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WO2025036479A1 - Inhibiteur de kinésine kif18a et son utilisation - Google Patents

Inhibiteur de kinésine kif18a et son utilisation Download PDF

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Publication number
WO2025036479A1
WO2025036479A1 PCT/CN2024/112662 CN2024112662W WO2025036479A1 WO 2025036479 A1 WO2025036479 A1 WO 2025036479A1 CN 2024112662 W CN2024112662 W CN 2024112662W WO 2025036479 A1 WO2025036479 A1 WO 2025036479A1
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alkyl
mmol
halogenated
compound
ring
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Chinese (zh)
Inventor
肖贻崧
谷晓辉
赖焜民
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Shanghai Apeiron Therapeutics Co Ltd
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Shanghai Apeiron Therapeutics Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • the present disclosure belongs to the field of medicine and relates to a class of kinesin KIF18A inhibitors and their use in inhibiting cancer cell proliferation and treating cancer.
  • KIF18A is a member of the kinesin-8 family of motor proteins. It can rely on the energy released by hydrolyzing ATP in cells to move toward the positive pole using microtubules as tracks. At the same time, KIF18A is located at the positive end of microtubules, which can regulate the dynamic instability of microtubules and exert activity similar to that of microtubule depolymerase. During mitosis, KIF18A can regulate the dynamics of spindle microtubules and chromosome amplitude, and plays a key role in the timely completion of chromosome alignment during mitosis, maintaining genome stability, and successfully completing mitosis.
  • the KIF18A gene belongs to the kinesin-8 subfamily of kinesins and is a plus-end directed motor. KIF18A is thought to affect the dynamics of the plus ends of centromeric microtubules to control correct chromosome positioning and spindle tension. Depletion of human KIF18A in HeLa cervical cancer cells leads to longer spindles, increased chromosome oscillation during metaphase, and activation of the mitotic spindle assembly checkpoint (MI Mayr et al., Current Biology 17, 488–98, 2007). KIF18A appears to be a viable target for cancer therapy.
  • KIF18A is overexpressed in multiple types of cancer, including but not limited to colon, breast, lung, pancreatic, prostate, bladder, head, neck, cervix, and ovarian cancers. Furthermore, in cancer cell lines, genetic deletion or knockout or KIF18A inhibition affects the mitotic spindle apparatus. In particular, inhibition of KIF18A has been found to induce mitotic cell arrest, a known weakness that can promote mitotic cell death by apoptosis, mitotic catastrophe, or multiphase-driven lethality or death after mitotic slippage in interphase. Therefore, there is a strong interest in finding inhibitors of the KIF18A protein. Therefore, inhibition of KIF18A ATPase activity is a promising approach for the development of new anticancer agents.
  • the present disclosure belongs to the field of medicine and relates to a class of kinesin KIF18A inhibitors, specifically to the compounds or their stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereomers or mixtures thereof or their pharmaceutically acceptable salts, cocrystals, metabolites, solvates, prodrugs or isotope labels, preparation methods thereof, and drug combinations containing the compounds and their use as therapeutic agents, especially as a method for inhibiting cancer cell proliferation and treating cancer.
  • the present disclosure provides a novel class of compounds for use in modulating KIF18A protein alone or in a binding complex with microtubules for treating KIF18A-mediated conditions and/or diseases, including cancer, inflammation or ciliary pathology.
  • the compounds provided by the present disclosure have MT-based KIF18A regulatory activity, in particular, KIF18A inhibitory activity.
  • the present disclosure also provides the use of these compounds and pharmaceutically acceptable salts thereof in the preparation and manufacture of pharmaceutical compositions or drugs for therapeutic, preventive, acute or chronic treatment of KIF18A-mediated diseases and disorders (including but not limited to cancer).
  • the present disclosure provides a compound having a structure of Formula I or a pharmaceutically acceptable salt, stereoisomer, or isotope isomer thereof:
  • X 1 represents CR W1 or N;
  • X 2 represents CR W2 or N;
  • X 3 represents CR W3 or N
  • L 1 represents -C(O)NH-, -NHC(O)- or triazolyl
  • Cy1 represents a 6-14 membered aryl or heteroaryl group; and the Cy1 may be arbitrarily substituted by 0-3 groups selected from the following substituents: halogen, C1 - C6 alkyl, C3 - C6 cycloalkyl, halogenated C1 - C6 alkyl, hydroxyl C1 - C6 alkyl, -ORa , -O-halogenated C1 - C6 alkyl , -SRa , -SF5, cyano, nitro, -NRaRb , -NRaC(O) Rb , -C (O) NRaRb , -OC(O) Ra , -C (O) ORa , -S(O) Ra , -S (O) 2Ra and -S ( O ) 2NRaRb ;
  • Cy2 represents a 3-12 membered saturated or unsaturated monocyclic or bicyclic ring, the ring may arbitrarily contain 0-3 heteroatoms selected from O, N and S, and the Cy2 may also be arbitrarily substituted by 0-3 groups selected from the following substituents: halogen, C1 - C6 alkyl, C3 - C6 cycloalkyl, halogenated C1 - C6 alkyl, hydroxyl C1 - C6 alkyl, -ORa , -O-halogenated C1 - C6 alkyl , -SRa, -SF5, cyano, nitro , -NRaRb , -NRaC(O) Rb , -C (O) NRaRb , -OC(O) Ra , -C (O) ORa , -S (O) Ra , -S(O) 2Ra and -S( O ) 2NRaRb ;
  • L2 represents a linear or branched C1 - C6 methylene group, and any hydrogen atom may be substituted by deuterium, hydroxyl, or halogen; or any two hydrogen atoms connected to the same carbon atom may form a structure;
  • Ra and Rb each independently represent hydrogen, C1 - C6 alkyl, or said Ra and Rb together with the atoms connected thereto form a 3-6 membered saturated or unsaturated ring, and said ring may also arbitrarily contain 0, 1, or 2 heteroatoms selected from O, S, and N;
  • RM and RN represent hydrogen, halogen, halogenated C1 - C6 alkyl, hydroxy-substituted C1 - C6 alkyl, C1 - C6 alkyl, or said RM , RN and the atoms to which they are connected together form a 3-6 membered saturated or unsaturated ring;
  • n 0, 1, 2, 3;
  • P 1 represents hydrogen or a protecting group
  • P 2 represents hydrogen or a protecting group
  • the premise is that P 1 and P 2 are not hydrogen at the same time.
  • L1 represents -C(O)NH- or -NHC(O)-.
  • L 1 represents a triazole group.
  • L 1 represents the following group: or
  • Cy 1 represents phenyl, pyridyl, pyrimidinyl, quinolyl, and the Cy 1 can be arbitrarily substituted by 0-3 groups selected from the following substituents: halogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, halogenated C 1 -C 6 alkyl, hydroxyl C 1 -C 6 alkyl, -OR a , -O-halogenated C 1 -C 6 alkyl, -SR a , -SF 5 , cyano, nitro, -NR a R b .
  • Cy 1 represents the following group:
  • Cy 1 can be arbitrarily substituted by 0-3 groups selected from the following substituents: halogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, halogenated C 1 -C 6 alkyl, hydroxyl C 1 -C 6 alkyl, -OR a , -O-halogenated C 1 -C 6 alkyl, -SR a , -SF 5 , cyano, nitro, -NR a R b .
