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WO2025226064A1 - Composé ligand pour ubiquitine ligase et molécule protac le comprenant - Google Patents

Composé ligand pour ubiquitine ligase et molécule protac le comprenant

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Publication number
WO2025226064A1
WO2025226064A1 PCT/KR2025/005577 KR2025005577W WO2025226064A1 WO 2025226064 A1 WO2025226064 A1 WO 2025226064A1 KR 2025005577 W KR2025005577 W KR 2025005577W WO 2025226064 A1 WO2025226064 A1 WO 2025226064A1
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Prior art keywords
alkyl
amino
benzamide
oxy
guanidinophenyl
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Pending
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PCT/KR2025/005577
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English (en)
Korean (ko)
Inventor
임현석
왕희명
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Camel Bioscience Inc
POSTECH Research and Business Development Foundation
Original Assignee
Camel Bioscience Inc
POSTECH Research and Business Development Foundation
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Publication of WO2025226064A1 publication Critical patent/WO2025226064A1/fr
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Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/12—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/18—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to carbon atoms of six-membered aromatic rings
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/12—Oxygen or sulfur atoms
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D307/10—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/14—Radicals substituted by nitrogen atoms not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/64—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/06—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring carbon atoms

Definitions

  • the present invention relates to a ligand compound of ubiquitin ligase and a PROTAC (proteolysis targeting chimera) molecule comprising the same, and more particularly, to a compound acting as a ligand of E3 ubiquitin ligase or a prodrug thereof, and a PROTAC heterobifunctional molecule comprising the same.
  • the N-end rule states that the lifetime of a protein depends on the characteristics of its N-terminal residue.
  • the N-terminal residue that destabilizes the protein is called an N-degron, and is divided into type 1 and type 2.
  • Type 1 N-degrons contain basic amino acid residues that can be positively charged, such as arginine (Arg), lysine (Lys), and histidine (His), while type 2 N-degrons contain hydrophobic amino acid residues, such as phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), leucine (Leu), or isoleucine (Ile) (Varshavsky, A. et al. Protein Science 2011, 20, 1298-1345).
  • UBR ubiquitin protein ligase E3 component n-recognin
  • the protein crystal structures of the UBR box of human UBR1, UBR2, and yeast UBR1 are known, and the UBR box contains three Zn cations and two zinc finger motifs that can coordinate Zn 2+ ions, and two binding pockets that can recognize the first and second amino acid residues of the N-degron (Choi, W. S. et al. Nat. Struct. Mol. Biol. 2010, 17, 1175-1181; Matta-Camacho, E. et al. Nat. Struct. Mol. Biol. 2010, 17, 1182-1187; Munoz-Escobar, J. Structure 2017, 25, 719-729).
  • the first binding pocket is composed of aspartic acid (Asp), threonine (Thr), and phenylalanine (Phe) residues, which form a negatively charged protein surface and allow strong interactions with type 1 N-degrons, which have positively charged basic residues, through hydrogen bonding and charge-charge interactions.
  • the second binding pocket also allows selective recognition of N-degrons through interactions with specific residues, such as serine (Ser), valine (Val), phenylalanine (Phe), and threonine (Thr).
  • the UBR box of the UBR protein recognizes N-degrons and plays a crucial role in cellular protein degradation.
  • Ligand compounds that bind to the UBR box can participate in protein degradation pathways.
  • UBR proteins are known to play essential regulatory roles in numerous signaling pathways, including G-protein signaling, apoptosis, and inflammation. Dysregulation in these signaling pathways can lead to disease states such as cancer and neurodegeneration.
  • GPCRs G-protein-coupled receptors
  • GPCRs G-protein-coupled receptors
  • GPCRs G-protein-coupled receptors
  • RGS Regulator of G-protein signaling (RGS) proteins, which play a key role in this signaling cascade, undergo arginylation of their N-terminal residues through metabolism, followed by ubiquitination by UBR1 and UBR2, leading to protein degradation (Lee, M. J. et al. Proc. Natl. Acad. Sci. USA 2005, 102, 15030-15035).
  • Inflammation is a protective response induced by the well-conserved innate immune system to protect against pathogens, damaged cells, or harmful stimuli.
  • Some inflammatory fragments contain unstable N-terminal residues, leading to their degradation via the N-degron pathway, such as UBR1, UBR2, UBR4, and UBR5.
  • UBR1, UBR2, UBR4, and UBR5 exhibit unstable N-terminal residues, leading to their degradation via the N-degron pathway, such as UBR1, UBR2, UBR4, and UBR5.
  • knockdown of UBR1, UBR2, UBR4, and UBR5 via siRNA significantly increased IL-1 ⁇ secretion (Leboeuf, D. Biomolecules 2020, 10, 903), suggesting that UBR E3 ligases are also involved in inflammatory responses.
  • a proteolysis targeting chimera is a heterobifunctional molecule consisting of a ligand for a target protein and a ligand that binds to an E3 ubiquitin ligase, linked via a linker.
  • PROTACs simultaneously bind to both proteins, bringing the target protein into close proximity to the E3 ubiquitin ligase. This allows the E3 ubiquitin ligase to recognize the target protein as a substrate, triggering polyubiquitination and subsequent proteasomal degradation. This principle allows for the effective removal of specific proteins from cells. Therefore, PROTACs can be used as chemical probes for studying the function of target proteins and, furthermore, hold great potential as therapeutic agents for diseases.
  • E3 ubiquitin ligases In the human body, only a few, such as cereblon (CRBN) and Von Hippel-Lindau tumor suppressor (VHL), are currently used as E3 ubiquitin ligases in the PROTAC design.
  • CBN cereblon
  • VHL Von Hippel-Lindau tumor suppressor
  • the present invention has as a technical problem a compound or a prodrug thereof that can efficiently act as a ligand of an E3 ubiquitin ligase by binding to the UBR box of a UBR protein.
  • the present invention has as another technical object to provide a PROTAC (proteolysis targeting chimera) heterobifunctional molecule comprising a ligand compound of the E3 ubiquitin ligase or a prodrug thereof.
  • PROTAC proteolysis targeting chimera
  • the present invention provides a compound of the following chemical formula 1 or a prodrug thereof:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , L 1 , L 2 , L 3 , m, n and p are as defined herein.
  • the present invention provides a chimeric compound of the following chemical formula 2:
  • a and B are as defined herein, and A and B are chemically linked by a linker.
