WO2021249533A1 - Estrogen receptor modulator compound and use thereof - Google Patents

Estrogen receptor modulator compound and use thereof Download PDF

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Publication number
WO2021249533A1
WO2021249533A1 PCT/CN2021/099653 CN2021099653W WO2021249533A1 WO 2021249533 A1 WO2021249533 A1 WO 2021249533A1 CN 2021099653 W CN2021099653 W CN 2021099653W WO 2021249533 A1 WO2021249533 A1 WO 2021249533A1
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Prior art keywords
group
compound
acceptable salt
pharmaceutically acceptable
cycloalkyl
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PCT/CN2021/099653
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French (fr)
Chinese (zh)
Inventor
张雁
杨圣伟
庞司林
丁海敏
李刚
唐锋
吴清萍
郝元斌
樊静
孙钰菖
彭少平
唐任宏
任晋生
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江苏先声药业有限公司
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Priority to CN202180041694.1A priority Critical patent/CN115697987A/en
Publication of WO2021249533A1 publication Critical patent/WO2021249533A1/en

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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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
    • C07D217/14—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to an estrogen receptor modulator compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the compound, and its use in the prevention or treatment of estrogen receptor-related diseases.
  • Estrogen (E2) and estrogen alpha receptor (ER ⁇ ) are important driving factors for the development of breast cancer. More than two-thirds of breast cancer patients express ER transcription factors, and in most ER-positive patients, ER is still a key driving factor even in tumors that progress after early endocrine therapy. Therefore, ER is A major target for breast cancer treatment (Pharmacology&Therapeutics 186(2018)1-24).
  • the purpose of endocrine therapy is to reduce ER activity.
  • SERMs selective estrogen receptor modulators
  • tamoxifen tamoxifen
  • aromatase inhibitors (aromatase inhibitors, AIs), by inhibiting the conversion of androgens to estrogen, reduce the level of estrogen in the body; and selective estrogen receptor downregulators, such as fulvestrant (fulvestrant) ), not only inhibits its activity as an ER antagonist, but also has the effect of inducing ER protein degradation.
  • endocrine therapy is the first choice for patients with estrogen receptor-positive breast cancer, about 30% of patients will relapse after treatment, and almost all patients with metastatic breast cancer will develop resistance and progress. There are two main mechanisms for the development of drug resistance in endocrine therapy.
  • Fulvestrant is the first and only clinically approved selective estrogen receptor down-regulator for the treatment of ER-positive, metastatic breast cancer in postmenopausal patients after the progression of tamoxifen or aromatase inhibitors.
  • receptor downregulators SERDs
  • AstraZeneca see patent application WO2018077630A1
  • Genentech see patent application WO2019245974A1
  • AstraZeneca see patent application WO2018077630A1
  • Genentech see patent application WO2019245974A1
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
  • X 1 , X 2 , X 3 , and X 4 are independently selected from CR 7 or N;
  • R 7 is selected from H, F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclyl, C 1 -C 6 alkoxy Group, C 3 -C 6 cycloalkyloxy or 3-6 membered heterocyclyloxy;
  • Het is selected from
  • R 1 , R 2 , and R 9 are independently selected from hydrogen, OH, F, Cl, Br, I, C 1 -C 6 alkyl or C 2 -C 8 alkenyl;
  • R 1 , R 2 and the carbon atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, the C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group group is optionally substituted with R a;
  • R 5 is selected from C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, the C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected from one or more of the following Substitution of groups: deuterium, F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
  • R 6 is selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, said C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected by one or more Substitution from the following groups: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
  • R 3 is selected from H, CN, COOH, C(O)OR b , OC(O)R b , CONH 2 , C(O)NR b R c , SO 2 R b , SO 2 NR b R c , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, C 3 -C 6 heterocyclyl, C 6 -C 10 aryl or C 5 -C 10 heteroaryl,
  • the C 1 -C 6 alkyl group, C 3 -C 6 cycloalkyl group, C 3 -C 6 heterocyclic group, C 6 -C 10 aryl group or C 5 -C 10 heteroaryl group are optionally selected by 1 or Multiple R d substitutions;
  • R 4 is selected from H, F, Cl, Br, I, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 heterocyclyl ,
  • the C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group, C 3 -C 6 cycloalkyl group or C 3 -C 6 heterocyclic group is optionally selected from one or more groups as follows Replacement: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
  • Ring Q is selected from a C 6 -C 10 aryl group or a 5-10 membered heteroaryl group, the C 6 -C 10 aryl group or a 5-10 membered heteroaryl group is optionally substituted by one or more R 8 ;
  • R 8 is selected from F, Cl, Br, I, OH, CN, COOH, C(O)OR b , OC(O)R b , CONH 2 , C(O)NR b R c , NR c C(O) R b , SO 2 R b , SO 2 NR b , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclic group, C 6- C 10 aryl group or 5-10 membered heteroaryl group, the C 3 -C 6 cycloalkyl group, 3-6 membered heterocyclic group, C 6 -C 10 aryl group or 5-10 membered heteroaryl group is optionally Substitution with one or more groups selected from the following: F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy;
  • R a is selected from halo, OH, CN, C 1-6 alkyl, C 1-6 alkoxy, C 3 - 6 cycloalkyl or 3-6 membered heterocyclyl, a C 1-6 alkyl group, C 1-6 alkoxy, C 3 - 6 cycloalkyl or 3-6 membered heterocyclyl group optionally substituted by halogen;
  • R b is independently selected from C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclyl, C 6 -C 10 aryl or 5-10 membered heteroaryl;
  • R c is independently selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclic group, C 6 -C 10 aryl or 5-10 membered heteroaryl;
  • R d is independently selected from F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or 3-6 membered heterocyclic group ,
  • the C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group, C 3 -C 6 cycloalkyl group or 3-6 membered heterocyclic group is optionally selected from one or more of F, Cl, Group substitution of Br, I or OH;
  • n 0, 1 or 2;
  • n 1, 2, 3 or 4;
  • p 1 or 2;
  • the conditions are: (1) when n is 0 or 1, R 1 is not H, F or methyl; (2) when m is 1 or 2, R 9 is not H.
  • R 5 is selected from C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, or 3-6 membered heterocycloalkane Group, the C 1 -C 6 alkyl group, C 2 -C 8 alkenyl group, C 2 -C 4 alkynyl group, C 3 -C 6 cycloalkyl group or 3-6 membered heterocycloalkyl group is optionally selected by one Or multiple groups selected from: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 .
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (Ia) or a pharmaceutically acceptable salt thereof:
  • the Het is selected from
  • the Het is selected from Wherein R 1 , R 2 and the carbon atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, the C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group R a is optionally substituted.
  • Het is selected from Wherein R 1 , R 2 and the carbon atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, the C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group optionally substituted with R a; n is the 1.
  • the Het is selected from Among them, R 1 , R 2 and the carbon atom to which they are connected together form cyclopropane.
  • the Het is selected from Wherein one of R 1 or R 2 forms a 3-6 membered heterocyclic group together with R 6 and C and N respectively connected.
  • the Het is selected from Wherein one of R 1 or R 2 forms a 3-6 membered heterocyclic group together with R 6 and the C and N respectively connected; the n is 1.
  • Het is selected from Wherein one of R 1 or R 2 forms a piperidine ring together with R 6 and C and N respectively connected.
  • the Het is selected from
  • the R 9 is selected from hydrogen, OH, F, Cl, Br, I, or C 1 -C 6 alkyl.
  • the R 9 is selected from hydrogen or CH 3 .
  • the m is selected from 1.
  • the m is selected from 3.
  • the m is selected from 4.
  • the Het is selected from
  • the p is 1.
  • the Het is selected from
  • the Het is selected from
  • the Het is selected from
  • the R 3 is selected from C 1 -C 6 alkyl, CN, COOH, C(O)OR b , OC(O)R b , CONH 2 , C(O)NR b R c or C 6 -C 10 aryl group; the C 1 -C 6 alkyl group or C 6 -C 10 aryl group is optionally substituted by one or more R d , and R d is independently selected from F, Cl, Br, I, OH , CN or C 1 -C 6 alkyl optionally substituted with one or more groups selected from F, Cl, Br, I or OH.
  • the R 3 is selected from C 1 -C 6 alkyl or C 6 -C 10 aryl; the C 1 -C 6 alkyl or C 6 -C 10 aryl is optionally selected by one or Multiple R d substitutions, R d is independently selected from F, Cl, Br, OH or a C 1 -C 6 alkyl group optionally substituted with one or more groups selected from F, Cl, Br or OH.
  • the R 3 is selected from C 1 -C 3 alkyl or phenyl; the C 1 -C 3 alkyl or phenyl is optionally substituted by one or more R d , and R d is independently selected From F, OH or trifluoromethyl.
  • the R 3 is selected from 4-trifluoromethylphenyl, -CH 2 CF 3 ,
  • the R 4 is selected from C 1 -C 6 alkyl optionally substituted by F, Cl, Br, or I.
  • the R 4 is methyl
  • the R 5 is a C 1 -C 6 alkyl group optionally substituted with deuterium, F, Cl, Br, or I.
  • the R 5 is a C 1 -C 6 alkyl optionally substituted with F, Cl, Br, or I.
  • the R 5 is 1,1-dideutero-3-fluoropropyl, 3-fluoropropyl, or 2-fluoroethyl.
  • the R 5 is 3-fluoropropyl or 2-fluoroethyl.
  • X 1 is selected from CR 7 or N, and X 2 , X 3 , and X 4 are all CR 7 .
  • the X 1 is selected from CH or N, and X 2 , X 3 , and X 4 are all CH.
  • the R 7 is selected from H, F, Cl, Br, or I.
  • the R 6 is selected from H or C 1 -C 6 alkyl.
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (II) or a pharmaceutically acceptable salt thereof:
  • Y is selected from NR 10 , O or S;
  • R 10 is selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, said C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected by one or more Substitution from the following groups: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 .
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IIa) or a pharmaceutically acceptable salt thereof:
  • Y is selected from NR 10 , O or S;
  • R 10 is selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, said C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected by one or more Substitution from the following groups: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 .
  • the Y is selected from NR 10 .
  • R 10 is selected from H, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, or 3-6 membered heterocycloalkyl.
  • R 10 is H.
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (III) or a pharmaceutically acceptable salt thereof:
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IIIa) or a pharmaceutically acceptable salt thereof:
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IV) or a pharmaceutically acceptable salt thereof:
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IVa) or a pharmaceutically acceptable salt thereof:
  • the R 8 in the compound of formula (IV) is selected from a C 6 -C 10 aryl group or a 5-10 membered heteroaryl group, the C 6 -C 10 aryl group or a 5-10 membered heteroaryl group
  • the aryl group is optionally substituted by one or more groups selected from the group consisting of F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy.
  • the R 8 in the compound of formula (IV) is selected from a phenyl group or a 5-6 membered heteroaryl group, and the phenyl group or a 5-6 membered heteroaryl group is optionally substituted by one or more Substitution selected from the following groups: F, Cl, Br, I, OH, C 1 -C 3 alkyl, C 1 -C 3 alkoxy.
  • the R 8 in the compound of formula (IV) is selected from pyrazole, and the pyrazole is optionally substituted with one or more methyl groups.
  • the R 8 in the compound of formula (IV) is selected from 1H-pyrazol-4-yl or 1-methyl-1H-pyrazol-4-yl.
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the compounds described in this application exist as racemic mixtures or in enantiomerically enriched or enantiomerically pure forms.
  • the compounds described in this application are prepared as their individual stereoisomers by reacting a racemic mixture of the compounds with an optically active resolving agent to form a pair of diastereomers The structure compound/salt separates the diastereomers and recovers the optically pure enantiomers.
  • the resolution of enantiomers is carried out using covalent diastereomeric derivatives of the compounds described in this application.
  • the diastereomers are separated by separation/resolution techniques based on differences in solubility.
  • the compounds described in this application are prepared into their individual stereoisomers by enzymatic resolution.
  • the resolution of individual stereoisomers is performed using lipase or esterase.
  • the resolution of individual stereoisomers is carried out by asymmetric deacylation catalyzed by lipase or esterase.
  • the separation of stereoisomers is carried out by chromatography, or by forming diastereomeric salts and separating by recrystallization or chromatography or any combination thereof.
  • stereoisomers are obtained by stereoselective synthesis.
  • the present invention also provides a pharmaceutical composition, which comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition is formulated for intravenous injection, subcutaneous injection, oral administration, or topical administration.
  • the pharmaceutical composition is a tablet, pill, capsule, liquid, suspension, gel, dispersion, solution, emulsion, ointment, or lotion.
  • the present invention relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutical composition thereof in the preparation of a medicine for preventing or treating estrogen receptor-related diseases.
  • the present invention relates to a compound represented by formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof, for the prevention or treatment of estrogen receptor related diseases.
  • the present invention relates to the use of the compound represented by formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the prevention or treatment of estrogen receptor-related diseases.
  • the present invention relates to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for preventing or treating estrogen receptor-related diseases.
  • the present invention also relates to a method for preventing or treating estrogen receptor-related diseases, the method comprising administering to a subject in need thereof a preventive or therapeutically effective dose of the compound represented by formula (I) of the present invention or its pharmaceutically Acceptable salt, or pharmaceutical composition thereof.
  • the estrogen receptor-related diseases include but are not limited to cancer and autoimmune diseases.
  • the estrogen receptor-related disease is preferably a tumor.
  • tautomer refers to an isomer of a functional group resulting from the rapid movement of an atom in two positions in a molecule.
  • the compounds of the present invention may exhibit tautomerism.
  • Tautomeric compounds can exist in two or more mutually convertible species.
  • Tautomers generally exist in an equilibrium form. An attempt to separate a single tautomer usually produces a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties of the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone type is dominant; in phenol, the enol type is dominant.
  • the present invention encompasses all tautomeric forms of the compound.
  • stereoisomer refers to the isomers produced by the different arrangements of atoms in the molecule in space, including cis and trans isomers, enantiomers, diastereomers and conformational isomers.
  • the compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so the compounds of the present application may exist in specific geometric or stereoisomeric forms.
  • Specific geometric or stereoisomeric forms can be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S )-Enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as enantiomers or diastereomers Enriched mixtures, all of the above isomers and their mixtures fall within the scope of the definition of compounds in this application.
  • the asymmetric atom-containing compound of this application can be separated in an optically active pure form or a racemic form.
  • the optically active pure form can be separated from the racemic mixture, or synthesized by using chiral raw materials or chiral reagents. .
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence of the specific atom is normal and the substituted compound is stable.
  • it means that two hydrogen atoms are replaced, and the oxo will not occur on the aromatic group.
  • the term “optional” or “optionally” means that the event or situation described later can occur or not occur, and the description includes occurrence of said event or situation and non-occurrence of said event or situation.
  • the ethyl group is "optionally" substituted by halogen, meaning that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (such as CH 2 CH 2 F), or polysubstituted (such as CHFCH 2 F, CH 2 CHF 2 etc.) or completely substituted (CF 2 CF 3 ).
  • CH 2 CH 3 unsubstituted
  • monosubstituted such as CH 2 CH 2 F
  • polysubstituted such as CHFCH 2 F, CH 2 CHF 2 etc.
  • CF 2 CF 3 completely substituted
  • C mn in this article means that the part has an integer number of carbon atoms in a given range.
  • C 1-6 means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
  • any variable such as R
  • its definition in each case is independent. For example, if a group is replaced by 2 Rs, each R has independent options.
  • linking group When the number of a linking group is 0, such as -(CH 2 ) 0 -, it means that the linking group is a bond.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues, but not Many toxicity, irritation, allergic reactions or other problems or complications are commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salts refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, and organic acids and bases.
  • pharmaceutically acceptable salts for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. can be mentioned. .
  • solvate refers to an association or complex of one or more solvent molecules with the compound of the present application.
  • solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate (EtOAc), acetic acid (AcOH), and ethanolamine.
  • pharmaceutical composition means a mixture of one or more of the compounds described in the text or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable carriers And excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present application to the organism.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence of the specific atom is normal and the substituted compound is stable.
  • it means that two hydrogen atoms are replaced, and the oxo will not occur on the aromatic group.
  • the term “optional” or “optionally” means that the event or situation described later can occur or not occur, and the description includes occurrence of said event or situation and non-occurrence of said event or situation.
  • the ethyl group is "optionally" substituted by halogen, meaning that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (such as CH 2 CH 2 F), or polysubstituted (such as CHFCH 2 F, CH 2 CHF 2 etc.) or completely substituted (CF 2 CF 3 ).
  • CH 2 CH 3 unsubstituted
  • monosubstituted such as CH 2 CH 2 F
  • polysubstituted such as CHFCH 2 F, CH 2 CHF 2 etc.
  • CF 2 CF 3 completely substituted
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • hydroxyl refers to the -OH group.
  • cyano refers to the -CN group.
  • mercapto refers to the -SH group.
  • amino refers to the -NH 2 group.
  • nitro refers to the -NO 2 group.
  • alkyl refers to a hydrocarbon group of the general formula C n H 2n+1.
  • the alkyl group may be linear or branched.
  • C 1 -C 6 alkyl should be understood to mean a linear or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms.
  • the alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl Group, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.
  • the alkyl portion (ie, alkyl) of the alkoxy group has the same definition as above.
  • alkenyl refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond.
  • alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, and the like.
  • C 2 -C 8 alkenyl should be understood to mean a linear or branched monovalent hydrocarbon group containing one or more double bonds and having 2, 3, 4, 5, 6, 7, 8 carbon atoms .
  • alkynyl refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one triple bond.
  • alkynyl groups include, but are not limited to, ethynyl (-C ⁇ CH), 1-propynyl (-C ⁇ C-CH 3 ), 2-propynyl (-CH 2 -C ⁇ CH), 1,3-Butadiynyl (-C ⁇ CC ⁇ CH) and so on.
  • C 2 -C 4 alkynyl should be understood to mean a linear or branched monovalent hydrocarbon group containing one or more triple bonds and having 2, 3, or 4 carbon atoms.
  • alkoxy refers to -O-alkyl.
  • suitable alkoxy groups are C 1-6 alkoxy groups, such as C 1-5 alkoxy groups, C 1-4 alkoxy groups, C 1-3 alkoxy groups, including methoxy and ethoxy. Group, propoxy, isopropoxy, isobutoxy, sec-butoxy, etc.
  • C 1 -C 6 alkoxy can be understood as “C 1 -C 6 alkyloxy” or "C 1 -C 6 alkyl-O-", such as "C 1 -C 6 alkoxy"" May include "C 1 -C 3 alkoxy".
  • cycloalkyl refers to a carbocyclic ring that is fully saturated and may exist as a single ring, a bridged ring, or a spiro ring. Unless otherwise indicated, the carbocyclic ring is usually a 3 to 10 membered ring.
  • Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantane Alkyl and so on.
  • C 3 -C 6 cycloalkyl should be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring, which has 3 to 6 carbon atoms.
  • cycloalkyloxy can be understood as “cycloalkyl-O-".
  • C 3 - 6 cycloalkyl group may be understood as “C 3 - 6 cycloalkyl group -O-.”
  • the heterocyclic ring is generally a 3 to 7 membered ring containing 1 to 3 heteroatoms (such as 1 or 2 heteroatoms) independently selected from sulfur, oxygen, and/or nitrogen.
  • heterocyclic groups include, but are not limited to, oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl , Pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl, etc.
  • the "3-6 membered heterocyclic group" in the term “3-6 membered heterocyclic group” or “3-6 membered heterocyclyloxy group” means a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring, It contains 1, 2 or 3 heteroatoms selected from N, O and S.
  • the heterocyclic group may include, but is not limited to: a 3-membered ring, such as an oxirane ring; a 4-membered ring, such as azetidinyl and oxetanyl; and a 5-membered ring, such as tetrahydrofuran.
  • the heterocyclic group is non-aromatic as a whole.
  • the "3-6 membered heterocyclic group” may include a “5-6 membered heterocyclic group”
  • the "3-6 membered heterocyclic group” may include a "5-6 membered heterocyclyloxy group”.
  • heterocycloalkyl refers to a monovalent cyclic group that is fully saturated and may exist as a monocyclic ring, a fused ring, a bridged ring, or a spiro ring. Unless otherwise indicated, the heterocyclic ring is generally a 3 to 7 membered ring containing 1, 2, or 3 heteroatoms (such as 1 or 2 heteroatoms) independently selected from sulfur, oxygen, and/or nitrogen.
  • 3-membered heterocycloalkyl groups include, but are not limited to, oxirane, sulfidene, and azaethylenyl groups
  • 4-membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetane
  • Examples of cyclic group, thibutyryl, and 5-membered heterocycloalkyl include but are not limited to tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidine
  • 6-membered heterocycloalkyl groups include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, Examples of 4-thiaxanyl, 1,4-dio
  • 3-6 membered heterocycloalkyl or “3-6 membered heterocycloalkyloxy” in the term “3-6 membered heterocycloalkyl” refers to all those with 3, 4, 5 or 6 ring atoms. ⁇ heterocycloalkyl.
  • the "3-6 membered heterocycloalkyl group” may include a "5-6 membered heterocycloalkyl group”
  • the "3-6 membered heterocycloalkyloxy group” may include a "5-6 membered heterocycloalkyloxy group”.
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic ring group with a conjugated ⁇ -electron system.
  • aryl groups can have 6-20 carbon atoms, 6-14 carbon atoms, or 6-12 carbon atoms.
  • Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, 1,2,3,4-tetralin and the like.
  • C 6 -C 10 aryl should be understood to mean a monovalent or partially aromatic monocyclic or bicyclic hydrocarbon ring having 6 to 10 carbon atoms.
  • C 6 aryl a ring having 6 carbon atoms
  • C 9 aryl a ring having 9 carbon atoms
  • 10 aryl a ring having three carbon atoms
  • heteroaryl refers to a monocyclic or condensed polycyclic ring system, which contains at least one ring atom selected from N, O, and S, the remaining ring atoms are C, and have at least one aromatic ring.
  • exemplary heteroaryl groups have a single 4 to 8 membered ring, especially a single 5 to 8 membered ring, or multiple fused rings containing 6 to 14, especially 6 to 10 ring atoms.
  • heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl , Tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.
  • the term "5-10 membered heteroaryl” should be understood to include monovalent monocyclic or bicyclic aromatic ring systems having 5-10 ring atoms and containing 1-5 independently selected from N, O and S Heteroatom.
  • 5-10 membered heteroaryl should be understood to include monovalent monocyclic or bicyclic aromatic ring systems having 5, 6, 7, 8, 9 or 10 ring atoms, especially 5 or 6 or 9 or 10 ring atoms, and it contains 1-5 or 1-3 heteroatoms independently selected from N, O, and S and, in addition, may be benzo-fused in each case.
  • the heteroaryl group is selected from the group consisting of thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 1, 2, 3 -Triazolyl, 1,2,4-triazolyl, thiadiazolyl, etc.
  • cinnoline Group phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pterridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, etc.
  • 5-6 membered heteroaryl refers to an aromatic ring system having 5 or 6 ring atoms, and it contains 1-3 or 1-2 heteroatoms independently selected from N, O and S, examples include But not limited to thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridine Group, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl.
  • the "5-10 membered heteroaryl group” may include a "5-6 membered heteroaryl group”.
  • terapéuticaally effective amount means (i) treatment or prevention of a specific disease, condition or disorder, (ii) reduction, amelioration or elimination of one or more symptoms of a specific disease, condition or disorder, or (iii) prevention or delay
  • the amount of the compound of the present invention that constitutes a “therapeutically effective amount” varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but it can be routinely determined by those skilled in the art. Determined by its own knowledge and the content of this disclosure.
  • excipients refers to pharmaceutically acceptable inert ingredients.
  • examples of types of the term “excipient” include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of the pharmaceutical preparation, that is, make the preparation more suitable for direct compression by increasing fluidity and/or adhesion.
  • examples of typical "pharmaceutically acceptable carriers” suitable for the above formulations are: sugars, starches, cellulose and its derivatives and other auxiliary materials commonly used in pharmaceutical formulations.
  • the present application also includes compounds of the present application that are the same as those described herein, but have one or more atoms replaced by an isotope-labeled atom having an atomic weight or mass number different from those generally found in nature.
  • isotopes that can be incorporated into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.
  • isotope-labeled compounds of the application can be used in compound and/or substrate tissue distribution analysis. Tritiated (i.e. 3 H) and carbon-14 (i.e. 14 C) isotopes are especially preferred due to their ease of preparation and detectability. Positron emission isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy.
  • PET positron emission tomography
  • the isotope-labeled compound of the present application can generally be prepared by the following procedures similar to those disclosed in the schemes and/or examples below, by replacing the non-isotopically-labeled reagent with an isotope-labeled reagent.
  • the pharmaceutical composition of the present application can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable excipients, for example, can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, and powders. , Granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.
  • Typical routes for administering the compound of the application or a pharmaceutically acceptable salt or pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, Intramuscular, subcutaneous, and intravenous administration.
  • the pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, emulsification methods, freeze-drying methods, and the like.
  • the pharmaceutical composition is in an oral form.
  • the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These auxiliary materials enable the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.
  • the solid oral composition can be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain tablets Or the core of the dragee.
  • suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents.
  • the pharmaceutical composition may also be suitable for parenteral administration, such as a sterile solution, suspension or lyophilized product in a suitable unit dosage form.
  • the daily dose is 0.01 to 200 mg/kg body weight, preferably 0.05 to 50 mg/kg body weight, more preferably 0.1 to 30 mg/kg body weight, either alone or The form of divided doses.
  • IC 50 is the half maximal inhibitory concentration, represents the concentration of a particular compound to obtain 50% inhibition in vitro biological process required.
  • the IC 50 value can be logarithmically converted into a pIC 50 value (-log IC 50 ), where a higher value indicates greater efficacy in an exponential manner.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • the unit of NMR shift is 10 -6 (ppm).
  • the solvent for NMR measurement is deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS).
  • TMS tetramethylsilane
  • IC 50 refers to the half maximal inhibitory concentration, refers to the maximum concentration at which half of the inhibitory effect.
  • Example 1 3-(((1R,3R)-1-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4] Heptane-7-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2, 2-Difluoropropane-1-ol
  • Step 1 Synthesis of tert-butyl (S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)carbamate
  • Step 3 3-(((1R,3R)-1-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hepta (Alk-7-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2 -Synthesis of difluoropropane-1-ol
  • Example 340 3-((1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-1,3,4,9-tetrahydro-2H- Pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol.
  • reaction solution was concentrated and diluted with 100mL of water. Adjust the pH to 8-9 with saturated sodium bicarbonate solution, extract with ethyl acetate, combine the organic layers, dry with anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography to obtain 3-((1R,3R)-1-(5-bromo -Pyridin-2-yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane -1-ol.
  • step 3 of Example 1 except that 3-((1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3 in Example 1 -Methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane-1-ol replaced with 3-( (1R,3R)-1-(5-Bromo-pyridin-2-yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole- 2-yl)-2,2-difluoropropane-1-ol, the title compound was prepared in the same way.
  • the preparation method is similar to that of Example 3, except that the starting material (R)-3-(((1-(1H-indol-3-yl)propan-2-yl)amino)-2 , 2-Difluoropropane-1-ol is replaced with (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine, the same Method to obtain the title compound.
  • Step 1 (7S)-N-(4-((6S,8R)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)- 8-Methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline-6- Yl)-3,5-difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane 7 amine
  • Step 2 3-((6S,8R)-6-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hepta- 7-yl)amino)phenyl)-8-methyl-3,6,8,9-tetrahydro-7H-pyrazolo[4,3-f]isoquinolin-7-yl)-2,2 -Synthesis of difluoropropane-1-ol
  • the starting material (6S, 8R)-6-(5-bromopyridin-2-yl)-7-(3-((tert-butyldiphenylsilyl)oxy) was prepared according to the preparation method of Example 16 of WO2018077630A1 -2,2-Difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4 ,3-f] isoquinoline.
  • Example 7 (S)-5-(3-fluoropropyl)-N-(6-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)- 6,7,8,9-Tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)pyridin-3-yl)-5-azaspiro[2.4]hept-7- amine
  • the starting material (6S, 8R)-6-(5-bromopyridin-2-yl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl) was prepared according to the preparation method of WO2018077630A1
  • Example 17 -7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline.
  • Example 8 (S)-N-(3,5-difluoro-4-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7 ,8,9-Tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4] Heptane-7-amine
  • the reaction solution was spin-dried, 100 mL of dichloromethane and dilute hydrochloric acid solution (2N, 100 mL) were added, and the reaction was completed after vigorous stirring for 2 hours. The layers were separated, the dichloromethane layer was discarded, and the aqueous layer was adjusted to pH with 5N aqueous sodium hydroxide solution. 10-11, extract with ethyl acetate, combine the organic layers, dry with anhydrous sodium sulfate, and concentrate the organic layer to obtain 12.0 g of crude oil.
  • Step 2 (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(2,2-difluoropropyl)-3,5-dimethyl-1,2 Synthesis of ,3,4-Tetrahydroisoquinoline-6-amine
  • Step 5 (S)-N-(4-((6S,8R)-7-(2,2-difluoropropyl)-8-methyl-6,7,8,9-tetrahydro-3H- Pyrazolo[4,3-f]isoquinolin-6-yl)-3,5-difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7 -Synthesis of amines
  • Step 2 Synthesis of (S)-2,6-difluoro-4-((5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-yl)amino)benzaldehyde
  • Step 3 (1S,3R)-1-(2,6-Difluoro-4-((S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7- Synthesis of (yl)amino)phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol
  • Step 4 (1S,3R)-1-(2,6-difluoro-4-((S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7- (Amino)phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-yltrifluoromethanesulfonic acid Synthesis of esters
  • Step 5 (S)-N-(3,5-Difluoro-4-((1S,3R)-3-methyl-6-(1H-pyrazol-4-yl)-2-(2,2 ,2-Trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro ring [2.4] Synthesis of heptane-7-amine
  • Example 11 3-((1R,3R)-1-(2,6-difluoro-4-((4aR,7aR)-6-(3-fluoropropyl)octahydro-1H-pyrrolo[3 ,4-b]pyridin-1-yl)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)- 2,2-Difluoropropane-1-ol
  • the preparation method is similar to Step 3 of Example 1, except that (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine in Step 3 of Example 1 is replaced with (4aR, 7aR)-6-(3-fluoropropyl)octahydro-1H-pyrrolo[3,4-b]pyridine, the title compound was obtained in the same way.