  • Cy2 represents a saturated, partially saturated or unsaturated 3-, 4-, 5-, 6-, 7-membered monocyclic ring, or a 3-, 4-, 5-, 6-, 7-membered cyclic ring, and the Cy2 may arbitrarily contain 0-3 N heteroatoms and 0-2 O or S heteroatoms, and the Cy2 may be arbitrarily substituted by 0-3 substituents selected from the following: halogen, cyano , nitro, hydroxyl C1 - C6 alkyl, C1 - C6 alkyl, C3 - C8 cycloalkyl, halogenated C1 - C6 alkyl, -ORa , -SO3Ra , -S(O) Ra , -Ohalogenated C1 - C6 alkyl , -SRa , -SF5 or NRaRb , wherein Ra and Rb each independently represent hydrogen, halogen, C1 - C6
  • Cy 2 represents any of the following groups:
  • the Cy2 may be arbitrarily substituted by 0-3 substituents selected from the following: halogen, cyano, nitro, hydroxy C1 - C6 alkyl, C1 - C6 alkyl, C3 - C8 cycloalkyl, halogenated C1 - C6 alkyl, -ORa , -SO3Ra , -S (O) Ra , -Ohalogenated C1 - C6 alkyl, -SRa , -SF5 or NRaRb , wherein Ra and Rb each independently represent hydrogen, halogen, C1 - C6 alkyl, C3 - C8 cycloalkyl, halogenated C1 - C6 alkyl or hydroxy C1 -C6 alkyl ; or Ra and Rb together with the atoms to which they are connected form a 3-6 membered ring containing 0-2 heteroatoms selected from O, N and S.
  • substituents selected from the
  • L2 represents -CH2CH2-, -CH( CH3) CH2- , -CH2CH ( CH3 )- , -CH(OH) CH2- , -CH2CH(OH)-, -CH( CH3 ) CH2- , -CH2CH(CH3) - , -CH (OH) CH2- , -CH2CH ( CH3 ) -, -CF2CH2- , -CH2CF2- , -CHFCH2-, -CH2CHF- ,
  • RM and RN each independently represent hydrogen, C1 - C6 alkyl, or RM , RN and the atoms connected thereto together form a 3-6 membered saturated or unsaturated ring.
  • P1 represents hydrogen, -C(O)R a , -C(O)(CR T R T' ) m NH 2 , -P(O)(OR a ) 2 , -P(O)(ONa) 2 , wherein R a represents a C 1 -C 6 alkyl group, wherein RT and RT ' each independently represent hydrogen, a substituted or unsubstituted C 1 -C 6 alkyl group, and the substituent is a halogen, a hydroxyl group, a phenyl group, or a 5-6 membered heteroaryl group.
  • P2 represents hydrogen, -C(O)R a , -C(O)(CR T R T' ) m NH 2 , -P(O)(OR a ) 2 , -P(O)(ONa) 2 , wherein R a represents a C 1 -C 6 alkyl group, wherein RT and RT ' each independently represent hydrogen, a substituted or unsubstituted C 1 -C 6 alkyl group, and the substituent is a halogen, a hydroxyl group, a phenyl group, or a 5-6 membered heteroaryl group.
  • the present disclosure provides a compound having the following structure:
  • the present disclosure also provides a pharmaceutical composition, which contains any one of the compounds in the technical scheme of the present disclosure or its pharmaceutically acceptable salt, stereoisomer, isotope isomer, prodrug, hydrate or solvate, and a pharmaceutically acceptable carrier thereof.
  • the present disclosure also provides a method for treating tumors by inhibiting KIF18A, comprising administering any one of the compounds of the present disclosure or a pharmaceutically acceptable salt, stereoisomer, isotope isomer, prodrug, hydrate or solvate thereof to an individual in need thereof.
  • the compounds of the present disclosure may be interpreted to include, in addition to the specific structures of the compounds, pharmaceutically acceptable salts of the compounds, their stereoisomers, isotope isomers (e.g., deuterated compounds), solvates, hydrates, prodrugs, and metabolites, that is, pharmaceutically acceptable salts of the compounds, their stereoisomers, isotope isomers, solvates, hydrates, prodrugs, and metabolites also fall within the protection scope of the compounds.
  • alkyl refers to a saturated aliphatic hydrocarbon group or linker, comprising 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms, straight chain and branched groups.
  • “Lower alkyl” refers in particular to alkyl groups having 1 to 4 carbon atoms. Examples of alkyl groups include -(CH 2 ) 3 -, methyl, trifluoromethyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, etc.
  • Alkyl groups may be substituted or unsubstituted.
  • Typical substituents include cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, C-carboxy, O-carboxy, nitro, silanyl, amino, and -NRxRy , wherein Rx and Ry are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl, and combined 5- or 6-membered heterocyclyl rings.
  • alkenyl refers to a straight or branched hydrocarbon group containing one or more double bonds and generally having a length of 2 to 20 carbon atoms.
  • C2 - C6 alkenyl contains two to six carbon atoms.
  • Alkenyl includes, but is not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
  • alkynyl refers to a straight or branched hydrocarbon group containing one or more triple bonds and typically 2 to 20 carbon atoms in length.
  • a "C2-C6 alkynyl” contains two to six carbon atoms.
  • Representative alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, etc.
  • alkoxy refers to -O-alkyl.
  • C1-C6 alkoxy (or alkyloxy) is intended to include C1, C2, C3, C4, C5, C6 alkoxy.
  • alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), and tert-butoxy.
  • alkylthio or “thioalkoxy” refers to an alkyl group as defined above connected by a sulfur bridge with a specified number of carbon atoms; for example, methyl-S- and ethyl-S-.
  • cycloalkyl refers to a 3-8 membered all-carbon monocyclic ring or bicyclic structure, an all-carbon 5-membered/6-membered or 6-membered/6-membered fused bicyclic ring, or a polycyclic fused ring (a "fused" ring system means that each ring in the system shares at least one adjacent carbon atom with other rings in the system) group, wherein one or more rings may contain one or more double bonds, but such rings do not have a complete conjugated ⁇ -electron system, or the bicyclic rings form a spirocycle by sharing one carbon.
  • cycloalkyls are (but not limited to) cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, adamantane, cycloheptane, cycloheptatriene, etc.
  • Bicyclic cyclic alkyls include bridged, spiro or fused ring cycloalkyls.
  • Illustrative examples of cycloalkyls are derived from (but not limited to) the following:
  • Aryl refers to an all-carbon monocyclic or fused-ring polycyclic group of 6 to 12 carbon atoms with a complete conjugated ⁇ -electron system. Examples of aryl are, but are not limited to, phenyl, naphthyl, and anthracenyl. Aryl may be substituted or unsubstituted.
  • Typical substituents include halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amide, sulfinyl, sulfonyl, amino, and -NR a R b , wherein R a and R b are as defined above.
  • Aryl-fused saturated or unsaturated cycloalkyl/saturated or unsaturated heterocycloalkyl can be considered as special substituents for aryl, typical examples of which include, but are not limited to:
  • Heteroaryl refers to a monocyclic or fused ring of 5 to 12 ring atoms, containing one, two, three or four ring heteroatoms selected from N, O and S, the remaining ring atoms being C, and furthermore, having a complete conjugated ⁇ -electron system.
  • heteroaryl groups are, but are not limited to, acridinyl, azetidinyl, azinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuranyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H
  • heteroaryl may also include a biaryl structure formed by the above-defined “aryl” and a monocyclic “heteroaryl”, such as but not limited to "-phenylbipyridyl-", “-phenylbipyrimidyl-”, “-pyridyl wherein the present disclosure also includes fused ring and spiro compounds containing, for example, the above-mentioned heterocycles.
  • a pharmaceutically acceptable heteroaryl group is one that is sufficiently stable to be attached to the compounds of the present disclosure, formulated into a pharmaceutical composition, and subsequently administered to a patient in need thereof.
  • substituents disclosed herein are independent of each other and not interrelated, for example, for Ra (or Rb) in a substituent, it is independent of each other in the definitions of different substituents. Specifically, when a definition is selected for Ra (or Rb) in a substituent, it does not mean that the Ra (or Rb) has the same definition in other substituents. More specifically, for example (listing only, not exhaustive), for NRaRb, when the definition of Ra (or Rb) is selected from hydrogen, it does not mean that Ra (or Rb) in -C(O)-NRaRb is necessarily hydrogen.
  • Halo or halogen includes fluorine, chlorine, bromine and iodine.