  • the compound according to the present invention or a prodrug thereof can act as a ligand for an efficient E3 ubiquitin ligase, thereby efficiently degrading a target protein by the ubiquitin-proteasome system of a cell in a PROTAC (proteolysis targeting chimera) technology utilizing the compound.
  • Figure 1 shows the seven isoforms of ubiquitin protein ligase E3 component n-recognin (UBR) proteins.
  • Figure 2 is a graph showing a competitive fluorescence polarization analysis method.
  • Figure 3 shows LC and Mass analysis data of PROTAC of Example 167.
  • Figure 4 is a graph showing the results of measuring the binding affinity of the compound of Example 113 and the PROTAC of Example 167 to the UBR1 protein in Experimental Example 2.
  • Figure 5 shows the results of evaluating the BRD4 protein decomposition ability of PROTAC of Example 167 in Experimental Example 3 (BRD4: bromodomain-containing protein 4, GAPDH: glyceraldehyde-3-phosphate dehydrogenase).
  • Figure 6 shows the results of evaluating the decomposition ability of BRD4 protein in Experimental Example 4 (JQ1: BET bromodomain inhibitor, MG132: proteasome inhibitor, BRD4: bromodomain-containing protein 4, GAPDH: glyceraldehyde-3-phosphate dehydrogenase).
  • Figure 7 shows LC and Mass analysis data of the PROTAC prodrug of Example 168.
  • Figure 8 shows the results of evaluating the ability of the PROTAC and the PROTAC prodrug of Example 168 to decompose BRD4 protein in which the compound of Example 100 and the BRD4 ligand were linked via a linker in Experimental Example 5 (BRD4: bromodomain-containing protein 4, GAPDH: glyceraldehyde-3-phosphate dehydrogenase).
  • a compound of the following chemical formula 1 or a prodrug thereof is provided:
  • R 1 and R 2 are each independently -H, -D, alkyl, deuterated alkyl or haloalkyl;
  • R 3 is -DEG; wherein D is a direct bond or alkylene; E is alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene, heterocycloalkylene, heterocycloalkenylene, or heteroarylene; wherein said alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene, heterocycloalkylene, heterocycloalkenylene or heteroarylene is optionally -D, halo, hydroxy, thiol (-SH), amino, nitro, cyano, alkyl, haloalkyl, deuterated alkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkoxyalkyl, alkylthio, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, aminocarbonyl, alkyla
  • R 4 and R 6 are each independently -H, -D, alkyl, deuterated alkyl or haloalkyl;
  • L 1 is -C-, -CH-, cycloalkylene, heterocyclylene, cycloalkenylene, heterocycloalkenylene, arylene or heteroarylene; or R 4 and the N atom to which L 1 is bonded may form a ring structure;
  • R 5 is -H, -D, halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkyl-alkyl, heterocycloalkyl-alkyl, aryl-alkyl, aryl-alkoxy or heteroaryl-alkyl; wherein said alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkyl-alkyl, heterocycloalkyl-alkyl, aryl-alkyl, aryl-alkoxy or heteroaryl-alkyl is optionally -D, hydroxy, thiol, amino, halo, nitro, cyano, alkyl,
  • L 2 may be a direct bond, or when n is 0, R 4 and L 2 may be connected to each other with the N atom to which they are bonded to form a ring structure; wherein said ring may be optionally substituted with one or more substituents selected from -D, hydroxy, amino, halo, nitro, cyano, alkyl, haloalkyl, deuterated alkyl, hydroxyalkyl, alkoxy, haloalkoxy, cycloalkyl-oxy, heterocycloalkyl-oxy, aryl-oxy, heteroaryl-oxy, alkoxyalkyl, aminoalkyl, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aminocarbonylalkyl, alkylaminocarbonylalkyl, dialkylaminocarbonylalkyl, dialkylaminocarbony
  • L 3 is a direct bond, -C-, -CH-, -CH 2 -, cycloalkylene, heterocycloalkylene, unsaturated carbocyclylene, unsaturated heterocyclylene, arylene or heteroarylene; or may form a ring structure together with the N atom to which R 6 and L 3 are bonded;
  • R 7 is halo, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl-oxy, cycloalkyl-alkyl, cycloalkyl-alkoxy, cycloalkenyl-oxy, cycloalkenyl-alkyl, cycloalkenyl-alkoxy, heterocycloalkyl-oxy, heterocycloalkyl-alkyl, heterocycloalkyl-alkoxy, heterocycloalkenyl-oxy, heterocycloalkenyl-alkyl, heterocycloalkenyl-alkoxy, aryl, aryl-oxy, aryl-alkyl, aryl-alkoxy, heteroaryl, heteroaryl-oxy, heteroaryl-alkyl, heteroaryl-alkoxy or and wherein R 14 and R 15 are each independently -H, alkyl, alkenyl, alky
  • R 7 and L 3 may be linked together to form cycloalkyl or heterocycloalkyl;
  • R 8 is -H, , , , , , , , or and wherein R 16 to R 31 are each independently -H, hydroxy, nitro, cyano, azido(-N 3 ), halo, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkylthio, alkylthioalkyl, haloalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, alkylamino, dialkylamino, dialkylaminoalkyl, aminoalkoxy-alkyl, alkylcarbonyl, aryl, heteroaryl, cycloalkyl-alkyl, cycloalkenyl-alkyl, heterocycloalkyl-alkyl, heterocycloalkenyl-alkyl, aryl-alkyl, heteroaryl-alkyl, partially unsaturated heterocycl
  • n 0 or 1
  • n 0, 1, or 2;
  • p 0, 1, 2, 3 or 4;
  • L 1 is -C- or -CH-
  • L 3 is not a direct bond or -CH-
  • L 2 and L 3 are not direct bonds at the same time
  • heterocycloalkylene, heterocycloalkenylene, heteroarylene, heterocycloalkyl, heteroaryl, heterocyclylene, heterocyclyl and partially unsaturated heterocyclyl have one or more heteroatoms selected from N, O and S.
  • halo or “halogen”, when used alone or in combination with other additional terms (e.g., haloalkyl), means fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).
  • hydroxy means -OH.
  • nitro group means -NO 2 .
  • cyano group means -CN.
  • thiol means -SH.
  • oxy means -O-.
  • amino in the present invention may refer to a primary, secondary, or tertiary amino group, alone or in combination, bonded via a nitrogen atom.
