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WO2025149076A1 - Dérivés phénoliques en tant que charge covalente ciblant la forme d'acide sulfenique de cystéine - Google Patents

Dérivés phénoliques en tant que charge covalente ciblant la forme d'acide sulfenique de cystéine

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Publication number
WO2025149076A1
WO2025149076A1 PCT/CN2025/071968 CN2025071968W WO2025149076A1 WO 2025149076 A1 WO2025149076 A1 WO 2025149076A1 CN 2025071968 W CN2025071968 W CN 2025071968W WO 2025149076 A1 WO2025149076 A1 WO 2025149076A1
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Prior art keywords
group
hydrogen
optionally substituted
compound
alkyl
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PCT/CN2025/071968
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English (en)
Inventor
Feng Ni
Songsen FU
Xiaoran MAO
Tong Chen
Bang HONG
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Ningbo Leadart Biotechnologies Co Ltd
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Ningbo Leadart Biotechnologies Co Ltd
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Publication of WO2025149076A1 publication Critical patent/WO2025149076A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C235/34—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/18—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring monocyclic with unsaturation outside the aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/20—Unsaturated compounds containing keto groups bound to acyclic carbon atoms
    • C07C49/24—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups
    • C07C49/245—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups containing six-membered aromatic rings
    • C07C49/248—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups containing six-membered aromatic rings having unsaturation outside the aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/04—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D233/28—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member 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
    • C07D233/30—Oxygen or sulfur atoms
    • C07D233/32—One oxygen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/022—Boron compounds without C-boron linkages
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine

Definitions

  • the present invention relates to the field of drug discovery, biological research and chemical pharmaceutical technology, specifically to novel phenolic derivative compounds or pharmaceutical acceptable salts, or stereoisomer thereof useful as covalent probe reagents, synthesis thereof, and use thereof as covalent warheads for targeting sulfenic acid form of Cys in protein labeling.
  • ABPP mainly consists of two key steps: (1) probe design and synthesis; (2) target capture and protein identification. It uses reactive groups in chemical molecular probes to covalently label a class of protein active sites in the proteome, and then enriches the labeled proteins using enriching groups (or a handle) on the probes. Subsequently, protein components and site information are identified through mass spectrometry of biomacromolecules, thereby directly obtaining information on the activity and functional state of these proteins in complex proteome systems.
  • a unique target space of a warhead can greatly expand the labeling space of cysteine residues, making it highly valuable for drug discovery and disease target identification. Development of corresponding covalent inhibitors from novel warhead has significant scientific impact.
  • phenolic derivative compounds that can selectively modify cysteine residues inside living cells, especially those in the state of sulfenic acid form (Cys-SOH) .
  • the present disclosure also provides synthesis and application of the phenolic derivative compounds.
  • the phenolic derivative compounds of the present disclosure are a valuable tool for selectively modifying protein Cys-SOH within living cells. This novel covalent warhead can capture a wide range of proteins with Cys-SOH sites and serves as a practical tool for drug target research and expanding the labeling space of cysteine, which has significant value in drug discovery and disease target identification.
  • the disclosure provides a compound of Formula (I) :
  • R 1 has a structure of Q- (CH 2 ) n -X- (CH 2 ) m -Y wherein
  • X is absent or selected from the group consisting of -CH 2 -, -NH-, and -O-;
  • Y is selected from the group consisting of
  • n is an integral of 0 to 5, or 0, 1, 2, 3, 4, or 5;
  • n is an integral of 1 to 5, or 1, 2, 3, 4, or 5;
  • R 2 is selected from the group consisting of hydrogen, halo, -NO 2 , -PO (OH) 2 , or a structure of -L-R 2 ’, where
  • L is a bivalent group selected from the group consisting of -CH 2 -, -NH-, -O-, -CONH-, -NHCO-, -SO-and -SO 2 -, and
  • R 2 ’ is selected from the group consisting of hydrogen, C 1-5 alkyl, and C 1-5 haloalkyl.
  • the disclosure provides a compound of Formula (Ia) :
  • R 1 and R 2 are defined as described herein.
  • R 1 and R 2 are defined as described herein.