  • Example 12 (S)-5-(3-fluoropropyl)-N-(6-((1S,3R)-3-methyl-6-(1H-pyrazol-4-yl)-2- (2,2,2-Trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)pyridin-3-yl)-5-azaspiro[2.4]heptane-7- amine
  • the preparation method is similar to that of Example 10, except that the starting material 5-bromo-2-(diethoxymethyl)-1,3-difluorobenzene in step 2 of Example 10 is replaced with 5-bromo- 2-(Diethoxymethyl)-pyridine, followed by step 2 and step 5 in the same way to obtain the title compound.
  • Step 3 (S)-N-(3,5-Difluoro-4-((1S,3R)-3-methyl-6-(1-methyl-1H-pyrazol-4-yl)-2 -(4-(Trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-aza Synthesis of Spiro[2.4]heptane-7-amine
  • the preparation method is similar to that in Example 10, except that (R)-3-(2-(((2,2,2-trifluoroethyl)amino)propyl)phenol in step 3 of Example 10 is replaced with (R)-3-(2-(((4-(Trifluoromethyl)phenyl)amino)propyl)phenol, and then proceed step 2 to step 5 in the same way to obtain the title compound.
  • Example 14 3-((1R,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)piperidin-3-yl)amino)benzene Yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane-1-ol
  • the preparation method is similar to Step 3 of Example 1, except that (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine in Step 3 of Example 1 is replaced with (S)-1-(3-Fluoropropyl)piperidin-3-amine to obtain the title compound.
  • Example 15 3-((1R,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)azacycloheptan-3-yl)amino )Phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane-1- alcohol
  • Example 2 The preparation method is similar to that in Example 1, except that (S)-(5-azaspiro[2.4]heptane-7-yl) t-butyl carbamate in step 1 of Example 1 is replaced with (S)- For tert-butyl azepan-3-yl carbamate, the reaction of step 1 to step 3 of Example 1 was carried out in the same manner to obtain the title compound.
  • Example 16 N-(3,5-Difluoro-4-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9 -Tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)phenyl)-1-(3-fluoropropyl)-3-methylazetidine-3- amine
  • step 1 and step 2 of Example 1 Refer to the method of step 1 and step 2 of Example 1, except that (S)-(5-azaspiro[2.4]heptane-7-yl) t-butyl carbamate in step 1 of Example 1 was replaced with 3-(Boc-amino)-3-methylazetidine, 1-(3-fluoropropyl)-3-amino-3-methylacridine was prepared in the same way.
  • Step 2 N-(3,5-Difluoro-4-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9- Tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)phenyl)-1-(3-fluoropropyl)-3-methylazetidine-3-amine Synthesis
  • Example 17 3-(((1R,3R)-1-(2,6-difluoro-4-((((1R,4R,5S)-2-(3-fluoropropyl)-2-nitrogen Heterobicyclo[2.1.1]hex-5-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2 -Base)-2,2-difluoropropane-1-ol
  • the preparation method is similar to that in Example 1, except that (7S)-5-(benzyl)-5-azaspiro[2.4]heptane-7-ylcarbamate in step 1 of Example 1 Replaced with ((1R, 4R, 5S)-2-azabicyclo[2.1.1] hexyl-5-yl) benzyl carbamate to prepare intermediate ((1R, 4R, 5S)-2-(3- Fluoropropyl)-2-azabicyclo[2.1.1]hexyl-5-yl) benzyl carbamate.
  • Step 3 of Example 1 except that (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine in Step 3 of Example 1 was replaced with (1R , 4R, 5S)-2-(3-fluoropropyl)-2-azabicyclo[2.1.1]hexyl-5-amine, the title compound was prepared in the same way.
  • Example 18 (S)-5-(3-fluoropropyl-1,1-d2)-N-(6-((1S,3R)-3-methyl-2-(2,2,2- Trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-yl)-5-azaspiro[2.4] Hept-7-amine
  • Step 1 Synthesis of tert-butyl (S)-(5-(3-fluoropropionyl)-5-azaspiro[2.4]heptane-7-yl)carbamate
  • Step 4 (S)-5-(3-fluoropropyl-1,1-d2)-N-(6-((1S,3R)-3-methyl-2-(2,2,2-tri Fluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-yl)-5-azaspiro[2.4]hepta Synthesis of -7-amine
  • Test Example 1 Detection of the degradation effect of the compound of the present invention on the estrogen receptor in MCF7 cells
  • Test principle Determine the degradation activity of the compound of the present invention on the endogenously expressed estrogen receptor in MCF7 cells, and evaluate the activity of the test compound based on the IC 50 and the maximum degradation efficiency.
  • MCF7 cells (purchased from ATCC, HTB-22) were cultured with DMEM (purchased from Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 384-well plate at a density of 3000 cells/well using complete medium, and cultured in a cell incubator at 37°C and 5% CO 2. The test compound was dissolved in DMSO at a storage concentration of 10 mM, diluted with Echo 550 (purchased from Labcyte Inc.) and added to the cell culture plate.
  • DMEM purchased from Gibco, 11995-065
  • the initial concentration of each compound treatment was 100 nM, 3-fold dilution, 9 concentration points, Set a blank control containing 0.5% DMSO, and set up a double-well control for each concentration point. Incubate for 24 hours in a 37°C, 5% CO 2 cell incubator. Add paraformaldehyde to each cell culture well to fix the cells at a final concentration of about 3.7%.
  • test compound has a good degradation activity on the ER level based on the cell level.
  • corresponding ER level activity test results of the test compounds are shown in Table 1.
  • Example 8 0.07 Example 9 0.08 Example 10 0.15 Example 11 0.21 Example 12 0.23 Example 14 0.11 Example 16 0.06 Example 17 0.15
  • Test Example 2 Detection of the inhibitory effect of the compound of the present invention on the proliferation of T47D cells
  • Test principle Determine the inhibitory effect of the compound of the present invention on the proliferation of T47D cells in vitro, and evaluate the activity of the compound based on the IC 50 and the maximum inhibitory efficiency.
  • T47D (T-47D) cells (purchased from ATCC, HTB-133) were cultured with RPMI-1640 (purchased from Gibco, A10491-01) complete medium containing 10% fetal bovine serum.
  • RPMI-1640 purchased from Gibco, A10491-01
  • T47D cells were seeded in a 384-well plate at a density of 500 cells/well in a complete medium at 37° C., and cultured overnight in a 5% CO 2 cell incubator.
  • the initial concentration of each compound in the cell It is 100nM, 3-fold gradient dilution, 10 concentration points, set a blank control containing 0.3% DMSO, and each concentration point is set up as a double-well control. Cultivate in a cell incubator at 37°C and 5% CO 2 for 7 days, and take out the cell culture plate on the eighth day. join in Luminescent Cell Viability Assay (purchased from Promega, G7573), placed at room temperature for 10 minutes, use a multi-label microplate reader EnVision (purchased from PerkinElmer) to read the luminescence signal value, and use XLfit to calculate the luminescence signal value of each compound according to the compound concentration and luminescence signal value. inhibitory activity IC 50.
  • test compound showed good inhibitory activity on T47D breast cancer cells.
  • results of the inhibitory activity of the test compounds on the proliferation of T47D cells are shown in Table 2.
  • Test compound T47D IC 50 (nM) Example 1 0.28 Example 2 0.44 Example 4 0.27 Example 5 0.04 Example 6 0.42 Example 7 0.57 Example 8 0.31 Example 9 0.42 Example 10 0.42 Example 11 0.21 Example 14 0.44 Example 16 0.21 Example 17 0.30
  • Test Example 3 Detection of the inhibitory effect of the compound of the present invention on the proliferation of MCF7 cells
  • Test principle Determination of Effect of compounds of this invention inhibit the proliferation of MCF7 cells, according to the IC 50 and maximum inhibition to evaluate the efficiency of the active compound.
  • MCF7 cells (ATCC, HTB-22) were cultured with DMEM (Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 384-well plate at a density of 500 cells/well in a complete medium at 37° C., and cultured overnight in a 5% CO 2 cell incubator. On the second day, add the test compound for drug treatment. Use Echo550 (Labcyte Inc.) to dilute the compound solution with a storage concentration of 10mM and transfer it to each cell culture well. The initial concentration of each compound in the cell is 100nM.
  • Test Example 4 Detection of the pharmacokinetic properties of the compound of the present invention
  • Test principle Using mice as test animals, the LC/MS/MS method was used to determine the drug concentration in plasma at different times after the mice were given the compound of the present invention intragastrically or intravenously. To study the pharmacokinetic behavior of the compound of the present invention in mice and evaluate its pharmacokinetic characteristics.
  • mice 18 healthy adult BALB/c mice, females, were equally divided into 6 groups, 3 in each group, gavage, the mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Zhejiang ) 2019-0001.
  • mice were fasted overnight and then administered by gavage.
  • the dose was 10 mg/kg and the volume was 1 mL/kg.
  • mice After intragastric administration of mice, 40 ⁇ L, 5 ⁇ L EDTA-K2 anticoagulant blood was collected from the orbit 5min, 15min, 30min, 1h, 2h, 4h, 24h after the administration, and the plasma was separated by centrifugation at 12000rpm, 4°C, 5 minutes at- Store at 20°C.
  • LC/MS/MS conditions mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile, column: ACE C18 5 ⁇ m (3.0mm*50mm), column temperature: 35°C, flow rate 0.5ml/min.
  • test compound showed good pharmacokinetic properties, and the results are shown in Table 4.
  • Test Example 5 Cerebral blood ratio test after oral administration of the compound of the present invention in mice
  • the brain penetration and cerebral blood ratio value of the drug were measured, and the brain penetration of the compound was evaluated.
  • GDC-9545 (CN107108611A compound 340 in Table 1, prepared according to the patent method), the compound of Example 4, and the compound of Example 8.
  • mice 18 healthy adult BALB/c mice, females, were equally divided into 6 groups, 3 in each group.
  • the specific grouping situation is as follows. The mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Zhejiang) 2019-0001.
  • test compound weigh a certain amount of the test compound, dissolve it in tetraethylene glycol 40% (7.5% Captisol) + 60% water, and prepare 1 mg/mL for intragastric gavage.
  • mice were fasted overnight and then administered by gavage. The dosage was 10mg/kg and the volume was 10mL/kg.
  • mice After the mice were given intragastric administration, the animals were euthanized at 2h and 4h after the administration, and brain tissue and whole blood were collected. The whole blood was anticoagulated with EDTA-K2, centrifuged at 12000 rpm, 4°C, 5 minutes to separate the plasma, and stored at -20°C. After the brain tissue samples were collected, PBS was added for homogenization, and acetonitrile protein precipitation was used to determine the concentration of the test compound in the brain tissue using liquid phase mass spectrometry. The linear range is 1 ⁇ 1000ng/mL.
  • the cerebral blood ratio parameters of each test compound are shown in Table 5.
  • the compound of the present invention can penetrate the blood-brain barrier of mice and enter the brain tissue.
  • Test Example 6 Determination of the plasma protein binding rate of the compound of the present invention
  • HTDialysis LLC Gales Ferry, CT, HTD96B
  • MWCO 12-14K #1101
  • Preparation of a buffer solution with a concentration of 100 mM sodium phosphate and 150 mM NaCl prepare an alkaline solution with a concentration of 14.2 g/L Na 2 HPO 4 and 8.77 g/L NaCl with ultra-pure water, and use ultra-pure water to prepare a concentration of An acidic solution of 12.0g/L NaH 2 PO 4 and 8.77g/L NaCl. Titrate the alkaline solution with an acidic solution to a pH of 7.4 to prepare a buffer solution with a concentration of 100 mM sodium phosphate and 150 mM NaCl.
  • Preparation of the dialysis membrane soak the dialysis membrane in ultrapure water for 60 minutes to separate the membrane into two pieces, then soak in 20% ethanol for 20 minutes, and finally soak in the buffer for dialysis for 20 minutes.
  • Preparation of plasma Thaw the frozen plasma quickly at room temperature, then centrifuge the plasma at 4°C and 3,220g for 10 minutes to remove clots, and collect the supernatant in a new centrifuge tube. Measure and record the pH value of plasma, using plasma with a pH value of 7-8.
  • Balanced dialysis step Assemble the dialysis device according to the operating instructions. Add 120 ⁇ L of plasma sample containing 1 ⁇ M compound on one side of the dialysis membrane, and add an equal volume of dialysate (phosphate buffer) on the other side. The experiment set up double samples. Seal the dialysis plate, put it into an incubator, and incubate for 6 hours at 37°C, 5% CO 2 and a rotation speed of about 100 rpm. After the incubation, remove the sealing membrane, and draw 50 ⁇ l from the buffer and plasma side of each well to different wells of the new plate.
  • dialysate phosphate buffer
  • the peak areas of the compound on the buffer side and the plasma side were measured.
  • the formula for calculating the plasma protein binding rate of the compound is as follows:
  • % Free rate (compound peak area to internal standard peak area ratio buffer side/compound peak area to internal standard peak area ratio plasma side) * 100%
  • Test Example 7 Determination of membrane permeability and transport characteristics of the compounds of the present invention
  • the membrane permeability and transport characteristics of the compounds of the present invention are determined by the following test methods.
  • HEPES Solarbio 804D049
  • Penicillin/Streptomycin Solarbio 20200109
  • Trypsin/EDTA Solarbio
  • PBS PBS
  • Fetal Bovine Serum (Sigma WXBD0055V), Fluorescent Yellow (Sigma MKCJ3738), NaHCO 3 (Sigma SLBZ4647)
  • Caco-2 cell culture medium high-sugar DMEM (containing L-glutamine) medium was prepared by adding FBS, penicillin, streptomycin, kanamycin and NEAA to contain 10% FBS, 0.1 mg/ mL streptomycin, 100 units of penicillin, 0.6 ⁇ g/mL kanamycin and 1 ⁇ NEAA cell culture medium.
  • the cells are cultured in a T-75 culture flask in an incubator at 37°C and 5% CO 2 and the culture medium is discarded when the cell growth reaches 80-90% density. Wash the cells with 5mL PBS, add 1.5mL Trypsin/EDTA, then incubate in a 37°C incubator for 5-10 minutes until the cells fall off as a quicksand, and finally neutralize the Trypsin/EDTA with a medium containing FBS.
  • TEER value TEER ( ⁇ ) measured value ⁇ film area (cm 2 )
  • the electrical resistance of the monolayer cell membrane is less than 230 ⁇ cm 2 , which indicates that the monolayer cell membrane has poor compactness and cannot be used in the test.
  • I acceptor refers to the fluorescence density on the receiving side (0.3 mL), and I donor refers to the fluorescence density on the dosing side (0.1 mL).
  • LY>1.0% means that the monolayer cell membrane has poor compactness, and the corresponding results will be excluded from the evaluation.
  • V A is the volume of the receiving end solution (A ⁇ B is 0.3mL, B ⁇ A is 0.1mL); Area is the membrane area of the Transwell-96-well plate (0.143cm 2 ); incubation time is the incubation time (unit: s); [drug] acceptor is the drug concentration on the receiving side, and [drug] initial donor is the drug concentration on the initial dosing side.
  • P app (BA) is the apparent permeability coefficient from the base end to the top
  • P app (AB) is the apparent permeability coefficient from the top end to the base end.
  • Test Example 8 The inhibitory effect of the compound of the present invention on the enzyme activity of CYP2C9, CYP2D6, and CYP3A4
  • the inhibition of CYP2C9, CYP2D6, and CYP3A4 enzyme activity by the compound of the present invention was determined by the following test method.
  • Table 8 The IC 50 values of the compounds of the present invention on CYP2C9, CYP2D6 and CYP3A4
  • Test Example 9 Growth inhibition experiment of compound on MCF-7 xenograft tumor
  • Human breast cancer MCF-7 cells ECACC-86012803
  • EMEM culture medium ATCC, Cat No.: 30-2003
  • Fetal Bovine Serum Hyclone; Cat No.: SV30087.03
  • Pancreatin-EDTA Gibco, Cat No.: 25200-072
  • mice Balb/c nude mice, female, 6-8 weeks old, weighing about 18-22 grams, the animals were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were kept in an SPF environment, each cage With separate air supply and exhaust, all animals have free access to standard-certified commercial laboratory food and free drinking water.
  • Cell culture Human breast cancer MCF-7 cell line is cultured in vitro, and the culture conditions are EMEM (cell culture medium) with 10% fetal bovine serum, 1% Antibiotic-Antimycotic, 37°C, 5% CO 2 incubator. Use 0.25% pancreatin-EDTA digestion solution twice a week for routine digestion and passage. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected and counted.
  • Cell inoculation 0.2ml/(containing 1 ⁇ 10 7 ) MCF-7 cell suspension (DPBS plus Matrigel, volume ratio 1:1) was subcutaneously inoculated on the right back of each mouse, and before cell inoculation 17 ⁇ -estradiol tablets were inoculated subcutaneously in two days. On the 6th day after cell inoculation, drugs were administered in random groups according to the tumor volume, and the day of grouping was Day 0.
  • the dosage of the compound is 3 mg/kg or 10 mg/kg, oral administration (PO), once a day (QD) x 3 weeks, PO, QD x 3 weeks. Each group has 6 mice.
  • the diameter of the tumor was measured with a vernier caliper twice a week.
  • the body weight of the mice was measured twice a week.
  • the anti-tumor efficacy of the compound was evaluated by the tumor growth inhibition rate TGI (%).
  • TGI(%) [(1-(Average tumor volume at the end of a certain treatment group-Average tumor volume at the start of the treatment group)/(Average tumor volume at the end of the solvent control group-When the solvent control group starts treatment Average tumor volume)] x 100%.
  • Test Example 10 Growth inhibition experiment of compound on T47D xenograft tumor
  • RPMI1640 culture medium Gibco, Cat No.: 22400-089
  • Fetal Bovine Serum Gbico; Cat No.: 10099-141C
  • Pancreatin-EDTA Gibco, Cat No.: 25200-072
  • D-PBS calcium-magnesium ion-free phosphate buffer
  • mice Female, 6-8 weeks old, weighing about 18-22 grams, the animals were purchased from Beijing Weitongda Biotechnology Co., Ltd. The mice were kept in an SPF environment, and each cage was sent out separately Wind, all animals have free access to standard certified commercial laboratory food and drinking water.
  • Cell culture human breast cancer T47D cell line is cultured in vitro, and the culture conditions are RPMI1640 (cell culture medium) with 10% fetal bovine serum, 8 ⁇ g/ml bovine insulin, 1% pen-strep, 37°C, 5% CO 2 incubator . Use 0.25% pancreatin-EDTA digestion solution for routine digestion and passage once a week. When the cell confluence is 80%-90% and the number reaches the requirement, the cells are collected and counted.
  • Cell inoculation 0.1ml/(containing 1 ⁇ 10 7 ) T47D cell suspension (DPBS plus Matrigel, volume ratio 1:1) subcutaneously inoculated on the right back of each mouse, and before cell inoculation 17 ⁇ -estradiol tablets were inoculated subcutaneously in two days. On the 29th day after cell inoculation, drugs were administered in random groups according to the tumor volume, and the day of grouping was Day 0.
  • Positive drug Fulvestrant Fulvestrant injection, AstraZeneca
  • SC subcutaneous injection
  • QW once a week
  • PO oral administration
  • QD once a day
  • PO QD x 4 weeks
  • Each group has 6 mice.
  • the diameter of the tumor was measured with a vernier caliper twice a week.
  • the body weight of the mice was measured twice a week.
  • the anti-tumor efficacy of the compound was evaluated by the tumor growth inhibition rate TGI (%).
  • TGI(%) [(1-(Average tumor volume at the end of a certain treatment group-Average tumor volume at the start of the treatment group)/(Average tumor volume at the end of the solvent control group-When the solvent control group starts treatment Average tumor volume)] x 100%.
  • Test compounds Example 4 compound, Example 8 compound, GDC-9545 (CN107108611A Table 1 compound 340, prepared according to the patent method), AZD-9833 (CN109843888A Example 17 compound, prepared according to the patent method).
  • the chest cavity was opened quickly, and the heart containing part of the superior vena cava and the longer aortic arch was immediately separated from the chest cavity of the guinea pig, and immersed in the pre-cooled bench-top solution (mM): 131NaCl, 4.0KCl, 1.8CaCl 2 , 1.2 MgSO 4 , 11.1 glucose, 24.9NaHCO 3 , 1.2KH 2 PO 4 (pH 7.4)), quickly insert the isolated guinea pig heart aorta into the perfusion needle of the Langendorff perfusion device and fix it with 36.5 ⁇ 1°C 95% O 2- 5% CO 2 saturated bench-top solution (pH 7.35 ⁇ 0.05) is used for reverse perfusion, the flow rate is controlled at 15-20ml/min, and the perfusion is maintained at a constant flow.
  • the two points of the recording electrode are placed on both sides of the ventricular membrane to generate a bipolar trans-ECG.
  • ECG bipolar epicardial electrocardiogram
  • All signals are amplified by an amplifier and digitally processed and recorded.
  • the sampling rate is 1kHz and saved in the hard disk for offline analysis. All data are recorded in real time by the PowerLab Isolated Heart System.
  • the ECG results show the overall electrical activity of the heart.

Abstract

The present application provides a compound having estrogen receptor modulating activity or function, or a pharmaceutically acceptable salt thereof. The compound has the structure of formula (I) and has substituents and structural features as described in the present application. The present application further provides a pharmaceutical composition containing the compound of formula (I) or the pharmaceutically acceptable salt thereof, and use of the compound of formula (I) or the pharmaceutically acceptable salt thereof in preparing a drug for preventing or treating estrogen receptor-related diseases.

Description

***受体调节剂化合物及其用途Estrogen receptor modulator compound and its use
本申请要求2020年6月12日向国家知识产权局提交的，专利申请号为202010536528.6，发明名称为“***受体调节剂化合物及其用途”、2020年6月22日向国家知识产权局提交的，专利申请号为202010572513.5，发明名称为“***受体调节剂化合物及其用途”以及2021年5月25日向国家知识产权局提交的，专利申请号为202110573837.5，发明名称为“***受体调节剂化合物及其用途”的三件在先申请的优先权。所述三件申请的全文通过引用的方式结合于本申请中。This application requires that it be submitted to the State Intellectual Property Office on June 12, 2020. The patent application number is 202010536528.6, and the title of the invention is "Estrogen Receptor Modulator Compounds and Their Uses." It is submitted to the State Intellectual Property Office on June 22, 2020. , The patent application number is 202010572513.5, the title of the invention is "estrogen receptor modulator compounds and their uses" and the patent application number is 202110573837.5 submitted to the State Intellectual Property Office on May 25, 2021, and the title of the invention is "estrogen receptor Priority rights of the three earlier applications of “modulator compounds and their uses”. The full texts of the three applications are incorporated into this application by reference.
技术领域Technical field
本发明涉及***受体调节剂化合物或其药学上可接受的盐，含有该化合物的药物组合物，以及其在预防或治疗***受体相关性疾病中的用途。The present invention relates to an estrogen receptor modulator compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the compound, and its use in the prevention or treatment of estrogen receptor-related diseases.
背景技术Background technique
***(E2)及***α受体(ERα)是乳腺癌发生发展的重要驱动因子。在乳腺癌患者中有超过2/3的患者表达ER转录因子，并且在大多数ER阳性患者中，即使经过早期的内分泌治疗后进展的肿瘤中，ER仍是一个关键的驱动因子，因此ER是乳腺癌治疗的一个主要靶点(Pharmacology&Therapeutics 186(2018)1-24)。内分泌治疗目的是降低ER活性，主要有三类，包括选择性***受体调节剂(selective estrogen receptor modulators,SERMs)，比如他莫昔芬(tamoxifen)，是ER的别构调节剂，同ER结合后抑制其转录活性；芳香化酶抑制剂(aromatase inhibitors,AIs),通过抑制雄激素转化为***，减低体内***水平；以及选择性***受体下调剂，比如氟维司群(fulvestrant)，不仅作为ER的拮抗剂抑制其活性，还具有诱导ER蛋白降解的作用。虽然内分泌治疗是***受体阳性乳腺癌患者的首选，但是约有30％的治疗后病人会发生复发，并且几乎所有的转移性乳腺癌患者都会产生耐药而发生进展。内分泌治疗产生耐药的机制主要有两类，一是集中在***受体信号通路本身，包括编码***受体的基因ESR1的激活突变，扩增，与其他基因的融合，***受体共调节因子和下游控制细胞周期因子的失调等；另一类机制包括与***受体信号通路有交叉反应的信号通路的激活，比如生长因子受体通路等(Oncol Ther，2017,5:17–29)。Estrogen (E2) and estrogen alpha receptor (ERα) are important driving factors for the development of breast cancer. More than two-thirds of breast cancer patients express ER transcription factors, and in most ER-positive patients, ER is still a key driving factor even in tumors that progress after early endocrine therapy. Therefore, ER is A major target for breast cancer treatment (Pharmacology&Therapeutics 186(2018)1-24). The purpose of endocrine therapy is to reduce ER activity. There are three main categories, including selective estrogen receptor modulators (SERMs), such as tamoxifen (tamoxifen), which is an allosteric modulator of ER and binds to ER. After inhibiting its transcriptional activity; aromatase inhibitors (aromatase inhibitors, AIs), by inhibiting the conversion of androgens to estrogen, reduce the level of estrogen in the body; and selective estrogen receptor downregulators, such as fulvestrant (fulvestrant) ), not only inhibits its activity as an ER antagonist, but also has the effect of inducing ER protein degradation. Although endocrine therapy is the first choice for patients with estrogen receptor-positive breast cancer, about 30% of patients will relapse after treatment, and almost all patients with metastatic breast cancer will develop resistance and progress. There are two main mechanisms for the development of drug resistance in endocrine therapy. One is focused on the estrogen receptor signaling pathway itself, including the activation mutation, amplification, and fusion with other genes of the gene encoding the estrogen receptor, ESR1, and the estrogen receptor. Co-regulators and downstream control of cell cycle factors, etc.; another type of mechanism includes the activation of signaling pathways that cross-react with the estrogen receptor signaling pathway, such as the growth factor receptor pathway (Oncol Ther, 2017, 5:17) –29).
氟维司群是首个也是唯一经临床批准用于他莫昔芬或芳香化酶抑制剂进展后治疗ER阳性、转移性乳腺癌的绝经后患者的选择性***受体下调剂(selective estrogen receptor downregulators,SERDs)类药物。此外，阿斯利康(参见专利申请WO2018077630A1)及基因泰克公司(参见专利申请WO2019245974A1)也公开了一系列结构新颖的SERD类化合物及相应的医药用途。多项研究数据显示经氟维司群治疗的患者体内并未能完全实现ER的降解，不过也可能是氟维司群的剂量(最高只能达到500mg，主要是其药效动力学特征和肌内给药途径限制了其可给予患者的最高剂量)限制了其药效。因此，具有更高生物利用度、对ER的拮抗活性更高、并能在更大程度上的ER降解、以及可以用于***水平较高的绝经前患者、方便的口服给药的SERD类药物是临床上迫切需要的。Fulvestrant is the first and only clinically approved selective estrogen receptor down-regulator for the treatment of ER-positive, metastatic breast cancer in postmenopausal patients after the progression of tamoxifen or aromatase inhibitors. receptor downregulators, SERDs) drugs. In addition, AstraZeneca (see patent application WO2018077630A1) and Genentech (see patent application WO2019245974A1) also disclosed a series of SERD compounds with novel structures and corresponding medical applications. Data from a number of studies have shown that patients treated with fulvestrant have not fully achieved ER degradation, but it may also be the dose of fulvestrant (the highest can only reach 500mg, mainly due to its pharmacodynamic characteristics and muscle The internal route of administration limits the maximum dose that can be given to patients), which limits its efficacy. Therefore, it has higher bioavailability, higher ER antagonistic activity, and a greater degree of ER degradation, and can be used in premenopausal patients with higher estrogen levels, and is convenient for oral administration. Drugs are urgently needed clinically.