  • the secondary amino group may refer to one having an alkyl substituent
  • the tertiary amino group may refer to one having two similar or different alkyl substituents.
  • the term “azido” means -N 3 .
  • alkyl when used alone or in combination with other additional terms (e.g., haloalkyl), means a radical of a straight or branched saturated aliphatic hydrocarbon group having, for example, 1 to 7 carbon atoms or 1 to 5 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, 2-methylbutyl, 1-ethylpropyl, and 1,2-dimethylpropyl.
  • deuterated alkyl means an alkyl group having one or more deuterium atoms.
  • alkenyl means a radical of an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, for example, having 2 to 7 carbon atoms or 2 to 5 carbon atoms.
  • alkynyl means a radical of an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond, for example, having 2 to 7 carbon atoms or 2 to 5 carbon atoms.
  • alkoxy means an alkyloxy (-O-alkyl group), for example, an alkyloxy having 1 to 7 carbon atoms or 1 to 5 carbon atoms.
  • alkylthio means an -S-alkyl group, for example, an -S-alkyl group having 1 to 7 or 1 to 5 carbon atoms.
  • alkylene means a radical of a divalent straight-chain or branched-chain saturated aliphatic hydrocarbon group having, for example, 1 to 7 carbon atoms or 1 to 5 carbon atoms.
  • alkynylene means a radical of an aliphatic hydrocarbon group containing at least one carbon-carbon double bond of two valences, for example, having 2 to 7 carbon atoms or 2 to 5 carbon atoms.
  • alkynylene means a radical of an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond of two valences, for example, having 2 to 7 carbon atoms or 2 to 5 carbon atoms.
  • hydroxyalkyl means an alkyl group substituted with hydroxy.
  • cycloalkyl refers to a saturated aliphatic hydrocarbon radical having, for example, 3 to 10 carbon atoms or 3 to 8 carbon atoms in a ring shape.
  • Typical examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • the cycloalkyl may include a bridged structure, a fused structure, or a spiro structure.
  • cycloalkenyl in the present invention means a radical of a partially unsaturated aliphatic hydrocarbon group having a cyclic shape, for example, 3 to 10 carbon atoms or 3 to 8 carbon atoms, and one carbon-carbon double bond.
  • heterocycloalkyl in the present invention means a radical of a saturated aliphatic hydrocarbon group containing one or more heteroatoms selected from N, O and S as a reducing group, for example, a 4 to 12 membered or a 4 to 10 membered group.
  • the heterocycloalkyl may include a bridged structure, a fused structure or a spiro structure.
  • partially unsaturated heterocyclyl as used herein means a cyclic, for example, 4 to 12-membered or 4 to 10-membered, partially unsaturated hydrocarbon group containing one or more heteroatoms selected from N, O and S as a reducing group.
  • the partially unsaturated heterocyclyl may include a bridged structure, a fused structure or a spiro structure.
  • aryl means an aromatic hydrocarbon having, for example, 6 to 12 or 6 to 10 carbon atoms, and specific examples include, but are not limited to, phenyl and naphthyl.
  • heteroaryl means, for example, a 5- to 12-membered or 5- to 10-membered aromatic hydrocarbon containing one or more heteroatoms selected from N, O and S as a reducing group and forming a single or fused ring that can be fused with benzo or cycloalkyl.
  • the term “prodrug” includes a form in which the compound of the above chemical formula 1 is converted in vivo to exhibit the same effect, and can be prepared according to a method known in the art, and there are no particular limitations thereto.
  • the prodrug can be provided in the form of, but is not limited to, carbamates, esters, N-Mannich adducts, phosphates, phosphonooxymethylethers, phosphoramidates, acylsulfonamides, amides, sulfenamides, imines, azo conjugates, N-acyloxyalkylamines, etc.
  • R 1 and R 2 are each independently -H, -D, C 1 -C 7 alkyl, deuterated C 1 -C 7 alkyl or halo-C 1 -C 7 alkyl;
  • R 3 is -DEG; wherein D is a direct bond or C 1 -C 7 alkylene; E is C 1 -C 7 alkylene, C 2 -C 7 alkenylene, C 2 -C 7 alkynylene, C 3 -C 10 cycloalkylene , C 3 -C 10 cycloalkenylene, C 6 -C 12 arylene, 4 to 12 membered heterocycloalkylene, 4 to 12 membered heterocycloalkenylene, or 5 to 12 membered heteroarylene; wherein said alkylene, alkenylene, alkynylene, cycloalkylene.
  • Cycloalkenylene, arylene, heterocycloalkylene, heterocycloalkenylene or heteroarylene is optionally -D, halo, hydroxy, thiol, amino, nitro, cyano, C 1 -C 7 alkyl, halo-C 1 -C 7 alkyl, deuterated C 1 -C 7 alkyl, hydroxy-C 1 -C 7 alkyl, C 1 -C 7 alkoxy, halo-C 1 -C 7 alkoxy, C 1 -C 7 alkoxy-C 1 -C 7 alkyl, C 1 -C 7 alkylthio, C 1 -C 7 alkylamino, di(C 1 -C 7 alkyl)amino, amino-C 1 -C 7 alkyl , C 1 -C 7 alkylamino-C 1 -C 7 alkyl, di(C 1 -C 7 Alkyl)amino-C 1 -C 7 alkyl, aminocarbon
