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WO2024233280A1 - Petites molécules hétérobifonctionnelles pour la déubiquitination et la stabilisation de protéines cibles par recrutement de usp7 - Google Patents

Petites molécules hétérobifonctionnelles pour la déubiquitination et la stabilisation de protéines cibles par recrutement de usp7 Download PDF

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WO2024233280A1
WO2024233280A1 PCT/US2024/027532 US2024027532W WO2024233280A1 WO 2024233280 A1 WO2024233280 A1 WO 2024233280A1 US 2024027532 W US2024027532 W US 2024027532W WO 2024233280 A1 WO2024233280 A1 WO 2024233280A1
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optionally substituted
membered
cycloalkyl
alkyl
cancer
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Jian Jin
Wenyi WEI
Yan Xiong
Jing Liu
Kaixiu LUO
Xiaoping Hu
Zhen Wang
Li Chen
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Beth Israel Deaconess Medical Center Inc
Icahn School of Medicine at Mount Sinai
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Beth Israel Deaconess Medical Center Inc
Icahn School of Medicine at Mount Sinai
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    • 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
    • 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
    • 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
    • C07D405/14—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 three or more hetero rings
    • 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
    • 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
    • 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

Definitions

  • the present disclosure is directed to heterobifunctional small-molecule compounds that recruit USP7, a deubiquitinase, to deubiquitinylate and stabilize a target protein such as AMPK, cGAS and CFTR, and to methods of use of the heterobifunctional small-molecule compounds for the treatment of AMPK, cGAS, or CFTR-mediated diseases in a subject in need thereof.
  • the disclosure also related to methods for identifying such heterobifunctional small-molecule compounds.
  • BACKGROUND Protein ubiquitination plays a critical role in a variety of cellular process, like protein degradation, quality control, trafficking, and signaling. Therefore, controlling the ubiquitination level of disease-relevant proteins can achieve substantial therapeutic effects.
  • Ubiquitin-specific-processing protease 7 (USP7), also known as herpesvirus-associated ubiquitin-specific protease (HAUSP), belongs to the deubiquitinase (DUB) family. USP7 is Attorney Docket No.: 27527-0219WO1 ubiquitously expressed in cells and has low tissue specificity. Therefore, USP7 is an excellent DUB to be recruited to achieve targeted protein stabilization (TPS).
  • TPS targeted protein stabilization
  • adenosine monophosphate-activated protein kinase also known as AMPK
  • AMPK 5 ⁇ adenosine monophosphate-activated protein kinase
  • cGAMP cyclic GMP-AMP synthase
  • the cGAS-stimulator of interferon genes (STING) pathway is a component of the innate immune system. cGAS detects the presence of cytosolic DNA and, in response, triggers expression of inflammatory genes that can lead to senescence or activation of defense mechanisms. In tumor cells, the cGAS-STING pathway exerts its antitumor effects through the induction of a robust type I IFN response, which activates immune cells, particularly dendritic cells (DCs), in the tumor micro environment (TME) (Samson and Ablasser, 2022). Besides AMPK and cGAS, cystic fibrosis transmembrane conductance regulator (CFTR) is another suitable protein target for TPS.
  • CFTR ?F508 mutation destabilizes CFTR, and leads to the cystic fibrosis phenotype (Ward et al., 1995). Therefore, deubiquitination of ?F508 mutant CFTR by heterobifunctional small-molecule compounds can be an effective strategy to prevent the cystic fibrosis process.
  • the targeted protein degradation (TPD) field has made great progress over the past 10 years.
  • the PROteolysis TArgeting Chimera (PROTAC) technology has been widely utilized in drug discovery field (Dale et al., 2022).
  • Deubiquitinase-targeting chimera which is a heterobifunctional small-molecule compound that hijacks a cellular deubiquitinase to remove the polyubiquitin attached to the target protein by inducing the close proximity between the target protein and deubiquitinase, recently emerged as an effective technology to improve target protein stability by reducing the proteasomal degradation of the target protein (Henning et al., 2022; Liu et al., 2022). To date, only OTUB1-recruiting DUBTACs have been reported (Henning et al., 2022; Liu et al., 2022).
  • DUBTACs or heterobifunctional small-molecule compounds that recruit USP7 to deubiquitinylate and stabilize a target protein are unprecedented.
  • Attorney Docket No.: 27527-0219WO1 SUMMARY The ubiquitination and degradation of many proteins is the main cause of numerous diseases. Therefore, de-ubiquitination and stabilization of these disease-relevant proteins by DUBTACs or heterobifunctional small-molecule compounds can provide a novel therapeutic strategy.
  • this disclosure provides a method of treating AMPK, cGAS, or ?F508-CFTR mediated diseases, the method including administering one or more AMPK, cGAS, or CFTR DUBTACs to a subject who has an AMPK, cGAS, or ?F508-CFTR -mediated disease, the AMPK, cGAS, or CFTR DUBTACs being heterobifunctional small-molecule compounds (or bivalent compounds in short) including an AMPK, cGAS, or CFTR ligand conjugated to a de- ubiquitination tag (such as a small-molecule ligand of USP7) via a linker, which would stabilize AMPK, cGAS, or CFTR.
  • a de- ubiquitination tag such as a small-molecule ligand of USP7
  • the AMPK, cGAS, or ?F508-CFTR -mediated disease can be a disease resulting from AMPK, cGAS, or CFTR destabilization.
  • the AMPK, cGAS, or ?F508-CFTR-mediated disease can have reduced AMPK, cGAS, or CFTR expression relative to a wild-type tissue of the same species and tissue type. More specifically, The invention disclosed here provides bivalent compounds that stabilize AMPK, cGAS, or CFTR by recruiting USP7.
  • the AMPK, cGAS, or CFTR DUBTACs have the form “PI-Linker-USP7 Recruiter”, as shown below: PI Linker USP7 Recruiter wherein PI (a ligand for a “protein of interest,” i.e., the protein to be de-ubiquitination) (e.g., a CFTR, AMPK or cGAS ligand), and USP7 recruiter comprises a de-ubiquitination tag by binding with USP7.
  • PI a ligand for a “protein of interest,” i.e., the protein to be de-ubiquitination
  • USP7 recruiter comprises a de-ubiquitination tag by binding with USP7.
  • exemplary CFTR, AMPK or cGAS ligands (PI), exemplary USP7 ligands (USP7 recruiter), and exemplary linkers (Linker) are illustrated below: PI Attorney Docket No.: 27527-0219WO1 AMPK ligand
  • the AMPK ligand can be an AMPK activator or AMPK inhibitor which potently binds to AMPK.
  • the AMPK ligand comprises Metformin (Xiao et al., 2020), AICAR (Nakamaru et al., 2005), ZMP, AMP, A-769662 (Sanders et al., 2007), 991 (Ngoei et al., 2018), PF-06685249 (Edmonds et al., 2018), PXL770 (Cusi et al., 2021), MK- 8722 (Myers et al., 2017), PF-739 (Aledavood et al., 2021), 4-azaindole, RSVA405 (Vingtdeux et al., 2011), COH-SR4 (Figarola and Rahbar, 2013), B10 (Sun et al., 2020), Dorsomorphin (Kim et al., 2011), BAY-3827 (Lemos et al., 2021), GSK-690693 (Rho
  • the AMPK ligand is a compound disclosed in one or more of WO2009124636, WO2009100130, WO2010036613, WO2011029855, WO2011080277, WO2011032320, WO2013116491, WO2014133008, WO2016008404, and WO2016001224, each of which is incorporated by reference in its entirety.
  • the AMPK ligand is an AMPK inhibitor.
  • the AMPK ligand is an ATP-competitive AMPK inhibitor.
  • the AMPK ligand is an AMPK activator.
  • the AMPK ligand is an AMPK activator, where the AMPK activator binds to an allosteric binding site. In some embodiments, the AMPK activator binds to an allosteric binding site located at the ?/? subunit interface. In some embodiments, the AMPK activator has different AMPK isoform selectivity. In some embodiments, the AMPK activator is a pan-AMPK isoform activator.
  • the AMPK ligand comprise the structure of Formula (B-I): , or a acceptable salt of the compound or the tautomer thereof, wherein A A is selected ; BA is selected ; Attorney Docket No.: 27527-0219WO1 denotes the point of attachment to Linker in Formula (A); CA is selected from N or CRA 4 ; Ring D A and Ring E A are independently selected from null, C 3 -C 12 cycloalkyl, 3-12- membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl; X A and Y A are independently selected from a bond, -O-, -S-, -NR A 9 -, -C(O)-, -C(O)O-, - C(O)NR A 9 -, -O-C(O)NR A 9 -, -NR A 10 C(O)NR A 9 -, -S(O)-, -S(O)
  • AA ; and BA is N.
  • the AMPK ligand comprise the structure of Formulae (B-I-a) and Formulae (B-I-b) : wherein AA and BA are independently selected from N, CRA 4 .
  • X A is selected from -O-, -S-, -NR A 9 -, -C(O)-, C 1 -C 6 alkylene, C 1 - C 6 haloalkylene, C 1 -C 6 heteroalkylene, C 6 -C 10 arylene, and 5-10 membered heteroarylene.
  • XA is selected from -O-, -S-, -NRA 9 -, -C(O)-, methylene, and halomethylene
  • X A is selected from -O-, -S-, -NH-, -C(O)-, CH 2 , and CF 2 .
  • XA is -O-.
  • YA is selected from a bond, -C(O)-, -C(O)O-, -C(O)NRA 9 -, -S(O)-, -S(O)2-, -S(O)NRA 9 -, -S(O)2NRA 9 -.
  • YA is selected from a bond, -C(O)-, - C(O)NR A 9 -, and -S(O) 2 NR A 9 -.
  • Y A is -C(O)NR A 9 -.
  • the AMPK ligand comprise the structure of Formulae (B-I-a1), (B- I-b1), and (B-I-b2): , , .
  • a A is N.
  • a A is CR A 4 .
  • BA is N.
  • BA is CRA 4 .
  • CA is N.
  • CA is CRA 4 .
  • Ring D A is selected from null, C 3 -C 12 cycloalkyl, 3-12-membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl.
  • Ring D A is C 3 - C12 cycloalkyl.
  • Ring DA is selected from cyclobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • Ring D A is 3-12- membered heterocyclic.
  • Ring DA is selected from tetrahydrofuryl, hexahydrofuro[3,2-b]furyl, azetidinyl, pyrrolidinyl, piperidinyl, and piperazinyl.
  • Ring DA is C6-C10 aryl.
  • Ring DA is phenyl.
  • Ring D A is 5-10 membered heteroaryl.
  • Ring D A is selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, thiazolyl, imidazolyl, furanyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, indolyl, and indazolyl.
  • Ring EA selected from null, C3-C12 cycloalkyl, 3-12-membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl.
  • Ring EA selected from null, C 6 -C 10 aryl, and 5-10 membered heteroaryl. In some embodiments, Ring E A selected from null. In some embodiments, Ring E A is C 6 -C 10 aryl. In some embodiments, Ring E A is phenyl. In some embodiments, Ring EA is 5-10 membered heteroaryl.
  • Ring EA is selected from thiophenyl, benzothiophenyl, tetrahydrobenzothiophenyl, thiazolyl, imidazolyl, Attorney Docket No.: 27527-0219WO1 furanyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, indolyl, and indazolyl.
  • the AMPK ligand comprise the structure of Formulae (B-I-a2), (B- I-a3), (B-I-a4), (B-I-b3), and (B-I-b4): wherein FA is selected from N or CRA 5 ; and GA is selected from N or CRA 7 .
  • R A 3 is selected from C 3 -C 12 cycloalkyl, 3-12 membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl.
  • R A 3 is selected from C 6 -C 10 aryl and 5-10 membered heteroaryl.
  • the AMPK ligand comprise the structure of Formulae (B-I-a5), (B- I-a6), (B-I-a7), (B-I-b5), and (B-I-b6):
  • ArA is selected from C6-C10 aryl and 5-10 membered heteroaryl. In some embodiments, ArA is selected from phenyl, naphthalene, indane, 5,6,7,8- tetrahydronaphthalene, biphenyl, pyrazole, pyridine, pyrazine, pyrimidine, thiazole, thiophene, benzoimidazole, quinoline, isoquinoline, indole, indazole, carbazole, benzotriazole, benzofuran, benzothiazole, benzo[b]thiophene, benzo[d]isooxazole, 3,4-dihydro-2H-benzo[1,4]oxazine, benzo[1,3]dioxole, benzo[1,4]dioxane, 1H-pyrrolo[2,3-b]pyridine, [1,2,4]triazolo[4,3-a]
  • Ar A is selected from phenyl, pyridine, biphenyl, and indole. In some embodiments, ArA is indole. In some embodiments, RA 6 is selected from a bond, -O-, -NRA 13 -, -C(O)-, -C(O)O-, - C(O)NR A 13 -, -S(O) 2 NR A 13 -, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, and C 1 -C 6 heteroalkylene. In some embodiments, RA 6 is selected from -C(O)-, -C(O)O-, and -C(O)NRA 13 -.
  • R A 6 is -C(O)NR A 13 -.
  • the N atom of the amide attaches two Linker-USP7 recruiter moieties.
  • RA 8 is selected from a bond, -O-, -NRA 13 -, -C(O)-, -C(O)O-, - C(O)NR A 13 -, -S(O) 2 NR A 13 -, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, and C 1 -C 6 heteroalkylene.
  • RA 1 is selected from H, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, RA 1 is selected from H and CH3. In some embodiments, RA 1 is H. In some embodiments, RA 1 is CH3. In some embodiments, R A 2 is selected from H, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • RA 2 is selected from halogen. In some embodiments, R A 2 selected from F or Cl. In some embodiments, R A 2 is F. In some embodiments, R A 2 is Cl. In some embodiments, each RA 4 is selected from H, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, R A 4 is H.
  • each R A 5 is selected from H, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each R A 5 is selected from H, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl. In some embodiments, R A 5 is H, CH 3 , CH 2 CH 3 , and CH 2 OH.