发明内容Summary of the invention
为了解决上述至少一个技术问题，本发明提供了一种式(I)所示化合物或其药学上可接受的盐：In order to solve at least one of the above technical problems, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021099653-appb-000001
Figure PCTCN2021099653-appb-000001
其中，in,
X 1、X 2、X 3、X 4独立地选自CR 7或N； X 1 , X 2 , X 3 , and X 4 are independently selected from CR 7 or N;
R 7选自H、F、Cl、Br、I、OH、CN、C 1-C 6烷基、C 3-C 6环烷基、3-6元杂环基、C 1-C 6烷氧基、C 3-C 6环烷基氧基或3-6元杂环基氧基； R 7 is selected from H, F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclyl, C 1 -C 6 alkoxy Group, C 3 -C 6 cycloalkyloxy or 3-6 membered heterocyclyloxy;
Het选自
Figure PCTCN2021099653-appb-000002
Het is selected from
Figure PCTCN2021099653-appb-000002
R 1、R 2、R 9独立地选自氢、OH、F、Cl、Br、I、C 1-C 6烷基或C 2-C 8烯基； R 1 , R 2 , and R 9 are independently selected from hydrogen, OH, F, Cl, Br, I, C 1 -C 6 alkyl or C 2 -C 8 alkenyl;
或者，R 1、R 2与其连接的碳原子共同形成C 3-C 6环烷基或3-6元杂环烷基，所述C 3-C 6环烷基或3-6元杂环烷基任选被R a取代； Alternatively, R 1 , R 2 and the carbon atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, the C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group group is optionally substituted with R a;
R 5选自C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基，所述C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基任选被1个或多个选自如下的基团取代：氘、F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3； R 5 is selected from C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, the C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected from one or more of the following Substitution of groups: deuterium, F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
R 6选自H、C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基，所述C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3； R 6 is selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, said C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected by one or more Substitution from the following groups: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
或者，R 1或R 2之一与R 6及其各自连接的C和N共同形成3-6元杂环基； Alternatively, one of R 1 or R 2 and R 6 and the C and N respectively connected to form a 3-6 membered heterocyclic group;
R 3选自H、CN、COOH、C(O)OR b、OC(O)R b、CONH 2、C(O)NR bR c、SO 2R b、SO 2NR bR c、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基、C 3-C 6杂环基、C 6-C 10芳基或C 5-C 10杂芳基，所述C 1-C 6烷基、C 3-C 6环烷基、C 3-C 6杂环基、C 6-C 10芳基或C 5-C 10杂芳基任选被1个或多个R d取代； R 3 is selected from H, CN, COOH, C(O)OR b , OC(O)R b , CONH 2 , C(O)NR b R c , SO 2 R b , SO 2 NR b R c , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, C 3 -C 6 heterocyclyl, C 6 -C 10 aryl or C 5 -C 10 heteroaryl, The C 1 -C 6 alkyl group, C 3 -C 6 cycloalkyl group, C 3 -C 6 heterocyclic group, C 6 -C 10 aryl group or C 5 -C 10 heteroaryl group are optionally selected by 1 or Multiple R d substitutions;
R 4选自H、F、Cl、Br、I、OH、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基或C 3-C 6杂环基，所述C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基或C 3-C 6杂环基任选被一个或多个选自如下的基团取代：F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3； R 4 is selected from H, F, Cl, Br, I, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 heterocyclyl , The C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group, C 3 -C 6 cycloalkyl group or C 3 -C 6 heterocyclic group is optionally selected from one or more groups as follows Replacement: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
环Q选自C 6-C 10芳基或5-10元杂芳基，所述C 6-C 10芳基或5-10元杂芳基任选被1个或多个R 8取代； Ring Q is selected from a C 6 -C 10 aryl group or a 5-10 membered heteroaryl group, the C 6 -C 10 aryl group or a 5-10 membered heteroaryl group is optionally substituted by one or more R 8 ;
R 8选自F、Cl、Br、I、OH、CN、COOH、C(O)OR b、OC(O)R b、CONH 2、C(O)NR bR c、NR cC(O)R b、SO 2R b、SO 2NR b、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基、3-6元杂环基、C 6-C 10芳基或5-10元杂芳基，所述C 3-C 6环烷基、3-6元杂环基、C 6-C 10芳基或5-10元杂芳基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、OH、CN、C 1-C 6烷基、C 1-C 6烷氧基； R 8 is selected from F, Cl, Br, I, OH, CN, COOH, C(O)OR b , OC(O)R b , CONH 2 , C(O)NR b R c , NR c C(O) R b , SO 2 R b , SO 2 NR b , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclic group, C 6- C 10 aryl group or 5-10 membered heteroaryl group, the C 3 -C 6 cycloalkyl group, 3-6 membered heterocyclic group, C 6 -C 10 aryl group or 5-10 membered heteroaryl group is optionally Substitution with one or more groups selected from the following: F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy;
R a选自卤素、OH、CN、C 1-6烷基、C 1-6烷氧基、C 3- 6环烷基或3-6元杂环基，所述C 1-6烷基、C 1-6烷氧基、C 3- 6环烷基或3-6元杂环基任选被卤素取代； R a is selected from halo, OH, CN, C 1-6 alkyl, C 1-6 alkoxy, C 3 - 6 cycloalkyl or 3-6 membered heterocyclyl, a C 1-6 alkyl group, C 1-6 alkoxy, C 3 - 6 cycloalkyl or 3-6 membered heterocyclyl group optionally substituted by halogen;
R b独立选自C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基、3-6元杂环基、C 6-C 10芳基或5-10元杂芳基； R b is independently selected from C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclyl, C 6 -C 10 aryl or 5-10 membered heteroaryl;
R c独立选自H、C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基、3-6元杂环基、C 6-C 10芳基或5-10元杂芳基； R c is independently selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclic group, C 6 -C 10 aryl or 5-10 membered heteroaryl;
R d独立选自F、Cl、Br、I、OH、CN、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基或3-6元杂环基，所述C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基或3-6元杂环基任选被1个或多个选自F、Cl、Br、I或OH的基团取代； R d is independently selected from F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or 3-6 membered heterocyclic group , The C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group, C 3 -C 6 cycloalkyl group or 3-6 membered heterocyclic group is optionally selected from one or more of F, Cl, Group substitution of Br, I or OH;
n为0、1或2；n is 0, 1 or 2;
m为1、2、3或4；m is 1, 2, 3 or 4;
p为1或2；p is 1 or 2;
条件是：(1)当n为0或1时，R 1不为H、F或甲基；(2)当m为1或2时，R 9不为H。 The conditions are: (1) when n is 0 or 1, R 1 is not H, F or methyl; (2) when m is 1 or 2, R 9 is not H.
在一些实施方案中，R 5选自C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基，所述C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3。 In some embodiments, R 5 is selected from C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, or 3-6 membered heterocycloalkane Group, the C 1 -C 6 alkyl group, C 2 -C 8 alkenyl group, C 2 -C 4 alkynyl group, C 3 -C 6 cycloalkyl group or 3-6 membered heterocycloalkyl group is optionally selected by one Or multiple groups selected from: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 .
在一些实施方案中，所述式(I)所示化合物或其药学上可接受的盐选自式(Ia)化合物或其药学上可接受的盐：In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (Ia) or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021099653-appb-000003
Figure PCTCN2021099653-appb-000003
在一些实施方案中，所述Het选自
Figure PCTCN2021099653-appb-000004
In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000004
在一些实施方案，所述Het选自
Figure PCTCN2021099653-appb-000005
其中R 1、R 2与其连接的碳原子共同形成C 3-C 6环烷基或3-6元杂环烷基，所述C 3-C 6环烷基或3-6元杂环烷基任选被R a取代。 In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000005
Wherein R 1 , R 2 and the carbon atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, the C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group R a is optionally substituted.
在一些实施方案中，Het选自
Figure PCTCN2021099653-appb-000006
其中R 1、R 2与其连接的碳原子共同形成C 3-C 6环烷基或3-6元杂环烷基，所述C 3-C 6环烷基或3-6元杂环烷基任选被R a取代；所述n为1。 In some embodiments, Het is selected from
Figure PCTCN2021099653-appb-000006
Wherein R 1 , R 2 and the carbon atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, the C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group optionally substituted with R a; n is the 1.
在一些实施方案中，所述Het选自
Figure PCTCN2021099653-appb-000007
其中R 1、R 2与其连接的碳原子共同形成环丙烷。 In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000007
Among them, R 1 , R 2 and the carbon atom to which they are connected together form cyclopropane.
在一些实施方案中，所述Het选自
Figure PCTCN2021099653-appb-000008
其中R 1或R 2之一与R 6以及各自连接的C和N一起形成3-6元杂环基。 In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000008
Wherein one of R 1 or R 2 forms a 3-6 membered heterocyclic group together with R 6 and C and N respectively connected.
在一些实施方案中，所述Het选自
Figure PCTCN2021099653-appb-000009
其中R 1或R 2之一与R 6以及各自连接的C和N一起形成3-6元杂环基；所述n为1。 In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000009
Wherein one of R 1 or R 2 forms a 3-6 membered heterocyclic group together with R 6 and the C and N respectively connected; the n is 1.
在一些实施方案中，Het选自
Figure PCTCN2021099653-appb-000010
其中R 1或R 2之一与R 6以及各自连接的C和N一起形成哌啶环。 In some embodiments, Het is selected from
Figure PCTCN2021099653-appb-000010
Wherein one of R 1 or R 2 forms a piperidine ring together with R 6 and C and N respectively connected.
在一些实施方案中，所述Het选自
Figure PCTCN2021099653-appb-000011
In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000011
在一些实施方案中，所述R 9选自氢、OH、F、Cl、Br、I或C 1-C 6烷基。 In some embodiments, the R 9 is selected from hydrogen, OH, F, Cl, Br, I, or C 1 -C 6 alkyl.
在一些实施方案中，所述R 9选自氢或CH 3。 In some embodiments, the R 9 is selected from hydrogen or CH 3 .
在一些实施方案中，所述m选自1。In some embodiments, the m is selected from 1.
在一些实施方案中，所述m选自3。In some embodiments, the m is selected from 3.
在一些实施方案中，所述m选自4。In some embodiments, the m is selected from 4.
在一些实施方案中，所述Het选自
Figure PCTCN2021099653-appb-000012
In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000012
在一些实施方案中，所述p为1。In some embodiments, the p is 1.
在一些实施方案中，所述Het选自
Figure PCTCN2021099653-appb-000013
In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000013
在一些实施方案中，所述Het选自
Figure PCTCN2021099653-appb-000014
In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000014
在一些实施方案中，所述Het选自
Figure PCTCN2021099653-appb-000015
In some embodiments, the Het is selected from
Figure PCTCN2021099653-appb-000015
在一些实施方案中，所述R 3选自C 1-C 6烷基、CN、COOH、C(O)OR b、OC(O)R b、CONH 2、C(O)NR bR c或C 6-C 10芳基；所述C 1-C 6烷基或C 6-C 10芳基任选被一个或多个R d取代，R d独立选自F、Cl、Br、I、OH、CN或者任选被1个或多个选自F、Cl、Br、I或OH基团取代的C 1-C 6烷基。 In some embodiments, the R 3 is selected from C 1 -C 6 alkyl, CN, COOH, C(O)OR b , OC(O)R b , CONH 2 , C(O)NR b R c or C 6 -C 10 aryl group; the C 1 -C 6 alkyl group or C 6 -C 10 aryl group is optionally substituted by one or more R d , and R d is independently selected from F, Cl, Br, I, OH , CN or C 1 -C 6 alkyl optionally substituted with one or more groups selected from F, Cl, Br, I or OH.
在一些实施方案中，所述R 3选自C 1-C 6烷基或C 6-C 10芳基；所述C 1-C 6烷基或C 6-C 10芳基任选被一个或多个R d取代，R d独立选自F、Cl、Br、OH或者任选被1个或多个选自F、Cl、Br或OH基团取代的C 1-C 6烷基。 In some embodiments, the R 3 is selected from C 1 -C 6 alkyl or C 6 -C 10 aryl; the C 1 -C 6 alkyl or C 6 -C 10 aryl is optionally selected by one or Multiple R d substitutions, R d is independently selected from F, Cl, Br, OH or a C 1 -C 6 alkyl group optionally substituted with one or more groups selected from F, Cl, Br or OH.
在一些实施方案中，所述R 3选自C 1-C 3烷基或苯基；所述C 1-C 3烷基或苯基任选被一个或多个R d取代，R d独立选自F、OH或三氟甲基。 In some embodiments, the R 3 is selected from C 1 -C 3 alkyl or phenyl; the C 1 -C 3 alkyl or phenyl is optionally substituted by one or more R d , and R d is independently selected From F, OH or trifluoromethyl.
在一些实施方案中，所述R 3选自4-三氟甲基苯基、-CH 2CF 3、
Figure PCTCN2021099653-appb-000016
In some embodiments, the R 3 is selected from 4-trifluoromethylphenyl, -CH 2 CF 3 ,
Figure PCTCN2021099653-appb-000016
在一些实施方案中，所述R 4选自任选被F、Cl、Br或I取代的C 1-C 6烷基。 In some embodiments, the R 4 is selected from C 1 -C 6 alkyl optionally substituted by F, Cl, Br, or I.
在一些实施方案中，所述R 4为甲基。 In some embodiments, the R 4 is methyl.
在一些实施方案中，所述R 5为任选被氘、F、Cl、Br或I取代的C 1-C 6烷基。 In some embodiments, the R 5 is a C 1 -C 6 alkyl group optionally substituted with deuterium, F, Cl, Br, or I.
在一些实施方案中，所述R 5为任选被F、Cl、Br或I取代的C 1-C 6烷基。 In some embodiments, the R 5 is a C 1 -C 6 alkyl optionally substituted with F, Cl, Br, or I.
在一些实施方案中，所述R 5为1,1-二氘代-3-氟丙基、3-氟丙基或2-氟乙基。 In some embodiments, the R 5 is 1,1-dideutero-3-fluoropropyl, 3-fluoropropyl, or 2-fluoroethyl.
在一些实施方案中，所述R 5为3-氟丙基或2-氟乙基。 In some embodiments, the R 5 is 3-fluoropropyl or 2-fluoroethyl.
在一些实施方案中，所述X 1选自CR 7或N，X 2、X 3、X 4均为CR 7。 In some embodiments, X 1 is selected from CR 7 or N, and X 2 , X 3 , and X 4 are all CR 7 .
在一些实施方案中，所述X 1选自CH或N，X 2、X 3、X 4均为CH。 In some embodiments, the X 1 is selected from CH or N, and X 2 , X 3 , and X 4 are all CH.
在一些实施方案中，所述R 7选自H、F、Cl、Br或I。 In some embodiments, the R 7 is selected from H, F, Cl, Br, or I.
在一些实施方案中，所述R 6选自H或C 1-C 6烷基。 In some embodiments, the R 6 is selected from H or C 1 -C 6 alkyl.
在一些实施方案中，当Het与NR 6相连的原子为手性碳时，其构型为(S)-构型。 In some embodiments, when the atom connecting Het to NR 6 is a chiral carbon, its configuration is the (S)-configuration.
在一些实施方案中，所述式(I)化合物或其药学上可接受的盐选自式(II)化合物或其药学上可接受的盐：In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (II) or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021099653-appb-000017
Figure PCTCN2021099653-appb-000017
其中X 1、X 2、X 3、X 4、Het、R 3、R 4、R 6如式(I)所定义； Wherein X 1 , X 2 , X 3 , X 4 , Het, R 3 , R 4 , R 6 are as defined in formula (I);
Y选自NR 10、O或S； Y is selected from NR 10 , O or S;
R 10选自H、C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基，所述C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3。 R 10 is selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, said C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected by one or more Substitution from the following groups: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 .
在一些实施方案中，所述式(I)化合物或其药学上可接受的盐选自式(IIa)化合物或其药学上可接受的盐：In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IIa) or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021099653-appb-000018
Figure PCTCN2021099653-appb-000018
其中X 1、X 2、X 3、X 4、Het、R 3、R 4、R 6如式(I)所定义； Wherein X 1 , X 2 , X 3 , X 4 , Het, R 3 , R 4 , R 6 are as defined in formula (I);
Y选自NR 10、O或S； Y is selected from NR 10 , O or S;
R 10选自H、C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基，所述C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3。 R 10 is selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, said C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected by one or more Substitution from the following groups: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 .
在一些实施方案中，所述Y选自NR 10。 In some embodiments, the Y is selected from NR 10 .
在一些实施方案中，R 10选自H、C 1-C 6烷基、C 3-C 6环烷基或3-6元杂环烷基。 In some embodiments, R 10 is selected from H, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, or 3-6 membered heterocycloalkyl.
在一些实施方案中，R 10为H。 In some embodiments, R 10 is H.
在一些实施方案中，所述式(I)化合物或其药学上可接受的盐选自式(III)化合物或其药学上可接受的盐：In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (III) or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021099653-appb-000019
Figure PCTCN2021099653-appb-000019
其中X 1、X 2、X 3、X 4、Het、R 3、R 4、R 6如式(I)所定义。 Wherein X 1 , X 2 , X 3 , X 4 , Het, R 3 , R 4 , R 6 are as defined in formula (I).
在一些实施方案中，所述式(I)化合物或其药学上可接受的盐选自式(IIIa)化合物或其药学上可接受的盐：In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IIIa) or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021099653-appb-000020
Figure PCTCN2021099653-appb-000020
其中X 1、X 2、X 3、X 4、Het、R 3、R 4、R 6如式(I)所定义。 Wherein X 1 , X 2 , X 3 , X 4 , Het, R 3 , R 4 , R 6 are as defined in formula (I).
在一些实施方案中，所述式(I)化合物或其药学上可接受的盐选自式(IV)化合物或其药学上可接受的盐：In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IV) or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021099653-appb-000021
Figure PCTCN2021099653-appb-000021
其中X 1、X 2、X 3、X 4、Het、R 3、R 4、R 6、R 8如式(I)所定义。 Wherein X 1 , X 2 , X 3 , X 4 , Het, R 3 , R 4 , R 6 , and R 8 are as defined in formula (I).
在一些实施方案中，所述式(I)化合物或其药学上可接受的盐选自式(IVa)化合物或其药学上可接受的盐：In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IVa) or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021099653-appb-000022
Figure PCTCN2021099653-appb-000022
其中X 1、X 2、X 3、X 4、Het、R 3、R 4、R 6、R 8如式(I)所定义。 Wherein X 1 , X 2 , X 3 , X 4 , Het, R 3 , R 4 , R 6 , and R 8 are as defined in formula (I).
在一些实施方案中，式(IV)的化合物中所述R 8选自C 6-C 10芳基或5-10元杂芳基，所述C 6-C 10芳基或5-10元杂芳基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、OH、CN、C 1-C 6烷基、C 1-C 6烷氧基。 In some embodiments, the R 8 in the compound of formula (IV) is selected from a C 6 -C 10 aryl group or a 5-10 membered heteroaryl group, the C 6 -C 10 aryl group or a 5-10 membered heteroaryl group The aryl group is optionally substituted by one or more groups selected from the group consisting of F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy.
在一些实施方案中，式(IV)的化合物中所述R 8选自苯基或5-6元杂芳基，所述苯基或5-6元杂芳基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、OH、C 1-C 3烷基、C 1-C 3烷氧基。 In some embodiments, the R 8 in the compound of formula (IV) is selected from a phenyl group or a 5-6 membered heteroaryl group, and the phenyl group or a 5-6 membered heteroaryl group is optionally substituted by one or more Substitution selected from the following groups: F, Cl, Br, I, OH, C 1 -C 3 alkyl, C 1 -C 3 alkoxy.
在一些实施方案中，式(IV)的化合物中所述R 8选自吡唑，所述吡唑任选被1个或多个甲基取代。 In some embodiments, the R 8 in the compound of formula (IV) is selected from pyrazole, and the pyrazole is optionally substituted with one or more methyl groups.
在一些实施方案中，式(IV)化合物中所述R 8选自1H-吡唑-4-基或1-甲基-1H-吡唑-4-基。 In some embodiments, the R 8 in the compound of formula (IV) is selected from 1H-pyrazol-4-yl or 1-methyl-1H-pyrazol-4-yl.
在一些实施方案中，式(I)化合物或其药学上可接受的盐选自以下化合物或其药学上可接受的盐：In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021099653-appb-000023
Figure PCTCN2021099653-appb-000023
Figure PCTCN2021099653-appb-000024
Figure PCTCN2021099653-appb-000024
Figure PCTCN2021099653-appb-000025
Figure PCTCN2021099653-appb-000025
在一个方面，本申请所述的化合物以外消旋混合物或以对映异构体富集的或对映异构体纯的形式存在。在某些实施方案中，本申请所述的化合物以它们的单独的立体异构体制备，其通过使所述化合物的外消旋混合物与光学活性拆分剂反应来形成一对非对映异构体化合物/盐，将所述非对映异构体分离并回收具有光学纯的对映异构体。在一些实施方案中，对映异构体的拆分使用本申请所述的化合物的共价非对映异构体衍生物来进行。在另一个实施方案中，通过基于溶解度差异的分离/拆分技术分离非对映异构体。在某些实施方案中，本申请所述的化合物通过酶法拆分制备为它们的单独的立体异构体。在一些实施方案中，单独的立体异构体的拆分使用脂酶或酯酶进行。在一些实施方案中，单独的立体异构体的拆分通过脂酶或酯酶催化的不对称脱酰作用来进行。在另一些实施方案中，通过色谱法，或者通过形成非对映异构体的盐并通过重结晶或色谱法或其任意组合来分离，进行立体异构体的分离。在一些实施方案中，通过立体选择性合成获得立体异构体。In one aspect, the compounds described in this application exist as racemic mixtures or in enantiomerically enriched or enantiomerically pure forms. In certain embodiments, the compounds described in this application are prepared as their individual stereoisomers by reacting a racemic mixture of the compounds with an optically active resolving agent to form a pair of diastereomers The structure compound/salt separates the diastereomers and recovers the optically pure enantiomers. In some embodiments, the resolution of enantiomers is carried out using covalent diastereomeric derivatives of the compounds described in this application. In another embodiment, the diastereomers are separated by separation/resolution techniques based on differences in solubility. In certain embodiments, the compounds described in this application are prepared into their individual stereoisomers by enzymatic resolution. In some embodiments, the resolution of individual stereoisomers is performed using lipase or esterase. In some embodiments, the resolution of individual stereoisomers is carried out by asymmetric deacylation catalyzed by lipase or esterase. In other embodiments, the separation of stereoisomers is carried out by chromatography, or by forming diastereomeric salts and separating by recrystallization or chromatography or any combination thereof. In some embodiments, stereoisomers are obtained by stereoselective synthesis.
本发明还提供药物组合物，其包含式(I)所示化合物或其药学上可接受的盐，以及药学上可接受的载体和/或赋形剂。The present invention also provides a pharmaceutical composition, which comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient.
在一些实施方案中，所述药物组合物配制用于静脉内注射、皮下注射、口服给药或局部给药。In some embodiments, the pharmaceutical composition is formulated for intravenous injection, subcutaneous injection, oral administration, or topical administration.
在一些实施方案中，所述药物组合物为片剂、丸剂、胶囊剂、液体、悬浮液、凝胶剂、分散剂、溶液、乳剂、软膏剂或洗剂。In some embodiments, the pharmaceutical composition is a tablet, pill, capsule, liquid, suspension, gel, dispersion, solution, emulsion, ointment, or lotion.
进一步地，本发明涉及式(I)所示的化合物或其药学上可接受的盐或者其药物组合物在制备预防或者治疗***受体相关性疾病的药物中的用途。Further, the present invention relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutical composition thereof in the preparation of a medicine for preventing or treating estrogen receptor-related diseases.
进一步地，本发明涉及式(I)所示的化合物或其药学上可接受的盐或其药物组合物，用于预防或者治疗***受体相关疾病。Further, the present invention relates to a compound represented by formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof, for the prevention or treatment of estrogen receptor related diseases.
进一步地，本发明涉及式(I)所示的化合物或其药学上可接受的盐或者其药物组合物在预防或者治疗***受体相关性疾病中的用途。Further, the present invention relates to the use of the compound represented by formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the prevention or treatment of estrogen receptor-related diseases.
进一步地，本发明涉及预防或者治疗***受体相关性疾病的式(I)所示的化合物或其药学上可接受的盐，或其药物组合物。Further, the present invention relates to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for preventing or treating estrogen receptor-related diseases.
本发明还涉及预防或者治疗***受体相关性疾病的方法，该方法包括向有此需要的对象施用预防或治疗有效剂量的本发明所述的式(I)所示的化合物或其药学上可接受的盐、或其药物组合物。The present invention also relates to a method for preventing or treating estrogen receptor-related diseases, the method comprising administering to a subject in need thereof a preventive or therapeutically effective dose of the compound represented by formula (I) of the present invention or its pharmaceutically Acceptable salt, or pharmaceutical composition thereof.
在本发明的一些实施方案中，所述的***受体相关性疾病包括但不限于癌症和自身免疫病等。In some embodiments of the present invention, the estrogen receptor-related diseases include but are not limited to cancer and autoimmune diseases.
在本发明的一些实施方案中，所述的***受体相关性疾病优选为肿瘤。In some embodiments of the present invention, the estrogen receptor-related disease is preferably a tumor.
术语定义和说明Definition and description of terms
除非另有说明，本申请中所用的术语具有下列含义，本申请中记载的基团和术语定义，包括其作为实例的定义、示例性的定义、优选的定义、表格中记载的定义、实施例中具体化合物的定义等，可以彼此之间任意组合和结合。一个特定的术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。Unless otherwise stated, the terms used in this application have the following meanings. The definitions of groups and terms described in this application include definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and examples The definitions of specific compounds in, etc., can be combined and combined arbitrarily with each other. A specific term should not be considered uncertain or unclear without a special definition, but should be understood according to the ordinary meaning in the field. When a trade name appears in this article, it is meant to refer to its corresponding commodity or its active ingredient.
本文中
Figure PCTCN2021099653-appb-000026
表示连接位点。 In this article
Figure PCTCN2021099653-appb-000026
Indicates the connection site.
本文中消旋体或者对映体纯的化合物的图示法来自Maehr,J.Chem.Ed.1985,62:114-120。除非另有说明，用楔形键和虚楔键
Figure PCTCN2021099653-appb-000027
表示一个立体中心的绝对构型，用黑实键和虚键
Figure PCTCN2021099653-appb-000028
表示一个立体中心的相对构型(如脂环化合物的顺反构型)。 The schematic diagram of the racemate or enantiomerically pure compound herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise specified, use wedge keys and virtual wedge keys
Figure PCTCN2021099653-appb-000027
Represents the absolute configuration of a three-dimensional center, with black real and virtual keys
Figure PCTCN2021099653-appb-000028
Represents the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).
术语“互变异构体”是指因分子中某一原子在两个位置迅速移动而产生的官能团异构体。本发明化合物可表现出互变异构现象。互变异构的化合物可以存在两种或多种可相互转化的种类。互变异构体一般以平衡形式存在，尝试分离单一互变异构体时通常产生一种混合物，其理化性质与化合物的混合物是一致的。平衡的位置取决于分子内的化学特性。例如，在很多脂族醛和酮如乙醛中，酮型占优势；而在酚中，烯醇型占优势。本发明包含化合物的所有互变异构形式。The term "tautomer" refers to an isomer of a functional group resulting from the rapid movement of an atom in two positions in a molecule. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds can exist in two or more mutually convertible species. Tautomers generally exist in an equilibrium form. An attempt to separate a single tautomer usually produces a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties of the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone type is dominant; in phenol, the enol type is dominant. The present invention encompasses all tautomeric forms of the compound.
术语“立体异构体”是指由分子中原子在空间上排列方式不同所产生的异构体，包括顺反异构体、对映异构体、非对应异构体和构象异构体。The term "stereoisomer" refers to the isomers produced by the different arrangements of atoms in the molecule in space, including cis and trans isomers, enantiomers, diastereomers and conformational isomers.
本发明的化合物可以具有不对称原子如碳原子、硫原子、氮原子、磷原子或不对称双键，因此本申请的化合物可以存在特定的几何或立体异构体形式。特定的几何或立体异构体形式可以是顺式和反式异构体、E型和Z型几何异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，以及其外消旋混合物或其它混合物，例如对映异构体或非对映体富集的混合物，以上所有这些异构体以及它们的混合物都属于本申请化合物的定义范围之内。烷基等取代基中可存在另外的不对称碳原子、不对称硫原子、不对称氮原子或不对称磷原子，所有取代基中涉及到的这些异构体以及它们的混合物，也均包括在本申请化合物的定义范围之内。本申请的含有不对称原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来，光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so the compounds of the present application may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms can be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S )-Enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as enantiomers or diastereomers Enriched mixtures, all of the above isomers and their mixtures fall within the scope of the definition of compounds in this application. There may be other asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms in substituents such as alkyl groups. These isomers and their mixtures involved in all substituents are also included in Within the scope of the definition of compounds in this application. The asymmetric atom-containing compound of this application can be separated in an optically active pure form or a racemic form. The optically active pure form can be separated from the racemic mixture, or synthesized by using chiral raw materials or chiral reagents. .
术语“被取代”是指特定原子上的任意一个或多个氢原子被取代基取代，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence of the specific atom is normal and the substituted compound is stable. When the substituent is oxo (ie =O), it means that two hydrogen atoms are replaced, and the oxo will not occur on the aromatic group.
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。例如，乙基“任选”被卤素取代，指乙基可以是未被取代的(CH 2CH 3)、单取代的(如CH 2CH 2F)、多取代的(如CHFCH 2F、CH 2CHF 2等)或完全被取代的(CF 2CF 3)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取代或取代模式。 The term "optional" or "optionally" means that the event or situation described later can occur or not occur, and the description includes occurrence of said event or situation and non-occurrence of said event or situation. For example, the ethyl group is "optionally" substituted by halogen, meaning that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (such as CH 2 CH 2 F), or polysubstituted (such as CHFCH 2 F, CH 2 CHF 2 etc.) or completely substituted (CF 2 CF 3 ). Those skilled in the art can understand that for any group containing one or more substituents, any substitution or substitution pattern that is impossible to exist in space and/or cannot be synthesized will not be introduced.
本文中的C m-n，是指该部分具有给定范围中的整数个碳原子。例如“C 1-6”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子或6个碳原子。 C mn in this article means that the part has an integer number of carbon atoms in a given range. For example, "C 1-6 "means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。例如，如果一个基团被2个R所取代，则每个R都有独立的选项。When any variable (such as R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. For example, if a group is replaced by 2 Rs, each R has independent options.
当一个连接基团的数量为0时，比如-(CH 2) 0-，表示该连接基团为键。 When the number of a linking group is 0, such as -(CH 2 ) 0 -, it means that the linking group is a bond.
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues, but not Many toxicity, irritation, allergic reactions or other problems or complications are commensurate with a reasonable benefit/risk ratio.
术语“药学上可接受的盐”是指药学上可接受的无毒酸或碱的盐，包括无机酸和碱、有机酸和碱的盐。作为药学上可接受的盐，例如，可以提及金属盐、铵盐、与有机碱形成的盐、与无机酸形成的盐、与有机酸形成的盐、与碱性或者酸性氨基酸形成的盐等。The term "pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, and organic acids and bases. As pharmaceutically acceptable salts, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. can be mentioned. .
术语“溶剂化物”是指一个或多个溶剂分子与本申请化合物的缔合物或络合物。形成溶剂化物的溶剂的实例包括但不限于水、异丙醇、乙醇、甲醇、DMSO、乙酸乙酯(EtOAc)、乙酸(AcOH)和乙醇胺。The term "solvate" refers to an association or complex of one or more solvent molecules with the compound of the present application. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate (EtOAc), acetic acid (AcOH), and ethanolamine.
术语“药物组合物”表示一种或多种文本所述化合物或其生理学/药学上可接受的盐或前体药物与其它化学组分的混合物，其它组分例如生理学/药学上可接受的载体和赋形剂。药物组合物的目的是促进化合物对生物体的给药。药物组合物的目的是有利于对有机体给予本申请的化合物。The term "pharmaceutical composition" means a mixture of one or more of the compounds described in the text or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable carriers And excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism. The purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present application to the organism.
术语“被取代”是指特定原子上的任意一个或多个氢原子被取代基取代，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence of the specific atom is normal and the substituted compound is stable. When the substituent is oxo (ie =O), it means that two hydrogen atoms are replaced, and the oxo will not occur on the aromatic group.