  • the disclosure provides a compound of Formula (II) :
  • R 1 ’ has a structure of - (CH 2 ) n -X- (CH 2 ) m -Y, wherein
  • X is selected from the group consisting of -CH 2 -, -NH-, and -O-;
  • Y is selected from the group consisting of
  • n is an integral of 0 to 5;
  • n is an integral of 1 to 5;
  • L is a bivalent group selected from the group consisting of -CH 2 -, -NH-, -O-, -CONH-, -NHCO-, -SO-and -SO 2 -, and
  • R 2 ’ is selected from the group consisting of hydrogen, C 1-5 alkyl, and C 1-5 haloalkyl.
  • the disclosure provides a compound of Formula (IIa) :
  • R 1 ’ and R 2 are defined as described herein.
  • the disclosure provides a compound of Formula (IIb) :
  • the disclosure provides a compound of Formula (III) :
  • R 1 has a structure of - (CH 2 ) m -Y, where
  • Y is selected from the group consisting of
  • n is an integral of 1 to 5;
  • R 2 is selected from the group consisting of hydrogen, halo, -NO 2 , -PO (OH) 2 , or a structure of -L-R 2 ’, where
  • L is a bivalent group selected from the group consisting of -CH 2 -, -NH-, -O-, -CONH-, -NHCO-, -SO-and -SO 2 -, and
  • R 2 ’ is selected from the group consisting of hydrogen, C 1-5 alkyl, C 1-5 haloalkyl.
  • the disclosure provides a compound of Formula (IIIa) :
  • R 1 and R 2 are defined as described herein.
  • the disclosure provides a compound of Formula (IIIb) :
  • R 1 and R 2 are defined as described herein.
  • the compound of Formula (I) , (II) or (III) is selected from Table 1.
  • the present disclosure provides use of a compound of Formula (I) , (II) or (III) or a pharmaceutically acceptable salt, or stereoisomer thereof as provided herein in labeling protein.
  • the present disclosure provides use of a compound of Formula (I) , (II) or (III) or a pharmaceutically acceptable salt, or stereoisomer thereof as provided herein in preparation of an agent for labeling protein.
  • the present disclosure provides a method for labeling protein, comprising a step of contacting a compound of Formula (I) , (II) or (III) or a pharmaceutically acceptable salt, or stereoisomer thereof as provided herein with the protein.
  • the protein is a protein in living cell.
  • the living cell is a cell derived from microorganism, plant or animal, especially mammal, for example human.
  • the compound selectively modifies Cys, especially those in the sulfenic acid form.
  • the method further comprises a step of enriching, isolating, purifying and/or identifying the labeled protein.
  • the method further comprises a step of detecting a detectable signal emitted by the labeled protein, especially, the detectable signal is a fluorescent signal.
  • FIG. 1 Exploration of the structure-activity relationships among CAA and its analog probes.
  • the image at the top is chemical structures of CAA and its analog probes.
  • the image on the bottom shows gel-based fluorescence imaging to assess the labeling efficiency of these probes (10 ⁇ M) on proteins at both live cell (left) and cell lysate (right) levels (FL: fluorescence labeling, CBB: Coomassie brilliant blue) .
  • FIG. 2 Investigation of CAA labeling performance.
  • the left is chemical structures of CAA and CAA’.
  • the right is gel-based fluorescence imaging showing CAA labeled proteins (from HeLa, HepG2, 293T) prepared under various conditions: the proteome in situ or based lysate; the order of competitor CAA’ addition (blank control, simultaneous administration group and pre-administered group) .
  • CAA and CAA’ were added simultaneously, and CAA was incubated for 30 min prior to the addition of CAA’.
  • FIG. 3 Gel-based competitive ABPP for CAA and Dyn-2. From left to right in order: structural formulas of CA, CAA, CAA’, dimedone (DO) , and Dyn-2; a gel-based fluorescence imaging shows competition of excess DO or CAA’ against CAA labeling; a gel-based fluorescence imaging depicts competition of excess DO, CA or CAA’ against CAA labeling.
  • FIG. 4 Screening of compounds for competitive ABPP experiments.