  • each RA 7 is selected from H, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R A 7 is selected from H, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C3-C12 cycloalkyl.
  • RA 7 is H, CH3, and CH2CH3.
  • each RA 9 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl. In some embodiments, each RA 9 is independently selected from H, C1- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, R A 9 is H.
  • each R A 10 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, C6-C10 aryl, and Attorney Docket No.: 27527-0219WO1 5-10 membered heteroaryl.
  • each R A 10 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • RA 10 is H.
  • each R A 11 and each R A 12 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, C 6 - C10 aryl, and 5-10 membered heteroaryl.
  • each RA 11 and each RA 12 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • R A 11 is selected from H and CH 3 .
  • RA 12 is selected from H and CH3. In some embodiments, RA 11 and RA 12 together with the atoms to which they are attached, optionally form 3-12 membered heterocyclic. In some embodiments, each RA 13 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl.
  • each R A 13 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R A 14 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl.
  • each RA 14 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each RA 15 is independently selected from each RA 15 is independently selected from H, halogen, cyano, ORA 11 , NRA 11 RA 12 , C(O)ORA 11 , C(O)NRA 11 RA 12 , S(O) 2 NR A 11 R A 12 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, 3-12 membered heterocyclic, C 6 -C 10 aryl and 5-10 membered heteroaryl.
  • Attorney Docket No.: 27527-0219WO1 In some embodiments, m A is selected from 0, 1, 2, 3, and 4. In some embodiments, m A is selected from 0, 1, and 2. In some embodiments, nA is selected from 0, 1, 2, 3, and 4. In some embodiments, nA is selected from 0, 1, and 2. In some embodiments, oA is selected from 0, 1, 2, 3, 4, and 5.
  • the AMPK ligand comprise the structure of Formulae (B-I), (B-I- a), (B-I-a1), (B-I-a2) and (B-I-a5). In some embodiments, the AMPK ligand comprise a derivative of following compounds:
  • the cGAS ligand is selected from a cGAS activator or cGAS inhibitor which potently binds with cGAS. In some embodiments, the cGAS ligand is cGAS activator. In some embodiments, the cGAS ligand is cGAS inhibitor.
  • the cGAS ligand comprises hydroxychloroquinine (HCQ), Quinacrine (QC), X6, RU114757, RU191752, RU100840, RU.365, RU.521, J001, G001, G108, G150, G140, CPD-25, asprin, CPD- C, PF-06928215(Zhao et al., 2022).
  • the cGAS ligand is a compound disclosed in one or more of U.S. 62/318,435, U.S. 62/355,403, U.S.
  • the cGAS ligand comprise the structure of Formula (C-I): acceptable salt of the compound or the tautomer wherein denotes the point of attachment to Linker in Formula (A); Attorney Docket No.: 27527-0219WO1 connects to RB 1b , R B 3 is R B 3a ; and when connects to R B 3b , R B 1 is R B 1a ; A B is selected from N and CR B 4 ; BB is selected from N, O, S, CRB 5 and NRB 5 ; CB is selected from N and C; D B is selected from N and C; X B is X B a -X B b ; XB a is selected from a bond, -C(O)-, -C(O)O-, -C(C(A)
  • the cGAS ligand comprise the structure of Formulae (C-I-a) and (C-I-b):
  • a B is CR B 4 .
  • B B is NR B 5 .
  • CB is C.
  • DB is C.
  • the cGAS ligand comprise the structure of Formulae (C-I-a1) and (C-I-b1): , .
  • RB 1a is selected from H, halogen, C3-C12 cycloalkyl, 3-12 membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl.
  • R B 1a is selected from C3-C12 cycloalkyl, 3-12 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, RB 1a is selected from C6-C10 aryl, and 5-10 membered heteroaryl. In some embodiments, R B 1a is 5-10 membered heteroaryl.
  • R B 1a is selected from furyl, thiophenyl, pyrrolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.
  • RB 1a is pyrazolyl.
  • RB 1a pyridinyl.
  • R B 1b is selected from C 3 -C 12 cycloalkylene, 3-12 membered heterocyclicene, C6-C10 arylene, and 5-10 membered heteroarylene. In some embodiments, RB 1b is selected from C6-C10 arylene, and 5-10 membered heteroarylene.
  • the cGAS ligand comprise the structure of Formulae (C-I-a2) and (C-I-b2):
  • each RB 11 is independently selected from H, halogen, cyano, ORB 7 , NRB 7 RB 8 , C(O)ORB 7 , C(O)NR B 7 R B 8 , S(O) 2 NR B 7 R B 8 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, C 3 -C 12 cycloalkyl, 3-12 membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl; or two R B 11 , together with the atoms to which they are attached, optionally form partially unsaturated C 3 -C 12 cycloalkyl, partially unsaturated 3-12 membered heteroaryl;
  • XB a is selected from -C(O)-, -S(O)-, and -S(O)2-. In some embodiments, XB a is -C(O)-. In some embodiments, XB b is selected from C1-C6 alkylene, C1-C6 haloalkylene, C 1 -C 6 heteroalkylene, C 3 -C 12 cycloalkylene, and 3-12 membered heterocyclicene.
  • the cGAS ligand comprise the structure of Formulae (C-I-a3) and (C-I-b3): each RB 12 is independently selected from H, halogen, ORB 7 , NRB 7 RB 8 , C1-C6 alkyl, C1-C6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic; or two R B 12 , R B 12 and R B 3a , and R B 12 and R B 3b , together with the atoms to which they are attached, optionally form C3-C12 cycloalkyl, 3-12 membered heterocyclic; and qB is slected from 0, 1, 2, 3, 4, and 5.
  • Ar B is 5-10 membered heteroaryl.
  • Ar B is selected from furyl, thiophenyl, pyrrolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, isoxazolyl, Attorney Docket No.: 27527-0219WO1 isothiazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.
  • ArB is pyrazolyl.
  • ArB is pyridinyl.
  • the cGAS ligand comprise the structure of Formulae (C-I-a4) and (C-I-b4): , .
  • each RB 2 is independently selected from H, C1-C3 alkyl, C1-C3 haloalkyl, C 1 -C 3 heteroalkyl, C 3 -C 6 cycloalkyl.
  • R B 2 is H.
  • R B 2 is C 1 -C 3 alkyl.
  • R B 2 is CH 3 .
  • two RB 2 together with the atoms to which they are attached, optionally form C3-C6 cycloalkyl or 3-6 membered heterocyclic.
  • R B 2 together with the atoms to which they are attached, optionally form C 3 -C 5 cycloalkyl.
  • RB 3a is selected from H, halogen, ORB 9 , NRB 9 RB 10 , C1-C6 alkyl, C1- C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • R B 3a is selected from H, OR B 9 and NR B 9 R B 10 .
  • R B 3a is selected from H, OH, OCH3, OCH2CH2OCH3, NH2, NHCH3, and NHC(O)CH2NH2.
  • RB 3a is OH.
  • R B 3b is selected from a bond, -O-, -NR B 9 -, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, C1-C6 heteroalkylene, C3-C12 cycloalkylene, and 3-12 membered heterocyclicene.
  • RB 3b is selected from a bond, -O-, -NRB 9 -.
  • RB 3b is selected from a bond, -O-, -NH-, and -N(CH 3 )-. In some embodiments, R B 3b is -O-. In some embodiments, each R B 4 is independently selected from H, halogen, cyano, OR B 7 , NRB 7 RB 8 , C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, and 3-6 membered heterocyclic. In some embodiments, each RB 4 is independently selected from H. In some embodiments, each R B 4 is independently selected from cyano.
  • each RB 4 is independently selected from halogen. In some embodiments, each RB 4 Attorney Docket No.: 27527-0219WO1 is independently selected from F. In some embodiments, each R B 4 is independently selected from Cl. In some embodiments, each RB 4 is independently selected from methyl, ethyl, propyl, isopropyl, cyclopropyl, trifluoromethyl, difluoromethyl, methoxy, ethoxy, cyclopropoxy, and azetidinyl. In some embodiments, each R B 4 is independently selected from H. In some embodiments, each R B 4 is independently selected from cyano. In some embodiments, each R B 4 is independently selected from halogen.
  • each RB 4 is independently selected from F. In some embodiments, each R B 4 is independently selected from Cl. In some embodiments, each R B 4 is independently selected from Br. In some embodiments, two RB 4 , together with the atoms to which they are attached, optionally form partially unsaturated C3-C6 cycloalkyl, partially unsaturated 3-6 membered heterocyclic, C 6 -C 10 aryl, or 5-10 membered heteroaryl. In some embodiments, RB 5 is selected from H, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, C3-C6 cycloalkyl, and 3-6 membered heterocyclic.
  • RB 5 is selected from H and C 1 -C 3 alkyl.
  • R B 5 is H.
  • R B 5 is CH3.
  • RB 6 , each RB 7 , each RB 8 , each RB 9 , and each RB 10 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • R B 6 , each R B 7 , each RB 8 , each RB 9 , and each RB 10 are independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, trifluoromethyl, and difluoromethyl.
  • RB 6 , each RB 7 , each RB 8 , each R B 9 , and each R B 10 are H.
  • R B 6 , each R B 7 , each R B 8 , each R B 9 , and each RB 10 are methyl.
  • each R B 11 is independently selected from H, halogen, cyano, OR B 7 , NRB 7 RB 8 , C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic.
  • R B 11 is H. In some embodiments, each R B 11 is CH3.
  • each R B 12 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each RB 12 is independently selected from H, and C1-C6 alkyl.
  • R B 12 is H. In some embodiments, two R B 12 , R B 12 and R B 3a , and R B 12 and R B 3b , together with the atoms to which they are attached, optionally form C3-C6 cycloalkyl, 3-6 membered heterocyclic.
  • m B is 1. In some embodiments, n B is 1. In some embodiments, n B is 2. In some embodiments, o B is 0. In some embodiments, o B is 1. In some embodiments, o B is 2. In some embodiments, pB is 0. In some embodiments, pB is 1. In some embodiments, pB is 2. In some embodiments, pB-1 is 0. In some embodiments, pB-1 is 1.
  • the cGAS ligand comprise the structure of Formulae (C-I), (C-I-a), (C-I-a1), (C-I-a2), (C-I-a3), and (C-I-a4).
  • the cGAS ligand may be a derivative of following compounds:
  • the CFTR ligand can be a CFTR potentiator which potently binds with either wild type CFTR or mutant CFTR.
  • the CFTR ligand comprises ivacftor, lumacaftor, tezacaftor, elexacafor, or icenticaftor, or derivative thereof.
  • the CFTR ligand is a compound disclosed in one or more of U.S. Patent No. 7,999,113; U.S. Patent No.8,247,436; U.S.8,410,274; WO 2011/133953; and WO2018/037350, each of which is incorporated by reference in its entirety.
  • the CFTR ligand comprise the structure of Formula (D-I): or a pharmaceutically acceptable salt of the wherein denotes the point of attachment to Linker in Formula (A).
  • CC are independently selected from O, S, or C(RC 8 )(RC 9 ); C(RC 10 )(RC 11 ) or NRC 12 ; Attorney Docket No.: 27527-0219WO1
  • Ar C 1 and Ar C 2 are independently selected from null, C 6 -C 10 aryl and 5-10 membered heteroaryl;
  • RC 1 and RC 2 are independently selected from H, halo, ORC 13 , NRC 13 RC 14 , C(O)ORC 13 , C(O)NR C 13 R C 14 , S(O) 2 NR C 13 R C 14 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 al
  • ArC 1 is 5-10 membered heteroaryl. In some embodiments, ArC 1 is selected from furyl, thiophenyl, pyrrolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, isoxazolyl, Attorney Docket No.: 27527-0219WO1 isothiazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl. In some embodiments, ArB is pyridinyl. In some embodiments, ArC 2 is C6-C10 aryl. In some embodiments, ArC 2 is phenyl.
  • the CFTR ligand comprise the structure of Formula (D-I-a): , or a pharmaceutically acceptable salt of the compound or the tautomer. In some embodiments, the CFTR ligand comprise the structure of Formula (D-I-a1): or a pharmaceutically acceptable salt of the compound or the tautomer.
  • AC is O.
  • BC is C(RC 10 )(RC 11 ). In some embodiments, CC is O.
  • the CFTR ligand comprise the structure of Formula (D-I-a2): a pharmaceutically acceptable salt of the
  • R C 1 is selected from H, C 1 -C 3 alkyl, C 2 -C 3 alkenyl, C 2 -C 3 alkynyl, C 3 -C 5 cycloalkyl, and 3-5 membered heterocyclic.
  • R C 2 is selected from H, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C5 cycloalkyl, and 3-5 membered heterocyclic.
  • R C 1 and R C 2 together with the atoms to which they are attached, optionally Attorney Docket No.: 27527-0219WO1 form C 3 -C 6 cycloalkyl.
  • R C 1 and R C 2 together with the atoms to which they are attached, optionally form cyclopropyl.
  • RC 3 is selected from H, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 heteroalkyl. In some embodiments, R C 3 is H.
  • each R C 4 is independently selected from H, halo, cyano, OR C 13 , NRC 13 RC 14 , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each R C 4 is independently selected from H and C 1 -C 6 alkyl. In some embodiments, each R C 4 is independently selected from H and CH 3 .
  • each RC 5 is independently selected from H, halo, cyano, ORC 13 , NRC 13 RC 14 , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R C 5 is independently selected from H and C 1 -C 6 alkyl.
  • RC 5 is H.
  • RC 6 is selected from H, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 heteroalkyl.
  • R C 6 is H.
  • R C 3 is CH 3 .
  • each RC 7 is independently selected from H, halogen, cyano, ORC 13 , NRC 13 RC 14 , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R C 7 is independently selected from H and C 1 -C 6 alkyl. In some embodiments, R C 7 is H.