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。例如，乙基“任选”被卤素取代，指乙基可以是未被取代的(CH 2CH 3)、单取代的(如CH 2CH 2F)、多取代的(如CHFCH 2F、CH 2CHF 2等)或完全被取代的(CF 2CF 3)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取 代或取代模式。 The term "optional" or "optionally" means that the event or situation described later can occur or not occur, and the description includes occurrence of said event or situation and non-occurrence of said event or situation. For example, the ethyl group is "optionally" substituted by halogen, meaning that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (such as CH 2 CH 2 F), or polysubstituted (such as CHFCH 2 F, CH 2 CHF 2 etc.) or completely substituted (CF 2 CF 3 ). Those skilled in the art can understand that for any group containing one or more substituents, any substitution or substitution pattern that is impossible to exist in space and/or cannot be synthesized will not be introduced.
术语“卤”或“卤素”是指氟、氯、溴和碘。The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.
术语“羟基”指-OH基团。The term "hydroxyl" refers to the -OH group.
术语“氰基”指-CN基团。The term "cyano" refers to the -CN group.
术语“巯基”指-SH基团。The term "mercapto" refers to the -SH group.
术语“氨基”指-NH 2基团。 The term "amino" refers to the -NH 2 group.
术语“硝基”指-NO 2基团。 The term "nitro" refers to the -NO 2 group.
术语“烷基”是指通式为C nH 2n+1的烃基。该烷基可以是直链或支链的。例如，术语“C 1-C 6烷基”应理解为表示具有1、2、3、4、5或6个碳原子的直链或支链饱和一价烃基。所述烷基是例如甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、2-甲基丁基、1-甲基丁基、1-乙基丙基、1，2-二甲基丙基、新戊基、1，1-二甲基丙基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、1-乙基丁基、3，3-二甲基丁基、2，2-二甲基丁基、1，1-二甲基丁基、2，3-二甲基丁基、1，3-二甲基丁基或1，2-二甲基丁基等。类似地，烷氧基的烷基部分(即烷基)具有上述相同定义。本文所述“C 1-6烷基”可以包含“C 1-3烷基”等范围。 The term "alkyl" refers to a hydrocarbon group of the general formula C n H 2n+1. The alkyl group may be linear or branched. For example, the term "C 1 -C 6 alkyl" should be understood to mean a linear or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl Group, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc. Similarly, the alkyl portion (ie, alkyl) of the alkoxy group has the same definition as above. The "C 1-6 alkyl group" described herein may include the range of "C 1-3 alkyl group" and the like.
术语“烯基”是指由碳原子和氢原子组成的直链或支链的具有至少一个双键的不饱和脂肪族烃基。烯基的非限制性实例包括但不限于乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、异丁烯基、1,3-丁二烯基等。术语“C 2-C 8烯基”应理解为表示直链或支链的一价烃基，其包含一个或多个双键并且具有2、3、4、5、6、7、8个碳原子。 The term "alkenyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, and the like. The term "C 2 -C 8 alkenyl" should be understood to mean a linear or branched monovalent hydrocarbon group containing one or more double bonds and having 2, 3, 4, 5, 6, 7, 8 carbon atoms .
术语“炔基”是指由碳原子和氢原子组成的直链或支链的具有至少一个三键的不饱和脂肪族烃基。炔基的非限制性实例包括但不限于乙炔基(-C≡CH)、1-丙炔基(-C≡C-CH 3)、2-丙炔基(-CH 2-C≡CH)、1,3-丁二炔基(-C≡C-C≡CH)等。术语“C 2-C 4炔基”应理解为表示直链或支链的一价烃基，其包含一个或多个三键并且具有2、3、4个碳原子。 The term "alkynyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), 1-propynyl (-C≡C-CH 3 ), 2-propynyl (-CH 2 -C≡CH), 1,3-Butadiynyl (-C≡CC≡CH) and so on. The term "C 2 -C 4 alkynyl" should be understood to mean a linear or branched monovalent hydrocarbon group containing one or more triple bonds and having 2, 3, or 4 carbon atoms.
术语“烷氧基”是指-O-烷基。根据本发明，合适的烷氧基为C 1-6烷氧基，如C 1-5烷氧基，C 1-4烷氧基，C 1-3烷氧基，包括甲氧基、乙氧基、丙氧基、异丙氧基、异丁氧基、仲丁氧基等。例如术语“C 1-C 6烷氧基”可理解为“C 1-C 6烷基氧基”或“C 1-C 6烷基-O-”，如“C 1-C 6烷氧基”可以包含“C 1-C 3烷氧基”。 The term "alkoxy" refers to -O-alkyl. According to the present invention, suitable alkoxy groups are C 1-6 alkoxy groups, such as C 1-5 alkoxy groups, C 1-4 alkoxy groups, C 1-3 alkoxy groups, including methoxy and ethoxy. Group, propoxy, isopropoxy, isobutoxy, sec-butoxy, etc. For example, the term "C 1 -C 6 alkoxy" can be understood as "C 1 -C 6 alkyloxy" or "C 1 -C 6 alkyl-O-", such as "C 1 -C 6 alkoxy""May include "C 1 -C 3 alkoxy".
术语“环烷基”指完全饱和的并且可以以呈单环、桥环或螺环存在的碳环。除非另有指示，该碳环通常为3至10元环。环烷基非限制性实例包括但不限于环丙基、环丁基、环戊基、环己基、降冰片基(双环[2.2.1]庚基)、双环[2.2.2]辛基、金刚烷基等。术语“C 3-C 6环烷基”应理解为表示饱和的一价单环或双环烃环，其具有3～6个碳原子。如环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基或环癸基，或者是双环烃基如十氢化萘环。 The term "cycloalkyl" refers to a carbocyclic ring that is fully saturated and may exist as a single ring, a bridged ring, or a spiro ring. Unless otherwise indicated, the carbocyclic ring is usually a 3 to 10 membered ring. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantane Alkyl and so on. The term "C 3 -C 6 cycloalkyl" should be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring, which has 3 to 6 carbon atoms. Such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon group such as decalin ring.
术语“环烷基氧基”可以理解为“环烷基-O-”。例如术语“C 3- 6环烷基氧基”可理解为“C 3- 6环烷基-O-”。 The term "cycloalkyloxy" can be understood as "cycloalkyl-O-". Example, the term "C 3 - 6 cycloalkyl group" may be understood as "C 3 - 6 cycloalkyl group -O-."
术语“杂环基”是指完全饱和的或部分不饱和的(整体上不是具有芳香性的杂芳族)一价单环、并环、桥环或螺环基团，其环原子中含有1-5个杂原子或杂原子团(即含有杂原子的原子团)，所述“杂原子或杂原子团”包括但不限于氮原子(N)、氧原子(O)、硫原子(S)、磷原子(P)、硼原子(B)，＝O,＝S,-O-N＝,-C(＝O)O-,-C(＝O)-,-C(＝S)-,-S(＝O) 2-,-S(＝O)-,以及任选被取代的-NH-,-S(＝O)(＝NH)-,-C(＝O)NH-,-C(＝NH)-,-S(＝O) 2NH-,S(＝O)NH-,-NHC(＝O)NH-等。除非另有指示，该杂环通常为含有1至3个独立地选自硫、氧和/或氮的杂原子(如1或2个杂原子)的3至7元环。杂环基的非限制性实例包括但不限于环氧乙烷基、四氢呋喃基、二氢呋喃基、吡咯烷基、N-甲基吡咯烷基、二氢吡咯基、哌啶基、哌嗪基、吡唑烷基、4H-吡喃基、吗啉基、硫代吗啉基、四氢噻吩基等。 The term "heterocyclyl" refers to a fully saturated or partially unsaturated (not aromatic heteroaromatic as a whole) monovalent monocyclic, fused, bridged or spiro ring group, the ring atoms of which contain 1 -5 heteroatoms or heteroatom groups (ie, atomic groups containing heteroatoms), the "heteroatoms or heteroatom groups" include but are not limited to nitrogen atoms (N), oxygen atoms (O), sulfur atoms (S), and phosphorus atoms (P), boron atom (B), =O,=S,-ON=,-C(=O)O-,-C(=O)-,-C(=S)-,-S(=O ) 2 -, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)- , -S(=O) 2 NH-, S(=O)NH-, -NHC(=O)NH- etc. Unless otherwise indicated, the heterocyclic ring is generally a 3 to 7 membered ring containing 1 to 3 heteroatoms (such as 1 or 2 heteroatoms) independently selected from sulfur, oxygen, and/or nitrogen. Non-limiting examples of heterocyclic groups include, but are not limited to, oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl , Pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl, etc.
术语“3-6元杂环基”或“3-6元杂环基氧基”中的“3-6元杂环基”意指饱和的或部分饱和的一价单环或双环烃环，其包含1、2或3个选自N、O和S的杂原子。特别地，所述杂环基可以包括但不限于：3元环，如环氧乙烷环；4元环，如氮杂环丁烷基、氧杂环丁烷基；5元环，如四氢呋喃基、二氧杂环戊烯基、吡咯烷基、咪唑烷基、吡唑烷基、吡咯啉基、4,5-二氢噁唑或2,5-二氢-1H-吡咯基；或6元环，如四氢吡喃基、哌啶基、吗啉基、二噻烷基、硫代吗啉基、哌嗪基或三噻烷基；或部分饱和的6元环如四氢吡啶基、哌啶基、吗啉基、二噻烷基、硫代吗啉基、哌嗪基、三噻烷基、四氢吡啶基或4H-[1,3,4]噻二嗪基。根据本发明，所述杂环基整体上是无芳香性的。“3-6元杂环基”可以包含“5-6元杂环基”，“3-6元杂环基氧基” 可以包含“5-6元杂环基氧基”。The "3-6 membered heterocyclic group" in the term "3-6 membered heterocyclic group" or "3-6 membered heterocyclyloxy group" means a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring, It contains 1, 2 or 3 heteroatoms selected from N, O and S. In particular, the heterocyclic group may include, but is not limited to: a 3-membered ring, such as an oxirane ring; a 4-membered ring, such as azetidinyl and oxetanyl; and a 5-membered ring, such as tetrahydrofuran. Group, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole or 2,5-dihydro-1H-pyrrolyl; or 6 Member ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithiaalkyl; or partially saturated 6-membered ring such as tetrahydropyridinyl , Piperidinyl, morpholinyl, dithiazinyl, thiomorpholinyl, piperazinyl, trithiaalkyl, tetrahydropyridinyl or 4H-[1,3,4]thiadiazinyl. According to the present invention, the heterocyclic group is non-aromatic as a whole. The "3-6 membered heterocyclic group" may include a "5-6 membered heterocyclic group", and the "3-6 membered heterocyclic group" may include a "5-6 membered heterocyclyloxy group".
术语“杂环烷基”是指完全饱和的并且可以以单环、并环、桥环或螺环存在的一价环状基团。除非另有指示，该杂环通常为含有1、2或3个独立地选自硫、氧和/或氮的杂原子(如1或2个杂原子)的3至7元环。3元杂环烷基的实例包括但不限于环氧乙烷基、环硫乙烷基、环氮乙烷基，4元杂环烷基的非限制性实例包括但不限于吖丁啶基、噁丁环基、噻丁环基，5元杂环烷基的实例包括但不限于四氢呋喃基、四氢噻吩基、吡咯烷基、异噁唑烷基、噁唑烷基、异噻唑烷基、噻唑烷基、咪唑烷基、四氢吡唑基，6元杂环烷基的实例包括但不限于哌啶基、四氢吡喃基、四氢噻喃基、吗啉基、哌嗪基、1,4-噻噁烷基、1,4-二氧六环基、硫代吗啉基、1,3-二噻烷基、1,4-二噻烷基，7元杂环烷基的实例包括但不限于氮杂环庚烷基、氧杂环庚烷基、硫杂环庚烷基。本发明前述形成的含B的5-6元杂环烷基的实例包括但不限于
Figure PCTCN2021099653-appb-000029
The term "heterocycloalkyl" refers to a monovalent cyclic group that is fully saturated and may exist as a monocyclic ring, a fused ring, a bridged ring, or a spiro ring. Unless otherwise indicated, the heterocyclic ring is generally a 3 to 7 membered ring containing 1, 2, or 3 heteroatoms (such as 1 or 2 heteroatoms) independently selected from sulfur, oxygen, and/or nitrogen. Examples of 3-membered heterocycloalkyl groups include, but are not limited to, oxirane, sulfidene, and azaethylenyl groups, and non-limiting examples of 4-membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetane Examples of cyclic group, thibutyryl, and 5-membered heterocycloalkyl include but are not limited to tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidine Examples of 6-membered heterocycloalkyl groups include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, Examples of 4-thiaxanyl, 1,4-dioxanyl, thiomorpholinyl, 1,3-dithianyl, 1,4-dithianyl, and 7-membered heterocycloalkyl include But it is not limited to azepanyl, oxepanyl, and thiepanyl. Examples of the aforementioned 5-6 membered heterocycloalkyl containing B formed in the present invention include but are not limited to
Figure PCTCN2021099653-appb-000029
术语“3-6元杂环烷基”或“3-6元杂环烷基氧基”中的“3-6元杂环烷基”是指环原子数为3、4、5或6的所述杂环烷基。“3-6元杂环烷基”可以包含“5-6元杂环烷基”，“3-6元杂环烷基氧基”可以包含“5-6元杂环烷基氧基”。The term "3-6 membered heterocycloalkyl" or "3-6 membered heterocycloalkyloxy" in the term "3-6 membered heterocycloalkyl" refers to all those with 3, 4, 5 or 6 ring atoms.述heterocycloalkyl. The "3-6 membered heterocycloalkyl group" may include a "5-6 membered heterocycloalkyl group", and the "3-6 membered heterocycloalkyloxy group" may include a "5-6 membered heterocycloalkyloxy group".
术语“芳基”是指具有共轭的π电子体系的全碳单环或稠合多环的芳香环基团。例如，芳基可以具有6-20个碳原子，6-14个碳原子或6-12个碳原子。芳基的非限制性实例包括但不限于苯基、萘基、蒽基和1,2,3,4-四氢化萘等。术语“C 6-C 10芳基”应理解为表示具有6～10个碳原子的一价芳香性或部分芳香性的单环或双环烃环。特别是具有6个碳原子的环(“C 6芳基”)，例如苯基；或者具有9个碳原子的环(“C 9芳基”)，例如茚满基或茚基，或者具有10个碳原子的环(“C 10芳基”)，例如四氢化萘基、二氢萘基或萘基。 The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group with a conjugated π-electron system. For example, aryl groups can have 6-20 carbon atoms, 6-14 carbon atoms, or 6-12 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, 1,2,3,4-tetralin and the like. The term "C 6 -C 10 aryl" should be understood to mean a monovalent or partially aromatic monocyclic or bicyclic hydrocarbon ring having 6 to 10 carbon atoms. In particular, a ring having 6 carbon atoms ("C 6 aryl"), such as phenyl; or a ring having 9 carbon atoms ("C 9 aryl"), such as indanyl or indenyl, or having 10 A ring of three carbon atoms ("C 10 aryl"), such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl.
术语“杂芳基”是指单环或稠合多环体系，其中含有至少一个选自N、O、S的环原子，其余环原子为C，并且具有至少一个芳香环。示例性的杂芳基具有单个4至8元环，尤其是单个5至8元环，或包含6至14个，尤其是6至10个环原子的多个稠合环。杂芳基的非限制性实例包括但不限于吡咯基、呋喃基、噻吩基、咪唑基、噁唑基、吡唑基、吡啶基、嘧啶基、吡嗪基、喹啉基、异喹啉基、四唑基、***基、三嗪基、苯并呋喃基、苯并噻吩基、吲哚基、异吲哚基等。术语“5-10元杂芳基”应理解为包括这样的一价单环或双环芳族环系：其具有5～10个环原子且包含1-5个独立选自N、O和S的杂原子。术语“5-10元杂芳基”应理解为包括这样的一价单环或双环芳族环系：其具有5、6、7、8、9或10个环原子，特别是5或6或9或10个环原子，且其包含1-5个或1-3个独立选自N、O和S的杂原子并且，另外在每一种情况下可为苯并稠合的。特别地，杂芳基选自噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑基、噁二唑基、1,2,3-***基、1,2,4-***基、噻二唑基等以及它们的苯并衍生物，例如苯并呋喃基、苯并噻吩基、苯并噻唑基、苯并噁唑基、苯并异噁唑基、苯并咪唑基、苯并***基、吲唑基、吲哚基、异吲哚基等；或吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基等，以及它们的苯并衍生物，例如喹啉基、喹唑啉基、异喹啉基等；或吖辛因基、吲嗪基、嘌呤基等以及它们的苯并衍生物；或噌啉基、酞嗪基、喹唑啉基、喹喔啉基、萘啶基、蝶啶基、咔唑基、吖啶基、吩嗪基、吩噻嗪基、吩噁嗪基等。术语“5-6元杂芳基”指具有5或6个环原子的芳族环系，且其包含1-3个或1-2个独立选自N、O和S的杂原子，实例包括但不限于噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑基、噁二唑基、***基、噻二唑基、吡啶基、哒嗪基、嘧啶基、吡嗪基或三嗪基。“5-10元杂芳基”可以包含“5-6元杂芳基”。The term "heteroaryl" refers to a monocyclic or condensed polycyclic ring system, which contains at least one ring atom selected from N, O, and S, the remaining ring atoms are C, and have at least one aromatic ring. Exemplary heteroaryl groups have a single 4 to 8 membered ring, especially a single 5 to 8 membered ring, or multiple fused rings containing 6 to 14, especially 6 to 10 ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl , Tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc. The term "5-10 membered heteroaryl" should be understood to include monovalent monocyclic or bicyclic aromatic ring systems having 5-10 ring atoms and containing 1-5 independently selected from N, O and S Heteroatom. The term "5-10 membered heteroaryl" should be understood to include monovalent monocyclic or bicyclic aromatic ring systems having 5, 6, 7, 8, 9 or 10 ring atoms, especially 5 or 6 or 9 or 10 ring atoms, and it contains 1-5 or 1-3 heteroatoms independently selected from N, O, and S and, in addition, may be benzo-fused in each case. In particular, the heteroaryl group is selected from the group consisting of thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 1, 2, 3 -Triazolyl, 1,2,4-triazolyl, thiadiazolyl, etc. and their benzo derivatives, such as benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, Benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl Etc., and their benzo derivatives, such as quinolinyl, quinazolinyl, isoquinolinyl, etc.; or azepinyl, indazinyl, purinyl, etc. and their benzo derivatives; or cinnoline Group, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pterridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, etc. The term "5-6 membered heteroaryl" refers to an aromatic ring system having 5 or 6 ring atoms, and it contains 1-3 or 1-2 heteroatoms independently selected from N, O and S, examples include But not limited to thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridine Group, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl. The "5-10 membered heteroaryl group" may include a "5-6 membered heteroaryl group".
术语“治疗有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本发明化合物的用量。构成“治疗有效量”的本发明化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。The term "therapeutically effective amount" means (i) treatment or prevention of a specific disease, condition or disorder, (ii) reduction, amelioration or elimination of one or more symptoms of a specific disease, condition or disorder, or (iii) prevention or delay The amount of the compound of the present invention for the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but it can be routinely determined by those skilled in the art. Determined by its own knowledge and the content of this disclosure.
术语“辅料”是指可药用惰性成分。术语“赋形剂”的种类实例非限制性地包括粘合剂、崩解剂、润滑剂、助流剂、稳定剂、填充剂和稀释剂等。赋形剂能增强药物制剂的操作特性，即通过增加流动性和/或粘着性使制剂更适于直接压缩。适用于上述制剂的典型的“药学上可接受的载体”的实例为：糖类，淀粉类，纤维素及其衍生物等在药物制剂中常用到的辅料。The term "excipients" refers to pharmaceutically acceptable inert ingredients. Examples of types of the term "excipient" include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of the pharmaceutical preparation, that is, make the preparation more suitable for direct compression by increasing fluidity and/or adhesion. Examples of typical "pharmaceutically acceptable carriers" suitable for the above formulations are: sugars, starches, cellulose and its derivatives and other auxiliary materials commonly used in pharmaceutical formulations.
词语“包括(comprise)”、“含有(contain)”或“包含(include)”及其英文变体例如comprises、contains、 includes、comprising、containing或including应理解为开放的、非排他性的意义，即“包括但不限于”。The words "comprise", "contain" or "include" and their English variants such as comprises, contains, includes, comprising, containing or including shall be understood as open, non-exclusive meanings, namely "including but not limited to".
本申请还包括与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本申请化合物。可结合到本申请化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为 2H、 3H、 11C、 13C、 14C、 13N、 15N、 15O、 17O、 18O、 31P、 32P、 35S、 18F、 123I、 125I和 36Cl等。 The present application also includes compounds of the present application that are the same as those described herein, but have one or more atoms replaced by an isotope-labeled atom having an atomic weight or mass number different from those generally found in nature. Examples of isotopes that can be incorporated into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.
某些同位素标记的本申请化合物(例如用 3H及 14C标记的那些)可用于化合物和/或底物组织分布分析中。氚化(即 3H)和碳-14(即 14C)同位素对于由于它们易于制备和可检测性是尤其优选的。正电子发射同位素，诸如 15O、 13N、 11C和 18F可用于正电子发射断层扫描(PET)研究以测定底物占有率。通常可以通过与公开于下文的方案和/或实施例中的那些类似的下列程序，通过同位素标记试剂取代未经同位素标记的试剂来制备同位素标记的本申请化合物。 Certain isotope-labeled compounds of the application (such as those labeled with 3 H and 14 C) can be used in compound and/or substrate tissue distribution analysis. Tritiated (i.e. 3 H) and carbon-14 (i.e. 14 C) isotopes are especially preferred due to their ease of preparation and detectability. Positron emission isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. The isotope-labeled compound of the present application can generally be prepared by the following procedures similar to those disclosed in the schemes and/or examples below, by replacing the non-isotopically-labeled reagent with an isotope-labeled reagent.
本申请的药物组合物可通过将本申请的化合物与适宜的药学上可接受的辅料组合而制备，例如可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等。The pharmaceutical composition of the present application can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable excipients, for example, can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, and powders. , Granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.
给予本申请化合物或其药学上可接受的盐或其药物组合物的典型途径包括但不限于口服、直肠、局部、吸入、肠胃外、舌下、***内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。Typical routes for administering the compound of the application or a pharmaceutically acceptable salt or pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, Intramuscular, subcutaneous, and intravenous administration.
本申请的药物组合物可以采用本领域众所周知的方法制造，如常规的混合法、溶解法、制粒法、乳化法、冷冻干燥法等。The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, emulsification methods, freeze-drying methods, and the like.
在一些实施方案中，药物组合物是口服形式。对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的辅料混合，来配制该药物组合物。这些辅料能使本申请的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，用于对患者的口服给药。In some embodiments, the pharmaceutical composition is in an oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These auxiliary materials enable the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.
可以通过常规的混合、填充或压片方法来制备固体口服组合物。例如，可通过下述方法获得：将所述的活性化合物与固体辅料混合，任选地碾磨所得的混合物，如果需要则加入其它合适的辅料，然后将该混合物加工成颗粒，得到了片剂或糖衣剂的核心。适合的辅料包括但不限于：粘合剂、稀释剂、崩解剂、润滑剂、助流剂、甜味剂或矫味剂等。The solid oral composition can be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain tablets Or the core of the dragee. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents.
药物组合物还可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。The pharmaceutical composition may also be suitable for parenteral administration, such as a sterile solution, suspension or lyophilized product in a suitable unit dosage form.
本文所述的通式(Ⅰ)化合物的所有施用方法中，每天给药的剂量为0.01到200mg/kg体重，优选为0.05到50mg/kg体重，更优选0.1到30mg/kg体重，以单独或分开剂量的形式。In all the methods of administration of the compound of general formula (I) described herein, the daily dose is 0.01 to 200 mg/kg body weight, preferably 0.05 to 50 mg/kg body weight, more preferably 0.1 to 30 mg/kg body weight, either alone or The form of divided doses.
术语“IC 50”是半数最大抑制浓度，表示获得体外生物学过程的50％抑制所需要的特定化合物的浓度。IC 50值可对数换算成pIC 50值(-log IC 50)，其中较高的值以指数方式表示较大的效力。 The term "IC 50" is the half maximal inhibitory concentration, represents the concentration of a particular compound to obtain 50% inhibition in vitro biological process required. The IC 50 value can be logarithmically converted into a pIC 50 value (-log IC 50 ), where a higher value indicates greater efficacy in an exponential manner.
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。The chemical reaction in the specific embodiment of the present invention is completed in a suitable solvent, and the solvent must be suitable for the chemical change of the present invention and the required reagents and materials. In order to obtain the compound of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction schemes on the basis of the existing embodiments.
为清楚起见，进一步用实施例来阐述本申请，但是实施例并非限制本申请的范围。本申请所使用的所有试剂是市售的，无需进一步纯化即可使用。For the sake of clarity, examples are further used to illustrate the application, but the examples do not limit the scope of the application. All reagents used in this application are commercially available and can be used without further purification.
具体实施方式detailed description
以下实施例详细说明发明的技术方案，但本发明的保护范围包括但不限于此。本文已经详细地描述了本发明，其中也公开了其具体实施方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种改变和改进将是显而易见的。本申请所使用的所有试剂是市售的，无需进一步纯化即可使用。The following examples illustrate the technical solutions of the invention in detail, but the protection scope of the invention includes but is not limited thereto. The present invention has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, various changes and improvements will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. It is obvious. All reagents used in this application are commercially available and can be used without further purification.
化合物的结构是通过核磁共振(NMR)和/或质谱(MS)来确定的。NMR位移的单位为10 -6(ppm)。NMR测定的溶剂为氘代二甲基亚砜、氘代氯仿、氘代甲醇等，内标为四甲基硅烷(TMS)。“IC 50”指半数抑制浓度，指达到最大抑制效果一半时的浓度。 The structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). The unit of NMR shift is 10 -6 (ppm). The solvent for NMR measurement is deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS). "IC 50" refers to the half maximal inhibitory concentration, refers to the maximum concentration at which half of the inhibitory effect.
实施例1：3-(((1R，3R)-1-(2,6-二氟-4-(((S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚烷-7-基)氨基)苯基) -3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇Example 1: 3-(((1R,3R)-1-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4] Heptane-7-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2, 2-Difluoropropane-1-ol
Figure PCTCN2021099653-appb-000030
Figure PCTCN2021099653-appb-000030
步骤1：(S)-(5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-基)氨基甲酸叔丁酯的合成Step 1: Synthesis of tert-butyl (S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)carbamate
Figure PCTCN2021099653-appb-000031
Figure PCTCN2021099653-appb-000031
将(S)-(5-氮杂螺[2.4]庚烷-7-基)氨基甲酸叔丁酯(2.12g，10.0mmol)加入到40mL乙腈(MeCN)中，加入碳酸钾(2.76g,20.0mmol)，3-氟-1-碘代丙烷(2.26g,12.0mmol)，加热至80℃搅拌6小时后反应完毕。将反应液过滤，滤饼用乙腈洗涤，合并滤液后柱层析得标题化合物。(S)-(5-Azaspiro[2.4]heptane-7-yl) tert-butyl carbamate (2.12g, 10.0mmol) was added to 40mL acetonitrile (MeCN), potassium carbonate (2.76g, 20.0 mmol), 3-fluoro-1-iodopropane (2.26g, 12.0mmol), heated to 80°C and stirred for 6 hours, and the reaction is complete. The reaction solution was filtered, the filter cake was washed with acetonitrile, and the combined filtrates were subjected to column chromatography to obtain the title compound.
步骤2：(S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺的合成Step 2: Synthesis of (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine
Figure PCTCN2021099653-appb-000032
Figure PCTCN2021099653-appb-000032
将(S)-(5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-基)氨基甲酸叔丁酯(1.00g，3.68mmol)加入到10mL二氯甲烷中，加入4mol/L的氯化氢二氧六环溶液5mL，室温搅拌2小时后反应完毕。将反应液浓缩后加入饱和碳酸钠水溶液调节pH至pH7-8，二氯甲烷萃取，合并有机层，无水硫酸钠干燥，浓缩后得标题化合物。(S)-(5-(3-Fluoropropyl)-5-azaspiro[2.4]hept-7-yl) tert-butyl carbamate (1.00g, 3.68mmol) was added to 10mL of dichloromethane, Add 5 mL of 4 mol/L hydrogen chloride dioxane solution, stir at room temperature for 2 hours, and then complete the reaction. After the reaction solution was concentrated, saturated aqueous sodium carbonate was added to adjust the pH to pH 7-8, extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the title compound.
步骤3：3-(((1R，3R)-1-(2,6-二氟-4-(((S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚烷-7-基)氨基)苯基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇的合成Step 3: 3-(((1R,3R)-1-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hepta (Alk-7-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2 -Synthesis of difluoropropane-1-ol
Figure PCTCN2021099653-appb-000033
Figure PCTCN2021099653-appb-000033
按照WO2016097072A1实施例340制备3-((1R，3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇。将3-((1R，3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇(470mg，1.00mmol)，甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯)(2-氨基-1,1-联苯-2-基)钯(II)(Brettphos Pd G3)(90.0mg，0.10mmol)，叔丁醇钠(t-BuONa)(192mg，2.00mmol)，(S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺(172mg，1.00mmol)加入到5mL甲苯中，氩气保护下加热至80℃搅拌4小时后反应完毕，将反应液加入到50mL水中，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，柱层析得标题化合物。According to WO2016097072A1 Example 340, 3-((1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-1,3,4,9-tetrahydro-2H- Pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol. The 3-((1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3, 4-b]Indol-2-yl)-2,2-difluoropropane-1-ol (470mg, 1.00mmol), methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy -2,4,6-Triisopropyl-1,1-biphenyl) (2-amino-1,1-biphenyl-2-yl)palladium(II) (Brettphos Pd G3) (90.0mg, 0.10mmol ), sodium tert-butoxide (t-BuONa) (192mg, 2.00mmol), (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine (172mg, 1.00mmol) ) Was added to 5mL of toluene, heated to 80°C under the protection of argon, and stirred for 4 hours. After the reaction was completed, the reaction solution was added to 50mL of water, extracted with ethyl acetate, combined the organic layers, dried over anhydrous sodium sulfate, and column chromatography to obtain the title Compound.