  • R 4 and R 6 are each independently -H, -D, C 1 -C 7 alkyl, deuterated C 1 -C 7 alkyl or halo-C 1 -C 7 alkyl;
  • L 1 is -C-, -CH-, C 3 -C 10 cycloalkylene, 4 to 12 membered heterocyclylene, C 3 -C 10 cycloalkenylene, 4 to 12 membered heterocycloalkenylene, C 6 -C 12 arylene or 5 to 12 membered heteroarylene; or may form a ring structure together with the N atom to which R 4 and L 1 are bonded;
  • R 5 is -H, -D, halo, C 1 -C 7 alkyl, halo-C 1 -C 7 alkyl, C 2 -C 7 alkenyl, C 2 -C 7 alkynyl, C 3 -C 10 cycloalkyl, C 3 -C 10 cycloalkenyl, 4 to 12 membered heterocycloalkyl, 4 to 12 membered heterocycloalkenyl, C 6 -C 12 aryl, 5 to 12 membered heteroaryl, C 3 -C 10 cycloalkyl-C 1 -C 7 alkyl, 4 to 12 membered heterocycloalkyl-C 1 -C 7 alkyl, C 6 -C 12 aryl-C 1 -C 7 alkyl or 5 to 12 membered heteroaryl-C 1 -C 7 alkyl; wherein said alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl
  • L 2 may be a direct bond, or when n is 0, R 4 and L 2 may be connected to each other with the N atom to which they are bonded to form a ring structure; wherein the ring is optionally -D, hydroxy, amino, halo, nitro, cyano, C 1 -C 7 alkyl, halo-C 1 -C 7 alkyl, deuterated C 1 -C 7 alkyl, hydroxy-C 1 -C 7 alkyl, C 1 -C 7 alkoxy, halo-C 1 -C 7 alkyl-oxy, C 3 -C 10 cycloalkyl-oxy, 4 to 12 membered heterocycloalkyl-oxy, C 6 -C 12 aryl-oxy, 5 to 12 membered heteroaryl-oxy, C 1 -C 7 alkoxy-C 1 -C 7 alkyl, amino-C 1 -C 7 alkyl, C 1 -C 7 alkylamino, di(C 1 -C
  • L 3 is a direct bond, -C-, -CH-, -CH 2 -, C 3 -C 10 cycloalkylene, 4 to 12 membered heterocycloalkylene, unsaturated C 3 -C 10 carbocyclylene, unsaturated 4 to 12 membered heterocyclylene, C 6 -C 12 arylene or 5 to 12 membered heteroarylene; or may form a ring structure together with the N atom to which R 6 and L 3 are bonded;
  • R 7 is halo, C 1 -C 7 alkyl, C 2 -C 7 alkenyl, C 2 -C 7 alkynyl, halo-C 1 -C 7 alkyl, C 1 -C 7 alkoxy, halo-C 1 -C 7 alkoxy, hydroxy-C 1 -C 7 alkyl, C 3 -C 10 cycloalkyl-oxy, C 3 -C 10 cycloalkyl-C 1 -C 7 alkyl, C 3 -C 10 cycloalkyl-C 1 -C 7 alkoxy, C 3 -C 10 cycloalkenyl-oxy, C 3 -C 10 cycloalkenyl-C 1 -C 7 alkyl, C 3 -C 10 cycloalkenyl-C 1 -C 7 alkoxy, 4 to 12 membered heterocycloalkyl-oxy, 4 to 12 membered Heterocycloalkyl-C 1 -C 7 alky
  • R 7 and L 3 may be linked together to form a C 3 -C 10 cycloalkyl or a 4 to 12 membered heterocycloalkyl;
  • R 8 is -H, , , , , , , , or and;
  • R 16 to R 31 are each independently -H, hydroxy, nitro, cyano, azido(-N 3 ), halo, C 1 -C 7 alkyl, C 2 -C 7 alkenyl, C 2 -C 7 alkynyl, C 1 -C 7 alkoxy, C 1 -C 7 alkoxy-C 1 -C 7 alkyl, C 1 -C 7 alkylthio, C 1 -C 7 alkylthio -C 1 -C 7 alkyl, halo-C 1 -C 7 alkyl, C 3 -C 10 cycloalkyl, C 3 -C 10 cycloalkenyl, 4 to 12 membered heterocycloalkyl, 4 to 12 membered heterocycloalkenyl, C 1 -C 7 alkylamino, di(C 1 -C 7 alkyl)
  • n 0 or 1
  • n 0, 1, or 2;
  • p 0, 1, 2, 3 or 4;
  • L 1 is -C- or -CH-
  • L 3 is not a direct bond or -CH-
  • L 2 and L 3 are not direct bonds at the same time
  • heterocycloalkylene, heterocycloalkenylene, heteroarylene, heterocycloalkyl, heteroaryl, heterocyclylene, heterocyclyl and partially unsaturated heterocyclyl have 1 to 4 heteroatoms selected from N, O and S.
  • R 1 and R 2 are each independently -H or C 1 -C 5 alkyl.
  • R 3 is -DEG; wherein D is C 1 -C 5 alkylene; E is C 1 -C 5 alkylene, C 6 -C 10 arylene or 5 to 10 membered heterocycloalkylene having 1 to 3 N atoms; G is amino, 5 to 10 membered heteroaryl, , or and; here R 7 , R 8 and R 9 is each independently -H, C 1 -C 5 alkyl or halo-C 1 -C 5 alkyl.
  • R 4 and R 6 are each independently -H or C 1 -C 5 alkyl.
  • L 1 is -C-, -CH-, C 4 -C 8 cycloalkylene, C 4 -C 8 cycloalkenylene or C 6 -C 10 arylene; or may form a 4 to 10 membered heterocycloalkyl together with the N atom to which R 4 and L 1 are bonded;
  • R 5 is halo, C 1 -C 5 alkyl, halo-C 1 -C 5 alkyl, C 6 -C 10 aryl, 5 to 8 membered heteroaryl, C 3 -C 8 cycloalkyl-C 1 -C 5 alkyl, C 6 -C 10 aryl-C 1 -C 5 alkyl or 5 to 8 membered heteroaryl-C 1 -C 5 alkyl; wherein said aryl may be optionally substituted with 1 to 3 substituents selected from hydroxy, halo, halo-C 1 -C 5 alkyl, C 1 -C 5 alkoxy and C 6 -C 10 aryl; when L 1 is -C- and m is 2, R 5 and L 1 may be linked together to form C 3 -C 8 cycloalkyl or 4 to 10 membered heterocycloalkyl.
  • L 2 is a direct bond, or when n is 0, R 4 and L 2 may be connected to each other together with the N atom to which they are bonded to form a 4 to 10 membered heterocycloalkyl, wherein the heterocycloalkyl may be optionally substituted with 1 to 3 substituents selected from C 1 -C 5 alkyl and C 6 -C 10 aryl-C 1 -C 5 alkyl.
  • L 3 is a direct bond, -CH-, -CH 2 -, C 4 -C 8 cycloalkylene, unsaturated C 4 -C 8 carbocyclylene or C 6 -C 10 arylene; or may form a 4 to 10 membered heterocycloalkyl together with the N atom to which R 6 and L 3 are bonded.