  • RC 8 and RC 9 are independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • R C 8 and R C 9 are independently selected from H and C 1 -C 6 alkyl
  • RC 10 and RC 11 are independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • R C 10 and R C 11 are independently selected from H, halogen and C 1 -C 6 alkyl. In some embodiments. R C 10 and R C 11 are independently selected from H, F, Cl, CH 3 , and CF 3 . In some embodiments, both RC 10 and RC 11 are F. In some embodiments, R C 12 is selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • RC 13 and RC 14 are independently selected from H, C1-C6 alkyl, C1- C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • R C 13 and R C 14 are independently selected from H and C 1 -C 6 alkyl.
  • RC 13 and RC 14 are independently selected from H and CH3.
  • RC 13 and RC 14 together with the atoms to which they are attached, optionally form 3-6 membered heterocyclic.
  • m C is 1.
  • n C is 0.
  • the CFTR ligand comprise the structure of Formulae (D-I-a3) and (D-I-a4): . .
  • the CFTR ligand may be a derivative of following compounds:
  • the AMPK, cGAS or CFTR ligand is conjugated to the de-ubiquitination tag through a linker.
  • the linker can include, for example, acyclic or cyclic saturated or unsaturated carbon, ethylene glycol, amide, amino, ether, urea, carbamate, aromatic, heteroaromatic, heterocyclic and/or carbonyl containing groups with different lengths.
  • the linker can be a moiety of Formula (L): wherein A, W and B, at each occurrence, are independently selected from null, or bivalent moiety selected from R ’ -R ’’ , R ’ COR ’’ , R ’ CO2R ’’ , R ’ C(O)N(R 1 )R ’’ , R ’ C(S)N(R 1 )R ’’ , R ’ OR ’’ , R ’ OC(O)R ’’ , R ’ OC(O)OR ’’ , R ’ OCON(R 1 )R ’’ , R ’ SR ’’ , R ’ SOR ’’ , R ’ SO 2 R ’’ , R ’ SO 2 N(R 1 )R ’’ , R ’ N(R 1 )R ’’ , R ’ NR 1 COR ’’ , R ’ NR 1 C(O
  • the linker moiety is of Formula (L-1)
  • R 1 , R 2 , R 3 and R 4 at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C 1 -C 8 alkoxyalkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, and optionally substituted C1-C8 alkylaminoC1-C8 alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted Attorney Docket No.: 27527-0219WO1 3-8 membered cycloalkoxy, optionally substituted 3-10 membere
  • the linker moiety is of Formula (L-2) wherein R 1 and R 2 , at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, and optionally substituted C1-C8 alkyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxy C1-C8 alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, C1- C 8 alkylaminoC 1 -C 8 alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted 3-8 membered cycloalkoxy, optionally substituted 3-10 membered carbocyclylamino, optionally substituted 4-10 membered heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl, or R 1 and R 2 together with the
  • the linker moiety is of Formula (L-3): Attorney Docket No.: 27527-0219WO1 Formula (L-3) wherein X is selected from O, NH, and NR 7 ; R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 , at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C1-C8 alkoxy C1-C8 alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 1 -C 8 alkylamino, optionally substituted C1-C8 alkylaminoC1-C8
  • m and n is 0 or 1, and p is 0 to 15;
  • X is selected from O and NH;
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, and optionally substituted C 1 -C 6 alkyl.
  • the linker moiety comprises a ring selected from the group consisting of a 3 to 13 membered ring, a 3 to 13 membered fused ring, a 3 to 13 membered bridged ring, and a 3 to13 membered spiro ring.
  • the linker moiety comprises a ring selected from the group consisting of Formula W1, W2, W3, W4 and W5: Attorney Docket No.: 27527-0219WO1
  • X ⁇ and Y ⁇ are independently selected from N and CR b ;
  • a 1 , B 1 , C 1 and D 1 are independently selected from null, O, CO, SO, SO2, NR b , and CR b R c ;
  • a 2 , B 2 , C 2 and D 2 at each occurrence, are independently selected from N and CR b ;
  • a 3 , B 3 , C 3 , D 3 , and E 3 at each occurrence, are independently selected from N, O, S, NR b , and CR b ;
  • R b and R c at each occurrence, are independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, optionally substituted
  • A, B, and W, at each occurrence, are independently selected from null, optionally substituted -(CH2)0-8-, optionally substituted -(CH2)0-8-CO-(CH2)0-8-, optionally Attorney Docket No.: 27527-0219WO1 substituted -(CH 2 ) 0-8 -NH-(CH 2 ) 0-8 -, optionally substituted -(CH 2 ) 0-8 -NH-CO-(CH 2 ) 0-8 -, optionally substituted -(CH2)0-8-CO-NH-(CH2)0-8-, optionally substituted -(CH2)0-3-NH-(CH2)0-3-CO-NH- (CH2)0-8-, optionally substituted -(CH2)0-3-NH-(CH2)1-3-NH-CO-(CH2)0-8-, optionally substituted - (CH 2 ) 0-8 -CO-NH-(CH 2 ) 1-3 -NH-(CH 2 ) 0-3
  • R r is of Formula W1, W2, W3, W4, or W5. In one embodiment, R r is selected from , , , , , , , , Attorney Docket No.: 27527-0219WO1 , , , , , , In another embodiment, the length of the linker is 0 to 20 atoms. In another embodiment, the length of the linker is 0 to 10 atoms. In another embodiment, the length of the linker is 0 to 40 atoms.
  • the linker is selected from null, optionally substituted -CO-(CH2)0- 10-, optionally substituted -(CH2)0-10-, optionally substituted -(CH2)1-2-(CO)NH-(CH2)0-10-, optionally substituted -(CH2)1-2-(CO)NH-(CH2)1-3-(OCH2CH2)1-7-, optionally substituted -(CH2)0- 1 -CO-(CH 2 ) 1-3 -(OCH 2 CH 2 ) 1-7 -, optionally substituted -CO-(CH 2 ) 0-3 -(alkenylene)-(CH 2 ) 0-3 -, optionally substituted -CO-(CH2)0-3-(alkynylene)-(CH2)0-3-, optionally substituted -CO-(CH2)0-3- (3-8 membered carbocyclyl)-(CH 2 ) 0-3 -, optionally substituted -CO-(CH 2 0-3 -(3-8 membered carbocycl
  • the USP7 recruiter can be a USP7 potentiator which potently binds with USP7.
  • the USP7 recruiter comprises P5019, GNE6776, Almac4, HBX19818, P217564, XL188, XL177A, FT671, USP-797, L55, ALM34, Parthenolide, or derivative thereof.
  • the USP7 recruiter is a compound disclosed in one or more of WO2019067503; WO2013030218; CN112047933; CN113801135; WO2016109480; WO2017212010; WO2017212012; US20200095260A1; US20190142834; WO2016150800; CN111808105; WO2018073602; WO2022048498; US20200095260; WO2021175234; and WO2018183587, each of which is incorporated by reference in its entirety.
  • QD 1 is selected from formulae QD-1, QD-2, QD-3, QD-4, QD-5, QD- 6 and Q D -7: Attorney Docket No.: 27527-0219WO1 wherein denotes the point of attachment to Linker in Formula (A); denotes the point of attachment to are selected from N or C, with the proviso that at least one of VD and WD is C; XD, YD, and ZD are independently selected from S, O, N, NRD 12 , and CRD 8 , with the proviso that when both V D and W D are C, one of X D , Y D , and Z D is S, O, or NR D 12 , and when one of V D and W D is N, none of X D , Y D , and Z D are S, O, or NR D 12 ; RD 7 is selected from hydrogen, halogen, cyano, C1-C3 alkyl, C1-C3 halo
  • VD is N and WD is C. In some embodiments, VD is C and WD is N. In some embodiments, VD is C and WD is C. In some embodiments, Q D -1 is selected from formulae Q D -8, Q D -9, and Q D -10: 15, QD-16, QD-17, QD-18, QD-19, QD-20, QD-21, QD-22, QD-23, QD-24, QD-25, QD-26, QD- 27, QD-28, QD-29, QD-30, QD-31, QD-32, QD-33, QD-34, QD-35, and QD-36: Attorney Docket No.: 27527-0219WO1 Attorney Docket No.: 27527-0219WO1 Formula Q D -35, and Formula Q D -36.
  • QD 1 is selected from formulae QD-1, QD-2, QD-8, QD-11, QD-12, and Q D -13. In some embodiments, QD 1 is selected from formulae QD-37, QD-38, QD-39, QD-40, QD-41, QD-42: In some embodiments, B D is N. In some embodiments, RD 7 is hydrogen, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, and C 3 -C 5 cycloalkyl. In some embodiments, R D 7 is selected from H, CH 3 , CF 3 , CH(CH 3 ) 2 , and CH(CH 2 ) 2 .
  • R D 7 is H.
  • QD 1 is selected from formulae QD-43, QD-44, QD-45, QD-46, QD-47, Q D -48: Attorney Docket No.: 27527-0219WO1
  • each RD 1 is independently selected from H, C1-C3 alkyl, and C1-C3 haloalkyl.
  • each R D 1 is independently selected from H, CH 3 , CF 3 , CH(CH 3 ) 2 , and CH(CH 2 ) 2 .
  • each R D 1 is independently selected from H.
  • R D L1 is selected from a bond, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene, C1-C6 heteroalkylene, C3-C12 cycloalkylene, 3-12 membered heterocyclicene, C6-C10 arylene, and Attorney Docket No.: 27527-0219WO1 5-10 membered heteroarylene.
  • R D L1 is selected from a , wherein (R D 13 ) 2 ; each RD 13 and RD 14 are independently selected from H, halogen, ORD 4 , NRD 4 RD 5 , OC(O)RD 6 , NRD 4 C(O)RD 6 , C(O)RD 6 , C(O)ORD 4 , C(O)NRD 4 RD 5 , S(O)2NRD 4 RD 5 , NRD 4 S(O)2RD 6 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, C 6 -C 10 aryl, and 5-10 membered heteroaryl, or RD 13 and RD 14 , or two RD 14 , together with the atom
  • each RD 13 is independently selected from H, halogen, C1-C6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R D 13 is independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl and C3-C12 cycloalkyl.
  • RD 13 is H.
  • R D 14 is selected from H, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 - C6 heteroalkyl, and C3-C12 cycloalkyl.
  • each RD 14 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl and C3-C12 cycloalkyl.
  • R D 14 is selected from H, CH 3 , CHF 2 and CF 3 .
  • R D 15 is selected from H, halogen, cyano, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • R D 15 is selected from H, F, and Cl. In some embodiments, R D 15 is H.
  • RD 16 is selected from H, C(O)RD 6 , C(O)ORD 4 , C(O)NRD 4 RD 5 , C1- C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, 3-12 membered heterocyclic, 5- 10 membered heteroaryl, and C 1 -C 6 alkyl-5-10 membered heteroaryl.
  • R D 16 is selected from hydrogen, C(O)ORD 4 , and C1-C6 alkyl-5-10 membered heteroaryl.
  • R D 16 is selected from H, C(O)OC(CH 3 ) 3 , .
  • RD L1 is selected from a , where each said C6-C10 aryl and 5-10 membered heteroaryl is optionally substituted with halogen, cyano, oxo, OR D 4 , NR D 4 R D 5 , C(O)OR D 4 , C(O)NR D 4 R D 5 , OC(O)R D 6 , NR D 4 C(O)R D 6 , S(O) 2 NR D 4 R D 5 , NR D 4 S(O) 2 R D 6 , C 1 -C 3 alkyl, C 1 -C 3 heteroalkyl, C 3 -C 7 cycloalkyl, 3-7 membered heterocyclic, C6-C10 aryl, or 5-10 membered heteroaryl.
  • RD 2 is selected Attorney Docket No.: 27527-0219WO1 from phenyl and 5-6 membered heteroaryl, where each said phenyl and 5-6 membered heteroaryl is optionally substituted with halogen, cyano, oxo, ORD 4 , NRD 4 RD 5 , C(O)ORD 4 , C(O)NRD 4 RD 5 , OC(O)RD 6 , NRD 4 C(O)RD 6 , S(O)2NRD 4 RD 5 , NRD 4 S(O)2RD 6 , C1-C3 alkyl, C1-C3 heteroalkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclic, C 6 -C 10 aryl, or 5-10 membered heteroaryl.
  • R D 2 is selected from phenyl and 5-6 membered heteroaryl, where each said phenyl and 5-6 membered heteroaryl is optionally substituted with halogen, C(O)ORD 4 , C(O)NRD 4 RD 5 , NR D 4 C(O)R D 6 , S(O) 2 NR D 4 R D 5 , NR D 4 S(O) 2 R D 6 , C 1 -C 3 alkyl, C 1 -C 3 heteroalkyl, C 3 -C 7 cycloalkyl, and 3-7 membered heterocyclic.
  • R D 2 is selected from phenyl, pyridinyl and pyrazolyl, where each said phenyl, pyridinyl and pyrazolyl is optionally substituted with halogen, C(O)ORD 4 , C(O)NRD 4 RD 5 , NRD 4 C(O)RD 6 , S(O)2NRD 4 RD 5 , NRD 4 S(O)2RD 6 , C1-C3 alkyl, C1-C3 heteroalkyl, C 3 -C 7 cycloalkyl, and 3-7 membered heterocyclic.
  • R D 2 is selected from , .
  • each R D 4 is selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R D 5 is selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R D 6 is selected from C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C2-C6 alkenyl, C3-C7 cycloalkyl, 3-7 membered heterocyclic, C6-C10 aryl, and 5-10 membered heteroaryl.
  • each R D 8 is selected from hydrogen, halogen, cyano, OR D 4 , NRD 4 RD 5 , NRD 4 C(O)RD 6 , C(O)NRD 4 RD 5 , S(O)2NRD 4 RD 5 , NRD 4 S(O)2RD 6 , C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R D 8 is selected from hydrogen, halogen, NR D 4 C(O)R D 6 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic. In some embodiments, each R D 8 is selected . In some embodiments, RD L2 is selected from a bond, -C(O)-, -C(O)O-, -C(O)NRD 4 -, - S(O)NRD 4 -, -S(O)2NRD 4 -, C6-C10 arylene, and 5-10 membered heteroarylene.
