtOH (7 mL) was added MeNH 2 (162.5 mg, 1.6 mmol, 30% purity, 1 eq).
  • Step 9 methyl 2-[tert-butoxycarbonyl-[2-[[5-(methylcarbamoyl)pyrazolo[1,5-a]pyridin-2- yl]amino]ethyl]amino]acetate
  • 2-amino-N-methyl-pyrazolo[1,5-a]pyridine-5-carboxamide 0.1 g, 525.8 ⁇ mol, 1 eq
  • MeOH 3 mL
  • Nitrogen a drop of AcOH (44.2 mg, 736.1 ⁇ mol, 42.14 ⁇ L, 1.4 eq) was added and then was stirred at 0 °C for 0.5 h.
  • Step 10 tert-butyl 4-[5-(methylcarbamoyl)pyrazolo[1,5-a]pyridin-2-yl]-3-oxo-piperazine-1- carboxylate
  • methyl 2-[tert-butoxycarbonyl-[2-[[5-(methylcarbamoyl)pyrazolo[1,5- a]pyridin-2-yl]amino]ethyl]amino]acetate 0.2 g, 493.3 ⁇ mol, 1 eq
  • MeOH 3 mL
  • K2CO3 204.5 mg, 1.5 mmol, 3 eq
  • Step 11 N-methyl-2-(2-oxopiperazin-1-yl)pyrazolo[1,5-a]pyridine-5-carboxamide
  • tert-butyl 4-[5-(methylcarbamoyl)pyrazolo[1,5-a]pyridin-2-yl]-3-oxo- piperazine-1-carboxylate (30 mg, 80.34 ⁇ mol, 1 eq) in TFA (460.50 mg, 4.04 mmol, 0.3 mL, 50.27 eq) and DCM (1 mL) was stirred at 20 °C for 1 h.
  • Step 12 N-methyl-2-(2-oxo-4-prop-2-enoyl-piperazin-1-yl)pyrazolo[1,5-a]pyridine-5- carboxamide
  • Example 51 Synthesis of Compound 187 Step 1: tert-butyl 3-oxo-4-prop-2-ynyl-piperazine-1-carboxylate A solution of tert-butyl 3-oxopiperazine-1-carboxylate (5 g, 24.97 mmol, 1 eq) in DMF (60 mL) was added NaH (1.50 g, 37.46 mmol, 60% purity, 1.5 eq) under N 2 , stirring at 25 °C for 1 h. And then, 3-bromoprop-1-yne (4.08 g, 27.47 mmol, 2.96 mL, 1.1 eq) was added. The reaction mixture was stirred at 25 °C for 0.5 h.
  • Step 2 tert-butyl 4-[[1-(2-methoxy-2-oxo-ethyl)triazol-4-yl]methyl]-3-oxo-piperazine-1- carboxylate
  • tert-butyl 3-oxo-4-prop-2-ynyl-piperazine-1-carboxylate 500 mg, 2.10 mmol, 1 eq
  • methyl 2-azidoacetate 241.5 mg, 2.1 mmol, 1 eq
  • the reaction mixture was stirred at 80 °C for 2 h.
  • Step 3 tert-butyl 4-[[1-[2-(methylamino)-2-oxo-ethyl]triazol-4-yl]methyl]-3-oxo-piperazine-1- carboxylate
  • Step 4 N-methyl-2-[4-[(2-oxopiperazin-1-yl)methyl]triazol-1-yl]acetamide
  • Step 5 N-methyl-2-[4-[(2-oxo-4-prop-2-enoyl-piperazin-1-yl)methyl]triazol-1-yl]acetamide
  • TEA 264.73 mg, 2.6 mmol, 364.1 ⁇ L, 3 eq
  • prop-2-enoyl chloride 78.9 mg, 872.1 ⁇ mol, 70.9 ⁇ L, 1 eq
  • Example 52 Gel-Based Activity-Based Protein Profiling (ABPP) to assess DUB recruiter binding to OTUB1 Recombinant OTUB1 from Sino Biological (12927-H07E) or produced and purified in- house (0.08 ⁇ g/sample) was pre-treated with either DMSO vehicle or covalent ligand or bifunctional compounds at 37°C for 30 min in 49 ⁇ L of PBS, and subsequently treated with of IA- Rhodamine (Setareh Biotech) at room temperature for 1 h. The reaction was stopped by addition of 4 ⁇ reducing Laemmli SDS sample loading buffer (Alfa Aesar).
  • ABPP Gel-Based Activity-Based Protein Profiling
  • Example 53 Intact-MS Assay via RapidFire-TOF Sample Preparation: Compound (1 ⁇ L of 5mM DMSO solution – final concentration 100 ⁇ M) was added to recombinant protein (1 ⁇ M, 49 ⁇ L) in buffer (25 mM Tris HCl; 10 mM NaCl; pH 7.5) in 96 well plates on ice. The reaction was mixed and incubated at 4C for 16 hours. Formic acid solution (1% in water, 50 ⁇ L) was added to quench the reaction and the samples were mixed, then centrifuged for 3 minutes at 3000xg and stored at 4C until analysis.
  • buffer 25 mM Tris HCl; 10 mM NaCl; pH 7.5
  • Sample Analysis A volume of 50 ⁇ L sample was aspirated from the 96-well plate by using an aspiration time of 300 ms, and 10 ⁇ L (limited by the sample loop size) was injected and concentrated on a C4 SPE cartridge (Agilent #G9203A, 20 ⁇ m, 4 ⁇ L).