  • R 7 is halo, C 1 -C 5 alkyl, halo-C 1 -C 5 alkyl, C 1 -C 5 alkoxy, hydroxy-C 1 -C 5 alkyl, C 3 -C 8 cycloalkyl-oxy, C 3 -C 8 cycloalkyl-C 1 -C 5 alkyl, C 3 -C 8 cycloalkyl-C 1 -C 5 alkoxy , C 6 -C 10 aryl, C 6 -C 10 aryl-C 1 -C 5 alkyl, C 6 -C 10 aryl-oxy, C 6 -C 10 aryl-C 1 -C 5 alkoxy, 5 to 8 membered heteroaryl-C 1 -C 5 alkyl, unsaturated 4 to 10 membered heterocyclyl or unsaturated C 4 -C 10 Carbocyclyl-oxy; wherein said aryl, aryl
  • R 8 is halo, hydroxy, 4 to 10 membered heterocycloalkyl, , , , , or and wherein R 17 to R 23 and R 31 are each independently -H, hydroxy, C 1 -C 7 alkyl, C 2 -C 5 alkenyl, halo-C 1 -C 5 alkyl, C 1 -C 5 alkoxy, C 1 -C 5 alkoxy-C 1 -C 5 alkyl, C 1 -C 5 alkylthio-C 1 -C 5 alkyl, di(C 1 -C 5 alkyl)amino-C 1 -C 5 alkyl, amino-C 1 -C 5 alkoxy-C 1 -C 5 alkyl, C 1 -C 5 alkylcarbonyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkyl-C 1 -C 5 alkyl, C 3
  • Representative compounds of the chemical formula 1 according to the present invention may include, but are not limited to, the following compounds:
  • a chimeric compound of the following chemical formula 2 is provided:
  • A is a ubiquitin ligase binding moiety (ULM) and is a compound of the above chemical formula 1 or a prodrug thereof;
  • B is a protein target moiety (PTM);
  • a and B are chemically linked by a linker.
  • any linker used in the production of PROTAC in the relevant field can be used, and there are no special limitations thereto.
  • the linker can be a compound having the structure of the following chemical formula 3,
  • Y 1 does not exist, or , , , , , , , , , , , , , , , , , cycloalkylene, cycloalkenylene, heterocycloalkylene, heterocycloalkenylene, arylene, heteroarylene, bridged cyclclylene, fused cyclylene, spiro-cyclylene, poly-cyclylene, bridged heterocyclclylene, fused heterocyclylene, spiro-heterocyclylene and poly-heterocyclylene; wherein R'" and R" are each independently -H, -D, halo, hydroxy, amino, cyano, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyalkyl, aminoalkyl, alkoxyalkyl, alkylthio, alkylamino, dialkylamino, alkylaminoalky
  • Y 2 is -not present, or , , , , , , , , , , , , , , , , , cycloalkylene, cycloalkenylene, heterocycloalkylene, heterocycloalkenylene, arylene, heteroarylene, bridged cyclclylene, fused cyclylene, spiro-cyclylene, poly-cyclylene, bridged heterocyclclylene, fused heterocyclylene, spiro-heterocyclylene and poly-heterocyclylene; wherein R'" and R" are each independently -H, -D, halo, hydroxy, amino, cyano, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyalkyl, aminoalkyl, alkoxyalkyl, alkylthio, alkylamino, dialkylamino, alkylamino
  • Y 3 does not exist, or , , , , , , , , , , , , , , , , , cycloalkylene, cycloalkenylene, heterocycloalkylene, heterocycloalkenylene, arylene, heteroarylene, bridged cyclclylene, fused cyclylene, spiro-cyclylene, poly-cyclylene, bridged heterocyclclylene, fused heterocyclylene, spiro-heterocyclylene and poly-heterocyclylene; wherein R'" and R" are each independently -H, -D, halo, hydroxy, amino, cyano, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyalkyl, aminoalkyl, alkoxyalkyl, alkylthio, alkylamino, dialkylamino, alkylaminoalky
  • a linker having the structure shown below may be used, and as another specific example, the linker is as described and defined in International Publication Nos. WO 2016/149668 A1, WO 2020/051564 A1, WO 2023/076161 A1, which are incorporated herein by reference, but are not limited thereto.
  • B which is a protein target moiety (PTM)
  • PTM protein target moiety
  • compounds targeting BET bromodomain-containing proteins such as those shown in Table 1 below may be used, but are not limited thereto.
  • kinase and phosphatase inhibitor compounds such as those shown in Table 2 below may be used, but are not limited thereto.
  • JAK Janus kinase family proteins
  • compounds targeting PARP-1 poly [ADP-ribose] polymerase 1
  • compounds targeting PARP-1 poly [ADP-ribose] polymerase 1) such as those shown in Table 4 below may be used, but are not limited thereto.
  • FAM focal adhesion kinase
  • RAF rapidly accelerated fibrosarcoma receptor
  • kinase kinase
  • compounds targeting the androgen receptor (AR) such as those shown in Table 7 below may be used, but are not limited thereto.
  • compounds targeting the estrogen receptor (ER) such as those shown in Table 8 below may be used, but are not limited thereto.
  • BTK Brunauer tyrosine kinase
  • HSP90 heat shock protein 90 inhibitors
  • Table 10 compounds targeting heat shock protein 90 (HSP90) inhibitors
  • HDM2/MDM2 human double minute 2/mouse double minute 2 inhibitors
  • Table 11 compounds targeting HDM2/MDM2 (human double minute 2/mouse double minute 2) inhibitors such as those shown in Table 11 below may be used, but are not limited thereto.
  • immunosuppressive compounds such as those shown in Table 12 below may be used, but are not limited thereto.
  • the UBR1 ligand was prepared through the following solid phase synthesis.
  • Rink amide MBHA resin 50 mg, 0.52 mmol/g loading, 0.026 mmol, 1.0 equiv.
  • DMF dimethyl methoxylate
  • the resin was treated with HATU (0.078 mmol, 3 equiv.), HOAt (0.078 mmol, 3 equiv.), DIPEA (0.156 mmol, 6 equiv.), and the Fmoc-protected amino acid (0.078 mmol, 3 equiv.) (Method AB).
  • the reaction mixture was discarded, and the resin was washed with DMF (3 ⁇ ), MeOH (3 ⁇ ), CH 2 Cl 2 (3 ⁇ ), and DMF (3 ⁇ ). This process was repeated to obtain the desired compound.
  • the compound on the resin was cleaved by treating with 1.0 mL of a cleavage cocktail (95% TFA, 2.5% TIPS, and 2.5% DDW) at room temperature for 3 h, and the compound was purified using reverse-phase column chromatography (C18 Silica Kelp, YL9100 GPC system) (solvent A: DDW with 0.1% TFA added, solvent B: ACN with 0.1% TFA added; gradient).