  • R D L2 is selected from a bond, -C(O)NR D 4 -, C 6 -C 10 arylene, and 5-10 membered heteroarylene.
  • R D L2 is selected from a bond, -NHC(O)-, and phenylene.
  • RD L3 is selected from a bond, -O-, -NRD 4 -, C1-C6 alkylene, C1-C6 haloalkylene, C 1 -C 6 heteroalkylene, C 3 -C 12 cycloalkylene, 3-12 membered heterocyclicene.
  • R D L3 is selected from a bond, -O-, -NH-, -CH 2 -, -CH 2 -CH 2 -, -CH 2 -NH-, and . embodimen 9 L2 L3 ts, each R D is independently selected from -R D -R D -.
  • each RD 9 is independently selected from a bond, -NHC(O)-, , . C1- RD 12 is selected from H and CH 3 .
  • m D is selected from 0 and 1.
  • m D is 0.
  • nD is selected from 0 and 1.
  • nD is 0.
  • nD is 1.
  • oD is selected from 0, 1 and 2. In some embodiments, oD is selected from 0 and 1. In some embodiments, o D is 0. In some embodiments, o D is 1. In some embodiments, Attorney Docket No.: 27527-0219WO1 p D is selected from 1 and 2. In some embodiments, p D is 2. In some embodiments, q D is selected from 0, 1, 2, 3, and 4. In some embodiments, qD is selected from 0, 1 and 2. In some embodiments, qD is 0. In some embodiments, rD is selected from 0, 1, 2, 3, and 4. In some embodiments, rD is selected from 0 and 1. In some embodiments, r D is 0.
  • the USP7 recruiter comprises the structure of formula (E-I-a). .
  • the USP7 recruiter has the structure of Formula (F-I): Or a tautomer, stereoisomers, or a pharmaceutically acceptable salt of the compound thereof, wherein denotes the point of attachment to Linker in Formula (A); Ring A E is selected from C 3 -C 12 cycloalkylene, 3-12 membered heterocyclicene, C 6 -C 10 arylene, and 5-10 membered heteroarylene; QE is selected from a bond, -O-, -NRE 5 -, -C(O)-, -C(O)O-, -C(O)NRE 5 -, -S(O)-, -S(O)2-, - S(O)NR E 5 -, -S(O) 2 NR E 5 -, C 1 -C 6 alkylene, C 1 -C 6 haloalkylene,
  • Ring AE is selected from C6-C10 arylene and 5-10 membered heteroarylene.
  • Ring A E is selected from phenylene, pyridinylene, pyrimidinylene, furylene, thiophenylene, pyrrolylene, pyrazolylene, 1H-benzo[d]imidazolylene, indolylene, benzo[b]thiophenylene, or indazolyene.
  • Ring AE is selected from phenylene, pyridinylene, and indazolyene.
  • Ring AE is phenylene.
  • Ring A E is pyridinylene.
  • Ring A E is indazolyene.
  • Q E is selected from a bond, -O-, -NR E 5 -, -C(O)NR E 5 -, -S(O)NR E 5 -, -S(O)2NRE 5 -, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 heteroalkylene, C3-C12 cycloalkylene, and 3-12 membered heterocyclicene.
  • QE is selected from a bond, - C(O)NR E 5 -.
  • Q E is selected from a bond, -C(O)NH-. In some embodiments, Q E is a bond. In some embodiments, Q E is -C(O)NH-.
  • the USP7 recruiter comprises the structure of Formulae (F-I-a) and (F-I-b): Or a tautomer, stereoisomers, or a pharmaceutically acceptable salt of the compound thereof, wherein denotes the point of attachment to Linker in Formula (A). wherein: X E is selected from C(R E 1 ) and N; and R E 1 , R E 2 , R E 3 , R E 4 , m E and n E are defined in Formula (F-I).
  • each RE 1 is independently selected from H, halogen, cyano, CF3, C1-C6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic, where each said C 1 - C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 12 cycloalkyl, and 3-12 membered heterocyclic is optionally substituted with halogen, cyano, oxo, ORE 8 , NRE 8 RE 9 , C(O)ORE 8 , C(O)NRE 8 RE 9 , OP(O)(OH)2, OP(O)(ORE 8 )(ORE 9 ), S(O)2RE 10 , S(O)2NRE 8 RE 9 , OC(O)RE 10 , NRE 8 C(O)RE 10 , NRE 8 S(O)2RE 10 , C1- C 6 alkyl, C 1 -C
  • each RE 1 is independently selected from H, halogen, cyano, CF3, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R E 1 is independently selected from H, halogen, cyano, CF3, C1-C6 alkyl, C1-C6 haloalkyl.
  • each RE 1 is independently selected from H, F, Cl, Br, CH3, CF3, and cyano.
  • RE 1 is H.
  • R E 1 is F, Cl, Br. In some embodiments R E 1 is CH 3 . In some embodiments, RE 1 is is CF3. In some embodiments RE 1 is cyano. In some embodiments, RE 2 is selected from H, cyano, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 heteroalkyl, or C 3 -C 6 cycloalkyl. In some embodiments, R E 2 is selected from H, CF 3 , CH 3 , CH 2 CH 3 , CH(CH 2 ) 2 , CH 2 CH 2 CH 3 , CH(CH 3 ) 2 , and CH 3 O.
  • R E 2 is H. In some embodiments, RE 2 is CH3. In some embodiments, RE 2 is CH2CH3. In some embodiments, RE 2 is CH(CH 2 ) 2 . In some embodiments, R E 2 is CF 3 . In some embodiments, R E 2 is CH 2 CH 2 CH 3 . In some embodiments, R E 2 is CH(CH 3 ) 2 .
  • R E 2 is CH 3 O
  • each RE 3 are independently selected from H, halogen, cyano, CF3, ORE 5 , NRE 5 RE 6 , C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C3-C12 cycloalkyl, and 3-12 membered heterocyclic.
  • each R E 3 are independently selected from H, OH, NH2, F, Cl, CN, CF3, CH3, CH(CH2)2, and CH(CH3)2.
  • RE 3 is OH.
  • RE 3 is NH2.
  • R E 4 is selected from H, C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, C 1 -C 3 heteroalkyl, or C3-C6 cycloalkyl.
  • RE 4 is selected from H, CH3, and CH2CH3.
  • RE 4 is H.
  • RE 4 is CH3.
  • RE 4 is CH 2 CH 3 .
  • the USP7 recruiter comprises the structure of Formulae (F-I-a1), (F-I-a2) and (F-I-b1):
  • the USP7 recruiter may be a derivative of following compounds: ALM34(Gavory et al., 2018), FT671(Turnbull et al., 2017), L55(Li et al., 2020), GNE- 6776(Kategaya et al., 2017), GNE-6640(Di Lello et al., 2017), HBX19818(Reverdy et al., 2012), XL177A(Schauer et al., 2020), FT827(Turnbull et al., 2017), NSC632839(Nicholson et al., 2008), compound 14(Weinstock et al., 2012), Example 14(Pepe and Mesecar, 2018), compound 5(O'Dowd et al., 2018
  • the USP7 recruiting AMPK DUBTACs are compounds selected from the following compounds, as identified in table 1 below: XH181-1, XH181-2, XH181-3, XH181-4, XH181-5, XH181-6, XH181-7, XH181-8, XH181-9, XH181-15, XH188-16, XH188-17, XH188- 18, XH188-19, KL186-1, KL186-2, KL186-3, KL186-4, KL186-5, KL186-6, KL186-7, KL186- 8, KL186-9, KL186-10, KL186-11, KL186-12, KL186-13, KL186-14, KL186-48, KL186-49, KL186-50, KL186-51, KL186-52, KL186-53, KL186-54, KL186-55, KL186-56, KL186-57, KL186-
  • the document provides a method of treating a AMPK-mediated diseases.
  • the method includes administering to a subject in need thereof with a AMPK-mediated diseases one or more compounds to stabilize and activate AMPK.
  • the AMPK-mediated disease may be a cancer or other diseases, including metabolic syndrome, type-2 diabetes, non-alcoholic fatty liver disease and cardiovascular disease .
  • the AMPK-mediated disease with lower expression level of AMPK or unactivated AMPK relative to a wild-type tissue of the same species and tissue type.
  • the AMPK-mediated diseases can have reduced AMPK enzymatic activity relative to a wildtype tissue of the same species and tissue type.
  • Non-limiting examples of AMPK-mediated cancer include mesothelioma, hepatocellular cancer, central nervous system neoplasm, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer, breast cancer (e.g., estrogen receptor positive (ER+) breast cancer), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, gastrointestinal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, leukemia, lymphoma, bladder cancer, renal cell cancer, brain stem glioma, pituitary cancer, adrenocortical cancer, gallbladder cancer, Attorney Docket No.: 27527-0219WO1 multiple myelo
  • the AMPK-mediated cancer can be a relapsed cancer.
  • the AMPK-mediated cancer can have been refractory to one or more previous treatments.
  • the USP7 recruiting cGAS DUBTAC are compounds selected from the following compounds, as identified in table 2 below: XH168-142, XH168-143, XH168-144, XH168-145, XH168-146, XH168-147, XH168-148, XH168-149, XH168-150, XH168-151, XH168-152, XH168-153, XH168-154, XH168-155, XH181-72, XH181-73, XH181-90, XH181- 91, XH181-92, XH181-93, XH181-94, XH181-95, XH181-96, XH181-97, XH181
  • cGAS–STING functions as an innate immune signaling pathway that recognizes perturbation of cellular homeostasis and triggers potent antitumor immune responses. Therefore, in some aspects, the document provides a method of treating various disease including cancer by activating cGAS-STING signaling pathway through the stabilization and activation of cGAS.
  • Non- limiting examples of cGAS-STING targeting cancer include mesothelioma, hepatocellular cancer, central nervous system neoplasm, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer, breast cancer (e.g., estrogen receptor positive (ER+) breast cancer), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, gastrointestinal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, Attorney Docket No.: 27527-0219WO1 testicular cancer, leukemia, lymphoma, bladder cancer, renal cell cancer, brain stem glioma, pituitary cancer, adrenocortical cancer, gallbladder cancer, multiple
  • the cGAS-STING targeting cancer can be a relapsed cancer.
  • the cGAS-STING targeting cancer can have been refractory to one or more previous treatments.
  • the USP7 recruiting CFTR DUBTAC are compounds selected from the following compounds, as identified in table 3 below: XH168-51, XH168-32, XH168-33, XH168- 59, XH168-66, XH168-67, XH168-68, XH168-69, XH168-70, XH168-58, XH168-47, XH168-48, XH168-49, XH168-50, KL180-124, KL180-125, KL180-126, KL180-127, KL180-128, KL180- 129, KL180-130, KL180-131, KL180-132, KL180-133, KL180-134, KL180-135, KL180-136, KL180-
  • the document provides a method of treating a CFTR-mediated diseases.
  • the method includes administering to a subject in need thereof with a CFTR-mediated diseases one or more compounds stabilizing CFTR.
  • the CFTR-mediated disease is associated with dysfunction of CFTR.
  • the CFTR-mediated disease may be cystic fibrosis, male infertility, polycystic kidney disease, obstructive lung disease, intestinal obstruction syndromes, liver dysfunction, exocrine and endocrine pancreatic dysfunction, or secretory diarrhea.
  • the compounds can be administered, e.g., orally, parenterally, intradermally, subcutaneously, topically, and/or rectally.
  • This disclosure additionally provides a method for identifying a bivalent compound which mediates de-ubiquitination/stabilization of AMPK, cGAS, or CFTR, the method including providing a heterobifunctional test compound including a AMPK, cGAS, or CFTR ligand conjugated to a de-ubiquitination tag (e.g. USP7 binder) via a linker, contacting the heterobifunctional test compound with a cell (e.g., a cell such as a AMPK, cGAS or CFTR- mediated disease cell) including a deubiquitinase (e.g., USP7) and AMPK, cGAS or CFTR protein.
  • a cell e.g., a cell such as a AMPK, cGAS or CFTR- mediated disease cell
  • a deubiquitinase e.g., USP7
  • any of the above-described methods can further include treating the subject with one or more additional therapeutic regimens for treating the target related disease.
  • the one or more additional therapeutic regimens for treating target related disease can be, e.g., one or more of surgery, chemotherapy, radiation therapy, hormone therapy, or immunotherapy.
  • Attorney Docket No.: 27527-0219WO1 As used herein, the terms "about” and “approximately” are defined as being within plus or minus 10% of a given value or state, preferably within plus or minus 5% of said value or state. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • Assays suitable for use in any or all of these steps are known in the art, and include, e.g., Western blotting, quantitative mass spectrometry (MS) analysis, flow cytometry, enzymatic inhibition, ITC, SPR, cell growth inhibition and xenograft and PDX models.
  • MS mass spectrometry
  • isotopic variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate isotopic variations of those reagents).
  • an isotopic variation is a compound in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature.
  • Useful isotopes are known in the art and include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine.
  • Exemplary isotopes thus include, e.g., 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 32 P, 35 S, 18 F, and 36 Cl.
  • Isotopic variations e.g., isotopic variations containing 2 H
  • certain isotopic variations can be used in drug or substrate tissue distribution studies.
  • the radioactive isotopes tritium ( 3 H) and carbon-14 ( 14 C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Pharmaceutically acceptable solvates of the compounds disclosed herein are contemplated.
  • a solvate can be generated, e.g., by substituting a solvent used to crystallize a compound disclosed Attorney Docket No.: 27527-0219WO1 herein with an isotopic variation (e.g., D 2 O in place of H 2 O, d 6 -acetone in place of acetone, or d 6 - DMSO in place of DMSO).
  • an isotopic variation e.g., D 2 O in place of H 2 O, d 6 -acetone in place of acetone, or d 6 - DMSO in place of DMSO.
  • Pharmaceutically acceptable fluorinated variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate fluorinated variations of those reagents).
  • a fluorinated variation is a compound in which at least one hydrogen atom is replaced by a fluoro atom. Fluorinated variations can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.