  • Haloalkyl is intended to include branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms and substituted with one or more halogens.
  • haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl and heptachloropropyl.
  • haloalkyl also include "fluoroalkyl” which is intended to include branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms and substituted with one or more fluorine atoms.
  • Haloalkoxy or "haloalkyloxy” means a haloalkyl group as defined above connected via an oxygen bridge with a specified number of carbon atoms.
  • C1-C6 haloalkoxy is intended to include C1, C2, C3, C4, C5, C6 haloalkoxy.
  • Examples of haloalkoxy include, but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, and pentafluoroethoxy.
  • haloalkylthio or “thiohaloalkoxy” means a haloalkyl group as defined above connected via a sulfur bridge with a specified number of carbon atoms; for example, trifluoromethyl-S- and pentafluoroethyl-S-.
  • Cx1-Cx2 when referring to some substituent groups, the expression Cx1-Cx2 is used, which means that the number of carbon atoms in the substituent group may be x1 to x2.
  • C0-C8 means that the group contains 0, 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms
  • C1-C8 means that the group contains 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms
  • C2-C8 means that the group contains 2, 3, 4, 5, 6, 7 or 8 carbon atoms
  • C3-C8 means that the group contains 3, 4, 5, 6, 7 or 8 carbon atoms
  • C4-C8 means that the group contains 4, 5, 6, 7 or 8 carbon atoms
  • C0-C6 means that the group contains 0, 1, 2, 3, 4, 5 or 6 carbon atoms
  • C1-C6 means that the group contains 1, 2, 3, 4, 5 or 6 carbon atoms
  • C2-C6 means that the group contains 2, 3, 4, 5 or 6 carbon atoms
  • C3-C6 means that the group contains 3, 4, 5 or 6
  • x1-x2 membered ring when referring to cyclic groups (such as aryl, heteroaryl, cycloalkyl and heterocycloalkyl), the expression "x1-x2 membered ring" is used, which means that the number of ring atoms of the group can be x1 to x2.
  • the 3-12 membered cyclic group can be a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11- or 12-membered ring, and the number of ring atoms thereof can be 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
  • the 3-6 membered ring means that the cyclic group can be a 3-, 4-, 5- or 6-membered ring, and the number of ring atoms thereof can be 3, 4, 5 or 6;
  • the 3-8 membered ring means that the cyclic group can be a 3-, 4-, 5-, 6-, 7- or 8-membered ring, and the number of ring atoms thereof can be 3, 4, 5, 6, 7 or 8;
  • the 3-9 membered ring means that the cyclic group can be a 3-, 4-, 5-, 6-, 7-, 8- or 9-membered ring, and the number of ring atoms thereof can be 3, 4, 5, 6, 7, 8 or 9; 4-7 membered ring means that the
  • the ring atoms can be carbon atoms or heteroatoms, such as heteroatoms selected from N, O and S.
  • the heterocycle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ring heteroatoms, such as heteroatoms selected from N, O and S.
  • one or more halogens may each independently be selected from fluorine, chlorine, bromine and iodine.
  • substituted means that at least one hydrogen atom is replaced by a non-hydrogen group, provided that normal valence is maintained and the substitution results in a stable compound.
  • nitrogen atoms e.g., amines
  • these nitrogen atoms can be converted to N-oxides by treatment with an oxidizing agent (e.g., mCPBA and/or hydrogen peroxide) to obtain other compounds of the present disclosure.
  • an oxidizing agent e.g., mCPBA and/or hydrogen peroxide
  • the nitrogen atoms shown and claimed are considered to encompass both the shown nitrogen and its N-oxide to obtain the disclosed derivatives.
  • any variable occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence.
  • the group may be optionally substituted with up to three R groups, and at each occurrence R is independently selected from the definition of R.
  • combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
  • patient refers to an organism treated by the methods of the present disclosure.
  • organisms preferably include, but are not limited to, mammals (e.g., mice, apes/monkeys, horses, cattle, pigs, dogs, cats, etc.) and most preferably refer to humans.
  • an effective amount means the amount of a drug or medicament (i.e., a compound of the present disclosure) that will cause a biological or medical response of a tissue, system, animal, or human being sought by, for example, a researcher or clinician.
  • therapeutically effective amount means an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the above amount.
  • An effective amount can be administered in one or more administrations, applications, or doses and is not intended to be limited by a specific formulation or route of administration.
  • the term also includes an effective amount that enhances normal physiological function within its scope.
  • treatment includes its broad meaning, covering therapeutic treatment and/or preventive treatment of a subject. Specifically, the “treatment” includes any treatment that leads to the alleviation, suppression, elimination and improvement and/or prevention of a condition, disease, disorder, etc., such as alleviating, reducing, regulating, improving, eliminating, preventing, preventing or improving its symptoms.
  • the therapeutic treatment includes alleviating, suppressing or improving the symptoms or conditions of the disease; suppressing the occurrence of complications; improving potential metabolic syndrome; suppressing the occurrence of the disease or symptoms, such as controlling the development of the disease or condition; alleviating the disease or symptoms; reducing the disease or symptoms; alleviating complications caused by the disease or symptoms, or treating signs caused by the disease or symptoms.
  • the preventive treatment includes prior treatment to prevent, block or delay, slow down the occurrence or development of a disease or condition or reduce the severity of the disease or condition.
  • therapeutic agent also includes agents or reagents that have therapeutic and/or prophylactic effects on a subject.
  • pharmaceutical or “pharmaceutically acceptable” are used herein to refer to those compounds, substances, compositions and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response and/or other problems or complications, and commensurate with a reasonable benefit/risk ratio.
  • cancer refers to an abnormal growth of cells that cannot be controlled and can metastasize (spread) under certain conditions.
  • This type of cancer includes, but is not limited to, solid tumors (such as bladder, intestine, brain, chest, uterus, heart, kidney, lung, lymphoid tissue (lymphoma), ovary, pancreas or other endocrine organs (such as thyroid), prostate, skin (melanoma) or blood tumors (such as non-leukemic leukemia).
  • combination administration refers to the administration of several selected therapeutic agents to one patient, in the same or different administration routes and at the same or different times.
  • enhancement refers to the ability to increase or prolong the potency or duration of the desired result. Therefore, in terms of enhancing the therapeutic effect of a drug, the term “enhancing” refers to the ability of the drug to increase or prolong the potency or duration in the system. "Efficacy value” as used herein refers to the ability to maximize the enhancement of another therapeutic drug in an ideal system.
  • immune disease refers to a disease or condition that is caused by an adverse or harmful response to endogenous or exogenous antigens. The result is usually cellular dysfunction, or the resulting damage and malfunction of, or destruction of, organs or tissues that may produce immune symptoms.
  • subject includes mammals and non-mammals.
  • Mammals include, but are not limited to, mammals: humans, non-human primates such as gorillas, apes and monkeys; agricultural animals such as cattle, horses, goats, sheep, pigs; livestock such as rabbits and dogs; experimental animals include rodents such as rats, mice and guinea pigs.
  • Non-mammalian animals include, but are not limited to, birds, fish, etc.
  • the selected mammal is a human.
  • a compound or pharmaceutical composition after administration, can improve a disease, symptom or condition, especially improve its severity, delay the onset, slow the progression of the disease, or reduce the duration of the disease. Whether fixed or temporary administration, continuous or intermittent administration, can be attributed to or related to the administration.
  • Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, auricular, nasal, and topical administration.
  • parenteral administration by way of example only, includes intramuscular, subcutaneous, intravenous, intramedullary, ventricular, intraperitoneal, intralymphatic, and intranasal injections.
  • the administration of the disclosed compounds can be a local administration.
  • the long-acting preparation is administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection.
  • the drug is administered by a targeted drug delivery system.
  • a liposome wrapped by an organ-specific antibody In this embodiment, the liposome is selectively directed to a specific organ and absorbed.