1H NMR(400MHz,DMSO-d 6)δ10.61(s,1H),7.46-7.45(m,1H),7.28-7.26(m,1H),7.09-7.00(m,2H),6.23-6.31(m,2H),5.36-5.33(m,1H),5.12(s,1H),4.65-4.62(m,1H),4.52-4.50(m,1H),3.80-3.69(m,2H),3.53-3.48(m,2H),3.40-3.37(m,1H),3.22-3.11(m,3H),2.92-2.87(m,1H),2.72-2.63(m,3H),2.50-2.42(m,3H),1.91-1.85(m,2H),1.16-1.14(m,3H),0.85-0.75(m,1H),0.72-0.68(m,2H),0.59-0.52(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 10.61 (s, 1H), 7.46-7.45 (m, 1H), 7.28-7.26 (m, 1H), 7.09-7.00 (m, 2H), 6.23-6.31 (m,2H),5.36-5.33(m,1H),5.12(s,1H),4.65-4.62(m,1H),4.52-4.50(m,1H),3.80-3.69(m,2H),3.53 -3.48(m,2H),3.40-3.37(m,1H),3.22-3.11(m,3H),2.92-2.87(m,1H),2.72-2.63(m,3H), 2.50-2.42(m, 3H), 1.91-1.85 (m, 2H), 1.16-1.14 (m, 3H), 0.85-0.75 (m, 1H), 0.72-0.68 (m, 2H), 0.59-0.52 (m, 1H).
LC/MS(m/z,MH +):563.3。 LC/MS (m/z, MH + ): 563.3.
实施例2：(S)-N-(3,5-二氟-4-((1R，3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)苯基)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺Example 2: (S)-N-(3,5-Difluoro-4-((1R,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3 ,4,9-Tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]hepta- 7-amine
Figure PCTCN2021099653-appb-000034
Figure PCTCN2021099653-appb-000034
按照WO2016097072A1实施例304制备(1R，3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚。然后参照上述实施例1步骤3，不同的是将实施例1步骤3中的3-((1R，3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇替换为(1R，3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚，同法制得标题化合物。Prepare (1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2 according to Example 304 of WO2016097072A1 ,3,4,9-Tetrahydro-1H-pyrido[3,4-b]indole. Then refer to step 3 of Example 1 above. The difference is that 3-((1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl in step 3 of Example 1 -1,3,4,9-Tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane-1-ol is replaced with (1R,3R)- 1-(4-Bromo-2,6-difluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H- Pyrido[3,4-b]indole, the title compound was prepared in the same way.
1H NMR(400MHz,DMSO-d 6)δ10.58(s,1H),7.39-7.38(m,1H),7.21-7.19(m,1H),7.02-6.92(m,3H),6.23-6.20(m,2H),5.09(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.75-3.70(m,1H),3.47-3.41(m,2H),3.35-3.32(m,1H),3.05-3.01(m,1H),2.97-2.91(m,1H),2.84-2.78(m,1H),2.62-2.55(m,2H),2.48-2.38(m,3H),1.85-1.74(m,2H),1.12-1.10(m,3H),0.74-0.70(m,1H),0.64-0.61(m,2H),0.48-0.43(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 10.58 (s, 1H), 7.39-7.38 (m, 1H), 7.21-7.19 (m, 1H), 7.02-6.92 (m, 3H), 6.23-6.20 (m,2H),5.09(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.75-3.70(m,1H),3.47-3.41(m,2H),3.35 -3.32(m,1H),3.05-3.01(m,1H),2.97-2.91(m,1H),2.84-2.78(m,1H),2.62-2.55(m,2H),2.48-2.38(m, 3H), 1.85-1.74 (m, 2H), 1.12-1.10 (m, 3H), 0.74-0.70 (m, 1H), 0.64-0.61 (m, 2H), 0.48-0.43 (m, 1H).
LC/MS(m/z,MH +):551.3。 LC/MS (m/z, MH + ): 551.3.
实施例3：2,2-二氟-3-((1S，3R)-1-(5-(((S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-基)氨基)吡啶-2-基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)丙-1-醇Example 3: 2,2-Difluoro-3-((1S,3R)-1-(5-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hepta -7-yl)amino)pyridin-2-yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propan- 1-alcohol
Figure PCTCN2021099653-appb-000035
Figure PCTCN2021099653-appb-000035
按照WO2016097072A1实施例340制备(R)-3-(((1-(1H-吲哚-3-基)丙-2-基)氨基)-2,2-二氟丙烷-1-醇。将(R)-3-(((1-(1H-吲哚-3-基)丙-2-基)氨基)-2,2-二氟丙烷-1-醇(2.68g,10.0mmol)和5-溴-2-吡啶甲醛(1.84g,10.0mmol)加入到20mL甲苯(Tol)中，加入1mL醋酸(AcOH)，加热至90℃搅拌12小时后反应完毕，将反应液浓缩后用100mL水稀释，饱和碳酸氢钠溶液调节pH至8-9，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，过滤浓缩后柱层析纯化得到3-((1R,3R)-1-(5-溴-吡啶-2-基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇。然后参照上述实施例1步骤3，不同的是将实施例1中的3-((1R，3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇替换为3-((1R,3R)-1-(5-溴-吡啶-2-基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇，同法制得标题化合物。(R)-3-(((1-(1H-indol-3-yl)prop-2-yl)amino)-2,2-difluoropropan-1-ol was prepared according to WO2016097072A1 Example 340. R)-3-(((1-(1H-indol-3-yl)prop-2-yl)amino)-2,2-difluoropropane-1-ol (2.68g, 10.0mmol) and 5- Bromo-2-pyridinecarbaldehyde (1.84g, 10.0mmol) was added to 20mL of toluene (Tol), 1mL of acetic acid (AcOH) was added, and the reaction was completed after heating to 90°C and stirring for 12 hours. The reaction solution was concentrated and diluted with 100mL of water. Adjust the pH to 8-9 with saturated sodium bicarbonate solution, extract with ethyl acetate, combine the organic layers, dry with anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography to obtain 3-((1R,3R)-1-(5-bromo -Pyridin-2-yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane -1-ol. Then refer to step 3 of Example 1 above, except that 3-((1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3 in Example 1 -Methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane-1-ol replaced with 3-( (1R,3R)-1-(5-Bromo-pyridin-2-yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole- 2-yl)-2,2-difluoropropane-1-ol, the title compound was prepared in the same way.
1H NMR(400MHz,DMSO-d 6)δ10.60(s,1H),7.91-7.90(m,1H),7.40-7.35(m,1H),7.25-7.20(m,2H),7.00-6.96(m,4H),5.10(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.79-3.69(m,2H),3.53-3.45(m,2H),3.40-3.32(m,1H),3.22-3.17(m,3H),2.92-2.80(m,1H),2.70-2.63(m,3H),2.50-2.46(m,3H),1.94-1.85(m,2H),1.16-1.10(m,3H),0.82-0.75(m,1H),0.75-0.68(m,2H),0.60-0.52(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 10.60 (s, 1H), 7.91-7.90 (m, 1H), 7.40-7.35 (m, 1H), 7.25-7.20 (m, 2H), 7.00-6.96 (m,4H),5.10(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.79-3.69(m,2H),3.53-3.45(m,2H),3.40 -3.32 (m, 1H), 3.22-3.17 (m, 3H), 2.92-2.80 (m, 1H), 2.70-2.63 (m, 3H), 2.50-2.46 (m, 3H), 1.94-1.85 (m, 2H), 1.16-1.10 (m, 3H), 0.82-0.75 (m, 1H), 0.75-0.68 (m, 2H), 0.60-0.52 (m, 1H).
LC/MS(m/z,MH +):528.3。 LC/MS (m/z, MH + ): 528.3.
实施例4：(S)-5-(3-氟丙基)-N-(6-((1S，3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)吡啶-3-基)-5-氮杂螺[2.4]庚-7-胺Example 4: (S)-5-(3-fluoropropyl)-N-(6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)- 2,3,4,9-Tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-yl)-5-azaspiro[2.4]hepta-7-amine
Figure PCTCN2021099653-appb-000036
Figure PCTCN2021099653-appb-000036
制备方法与实施例3类似，不同的是将实施例3的起始物料(R)-3-(((1-(1H-吲哚-3-基)丙-2-基)氨基)-2,2-二氟丙烷-1-醇替换为(R)-1-(1H-吲哚-3-基)-N-(2,2,2-三氟乙基)丙-2-胺，同法制得标题化合物。The preparation method is similar to that of Example 3, except that the starting material (R)-3-(((1-(1H-indol-3-yl)propan-2-yl)amino)-2 , 2-Difluoropropane-1-ol is replaced with (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine, the same Method to obtain the title compound.
1H NMR(400MHz,DMSO-d 6)δ10.58(s,1H),7.90-7.88(m,1H),7.42-7.40(m,1H),7.27-7.25(m,1H),7.05-6.96(m,4H),4.89(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.75-3.70(m,1H),3.47-3.41(m,1H),3.35-3.32(m,1H),3.04-3.00(m,1H),2.96-2.89(m,1H),2.84-2.78(m,1H),2.62-2.55(m,2H),2.48-2.38(m,3H),1.85-1.74(m,2H),1.14-1.13(m,3H),0.75-0.73(m,1H),0.64-0.61(m,2H),0.48-0.43(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 10.58 (s, 1H), 7.90-7.88 (m, 1H), 7.42-7.40 (m, 1H), 7.27-7.25 (m, 1H), 7.05-6.96 (m, 4H), 4.89 (s, 1H), 4.56-4.53 (m, 1H), 4.44-4.41 (m, 1H), 3.75-3.70 (m, 1H), 3.47-3.41 (m, 1H), 3.35 -3.32(m,1H),3.04-3.00(m,1H),2.96-2.89(m,1H),2.84-2.78(m,1H),2.62-2.55(m,2H),2.48-2.38(m, 3H), 1.85-1.74 (m, 2H), 1.14-1.13 (m, 3H), 0.75-0.73 (m, 1H), 0.64-0.61 (m, 2H), 0.48-0.43 (m, 1H).
13C NMR(400MHz,DMSO-d 6)δ147.24,143.56,136.57,133.91,132.84,126.62,126.51(q,J＝278.2Hz),122.64,120.60,118.67,118.15,117.57,111.09,107.61,82.29(d,J＝161.4Hz),64.30,62.33,61.42,55.99,51.69(d,J＝5.7Hz),48.66,48.24(q,J＝29.9Hz),28.95(d,J＝19.3Hz),26.16,25.32,17.64,13.28,7.94. 13 C NMR (400MHz, DMSO-d 6 ) δ147.24,143.56,136.57,133.91,132.84,126.62,126.51(q,J=278.2Hz),122.64,120.60,118.67,118.15,117.57,111.09,107.61,82.29(d ,J=161.4Hz),64.30,62.33,61.42,55.99,51.69(d,J=5.7Hz),48.66,48.24(q,J=29.9Hz),28.95(d,J=19.3Hz),26.16,25.32 , 17.64, 13.28, 7.94.
LC/MS(m/z,MH +):516.3。 LC/MS (m/z, MH + ): 516.3.
实施例5：3-((6S，8R)-6-(2,6-二氟-4-(((S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-基)氨基)苯基)-8-甲基-3,6,8,9-四氢-7H-吡唑并[4,3-f]异喹啉-7-基)-2,2二氟丙烷-1-醇Example 5: 3-((6S,8R)-6-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptan -7-yl)amino)phenyl)-8-methyl-3,6,8,9-tetrahydro-7H-pyrazolo[4,3-f]isoquinolin-7-yl)-2, 2Difluoropropane-1-ol
Figure PCTCN2021099653-appb-000037
Figure PCTCN2021099653-appb-000037
步骤1：(7S)-N-(4-((6S，8R)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉-6-基)-3,5-二氟苯基)-5-(3-氟丙基)-5-氮杂螺[2.4]庚7胺的合成Step 1: (7S)-N-(4-((6S,8R)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)- 8-Methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline-6- Yl)-3,5-difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane 7 amine
Figure PCTCN2021099653-appb-000038
Figure PCTCN2021099653-appb-000038
按照WO2018077630A1实施例5制备方法制备(6S，8R)-6-(4-溴-2,6-二氟苯基)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉。然后，将(6S，8R)-6-(4-溴-2,6-二氟苯基)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉(793mg，1.00mmol)，Brettphos Pd G3(90.0mg，0.10mmol)，叔丁醇钠(192mg，2.00mmol)，(S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺(172mg，1.00mmol)加入到5mL甲苯中，氩气保护下加热至80℃搅拌6小时后反应完毕，将反应液加入到50mL水中，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，柱层析得标题化合物。Prepare (6S, 8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-((tert-butyldiphenylsilyl)oxy group according to the preparation method of Example 5 of WO2018077630A1 )-2,2-Difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[ 4,3-f] Isoquinoline. Then, (6S, 8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2 -Difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f ]Isoquinoline (793mg, 1.00mmol), Brettphos Pd G3 (90.0mg, 0.10mmol), sodium tert-butoxide (192mg, 2.00mmol), (S)-5-(3-fluoropropyl)-5-nitrogen Heterspiro[2.4]heptan-7-amine (172mg, 1.00mmol) was added to 5mL of toluene, heated to 80°C under argon protection, and stirred for 6 hours. After the reaction was completed, the reaction solution was added to 50mL of water and extracted with ethyl acetate. Combine the organic layers, dry with anhydrous sodium sulfate, and column chromatography to obtain the title compound.
步骤2：3-((6S，8R)-6-(2,6-二氟-4-(((S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-基)氨基)苯基)-8-甲基-3,6,8,9-四氢-7H-吡唑并[4,3-f]异喹啉-7-基)-2,2-二氟丙烷-1-醇的合成Step 2: 3-((6S,8R)-6-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hepta- 7-yl)amino)phenyl)-8-methyl-3,6,8,9-tetrahydro-7H-pyrazolo[4,3-f]isoquinolin-7-yl)-2,2 -Synthesis of difluoropropane-1-ol
Figure PCTCN2021099653-appb-000039
Figure PCTCN2021099653-appb-000039
将(7S)-N-(4-((6S，8R)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉-6-基)-3,5-二氟苯基)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺(400mg，0.50mmol)加入到10mL甲醇(MeOH)中，加入4mol/L的氯化氢二氧六环溶液5mL，室温搅拌1小时后反应完毕。将反应液浓缩后加入饱和碳酸钠水溶液调节pH至7-8，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，浓缩后柱层析得标题化合物。(7S)-N-(4-((6S,8R)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8- Methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl) -3,5-Difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine (400mg, 0.50mmol) was added to 10mL methanol (MeOH), added 5 mL of 4mol/L hydrogen chloride dioxane solution was stirred at room temperature for 1 hour and the reaction was completed. After the reaction solution was concentrated, saturated aqueous sodium carbonate solution was added to adjust the pH to 7-8, extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatographed to obtain the title compound.
1H NMR(400MHz,DMSO-d 6)δ12.97(s,1H),8.05(s,1H),7.22-7.20(m,1H),6.71-6.69(m,1H),6.20-6.14(m,3H),5.28-5.25(m,1H),5.07(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.72-3.64(m,2H),3.54-3.50(m,1H),3.18-3.00(m,3H),2.90-2.85(m,1H),2.67-2.60(m,2H),2.48-2.37(m,6H),1.85-1.74(m,2H),1.03-1.02(m,3H),0.75-0.70(m,1H),0.64-0.60(m,2H),0.47-0.42(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 12.97 (s, 1H), 8.05 (s, 1H), 7.22-7.20 (m, 1H), 6.71-6.69 (m, 1H), 6.20-6.14 (m ,3H),5.28-5.25(m,1H),5.07(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.72-3.64(m,2H),3.54-3.50 (m, 1H), 3.18-3.00 (m, 3H), 2.90-2.85 (m, 1H), 2.67-2.60 (m, 2H), 2.48-2.37 (m, 6H), 1.85-1.74 (m, 2H) ,1.03-1.02(m,3H),0.75-0.70(m,1H),0.64-0.60(m,2H),0.47-0.42(m,1H).
LC/MS(m/z,MH +):564.3。 LC/MS (m/z, MH + ): 564.3.
实施例6：2,2-二氟-3-(((6S，8R)-6-(5-(((S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚基-7-基)氨基]吡啶]-2-基)-8-甲基-3,6,8,9-四氢-7H-吡唑并[4,3-f]异喹啉-7-基)丙-1-醇Example 6: 2,2-Difluoro-3-(((6S,8R)-6-(5-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4] Heptyl-7-yl)amino]pyridine]-2-yl)-8-methyl-3,6,8,9-tetrahydro-7H-pyrazolo[4,3-f]isoquinoline-7 -Base) propan-1-ol
Figure PCTCN2021099653-appb-000040
Figure PCTCN2021099653-appb-000040
Figure PCTCN2021099653-appb-000041
Figure PCTCN2021099653-appb-000041
按照WO2018077630A1实施例16制备方法制备起始物料(6S，8R)-6-(5-溴吡啶-2-基)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉。然后参考实施例5的制备方法，不同的是将实施例5步骤1中的(6S，8R)-6-(4-溴-2,6-二氟苯基)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉替换为(6S，8R)-6-(5-溴吡啶-2-基)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉，同法制得标题化合物。The starting material (6S, 8R)-6-(5-bromopyridin-2-yl)-7-(3-((tert-butyldiphenylsilyl)oxy) was prepared according to the preparation method of Example 16 of WO2018077630A1 -2,2-Difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4 ,3-f] isoquinoline. Then refer to the preparation method of Example 5, except that (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-(( Tert-Butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8 ,9-Tetrahydro-3H-pyrazolo[4,3-f]isoquinoline is replaced with (6S,8R)-6-(5-bromopyridin-2-yl)-7-(3-((tert (Butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8, 9-Tetrahydro-3H-pyrazolo[4,3-f]isoquinoline, the title compound was prepared in the same way.
1H NMR(400MHz,DMSO-d 6)δ13.06(s,1H),8.04(s,1H),7.84-7.83(m,1H),7.24-7.22(m,1H),6.89-6.79(m,3H),5.70-5.68(m,1H),4.92(s,1H),4.55-4.52(m,1H),4.43-4.40(m,1H),3.72-3.64(m,2H),3.54-3.50(m,1H),3.18-3.00(m,3H),2.90-2.85(m,1H),2.67-2.60(m,2H),2.48-2.37(m,6H),1.85-1.74(m,2H),1.03-1.02(m,3H),0.75-0.70(m,1H),0.64-0.60(m,2H),0.47-0.42(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 13.06 (s, 1H), 8.04 (s, 1H), 7.84-7.83 (m, 1H), 7.24-7.22 (m, 1H), 6.89-6.79 (m ,3H),5.70-5.68(m,1H),4.92(s,1H),4.55-4.52(m,1H),4.43-4.40(m,1H),3.72-3.64(m,2H),3.54-3.50 (m, 1H), 3.18-3.00 (m, 3H), 2.90-2.85 (m, 1H), 2.67-2.60 (m, 2H), 2.48-2.37 (m, 6H), 1.85-1.74 (m, 2H) ,1.03-1.02(m,3H),0.75-0.70(m,1H),0.64-0.60(m,2H),0.47-0.42(m,1H).
LC/MS(m/z,MH +):529.3。 LC/MS (m/z, MH + ): 529.3.
实施例7：(S)-5-(3-氟丙基)-N-(6-((6S，8R)-8-甲基-7-(2,2,2-三氟乙基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉-6-基)吡啶-3-基)-5-氮杂螺[2.4]庚-7-胺Example 7: (S)-5-(3-fluoropropyl)-N-(6-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)- 6,7,8,9-Tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)pyridin-3-yl)-5-azaspiro[2.4]hept-7- amine
Figure PCTCN2021099653-appb-000042
Figure PCTCN2021099653-appb-000042
按照WO2018077630A1实施例17的制备方法制备起始物料(6S，8R)-6-(5-溴吡啶-2-基)-8-甲基-3-(四氢-2H-吡喃-2-基)-7-(2,2,2-三氟乙基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉。参考上述实施例5的制备方法，不同的是将实施例5步骤1中的(6S，8R)-6-(4-溴-2,6-二氟苯基)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉替换为(6S，8R)-6-(5-溴吡啶-2-基)-8-甲基-3-(四氢-2H-吡喃-2-基)-7-(2,2,2-三氟乙基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉，同法制得标题化合物。The starting material (6S, 8R)-6-(5-bromopyridin-2-yl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl) was prepared according to the preparation method of WO2018077630A1 Example 17 )-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline. With reference to the preparation method of Example 5 above, the difference is that (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-(( Tert-Butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8 ,9-Tetrahydro-3H-pyrazolo[4,3-f]isoquinoline is replaced by (6S,8R)-6-(5-bromopyridin-2-yl)-8-methyl-3-( Tetrahydro-2H-pyran-2-yl)-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f ] Isoquinoline, the title compound was obtained by the same method.
1H NMR(400MHz,DMSO-d 6)δ12.99(s,1H),8.06(s,1H),7.83(s,1H),7.23-7.21(m,1H),6.98-6.88(m,2H),6.81-6.79(m,1H),5.71-5.69(m,1H),4.92(s,1H),4.55-4.52(m,1H),4.43-4.40(m,1H),3.75-3.70(m,1H),3.52-3.45(m,2H),3.09-3.05(m,2H),3.00-2.94(m,1H),2.87-2.81(m,1H),2.65-2.60(m,1H),2.48-2.45(m,3H),2.39-2.35(m,1H),1.85-1.72(m,2H),1.09-1.08(m,3H),0.77-0.72(m,1H),0.64-0.59(m,2H),0.45-0.40(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 12.99 (s, 1H), 8.06 (s, 1H), 7.83 (s, 1H), 7.23-7.21 (m, 1H), 6.98-6.88 (m, 2H) ),6.81-6.79(m,1H),5.71-5.69(m,1H),4.92(s,1H),4.55-4.52(m,1H),4.43-4.40(m,1H),3.75-3.70(m , 1H), 3.52-3.45 (m, 2H), 3.09-3.05 (m, 2H), 3.00-2.94 (m, 1H), 2.87-2.81 (m, 1H), 2.65-2.60 (m, 1H), 2.48 -2.45(m,3H),2.39-2.35(m,1H),1.85-1.72(m,2H),1.09-1.08(m,3H),0.77-0.72(m,1H),0.64-0.59(m, 2H), 0.45-0.40 (m, 1H).
LC/MS(m/z,MH +):517.3。 LC/MS (m/z, MH + ): 517.3.
实施例8：(S)-N-(3,5-二氟-4-((6S，8R)-8-甲基-7-(2,2,2-三氟乙基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉-6-基)苯基)-5-(3-氟丙基)-5-氮杂螺[2.4]庚烷-7-胺Example 8: (S)-N-(3,5-difluoro-4-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7 ,8,9-Tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4] Heptane-7-amine
Figure PCTCN2021099653-appb-000043
Figure PCTCN2021099653-appb-000043
按照WO2019223715A1实施例27第一步、第二步制备方法制备(6S,8R)-6-(4-溴-2，6-二氟苯基)-8-甲基-3-(四氢-2H-吡喃-2-基)-7-(2,2,2-三氟乙基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉。然后，参考实施例5的制备方法，不同的是将实施例5步骤1中的(6S，8R)-6-(4-溴-2,6-二氟苯基)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉替换为(6S，8R)-6-(4-溴-2,6-二氟苯基)-8-甲基-3-(四氢-2H-吡喃-2-基)-7-(2,2,2-三氟乙基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉，同法制得标题化合物。Prepare (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-8-methyl-3-(tetrahydro-2H) according to the first and second preparation methods of Example 27 of WO2019223715A1 -Pyran-2-yl)-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline . Then, referring to the preparation method of Example 5, the difference is that (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-( (Tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7, Replaced 8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline with (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-8-methan Base-3-(tetrahydro-2H-pyran-2-yl)-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[ 4,3-f] isoquinoline, the title compound was obtained in the same way.
1H NMR(400MHz,DMSO-d 6)δ13.05(s,1H),8.11(s,1H),7.29-7.27(m,1H),6.80-6.78(m,1H),6.26-6.21(m,3H),5.20(s,1H),4.62-4.59(m,1H),4.50-4.47(m,1H),3.76-3.73(m,1H),3.54-3.51(m,1H),3.27-3.20(m,2H),3.08-3.06(m,1H),2.97-2.93(m,2H),2.68-2.66(m,1H),2.54-2.42(m,4H),1.89-1.79(m,2H),1.14-1.13(m,3H),0.78-0.75(m,1H),0.70-0.66(m,2H),0.53-0.50(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 13.05 (s, 1H), 8.11 (s, 1H), 7.29-7.27 (m, 1H), 6.80-6.78 (m, 1H), 6.26-6.21 (m ,3H),5.20(s,1H),4.62-4.59(m,1H),4.50-4.47(m,1H),3.76-3.73(m,1H),3.54-3.51(m,1H),3.27-3.20 (m,2H),3.08-3.06(m,1H),2.97-2.93(m,2H),2.68-2.66(m,1H),2.54-2.42(m,4H),1.89-1.79(m,2H) , 1.14-1.13 (m, 3H), 0.78-0.75 (m, 1H), 0.70-0.66 (m, 2H), 0.53-0.50 (m, 1H).
LC/MS(m/z,MH +):552.2。 LC/MS (m/z, MH + ): 552.2.
实施例9：(S)-N-(4-((6S，8R)-7-(2,2-二氟丙基)-8-甲基-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉-6-基)-3,5-二氟苯基)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺Example 9: (S)-N-(4-((6S,8R)-7-(2,2-difluoropropyl)-8-methyl-6,7,8,9-tetrahydro-3H -Pyrazolo[4,3-f]isoquinolin-6-yl)-3,5-difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]hepta- 7-amine
Figure PCTCN2021099653-appb-000044
Figure PCTCN2021099653-appb-000044
步骤1：(R)-3-(2-(((2,2-二氟丙基)氨基)丙基)-2-甲基苯胺的合成Step 1: Synthesis of (R)-3-(2-(((2,2-difluoropropyl)amino)propyl)-2-methylaniline
Figure PCTCN2021099653-appb-000045
Figure PCTCN2021099653-appb-000045
将(R)-N-(1-(3-溴-2-甲基苯基)丙-2-基)-2,2-二氟丙-1-胺(14.3g)，二苯甲酮亚胺(10.0g)，三(二亚苄基丙酮)二钯(Pd 2(dba) 3)(0.42g)，1,1'-联萘-2,2'-双二苯膦(BINAP)(0.57g)，叔丁醇钠(6.60g)加入到60mL甲苯中，氩气保护下加热至80℃搅拌5小时后反应完毕。将反应液旋干，加入100mL二氯甲烷和稀盐酸溶液(2N，100mL)，剧烈搅拌2小时后反应完毕，分层，二氯甲烷层弃去，水层用5N氢氧化钠水溶液调节pH至10-11，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，有机层浓缩后得粗品油状物12.0g。 Add (R)-N-(1-(3-bromo-2-methylphenyl)prop-2-yl)-2,2-difluoroprop-1-amine (14.3g), benzophenone sub Amine (10.0g), tris(dibenzylideneacetone)dipalladium (Pd 2 (dba) 3 ) (0.42g), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (BINAP) ( 0.57g), sodium tert-butoxide (6.60g) was added to 60mL of toluene, heated to 80°C under the protection of argon, stirred for 5 hours, and then the reaction was completed. The reaction solution was spin-dried, 100 mL of dichloromethane and dilute hydrochloric acid solution (2N, 100 mL) were added, and the reaction was completed after vigorous stirring for 2 hours. The layers were separated, the dichloromethane layer was discarded, and the aqueous layer was adjusted to pH with 5N aqueous sodium hydroxide solution. 10-11, extract with ethyl acetate, combine the organic layers, dry with anhydrous sodium sulfate, and concentrate the organic layer to obtain 12.0 g of crude oil.
步骤2：(1S，3R)-1-(4-溴-2,6-二氟苯基)-2-(2,2-二氟丙基)-3,5-二甲基-1,2,3,4-四氢异喹啉-6-胺的合成Step 2: (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(2,2-difluoropropyl)-3,5-dimethyl-1,2 Synthesis of ,3,4-Tetrahydroisoquinoline-6-amine
Figure PCTCN2021099653-appb-000046
Figure PCTCN2021099653-appb-000046
将(R)-3-(2-(((2,2-二氟丙基)氨基)丙基)-2-甲基苯胺(12.0g)，4-溴-2,6-二氟苯甲醛(13.0g)加入到50mL醋酸中，加入5mL水，60℃搅拌，TLC检测反应，反应完毕后将反应液旋干，加入100ml水，加入饱和碳酸氢钠水溶液调节pH至8～9，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，浓缩后柱层析石油醚：乙酸乙酯＝10:1(V:V)得油状物13.9g。(R)-3-(2-(((2,2-difluoropropyl)amino)propyl)-2-methylaniline (12.0g), 4-bromo-2,6-difluorobenzaldehyde (13.0g) was added to 50mL of acetic acid, 5mL of water was added, stirred at 60℃, and the reaction was detected by TLC. After the reaction was completed, the reaction solution was spin-dried, 100ml of water was added, and saturated aqueous sodium bicarbonate solution was added to adjust the pH to 8-9, ethyl acetate Ester extraction, combined organic layers, dried over anhydrous sodium sulfate, concentrated, and column chromatography petroleum ether: ethyl acetate = 10:1 (V:V) to obtain 13.9 g of oil.
步骤3：(6S，8R)-6-(4-溴-2,6-二氟苯基)-7-(2,2-二氟丙基)-8-甲基-6,7,8,9-四氢-3H-吡唑并[4，3-f]异喹啉的合成Step 3: (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(2,2-difluoropropyl)-8-methyl-6,7,8, Synthesis of 9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline
Figure PCTCN2021099653-appb-000047
Figure PCTCN2021099653-appb-000047
将(1S，3R)-1-(4-溴-2,6-二氟苯基)-2-(2,2-二氟丙基)-3,5-二甲基-1,2,3,4-四氢异喹啉-6-胺(3.56g)加入到40mL丙酸中，冷却至-20℃，滴加2N亚硝酸钠的水溶液(560mg)，搅拌1小时后反应完毕，向反应液中加入50mL乙酸乙酯，随后缓慢滴加饱和碳酸氢钠溶液调节pH至8～9，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，浓缩后得油状物4.00g。Add (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(2,2-difluoropropyl)-3,5-dimethyl-1,2,3 ,4-Tetrahydroisoquinoline-6-amine (3.56g) was added to 40mL propionic acid, cooled to -20°C, 2N sodium nitrite aqueous solution (560mg) was added dropwise, and the reaction was completed after stirring for 1 hour. Add 50 mL of ethyl acetate to the solution, then slowly dropwise add saturated sodium bicarbonate solution to adjust the pH to 8-9, extract with ethyl acetate, combine the organic layers, dry with anhydrous sodium sulfate, and concentrate to obtain 4.00 g of oil.