  • Pharmaceutically acceptable prodrugs of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (e.g., converting hydroxyl groups or carboxylic acid groups to ester groups).
  • a "prodrug” refers to a compound that can be converted via some chemical or physiological process (e.g., enzymatic processes and metabolic hydrolysis) to a therapeutic agent.
  • prodrug also refers to a precursor of a biologically active compound that is pharmaceutically acceptable.
  • a prodrug may be inactive when administered to a subject, i.e. an ester, but is converted in vivo to an active compound, for example, by hydrolysis to the free carboxylic acid or free hydroxyl.
  • the prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in an organism.
  • prodrug is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a subject.
  • Prodrugs of an active compound may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound.
  • Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation.
  • An alkyl may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms.
  • an alkyl comprises one to fifteen carbon atoms (e.g., C1-C15 alkyl).
  • an alkyl comprises one to thirteen carbon atoms (e.g., C 1 -C 13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., C 1 -C 8 alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., C5-C8 alkyl).
  • alkyl is attached to the rest of the molecule by a single bond, for example, methyl (Me), ethyl (Et), n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), pentyl, 3-methylhexyl, 2-methylhexyl, and the like.
  • Alkylene refers to a bivalent saturated aliphatic radical (such as ethylene) regarded as derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from different carbon atoms.
  • Alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond.
  • An alkenyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms.
  • an alkenyl comprises two to twelve carbon atoms (e.g., C 2 -C 12 alkenyl).
  • an alkenyl comprises two to eight carbon atoms (e.g., C2-C8 alkenyl).
  • an alkenyl comprises two to six carbon atoms (e.g., C2- C6 alkenyl).
  • an alkenyl comprises two to four carbon atoms (e.g., C2-C4 alkenyl).
  • the alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.
  • alkynylene refers to a linear or branched divalent hydrocarbon radical of two to eight carbon atoms (C 2 -C 8 ) with at least one site of unsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylene radical may be optionally substituted independently with one or more substituents described herein. Examples include, but are not limited to ,ethynylene (-C?C-), propynylene (propargylene, -CH 2 C?C-), and the like.
  • alkynyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond.
  • An alkynyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms.
  • an alkynyl comprises two to twelve carbon atoms (e.g., C 2 -C 12 alkynyl).
  • an alkynyl comprises two to eight carbon atoms (e.g., C 2 -C 8 alkynyl).
  • an alkynyl has two to six carbon atoms (e.g., C2-C6 alkynyl). In other embodiments, an alkynyl has two to four carbon atoms (e.g., C2-C4 alkynyl).
  • the alkynyl is attached to the rest of the molecule by a single bond. Examples of such groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and the like.
  • alkoxy means an alkyl group as defined herein which is attached to the rest of the molecule via an oxygen atom. Examples of such groups include, but are not limited to, methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butoxy, iso-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.
  • aryl refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon atoms.
  • An aryl may comprise from six to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ?–electron system in accordance with the Hückel theory.
  • an aryl comprises six to fourteen carbon atoms (C6-C14 aryl).
  • an aryl comprises six to ten carbon atoms (C6-C10 aryl). Examples of such groups include, but are not limited to, phenyl, fluorenyl and naphthyl.
  • arylene means a divalent aromatic hydrocarbon radical of 6-20 carbon atoms (C6-C20) derived by the removal of two hydrogen atom from a two carbon atoms of a parent aromatic ring system. Some arylene groups are represented in the exemplary structures as “Ar”. Arylene includes bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic ring.
  • Typical arylene groups include, but not limited to, radicals derived from benzene (phenylene), substituted benzenes, naphthalene, anthracene, biphenylene indenylene, indaylene, 1,2-dihydronaphthalene, 1,2,3,4,-tetrahydronaphthyl, and the like.
  • Arylene groups are optionally substituted with one or more substituents described herein.
  • heteroaryl refers to a radical derived from a 3- to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur.
  • the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ?–electron system in accordance with the Hückel theory.
  • Heteroaryl includes fused or bridged ring systems.
  • a heteroaryl refers to a radical derived from a 3- to 10-membered aromatic ring radical (3-10 membered heteroaryl).
  • a heteroaryl refers to a radical derived from 5- to 7-membered aromatic ring (5-7 membered heteroaryl).
  • Heteroaryl includes fused or bridged ring systems.
  • the heteroatom(s) in the heteroaryl radical is optionally oxidized.
  • One or more nitrogen atoms, if present, are optionally quaternized.
  • the heteroaryl is attached to the rest of the molecule through any atom of the ring(s).
  • Examples of such groups include, but not limited to, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl
  • an heteroaryl is attached to the rest of the molecule via a ring carbon atom. In certain embodiments, an heteroaryl is attached to the rest of the molecule via a nitrogen atom (N-attached) or a carbon atom (C-attached).
  • N-attached nitrogen atom
  • C-attached carbon atom
  • a group derived from pyrrole may be pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached).
  • a group derived from imidazole may be imidazol-1-yl (N-attached) or imidazol-3-yl (C-attached).
  • heteroalkyl means a saturated or unsaturated chain of carbon atoms and at least one heteroatom, wherein no two heteroatoms are adjacent.
  • heterocyclic means a non-aromatic, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 atoms in its ring system, and containing from 3 to 12 carbon atoms and from 1 to 4 heteroatoms each independently selected from O, S and N, and with the proviso that the ring of said group does not contain two adjacent O atoms or two adjacent S atoms.
  • a heterocyclic group may include fused, bridged or spirocyclic ring systems.
  • a heterocyclic group comprises 3 to 10 ring atoms (3-10 membered heterocyclic).
  • a heterocyclic group comprises 3 to 8 ring atoms (3-8 membered heterocyclic).
  • a heterocyclic group comprises 4 to 8 ring atoms (4-8 membered heterocyclic).
  • a heterocyclic group comprises 3 to 6 ring atoms (3-6 membered heterocyclic).
  • a heterocyclic group may contain an oxo substituent at any available atom that will result in a stable compound. For example, such a group may contain an oxo atom at an available carbon or nitrogen atom.
  • Such a group may contain more than one oxo substituent if chemically feasible.
  • a heterocyclic group contains a sulfur atom, said sulfur atom may be oxidized with one or two oxygen atoms to afford either a sulfoxide or sulfone.
  • An example of a 4 membered heterocyclic group is azetidinyl (derived from azetidine).
  • An example of a 5 membered cycloheteroalkyl group is pyrrolidinyl.
  • An example of a 6 membered cycloheteroalkyl group is piperidinyl.
  • An example of a 9 membered cycloheteroalkyl group is indolinyl.
  • An example of a 10 membered cycloheteroalkyl group is 4H-quinolizinyl.
  • Such heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3- pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dio
  • a heteroaryl group may be attached to the rest of molecular via a carbon atom (C-attached) or a Attorney Docket No.: 27527-0219WO1 nitrogen atom (N-attached).
  • a group derived from piperazine may be piperazin-1-yl (N-attached) or piperazin-2-yl (C-attached).
  • cycloalkyl or “carbocyclyl” means a saturated, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms in its ring system.
  • a cycloalkyl may be fused, bridged or spirocyclic.
  • a cycloalkyl comprises 3 to 8 carbon ring atoms (C3-C8 cycloalkyl). In certain embodiments, a cycloalkyl comprises 3 to 6 carbon ring atoms (C 3 -C 6 cycloalkyl). Examples of such groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, adamantyl, and the like.
  • the term “cycloalkylene” or “carbocyclylene” is a bidentate radical obtained by removing a hydrogen atom from a cycloalkyl ring as defined above.
  • spirocyclic as used herein has its conventional meaning, that is, any ring system containing two or more rings wherein two of the rings have one ring carbon in common.
  • Each ring of the spirocyclic ring system independently comprises 3 to 20 ring atoms. Preferably, they have 3 to 10 ring atoms.
  • Non-limiting examples of a spirocyclic system include spiro[3.3]heptane, spiro[3.4]octane, and spiro[4.5]decane.
  • the term cyano refers to a -C?N group.
  • An "aldehyde” group refers to a –C(O)H group.
  • An "alkoxy” group refers to both an –O-alkyl, as defined herein.
  • An “alkoxycarbonyl” refers to a -C(O)-alkoxy, as defined herein.
  • An "alkylaminoalkyl” group refers to an -alkyl-NR-alkyl group, as defined herein.
  • alkylsulfonyl refer to a -SO 2 alkyl, as defined herein.
  • An “amino” group refers to an optionally substituted -NH 2 .
  • An “aminoalkyl” group refers to an —alky-amino group, as defined herein.
  • An “aminocarbonyl” refers to a -C(O)-amino, as defined herein.
  • arylalkyl refers to -alkylaryl, where alkyl and aryl are defined herein.
  • An “aryloxy” group refers to both an –O-aryl and an –O-heteroaryl group, as defined herein.
  • aryloxycarbonyl refers to -C(O)-aryloxy, as defined herein.
  • An “arylsulfonyl” group refers to a -SO 2 aryl, as defined herein. Attorney Docket No.: 27527-0219WO1
  • a "carbonyl” group refers to a -C(O)- group, as defined herein.
  • a “carboxylic acid” group refers to a –C(O)OH group.
  • cycloalkoxy refers to a –O-cycloalkyl group, as defined herein.
  • a “halo" or “halogen” group refers to fluorine, chlorine, bromine or iodine.
  • a “haloalkyl” group refers to an alkyl group substituted with one or more halogen atoms.
  • a "hydroxy” group refers to an -OH group.
  • a “nitro” group refers to a -NO 2 group.
  • a “trihalomethyl” group refers to a methyl substituted with three halogen atoms.
  • a “electron withdrawing” group refers to an atom or group that draws electron density from neighboring atoms towards itself.
  • substituted means that the specified group or moiety bears one or more substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C 1 -C 4 alkyl-, C 1 -C 4 haloalkyl, -OC 1 -C 4 alkyl, -OC 1 -C 4 alkylphenyl, -C 1 -C 4 alkyl-OH, -OC1-C4 haloalkyl, halo, -OH, -NH2, -C1-C4 alkyl-NH2, -N(C1-C4 alkyl)(C1-C4 alkyl), -NH(C1-C4 alkyl), -N(C1-C4 alkyl)(C1-C4 alkylphenyl), -NH(C1-C4 alkylphenyl), -NH(C1-C4 alkylphenyl),
  • null means the absence of an atom or moiety, and there is a bond between adjacent atoms in the structure.
  • optionally substituted means that the specified group may be either unsubstituted or substituted by one or more substituents as defined herein. It is to be understood that in the compounds of the present invention when a group is said to be “unsubstituted,” or is “substituted” with fewer groups than would fill the valencies of all the atoms in the compound, the remaining valencies on such a group are filled by hydrogen.
  • a C 6 aryl group in the present compounds is said to be “disubstituted,” one of ordinary skill in the art would understand it to mean that the C 6 aryl has 3 carbon atoms remaining that are unsubstituted. Those three unsubstituted carbon atoms are each bound to one hydrogen atom to fill their valencies.
  • m or n or x
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • a pharmaceutically acceptable salt of any one of the bivalent compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms.
  • Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like.
  • salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc.
  • acetic acid trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, Attorney Docket No.: 27527-0219WO1 tartrates, methanesulfonates, and the like.
  • salts of amino acids such as arginates, gluconates, and galacturonates
  • Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
  • “Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid.
  • Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • compositions and methods described herein include the manufacture and use of pharmaceutical compositions and medicaments that include one or more bivalent compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.
  • the compositions disclosed herein can include other compounds, drugs, or agents used for the treatment of cancer.
  • pharmaceutical compositions disclosed herein can be combined with one or more (e.g., one, two, three, four, five, or less than ten) compounds.
  • additional compounds can include, e.g., conventional chemotherapeutic agents known in the art.
  • USP2 inhibitors disclosed Attorney Docket No.: 27527-0219WO1 herein can operate in conjunction with conventional chemotherapeutic agents to produce mechanistically additive or synergistic therapeutic effects.
  • the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the USP2 inhibitors or its delivery form.
  • Pharmaceutical compositions typically include a pharmaceutically acceptable carrier, adjuvant, or vehicle.
  • pharmaceutically acceptable refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • a pharmaceutically acceptable carrier, adjuvant, or vehicle is a composition that can be administered to a patient, together with a compound of the invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • exemplary conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles include saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • pharmaceutically acceptable carriers, adjuvants, and vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d- ?-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes,
  • Cyclodextrins such as ?-, ?-, and ?-cyclodextrin, may also be advantageously used to enhance delivery of compounds of the formulae described herein.
  • the USP7 recruiting DUBTAC disclosed herein are defined to include pharmaceutically acceptable derivatives or prodrugs thereof.
  • a “pharmaceutically acceptable Attorney Docket No.: 27527-0219WO1 derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, e.g., carbamate, ester, phosphate ester, salt of an ester, or other derivative of a compound or agent disclosed herein, which upon administration to a recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof.
  • Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds disclosed herein when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
  • Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. Such derivatives are recognizable to those skilled in the art without undue experimentation.
  • USP7 recruiting DUBTAC include pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated derivatives thereof.
  • USP2 inhibitors disclosed herein include, e.g., those derived from pharmaceutically acceptable inorganic and organic acids and bases.
  • Suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, trifluoromethylsulfonate, and undecanoate.
  • Salts derived from appropriate bases include, e.g., alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4+ salts.
  • alkali metal e.g., sodium
  • alkaline earth metal e.g., magnesium
  • ammonium e.g., ammonium
  • N-(alkyl)4+ salts e.g., sodium
  • alkaline earth metal e.g., magnesium
  • ammonium e.g., sodium
  • N-(alkyl)4+ salts e.g., sodium
  • the invention also envisions the quaternization of any basic nitrogen-containing groups of the USP2 inhibitors disclosed herein. Water or oil-soluble or dispersible products can be obtained by such quaternization.
  • the pharmaceutical compositions disclosed herein can include an effective amount of one or more USP7 recruiting DUBTAC.