  • the phrase "pharmaceutically acceptable carrier” means a pharmaceutical substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc stearate or stearic acid) or solvent encapsulating substance, which is involved in carrying or transporting the subject compound from one organ or part of the body to another.
  • a pharmaceutical substance, composition or vehicle such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc stearate or stearic acid) or solvent encapsulating substance, which is involved in carrying or transporting the subject compound from one organ or part of the body to another.
  • manufacturing aid e.g., lubricant, talc, magnesium, calcium or zinc stearate or stearic acid
  • solvent encapsulating substance e.g., solvent encapsulating substance, which is involved
  • composition means a composition comprising a compound of the present disclosure and optionally other pharmaceutically acceptable carriers.
  • “Pharmaceutically acceptable carrier” refers to a medium generally accepted in the art for delivering biologically active agents to animals (particularly mammals), including (i.e.) adjuvants, excipients or vehicles, such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatics, antibacterial agents, antifungal agents, lubricants and dispersants, depending on the nature of the mode of administration and dosage form.
  • adjuents such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatics, antibacterial agents, antifungal agents, lubricants and dispersants, depending on the nature of the mode of administration and dosage form.
  • the pharmaceutical composition of the present disclosure may include a therapeutically effective amount of one or more compounds of the present disclosure formulated with one or more optional pharmaceutically acceptable carriers (additives) and/or diluents, and one or more other therapeutic agents.
  • the compounds of the present disclosure may be administered in any suitable manner for any of the above-mentioned uses, for example, orally, such as tablets, pills, powders, granules, elixirs, tinctures, suspensions (including nanosuspensions, microsuspensions, spray-dried dispersions), syrups and emulsions; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques (e.g., in the form of sterile injectable aqueous or non-aqueous solutions or suspensions); nasally, including administration to the nasal membrane, such as by inhalation spray; topically, such as in the form of creams or ointments; or rectally
  • Pharmaceutically acceptable carriers are formulated based on a number of factors that are within the purview of those skilled in the art. These factors include, but are not limited to: the type and nature of the active agent being formulated; the subject to whom the composition containing the active agent is to be administered; the intended route of administration of the composition; and the targeted therapeutic indication. Pharmaceutically acceptable carriers include aqueous and non-aqueous liquid media and various solid and semisolid dosage forms.
  • Such carriers may include a variety of different ingredients and additives in addition to the active agent, which are included in the formulation for a variety of reasons known to those skilled in the art, such as stabilizing the active agent, binders, etc.
  • suitable pharmaceutical carriers and factors involved in carrier selection can be found in a number of readily available sources, such as Allen L.V.Jr. et al. Remington: The Science and Practice of Pharmacy (2 Volumes), 22nd Edition (2012), Pharmaceutical Press.
  • the dosage regimen of the disclosed compounds varies depending on known factors, such as the pharmacodynamic properties of the specific agent and its mode and route of administration; the species, age, sex, health condition, medical condition and weight of the recipient; the nature and extent of the symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and liver function and the desired effect.
  • the daily oral dose of each active ingredient should be about 0.001 mg/day to about 10-5000 mg/day, preferably about 0.01 mg/day to about 1000 mg/day, and most preferably about 0.1 mg/day to about 250 mg/day.
  • the most preferred intravenous dose should be about 0.01 mg/kg/minute to about 10 mg/kg/minute.
  • the disclosed compounds can be administered in a single daily dose, or the total daily dose can be administered in divided doses twice, three times or four times a day.
  • the compound is usually administered in a mixture with a suitable pharmaceutical diluent, excipient or carrier (collectively referred to herein as a pharmaceutical carrier) appropriately selected according to the intended administration form (eg, oral tablets, capsules, elixirs and syrups) and in accordance with conventional pharmaceutical practice.
  • a suitable pharmaceutical diluent, excipient or carrier collectively referred to herein as a pharmaceutical carrier
  • Dosage forms suitable for administration may contain from about 1 mg to about 2000 mg of active ingredient per dosage unit.
  • the active ingredient will generally be present in an amount of about 0.1-95% by weight based on the total weight of the composition.
  • compositions comprising (alone or in combination with a pharmaceutically acceptable carrier) a therapeutically effective amount of at least one compound of the disclosure as an active ingredient.
  • the compounds of the disclosure may be used alone, in combination with other compounds of the disclosure, or in combination with one or more other therapeutic agents (e.g., anticancer agents or other pharmaceutically active substances).
  • the compounds of the present invention (which may be used in a suitable hydrated form) and/or the pharmaceutical compositions of the present disclosure are formulated into pharmaceutical dosage forms by conventional methods known to those skilled in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors, including the activity of the particular compound of the present disclosure employed, or its ester, salt or amide thereof; the route of administration; the time of administration; the rate of excretion of the particular compound employed; the rate and extent of absorption; the duration of the treatment; other drugs, compounds and/or substances used in combination with the particular compound employed; and the age, sex, weight, condition, general health and prior medical history of the patient being treated, and other factors well known in the medical art.
  • a doctor or veterinarian with ordinary skills in the art can easily determine and prescribe an effective amount of the desired pharmaceutical composition.
  • a physician or veterinarian can start the dosage of the disclosed compound used in the pharmaceutical composition at a level lower than the desired level, and gradually increase the dosage until the desired effect is achieved.
  • the amount of the disclosed compound of a suitable daily dose will be the amount of the compound at the lowest dose that effectively produces a therapeutic effect.
  • Such an effective dose generally depends on the above factors.
  • the range of the disclosed compound for oral, intravenous, intracerebroventricular and subcutaneous doses for patients is about 0.01 to about 50 mg/kg body weight/day.
  • the active compound of the effective daily dose can be administered separately at appropriate intervals in two, three, four, five, six or more subdoses in the middle of the day, optionally in a unit dosage form.
  • medication is administered once a day.
  • composition While it is possible for a compound of the present disclosure to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
  • Kits/product packages are also described herein for use in the treatment of the above-mentioned indications.
  • These kits may consist of a conveyor, a medicine bag or a container box, which may be divided into multiple compartments to accommodate one or more containers, such as vials, test tubes and the like, each of which contains a single component of the method.
  • Suitable containers include bottles, vials, syringes and test tubes.
  • the container is made of acceptable materials such as glass or plastic.
  • a container may contain one or more compounds described herein, which may be present as a pharmaceutical composition or in admixture with other ingredients described herein.
  • the container may have a sterile outlet (e.g., the container may be an intravenous infusion bag or bottle, the stopper of which may be pierced by a hypodermic syringe needle).
  • a kit may include a compound and instructions, labels, or operating instructions for use as described herein.
  • a typical kit may include one or more containers, each containing one or more materials (such as reagents, concentrated stock solutions, and/or instruments) to suit the needs of commercial promotion and user use of the compound.
  • materials include but are not limited to buffers, diluents, filters, needles, syringes, delivery devices, bags, containers, bottles and/or test tubes, with a list of contents and/or instructions for use, and the inner packaging also comes with instructions. The entire set of instructions must be included.
  • the label may be displayed on the container or closely associated with the container.
  • the label may be on the container when the label letters, numbers or other features are affixed, molded, or engraved on the container; the label may also be present in a container box or shipping box containing multiple containers, such as in a product insert.
  • a label may be used to indicate a specific therapeutic use of the contents.
  • the label may also indicate instructions for use of the contents, such as described in the above method.
  • NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvent used for the measurements is indicated in the spectrum analysis.
  • MS was determined using Agilent 1200-G1956A/1200-6110A/1200-6140A/1260-6125B/Prime-6125B/1260-6120 LC/MS, SHIMADZU 20A-2010/20A-2020 LC/MS, and Waters ACQ-QDA LC/MS.
  • HPLC analysis was performed using a SHIMADZU 20A high performance liquid chromatograph.
  • SFC separation uses The Berger MG II, MG III, Sepiatec's Prep SFC 100 system, Waters Prep 80Q SFC SYSTEM, Prep 150 AP SFC SYSTEM, Prep 200 SFC SYSTEM, Prep 350 SFC SYSTEM.