  • the left side of the image depicts chemical structures of competitors 9 ⁇ 16, and the right displays screening of compounds 9 ⁇ 16 for competitive ABPP experiments, which the concentration of CAA and competitors are 10 ⁇ M and 100 ⁇ M, respectively.
  • FIG. 5 Competition gel experiment of curcumin and its derivatives. Structural formula of curcumin and its derivatives: curcumin (CUR) , tetrahydro-curcumin (THC) , curcumin (MC) and dimethyl curcumin (DMC) (left) . Results of the SDS-PAGE experiment (right) . Steps of the experiment are after incubation with 100 ⁇ M curcumin and its derivatives (CUR, THC, MC and DMC) for 4h, HeLa cells were recovered for 30min and incubated with 10 ⁇ M CAA for 1h.
  • CUR curcumin
  • THC tetrahydro-curcumin
  • MC curcumin
  • DMC dimethyl curcumin
  • FIG. 6A-6E Investigation on proteome-wide reactivity of CAA and representative MS/MS spectra with diagnostic ions (DI-1 ⁇ DI-3) .
  • FIG. 7 Generation Pathway of Diagnostic Ions from Precursor-1 via HCD Fragmentation. Diagnostic ions DI-1 to DI-3 are produced from precursor-1, while precursor-2 and precursor-3 do not generate DI-1 or DI-2.
  • FIG. 8 schematic diagram illustrates the labeling of protein Cys-SOH by CAA and the generation pathway of precursor-1 and diagnostic ions.
  • FIG. 9A-9B Analysis of the overlap of proteins and sites labeled by CAA and BTD.
  • At the top is a table showing experimental conditions for CAA and BTD labeling proteins (HeLa)
  • HeLa CAA and BTD labeling proteins
  • venn diagrams illustrating the overlap between CAA and BTD regarding labeled proteins (9A) and sites (9B) , respectively.
  • FIG. 10A-10C Chemical proteomics-based competitive ABPP for CAA.
  • FIG. 10A table shows the chemical structures of curcumin (CUR) , tetrahydrocurcumin (THC) , curcumin (MC) and dimethyl curcumin (DMC) , and the number of reactive moieties, targeted Cys-SOHs and proteins of them. The moieties reacting with Cys-SOH was highlighted by green and yellow circles, respectively.
  • FIG. 10B is a venn graph showing the overlap of targeted Cys-SOH sites across four compounds.
  • FIG. 10C categorizes targeted proteins of these four compounds into two groups based on presence or absence in DrugBank.
  • R 1 has a structure of Q- (CH 2 ) n -X- (CH 2 ) m -Y where
  • X is absent or selected from the group consisting of -CH 2 -, -NH-, and -O-;
  • Y is selected from the group consisting of
  • n is an integral of 0 to 5, or 0, 1, 2, 3, 4, or 5;
  • n is an integral of 1 to 5, or 1, 2, 3, 4, or 5;
  • R 2 is selected from the group consisting of hydrogen, halo, -NO 2 , -PO (OH) 2 , or a structure of -L-R 2 ’, where
  • L is a bivalent group selected from the group consisting of -CH 2 -, -NH-, -O-, -CONH-, -NHCO-, -SO-and -SO 2 -, and
  • R 2 ’ is selected from the group consisting of hydrogen, C 1-5 alkyl, and C 1-5 haloalkyl.
  • the position of the group R 2 is not particularly limited and may be located adjacent or interstitial to the phenol hydroxyl group.
  • the disclosure provides a compound of Formula (Ia) :
  • R 1 and R 2 are defined as described herein.
  • R 1 and R 2 are defined as described herein.
  • the disclosure provides a compound of Formula (II) :
  • R 1 ’ has a structure of - (CH 2 ) n -X- (CH 2 ) m -Y, wherein
  • Y is selected from the group consisting of
  • n is an integral of 1 to 5;
  • R 2 is selected from the group consisting of hydrogen, halo, -NO 2 , -PO (OH) 2 , or a structure of -L-R 2 ’, where
  • L is a bivalent group selected from the group consisting of -CH 2 -, -NH-, -O-, -CONH-, -NHCO-, -SO-and -SO 2 -, and
  • R 2 ’ is selected from the group consisting of hydrogen, C 1-5 alkyl, and C 1-5 haloalkyl.