  • phrases “effective amount” and “effective Attorney Docket No.: 27527-0219WO1 to treat,” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer).
  • pharmaceutical compositions can further include one or more additional compounds, drugs, or agents used for the treatment of cancer (e.g., conventional chemotherapeutic agents) in amounts effective for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer).
  • the pharmaceutical compositions disclosed herein can be formulated for sale in the United States, import into the United States, or export from the United States.
  • Administration of Pharmaceutical Compositions The pharmaceutical compositions disclosed herein can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA Data Standards Manual (DSM) (available at http://www.fda.gov/Drugs/DevelopmentApprovalProcess/ FormsSubmissionRequirements/ElectronicSubmissions/DataStandardsManualmonographs).
  • DSM Food and Drug Administration
  • the pharmaceutical compositions can be formulated for and administered via oral, parenteral, or transdermal delivery.
  • parenteral includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • the pharmaceutical compositions disclosed herein can be administered, e.g., topically, rectally, nasally (e.g., by inhalation spray or nebulizer), buccally, vaginally, subdermally (e.g., by injection or via an implanted reservoir), or ophthalmically.
  • compositions of this invention can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
  • carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include Attorney Docket No.: 27527-0219WO1 lactose and dried corn starch.
  • compositions of this invention can be administered in the form of suppositories for rectal administration.
  • suppositories for rectal administration.
  • suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
  • the pharmaceutical compositions of this invention can be administered by nasal aerosol or inhalation.
  • compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art.
  • the pharmaceutical compositions of this invention can be administered by injection (e.g., as a solution or powder).
  • Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) 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, e.g., as a solution in 1,3-butanediol.
  • a non-toxic parenterally acceptable diluent or solvent e.g., as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents e.g., mannitol, 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 can be employed, including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, e.g., olive oil or castor oil, especially in their polyoxyethylated versions.
  • oils e.g., olive oil or castor oil
  • These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
  • Other commonly used surfactants such as Tweens, Spans, or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.
  • an effective dose of a pharmaceutical composition of this invention can include, but is not limited to, e.g., about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, or 10000 mg/kg/day, or according to the requirements of the particular pharmaceutical composition.
  • both the compound and the additional compound should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen.
  • the additional agents can be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents can be part of a single dosage form, mixed together with the compounds of this invention in a single composition.
  • the pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration.
  • Methods of Treatment contemplate administration of an effective amount of a compound or composition to achieve the desired or stated effect.
  • the compounds or compositions of the invention will be administered from about 1 to about 6 times per day or, alternately or in addition, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations can contain from about 20% to about 80% active compound.
  • the present disclosure provides methods for using a composition comprising a USP7 recruiting DUBTAC, including pharmaceutical compositions (indicated below as ‘X’) disclosed herein in the following methods:
  • Substance X for use as a medicament in the treatment of one or more diseases or conditions disclosed herein e.g., cancer, referred to in the following examples as ‘Y’).
  • the methods disclosed include the administration of a therapeutically effective amount of one or more of the compounds or compositions described herein to a subject (e.g., a mammalian subject, e.g., a human subject) who is in need of, or who has been determined to be in need of, such treatment.
  • the methods disclosed include selecting a subject and administering to the subject an effective amount of one or more of the compounds or compositions described herein, and optionally repeating administration as required for the prevention or treatment of cancer.
  • subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection.
  • the subject can be confirmed or identified, e.g.
  • suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease).
  • exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, or detecting an indication of a positive immune response.
  • multiple parties can be included in subject selection.
  • a first party can obtain a sample from a candidate subject and a second party can test the sample.
  • subjects can be selected or referred by a medical practitioner (e.g., a general practitioner).
  • subject selection can include obtaining a sample from a selected subject and storing the sample or using the in the methods disclosed herein. Samples can include, e.g., cells or populations of cells.
  • methods of treatment can include a single administration, multiple administrations, and repeating administration of one or more compounds disclosed herein as required for the prevention or treatment of the disease or condition from which the subject is suffering (e.g., an AMPK, cGAS or CFTR-mediated disease).
  • methods of treatment can include assessing a level of disease in the subject prior to treatment, during treatment, or after treatment. In some aspects, treatment can continue until a decrease in the level of disease in the subject is detected.
  • subject refers to any animal. In some instances, the subject is a mammal.
  • the term “subject,” as used herein, refers to a human (e.g., a man, a woman, or a child).
  • the terms “administer,” “administering,” or “administration,” as used herein, refer to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of form.
  • the methods disclosed herein include administration of an effective amount of a compound or composition to achieve the desired or stated effect.
  • the terms “treat”, “treating,” or “treatment,” as used herein, refer to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering.
  • amelioration of the symptoms of a particular disorder refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the compositions and methods of the present invention.
  • treatment can promote or result in, for example, a decrease in the number of tumor cells (e.g., in a subject) relative to the number of tumor cells prior to treatment; a decrease in the viability (e.g., the average/mean viability) of tumor cells (e.g., in a subject) relative to the viability of tumor cells prior to treatment; a decrease in the rate of growth of tumor cells; a decrease in the rate of local or distant tumor metastasis; or reductions in one or more symptoms associated with one or more tumors in a subject relative to the subject’s symptoms prior to treatment.
  • a decrease in the number of tumor cells e.g., in a subject
  • a decrease in the viability e.g., the average/mean viability
  • the rate of growth of tumor cells e.g., in a subject
  • a decrease in the rate of local or distant tumor metastasis e.g., the rate of local or distant tumor metastasis
  • the term “treating cancer” means causing a partial or complete decrease in the rate of growth of a tumor, and/or in the size of the tumor and/or in the rate of local or distant tumor metastasis, and/or the overall tumor burden in a subject, and/or any decrease in tumor Attorney Docket No.: 27527-0219WO1 survival, in the presence of an bivalent compound (e.g., an USP7 recruiting DUBTAC) described herein.
  • an bivalent compound e.g., an USP7 recruiting DUBTAC
  • virus infection means when an organism’s body is invaded by pathogenic viruses and infectious virus particle attach to and enter susceptible cells.
  • prevent shall refer to a decrease in the occurrence of a disease or decrease in the risk of acquiring a disease or its associated symptoms in a subject.
  • the prevention may be complete, e.g., the total absence of disease or pathological cells in a subject.
  • the prevention may also be partial, such that the occurrence of the disease or pathological cells in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the present invention.
  • Exemplary AMPK, cGAS or CFTR-mediated diseases that can be treated with USP7 recruiting bivalent compounds include, for example, cystic fibrosis, breast cancer, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, bladder cancer, liver cancer and cervical cancer.
  • preventing a disease e.g., preventing cancer
  • the term “preventing a disease” means for example, to stop the development of one or more symptoms of a disease in a subject before they occur or are detectable, e.g., by the patient or the patient’s doctor.
  • the disease e.g., cancer
  • the disease does not develop at all, i.e., no symptoms of the disease are detectable.
  • an effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a therapeutic compound depends on the therapeutic compounds selected.
  • treatment of a subject with a therapeutically effective amount of the compounds or compositions described herein can include a single treatment or a series of treatments.
  • effective amounts can be administered at least once.
  • the compositions can be administered from one or more times per day to one or more Attorney Docket No.: 27527-0219WO1 times per week; including once every other day.
  • the skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.
  • the subject can be evaluated to detect, assess, or determine their level of disease.
  • treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected.
  • a maintenance dose of a compound, or composition disclosed herein can be administered, if necessary.
  • the dosage or frequency of administration, or both can be reduced, e.g., as a function of the symptoms, to a level at which the improved condition is retained.
  • Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • the present disclosure is also described and demonstrated by way of the following examples.
  • Flash column chromatography was performed on Teledyne ISCO CombiFlash Rf+ instrument equipped with a 220/254/280 nm wavelength UV detector and a fraction collector. Normal phase column chromatography was conducted on silica gel columns with either hexane/ethyl acetate or dichloromethane/methanol as eluent. Reverse phase column chromatography was conducted on HP C18 RediSep Rf columns, and the gradient was set to 10% of acetonitrile in H2O containing 0.1% TFA progressing to 100% of acetonitrile.
  • linker 1 -2- methylbenzoyl)glycine To a solution of activator 991 (prepared according to previous published paper) (Ngoei et al., 2018) (50 mg, 0.12 mmol) in DMF (1 mL) were added tert-butyl (2- aminoethyl)carbamate (19.2 mg, 0.12 mmol, 1.0 equiv), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI, 34.3 mg, 0.18 mmol, 1.5 equiv), 1-hydroxy-7- azabenzo-triazole (HOAt, 24.5 mg, 0.18 mmol, 1.5 equiv), and N-methylmorpholine (NMM, 36.4 mg, 0.36 mmol, 3.0 equiv).
  • Example 2 (R)-5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol-2-yl)oxy)-N- (3-(2-((4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7- dihydro-2H-pyrazolo[4,3-d]pyrimidin-3-yl)benzyl)amino)acetamido)propyl)-2- methylbenzamide (Xh181-2)
  • Example 2 was synthesized following the same procedure for preparing example 1 from linker 15.
  • Example 8 (R)-5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol-2-yl)oxy)-N- (9-(2-((4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7- dihydro-2H-pyrazolo[4,3-d]pyrimidin-3-yl)benzyl)amino)acetamido)nonyl)-2- methylbenzamide (XH181-8)
  • Example 8 was synthesized following the same procedure for preparing example 1 from intermediate 3 and linker 21.
  • Example 19 6'-amino-N-(3-((6-(5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H- benzo[d]imidazol-2-yl)oxy)-2-methylbenzamido)hexyl)amino)-3-oxopropyl)-4'-ethyl-5'-(4- hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-5)
  • Example 19 was synthesized following similar procedure for preparing example 15 from linker 18 and intermediate 7.
  • Example 26 6'-amino-N-(1-(5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol- 2-yl)oxy)-2-methylphenyl)-1,15-dioxo-5,8,11-trioxa-2,14-diazaheptadecan-17-yl)-4'-ethyl-5'- (4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-12)
  • Example 26 was synthesized following similar procedure for preparing example 15 from linker 25 and intermediate 7.
  • linker 1 (4.9 mg, 0.01 mmol, 1.0 equiv)
  • EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)
  • HOAt (1- hydroxy-7-azabenzo-triazole)
  • NMM N-Methylmorpholine
  • Example 30 6'-amino-N-(2-(3-(5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H- benzo[d]imidazol-2-yl)oxy)-2-methylbenzamido)propanamido)ethyl)-4'-ethyl-5'-(4- hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-49)
  • Example 30 was synthesized following similar procedure for preparing example 29 from linker 2 and intermediate 6.
  • Example 32 6'-amino-N-(2-(5-(5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H- benzo[d]imidazol-2-yl)oxy)-2-methylbenzamido)pentanamido)ethyl)-4'-ethyl-5'-(4- hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-51)
  • Example 32 was synthesized following similar procedure for preparing example 29 from linker 4 and intermediate 6.
  • Example 37 6'-amino-N-(2-(3-(2-(5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H- benzo[d]imidazol-2-yl)oxy)-2-methylbenzamido)ethoxy)propanamido)ethyl)-4'-ethyl-5'-(4- hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-56)
  • Example 37 was synthesized following similar procedure for preparing example 29 from linker 9 and intermediate 6. Pink solid, 5.3 mg, 59% yield.
  • Example 39 6'-amino-N-(1-(5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol- 2-yl)oxy)-2-methylphenyl)-1,14-dioxo-5,8,11-trioxa-2,15-diazaheptadecan-17-yl)-4'-ethyl-5'- (4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-58)
  • Example 39 was synthesized following similar procedure for preparing example 29 from linker 11 and intermediate 6.
  • linker 42 NH NH 2 2 HN O mmol, 1.5 eq
  • DMF/DCM (1:2, 3 mL) was added NMM (0.5 mmol, 5.0 eq), intermediate 11 (0.1 mmol, 1.0 eq), HOAt (0.12 mmol, 1.2 eq), and EDCI (0.12 mmol, 1.2eq) at 0 °C.
  • the mixture Attorney Docket No.: 27527-0219WO1 was stirred at 0 °C for 6 h before being stirred at room temperature overnight followed by concentrated.
  • the resulted residue was redissolved in DCM/TFA (2:1, 3 mL), and stirred at room temperature for 1 h followed by concentration.
  • intermediate 14 was purified by prep-HPLC (acid condition) to give intermediate 14 as a yellow solid, 50% yield.
  • tert-butyl (2- aminoethyl)carbamate (19.2 mg, 0.12 mmol, 1.0 equiv)
  • 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide EDCI, 34.3 mg, 0.18 mmol, 1.5 equiv
  • 1-hydroxy-7- azabenzo-triazole HOAt, 24.5 mg, 0.18 mmol, 1.5 equiv
  • NMM N-methylmorpholine
  • intermediate 3 8.7 mg, 0.015 mmol, 1.0 equiv
  • 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide EDCI, 4.3 mg, 0.0225 mmol, 1.5 equiv
  • 1- hydroxy-7-azabenzo-triazole HOAt, 3.1 mg, 0.0225 mmol, 1.5 equiv
  • NMM N-methylmorpholine
  • Example 43 (R)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-1H-pyrazol-1-yl)-N-(3-(2-((4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4- yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-3- yl)benzyl)amino)acetamido)propyl)acetamide (XH168-143)
  • Example 43 was synthesized following the same procedure for preparing example 42 from linker 58 and intermediate 3.
  • Example 44 was synthesized following the same procedure for preparing example 42 from linker 59 and intermediate 3. (white solid, 6.9 mg, yield 40%).