  • the compound was analyzed by LC (Agilent 1220 LC system Ontario, CA, USA) and MALDI-TOF MS (Autoflex Speed LRF, Bruker, Billerica, MA, USA).
  • the purified compound was lyophilized to obtain a white solid, and Examples 1 to 26 were synthesized using the above synthetic method.
  • Rink amide MBHA resin 50 mg, 0.52 mmol/g loading, 0.026 mmol, 1.0 equiv.
  • DMF 5.0 mL fritted syringe
  • the resin was treated with HATU (0.078 mmol, 3 equiv.), HOAt (0.078 mmol, 3 equiv.), DIPEA (0.156 mmol, 6 equiv.), and fluoronitrobenzoic acid (0.078 mmol, 3 equiv.) (Method C).
  • the amide coupling reaction was carried out by treating the beads with HOAt (0.078 mmol, 3 equiv.), EDC (0.078 mmol, 3 equiv.), 2,6-lutidine (0.156 mmol, 6 equiv.), Fmoc-protected amino acid (0.078 mmol, 3 equiv.) in DMF (0.5 mL) at room temperature for 2 h.
  • the resin was treated with HATU (0.078 mmol, 3 equiv.), HOAt (0.078 mmol, 3 equiv.), DIPEA (0.156 mmol, 6 equiv.), Fmoc-protected amino acid (0.078 mmol, 3 equiv.).
  • the compound on the resin was cleaved by treating with 1.0 mL of a cleavage cocktail (95% TFA, 2.5% TIPS, and 2.5% DDW) at room temperature for 3 hours, and the compound was purified using reverse-phase column chromatography (C18 Silica Kelp, YL9100 GPC system) (solvent A: DDW with 0.1% TFA added, solvent B: ACN with 0.1% TFA added; gradient).
  • solvent A DDW with 0.1% TFA added
  • solvent B ACN with 0.1% TFA added; gradient
  • Rink amide MBHA resin 50 mg, 0.52 mmol/g loading, 0.026 mmol, 1.0 equiv.
  • the Fmoc protecting group was removed with 20% piperidine (v/v) in DMF (0.5 mL) for 20 min, followed by treatment with BAA (0.52 mmol, 20 equiv.) and DIC (0.52 mmol, 20 equiv.) in DMF (0.5 mL) at room temperature for 30 min (Method D).
  • the amine substitution reaction was performed by treatment with R 1 -substituted piperazine (0.52 mmol, 20 equiv.) in DMF for 2 h at room temperature.
  • DMF 3x
  • MeOH 3x
  • CH 2 Cl 2 3x
  • DMF 3x
  • HATU 0.078 mmol, 3 equiv.
  • HOAt 0.078 mmol, 3 equiv.
  • DIPEA 0.156 mmol, 6 equiv.
  • Fmoc-protected amino acid 0.078 mmol, 3 equiv.
  • the compound on the resin was cleaved by treating with 1.0 mL of cleavage cocktail (95% TFA, 2.5% TIPS, and 2.5% DDW) for 3 h at room temperature, and the compound was purified using reversed-phase column chromatography (C18 Silica Kelp, YL9100 GPC system) (Solvent A: DDW with 0.1% TFA, Solvent B: ACN with 0.1% TFA; Gradient).
  • cleavage cocktail 95% TFA, 2.5% TIPS, and 2.5% DDW
  • solvent B ACN with 0.1% TFA; Gradient
  • PAL resin 50 mg, 0.92 mmol/g loading, 0.046 mmol, 1.0 equiv.
  • THF 4.0 mL vial
  • primary amine 0.23 mmol, 5.0 equiv.
  • AcOH 0.46 mmol, 10 equiv.
  • the reaction mixture was transferred to a 5.0 mL fritted syringe, and the resin was washed with THF (3 ⁇ ), MeOH (3 ⁇ ), CH 2 Cl 2 (3 ⁇ ), and DMF (3 ⁇ ).
  • HATU (0.138 mmol, 3.0 equiv.
  • HOAt (0.138 mmol, 3.0 equiv.
  • DIPEA 0.276 mmol, 6.0 equiv.
  • 4-fluoro nitrobenzoic acid (0.138 mmol, 3.0 equiv.) were treated on the resin.
  • the reaction mixture was discarded, and the resin was washed with DMF (3x), MeOH (3x), CH 2 Cl 2 (3x), and DMF (3x).
  • the compound on the resin was cleaved by treating with 1.0 mL of a cleavage cocktail (95% TFA, 2.5% TIPS, and 2.5% DDW) at room temperature for 3 h, and the compound was purified using reverse-phase column chromatography (C18 Silica Kelp, YL9100 GPC system) (solvent A: DDW with 0.1% TFA added, solvent B: ACN with 0.1% TFA added; gradient).
  • the compound was analyzed by LC (Agilent 1220 LC system Ontario, CA, USA) and MALDI-TOF MS (Autoflex Speed LRF, Bruker, Billerica, MA, USA).
  • the purified compound was lyophilized to obtain a white solid, and Examples 116 to 155 were synthesized using the above synthetic method.
  • Iron powder Fe, 7.0 equiv.
  • ammonium chloride NH4Cl, 10 equiv.
  • 3-bromo-1-nitrobenzene 1.0 equiv.
  • 4-bromo-1-nitrobenzene 1.0 equiv.
  • the compound was analyzed by LC (Agilent 1220 LC system Ontario, CA, USA) and MALDI-TOF MS (Autoflex Speed LRF, Bruker, Billerica, MA, USA).
  • the purified compound was freeze-dried to obtain a white solid, and Examples 158 and 159 were synthesized using the above synthetic method.
  • Iron powder (Fe, 7.0 equiv.) and ammonium chloride (NH4Cl, 10.0 equiv.) were added to 4-nitrophenol (1.0 equiv.), and reduced in a solvent of ethanol and water (1:1, v/v) at 80°C for 4 h by stirring.
  • Fmoc-Phe(4-Boc 2 -guanidino)-OH (1.2 equiv.), HATU (1.2 equiv.), and DIEA (3.0 equiv.) were added in a solvent of DMF, and the reaction was carried out at room temperature for 2 h to synthesize an amide derivative.