  • the position of the group R 2 is not particularly limited and may be located adjacent or interstitial to the phenol hydroxyl group.
  • the disclosure provides a compound of Formula (IIa) :
  • R 1 ’ and R 2 are defined as described herein.
  • the disclosure provides a compound of Formula (IIb) :
  • Y is selected from the group consisting of
  • Y is selected from the group consisting of -C ⁇ CH,
  • the disclosure provides a compound of Formula (Ia) :
  • R 1 and R 2 are defined as described herein.
  • R 1 and R 2 are defined as described herein.
  • the compound of Formula (I) is selected from:
  • the phenol derivative compounds of the present disclosure are capable of selectively modifying a wide variety of active cysteines within living cells, and in particular, modifying cysteines in the sulfenic acid form.
  • the living cell is a cell derived from microorganism, plant or animal, especially mammal, for example human.
  • the method further comprises a step of enriching, isolating, purifying and/or identifying the labeled protein.
  • the method further comprises a step of detecting a detectable signal emitted by the labeled protein, especially, the detectable signal is a fluorescent signal.
  • a reactant carrying a biotin, a desthiobiotin, or a fluorescent group can be added sequentially or simultaneously to the reaction system.
  • the compound of the present disclosure is first labeled to a Cys residue of a protein, and then biotin, desthiobiotin, or a fluorescent group, etc. is attached to the compound of the present disclosure by click chemistry; (2) the compound of the present disclosure is first attached to biotin, desthiobiotin, or a fluorescent group, etc., by click chemistry, and then labeled to a Cys residue of a protein; or (3) the reaction in which the compound of the present disclosure labels a Cys residue of a protein and the reaction in which the compound of the present disclosure is attached to biotin, desulfobiotin, or a fluorescent group, etc., by click chemistry proceed simultaneously.
  • a compound of the present disclosure When a compound of the present disclosure carries a biotin, desthiobiotin, or fluorescent group is used, it can be used directly to label Cys in proteins and for subsequent enrichment, isolation, purification and/or identification without the need to add other biotin-carrying, desthiobiotin-carrying, fluorescent group-containing reactants.
  • Such kind of compounds of the present disclosure can be obtained by click chemistry of the compounds of the present disclosure carrying a reactive end group such as an azide group, a terminal alkynyl group, with a reactant carrying a biotin moiety, a desthiobiotin moeity, or a fluorescent group.
  • C 1-6 is intended to encompass, C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1- 5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 .
  • a heteroaromatic ring described as containing from “1 to 4 heteroatoms” means that the ring can contain 1, 2, 3 or 4 heteroatoms.
  • any variable occurs more than one time in any constituent or in Formula (I) or in any other formula depicting and describing the compounds of the present disclosure, its definition at each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
  • alkyl refers to a linear or branched chain saturated hydrocarbon group.
  • C i-j alkyl refers to an alkyl having i to j carbon atoms.
  • Alkyl groups may contain 1 to 10 carbon atoms, unless otherwise stated. In certain embodiments, alkyl groups contain 1 to 6 carbon atoms (C 1-6 ) , such as, 1, 2, 3, 4, or 5 carbon atoms (C 1-5 ) , 1 to 4 carbon atoms (C 1-4 ) , 1 to 3 carbon atoms (C 1-3 ) , or 1 to 2 carbon atoms (C 1-2 ) .
  • alkoxy refers to -O-alkyl and alkyl is as defined herein.
  • alkyl groups include methoxy, ethoxy, n-and iso-propoxy, n-, sec-, iso-, and tert-butoxy, neopentyloxy, and the like.
  • aryl refers to a mono-, bicyclic, or multicyclic carbocyclic ring system having at least one aromatic rings.
  • Aryl groups may be 6-to 10-membered, unless otherwise stated.
  • aryl groups may contain 6 ring forming carbon atoms. All ring forming atoms within a carbocyclic aryl group are carbon atoms.