  • 1 H NMR 400 MHz, Methanol-d4) ? 7.98 – 7.88 (m, 1H), 7.75 – 7.56 (m, 5H), 7.32 - 7.14 (m, 6H), 6.55 - 6.52 (m, 1H), 4.94 - 4.92 (m, 2H), 4.62 (s, 1H), 4.43 (s, 1H), 4.33 (s, 2H), 4.26 – 4.18 3H), 4.11 – 4.00 (m, 4H), 3.93 - 3.89 (m, 2H), 3.82 - 3.76 (m, 2H), 3.69 – 3.59 (m, 2H), 3.34 (s, 1H), 3.26 – 3.14 (m, 6H), 3.04 – 2.84 (m, 3H), 2.80 - 2.69 (m, 1H), 2.61 – 2.44 (m, 1H), 1.56 - 1.47
  • Example 45 (R)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-1H-pyrazol-1-yl)-N-(5-(2-((4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4- yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-3- yl)benzyl)amino)acetamido)pentyl)acetamide (XH168-145)
  • Example 45 was synthesized following the same procedure for preparing example 42 from linker 60 and intermediate 3.
  • Example 47 (R)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-1H-pyrazol-1-yl)-N-(7-(2-((4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4- yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-3- yl)benzyl)amino)acetamido)heptyl)acetamide (XH168-147)
  • Example 47 was synthesized following the same procedure for preparing example 42 from linker 62 and intermediate 3.
  • Example 49 (R)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-1H-pyrazol-1-yl)-N-(9-(2-((4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4- yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-3- yl)benzyl)amino)acetamido)nonyl)acetamide (XH168-149)
  • Example 49 was synthesized following the same procedure for preparing example 42 from linker 64 and intermediate 3.
  • Example 50 was synthesized following the same procedure for preparing example 42 from linker 65 and intermediate 3.
  • Example 51 (R)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-1H-pyrazol-1-yl)-N-(2-(2-((4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin- 4-yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-3- yl)benzyl)amino)acetamido)ethoxy)ethyl)acetamide (XH168-151)
  • Example 51 was synthesized following the same procedure for preparing example 42 from linker 66 and intermediate 3.
  • Example 53 (R)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-1H-pyrazol-1-yl)-N-(1-(4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4- yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-3-yl)phenyl)-4-oxo- 8,11,14-trioxa-2,5-diazahexadecan-16-yl)acetamide (XH168-153)
  • Example 53 was synthesized following the same procedure for preparing example 42 from linker 68 and intermediate 3.
  • Example 55 (R)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3- b]indol-9-yl)-1H-pyrazol-1-yl)-N-(1-(4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4- yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-3-yl)phenyl)-4-oxo- 8,11,14,17,20-pentaoxa-2,5-diazadocosan-22-yl)acetamide (XH168-155)
  • Example 55 was synthesized following the same procedure for preparing example 42 from linker 70 and intermediate 3.
  • Example 58 (R)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)ethyl)-4-(2-((4-(6-((4-hydroxy-1-(3- phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3- d]pyrimidin-3-yl)benzyl)amino)acetamido)butanamide (XH181-90)
  • Example 58 was synthesized following the same procedure for preparing example 56 from linker 44 and intermediate 3.
  • Example 59 was synthesized following the same procedure for preparing example 56 from linker 45 and intermediate 3. (white solid, 7.2 mg, yield 61%).
  • 1 H NMR (400 MHz, Methanol-d4) ? 7.98 – 7.93 (m, 1H), 7.72 – 7.62 (m, 5H), 7.30 – 7.15 (m, 6H), 6.51 – 6.42 (m, 1H), 4.55 (s, 2H), 4.37 – 4.28 (m, 5H), 4.21 - 4.18 (m, 1H), 4.11 – 4.02 (m, 4H), 3.96 – 3.82 (m, 2H), 3.78 – 3.60 (m, 6H), 3.26 - 3.14 (m, 4H), 3.04 - 2.94 (m, 3H), 2.81 – 2.68 (m, 1H), 2.61 – 2.44 (m, 1H), 2.22 - 2.19 (m, 2H), 1.60 - 1.47 (m, 5H), 1.42 – 1.27 (m, 6H), 0.
  • Example 62 (R)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)ethyl)-8-(2-((4-(6-((4-hydroxy-1-(3- phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3- d]pyrimidin-3-yl)benzyl)amino)acetamido)octanamide (XH181-94)
  • Example 62 was synthesized following the same procedure for preparing example 56 from linker 48 and intermediate 3.
  • Example 64 (R)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)ethyl)-10-(2-((4-(6-((4-hydroxy-1-(3- phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3- d]pyrimidin-3-yl)benzyl)amino)acetamido)decanamide (XH181-96)
  • Example 64 was synthesized following the same procedure for preparing example 56 from linker 50 and intermediate 3.
  • Example 68 (R)-N-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)ethyl)-3-((1-(4-(6-((4-hydroxy-1-(3- phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3- d]pyrimidin-3-yl)phenyl)-4-oxo-8,11-dioxa-2,5-diazatridecan-13-yl)oxy)propenamide (XH181-101)
  • Example 68 was synthesized following the same procedure for preparing example 56 from
  • Example 70 was synthesized following the same procedure for preparing example 56 from linker 66 and intermediate 3. (white solid, 7.4 mg, yield 54%).
  • 1 H NMR (400 MHz, Methanol-d 4 ) ? 7.97 - 7.92 (m, 1H), 7.72 – 7.65 (m, 5H), 7.30 – 7.16 (m, 6H), 6.51 - 6.44 (m, 1H), 4.57 - 4.54 (m, 2H), 4.34 – 4.30 (m, 5H), 4.20 (s, 1H), 4.10 - 4.01 (m, 4H), 3.94 - 3.91 (m, 1H), 3.82 (s, 2H), 3.88 -3.86 (m, 6H), 3.56 – 3.53 (m, 18H), 3.41 - 3.40 (m, 2H), 3.26 - 3.13 (m, 2H), 3.05 – 2.87 (m, 3H), 2.80 – 2.68 (m, 1H), 2.61 – 2.39 (m, 3H), 1.63
  • Example 110 6'-amino-N-(1-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19- diazahenicosan-21-yl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-127)
  • Example 110 was synthesized following similar procedure for preparing example 97 from intermediate 6 and linker 84.
  • linker 57 (4.6 mg, 0.01 mmol, 1.0 equiv)
  • EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mg, 0.015 mmol, 1.5 equiv)
  • HOAt (1-hydroxy-7-azabenzo-triazole) (2.0 mg, 0.015 mmol, 1.5 equiv)
  • NMM N- Methylmorpholine
  • Example 113 6'-amino-N-(3-((3-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro- 1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)acetamido)propyl)- amino)-3-oxopropyl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-130)
  • Example 113 was synthesized following similar procedure for preparing example 112 from intermediate 7 and linker 58.
  • Example 115 6'-amino-N-(3-((5-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro- 1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)acetamido)pentyl)- amino)-3-oxopropyl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-132)
  • Example 115 was synthesized following similar procedure for preparing example 112 from intermediate 7 and linker 60.
  • Example 117 6'-amino-N-(3-((7-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro- 1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)acetamido)heptyl)amino) -3-oxopropyl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-134)
  • Example 117 was synthesized following similar procedure for preparing example 112 from intermediate 7 and linker 62.
  • Example 119 6'-amino-N-(3-((9-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro- 1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)acetamido)nonyl)amino) -3-oxopropyl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-136)
  • Example 119 was synthesized following similar procedure for preparing example 112 from intermediate 7 and linker 64.
  • Example 121 6'-amino-N-(3-((2-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5- tetrahydro-1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)acetamido)ethoxy)- ethyl)amino)-3-oxopropyl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-138)
  • Example 121 was synthesized following similar procedure for preparing example 112 from intermediate 7 and linker 66.
  • Example 122 was synthesized following similar procedure for preparing example 112 from intermediate 7 and linker 67. Light yellow solid, 5.4 mg, 57% yield.
  • Example 123 6'-amino-N-(1-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)-2,16-dioxo-6,9,12-trioxa-3,15-diazaoctadecan-18- yl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-140)
  • Example 123 was synthesized following similar procedure for preparing example 112 from intermediate 7 and linker 68.
  • Example 125 6'-amino-N-(1-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)-2,22-dioxo-6,9,12,15,18-pentaoxa-3,21- diazatetracosan-24-yl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-142)
  • Example 125 was synthesized following similar procedure for preparing example 112 from intermediate 7 and linker 70.
  • linker 42 (4.6 mg, Attorney Docket No.: 27527-0219WO1 0.01 mmol, 1.0 equiv)
  • EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mg, 0.015 mmol, 1.5 equiv)
  • HOAt (1-hydroxy-7-azabenzo-triazole) (2.0 mg, 0.015 mmol, 1.5 equiv)
  • NMM N-Methylmorpholine
  • Example 128 6'-amino-N-(3-((4-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5- tetrahydro-1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)ethyl)amino)-4-oxobutyl)amino)-3- oxopropyl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-146)
  • Example 128 was synthesized following similar procedure for preparing example 126 from intermediate 7 and linker 44.
  • Example 130 6'-amino-N-(3-((6-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5- tetrahydro-1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)ethyl)amino)-6-oxohexyl)amino)-3- oxopropyl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-148)
  • Example 130 was synthesized following similar procedure for preparing example 126 from intermediate 7 and linker 46.
  • Example 132 6'-amino-N-(3-((8-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5- tetrahydro-1H-pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)ethyl)amino)-8-oxooctyl)amino)-3- oxopropyl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL186-150)
  • Example 132 was synthesized following similar procedure for preparing example 126 from intermediate 7 and linker 48.
  • Example 139 N 1 -(2-(6'-amino-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6- carboxamido)ethyl)-N 4 -(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)ethyl)succinamide (KL186-158).
  • linker 29 (5.1 mg, 0.01 mmol, 1.0 equiv)
  • EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mg, 0.015 mmol, 1.5 equiv)
  • HOAt (1-hydroxy-7-azabenzo-triazole) (2.0 mg, 0.015 mmol, 1.5 equiv)
  • NMM N- Methylmorpholine
  • Example 114 N 1 -(2-(6'-amino-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6- carboxamido)ethyl)-N 5 -(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-1H- pyrido[4,3-b]indol-9-yl)-1H-pyrazol-1-yl)ethyl)glutaramide (KL186-159)
  • Example 140 was synthesized following similar procedure for preparing example 139 from intermediate 6 and linker 30.
  • Linker 87 N-(5-aminopentyl)-3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 65% yield. 1 H NMR (400 MHz, CD3OD) ?
  • Linker 91 N-(9-aminononyl)-3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1- carboxamido)-3-methylpyridin-2-yl)benzamide White solid, 59% yield. 1 H NMR (400 MHz, CD 3 OD) ?
  • Linker 95 N-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6-(1-(2,2- difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1-carboxamido)-3-methylpyridin-2- yl)benzamide White solid, 61% yield. 1 H NMR (400 MHz, CD 3 OD) ?
  • Linker 98 Attorney Docket No.: 27527-0219WO1
  • Linker 98 3-(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1-carboxamido)-3- methylpyridin-2-yl)benzamido)propanoic acid 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5- yl)cyclopropane-1-carboxamido)-3-methylpyridin-2-yl)benzoic acid (100 mg, 0.22 mmol, 1.0 eq), tert-butyl 3-aminopropanoate (31.5 mg, 0.24 mmol, 1.1 eq), EDC ⁇ HCl (63 mg, 0.33 mmol, 1.5 eq), HOAt (45 mg, 0.33 mmol, 1.5 eq), NMM (67 mg, 0.66
  • Linker 109 1-(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1-carboxamido)- 3-methylpyridin-2-yl)phenyl)-1-oxo-5,8,11,14-tetraoxa-2-azaheptadecan-17-oic acid White solid, 65% yield. 1 H NMR (400 MHz, CD3OD) ?
  • linker 85 (10 mg, 0.013 mmol) in DMF (0.5 mL) were added intermediate 3 (2, 8.5 mg, 0.013 mmol, 1.0 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 2.7 mg, 0.0195 mmol, 1.5 equiv), 1-hydroxy-7-azabenzo-triazole (HOAt, 3.7 mg, 0.0195 mmol, 1.5 equiv), and N- methylmorpholine (NMM, 3.9 mg, 0.039 mmol, 3.0 equiv). After being stirred for overnight at rt, the resulting mixture was purified by preparative HPLC to afford crude product.
  • intermediate 3 (2, 8.5 mg, 0.013 mmol, 1.0 equiv)
  • EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • HOAt 1-hydroxy-7-azabenzo-triazole
  • Example 154 was synthesized following the same procedure for preparing example 153 from linker 84 and intermediate 3. (white solid, 9.3 mg, yield 55%).
  • 1 H NMR 400 MHz, Methanol-d 4 ) ? 8.39 (s, 2H), 8.09 - 8.08 (m, 3H), 7.77 - 7.75 (m, 6H), 7.40 – 7.22 (m, 8H), 4.39 (s, 2H), 4.23 - 4.01 (m, 5H), 3.94 - 3.90 (m, 2H), 3.73 – 3.57 (m, 4H), 3.43 (s, 2H), 3.34 (s, 2H), 3.24 - 3.23(m, 2H), 3.05 - 2.91 (m, 1H), 2.86 - 2.75 (m, 1H), 2.63 – 2.50 (m, 1H), 2.41 (s, 3H), 1.78 (s, 2H), 1.62 - 1.58 (m, 1H), 1.39 - 1.27 (m, 8H).
  • Example 155 was synthesized following the same procedure for preparing example 153 from linker 86 and intermediate 3. (white solid, 9.8 mg, yield 58%).
  • Example 156 was synthesized following the same procedure for preparing example 153 from linker 87 and intermediate 3. (white solid, 13.9 mg, yield 81%).
  • 1 H NMR (400 MHz, Methanol-d4) ? 8.49 - 8.36 (m, 2H), 8.03 (s, 3H), 7.74 - 7.69 (m, 6H), 7.36 - 7.18 (m, 8H), 4.35 (s, 2H), 4.18 - 3.99 (m, 5H), 3.92 - 3.83 (m, 3H), 3.69 – 3.53 (m, 3H), 3.35 (s, 2H), 3.26 - 3.23 (m, 3H), 2.99 - 2.92 (m, 1H), 2.80 - 2.74 (m, 1H), 2.60 - 2.51 (m, 1H), 2.38 (s, 3H), 1.75 (s, 2H), 1.62 - 1.55 (m, 4H), 1.41 – 1.24 (m, 10H).