  • the compound was purified using reverse-phase column chromatography (C18 silica gel, YL9100 GPC system) (solvent A: DDW with 0.1% TFA, solvent B: ACN with 0.1% TFA; gradient).
  • solvent A DDW with 0.1% TFA
  • solvent B ACN with 0.1% TFA; gradient.
  • the compound was analyzed by LC (Agilent 1220 LC system Ontario, CA, USA) and MALDI-TOF MS (Autoflex Speed LRF, Bruker, Billerica, MA, USA).
  • the purified compound was freeze-dried to obtain a white solid, and Example 160 was synthesized using the above synthetic method.
  • Example 161 was synthesized using the above synthetic method.
  • the intermediate was added to a mixed solvent of DCM and water (1:1, v/v) together with K 2 CO 3 (15.0 equiv.), Na 2 S 2 O 4 (10.0 equiv.), and tetrabutylammonium hydrogen sulfate (TBAHS, 0.2 equiv.), and stirred three times for 1 hour each at room temperature to reduce the nitro group to obtain an aryl amine compound.
  • K 2 CO 3 (15.0 equiv.
  • Na 2 S 2 O 4 (10.0 equiv.
  • TSAHS tetrabutylammonium hydrogen sulfate
  • the compound was analyzed by LC (Agilent 1220 LC system Ontario, CA, USA) and MALDI-TOF MS (Autoflex Speed LRF, Bruker, Billerica, MA, USA).
  • the purified compound was freeze-dried to obtain a white solid, and Examples 156 and 162 were synthesized using the above synthetic method.
  • This compound was treated with trifluoroacetic acid (50% in DCM) to remove the Boc protecting group, and then acetylated by reacting with 5% acetic anhydride in DCM at room temperature for 30 min. Subsequently, K 2 CO 3 (15.0 equiv.), Na 2 S 2 O 4 (10.0 equiv.), and TBAHS (0.2 equiv.) were added to a DCM/H2O mixed solvent, and the nitro group was reduced three times for 1 hour each at room temperature.
  • Example 157 was synthesized using the above synthetic method.
  • Alkyl aryl ether derivatives were synthesized by adding (2-bromoethyl)cyclohexane (1.5 equiv.) and K 2 CO 3 (3.0 equiv.) to 3-bromo-5-nitrophenol (1.0 equiv.) in DMF solvent and stirring at 50°C for 16 h.
  • the Fmoc and Pbf protecting groups were removed under basic and acidic conditions, respectively.
  • the compound was purified using reverse-phase column chromatography (C18 silica gel, YL9100 GPC system) (solvent A: DDW with 0.1% TFA added, solvent B: ACN with 0.1% TFA added; gradient).
  • the compound was analyzed by LC (Agilent 1220 LC system Ontario, CA, USA) and MALDI-TOF MS (Autoflex Speed LRF, Bruker, Billerica, MA, USA).
  • the purified compound was lyophilized to obtain a white solid, and Examples 163, 164, and 165 were synthesized using the above synthetic method.
  • alkyl aryl ether derivative was synthesized by adding (2-bromoethyl)cyclohexane (1.5 equiv.) and K 2 CO 3 (3.0 equiv.) to 3-bromo-5-nitrophenol (1.0 equiv.) in DMF solvent and stirring at 50 °C for 16 h.
  • the Sonogashira reaction was performed by reacting the resulting aryl ether compound with tert -butyl (2-(prop-2-yn-1-yloxy)ethyl)carbamate (1.2 equiv.), CuI (0.05 equiv.), Pd 2 (PPh 3 ) 2 Cl 2 (0.03 equiv.), and PPh3 (0.06 equiv.) as a catalyst in a triethylamine/DMF (3:1, v/v) mixed solvent at 70 °C for 12 h.
  • the Fmoc and Pbf protecting groups were removed under basic and acidic conditions, respectively.
  • the compound was purified using reverse-phase column chromatography (C18 silica gel, YL9100 GPC system) (solvent A: DDW with 0.1% TFA added, solvent B: ACN with 0.1% TFA added; gradient).
  • the compound was analyzed by LC (Agilent 1220 LC system Ontario, CA, USA) and MALDI-TOF MS (Autoflex Speed LRF, Bruker, Billerica, MA, USA).
  • the purified compound was freeze-dried to obtain a white solid, and Example 166 was synthesized using the above synthetic method.
  • Example 167 Synthesis of PROTAC in which the compound of Example 113 and a bromodomain-containing protein 4 (BRD4) ligand are linked via a linker
  • PAL resin 100 mg, 0.91 mmol/g loading, 0.091 mmol, 1.0 equiv.
  • 13-amino-5,8,11-trioxa-2-azatridecanoic acid 1,1-dimethylethyl ester 0.55 mmol, 5 equiv.
  • AcOH 0.55 mmol, 5 equiv.
  • NaBH(OAc) 3 0.55 mmol, 5 equiv.
  • the amide coupling reaction was performed by treating the beads with HBTU (0.455 mmol, 5 equiv.), HOBt (0.455 mmol, 5 equiv.), DIPEA (0.91 mmol, 10 equiv.), 2-fluoro-4-nitrobenzoic acid (0.455 mmol, 5 equiv.) in DMF (1 mL) at room temperature for 3 h.
  • the resin was washed with DMF (3x), MeOH (3x), DCM (3x), and DMF (3x), and then the alcohol substitution reaction was performed by treating 2-cyclohexylethanol (3.64 mmol, 40 equiv.), Cs 2 CO 3 (3.64 mmol, 40 equiv.) in DMF (1 mL) at 60 °C for 6 h. Next, SnCl 2 (5.46 mmol, 60 equiv.) in DMF (1 mL) was reacted overnight at room temperature.
  • the purified compound was freeze-dried to obtain a light brown solid.
  • the obtained compound was treated with beads at room temperature for 2 hours with a solution of HATU (0.182 mmol, 2.0 eq), HOAt (0.182 mmol, 2.0 eq), DIPEA (0.364 mmol, 4.0 eq), and JQ1 carboxylic acid (0.091 mmol, 1.0 eq) in DMF, and then piperidine was added to obtain a final 20% piperidine in DMF solution. After reacting at room temperature for 30 minutes, purification was performed using reverse-phase HPLC.
  • PC-3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • PC-3 cells were treated with DMSO or various concentrations of PROTAC compounds.