  • Flash column chromatography separations were performed using a Biotage IsoleraOne flash preparative chromatograph.
  • the thin layer chromatography silica gel plate used was GF254 acrylic adhesive silica gel plate produced by Anhui Liangchen Silicon Source Material Co., Ltd.
  • the specification of the silica gel plate used in thin layer chromatography (TLC) was 0.25 mm, and the specification of the thin layer chromatography separation and purification product was 0.5 mm.
  • Microwave reactions were performed using a Biotage Initiator+ microwave synthesizer.
  • the glove box is customized by DELLIX.
  • Step 1 Add 6-azaspiro [2.5] octane hydrochloride (1.33 g, 12.0 mmol) to a solution of 2-fluoro-4-iodobenzaldehyde (2.00 g, 8.00 mmol) and potassium carbonate (3.31 g, 24.0 mmol) in dimethyl sulfoxide (20.0 mL). The mixture was heated to 120 ° C and stirred for 16 hours. Water (20 mL) was added to the reaction mixture and extracted with ethyl acetate (20 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was dried to obtain a residue.
  • Step 2 Add dimethyl (1-diazo-2-oxypropylidene) phosphonate (1.15 g, 5.98 mmol) to a solution of 4-iodo-2-(6-azaspiro [2.5] octane-6-yl) benzaldehyde (1.70 g, 4.98 mmol) and potassium carbonate (1.37 g, 9.97 mmol) in methanol (20.0 mL). The mixture was stirred at 25 ° C for 16 hours. Water (20 mL) was added to the reaction mixture and extracted with ethyl acetate (20 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was dried to obtain a residue.
  • Step 3 Add tert-butyl nitrite (599 mg, 5.70 mmol) to a solution of 8-(4,4-difluoropiperidin-1-yl)quinolin-6-amine (600 mg, 2.28 mmol) in acetonitrile (5.00 mL). Then add trimethylsilyl azide (525 mg, 4.56 mmol) to the reaction solution, and stir the mixture at 25 ° C for 16 hours. Pour the reaction solution into water (10 mL) and extract with ethyl acetate (10 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and spin-dry the filtrate to obtain the residue.
  • Step 4 Add copper sulfate (88.3 mg, 0.55 mmol) and sodium ascorbate (110 mg, 0.55 mmol) to a solution of 6-azido-8-(4,4-difluoropiperidin-1-yl)quinoline (160 mg, 0.55 mmol), 6-(2-ethynyl-5-iodophenyl)-6-azaspiro[2.5]octane (224 mg, 0.66 mmol) in tert-butyl alcohol (5.00 mL) and water (5.00 mL). Stir the mixture at 25 °C for 16 hours. Add water (20 mL) to the reaction solution and extract with ethyl acetate (20 mL x 3).
  • Step 5 To a solution of 2-hydroxyethanesulfonamide (47.9 mg, 0.38 mmol), (1R,2R)-(-)-N,N-dimethylcyclohexane-1,2-diamine (27.5 mg, 0.19 mmol), potassium phosphate (163 mg, 0.77 mmol) in N,N-dimethylformamide (5.0 mL) was added cuprous iodide (18.6 mg, 0.10 mmol). The mixture was stirred at 50° C. for 5 minutes.
  • 6-[4-(2- ⁇ 6-azaspiro[2.5]octan-6-yl ⁇ -4-iodophenyl)-1H-1,2,3-triazol-1-yl]-8-(4,4-difluoropiperidin-1-yl)quinoline 120 mg, 0.19mmol
  • Water (10mL) was added to the reaction solution and extracted with ethyl acetate (10mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was spin-dried to obtain a residue.
  • Step 1 Add 4,4-difluoropiperidine hydrochloride (5.22 g, 33.2 mmol) and potassium carbonate (9.36 g, 66.4 mmol) to a solution of 6-bromo-8-fluoroquinoline (5.00 g, 22.1 mmol) in 1-methyl-2-pyrrolidone (50.0 mL). Heat the mixture to 180 ° C and stir for 50 hours. Add water (20 mL) to the reaction solution and extract with ethyl acetate (20 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and spin-dry the filtrate to obtain the residue.
  • Step 2 Add dichlorobis(triphenylphosphine)palladium(II) (0.87 g, 1.22 mmol) to a solution of 6-bromo-8-(4,4-difluoropiperidin-1-yl)quinoline (4.00 g, 12.2 mmol), ethynyltrimethylsilane (1.32 g, 13.4 mmol), cuprous iodide (0.24 g, 1.22 mmol) and triphenylphosphine (0.32 g, 1.22 mmol) in triethylamine (30.0 mL) and tetrahydrofuran (20.0 mL), replace with nitrogen three times, and heat the mixture to 85 ° C and stir for 16 hours.
  • Step 3 Add copper sulfate (225 mg, 1.41 mmol) and sodium ascorbate (279 mg, 1.41 mmol) to a solution of 6-(2-azido-5-iodophenyl)-6-azaspiro[2.5]octane (500 mg, 1.41 mmol), 8-(4,4-difluoropiperidin-1-yl)-6-((trimethylsilyl)ethynyl)quinoline (490 mg, 1.41 mmol) in tert-butyl alcohol (5 mL), tetrahydrofuran (5 mL) and water (5 mL). Stir the mixture at 25 °C for 16 hours.
  • Step 4 To a solution of 2-hydroxyethanesulfonamide (120 mg, 0.96 mmol), sarcosine (43.5 mg, 0.48 mmol), potassium phosphate (415 mg, 1.92 mmol) in N,N-dimethylformamide (6 mL) was added cuprous iodide (93.1 mg, 0.48 mmol). The mixture was stirred at 50 ° C for 5 minutes.
  • Step 1 Add diisobutylaluminum hydride 1M toluene solution (25.2mL, 25.2mmol) to a tetrahydrofuran solution of 2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidine-4-carbonitrile (2.00g, 8.39mmol) at -40°C. Slowly warm the mixture to 25°C and stir for 1 hour. Add the mixture to ice water (20mL) to quench, and extract with ethyl acetate (20mL x 2). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and spin-dry the filtrate to obtain a residue.
  • Step 2 Add dimethyl (1-diazo-2-oxypropylidene) phosphonate (0.33 g, 1.74 mmol) to a solution of 2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidine-4-carboxaldehyde (0.35 g, 1.45 mmol) and potassium carbonate (0.40 g, 2.90 mmol) in methanol (7 mL). Stir the mixture at 25 ° C for 16 hours. Add water (20 mL) to the reaction mixture and extract with ethyl acetate (20 mL x 2).
  • Step 3 Add 6-azaspiro [2.5] octane hydrochloride (1.82 g, 12.3 mmol) to a solution of 2-fluoro-4-iodo-1-nitrobenzene (3.00 g, 11.2 mmol) and potassium carbonate (4.65 g, 33.7 mmol) in dimethyl sulfoxide (45 mL). Heat the mixture to 140 ° C and stir for 16 hours. Pour water (100 mL) into the reaction solution and extract with ethyl acetate (100 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and spin-dry the filtrate to obtain a residue.
  • Step 4 Add ammonium chloride (1.36 g, 25.1 mmol) and iron powder (4.73 g, 83.7 mmol) to a solution of 6-(5-iodo-2-nitrophenyl)-6-azaspiro[2.5]octane (3.00 g, 8.37 mmol) in methanol (60 mL) and water (20 mL). Heat the mixture to 60 ° C and stir for 16 hours. Filter the reaction solution through diatomaceous earth, and extract the filtrate with ethyl acetate (100 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and spin-dry the filtrate to obtain a residue.
  • Step 5 Add tert-butyl nitrite (1.09 g, 10.3 mmol) to a solution of 4-iodo-2-(6-azaspiro[2.5]octane-6-yl)aniline (2.26 g, 6.89 mmol) in acetonitrile (40 mL) at 0 ° C. Then add trimethylsilyl azide (1.19 g, 10.3 mmol) to the reaction solution, and stir the mixture at 25 ° C for 16 hours. Pour the reaction solution into water (40 mL) and extract with ethyl acetate (40 mL x 3).