  • Example 158 (R)-3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1- carboxamido)-3-methylpyridin-2-yl)-N-(7-(2-((4-(6-((4-hydroxy-1-(3- phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3- d]pyrimidin-3-yl)benzyl)amino)acetamido)heptyl)benzamide (XH168-67)
  • Example 158 was synthesized following the same procedure for preparing example 153 from linker 89 and intermediate 3.
  • Example 160 (R)-3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1- carboxamido)-3-methylpyridin-2-yl)-N-(9-(2-((4-(6-((4-hydroxy-1-(3- phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3- d]pyrimidin-3-yl)benzyl)amino)acetamido)nonyl)benzamide (XH168-69)
  • Example 160 was synthesized following the same procedure for preparing example 153 from linker 91 and intermediate 3.
  • Example 165 was synthesized following the same procedure for preparing example 153 from linker 96 and intermediate 3. (white solid, 15.7 mg, yield 93%).
  • 1 H NMR (400 MHz, Methanol-d4) ? 8.52 – 8.40 (m, 2H), 8.07 (s, 3H), 7.78 - 7.74 (m, 6H), 7.40 – 7.17 (m, 8H), 4.39 (s, 2H), 4.23 - 4.03 (m, 5H), 3.96 - 3.88 (m, 3H), 3.73 – 3.58 (m, 20H), 3.43 (s, 2H), 3.34 (m, 2H), 3.23 (s, 1H), 3.03 - 2.95 (m, 1H), 2.87 - 2.76 (m, 1H), 2.64 – 2.53 (m, 1H), 2.41 (s, 3H), 1.78 (s, 2H), 1.60 (s, 1H), 1.39 – 1.28 (m, 6H).
  • Example 166 was synthesized following the same procedure for preparing example 153 from linker 97 and intermediate 3. (white solid, 16.1 mg, yield 92%) 1 H NMR (400 MHz, Methanol-d 4 ) ? 8.07 – 7.97 (m, 2H), 7.89 – 7.84 (m, 2H), 7.78 – 7.68 (m, 5H), 7.58 – 7.52 (m, 2H), 7.36 – 7.14 (m, 8H), 4.35 (s, 2H), 4.22 – 4.12 (m, 4H), 4.08 – 3.99 (m, 1H), 3.96 – 3.89 (m, 1H), 3.84 (s, 2H), 3.62 – 3.55 (m, 22H), 3.43 - 3.42 (m, 2H), 3.25 - 3.15 (m, 3H), 3.05 - 2.89 (m, 1H), 2.81 – 2.69 (m, 1H), 2.61 – 2.45 (m, 1H), 2.23 (s, 3H), 1.67 (s, 2H
  • linker 85 4.9 mg, 0.01 mmol, 1.0 equiv
  • EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • HOAt 1-hydroxy-7-azabenzo-triazole
  • NMM N-Methylmorpholine
  • Example 177 6'-amino-N-(1-(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1- carboxamido)-3-methylpyridin-2-yl)phenyl)-1,12-dioxo-5,8-dioxa-2,11-diazatetradecan-14- yl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL180-134)
  • Example 177 was synthesized following similar procedure for preparing example 167 from linker 94 and intermediate 7.
  • Example 179 6'-amino-N-(1-(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1- carboxamido)-3-methylpyridin-2-yl)phenyl)-1,18-dioxo-5,8,11,14-tetraoxa-2,17- diazaicosan-20-yl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL180- 136)
  • Example 179 was synthesized following similar procedure for preparing example 167 from linker 96 and intermediate 7.
  • linker 98 5.1 mg, 0.01 mmol, 1.0 equiv
  • EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • HOAt 1-hydroxy-7-azabenzo-triazole
  • NMM N- Methylmorpholine
  • Example 189 6'-amino-N-(2-(3-(2-(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5- yl)cyclopropane-1-carboxamido)-3-methylpyridin-2-yl)benzamido)ethoxy)propanamido)- ethyl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL180-170)
  • Example 189 was synthesized following similar procedure for preparing example 181 from intermediate 6 and linker 106.
  • Example 191 6'-amino-N-(1-(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1- carboxamido)-3-methylpyridin-2-yl)phenyl)-1,14-dioxo-5,8,11-trioxa-2,15- diazaheptadecan-17-yl)-4'-ethyl-5'-(4-hydroxyphenyl)-[3,3'-bipyridine]-6-carboxamide (KL180-172)
  • Example 191 was synthesized following similar procedure for preparing example 181 from intermediate 6 and linker 108.
  • Example AMPK based DUBTACs are set forth in Table 1 below. Table 1.
  • Example - az - 4- - y - 3- in et mi ro- H- - azo (3- yl) - H- - no) op Attorney Docket No.: 27527-0219WO1 mid ro- H- - azo (4- yl) - H- - no) tyl mid ro- H- - azo (5- yl) - H- - no) nty mid ro- H- - azo (6- yl) - H- - no) xyl
  • HCT116 cells were treated with AMPK DUBTAC XH181-16, XH181-17, or XH181-18 (MS8118) for 24 h.
  • F HeLa cells were treated with 10 ?M AMPK DUBTAC XH181-16, XH181-17, or XH181-18 (MS8118) for 24 h, followed by Oil Red O staining.
  • n 3. **: p ⁇ 0.01. ns: not significant.
  • G Representative images of the colony formation assay.
  • (H) AMPK DUBTAC XH181-16, XH181-17, or XH181-18 (MS8118) do not affect the mRNA levels of AMPK?1 in cells.
  • Hela cells were treated with 10 ?M indicated compounds for 24 h, followed by RT-qPCR analysis for the mRNA levels of AMPK?1.
  • Example 197 Example 197.
  • Example 200 Assessment of the effect of exemplary cGAS based bivalent compounds (KL series) on stabilization of the cGAS protein level in Hela cells ( Figure 7) The Hela cells was treated with DMSO, or selected bivalent compound at 10 ?M for 24 h. The Western blot results showed that multiple compounds can increase cGAS and its downstream target STING proteins level.
  • Example 201 Assessment of the effect of exemplary cGAS based bivalent compounds (KL series) on stabilization of the cGAS protein level in Hela cells ( Figure 7) The Hela cells was treated with DMSO, or selected bivalent compound at 10 ?M for 24 h. The Western blot results showed that multiple compounds can increase cGAS and its downstream target STING proteins level.
  • Example 201 Assessment of the effect of exemplary cGAS based bivalent compounds (KL series) on stabilization of the cGAS protein level in Hela cells ( Figure 7) The Hela cells was treated with DMSO, or selected bivalent compound at 10 ?
  • Hela cells were treated with KL186-141 at indicated concentrations for 24 h.
  • D KL186-141, but not cGAS agonist G140 effective suppressed Hela cells proliferation. Hela cells were treated with DMSO, G140 or KL186-141 (10 ?M).
  • E IC 50 determination of KL186-141 in Hela cells. Hela cells were treated with DMSO, G140 or KL186-141 at indicated concentrations for 3 days.
  • Example 202 KL186-141, but not cGAS agonist G140 effective suppressed Hela cells proliferation. Hela cells were treated with DMSO, G140 or KL186-141 (10 ?M).
  • E IC 50 determination of KL186-141 in Hela cells. Hela cells were treated with DMSO, G140 or KL186-141 at indicated concentrations for 3 days. Example 202.
  • the Western blot results showed that multiple compounds can increase CFTR protein level; Right, the CFBE41o- 4.7 ?F508-CFTR human cystic fibrosis bronchial epithelial cells were treated with DMSO, positive control (NJH-2-057) or selected bivalent compound at 10 or 20 ?M for 24 h. (b) CFBE41o- 4.7 ?F508-CFTR human cystic fibrosis bronchial epithelial cells was treated with DMSO, positive control (NJH-2-057) or selected bivalent compound at indicated concentrations (10??M) for 24 h. The Western blot results showed that the compounds can effectively increase CFTR protein level.
  • Example 203 The Western blot results showed that the compounds can effectively increase CFTR protein level.
  • C IB analysis of CFTR-IP and WCL derived from HEK293 cells transfected with HA-Ub and CFTR expression plasmids. Cells were treated with or without 20 ?M MS8118 for 24 h and 30 ?M proteasome inhibitor MG132 for 8 h before harvesting.
  • D CFBE41o- 4.7 ?F508-CFTR cells were treated with the indicated concentrations of MS6869 for 24 h, followed by Western blot analysis.
  • E CFBE41o-4.7 ?F508-CFTR cells were treated with 10 ?M MS6869 for the indicated hours, followed by Western blot analysis.
  • CFBE41o-4.7 ?F508-CFTR cells were treated with 10 ?M MS6869 for 24 h, followed by the cellular component fraction for cytoplasm (Cyto) and plasma membrane (Mem).
  • G CFBE41o- 4.7 ?F508-CFTR cells were treated with MS6869, with or without the co-treatment with USP7 ligand #1 or CFTR ligand Lumacaftor for 24 h, followed by Western blot analysis.
  • H CFBE41o- 4.7 ?F508-CFTR cells were depleted of USP7 and then treated with 10 ?M MS6869 for 24 h, followed by Western blot analysis.
  • CFBE41o- 4.7 ?F508- CFTR cells were treated with 10 ?M of MS6869 for 24 h, followed by RT-qPCR analysis for the mRNA levels of CFTR.
  • Materials and Methods Cell culture The CFBE41o- 4.7 ?F508-CFTR human cystic fibrosis bronchial epithelial cells were purchased from Millipore Sigma (SCC159). CFBE41o- 4.7 ?F508-CFTR cells are derived from the parental CFBE41o- cells by introduced the ?F508-CFTR construct. CFBE41o- 4.7 ?F508-CFTR cells were cultured in ?-MEM media (Sigma.
  • HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 Units of penicillin and 100 ⁇ g/ml streptomycin.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • cells in 6-well plate were treated with indicated compounds.
  • HT-DNA Sigma, D6898
  • PEI Polysciences, # 23966
  • free ligand competition assay the free ligand, USP7 ligand, cGAS ligand or AMPK ligand in DMSO were co-treated with indicated DUBTAC compounds.
  • CFTR (#78335, 1:1,000) antibody was purchased from Cell Signaling Technologies.
  • Anti- Actin (A2228, 1:50,000), anti-vinculin antibody (V-4505, 1:50,000), peroxidase-conjugated anti- mouse secondary antibody (A-4416, 1:3000) and peroxidase-conjugated anti-rabbit secondary Attorney Docket No.: 27527-0219WO1 antibody (A-4914, 1:3,000) were purchased from Sigma. All primary antibodies were diluted in 5% bovine serum albumin (BSA) in TBST buffer, and secondary antibodies were diluted in 5% non-fat milk in TBST buffer.
  • BSA bovine serum albumin
  • the potent AMPK inhibitor BAY-3827 shows strong efficacy in androgen-dependent prostate cancer models.
  • AMP-activated protein kinase AMP-activated protein kinase
  • RSVA314 and RSVA405 Small-molecule activators of AMP-activated protein kinase (AMPK), RSVA314 and RSVA405, inhibit adipogenesis. Mol. Med.17, 1022-1030.10.2119/molmed.2011.00163. Ward, C.L., Omura, S., and Kopito, R.R. (1995). Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83, 121-127.10.1016/0092-8674(95)90240-6.

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Abstract

Sont divulgués une méthodes et des composés pour traiter des maladies médiées par AMPK, cGAS ou ΔF508-CFTR, la méthode comprenant l'administration d'une ou de plusieurs maladies médiées par AMPK, cGAS ou CFTR à un sujet atteint d'une maladie médiée par AMPK, cGAS ou ΔF508-CFTR, l'AMPK, cGAS, ou CFTR DUBTACs étant des composés à petites molécules hétérobifonctionnels (ou des composés bivalents en abrégé) comprenant un ligand AMPK, cGAS ou CFTR conjugué à une étiquette de déubiquitination (telle qu'un ligand à petites molécules de USP7) par l'intermédiaire d'un lieur, qui stabilise l'AMPK, le cGAS ou le CFTR. L'AMPK, le cGAS ou la maladie médiée par ΔF508-CFTR peuvent être une maladie résultant de l'AMPK, du cGAS ou de la déstabilisation de CFTR. L'AMPK, la cGAS ou la maladie médiée par ΔF508-CFTR peuvent avoir une expression d'AMPK, de cGAS ou de CFTR réduite par rapport à un tissu de type sauvage de la même espèce et du même type de tissu. Les composés bivalents divulgués stabilisent l'AMPK, le cGAS ou le CFTR par recrutement de USP7.
PCT/US2024/027532 2023-05-05 2024-05-02 Petites molécules hétérobifonctionnelles pour la déubiquitination et la stabilisation de protéines cibles par recrutement de usp7 Pending WO2024233280A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021146390A1 (fr) * 2020-01-14 2021-07-22 The Trustees Of Columbia University In The City Of New York Compositions et méthodes de stabilisation de protéines ciblées par réorientation de désubiquitinases endogènes
WO2022232634A1 (fr) * 2021-04-29 2022-11-03 Novartis Ag Chimères ciblant la désubiquitinase et procédés associés

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021146390A1 (fr) * 2020-01-14 2021-07-22 The Trustees Of Columbia University In The City Of New York Compositions et méthodes de stabilisation de protéines ciblées par réorientation de désubiquitinases endogènes
WO2022232634A1 (fr) * 2021-04-29 2022-11-03 Novartis Ag Chimères ciblant la désubiquitinase et procédés associés

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAMATAM REKHA, SHIN DONGYUN: "Emerging Strategies in Proteolysis-Targeting Chimeras (PROTACs): Highlights from 2022", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 24, no. 6, 5190, 8 March 2023 (2023-03-08), CH, pages 1 - 21, XP093233241, ISSN: 1422-0067, DOI: 10.3390/ijms24065190 *

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