  • PC-3 cells were seeded in 6-well plates and cultured at 37°C for 24 hours, and then treated with DMSO or PROTAC compounds in Opti-MEM medium for 48 hours. Before cell lysis, cells were washed twice with cold Dulbecco's phosphate-buffered saline (DPBS).
  • DPBS cold Dulbecco's phosphate-buffered saline
  • lysis buffer 50 mM Tris ⁇ HCl pH 7.4, 150 mM NaCl, 1% Triton X, 0.1% SDS, 0.5% sodium deoxycholate, 1x protease inhibitor cocktail
  • the cell lysate was then centrifuged at 13,000 rpm for 15 minutes at 4°C. The supernatant was collected, and the protein concentration was measured using Pierce TM 660 nm Assay Reagent.
  • 6x SDS loading buffer was added to the cell lysate and heated at 95°C for 5 minutes. Equal amounts of protein were loaded onto SDS-PAGE and transferred to PVDF membranes.
  • the PVDF membranes were blocked with 5% skim milk in TBST (Tris-buffered saline containing 0.01% Tween-20) and treated with primary antibodies at 4°C for 15 hours. After incubation with HRP-conjugated secondary antibodies for 1 hour at room temperature, Western blot images were obtained with ECL solution.
  • the PROTAC of Example 167 reduced the intracellular BRD4 protein expression level by approximately 50% at a concentration of approximately 50 nM, and exhibited stronger BRD4 protein degradation activity as the concentration increased (Fig. 5).
  • Example 167 To determine whether the PROTAC of Example 167 induces BRD4 protein degradation via the proteasome-mediated pathway, the PROTAC of Example 167 (500 nM) was treated alone or in combination with the proteasome inhibitor MG-132 (5 ⁇ M).
  • PC-3 cells were seeded in 6-well plates and cultured at 37°C for 24 hours, followed by compound treatment in Opti-MEM medium. Cells were then washed twice with cold DPBS to lyse the cells, and lysis buffer was added to the cells, which were then placed on ice. The cell lysate was centrifuged at 13,000 rpm for 15 minutes at 4°C, the supernatant was collected, and the protein concentration was measured using Pierce TM 660 nm Assay Reagent.
  • Example 167 In addition, to confirm whether the PROTAC of Example 167 induces BRD4 protein degradation by forming a ternary complex between the BRD4 protein and the UBR1 protein, PC-3 cells were treated together with the PROTAC of Example 167 (500 nM) and JQ1 (10 ⁇ M) or the compound of Example 113 (10 ⁇ M), and then the amount of intracellular BRD4 was measured by Western blot. When PROTAC of Example 167 was treated with JQ1 (10 ⁇ M) or the compound of Example 113 (10 ⁇ M), no BRD4 degradation effect was observed, thereby proving that the degradation of BRD4 by PROTAC of Example 167 was due to the formation of a ternary complex with BRD4 protein and UBR1 protein (Fig. 6).
  • Example 168 Prodrug synthesis of PROTAC in which the compound of Example 100 and a bromodomain-containing protein 4 (BRD4) ligand are linked via a linker
  • BBD4 bromodomain-containing protein 4
  • PAL resin 100 mg, 0.91 mmol/g loading, 0.091 mmol, 1.0 equiv.
  • 13-amino-5,8,11-trioxa-2-azatridecanoic acid 1,1-dimethylethyl ester 13-amino-5,8,11-trioxa-2-azatridecanoic acid 1,1-dimethylethyl ester
  • AcOH 0.46 mmol, 5.0 equiv.
  • the resin was washed with DMF (3x), MeOH (3x), DCM (3x), and DMF (3x), and then treated with 4-fluorobenzyl alcohol (3.6 mmol, 40 equiv.), Cs 2 CO 3 (3.6 mmol, 40 equiv.) in DMF (1.0 mL) at 60°C for 6 h to carry out the alcohol substitution reaction.
  • SnCl 2 5.5 mmol, 60 equiv.
  • DMF 1.0 mL
  • the compound on the resin was cleaved by treating with 2.0 mL of cleavage cocktail (95% TFA, 2.5% TIPS, 2.5% DDW), and purified by reverse phase HPLC.
  • the purified compound was lyophilized and obtained as a light brown solid.
  • the obtained compound was treated with beads at room temperature for 2 hours with a solution of HATU (0.18 mmol, 2.0 eq), HOAt (0.18 mmol, 2.0 eq), DIPEA (0.36 mmol, 4.0 eq), and JQ1 carboxylic acid (0.091 mmol, 1.0 eq) in DMF, and then piperidine was added to obtain a final 20% piperidine in DMF solution.
  • PC-3 cells were treated with DMSO or various concentrations of protac compounds.
  • PC-3 cells were seeded in 6-well plates and cultured at 37°C for 24 hours, and then treated with DMSO or protac compounds in Opti-MEM medium for 48 hours. Before cell lysis, cells were washed twice with cold Dulbecco's phosphate-buffered saline (DPBS).
  • DPBS cold Dulbecco's phosphate-buffered saline
  • lysis buffer 50 mM Tris ⁇ HCl pH 7.4, 150 mM NaCl, 1% Triton X, 0.1% SDS, 0.5% sodium deoxycholate, 1x protease inhibitor cocktail
  • the cell lysate was then centrifuged at 13,000 rpm for 15 minutes at 4°C. The supernatant was collected, and the protein concentration was measured using Pierce TM 660 nm Assay Reagent.
  • 6x SDS loading buffer was added to the cell lysate and heated at 95°C for 5 minutes. Equal amounts of protein were loaded onto SDS-PAGE and transferred to PVDF membranes.
  • PVDF membranes were blocked with 5% skim milk in TBST (Tris-buffered saline containing 0.01% Tween-20) and treated with primary antibodies for 15 hours at 4°C. After incubation with HRP-conjugated secondary antibodies for 1 hour at room temperature, Western blot images were obtained with ECL solution (Fig. 8).

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Abstract

La présente invention concerne un composé ligand pour une ubiquitine ligase et une molécule chimère ciblant la protéolyse (PROTAC) le comprenant et, plus particulièrement, un composé agissant en tant que ligand pour une ubiquitine ligase E3 ou un promédicament de celle-ci, et une molécule hétérobifonctionnelle PROTAC le comprenant.
PCT/KR2025/005577 2024-04-25 2025-04-24 Composé ligand pour ubiquitine ligase et molécule protac le comprenant Pending WO2025226064A1 (fr)

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KR20240055604 2024-04-25

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