  • Non-limiting examples of aryl groups include phenyl, naphthyl, 1, 2-dihydronaphthyl, 1, 2, 3, 4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, etc.
  • aryl is phenyl or naphthyl.
  • aryl is phenyl.
  • Aryl groups may be unsubstituted or substituted.
  • An optionally substituted aryl group may be an aryl optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylsulfenyl; alkylsulfonyl; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halogen; heteroalkyl; heteroalkenyl; heteroalkynyl; heterocyclyl; (heterocyclyl) oxy; heteroaryl; hydroxy; nitro; thiol; silyl; - (CH
  • Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent (s) defined herein for each respective group.
  • aryl groups may be optionally substituted with one or more substitutes selected from 4-to 10-membered heterocyclyl, 6-to 10-membered aryl, and 5-to 10-membered heteroaryl.
  • haloalkyl refers to an alkyl group as described herein in which one or more of hydrogen atoms have been replaced with one or more halogen atoms independently selected from the group consisting of fluoride, chloride, bromide, and iodide. When a haloalkyl contains more than one halogen atom, the halogen atoms can be the same or be different from each other.
  • Non-limiting examples of haloalkyl groups include -CH 2 F, -CHF 2 , -CF 3 , -CF 2 Cl, -CH 2 CF 3 , -CF 2 CF 3 , etc.
  • haloalkyl groups may be perhaloalkyl groups, such as perfluoroalkyl.
  • the term “optionally substituted” means that the chemical group may have no substituents (i.e., unsubstituted) or may have one or more substituents (i.e., substituted) . It is to be understood that substitution at a given atom is limited by valency.
  • bonds represented by solid wedge lines and dashed wedge lines are used to indicate absolute configuration of a chiral center
  • bonds represented by solid lines and dashed lines are used to indicate relative configuration of a chiral center
  • a bond represented by a wavy line is used to indicate (a) a solid wedge line or a dashed wedge line or (b) a solid line or a dashed line
  • atropisomer refers to a stereoisomer resulting from restricted rotation about single bonds where the rotation barrier is high enough to permit isolation of the isomeric species. Typically, rotation about the single bond in the molecule is prevented, or greatly slowed, as a result of steric interactions with other parts of the molecule and the substituents at both ends of the single bond are unsymmetrical.
  • the compound may comprise nearly 100 mole%or 100 mole%of the specified atropisomer of the compound.
  • the compound is substantially atropisomerically pure.
  • the term “substantially pure” means that the compound, i.e., mixture of atropisomers, comprises at least 90 mole%, optionally at least 95 mole%, more optionally at least 98 mole%, and even more optionally at least 99 mole%of one atropisomer.
  • substantially free means that the compound comprises less than 10 mole%, optionally less than 5 mole%, more optionally less than 2 mole%, and even more optionally less than 1 mole%of one atropisomer.
  • the term “pharmaceutically acceptable salt” includes salts that retain the biological effectiveness of the free acid/base form of the specified compound and that are not biologically or otherwise undesirable.
  • Contemplated pharmaceutically acceptable salt forms include, but are not limited to, mono, bis, tris, tetrakis, and so on.
  • Pharmaceutically acceptable salts are non-toxic in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of a compound without preventing it from exerting its physiological effect. Useful alterations in physical properties may include, for example, increasing the solubility to facilitate administering higher concentrations of the drug.
  • Pharmaceutically acceptable salts of the compounds of Formula (I) , (II) , or (III) include acid addition and base salts. Suitable acid addition salts can be formed from acids which form non-toxic salts. Non-limiting examples may include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxa
  • Suitable base salts are formed from bases which form non-toxic salts.
  • Non-limiting examples may include the aluminium, arginine, benzathine, calcium, choline, diethylamine, bis (2-hydroxyethyl) amine (diolamine) , glycine, lysine, magnesium, meglumine, 2-aminoethanol (olamine) , potassium, sodium, 2-Amino-2- (hydroxymethyl) propane-1, 3-diol (tris or tromethamine) and zinc salts.
  • Hemisalts of acids and bases may also be formed, for example, hemisulfate and hemicalcium salts.
  • suitable salts see, Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002) .