步骤4：(6S，8R)-6-(4-溴-2,6-二氟苯基)-7-(2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉的合成Step 4: (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(2,2-difluoropropyl)-8-methyl-3-(tetrahydro- Synthesis of 2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline
Figure PCTCN2021099653-appb-000048
Figure PCTCN2021099653-appb-000048
将(6S，8R)-6-(4-溴-2,6-二氟苯基)-7-(2,2-二氟丙基)-8-甲基-6,7,8,9-四氢-3H-吡唑并[4，3-f]异 喹啉(4.00g)加入到100mL二氯甲烷中，加入4-甲基苯磺酸吡啶(PPTS)(100mg)，加入6mL 3,4-二氢-2H-吡喃(DHP)，加热至50℃搅拌10小时后反应完毕，将反应液浓缩后柱层析石油醚：乙酸乙酯＝10:1(V:V)得油状物1.90g。Add (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(2,2-difluoropropyl)-8-methyl-6,7,8,9- Tetrahydro-3H-pyrazolo[4,3-f]isoquinoline (4.00g) was added to 100mL dichloromethane, 4-methylbenzenesulfonic acid pyridine (PPTS) (100mg) was added, 6mL 3, 4-Dihydro-2H-pyran (DHP), heated to 50°C and stirred for 10 hours, the reaction is complete, the reaction solution is concentrated and column chromatography petroleum ether: ethyl acetate = 10:1 (V: V) to obtain an oil 1.90g.
步骤5：(S)-N-(4-((6S，8R)-7-(2,2-二氟丙基)-8-甲基-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉-6-基)-3,5-二氟苯基)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺的合成Step 5: (S)-N-(4-((6S,8R)-7-(2,2-difluoropropyl)-8-methyl-6,7,8,9-tetrahydro-3H- Pyrazolo[4,3-f]isoquinolin-6-yl)-3,5-difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7 -Synthesis of amines
Figure PCTCN2021099653-appb-000049
Figure PCTCN2021099653-appb-000049
参考实施例5的制备方法，不同的是将实施例5步骤1中的(6S，8R)-6-(4-溴-2,6-二氟苯基)-7-(3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-8-甲基-3-(四氢-2H-吡喃-2-基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉替换为(6S,8R)-6-(4-溴-2，6-二氟苯基)-8-甲基-3-(四氢-2H-吡喃-2-基)-7-(2,2-二氟丙基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉，同法制得标题化合物。Refer to the preparation method of Example 5, except that the (6S, 8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-((tert (Butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8, Replaced 9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline with (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-8-methyl- 3-(Tetrahydro-2H-pyran-2-yl)-7-(2,2-difluoropropyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3- f] Isoquinoline, the title compound was obtained by the same method.
1H NMR(400MHz,DMSO-d 6)δ12.97(s,1H),8.05(s,1H),7.23-7.21(m,1H),6.72-6.70(m,1H),6.22-6.19(m,3H),5.07(s,1H),4.57-4.52(m,1H),4.42-4.39(m,1H),3.75-3.70(m,1H),3.52-3.45(m,2H),3.09-3.05(m,2H),3.00-2.94(m,1H),2.87-2.81(m,1H),2.65-2.60(m,1H),2.48-2.45(m,3H),2.39-2.35(m,1H),1.85-1.72(m,2H),1.46-1.36(m,3H),1.03-1.01(m,3H),0.70-0.58(m,3H),0.45-0.42(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 12.97 (s, 1H), 8.05 (s, 1H), 7.23-7.21 (m, 1H), 6.72-6.70 (m, 1H), 6.22-6.19 (m ,3H),5.07(s,1H),4.57-4.52(m,1H),4.42-4.39(m,1H),3.75-3.70(m,1H),3.52-3.45(m,2H),3.09-3.05 (m, 2H), 3.00-2.94 (m, 1H), 2.87-2.81 (m, 1H), 2.65-2.60 (m, 1H), 2.48-2.45 (m, 3H), 2.39-2.35 (m, 1H) ,1.85-1.72(m,2H),1.46-1.36(m,3H),1.03-1.01(m,3H),0.70-0.58(m,3H),0.45-0.42(m,1H).
LC/MS(m/z,MH +):548.3。 LC/MS (m/z, MH + ): 548.3.
实施例10：(S)-N-(3,5-二氟-4-((1S，3R)-3-甲基-6-(1H-吡唑-4-基)-2-(2,2,2-三氟乙基)-1,2,3,4-四氢异喹啉-1-基)苯基)-5-(3-氟丙基)-5-氮杂螺环[2.4]庚烷-7-胺Example 10: (S)-N-(3,5-Difluoro-4-((1S,3R)-3-methyl-6-(1H-pyrazol-4-yl)-2-(2, 2,2-Trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-azaspirocyclic ring [2.4 ]Heptane-7-amine
Figure PCTCN2021099653-appb-000050
Figure PCTCN2021099653-appb-000050
步骤1：5-溴-2-(二乙氧基甲基)-1,3-二氟苯的合成Step 1: Synthesis of 5-bromo-2-(diethoxymethyl)-1,3-difluorobenzene
Figure PCTCN2021099653-appb-000051
Figure PCTCN2021099653-appb-000051
将4-溴-2,6-二氟苯甲醛(1.69g，7.66mmol)加入到17mL乙醇中，加入三乙氧基甲烷(1.13g，7.66mmol)，浓硫酸1滴，加热至60℃搅拌2小时后反应完毕。将反应液旋干得标题化合物。Add 4-bromo-2,6-difluorobenzaldehyde (1.69g, 7.66mmol) to 17mL ethanol, add triethoxymethane (1.13g, 7.66mmol), 1 drop of concentrated sulfuric acid, heat to 60℃ and stir The reaction was complete after 2 hours. The reaction solution was spin-dried to obtain the title compound.
步骤2：(S)-2,6-二氟-4-((5-(3-氟丙基)-5-氮杂螺环[2.4]庚烷-7-基)氨基)苯甲醛的合成Step 2: Synthesis of (S)-2,6-difluoro-4-((5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-yl)amino)benzaldehyde
Figure PCTCN2021099653-appb-000052
Figure PCTCN2021099653-appb-000052
将5-溴-2-(二乙氧基甲基)-1,3-二氟苯(2.26g，7.66mmol)，5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺(1.1g，6.4mmol)，Brettphos Pd G3(580.0mg，0.06mmol)，叔丁醇钠(1.47g，15.31mmol)，加入到30mL甲苯中，氩气保护下加热至80℃搅拌15小时后反应完毕，将反应液加入到50mL水中，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，有机层浓缩后加入20mL四氢呋喃，加入2mol/L稀盐酸溶液，室温搅拌2小时后反应完毕。加入50mL水，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，浓缩后柱层析得标题化合物。The 5-bromo-2-(diethoxymethyl)-1,3-difluorobenzene (2.26g, 7.66mmol), 5-(3-fluoropropyl)-5-azaspiro[2.4]heptane -7-amine (1.1g, 6.4mmol), Brettphos Pd G3 (580.0mg, 0.06mmol), sodium tert-butoxide (1.47g, 15.31mmol), add to 30mL toluene, heat to 80℃ under argon protection and stir After 15 hours, the reaction was completed. The reaction solution was added to 50 mL of water, extracted with ethyl acetate, and the organic layers were combined and dried with anhydrous sodium sulfate. After the organic layer was concentrated, 20 mL of tetrahydrofuran was added, and 2mol/L dilute hydrochloric acid solution was added. After stirring for 2 hours at room temperature The reaction is complete. Add 50 mL of water, extract with ethyl acetate, combine the organic layers, dry with anhydrous sodium sulfate, concentrate and column chromatography to obtain the title compound.
步骤3：(1S，3R)-1-(2,6-二氟-4-((S)-(5-(3-氟丙基)-5-氮杂螺[2.4]庚烷-7-基)氨基)苯基)-3-甲基-2-(2,2,2-三氟乙基)-1,2,3,4-四氢异喹啉-6-醇的合成Step 3: (1S,3R)-1-(2,6-Difluoro-4-((S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7- Synthesis of (yl)amino)phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol
Figure PCTCN2021099653-appb-000053
Figure PCTCN2021099653-appb-000053
将2,6-二氟-4-((5-(3-氟丙基)-5-氮杂螺环[2.4]庚烷-7-基)氨基)苯甲醛(0.95g，3.05mmol)，(R)-3-(2-(((2,2,2-三氟乙基)氨基)丙基)苯酚(0.71g，3.05mmol)加入到20mL甲苯/醋酸＝10/1体系中，加热至80℃搅拌15小时后反应完毕，将反应液浓缩后柱层析(石油醚/乙酸乙酯梯度洗脱)得标题化合物。2,6-Difluoro-4-((5-(3-fluoropropyl)-5-azaspirocyclo[2.4]heptane-7-yl)amino)benzaldehyde (0.95g, 3.05mmol), (R)-3-(2-(((2,2,2-trifluoroethyl)amino)propyl)phenol (0.71g, 3.05mmol) was added to 20mL toluene/acetic acid=10/1 system, heated After stirring at 80°C for 15 hours, the reaction was completed. The reaction solution was concentrated and column chromatography (petroleum ether/ethyl acetate gradient elution) was used to obtain the title compound.
步骤4：(1S，3R)-1-(2,6-二氟-4-((S)-(5-(3-氟丙基)-5-氮杂螺[2.4]庚烷-7-基)氨基)苯基)-3-甲基-2-(2,2,2-三氟乙基)-1,2,3,4-四氢异喹啉-6-基三氟甲磺酸酯的合成Step 4: (1S,3R)-1-(2,6-difluoro-4-((S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7- (Amino)phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-yltrifluoromethanesulfonic acid Synthesis of esters
Figure PCTCN2021099653-appb-000054
Figure PCTCN2021099653-appb-000054
将(1S，3R)-1-(2,6-二氟-4-((5-(3-氟丙基)-5-氮杂螺[2.4]庚烷-7-基)氨基)苯基)-3-甲基-2-(2,2,2-三氟乙基)-1,2,3,4-四氢异喹啉-6-醇(1.10g，2.09mmol)，双(三氟甲磺酰基)苯胺(PhNTf 2)(1.49g，4.17mmol)，三乙胺(TEA)(0.63g，6.26mmol)加入到10mL二氯甲烷(DCM)中，室温搅拌12小时后反应完毕。将100mL水加入到反应液中，乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，有机层浓缩后柱层析得标题化合物。 (1S,3R)-1-(2,6-difluoro-4-((5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-yl)amino)phenyl )-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol (1.10g, 2.09mmol), bis(trifluoroethyl) Fluoromethylsulfonyl)aniline (PhNTf 2 ) (1.49 g, 4.17 mmol), triethylamine (TEA) (0.63 g, 6.26 mmol) were added to 10 mL of dichloromethane (DCM), and the reaction was completed after stirring at room temperature for 12 hours. 100 mL of water was added to the reaction solution, extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, the organic layer was concentrated and column chromatography was used to obtain the title compound.
步骤5：(S)-N-(3,5-二氟-4-((1S，3R)-3-甲基-6-(1H-吡唑-4-基)-2-(2,2,2-三氟乙基)-1,2,3,4-四氢异喹啉-1-基)苯基)-5-(3-氟丙基)-5-氮杂螺环[2.4]庚烷-7-胺的合成Step 5: (S)-N-(3,5-Difluoro-4-((1S,3R)-3-methyl-6-(1H-pyrazol-4-yl)-2-(2,2 ,2-Trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro ring [2.4] Synthesis of heptane-7-amine
Figure PCTCN2021099653-appb-000055
Figure PCTCN2021099653-appb-000055
将(1S，3R)-1-(2,6-二氟-4-((5-(3-氟丙基)-5-氮杂螺[2.4]庚烷-7-基)氨基)苯基)-3-甲基-2-(2,2,2-三氟乙基)-1,2,3,4-四氢异喹啉-6-基三氟甲磺酸(200mg，0.30mmol)，1,1'-双(二苯基膦基)二茂铁]二氯化钯(Pd(dppf)Cl 2)(22.0mg，0.03mmol)，碳酸铯(198mg，0.61mmol)，4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-1H-吡唑-1-甲酸叔丁酯(89mg，.30mmol)加入到5mL1,4-二氧六环/水＝10/1体系中，氩气保护下加热至90℃搅拌14小时后反应完毕，将反应液浓缩后加入10mL二氯甲烷，随后加入2mL三氟乙酸，室温搅拌2小时后反应完毕，将反应液浓缩后柱层析得标题化合物。 (1S,3R)-1-(2,6-difluoro-4-((5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-yl)amino)phenyl )-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-yltrifluoromethanesulfonic acid (200mg, 0.30mmol) , 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (Pd(dppf)Cl 2 ) (22.0mg, 0.03mmol), cesium carbonate (198mg, 0.61mmol), 4-( 4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)-1H-pyrazole-1-carboxylic acid tert-butyl ester (89mg, .30mmol) was added to 5mL1, In the 4-dioxane/water=10/1 system, heat to 90°C and stir for 14 hours under the protection of argon, and then the reaction is complete. After concentrating the reaction solution, add 10 mL of dichloromethane, and then add 2 mL of trifluoroacetic acid, and stir at room temperature. After 2 hours, the reaction is complete, and the reaction solution is concentrated and column chromatography is used to obtain the title compound.
1H NMR(400MHz,DMSO-d 6)δ8.20(s,1H),7.99(s,2H),7.33-7.26(m,2H),6.71-6.69(m,1H),6.22-6.16(m,3H),5.04(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.73-3.68(m,2H),3.18-3.02(m,3H),2.83-2.92(m,2H),2.68-2.56(m,2H),2.47-2.36(m,3H),1.84-1.74(m,2H),1.03-1.02(m,3H),0.72-0.69(m,1H),0.64-0.60(m,2H),0.47-0.45(m,1H). 1 H NMR(400MHz,DMSO-d 6 )δ8.20(s,1H),7.99(s,2H),7.33-7.26(m,2H),6.71-6.69(m,1H),6.22-6.16(m ,3H),5.04(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.73-3.68(m,2H),3.18-3.02(m,3H),2.83-2.92 (m, 2H), 2.68-2.56 (m, 2H), 2.47-2.36 (m, 3H), 1.84-1.74 (m, 2H), 1.03-1.02 (m, 3H), 0.72-0.69 (m, 1H) , 0.64-0.60 (m, 2H), 0.47-0.45 (m, 1H).
LC/MS(m/z,MH +):578.2。 LC/MS (m/z, MH + ): 578.2.
实施例11：3-((1R，3R)-1-(2,6-二氟-4-((4aR，7aR)-6-(3-氟丙基)八氢-1H-吡咯并[3,4-b]吡啶-1-基)苯基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇Example 11: 3-((1R,3R)-1-(2,6-difluoro-4-((4aR,7aR)-6-(3-fluoropropyl)octahydro-1H-pyrrolo[3 ,4-b]pyridin-1-yl)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)- 2,2-Difluoropropane-1-ol
Figure PCTCN2021099653-appb-000056
Figure PCTCN2021099653-appb-000056
制备方法与实施例1步骤3类似，不同的是将实施例1步骤3中的(S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺替换为(4aR，7aR)-6-(3-氟丙基)八氢-1H-吡咯并[3,4-b]吡啶，同法制得标题化合物。The preparation method is similar to Step 3 of Example 1, except that (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine in Step 3 of Example 1 is replaced with (4aR, 7aR)-6-(3-fluoropropyl)octahydro-1H-pyrrolo[3,4-b]pyridine, the title compound was obtained in the same way.
1H NMR(400MHz,DMSO-d 6)δ10.56(s,1H),7.40-7.38(m,1H),7.20-7.18(m,1H),7.02-6.92(m,2H),6.49-6.46(m,2H),5.30-5.26(m,1H),5.11(s,1H),4.53-4.50(m,1H),4.41-4.31(m,2H),3.74-3.62(m,1H),3.49-3.41(m,3H),3.18-3.05(m,1H),2.85-2.80(m,2H),2.77-2.60(m,4H),2.58-2.56(m,2H),2.48-2.40(m,2H),2.24-2.20(m,1H),1.80-1.68(m,4H),1.55-1.49(m,2H),1.09-1.07(m,3H). 1 H NMR (400MHz, DMSO-d 6 ) δ 10.56 (s, 1H), 7.40-7.38 (m, 1H), 7.20-7.18 (m, 1H), 7.02-6.92 (m, 2H), 6.49-6.46 (m,2H),5.30-5.26(m,1H),5.11(s,1H),4.53-4.50(m,1H),4.41-4.31(m,2H),3.74-3.62(m,1H),3.49 -3.41(m,3H),3.18-3.05(m,1H),2.85-2.80(m,2H),2.77-2.60(m,4H),2.58-2.56(m,2H),2.48-2.40(m, 2H), 2.24-2.20 (m, 1H), 1.80-1.68 (m, 4H), 1.55-1.49 (m, 2H), 1.09-1.07 (m, 3H).
LC/MS(m/z,MH +):577.3。 LC/MS (m/z, MH + ): 577.3.
实施例12：(S)-5-(3-氟丙基)-N-(6-((1S，3R)-3-甲基-6-(1H-吡唑-4-基)-2-(2,2,2-三氟乙基)-1,2,3,4-四氢异喹啉-1-基)吡啶-3-基)-5-氮杂螺[2.4]庚-7-胺Example 12: (S)-5-(3-fluoropropyl)-N-(6-((1S,3R)-3-methyl-6-(1H-pyrazol-4-yl)-2- (2,2,2-Trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)pyridin-3-yl)-5-azaspiro[2.4]heptane-7- amine
Figure PCTCN2021099653-appb-000057
Figure PCTCN2021099653-appb-000057
Figure PCTCN2021099653-appb-000058
Figure PCTCN2021099653-appb-000058
制备方法与实施例10类似，不同的是将实施例10步骤2中的起始物料5-溴-2-(二乙氧基甲基)-1,3-二氟苯替换为5-溴-2-(二乙氧基甲基)-吡啶，依次进行步骤2-步骤5同法反应，制得标题化合物。The preparation method is similar to that of Example 10, except that the starting material 5-bromo-2-(diethoxymethyl)-1,3-difluorobenzene in step 2 of Example 10 is replaced with 5-bromo- 2-(Diethoxymethyl)-pyridine, followed by step 2 and step 5 in the same way to obtain the title compound.
1H NMR(400MHz,DMSO-d 6)δ8.73(s,1H),7.85(s,2H),7.67-7.59(m,2H),7.39-7.26(m,2H),6.71-6.69(m,1H),6.22-6.16(m,2H),4.94(s,1H),4.55-4.41(m,2H),3.73-3.68(m,2H),3.18-3.02(m,3H),2.83-2.92(m,2H),2.68-2.56(m,2H),2.47-2.36(m,3H),1.86-1.72(m,3H),1.05-0.96(m,1H),0.75-0.57(m,2H),0.38-0.49(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 8.73 (s, 1H), 7.85 (s, 2H), 7.67-7.59 (m, 2H), 7.39-7.26 (m, 2H), 6.71-6.69 (m ,1H),6.22-6.16(m,2H),4.94(s,1H),4.55-4.41(m,2H),3.73-3.68(m,2H),3.18-3.02(m,3H),2.83-2.92 (m, 2H), 2.68-2.56 (m, 2H), 2.47-2.36 (m, 3H), 1.86-1.72 (m, 3H), 1.05-0.96 (m, 1H), 0.75-0.57 (m, 2H) ,0.38-0.49(m,1H).
LC/MS(m/z,MH +):543.2。 LC/MS (m/z, MH + ): 543.2.
实施例13：(S)-N-(3,5-二氟-4-((1S，3R)-3-甲基-6-(1-甲基-1H-吡唑-4-基)-2-(4-(三氟甲基)苯基)-1,2,3,4-四氢异喹啉-1-基)苯基)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺Example 13: (S)-N-(3,5-Difluoro-4-((1S,3R)-3-methyl-6-(1-methyl-1H-pyrazol-4-yl)- 2-(4-(Trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-nitrogen Heterospiro[2.4]heptane-7-amine
Figure PCTCN2021099653-appb-000059
Figure PCTCN2021099653-appb-000059
步骤1：(R)-N-(1-(3-(苯氧基苯基)丙-2-基)-4-(三氟甲基)苯胺的合成Step 1: Synthesis of (R)-N-(1-(3-(phenoxyphenyl)prop-2-yl)-4-(trifluoromethyl)aniline
Figure PCTCN2021099653-appb-000060
Figure PCTCN2021099653-appb-000060
将(R)-1-(3-苯氧基苯基)丙烷-2-胺(2.27g)，对溴三氟甲苯(2.25g)，三(二亚苄基丙酮)二钯(900mg)，2-二环己基膦-2',4',6'-三异丙基联苯(Xphos)(920mg)，碳酸铯(6.50g)加入到50mL1,4-二氧六环中，氮气保护下加热至100℃，6小时反应完毕。反应液柱层析(石油醚/乙酸乙酯梯度洗脱)制得标题 化合物。(R)-1-(3-phenoxyphenyl)propane-2-amine (2.27g), p-bromobenzotrifluoride (2.25g), tris(dibenzylideneacetone)dipalladium (900mg), 2-Dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (Xphos) (920mg), cesium carbonate (6.50g) was added to 50mL 1,4-dioxane, under nitrogen protection Heat to 100°C, and the reaction is complete in 6 hours. The reaction solution was subjected to column chromatography (petroleum ether/ethyl acetate gradient elution) to obtain the title compound.
步骤2：(R)-N-(1-(3-(苯氧基苯基)丙-2-基)-4-(三氟甲基)苯胺的合成Step 2: Synthesis of (R)-N-(1-(3-(phenoxyphenyl)prop-2-yl)-4-(trifluoromethyl)aniline
Figure PCTCN2021099653-appb-000061
Figure PCTCN2021099653-appb-000061
将(R)-N-(1-(3-(苯氧基苯基)丙-2-基)-4-(三氟甲基)苯胺(1.00g)加入到20mL甲醇中，加入钯碳(100mg)，通入氢气后室温搅拌，4小时后反应完毕，抽滤，滤液浓缩后制得标题化合物。(R)-N-(1-(3-(phenoxyphenyl)propan-2-yl)-4-(trifluoromethyl)aniline (1.00g) was added to 20mL methanol, and palladium carbon ( 100mg), hydrogen gas was introduced and stirred at room temperature. After 4 hours, the reaction was completed, filtered with suction, and the filtrate was concentrated to obtain the title compound.
步骤3：(S)-N-(3,5-二氟-4-((1S，3R)-3-甲基-6-(1-甲基-1H-吡唑-4-基)-2-(4-(三氟甲基)苯基)-1,2,3,4-四氢异喹啉-1-基)苯基)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺的合成Step 3: (S)-N-(3,5-Difluoro-4-((1S,3R)-3-methyl-6-(1-methyl-1H-pyrazol-4-yl)-2 -(4-(Trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-aza Synthesis of Spiro[2.4]heptane-7-amine
Figure PCTCN2021099653-appb-000062
Figure PCTCN2021099653-appb-000062
制备方法与实施例10类似，不同的是将实施例10步骤3中的(R)-3-(2-(((2,2,2-三氟乙基)氨基)丙基)苯酚替换为(R)-3-(2-(((4-(三氟甲基)苯基)氨基)丙基)苯酚，然后依次进行步骤2-步骤5同法反应，制得标题化合物。The preparation method is similar to that in Example 10, except that (R)-3-(2-(((2,2,2-trifluoroethyl)amino)propyl)phenol in step 3 of Example 10 is replaced with (R)-3-(2-(((4-(Trifluoromethyl)phenyl)amino)propyl)phenol, and then proceed step 2 to step 5 in the same way to obtain the title compound.
1H NMR(400MHz,DMSO-d 6)δ8.10(s,1H),7.83(s,1H),7.48-7.36(m,4H),7.17-7.15(m,1H),7.01-6.99(m,2H),6.53(s,1H),6.19-6.15(m,2H),5.92(s,1H),4.52-4.51(m,1H),4.40-4.37(m,1H),3.85(s,1H),3.54-3.47(m,4H),2.79-2.50(m,3H),2.34-2.33(m,2H),1.91-1.79(m,2H),0.92-0.91(m,2H),0.65-0.64(m,1H),0.590.57(m,2H),0.43-0.42(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 8.10 (s, 1H), 7.83 (s, 1H), 7.48-7.36 (m, 4H), 7.17-7.15 (m, 1H), 7.01-6.99 (m , 2H), 6.53 (s, 1H), 6.19-6.15 (m, 2H), 5.92 (s, 1H), 4.52-4.51 (m, 1H), 4.40-4.37 (m, 1H), 3.85 (s, 1H) ),3.54-3.47(m,4H),2.79-2.50(m,3H),2.34-2.33(m,2H),1.91-1.79(m,2H),0.92-0.91(m,2H),0.65-0.64 (m,1H),0.590.57(m,2H),0.43-0.42(m,1H).
LC/MS(m/z,MH +):654.3。 LC/MS (m/z, MH + ): 654.3.
实施例14：3-((1R，3R)-1-(2,6-二氟-4-(((S)-1-(3-氟丙基)哌啶-3-基)氨基)苯基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇Example 14: 3-((1R,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)piperidin-3-yl)amino)benzene Yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane-1-ol
Figure PCTCN2021099653-appb-000063
Figure PCTCN2021099653-appb-000063
制备方法与实施例1步骤3类似，不同的是将实施例1步骤3中的(S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺替换为(S)-1-(3-氟丙基)哌啶-3-胺，制得标题化合物。The preparation method is similar to Step 3 of Example 1, except that (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine in Step 3 of Example 1 is replaced with (S)-1-(3-Fluoropropyl)piperidin-3-amine to obtain the title compound.
1H NMR(400MHz,DMSO-d 6)δ10.66(s,1H),7.43-7.37(m,2H),7.03-6.90(m,2H),6.25-6.17(m,3H),5.79-5.75(m,1H),5.35(s,1H),4.55-4.52(m,1H),4.43-4.39(m,1H),3.60-3.57(m,2H),3.31-3.30(m,3H),2.98-2.78(m,4H),2.72-2.60(m,2H),2.39-2.32(m,2H),2.05-1.95(m,1H),1.84-1.64(m,6H),0.89—0.77(m,3H). 1 H NMR (400MHz, DMSO-d 6 ) δ10.66 (s, 1H), 7.43-7.37 (m, 2H), 7.03-6.90 (m, 2H), 6.25-6.17 (m, 3H), 5.79-5.75 (m, 1H), 5.35 (s, 1H), 4.55-4.52 (m, 1H), 4.43-4.39 (m, 1H), 3.60-3.57 (m, 2H), 3.31-3.30 (m, 3H), 2.98 -2.78(m,4H),2.72-2.60(m,2H),2.39-2.32(m,2H),2.05-1.95(m,1H),1.84-1.64(m,6H),0.89-0.77(m, 3H).
LC/MS(m/z,MH +):551.2 LC/MS(m/z,MH + ): 551.2
实施例15：3-((1R，3R)-1-(2,6-二氟-4-(((S)-1-(3-氟丙基)氮杂环庚-3-基)氨基)苯基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇Example 15: 3-((1R,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)azacycloheptan-3-yl)amino )Phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane-1- alcohol
Figure PCTCN2021099653-appb-000064
Figure PCTCN2021099653-appb-000064
制备方法与实施例1类似，不同的是将实施例1步骤1中的(S)-(5-氮杂螺[2.4]庚烷-7-基)氨基甲酸叔丁酯替换为(S)-氮杂环庚-3-基氨基甲酸叔丁酯，同法进行实施例1的步骤1-步骤3反应制得标题化合物。The preparation method is similar to that in Example 1, except that (S)-(5-azaspiro[2.4]heptane-7-yl) t-butyl carbamate in step 1 of Example 1 is replaced with (S)- For tert-butyl azepan-3-yl carbamate, the reaction of step 1 to step 3 of Example 1 was carried out in the same manner to obtain the title compound.
1H NMR(400MHz,DMSO-d 6)δ10.53(s,1H),7.38-7.36(m,1H),7.20-7.18(m,1H),7.00-6.91(m,2H),6.19-6.06(m,3H),5.27-5.24(m,1H),5.04(s,1H),4.55-4.53(m,1H),4.43-4.42(m,1H),3.44-3.38(m,3H),2.83-2.80(m,2H),2.72-2.50(m,6H),1.99-1.96(m,2H),1.80-1.60(m,7H),1.55-1.50(m,3H),1.07-1.06(m,3H). 1 H NMR (400MHz, DMSO-d 6 ) δ 10.53 (s, 1H), 7.38-7.36 (m, 1H), 7.20-7.18 (m, 1H), 7.00-6.91 (m, 2H), 6.19-6.06 (m,3H),5.27-5.24(m,1H),5.04(s,1H),4.55-4.53(m,1H),4.43-4.42(m,1H),3.44-3.38(m,3H),2.83 -2.80(m,2H),2.72-2.50(m,6H),1.99-1.96(m,2H),1.80-1.60(m,7H),1.55-1.50(m,3H),1.07-1.06(m, 3H).
LC/MS(m/z,MH +):565.2。 LC/MS (m/z, MH + ): 565.2.