  • the sample load/wash time was 8000 ms at a flow rate of 0.6 mL/min (10% acetonitrile with 0.1% formic acid); elution time was 7000 ms at a flow rate of 0.5 mL/min (80% acetonitrile, 0.1% formic acid); re-equilibration time was 500 ms at a flow rate of 0.6 mL/min (10% acetonitrile, 0.1% formic acid).
  • An Agilent 6230 TOF mass spectrometer was operated with a dual Agilent Jet Spray (AJS) ion source in the positive ionization mode and 3200 m/z range.
  • AJS Agilent Jet Spray
  • the source parameters were as follows: gas temperature 325 °C, drying gas 8 L/min, nebulizer 35 psi, sheath gas 350 °C, sheath gas flow 11 L/min, capillary 3500V, nozzle 1000 V, fragmentor 175 V, and skimmer 65 V.
  • Data Analysis Data files were parsed into individual injections using the RapidFire UI software. In Agilent BioConfirm 12.0 TICs for individual injection files were integrated (with only one peak allowed per injection) and MS spectra extracted, using a designated background time range of 0.0 – 0.05 min.
  • Decovolution for OTUB1 samples was performed using the maximum entropy method, filtering for peak signal-to-noise of >30, calculating average mass using the top 25% of peak height, using an explicit output mass range of 28,000 – 40,000 Da and a mass step of 0.2 Da, using a limited input range of 850 – 1800 m/z, and subtracting baseline with a baseline factor of 1.0.
  • Biomolecule filters were set to a minimum consecutive charge states of 4, minimum fit score of 2, and minimum height threshold of 5%. Deconvolution was followed by bulk export of “Biomolecules” csv lists.
  • Peaks present in the Biomolecules lists were compared to expected masses of protein + compound adducts for up to 5 modifications allowing for a window of +/- 2 amu in Excel. Percent modification values were calculated by comparing the peak height for a given species (i.e. a given degree of modification) versus the total peak height of unmodified and modified protein peaks in the deconvoluted spectrum. These results are summarized in Table 4, wherein compounds that demonstrated less than 20% modification for a single cysteine are annotated “ ; and compounds that demonstrated more than 20% modification for a single cysteine are annotated “++”. Table 4.
  • Example 53 Deubiquitinase Activity Assay Recombinant OTUB1 (500 nM) was pre-incubated with DMSO or Compound 100 (50 mM) for 1 hr. To initiate assay pre-treated OTUB1 enzyme was mixed 1:1 with di-Ub reaction mix for final concentrations of 250 nM OTUB1, 1.5 ⁇ M di-Ub, 12.5 ⁇ M UBE2D1 and 5 mM DTT. The appearance of mono-Ub was monitored by Western blotting over time by removing a portion of the reaction mix and adding Laemmli’s buffer to terminate the reaction.
  • TBS-T Tris- buffered saline containing Tween 20
  • the membranes were incubated in the dark with IR680- or IR800- conjugated secondary antibodies at 1:10,000 dilution in 5 % BSA in TBS-T at room temperature for 1 h. After 3 additional washes with TBST, blots were visualized using an Odyssey CLx Li-Cor fluorescent scanner. The membranes were stripped using ReBlot Plus Strong Antibody Stripping Solution (EMD Millipore) when additional primary antibody incubations were performed.
  • EMD Millipore ReBlot Plus Strong Antibody Stripping Solution
  • Antibodies used in this study were CFTR (Cell Signaling Technologies, Rb mAb #78335), GAPDH (Proteintech, Ms mAb, #60004-1-Ig), OTUB1 (Abcam, Rb mAb, #ab175200, [EPR13028(B)]).
  • target protein e.g. CFTR
  • target protein e.g. CFTR
  • Simple Western on a Jess instrument Protein Simple
  • DMTP01 total protein detection kit
  • Example 54 Quantitative TMT Proteomics Analysis Quantitative TMT-based proteomic analysis will be performed to assess relative protein abundance.
  • CFBE41o-4.7 Assessing protein abundance in ⁇ F508-CFTR human cystic fibrosis bronchial epithelial cells CFBE41o-4.7 with treatment of exemplary bifunctional compounds. CFBE41o-4.7 cells were seeded at 600k cells per 6cm 2 plate and were treated with either DMSO vehicle or 10uM DUBTAC (total of 0.1% DMSO) for 24 hours, then harvested, lysed, and cell lysate normalized to total protein content. Proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes.
  • Membranes were blocked with 5% BSA in Tris-buffered saline containing Tween 20 (TBS-T) solution for 30 min at room temperature, washed in TBS-T and probed with primary antibody against CFTR (Cell Signaling Technologies, rabbit monoclonal antibody 78335, 1:1,000 dilution in 5% BSA) and GAPDH (Proteintech, mouse monoclonal antibody, 1:10,000 dilution in 5% BSA, 60004-1-Ig), and incubated overnight at 4 °C.
  • TBS-T Tris-buffered saline containing Tween 20
  • the membranes were incubated in the dark with IR680- or IR800-conjugated secondary antibodies at a 1:10,000 dilution in 5% BSA in TBS-T at room temperature for 1 h.
  • blots were visualized using an Odyssey CLx Li-Cor fluorescent scanner. CFTR abundance was quantified relative to GAPDH control.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des composés bifonctionnels, ainsi que des sels, des hydrates, des solvates, des promédicaments, des stéréoisomères ou des tautomères pharmaceutiquement acceptables de ceux-ci, qui fonctionnent pour recruter certaines désubiquitinases au niveau d'une protéine cible à des fins de modulation (par exemple, stabilisation) de la protéine cible, ainsi que des procédés d'utilisation de ceux-ci.
PCT/US2023/036699 2022-11-02 2023-11-02 Chimères ciblant la désubiquitinase et procédés associés WO2024097355A1 (fr)

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