  • Step 6 To a solution of 6-(2-azido-5-iodophenyl)-6-azaspiro[2.5]octane (500 mg, 1.41 mmol), 2-(4,4-difluoropiperidin-1-yl)-4-ethynyl-6-methylpyrimidine (334 mg, 1.41 mmol) in tert-butanol (5 mL) and water (5 mL) was added copper sulfate (225 mg, 1.41 mmol) and sodium ascorbate (279 mg, 1.41 mmol). The mixture was stirred at 25 ° C for 16 hours. Water (20 mL) was added to the reaction solution and extracted with ethyl acetate (20 mL x 3).
  • Step 7 To a solution of 2-hydroxyethanesulfonamide (82.5 mg, 0.66 mmol), 2-(methylamino)acetic acid (45.2 mg, 0.51 mmol), potassium phosphate (439 mg, 2.03 mmol) in N,N-dimethylformamide (6 mL) was added cuprous iodide (49.2 mg, 0.25 mmol). The mixture was stirred at 50 ° C for 5 minutes.
  • 6-(2-(4-(2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)-5-iodophenyl)-6-azaspiro[2.5]octane 300 mg, 0.51 mmol was added to the reaction mixture at 50 ° C, and the mixture was heated to 100 ° C under nitrogen and stirred for 16 hours.
  • Water (10 mL) was added to the reaction solution and extracted with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was dried to obtain a residue.
  • Step 1 A mixed solution containing 3-fluoro-2-iodo-1-nitrobenzene (9.50 g, 35.58 mmol), iron powder (10.00 g, 177.90 mmol), ammonium chloride (9.60 g, 177.90 mmol), ethanol (100 mL) and water (20 mL) was stirred at 60°C for 1 hour. The reaction solution was filtered while hot and concentrated, then extracted with ethyl acetate (50 mL x 3).
  • Step 2 Add N-bromosuccinimide (5.0 g, 28.27 mmol) to a mixed solution containing 3-fluoro-2-iodoaniline (6.70 g, 28.27 mmol) and N,N-dimethylformamide (60 mL), stir the reaction solution at 20°C for 1 hour, pour the reaction solution into water (300 mL) and extract with ethyl acetate (100 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography (silica gel, 0-30% gradient ethyl acetate/petroleum ether) to obtain compound 4-bromo-3-fluoro-2-iodoaniline (7.00 g, 78.7%) as a gray solid.
  • LCMS (ESI): [M+H] + 317.9.
  • Step 3 Add glycerol (5.60 g, 59.83 mmol) and 3-nitrobenzenesulfonic acid sodium salt (5.40 g, 23.93 mmol) to a solution containing concentrated sulfuric acid (20 mL) and water (15 mL), heat the mixture to 110°C, then slowly add 4-bromo-3-fluoro-2-iodoaniline (6.30 g, 19.94 mmol), and stir the resulting mixture at 130°C for 12 hours.
  • Step 4 Under nitrogen atmosphere, 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (336 mg, 0.57 mmol) and tris(dibenzylideneacetone)dipalladium (531 mg, 0.57 mmol) were added to a mixed solution containing 6-bromo-7-fluoro-8-iodoquinoline (2.00 g, 5.68 mmol), 4,4-difluoropiperidine hydrochloride (1.00 g, 6.25 mmol), cesium carbonate (5.70 g, 17.05 mmol) and dioxane (20 mL). The resulting mixture was stirred at 100° C.
  • Step 5 Under nitrogen atmosphere, 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (137 mg, 0.23 mmol) and tris(dibenzylideneacetone)dipalladium (217 mg, 0.23 mol) were added to a mixture containing 8-(4,4-difluoropropiperidinyl)-6-bromo-7-fluoroquinoline (800 mg, 2.32 mmol), tert-butyl carbamate (329 mg, 2.78 mmol), cesium carbonate (2.30 g, 6.95 mmol) and dioxane (10 mL), the resulting mixture was stirred at 100 ° C for 12 hours, the reaction solution was poured into water (30 mL) and extracted with ethyl acetate (30 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by column chromatography (silica gel, 0-30% gradient of e
  • Step 6 A mixed solution containing N-[8-(4,4-difluoropiperidinyl)-7-fluoro(6-quinolyl)](tert-butoxy)formamide (600 mg, 1.57 mmol) and trifluoroacetic acid (4 mL) was stirred at 25°C for 30 minutes. After the reaction solution was concentrated, a saturated sodium bicarbonate aqueous solution (30 mL) was added to adjust the pH to close to 8, and then extracted with ethyl acetate (30 mL x 3).
  • Step 7 2-(6-azaspiro[2.5]octan-6-yl)-4-bromobenzoyl chloride (631 mg, 1.92 mmol) was added to a mixed solution containing 8-(4,4-difluoropiperidinyl)-7-fluoro-6-quinolinamine (450 mg, 1.60 mmol), N,N-diisopropylethylamine (1.10 g, 8.00 mmol) and tetrahydrofuran (5 mL). The reaction solution was stirred at 25°C for 1 hour, poured into water (30 mL) and washed with ethyl acetate (30 mL x 10 mmol).
  • Step 8 Under a nitrogen atmosphere, 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (37 mg, 0.087 mmol) and tris(dibenzylideneacetone)dipalladium (81 mg, 0.087 mmol) were added to a mixed solution containing [2-(6-azaspiro[2.5]octan-6-yl)-4-bromophenyl]-N-[8-(4,4-difluoropiperidinyl)-7-fluoro(6-quinolyl)]formamide (500 mg, 0.87 mmol), 2-hydroxyethanesulfonamide (137 mg, 1.05 mmol), potassium phosphate (567 mg, 2.62 mmol) and dioxane (10 mL), and the reaction solution was stirred at 100°C for 12 hours.
  • Step 1 Dissolve N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)-2-hydroxyethane-1-sulfonamide (400 mg, 0.64 mmol) and (tert-butyloxycarbonyl)-L-valine (696 mg, 3.20 mmol) in tetrahydrofuran (8.0 mL), add N,N-diisopropylethylamine (9.92 mg, 0.05 mmol) and 3-nitro-1H-1,2,4-triazole (169 mg, 1.28 mmol), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (408 mg, 1.60 mmol), and stir at 25 °C for 16 hours.
  • Step 2 Dissolve 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)-L-valine ester (350 mg, 0.42 mmol) in dichloromethane (8.00 mL) and trifluoroacetic acid (1.6 mL) and stir at 25 ° C for 1 hour.
  • reaction solution was poured into a sodium bicarbonate aqueous solution (20.0 mL), extracted with ethyl acetate (50 mL x 2), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by preparative HPLC (C18, 10%-50% gradient of water (hydrochloric acid)/acetonitrile) and freeze-dried to give a white solid 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl L-valine ester hydrochloride (11.0 mg, 0.14 mmol, 3.42% yield).
  • Step 1 Dissolve N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)-2-hydroxyethane-1-sulfonamide (400 mg, 0.64 mmol) and tetrazole (220 mg, 3.21 mmol) in dichloromethane (8.00 mL), add di-tert-butyl diethylphosphoramide subsalt (711 mg, 2.56 mmol), and stir the mixture at 25 ° C for 1 hour.
  • Step 2 Dissolve di-tert-butyl (2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl)phosphate (200 mg, 0.24 mmol) in dichloromethane (4.00 mL) and trifluoroacetic acid (1.00 mL) and stir at 25 ° C for 1 hour. The reaction solution was dried and concentrated using a nitrogen stream.