实施例16：N-(3,5-二氟-4-((6S，8R)-8-甲基-7-(2,2,2-三氟乙基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉-6-基)苯基)-1-(3-氟丙基)-3-甲基氮杂环丁烷-3-胺Example 16: N-(3,5-Difluoro-4-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9 -Tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)phenyl)-1-(3-fluoropropyl)-3-methylazetidine-3- amine
Figure PCTCN2021099653-appb-000065
Figure PCTCN2021099653-appb-000065
步骤1：1-(3-氟丙基)-3-甲基氮杂环丁烷-3-胺的合成Step 1: Synthesis of 1-(3-fluoropropyl)-3-methylazetidine-3-amine
Figure PCTCN2021099653-appb-000066
Figure PCTCN2021099653-appb-000066
参照实施例1步骤1和步骤2的方法，不同的是将实施例1步骤1中的(S)-(5-氮杂螺[2.4]庚烷-7-基)氨基甲酸叔丁酯替换为3-(Boc-氨基)-3-甲基氮杂环丁烷，同法制备1-(3-氟丙基)-3-氨基-3-甲基吖啶。Refer to the method of step 1 and step 2 of Example 1, except that (S)-(5-azaspiro[2.4]heptane-7-yl) t-butyl carbamate in step 1 of Example 1 was replaced with 3-(Boc-amino)-3-methylazetidine, 1-(3-fluoropropyl)-3-amino-3-methylacridine was prepared in the same way.
步骤2：N-(3,5-二氟-4-((6S，8R)-8-甲基-7-(2,2,2-三氟乙基)-6,7,8,9-四氢-3H-吡唑并[4,3-f]异喹啉-6-基)苯基)-1-(3-氟丙基)-3-甲基氮杂环丁烷-3-胺的合成Step 2: N-(3,5-Difluoro-4-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9- Tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)phenyl)-1-(3-fluoropropyl)-3-methylazetidine-3-amine Synthesis
Figure PCTCN2021099653-appb-000067
Figure PCTCN2021099653-appb-000067
参照实施例8的制备方法制备，不同的是将实施例8中的(S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺替换为1-(3-氟丙基)-3-氨基-3-甲基吖啶，同法进行实施例8的最后2步反应制得标题化合物。It was prepared with reference to the preparation method of Example 8, except that (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine in Example 8 was replaced with 1-( 3-Fluoropropyl)-3-amino-3-methylacridine, the last two steps of the reaction in Example 8 were carried out in the same way to obtain the title compound.
1H NMR(400MHz,DMSO-d 6)δ13.00(s,1H),8.06(s,1H),7.24-7.22(m,1H),6.77-6.75(m,1H),6.63(s,1H),5.94-5.90(m,2H),5.17(s,1H),4.52-4.49(m,1H),4.40-4.37(m,1H),3.51-3.44(m,2H),3.32-3.30(m,3H),3.19-3.14(m,1H),2.98-2.88(m,4H),2.48-2.46(m,1H),1.69-1.60(m,2H),1.46(s,3H),1.10-1.08(m,3H). 1 H NMR (400MHz, DMSO-d 6 ) δ 13.00 (s, 1H), 8.06 (s, 1H), 7.24-7.22 (m, 1H), 6.77-6.75 (m, 1H), 6.63 (s, 1H) ), 5.94-5.90 (m, 2H), 5.17 (s, 1H), 4.52-4.49 (m, 1H), 4.40-4.37 (m, 1H), 3.51-3.44 (m, 2H), 3.32-3.30 (m ,3H),3.19-3.14(m,1H),2.98-2.88(m,4H),2.48-2.46(m,1H),1.69-1.60(m,2H),1.46(s,3H),1.10-1.08 (m,3H).
LC/MS(m/z,MH +):526.2。 LC/MS (m/z, MH + ): 526.2.
实施例17：3-(((1R，3R)-1-(2,6-二氟-4-((((1R，4R，5S)-2-(3-氟丙基)-2-氮杂双环[2.1.1]己-5-基)氨基)苯基)-3-甲基-1,3,4,9-四氢-2H-吡啶并[3,4-b]吲哚-2-基)-2,2-二氟丙烷-1-醇Example 17: 3-(((1R,3R)-1-(2,6-difluoro-4-((((1R,4R,5S)-2-(3-fluoropropyl)-2-nitrogen Heterobicyclo[2.1.1]hex-5-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2 -Base)-2,2-difluoropropane-1-ol
Figure PCTCN2021099653-appb-000068
Figure PCTCN2021099653-appb-000068
制备方法与实施例1类似，不同的是将实施例1步骤1中的(7S)-5-(苯甲基)-5-氮杂螺[2.4]庚烷-7-基氨基甲酸叔丁酯替换为((1R，4R，5S)-2-氮杂双环[2.1.1]己基-5-基)氨基甲酸苄酯，制得中间体((1R，4R，5S)-2-(3-氟丙基)-2-氮杂双环[2.1.1]己基-5-基)氨基甲酸苄酯。将((1R，4R，5S)-2-(3-氟丙基)-2-氮杂双环[2.1.1]己基-5-基)氨基甲酸苄酯(290mg，1.00mmol)加入到10mL甲醇中，加入10mg Pd/C，通入氢气后室温搅拌5小时后反应完毕，将反应液抽滤浓缩后得中间体(1R，4R，5S)-2-(3-氟丙基)-2-氮杂双环[2.1.1]己-5-胺。然后参照实施例1步骤3，不同的是将实施例1步骤3中的(S)-5-(3-氟丙基)-5-氮杂螺[2.4]庚-7-胺替换为(1R，4R，5S)-2-(3-氟丙基)-2-氮杂双环[2.1.1]己-5-胺，同法制得标题化合物。The preparation method is similar to that in Example 1, except that (7S)-5-(benzyl)-5-azaspiro[2.4]heptane-7-ylcarbamate in step 1 of Example 1 Replaced with ((1R, 4R, 5S)-2-azabicyclo[2.1.1] hexyl-5-yl) benzyl carbamate to prepare intermediate ((1R, 4R, 5S)-2-(3- Fluoropropyl)-2-azabicyclo[2.1.1]hexyl-5-yl) benzyl carbamate. Benzyl ((1R, 4R, 5S)-2-(3-fluoropropyl)-2-azabicyclo[2.1.1]hexyl-5-yl)carbamate (290mg, 1.00mmol) was added to 10mL methanol Add 10mg Pd/C, add hydrogen and stir at room temperature for 5 hours, then the reaction is complete, the reaction solution is filtered and concentrated to obtain the intermediate (1R, 4R, 5S)-2-(3-fluoropropyl)-2- Azabicyclo[2.1.1]hexyl-5-amine. Then refer to Step 3 of Example 1, except that (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine in Step 3 of Example 1 was replaced with (1R , 4R, 5S)-2-(3-fluoropropyl)-2-azabicyclo[2.1.1]hexyl-5-amine, the title compound was prepared in the same way.
1H NMR(400MHz,DMSO-d 6)δ10.39(s,1H),7.37-7.32(m,2H),6.98-6.88(m,2H),6.35(s,1H),6.21-6.18(m,1H),5.95(s,1H),5.67-5.65(m,1H),4.66-4.63(m,1H),4.54-4.51(m,1H),4.34-4.36(m,1H),4.27-4.20(m,1H),3.41-3.40(m,1H),3.31-3.24(m,2H),2.93-2.91(m,1H),2.81-2.67(m,4H),2.59-2.55(m,3H),2.19-2.16(m,1H),1.88-1.77(m,2H),1.29-1.20(m,2H),1.07-1.06(m,3H). 1 H NMR (400MHz, DMSO-d 6 ) δ 10.39 (s, 1H), 7.37-7.32 (m, 2H), 6.98-6.88 (m, 2H), 6.35 (s, 1H), 6.21-6.18 (m ,1H),5.95(s,1H),5.67-5.65(m,1H),4.66-4.63(m,1H),4.54-4.51(m,1H),4.34-4.36(m,1H),4.27-4.20 (m,1H),3.41-3.40(m,1H),3.31-3.24(m,2H),2.93-2.91(m,1H),2.81-2.67(m,4H),2.59-2.55(m,3H) , 2.19-2.16 (m, 1H), 1.88-1.77 (m, 2H), 1.29-1.20 (m, 2H), 1.07-1.06 (m, 3H).
LC/MS(m/z,MH +):549.2。 LC/MS (m/z, MH + ): 549.2.
实施例18：(S)-5-(3-氟丙基-1,1-d2)-N-(6-((1S，3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3，4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)吡啶-3-基)-5-氮杂螺[2.4]庚-7-胺Example 18: (S)-5-(3-fluoropropyl-1,1-d2)-N-(6-((1S,3R)-3-methyl-2-(2,2,2- Trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-yl)-5-azaspiro[2.4] Hept-7-amine
Figure PCTCN2021099653-appb-000069
Figure PCTCN2021099653-appb-000069
步骤1：(S)-(5-(3-氟丙酰基)-5-氮杂螺[2.4]庚烷-7-基)氨基甲酸叔丁酯的合成Step 1: Synthesis of tert-butyl (S)-(5-(3-fluoropropionyl)-5-azaspiro[2.4]heptane-7-yl)carbamate
Figure PCTCN2021099653-appb-000070
Figure PCTCN2021099653-appb-000070
将(S)-(5-氮杂螺[2.4]庚烷-7-基)氨基甲酸叔丁酯(1.06g)加入到10mL N,N-二甲基甲酰胺中，加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.60g)，3-氟丙酸(1.00g)，N,N-二异丙基乙胺(2.50g)，室温搅拌4小时后反应完毕，将反应液加入到100mL水中，乙酸乙酯萃取，合并有机层，浓缩后柱层析(二氯甲烷/甲醇梯度洗脱)得标题化合物。Add (S)-(5-azaspiro[2.4]heptane-7-yl) tert-butyl carbamate (1.06g) to 10mL N,N-dimethylformamide, add 2-(7- Azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.60g), 3-fluoropropionic acid (1.00g), N,N-diisopropyl Ethylamine (2.50g) was stirred at room temperature for 4 hours and the reaction was complete. The reaction solution was added to 100mL of water, extracted with ethyl acetate, the organic layers were combined, concentrated, and column chromatography (dichloromethane/methanol gradient elution) to obtain the title compound .
步骤2：(S)-1-(7-氨基-5-氮杂螺[2.4]庚基-5-基)-3-氟丙烷-1-酮的合成Step 2: Synthesis of (S)-1-(7-amino-5-azaspiro[2.4]heptyl-5-yl)-3-fluoropropan-1-one
Figure PCTCN2021099653-appb-000071
Figure PCTCN2021099653-appb-000071
将(S)-(5-(3-氟丙酰基)-5-氮杂螺[2.4]庚烷-7-基)氨基甲酸叔丁酯(1.00g)加入到4mol/L的氯化氢1,4-二氧六环溶液10mL中，室温搅拌1小时后反应完毕，将反应液浓缩，加入饱和碳酸氢钠溶液后乙酸乙酯萃取，合并有机层，无水硫酸钠干燥，有机层浓缩后直接投入下一步中。Add (S)-(5-(3-fluoropropionyl)-5-azaspiro[2.4]heptane-7-yl) tert-butyl carbamate (1.00g) to 4mol/L hydrogen chloride 1,4 -In 10 mL of dioxane solution, stir at room temperature for 1 hour and then the reaction is complete. The reaction solution is concentrated, saturated sodium bicarbonate solution is added and ethyl acetate is extracted, the organic layers are combined, dried over anhydrous sodium sulfate, and the organic layer is concentrated and put directly into In the next step.
步骤3：(S)-5-(3-氟丙基-1,1-d2)-5-氮杂螺[2.4]庚-7-胺的合成Step 3: Synthesis of (S)-5-(3-fluoropropyl-1,1-d2)-5-azaspiro[2.4]heptane-7-amine
Figure PCTCN2021099653-appb-000072
Figure PCTCN2021099653-appb-000072
将(S)-1-(7-氨基-5-氮杂螺[2.4]庚基-5-基)-3-氟丙烷-1-酮(400mg)加入到20mL无水四氢呋喃中，加入氘代氢化铝锂(AlD 4Li)(100mg)，氮气保护下加热至70℃，4小时后反应完毕，将反应液浓缩，直接用于下一步中。 Add (S)-1-(7-amino-5-azaspiro[2.4]heptyl-5-yl)-3-fluoropropan-1-one (400mg) to 20mL of anhydrous tetrahydrofuran, add deuterium Lithium aluminum hydride (AlD 4 Li) (100 mg) was heated to 70° C. under the protection of nitrogen. After 4 hours, the reaction was completed. The reaction solution was concentrated and used directly in the next step.
步骤4：(S)-5-(3-氟丙基-1,1-d2)-N-(6-((1S，3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3，4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)吡啶-3-基)-5-氮杂螺[2.4]庚-7-胺的合成Step 4: (S)-5-(3-fluoropropyl-1,1-d2)-N-(6-((1S,3R)-3-methyl-2-(2,2,2-tri Fluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-yl)-5-azaspiro[2.4]hepta Synthesis of -7-amine
Figure PCTCN2021099653-appb-000073
Figure PCTCN2021099653-appb-000073
将(1S，3R)-1-(5-溴吡啶-2-基)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶基[3，4-b]吲哚(200mg)，甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯)(2-氨基-1,1-联苯-2-基)钯(II)(Brettphos Pd G3)(45.0mg)，叔丁醇钠(192mg)，(S)-5-(3-氟丙基-1,1-d2)-5-氮杂螺[2.4]庚-7-胺(100mg)加入到5mL甲苯中，氩气保护下加热至80℃搅拌4小时后反应完毕，反应液柱层析(二氯甲烷/甲醇梯度洗脱)得标题化合物。Add (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro -1H-pyridyl[3,4-b]indole (200mg), methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl- 1,1-Biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II)(BrettphosPdG3)(45.0mg), sodium tert-butoxide (192mg), (S)-5 -(3-Fluoropropyl-1,1-d2)-5-azaspiro[2.4]heptane-7-amine (100mg) was added to 5mL of toluene, heated to 80°C under argon protection, stirred for 4 hours, and then reacted After completion, the reaction solution was subjected to column chromatography (dichloromethane/methanol gradient elution) to obtain the title compound.
1H NMR(400MHz,DMSO-d 6)δ10.58(s,1H),7.90-7.88(m,1H),7.42-7.40(m,1H),7.27-7.25(m,1H),7.05-6.96(m,4H),5.75(d,J＝8.8Hz,1H),4.89(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.77-3.70(m,1H),3.59-3.41(m,1H),3.35-3.25(m,1H),3.04-2.89(m,2H),2.75-2.36(m,5H),1.85-1.71(m,2H),1.14-1.12(m,3H),0.75-0.73(m,1H),0.64-0.61(m,2H),0.48-0.43(m,1H). 1 H NMR (400MHz, DMSO-d 6 ) δ 10.58 (s, 1H), 7.90-7.88 (m, 1H), 7.42-7.40 (m, 1H), 7.27-7.25 (m, 1H), 7.05-6.96 (m,4H),5.75(d,J=8.8Hz,1H),4.89(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.77-3.70(m,1H) ),3.59-3.41(m,1H),3.35-3.25(m,1H),3.04-2.89(m,2H),2.75-2.36(m,5H),1.85-1.71(m,2H),1.14-1.12 (m, 3H), 0.75-0.73 (m, 1H), 0.64-0.61 (m, 2H), 0.48-0.43 (m, 1H).
LC/MS(m/z,MH +):518.3。 LC/MS (m/z, MH + ): 518.3.
生物学活性及相关性质测试例Test examples of biological activity and related properties
测试实施例1：本发明化合物对MCF7细胞内***受体降解效果检测Test Example 1: Detection of the degradation effect of the compound of the present invention on the estrogen receptor in MCF7 cells
试验原理：测定本发明化合物对MCF7细胞内内源表达的***受体的降解活性，根据IC 50及最大降解效率评价待测化合物的活性。 Test principle: Determine the degradation activity of the compound of the present invention on the endogenously expressed estrogen receptor in MCF7 cells, and evaluate the activity of the test compound based on the IC 50 and the maximum degradation efficiency.
试验方法：experiment method:
MCF7细胞(购于ATCC,HTB-22)用含10％胎牛血清的DMEM(购于Gibco，11995-065)完全培养基进行培养。实验第一天，使用完全培养基将MCF7细胞以3000个/孔的密度种于384孔板，37℃，5％CO 2细胞培养箱培养。待测化合物溶解于DMSO,储存浓度为10mM,用Echo 550(购于Labcyte Inc.)稀释并加入细胞培养板内，各化合物处理的起始浓度为100nM，3倍梯度稀释，9个浓度点，设置含0.5％DMSO的空白对照，各浓度点设双复孔对照。37℃，5％CO 2细胞培养箱培养24小时。各细胞培养孔内加入多聚甲醛至细胞培养液内，终浓度约3.7％固定细胞，作用30分钟后，弃上清，加入50μL PBS每孔洗涤一次；加入PBS含0.5％v/v Tween-20处理细胞30分钟，PBS洗涤一次；加入封闭液(自制，PBS内含5％BSA，0.05％Tween-20)室温孵育1小时；去封闭液加入一抗混合液(抗-ER单抗，Estrogen Receptorα(D8H8)Rabbit mAb,购于CST,#8644S,1：1000稀释；抗-GAPDH单抗，GAPDH(D4C6R)Mouse mAb,购于CST,#97166S，1：2000稀释)室温孵育3小时；用PBST(自制，PBS内含0.05％Tween-20)洗涤3次；加入检测二抗(800CW-羊抗兔IgG，购于LI-COR，P/N:926-32211,1:1000稀释；680RD-羊抗鼠IgG，购于LI-COR，#925-68070，1：1000稀释)，室温，避光孵育45分钟；PBST洗涤3次，使用Odyssey CLx读取各孔荧光信号。数据处理用XLfit，根据化合物的浓度和荧光信号值计算各化合物的降解活性DC 50。 MCF7 cells (purchased from ATCC, HTB-22) were cultured with DMEM (purchased from Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 384-well plate at a density of 3000 cells/well using complete medium, and cultured in a cell incubator at 37°C and 5% CO 2. The test compound was dissolved in DMSO at a storage concentration of 10 mM, diluted with Echo 550 (purchased from Labcyte Inc.) and added to the cell culture plate. The initial concentration of each compound treatment was 100 nM, 3-fold dilution, 9 concentration points, Set a blank control containing 0.5% DMSO, and set up a double-well control for each concentration point. Incubate for 24 hours in a 37°C, 5% CO 2 cell incubator. Add paraformaldehyde to each cell culture well to fix the cells at a final concentration of about 3.7%. After 30 minutes, discard the supernatant and add 50μL PBS to each well for washing; add PBS containing 0.5% v/v Tween- 20 Treat cells for 30 minutes, wash once with PBS; add blocking solution (homemade, PBS containing 5% BSA, 0.05% Tween-20) and incubate at room temperature for 1 hour; remove blocking solution and add primary antibody mixture (anti-ER monoclonal antibody, Estrogen Receptorα(D8H8) Rabbit mAb, purchased from CST, #8644S, 1:1000 dilution; anti-GAPDH monoclonal antibody, GAPDH(D4C6R) Mouse mAb, purchased from CST, #97166S, 1:2000 dilution) Incubate at room temperature for 3 hours; Wash 3 times with PBST (homemade, PBS containing 0.05% Tween-20); add detection secondary antibody (800CW-goat anti-rabbit IgG, purchased from LI-COR, P/N: 926-32211, 1:1000 dilution; 680RD- Goat anti-mouse IgG, purchased from LI-COR, #925-68070, 1:1000 dilution), incubated at room temperature, protected from light for 45 minutes; washed 3 times with PBST, and read the fluorescence signal of each well with Odyssey CLx. XLfit is used for data processing, and the degradation activity DC 50 of each compound is calculated based on the concentration of the compound and the fluorescence signal value.
试验结果：test results:
在本实验条件下，测试化合物对基于细胞水平的ER水平具有良好的降解活性。测试化合物相应的ER水平活性测试结果具体见表1。Under the experimental conditions, the test compound has a good degradation activity on the ER level based on the cell level. The corresponding ER level activity test results of the test compounds are shown in Table 1.
表1测试化合物体外基于细胞水平的ER水平活性测试结果Table 1 In vitro cell-based ER activity test results of test compounds
测试化合物Test compound ER水平DC 50(nM) ER level DC 50 (nM)
实施例1Example 1 0.120.12
实施例2Example 2 0.120.12
实施例3Example 3 0.190.19
实施例4Example 4 0.210.21
实施例5Example 5 0.040.04
实施例6Example 6 0.370.37
实施例7Example 7 0.090.09
实施例8Example 8 0.070.07
实施例9Example 9 0.080.08
实施例10Example 10 0.150.15
实施例11Example 11 0.210.21
实施例12Example 12 0.230.23
实施例14Example 14 0.110.11
实施例16Example 16 0.060.06
实施例17Example 17 0.150.15
测试实施例2：本发明化合物对T47D细胞增殖的抑制效果检测Test Example 2: Detection of the inhibitory effect of the compound of the present invention on the proliferation of T47D cells
试验原理：测定本发明化合物对T47D细胞体外增殖的抑制影响，根据IC 50及最大抑制效率评价化合物的活性。 Test principle: Determine the inhibitory effect of the compound of the present invention on the proliferation of T47D cells in vitro, and evaluate the activity of the compound based on the IC 50 and the maximum inhibitory efficiency.
试验方法：experiment method:
T47D(T-47D)细胞(购于ATCC,HTB-133)用含10％胎牛血清的RPMI-1640(购于Gibco，A10491-01)完全培养基进行培养。实验第一天，使用完全培养基将T47D细胞以500个/孔的密度种于384孔板，37℃，5％CO 2细胞培养箱过夜培养。第二天，加入待测化合物进行药物处理，采用Echo550(购于Labcyte Inc.)将储存浓度为10mM的化合物溶液进行稀释及转移至各细胞培养孔内，各化合物在细胞内的处理起始浓度为100nM，3倍梯度稀释，10个浓度点，设置含0.3％DMSO的空白对照，各浓度点设双复孔对照。37℃，5％CO 2细胞培养箱培养7天，第八天，取出细胞培养板。加入
Figure PCTCN2021099653-appb-000074
Luminescent Cell Viability Assay(购于Promega,G7573),室温放置10分钟后，使用多标记酶标仪EnVision(购于PerkinElmer)读取发光信号值，用XLfit根据化合物的浓度和发光信号值计算各化合物的抑制活性IC 50。 T47D (T-47D) cells (purchased from ATCC, HTB-133) were cultured with RPMI-1640 (purchased from Gibco, A10491-01) complete medium containing 10% fetal bovine serum. On the first day of the experiment, T47D cells were seeded in a 384-well plate at a density of 500 cells/well in a complete medium at 37° C., and cultured overnight in a 5% CO 2 cell incubator. The next day, add the test compound for drug treatment, use Echo550 (purchased from Labcyte Inc.) to dilute the compound solution with a storage concentration of 10mM and transfer it to each cell culture well. The initial concentration of each compound in the cell It is 100nM, 3-fold gradient dilution, 10 concentration points, set a blank control containing 0.3% DMSO, and each concentration point is set up as a double-well control. Cultivate in a cell incubator at 37°C and 5% CO 2 for 7 days, and take out the cell culture plate on the eighth day. join in
Figure PCTCN2021099653-appb-000074
Luminescent Cell Viability Assay (purchased from Promega, G7573), placed at room temperature for 10 minutes, use a multi-label microplate reader EnVision (purchased from PerkinElmer) to read the luminescence signal value, and use XLfit to calculate the luminescence signal value of each compound according to the compound concentration and luminescence signal value. inhibitory activity IC 50.
试验结果：test results:
在本实验条件下，测试化合物对T47D乳腺癌细胞表现出了良好的抑制活性。测试化合物对T47D细胞增殖的抑制活性结果具体见表2。Under the experimental conditions, the test compound showed good inhibitory activity on T47D breast cancer cells. The results of the inhibitory activity of the test compounds on the proliferation of T47D cells are shown in Table 2.
表2化合物对T47D细胞增殖抑制活性Table 2 Compounds inhibiting the proliferation of T47D cells
测试化合物Test compound T47D IC 50(nM) T47D IC 50 (nM)
实施例1Example 1 0.280.28
实施例2Example 2 0.440.44
实施例4Example 4 0.270.27
实施例5Example 5 0.040.04
实施例6Example 6 0.420.42
实施例7Example 7 0.570.57
实施例8Example 8 0.310.31
实施例9Example 9 0.420.42
实施例10Example 10 0.420.42
实施例11Example 11 0.210.21
实施例14Example 14 0.440.44
实施例16Example 16 0.210.21
实施例17Example 17 0.300.30
测试实施例3：本发明化合物对MCF7细胞增殖的抑制效果检测Test Example 3: Detection of the inhibitory effect of the compound of the present invention on the proliferation of MCF7 cells
试验原理：测定本发明化合物对MCF7细胞体外增殖的抑制影响，根据IC 50及最大抑制效率以评价化合物的活性。 Test principle: Determination of Effect of compounds of this invention inhibit the proliferation of MCF7 cells, according to the IC 50 and maximum inhibition to evaluate the efficiency of the active compound.
试验方法：experiment method:
MCF7细胞(ATCC,HTB-22)用含10％胎牛血清的DMEM(Gibco，11995-065)完全培养基进行培养。实验第一天，使用完全培养基将MCF7细胞以500个/孔的密度种于384孔板，37℃，5％CO 2细胞培养箱过夜培养。第二天，加入待测化合物进行药物处理，采用Echo550(Labcyte Inc.)将储存浓度为10mM的化合物溶液进行稀释及转移至各细胞培养孔内，各化合物在细胞内的处理起始浓度为100nM，3倍梯度稀释，9个浓度点，设置含0.3％DMSO的空白对照，各浓度点设双复孔对照。37℃，5％CO 2细胞培养箱培养7天，第八天，取出细胞培养板。加入
Figure PCTCN2021099653-appb-000075
Luminescent Cell Viability Assay(Promega,G7573), 室温放置10分钟后，使用多标记酶标仪EnVision(PerkinElmer)读取发光信号值，用XLfit根据化合物的浓度和发光信号值计算各化合物的抑制活性IC 50及最大抑制率I max，结果如表3所示。 MCF7 cells (ATCC, HTB-22) were cultured with DMEM (Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 384-well plate at a density of 500 cells/well in a complete medium at 37° C., and cultured overnight in a 5% CO 2 cell incubator. On the second day, add the test compound for drug treatment. Use Echo550 (Labcyte Inc.) to dilute the compound solution with a storage concentration of 10mM and transfer it to each cell culture well. The initial concentration of each compound in the cell is 100nM. , 3-fold gradient dilution, 9 concentration points, set a blank control containing 0.3% DMSO, and each concentration point set a double-well control. Cultivate in a cell incubator at 37°C and 5% CO 2 for 7 days, and take out the cell culture plate on the eighth day. join in
Figure PCTCN2021099653-appb-000075
Luminescent Cell Viability Assay (Promega, G7573), after standing at room temperature for 10 minutes, use the multi-label microplate reader EnVision (PerkinElmer) to read the luminescence signal value, and use XLfit to calculate the inhibitory activity IC 50 of each compound based on the compound concentration and luminescence signal value And the maximum inhibition rate I max , the results are shown in Table 3.
表3化合物对MCF7细胞增殖抑制活性Table 3 Compounds inhibiting the proliferation of MCF7 cells
化合物编号Compound number MCF-7细胞增殖抑制IC 50(nM) MCF-7 cell proliferation inhibition IC 50 (nM) 最大抑制率(％)Maximum inhibition rate (%)
实施例4Example 4 0.070.07 105.79105.79
实施例8Example 8 0.080.08 105.71105.71
测试实施例4：本发明化合物的药代动力学性质检测Test Example 4: Detection of the pharmacokinetic properties of the compound of the present invention
试验原理：以小鼠为受试动物，应用LC/MS/MS法测定小鼠灌胃和静脉给予本发明化合物后，不同时刻血浆中的药物浓度。研究本发明化合物在小鼠体内的药代动力学行为，评价其药动学特征。Test principle: Using mice as test animals, the LC/MS/MS method was used to determine the drug concentration in plasma at different times after the mice were given the compound of the present invention intragastrically or intravenously. To study the pharmacokinetic behavior of the compound of the present invention in mice and evaluate its pharmacokinetic characteristics.
试验方法：experiment method:
4.1试验动物4.1 Experimental animals
健康成年BALB/c小鼠18只，雌性，平均分为6组，每组3只，灌胃，小鼠购自北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(浙)2019-0001。18 healthy adult BALB/c mice, females, were equally divided into 6 groups, 3 in each group, gavage, the mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Zhejiang ) 2019-0001.
4.2药物配制4.2 Drug preparation
称取一定量药物，溶于DMSO 5％+PG 20％+无水乙醇5％+solutol 10％+水60％，配制成10mg/ml，用于灌胃。Weigh a certain amount of medicine, dissolve it in DMSO 5% + PG 20% + anhydrous ethanol 5% + solutol 10% + water 60%, make 10mg/ml, and use for intragastric gavage.
4.3给药4.3 Administration
BALB/c小鼠禁食过夜后灌胃给药，给药剂量均为10mg/kg，给药体积均为1mL/kg。BALB/c mice were fasted overnight and then administered by gavage. The dose was 10 mg/kg and the volume was 1 mL/kg.
4.4操作4.4 Operation
小鼠灌胃给药后，于给药后5min，15min，30min，1h，2h，4h，24h由眼眶采血40μL，5μL EDTA-K2抗凝，12000rpm，4℃，5分钟离心分离血浆，于-20℃保存。After intragastric administration of mice, 40μL, 5μL EDTA-K2 anticoagulant blood was collected from the orbit 5min, 15min, 30min, 1h, 2h, 4h, 24h after the administration, and the plasma was separated by centrifugation at 12000rpm, 4℃, 5 minutes at- Store at 20°C.
测定不同浓度的药物灌胃给药后小鼠血浆中待测化合物含量：取样品室温融解，涡旋1min；定量转移15μL至2ml 96孔板中，加入150μL内标沉淀剂，振荡(1200rpm*3min)；离心(4000rpm*15min)，转移上清100μL至1ml 96孔板中；氮气吹干，加入100μL复溶液(乙腈水1：9)，振荡摇匀(900rpm*3min),20μL进样分析。LC/MS/MS条件：流动相A:0.1％甲酸水溶液，流动相B：0.1％甲酸乙腈，色谱柱：ACE C18 5μm(3.0mm*50mm)，柱温：35℃，流速0.5ml/min。Determine the content of the test compound in mouse plasma after gavage of different concentrations of drugs: take the sample to melt at room temperature, vortex for 1 min; quantitatively transfer 15 μL to a 2 ml 96-well plate, add 150 μL of internal standard precipitant, and shake (1200rpm*3min) ); Centrifuge (4000rpm*15min), transfer 100μL of supernatant to a 1ml 96-well plate; blow dry with nitrogen, add 100μL of reconstituted solution (acetonitrile water 1:9), shake and shake (900rpm*3min), 20μL injection analysis. LC/MS/MS conditions: mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile, column: ACE C18 5μm (3.0mm*50mm), column temperature: 35°C, flow rate 0.5ml/min.