  • Pharmaceutically acceptable salts of the compound of Formula (I) may be prepared by one or more of three methods: (i) by reacting the compound of Formula (I) with the desired acid or base; (ii) by removing an acid-or base-labile protecting group from a suitable precursor of the compound of Formula (I) or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or (iii) by converting one salt of the compound of Formula (I) to another by a reaction with an appropriate acid or base or by means of a suitable ion exchange column.
  • the three reactions may be typically carried out in solution.
  • the resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent.
  • the degree of ionization in the resulting salt may vary from completely ionized to almost non-ionized.
  • the compounds of Formula (I) , (II) , or (III) may have one or more chiral (asymmetric) centers.
  • the present disclosure encompasses all stereoisomeric forms of the compounds of Formula (I) , (II) , or (III) . Centers of asymmetry that are present in the compounds of Formula (I) , (II) , or (III) can all independently of one another have (R) or (S) configuration.
  • the present disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all ratios.
  • enantiomers are a subject of the present disclosure in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios.
  • the structures depicted herein are also meant to include the compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, the compounds wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature. Such compounds are referred to as a “isotopic variant” .
  • the present disclosure is intended to include all pharmaceutically acceptable isotopic variants of the compounds of Formula (I) , (II) , or (III) .
  • isotopes suitable for inclusion in the compounds of the present disclosure include, but not limited to, isotopes of hydrogen, such as 2 H and 3 H; carbon, such as 11 C, 13 C and 14 C; chlorine, such as 36 Cl; fluorine, such as 18 F; iodine, such as 123 I and 125 I; nitrogen, such as 13 N and 15 N; oxygen, such as 15 O, 17 O and 18 O; phosphorus, such as 32 P; and sulfur, such as 35 S.
  • isotopic variants of the compounds of Formula (I) , (II) , or (III) for example those incorporating a radioactive isotope, may be useful in drug and/or substrate tissue distribution studies.
  • Isotopic variants of compounds of Formula (I) , (II) , or (III) can generally be prepared by conventional techniques known to one skilled in the art or by processes analogous to those described in the accompanying examples and synthesis using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
  • isotopic variants of compounds of the present disclosure are deuterated variants.
  • peptide As used herein, the terms “peptide” , “polypeptide” , and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide contains at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • the compounds of the present disclosure may be prepared by the general and specific methods described below, using the common general knowledge of those skilled in the art of synthetic organic chemistry. Such common general knowledge can be found in standard reference books, e.g., Barton and Ollis (Ed. ) , Comprehensive Organic Chemistry, Elsevier; Richard Larock, Comprehensive Organic Transformations: A Guide to Functional Group Preparations, John Wiley and Sons; and Compendium of Organic Synthetic Methods, Vol. I-XII, Wiley-Interscience.
  • the starting materials used herein are commercially available or may be prepared by routine methods known in the art.
  • the compounds of the present disclosure can be readily prepared according to the following reaction schemes and examples, or modifications thereof, using readily available starting materials, reagents, and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are themselves known to those skilled in the art, but are not mentioned in greater detail. Furthermore, other methods for preparing the compounds of the disclosure will be readily apparent to those skilled in the art in light of the reaction schemes and examples as described herein. Unless otherwise indicated, all variables are as defined above.
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • Known starting materials for the present invention could be either synthesized according to known methods in the field or purchased from companies such as AnaSpec, McLane Laboratories, Bide Pharmatech Ltd., China National Pharmaceutical Group Corporation (Sinopharm) , PerkinElmer, Sigma-Aldrich, and others.
  • the crude cis-p-coumaric acid (0.5 mmol) obtained from the previous step was dissolved in DCM (5 mL) , followed by the addition of DIPEA (1.5 mmol) , HATU (0.5 mmol) , and oct-7-yn-1-amine (0.55 mmol) .
  • the reaction mixture was stirred at room temperature for 16 hours.
  • the reaction was then diluted with EtOAc (10 mL) and washed with 1M HCl solution and saturated aqueous NaHCO 3 .
  • TIPS-protected intermediate was dissolved in anhydrous THF (1.5 mL) and added TBAF (0.4 mL, 0.4 mmol, 1M in THF) at 0°C.