  • Step 1 Dissolve N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)-2-hydroxyethane-1-sulfonamide (200 mg, 0.32 mmol) and dimethylglycine (165 mg, 1.6 mmol) in tetrahydrofuran (4.00 mL), add N,N-diisopropylethylamine (105 mg, 0.8 mmol) and 3-nitro-1H-1,2,4-triazole (91.4 mg, 0.8 mmol), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (204 mg, 0.8 mmol), and stir at 25°C for 2 hours.
  • reaction solution was diluted with water (10 mL), extracted with ethyl acetate (30 mL x 3), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by preparative HPLC (C18, 6%-46% gradient of water (hydrochloric acid)/acetonitrile) to give 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl dimethylglycinate hydrochloride (210 mg, 0.29 mmol, 92%) as a yellow solid.
  • Step 1 Dissolve N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)-2-hydroxyethane-1-sulfonamide (200 mg, 0.32 mmol) and (tert-butyloxycarbonyl)glycine (281 mg, 1.6 mmol) in tetrahydrofuran (4.00 mL), add N,N-diisopropylethylamine (105 mg, 0.8 mmol) and 3-nitro-1H-1,2,4-triazole (91.4 mg, 0.8 mmol), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (204 mg, 0.8 mmol), and stir at 25 °C for 2 hours.
  • Step 2 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)glycine ester (300 mg, 0.38 mmol) was dissolved in dichloromethane (5.00 mL) and trifluoroacetic acid (1.00 mL) and stirred at 25°C for 1 hour. The reaction solution was dried and concentrated using a nitrogen stream.
  • Step 1 Dissolve N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)-2-hydroxyethane-1-sulfonamide (220 mg, 0.35 mmol) and (tert-butyloxycarbonyl)histidine (333 mg, 1.76 mmol) in tetrahydrofuran (4.00 mL), add N,N-diisopropylethylamine (116 mg, 0.88 mmol) and 3-nitro-1H-1,2,4-triazole (100 mg, 0.88 mmol), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (224 mg, 0.88 mmol), and stir at 25 °C for 16 hours.
  • reaction solution was diluted with water (100 mL), extracted with ethyl acetate (100 mL x 3), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by flash column chromatography (silica gel, 0-52% gradient of tetrahydrofuran/petroleum ether) to obtain a yellow solid 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)histidine ester (330 mg, 0.35 mmol, crude).
  • Step 2 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)histidine ester (310 mg, 0.36 mmol) was dissolved in dichloromethane (6.00 mL) and trifluoroacetic acid (1.20 mL) and stirred at 25°C for 1 hour. The reaction solution was dried and concentrated using a nitrogen stream.
  • Step 1 Dissolve N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)-2-hydroxyethane-1-sulfonamide (220 mg, 0.35 mmol) and (tert-butyloxycarbonyl)alanine (333 mg, 1.76 mmol) in tetrahydrofuran (4.00 mL), add N,N-diisopropylethylamine (116 mg, 0.88 mmol) and 3-nitro-1H-1,2,4-triazole (100 mg, 0.88 mmol), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (224 mg, 0.88 mmol), and stir at 25 °C for 1 hour.
  • reaction solution was diluted with water (100 mL), extracted with ethyl acetate (100 mL x 3), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by flash column chromatography (silica gel, 0-26% gradient of tetrahydrofuran/petroleum ether) to obtain a yellow solid 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)alaninate (300 mg, 0.35 mmol, crude).
  • Step 2 Dissolve 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butoxycarbonyl)alaninate (280 mg, 0.35 mmol) in dichloromethane (5.00 mL) and trifluoroacetic acid (1.00 mL) and stir at 25°C for 1 hour. The reaction solution was dried and concentrated using a nitrogen stream.
  • Step 1 Dissolve (tert-butyloxycarbonyl)serine (5.00 g, 24.3 mmol) in N,N-dimethylformamide (50.0 mL), add imidazole (5.31 g, 77.9 mmol), stir at 25 ° C for 30 minutes, add tert-butyldimethylsilyl chloride (5.51 g, 36.5 mmol) dropwise at 0 ° C, and stir at 25 ° C for 16 hours.
  • the reaction solution was diluted with ethyl acetate (60.0 mL) and added to a stirred dilute hydrochloric acid aqueous solution (1.2 M) (200 mL), stirred for 30 minutes, and after separation, the aqueous phase was extracted with ethyl acetate (200 mL x 2), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by flash column chromatography (silica gel, 0-5% gradient tetrahydrofuran/petroleum ether) to obtain colorless oily N-(tert-butyloxycarbonyl)-O-(tert-butyldimethylsilyl)serine (3.70 g, 11.7 mmol, 48.0% yield).
  • Step 2 Dissolve N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)-2-hydroxyethane-1-sulfonamide (500 mg, 0.80 mmol) and N-(tert-butyloxycarbonyl)-O-(tert-butyldimethylsilyl)serine (1.28 g, 4.01 mmol) in tetrahydrofuran (10.0 mL), add N,N-diisopropylethylamine (264 mg, 2.00 mmol) and 3-nitro-1H-1,2,4-triazole (228 mg, 2.00 mmol), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (510 mg, 2.00 mmol), and stir at 25°C for 1 hour.
  • reaction solution was diluted with water (100 mL), extracted with ethyl acetate (100 mL x 3), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by flash column chromatography (silica gel, 0-28% gradient of tetrahydrofuran/petroleum ether) to give yellow 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl N-(tert-butyloxycarbonyl)-O-(tert-butyldimethylsilyl)serine ester (390 mg, 0.42 mmol, 53.0% yield) was obtained as a colored oil.
  • Step 3 Dissolve 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl N-(tert-butyloxycarbonyl)-O-(tert-butyldimethylsilyl)serine ester (390 mg, 0.42 mmol) in tetrahydrofuran (8.00 mL), add tetrabutylammonium fluoride (tetrahydrofuran solution) (0.63 mL, 0.63 mmol), and stir at 25 ° C for 2 hours.
  • reaction solution was quenched by adding saturated ammonium chloride solution (100 mL), extracted with ethyl acetate (100 mL x 2), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by flash column chromatography (silica gel, 0-32% gradient of tetrahydrofuran/petroleum ether) to give 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)serine ester (250 mg, 0.23 mmol, 54.0% yield) as a yellow oil.
  • Step 4 Dissolve 2-(N-(4-(1-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butoxycarbonyl)serine ester (250 mg, 0.31 mmol) in dioxane (2.00 mL), add dioxane hydrochloride (2.00 mL), and stir at 25 °C for 1 hour.
  • Step 1 Dissolve N-(8-(4,4-difluoropiperidin-1-yl)-7-fluoroquinolin-6-yl)-4-((2-hydroxyethyl)sulfonylamino)-2-(6-azaspiro[2.5]octan-6-yl)benzamide (500 mg, 0.81 mmol) and (tert-butyloxycarbonyl)-L-valine (879 mg, 4.05 mmol) in tetrahydrofuran (10.0 mL), add N,N-diisopropylethylamine (9.92 mg, 0.05 mmol) and 3-nitro-1H-1,2,4-triazole (230 mg, 2.02 mmol), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (515 mg, 2.02 mmol), and stir at 25°C for 16 hours.
  • reaction solution was diluted with water (50.0 mL), extracted with ethyl acetate (100 mL x 2), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by flash column chromatography (silica gel, 0-23% gradient of tetrahydrofuran/petroleum ether) to give 2-(N-(4-((8-(4,4-difluoropiperidin-1-yl)-7-fluoroquinolin-6-yl)aminocarbonyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)-L-valine ester (340 mg, 0.38 mmol, 47.0% yield) as a yellow solid.
  • Step 2 Dissolve 2-(N-(4-((8-(4,4-difluoropiperidin-1-yl)-7-fluoroquinolin-6-yl)aminocarbonyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)-L-valine ester (320 mg, 0.39 mmol) in dichloromethane (8.00 mL) and trifluoroacetic acid (1.60 mL) and stir at 25 ° C for 1 hour.