  • the reaction mixture was stirred at room temperature for 5 hours, and then concentrated under reduced pressure to afford crude product, which was purified by flash column chromatography (SiO 2 , 100%petroleum ether to 40%EtOAc in petroleum ether) to afford probe 4.
  • Probe 4 was further purified by prep-HPLC (40%-95% (H 2 O/NH 3 H 2 O) /ACN in a 10 min gradient) ) to afford 4 as a white solid (17 mg, 35%) .
  • ⁇ Cell lysates were prepared by adding a protease inhibitor-containing cell lysis buffer (1 ⁇ PBS, 0.1%TritonX-100 (v/v) , 1 ⁇ Protease inhibitor cocktails without EDTA) to the cells, followed by sonication in an ice bath. The supernatant containing proteins was obtained after centrifugation. Protein concentration was measured using the BCA protein assay, and the protein solution was diluted to 2 mg/mL with PBS.
  • a protease inhibitor-containing cell lysis buffer (1 ⁇ PBS, 0.1%TritonX-100 (v/v) , 1 ⁇ Protease inhibitor cocktails without EDTA)
  • Hela cells were cultivated in a 10 cm dish until they reached approximately 90% confluence. The culture medium was removed, and the cells were washed twice with 2 mL of PBS. Then, 4 mL of DMEM containing the CAA probe solution in DMSO (final probe concentration of 30 ⁇ M or 300 ⁇ M, final DMSO concentration of 1%) was added, two biological replicates per group. The cells were incubated at 37°C in a CO 2 incubator for 60 minutes. After washing the culture dish twice with 2 mL of cold PBS, the cells were collected in a 5 mL centrifuge tube. The dish was again washed twice with 1 mL of cold PBS, and the wash solution was combined with the cell suspension.
  • the protein concentration was adjusted to 2 mg/mL, a freshly prepared click reagent mixture (containing isoDTB-L or isoDTB-H) was added to each tube, and the mixture was shaken on an oscillator (25°C, 1400 rpm) for 2 hours.
  • the components of the click reagent mixture included TBTA dissolved in DMSO at a final concentration of 51 ⁇ M, CuSO 4 dissolved in H 2 O at a final concentration of 1 mM, isoDTB-L or H dissolved in DMSO at a final concentration of 150 ⁇ M, and TCEP dissolved in H 2 O at a final concentration of 1mM.
  • streptavidin beads were washed with double-distilled water three times and 0.1 M TEAB once, then dispersed in 1.2 mL of 0.2%NP40 in 0.1 M TEAB solution. These washed beads were added to the samples and incubated at room temperature for 2 hours with rotation. After incubation, the supernatant was discarded following centrifugation, and the beads were resuspended in 600 ⁇ L of 0.1%NP40/PBS. The beads were then washed twice with 600 ⁇ L of 0.1%NP40 in PBS, followed by three washes with 1 ⁇ PBS and three washes with ddH 2 O.
  • MS1 spectra were acquired at 60-K resolving power with a maximum of 50-ms ion injection time in the Orbitrap.
  • MS2 spectra were acquired by selecting the top ten most abundant features via high-energy collisional induced dissociation (HCD) in the Orbitrap at 15-K resolution using an automatic gain control (AGC) setting of 50 K, a collision energy of 35%, a quadrupole isolation window of 1.2 m/z, and a maximum ion injection time of 22ms.
  • HCD collisional induced dissociation
  • Mass spectra were analyzed using the FragPipe pipeline's open search workflow, identifying mass shifts of 754.4126 and 760.4198 Da, which exceed the theoretical masses of CAA-DTB adducts by 16 Da, indicating an extra oxygen atom (FIG. 6A) .
  • Subsequent offset search analysis revealed high selectivity for cysteine residues (FIG. 6B) .
  • Diagnostic ion mining identified three significant diagnostic ions, particularly DI-1 (771.3992 for light, 777.4084 for heavy) , suggesting the inclusion of cysteine atoms (FIG. 6C, 6D and 6E) .