  • reaction solution was poured into sodium bicarbonate aqueous solution (50.0 mL), extracted with ethyl acetate (100 mL x 2), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by preparative HPLC (C18, 6%-46% gradient of water (formic acid)/acetonitrile) and freeze-dried to give 2-(N-(4-((8-(4,4-difluoropiperidin-1-yl)-7-fluoroquinolin-6-yl)aminocarbonyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl L-valine ester (104.92 mg, 0.14 mmol, 36.0% yield) as a white solid.
  • Step 1 Dissolve N-(4-(4-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-1-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)-2-hydroxyethane-1-sulfonamide (400 mg, 0.64 mmol) and (tert-butyloxycarbonyl)-L-valine (696 mg, 3.20 mmol) in tetrahydrofuran (8.0 mL), add N,N-diisopropylethylamine (9.92 mg, 0.05 mmol) and 3-nitro-1H-1,2,4-triazole (169 mg, 1.28 mmol), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (408 mg, 1.60 mmol), and stir at 25 °C for 2 hours.
  • Step 2 Dissolve 2-(N-(4-(4-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-1-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)-L-valine ester (200 mg, 0.24 mmol) in dichloromethane (4.00 mL) and trifluoroacetic acid (0.8 mL) and stir at 25 ° C for 2 hours.
  • reaction solution was poured into a sodium bicarbonate aqueous solution (20.0 mL), extracted with dichloromethane (50 mL x 2), and the organic phase was dried, filtered, and concentrated.
  • the residue was purified by preparative HPLC (C18, 6%-46% gradient of water (formic acid)/acetonitrile) and freeze-dried to give 2-(N-(4-(4-(8-(4,4-difluoropiperidin-1-yl)quinolin-6-yl)-1H-1,2,3-triazol-1-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl L-valine ester formate (39.0 mg, 0.09 mmol, 39.2% yield) as a white solid.
  • Step 1 Dissolve N-(4-(4-(2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)-2-hydroxyethane-1-sulfonamide (500 mg, 0.85 mmol) in dichloromethane (10.0 mL), add (tert-butyloxycarbonyl)-L-valine (184 mg, 0.85 mmol) and triethylamine (263 mg, 2.55 mmol) and 1-hydroxybenzotriazole (34.4 mg, 0.25 mmol), add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (244 mg, 1.27 mmol), and stir at 25 ° C for 16 hours.
  • Step 2 2-(N-(4-(4-(2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl(tert-butyloxycarbonyl)-L-valine ester (520 mg, 0.66 mmol) was dissolved in dichloromethane (10.0 mL) and trifluoroacetic acid (2.00 mL) and stirred at 25 ° C for 1 hour.
  • Human KIF18A (amino acid sequence 1-417) was purchased from Shanghai Via Biotechnology Co., Ltd.; ADP-Glo TM protein kinase kit was purchased from Promega, USA; tubulin was purchased from Cytoskeleton, USA; 384-well detection plate and multi-function microplate reader Envision were purchased from PerkinElmer, USA.
  • Enzyme activity detection Use DMSO to dissolve the compound powder into a 10mM storage solution; dilute the compound in a gradient in a microplate to a final concentration of 0-10 ⁇ M. Then add 2.5 ⁇ L of tubulin, compound, ATP, and KIF18A protein to the microplate in turn and react at room temperature for 120 minutes. The final concentration of the enzyme reaction is 60 ⁇ g/mL for tubulin, 25 ⁇ M for ATP, and 2.5nM for KIF18A protein. After the enzyme reaction is completed, add 10 ⁇ l of ADP-GLO reaction reagent to each well and incubate at room temperature for 30 minutes. After that, add 20 ⁇ l of detection reagent to each well and incubate at room temperature in the dark for 30 minutes. Finally, use PerkinElmer Envision for chemiluminescence detection.
  • OVCAR3 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • RPMI-1640 medium, fetal bovine serum, and CyQUANT Direct Cell Proliferation Assay kit were purchased from Thermo Fisher Scientific, USA;
  • 96-well cell culture plates were purchased from Corning, USA.
  • OVCAR3 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and placed in an incubator at 37°C and 5% CO 2. Cells in the logarithmic growth phase were used for the experiment.
  • OVCAR3 cells were inoculated in 96-well cell culture plates, 90 ⁇ l per well, and cultured overnight in a 37°C, 5% CO2 incubator.
  • Compound powder was dissolved into a 10 mM stock solution using DMSO; the compound was graded diluted in a microplate to a final concentration of 0-10 ⁇ M.
  • 10 ⁇ L of cell culture solution containing the compound was added to each well to a final DMSO content of 0.2%.
  • the cell plate was cultured in a 37°C, 5% CO2 incubator for 3 days. 100 ⁇ l of CyQUANT detection reagent was added to each well, reacted at 37°C for 60 minutes, and fluorescence detection was performed using PerkinElmer's Envision.
  • HT29 cells were purchased from Nanjing Kebai Biotechnology; RPMI-1640 culture medium was purchased from ThermoFisher (USA); fetal bovine serum purchased from ThermoFisher (USA); Trypsin-EDTA (0.25%) purchased from ThermoFisher (USA); DMSO purchased from SIGMA (USA); 96-well plate purchased from ThermoFisher (USA); CyQuant reagent purchased from ThermoFisher (USA).
  • HT29 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO 2. Cells in the logarithmic growth phase were used for the experiment.
  • CyQuant reagent was used to detect the inhibitory activity of compounds on HT29 cells. Adjust the cell density, inoculate 100 ⁇ l per well of a 96-well plate (2000/well for HT29), and culture overnight at 37°C and 5% CO2. Add compounds of each concentration (starting concentration 3000 nM, 3-fold dilution, 9 concentration gradients), and the DMSO content is 0.2%. The cell plate was incubated at 37°C and 5 % CO2 for 3 days. Add CyQuant reagent and incubate for 1 hour, read the plate with Envision, and calculate IC50 using XLFIT.

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Abstract

L'invention concerne un inhibiteur de KIF18 et son procédé de synthèse. L'invention concerne plus particulièrement un composé tel que représenté dans la formule (I). Le composé peut ajuster la protéine KIF18A, affectant ainsi le cycle cellulaire et le procédé de prolifération cellulaire pour traiter des cancers et des maladies associées au cancer. L'invention concerne en outre une composition pharmaceutique contenant le composé et une méthode de traitement d'états associés à l'activité de KIF18A.
PCT/CN2024/112662 2023-08-16 2024-08-16 Inhibiteur de kinésine kif18a et son utilisation Pending WO2025036479A1 (fr)

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WO2020132653A1 (fr) * 2018-12-20 2020-06-25 Amgen Inc. Amides d'hétéroaryle utiles en tant qu'inhibiteurs de kif18a
CN114302880A (zh) * 2019-08-02 2022-04-08 美国安进公司 Kif18a抑制剂
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CN114401953A (zh) * 2019-08-02 2022-04-26 美国安进公司 Kif18a抑制剂
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WO2024002328A1 (fr) * 2022-06-30 2024-01-04 勤浩医药（苏州）有限公司 Composé contenant de l'azote et son utilisation
WO2024039829A1 (fr) * 2022-08-18 2024-02-22 Accent Therapeutics, Inc. Inhibiteurs de kif18a et leurs utilisations
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WO2024078569A1 (fr) * 2022-10-13 2024-04-18 浙江海正药业股份有限公司 Dérivé d'amide aromatique, son procédé de préparation et son utilisation
WO2024099398A1 (fr) * 2022-11-10 2024-05-16 南京明德新药研发有限公司 Classe de composés sulfonamide contenant un hétérocycle ortho-condensé et leur utilisation
WO2024146593A1 (fr) * 2023-01-05 2024-07-11 浙江海正药业股份有限公司 Dérivé d'amide aromatique, son procédé de préparation et son utilisation médicale
WO2024149189A1 (fr) * 2023-01-09 2024-07-18 浙江海正药业股份有限公司 Dérivé d'amide aromatique, son procédé de préparation et son utilisation

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