  • the analysis points to three potential mechanisms for incorporating the extra oxygen atom into the probe's structure, with precursor-1 as the most viable candidate (FIG.
  • ⁇ Cell lysates were prepared by adding a protease inhibitor-containing cell lysis buffer (1 ⁇ PBS, 0.1%TritonX-100 (v/v) , 1 ⁇ Protease inhibitor cocktails without EDTA) to the cells, followed by sonication in an ice bath. The supernatant containing proteins was obtained after centrifugation. Protein concentration was measured using the BCA protein assay, and the protein solution was diluted to 2 mg/mL with PBS.
  • a protease inhibitor-containing cell lysis buffer (1 ⁇ PBS, 0.1%TritonX-100 (v/v) , 1 ⁇ Protease inhibitor cocktails without EDTA)
  • the protein concentration was adjusted to 2 mg/mL.
  • a freshly prepared click reagent mixture (containing isoDTB) was added to each tube, and the mixture was shaken on an oscillator (25°C, 1400 rpm) for 2 hours.
  • the components of the click reagent mixture included TBTA dissolved in DMSO at a final concentration of 51 ⁇ M, CuSO 4 dissolved in H 2 O at a final concentration of 1 mM, isoDTB-L or H dissolved in DMSO at a final concentration of 150 ⁇ M, and TCEP dissolved in H 2 O at a final concentration of 1mM.
  • the samples were transferred to 15 mL centrifuge tubes, and 5 mL of cold methanol was added. The mixture was vortexed for 20 seconds and then placed at -20°C overnight to allow complete protein precipitation.
  • FIG. 10A and 10B are overview of targets and Cys sites of the selected compounds CUR, THC, MC and DMC. All the protein targets were queried against the DrugBank database (https: //go. drugbank. com/contact) and fractionated into DrugBank and non-DrugBank proteins (see FIG. 10C) .

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Abstract

L'invention concerne une classe de nouveaux composés dérivés phénoliques de formule (I), ou des sels pharmaceutiquement acceptables, ou un stéréoisomère de ceux-ci utiles en tant que réactifs de sonde covalente, leur synthèse, et leur utilisation en tant que charges covalentes pour cibler la forme d'acide sulfénique de Cys dans le marquage de protéines. Les composés dérivés phénoliques selon l'invention peuvent modifier sélectivement Cys, en particulier ceux sous la forme d'acide sulfénique, dans des cellules vivantes. Cette nouvelle charge covalente peut capturer une large gamme de protéines avec des sites Cys réactifs et sert d'outil pratique pour la recherche et le développement de cibles médicamenteuses de ligands covalents ayant une réactivité souhaitée.
PCT/CN2025/071968 2024-01-12 2025-01-13 Dérivés phénoliques en tant que charge covalente ciblant la forme d'acide sulfenique de cystéine Pending WO2025149076A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN114805385A (zh) * 2022-06-07 2022-07-29 南京农业大学 一种基于分子探针技术制备乳清蛋白-咖啡酸结合物的方法
CN115160170A (zh) * 2022-07-28 2022-10-11 中国中医科学院中药研究所 一种咖啡酸小分子活性探针及其制备方法和用途

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114805385A (zh) * 2022-06-07 2022-07-29 南京农业大学 一种基于分子探针技术制备乳清蛋白-咖啡酸结合物的方法
CN115160170A (zh) * 2022-07-28 2022-10-11 中国中医科学院中药研究所 一种咖啡酸小分子活性探针及其制备方法和用途

Non-Patent Citations (1)

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Title
JÉRÉMIE A DOIRON; LUC M LEBLANC; MARTIN J G HÉBERT; NATALIE A LEVESQUE; AURÉLIE F PARÉ; JACQUES JEAN-FRANÇOIS; MARC CORMIER; MARC : "Structure-activity relationship of caffeic acid phenethyl ester analogs as new 5-lipoxygenase inhibitors", CHEMICAL BIOLOGY & DRUG DESIGN, vol. 89, no. 4, 1 April 2017 (2017-04-01), Hoboken, USA, pages 514 - 528, XP055419740, ISSN: 1747-0277, DOI: 10.1111/cbdd.12874 *

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