试验结果：test results:
在本实验条件下，测试化合物表现出较好的药代动力学性质，结果具体见表4。Under the experimental conditions, the test compound showed good pharmacokinetic properties, and the results are shown in Table 4.
表4小鼠单次灌胃给予化合物后药代动力学参数Table 4 The pharmacokinetic parameters of mice after a single intragastric administration of compounds
Figure PCTCN2021099653-appb-000076
Figure PCTCN2021099653-appb-000076
测试实施例5：本发明化合物小鼠口服给药后的脑血比测试Test Example 5: Cerebral blood ratio test after oral administration of the compound of the present invention in mice
1.实验目的：1. The purpose of the experiment:
测定本发明化合物小鼠口服给药后，药物入脑情况及脑血比数值，评价化合物透脑情况。After oral administration of the compound of the present invention to mice, the brain penetration and cerebral blood ratio value of the drug were measured, and the brain penetration of the compound was evaluated.
2.实验方法：2. Experimental method:
2.1试验药品2.1 Experimental drugs
GDC-9545(CN107108611A表1化合物340，按专利方法制备)、实施例4化合物、实施例8化合物。GDC-9545 (CN107108611A compound 340 in Table 1, prepared according to the patent method), the compound of Example 4, and the compound of Example 8.
2.2试验动物2.2 Experimental animals
健康成年BALB/c小鼠18只，雌性，平均分为6组，每组3只，具体分组情况如下表，小鼠购自北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(浙)2019-0001。18 healthy adult BALB/c mice, females, were equally divided into 6 groups, 3 in each group. The specific grouping situation is as follows. The mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Zhejiang) 2019-0001.
Figure PCTCN2021099653-appb-000077
Figure PCTCN2021099653-appb-000077
2.3药物配制2.3 Drug preparation
称取一定量受试化合物，溶于四甘醇40％(7.5％Captisol)+水60％，配制成1mg/mL，用于灌胃。Weigh a certain amount of the test compound, dissolve it in tetraethylene glycol 40% (7.5% Captisol) + 60% water, and prepare 1 mg/mL for intragastric gavage.
2.4给药2.4 Administration
BALB/c小鼠禁食过夜后灌胃给药，给药剂量均为10mg/kg，给药体积均为10mL/kg。BALB/c mice were fasted overnight and then administered by gavage. The dosage was 10mg/kg and the volume was 10mL/kg.
2.5样品收集及检测2.5 Sample collection and testing
小鼠灌胃给药后，于给药后2h、4h安乐死动物，取脑组织、全血。全血使用EDTA-K2抗凝，12000rpm，4℃，5分钟离心分离血浆，于-20℃保存。脑组织样品收集后，加入PBS匀浆，经乙腈蛋白沉淀，使用液相质谱联用法测定脑组织中待测化合物浓度。线性范围1～1000ng/mL。After the mice were given intragastric administration, the animals were euthanized at 2h and 4h after the administration, and brain tissue and whole blood were collected. The whole blood was anticoagulated with EDTA-K2, centrifuged at 12000 rpm, 4°C, 5 minutes to separate the plasma, and stored at -20°C. After the brain tissue samples were collected, PBS was added for homogenization, and acetonitrile protein precipitation was used to determine the concentration of the test compound in the brain tissue using liquid phase mass spectrometry. The linear range is 1～1000ng/mL.
2.6实验结果2.6 Experimental results
各受试化合物的脑血比参数如表5所示。The cerebral blood ratio parameters of each test compound are shown in Table 5.
表5受试化合物的脑血比参数Table 5 Cerebral blood ratio parameters of test compounds
Figure PCTCN2021099653-appb-000078
Figure PCTCN2021099653-appb-000078
从以上结果可以看出，本发明化合物能穿透小鼠血脑屏障，进入脑组织。It can be seen from the above results that the compound of the present invention can penetrate the blood-brain barrier of mice and enter the brain tissue.
测试实施例6：本发明化合物的血浆蛋白结合率测定Test Example 6: Determination of the plasma protein binding rate of the compound of the present invention
一、试验材料及仪器1. Test materials and instruments
1.CD-1小鼠血浆(BioIVT)1. CD-1 mouse plasma (BioIVT)
2. 96孔平衡透析板(HTDialysis LLC,Gales Ferry,CT，HTD96B)，平衡透析膜(MWCO 12-14K,#1101)2. 96-well balanced dialysis plate (HTDialysis LLC, Gales Ferry, CT, HTD96B), balanced dialysis membrane (MWCO 12-14K, #1101)
3.阳性对照化合物华法林3. Positive control compound warfarin
4.ABI QTrap 5500液质联用仪4. ABI QTrap 5500 LC/MS instrument
二、试验步骤Second, the test steps
1.浓度为100mM磷酸钠盐和150mM NaCl的缓冲液的配制：用超纯水配制浓度为14.2g/L Na 2HPO 4和8.77g/L NaCl的碱性溶液，用超纯水配制浓度为12.0g/L NaH 2PO 4和8.77g/L NaCl的酸性溶液。用酸性溶液滴定碱性溶液至pH值为7.4配制成浓度为100mM磷酸钠盐和150mM NaCl的缓冲液。 1. Preparation of a buffer solution with a concentration of 100 mM sodium phosphate and 150 mM NaCl: prepare an alkaline solution with a concentration of 14.2 g/L Na 2 HPO 4 and 8.77 g/L NaCl with ultra-pure water, and use ultra-pure water to prepare a concentration of An acidic solution of 12.0g/L NaH 2 PO 4 and 8.77g/L NaCl. Titrate the alkaline solution with an acidic solution to a pH of 7.4 to prepare a buffer solution with a concentration of 100 mM sodium phosphate and 150 mM NaCl.
2.透析膜的准备：将透析膜浸泡在超纯水中60分钟以便将膜分离成两片，然后用20％乙醇浸泡20分钟，最后用透析所用缓冲液浸泡20分钟。2. Preparation of the dialysis membrane: soak the dialysis membrane in ultrapure water for 60 minutes to separate the membrane into two pieces, then soak in 20% ethanol for 20 minutes, and finally soak in the buffer for dialysis for 20 minutes.
3.血浆的准备：将冷冻的血浆迅速在室温下解冻，然后将血浆在4℃、3,220g下离心10分钟去除凝块，并将上清收集到新的离心管中。测定和记录血浆的pH值，使用pH值为7-8的血浆。3. Preparation of plasma: Thaw the frozen plasma quickly at room temperature, then centrifuge the plasma at 4°C and 3,220g for 10 minutes to remove clots, and collect the supernatant in a new centrifuge tube. Measure and record the pH value of plasma, using plasma with a pH value of 7-8.
4.含化合物的血浆样品的配制：用DMSO稀释10mM的本发明化合物或阳性对照化合物的储备液得到200μM的工作液。597μl小鼠血浆中加入3μl 200μM的化合物工作液得到终浓度为1μM的血浆样品。4. Preparation of compound-containing plasma samples: diluted 10 mM stock solution of the compound of the present invention or positive control compound with DMSO to obtain 200 μM working solution. Add 3μl 200μM compound working solution to 597μl mouse plasma to obtain a plasma sample with a final concentration of 1μM.
5.平衡透析步骤：按照操作说明将透析装置组装起来。在透析膜的一侧加入120μL含1μM化合物的血浆样品，另一侧加入等体积的透析液(磷酸盐缓冲液)。试验设双样本。封上透析板，放入孵育装置，在37℃、5％CO 2及约100rpm转速下孵育6小时。孵育结束后，去除封膜，从每个孔的缓冲液和血浆侧吸取50μl到新板的不同孔中。在磷酸盐缓冲液样品中加入50μl空白血浆，在血浆样品中加入等体积的空白磷酸盐缓冲液，然后加入300μl含内标的乙腈沉淀蛋白。涡旋5分钟，在4℃、3,220g下离心30分钟。取100μl上清液至进样板，加入100μL超纯水混匀，用于LC-MS/MS分析。 5. Balanced dialysis step: Assemble the dialysis device according to the operating instructions. Add 120 μL of plasma sample containing 1 μM compound on one side of the dialysis membrane, and add an equal volume of dialysate (phosphate buffer) on the other side. The experiment set up double samples. Seal the dialysis plate, put it into an incubator, and incubate for 6 hours at 37°C, 5% CO 2 and a rotation speed of about 100 rpm. After the incubation, remove the sealing membrane, and draw 50μl from the buffer and plasma side of each well to different wells of the new plate. Add 50μl of blank plasma to the phosphate buffer sample, add an equal volume of blank phosphate buffer to the plasma sample, and then add 300μl of acetonitrile containing internal standard to precipitate the protein. Vortex for 5 minutes and centrifuge at 3,220g for 30 minutes at 4°C. Take 100μl of supernatant to the sample plate, add 100μL of ultrapure water to mix, and use for LC-MS/MS analysis.
测定化合物在缓冲液侧和血浆侧的峰面积。计算化合物的血浆蛋白结合率公式如下：The peak areas of the compound on the buffer side and the plasma side were measured. The formula for calculating the plasma protein binding rate of the compound is as follows:
％游离率＝(化合物峰面积与内标峰面积比值缓冲液侧/化合物峰面积与内标峰面积比值血浆侧)*100％% Free rate = (compound peak area to internal standard peak area ratio buffer side/compound peak area to internal standard peak area ratio plasma side) * 100%
％结合率＝1-％游离率% Binding rate = 1-% free rate
所有的数据均通过Microsoft Excel软件进行计算。计算得到的本发明化合物的血浆蛋白结合率值见表6。All data are calculated by Microsoft Excel software. The calculated plasma protein binding rate value of the compound of the present invention is shown in Table 6.
表6本发明化合物在CD-1小鼠血浆中的蛋白结合率值Table 6 The protein binding rate value of the compound of the present invention in CD-1 mouse plasma
实施例编号Example number ％结合率% Binding rate
实施例4Example 4 98.7798.77
测试实施例7、本发明化合物的膜渗透性及转运特性测定Test Example 7. Determination of membrane permeability and transport characteristics of the compounds of the present invention
本发明化合物的膜渗透性及转运特性采用如下试验方法测定。The membrane permeability and transport characteristics of the compounds of the present invention are determined by the following test methods.
一、试验材料及仪器1. Test materials and instruments
1.Caco-2细胞(ATCC)1.Caco-2 cells (ATCC)
2.HEPES(Solarbio 804D049)、青霉素/链霉素(Solarbio 20200109)和Trypsin/EDTA(Solarbio)、PBS(Solarbio 20200620)2. HEPES (Solarbio 804D049), Penicillin/Streptomycin (Solarbio 20200109) and Trypsin/EDTA (Solarbio), PBS (Solarbio 20200620)
3.胎牛血清(FBS)(Sigma WXBD0055V)、荧光黄(Sigma MKCJ3738)、NaHCO 3(Sigma SLBZ4647) 3. Fetal Bovine Serum (FBS) (Sigma WXBD0055V), Fluorescent Yellow (Sigma MKCJ3738), NaHCO 3 (Sigma SLBZ4647)
4.Hank’s平衡盐溶液(Gibco 2085528)和非必需氨基酸(NEAA)(Gibco 2211548)、Trypsin/EDTA(Gibco 2120732)4. Hank’s balanced salt solution (Gibco 2085528) and non-essential amino acids (NEAA) (Gibco 2211548), Trypsin/EDTA (Gibco 2120732)
5.高糖DMEM(Corning 20319014)5. High sugar DMEM (Corning 20319014)
6.HTS Transwell-96 Well Permeable(Corning,3391)6.HTS Transwell-96 Well Permeable (Corning, 3391)
7.电阻检测仪(Millipore，
Figure PCTCN2021099653-appb-000079
ERS-2) 7. Resistance detector (Millipore,
Figure PCTCN2021099653-appb-000079
ERS-2)
8.
Figure PCTCN2021099653-appb-000080
Vision(Nexcelom Bioscience) 8.
Figure PCTCN2021099653-appb-000080
Vision (Nexcelom Bioscience)
9.Infinite 200 PRO酶标仪(Tecan，Infinite M200PRO)9. Infinite 200 PRO microplate reader (Tecan, Infinite M200PRO)
10.阳性对照化合物Metoprolol(Sinopharm 100084-201403)、Erythromycin(MCE 84550)和Cimetidine(Sinopharm 100158-201406)10. Positive control compounds Metoprolol (Sinopharm 100084-201403), Erythromycin (MCE 84550) and Cimetidine (Sinopharm 100158-201406)
11.ABI QTrap 5500液质联用仪11.ABI QTrap 5500 LC/MS instrument
二、试验步骤Second, the test steps
1.Caco-2细胞培养1. Caco-2 cell culture
1)转运缓冲液(含25mM HEPES的HBSS，pH 7.4)的配制：精确称量5.958g HEPES和0.35g NaHCO 3，加900mL纯水让其溶解，然后加100mL 10×HBSS搅拌均匀，调pH至7.4，过滤。 1) Preparation of transport buffer (HBSS containing 25mM HEPES, pH 7.4): accurately weigh 5.958g HEPES and 0.35g NaHCO 3 , add 900mL pure water to dissolve it, then add 100mL 10×HBSS and stir well, adjust the pH to 7.4, filter.
2)Caco-2细胞培养基的配制：高糖DMEM(含有L-谷氨酰胺)培养基中加入FBS、青霉素、链霉素、卡那霉素和NEAA配制成含10％FBS、0.1mg/mL链霉素、100单位青霉素、0.6μg/mL卡那霉素和1×NEAA的细胞培养基。2) Preparation of Caco-2 cell culture medium: high-sugar DMEM (containing L-glutamine) medium was prepared by adding FBS, penicillin, streptomycin, kanamycin and NEAA to contain 10% FBS, 0.1 mg/ mL streptomycin, 100 units of penicillin, 0.6μg/mL kanamycin and 1×NEAA cell culture medium.
3)在37℃、5％CO 2的培养箱中用T-75培养瓶培养细胞，细胞生长达到80-90％密度时弃去培养基。用5mL PBS冲洗细胞，加入1.5mL Trypsin/EDTA，然后在37℃培养箱中孵育5-10分钟直至细胞呈流沙状脱落，最后用含FBS的培养基中和Trypsin/EDTA。 3) The cells are cultured in a T-75 culture flask in an incubator at 37°C and 5% CO 2 and the culture medium is discarded when the cell growth reaches 80-90% density. Wash the cells with 5mL PBS, add 1.5mL Trypsin/EDTA, then incubate in a 37°C incubator for 5-10 minutes until the cells fall off as a quicksand, and finally neutralize the Trypsin/EDTA with a medium containing FBS.
4)细胞混悬液在120xg下离心10分钟，弃去上清液。4) The cell suspension was centrifuged at 120xg for 10 minutes, and the supernatant was discarded.
5)加细胞培养基重悬细胞，调至密度为6.86×10 5cells/mL的细胞悬浮液。 5) Add cell culture medium to resuspend the cells and adjust to a cell suspension with a density of 6.86×10 5 cells/mL.
2.Caco-2细胞接种2. Caco-2 cell inoculation
1)Transwell小室每孔加入50μL培养基，下层加入25mL培养基，置于37℃，5％CO 2培养箱中预热1小时。 1) Add 50μL of medium to each well of the Transwell chamber, add 25mL of medium to the lower layer, and place it in a 37°C, 5% CO 2 incubator for preheating for 1 hour.
2)预热的Transwell小室每孔加入50μL细胞悬浮液，最终接种密度为2.4×10 5cells/cm 2。 2) Add 50μL of cell suspension to each well of the preheated Transwell cell, and the final seeding density is 2.4×10 5 cells/cm 2 .
3)培养14-18天，隔一天换一次培养基，在最初种板以后的48小时之内更换培养基。实验前一天培养基必须更换。3) Cultivate for 14-18 days, change the medium every other day, and change the medium within 48 hours after the initial seeding. The medium must be changed the day before the experiment.
3.评估单层细胞膜完整性3. Assess the integrity of the monolayer cell membrane
1)细胞培养14天后融合并且分化，准备进行转运实验。1) The cells are fused and differentiated after 14 days of culture, ready for transport experiments.
2)用电阻仪测量单层膜电阻，记录每孔电阻。2) Measure the resistance of the single-layer film with a resistance meter, and record the resistance of each hole.
3)测量完毕后，将Transwell培养板重新孵育3) After the measurement, re-incubate the Transwell culture plate
4)计算TEER值：TEER值＝TEER(Ω)测量值×膜面积(cm 2) 4) Calculate TEER value: TEER value = TEER (Ω) measured value × film area (cm 2 )
单层细胞膜的电阻<230Ω·cm 2，指示单层细胞膜致密性差，不能用于试验。 The electrical resistance of the monolayer cell membrane is less than 230Ω·cm 2 , which indicates that the monolayer cell membrane has poor compactness and cannot be used in the test.
4.转运实验4. Transport experiment
1)用DMSO稀释10mM的本发明化合物或阳性对照化合物的储备液得到2mM的储备液，然后用转运缓冲液稀释2mM的储备液得到10μM的本发明化合物或阳性对照化合物的工作液。1) Dilute 10 mM stock solution of the compound of the present invention or positive control compound with DMSO to obtain a 2 mM stock solution, and then dilute the 2 mM stock solution with transport buffer to obtain a working solution of 10 μM compound of the present invention or positive control compound.
2)从培养箱中取出Caco-2细胞板，然后用预热的转运缓冲液清洗Transwell培养板两次，再置于37℃培养箱孵育30分钟。2) Take out the Caco-2 cell plate from the incubator, then wash the Transwell culture plate twice with pre-warmed transport buffer, and then place it in the 37°C incubator and incubate for 30 minutes.
3)为测定化合物从顶端到基底端(A→B)的转运速率，加108μL化合物的工作液到Transwell小室(顶端)，同时立即从顶端取出8μL样品至72μL转运缓冲液中，加入240μL含内标的终止液终止转运以作为初始顶端样品。同时，接收端(基底端)加入300μL转运缓冲液。试验设双样本。3) To determine the transfer rate of the compound from the top to the basal end (A→B), add 108μL of the working solution of the compound to the Transwell chamber (top), and at the same time immediately take out 8μL of the sample from the top to 72μL of the transport buffer, and add 240μL containing The target stop solution was used as the initial tip sample to terminate the transport. At the same time, 300 μL of transport buffer was added to the receiving end (basal end). The experiment set up double samples.
4)为测定化合物从基底端到顶端(B→A)的转运速率，加308μL化合物的工作液到接收端(基底端)，同时立即从基底端取出8μL样品至72μL转运缓冲液中，加入240μL含内标的终止液终止转运以作为初始基底端样品。同时，Transwell小室(顶端)加入300μL转运缓冲液。试验设双样本。4) To determine the transfer rate of the compound from the basal end to the top (B→A), add 308 μL of the compound working solution to the receiving end (basal end), and at the same time immediately take out 8 μL of the sample from the basal end to 72 μL of transport buffer, and add 240 μL The stop solution containing the internal standard was used as the initial basal end sample to terminate the transport. At the same time, 300 μL of transport buffer was added to the Transwell chamber (top). The experiment set up double samples.
5)将细胞培养板置于37℃ CO 2培养箱中孵育2小时。 5) Place the cell culture plate in a 37°C CO 2 incubator and incubate for 2 hours.
6)转运实验结束后，从给药端(即A→B方向的顶端和B→A方向的基底端)取8μL样品至72μL转运缓冲液中，然后加入240μL含内标的终止液终止转运。从接收端(即A→B方向的基底端和B→A方向的顶端)取80μL样品至240μL含内标的终止液中，1000rpm下涡旋10分钟，3,220g下离心30分钟。取100μl上清液至进样板，加入100μL超纯水混匀，用于LC-MS/MS分析。6) After the end of the transfer experiment, take 8 μL of sample from the drug delivery end (the top end in the A→B direction and the basal end in the B→A direction) to 72 μL of the transfer buffer, and then add 240 μL of the stop solution containing the internal standard to stop the transfer. From the receiving end (ie the basal end in the A→B direction and the top in the B→A direction), take 80 μL of the sample into 240 μL of the stop solution containing the internal standard, vortex at 1000 rpm for 10 minutes, and centrifuge at 3,220 g for 30 minutes. Take 100μl of supernatant to the sample plate, add 100μL of ultrapure water to mix, and use for LC-MS/MS analysis.
7)转运实验结束后测量荧光值，用水配制10mM荧光黄储备液，然后用转运缓冲溶液稀释至100μM。往Transwell小室(顶端)中加入100μL荧光黄溶液，基底端加入300μL转运缓冲溶液，置于37℃的CO 2培养箱中孵育30分钟。从基底端取80μL溶液至96孔板中，在激发波长为485nm及发射波长为530nm下用酶标仪测量细胞荧光值(检测膜完整性)。 7) Measure the fluorescence value after the end of the transport experiment, prepare a 10mM fluorescent yellow stock solution with water, and then dilute it to 100μM with transport buffer solution. Add 100 μL of fluorescent yellow solution to the Transwell chamber (top) and 300 μL of transport buffer solution to the basal end, and incubate in a CO 2 incubator at 37°C for 30 minutes. Take 80 μL of the solution from the base end to a 96-well plate, and measure the cell fluorescence value with a microplate reader under the excitation wavelength of 485nm and emission wavelength of 530nm (to detect membrane integrity).
用以下公式计算Caco-2细胞单层膜的荧光值：Use the following formula to calculate the fluorescence value of the Caco-2 cell monolayer:
LY Leakage＝{I acceptor×0.3/(I acceptor×0.3+I donor×0.1)}×100％ LY Leakage={I acceptor ×0.3/(I acceptor ×0.3+I donor ×0.1)}×100%
I acceptor指接收侧(0.3mL)的荧光密度，I donor指给药侧(0.1mL)的荧光密度。LY>1.0％表示单层细胞膜致密性差，相应的结果将从评估中排除。 I acceptor refers to the fluorescence density on the receiving side (0.3 mL), and I donor refers to the fluorescence density on the dosing side (0.1 mL). LY>1.0% means that the monolayer cell membrane has poor compactness, and the corresponding results will be excluded from the evaluation.
测定化合物在给药侧和接收侧的峰面积。计算化合物的表观渗透系数(P app，单位：cm/s)和外排比(Efflux ratio,ER)： The peak areas of the compound on the dosing side and the receiving side are measured. Calculate the apparent permeability coefficient (P app , unit: cm/s) and Efflux ratio (ER) of the compound:
P app＝{V A×[drug] acceptor/(Area×incubation time×[drug] initial donor} P app ＝{V A ×[drug] acceptor /(Area×incubation time×[drug] initial donor }
V A为接收端溶液的体积(A→B是0.3mL，B→A是0.1mL)；Area为Transwell-96孔板膜面积(0.143cm 2)；incubation time为孵育时间(单位：s)；[drug] acceptor为接收侧药物浓度，[drug] initial donor为初始给药侧药物浓度。 V A is the volume of the receiving end solution (A→B is 0.3mL, B→A is 0.1mL); Area is the membrane area of the Transwell-96-well plate (0.143cm 2 ); incubation time is the incubation time (unit: s); [drug] acceptor is the drug concentration on the receiving side, and [drug] initial donor is the drug concentration on the initial dosing side.
Figure PCTCN2021099653-appb-000081
Figure PCTCN2021099653-appb-000081
P app(B-A)为由基底端到顶端的表观渗透系数；P app(A-B)为由顶端到基底端的表观渗透系数。 P app (BA) is the apparent permeability coefficient from the base end to the top; P app (AB) is the apparent permeability coefficient from the top end to the base end.
计算得到的本发明化合物的表观渗透系数和外排比值见表7。The calculated apparent permeability coefficient and efflux ratio of the compound of the present invention are shown in Table 7.
表7本发明化合物的表观渗透系数和外排比值Table 7 Apparent permeability coefficient and efflux ratio of the compound of the present invention
Figure PCTCN2021099653-appb-000082
Figure PCTCN2021099653-appb-000082
测试实施例8、本发明化合物对CYP2C9、CYP2D6、CYP3A4酶活性的抑制作用Test Example 8. The inhibitory effect of the compound of the present invention on the enzyme activity of CYP2C9, CYP2D6, and CYP3A4
本发明化合物对CYP2C9、CYP2D6、CYP3A4酶活性的抑制采用如下试验方法测定。The inhibition of CYP2C9, CYP2D6, and CYP3A4 enzyme activity by the compound of the present invention was determined by the following test method.
一、试验材料及仪器1. Test materials and instruments
1.人肝微粒体(Corning 452117)1. Human liver microsomes (Corning 452117)
2.Na 2HPO 4(Sigma SLBZ6180) 2.Na 2 HPO 4 (Sigma SLBZ6180)
3.KH 2PO 4(Sigma SLBT6559) 3.KH 2 PO 4 (Sigma SLBT6559)
4.NADPH(Solarbio 705Y021)4.NADPH (Solarbio 705Y021)
5.NADPH(Solarbio 705Y021)5.NADPH (Solarbio 705Y021)
6.阳性底物双氯芬酸(Sigma SLBV3438)、右美沙芬(TRC 3-EDO-175-1)和咪达***(Cerilliant FE01161704)6. Positive substrates: Diclofenac (Sigma SLBV3438), Dextromethorphan (TRC 3-EDO-175-1) and Midazolam (Cerilliant FE01161704)
7.阳性抑制剂磺胺苯吡唑(D.Ehrenstorfer GmbH 109012)、奎尼丁(TCI WEODL-RE)和酮康唑(Sigma 100M1091V)7. Positive inhibitors Sulfafenpyrazole (D.Ehrenstorfer GmbH 109012), Quinidine (TCI WEODL-RE) and Ketoconazole (Sigma 100M1091V)
8.AB Sciex Triple Quad 5500液质联用仪8.AB Sciex Triple Quad 5500 LC/MS
二、试验步骤Second, the test steps
1. 100mM磷酸缓冲液(PBS)的配制：称取7.098g Na 2HPO 4，加入500mL纯水超声溶解，作为溶液A。称取3.400g KH 2PO 4，加入250mL纯水超声溶解，作为溶液B。将A溶液放置在搅拌器上缓慢加入B溶液直到pH值达到7.4配制成100mM的PBS缓冲液。 1. Preparation of 100mM phosphate buffer (PBS): Weigh 7.098g Na 2 HPO 4 , add 500mL pure water and dissolve it ultrasonically, as solution A. Weigh 3.400 g of KH 2 PO 4 , add 250 mL of pure water and dissolve it ultrasonically, as solution B. Place the A solution on a stirrer and slowly add the B solution until the pH value reaches 7.4 to prepare a 100 mM PBS buffer.
2.用100mM的PBS缓冲液配制10mM的NADPH溶液。用DMSO稀释10mM的本发明化合物储备液得到200×浓度的化合物工作液(6000、2000、600、200、60、20、0μM)。用DMSO稀释阳性抑制剂储备液得到200×浓度的阳性抑制剂工作液(磺胺苯吡唑，1000、300、100、30、10、3、0μM；奎尼丁/酮康唑，100、30、10、3、1、0.3、0μM)。用水、乙腈或乙腈/甲醇配制200×浓度的底物工作液(120μM双氯芬酸、400μM右美沙芬和200μM咪达***)。2. Prepare 10 mM NADPH solution with 100 mM PBS buffer. The 10 mM stock solution of the compound of the present invention was diluted with DMSO to obtain a 200× concentration compound working solution (6000, 2000, 600, 200, 60, 20, 0 μM). Dilute the positive inhibitor stock solution with DMSO to obtain a 200×concentration positive inhibitor working solution (Sulfapyrazole, 1000, 300, 100, 30, 10, 3, 0μM; Quinidine/ketoconazole, 100, 30, 10, 3, 1, 0.3, 0μM). Prepare a 200× concentration substrate working solution (120μM diclofenac, 400μM dextromethorphan and 200μM midazolam) with water, acetonitrile or acetonitrile/methanol.
3.取2μl 20mg/ml的肝微粒体溶液、1μl底物工作液、1μl化合物工作液和176μl PBS缓冲液，混合均匀，置于37℃水浴中预孵育15分钟。阳性对照组加入1μl双氯芬酸、右美沙芬或咪达***工作液代替化合物工作液。同时将10mM的NADPH溶液一起在37℃水浴中预孵育15分钟。15分钟后，取20μl NADPH加入到各个孔中，启动反应，37℃下孵育5分钟(CYP2C9)、20分钟(CYP2D6)或5分钟(CYP3A4)。所有孵育样品设双样本。孵育相应时间后向所有样本中加入400μl含内标的冰甲醇终止反应。涡旋混匀，3220g、4℃离心40分钟。离心结束后转移100μL上清液到进样板，加入100μL超纯水混匀，用于LC-MS/MS分析。3. Take 2μl of 20mg/ml liver microsome solution, 1μl of substrate working solution, 1μl of compound working solution and 176μl of PBS buffer, mix well, and place in a 37℃ water bath to pre-incubate for 15 minutes. In the positive control group, 1 μl of diclofenac, dextromethorphan or midazolam working solution was added to replace the compound working solution. At the same time, the 10mM NADPH solution was pre-incubated in a 37°C water bath for 15 minutes. After 15 minutes, add 20μl NADPH to each well to start the reaction, and incubate at 37°C for 5 minutes (CYP2C9), 20 minutes (CYP2D6) or 5 minutes (CYP3A4). All incubation samples have dual samples. After incubating for the corresponding time, add 400μl of ice methanol containing internal standard to all samples to terminate the reaction. Vortex to mix, centrifuge at 3220g for 40 minutes at 4°C. After centrifugation, transfer 100μL of supernatant to the sample plate, add 100μL of ultrapure water to mix, and use for LC-MS/MS analysis.
数值经Excel XLfit 5.3.1.3计算得到本发明化合物对CYP2C9、CYP2D6和CYP3A4的IC 50值见表8。 The values are calculated by Excel XLfit 5.3.1.3 to obtain the IC 50 values of the compounds of the present invention for CYP2C9, CYP2D6 and CYP3A4, as shown in Table 8.
表8本发明化合物对CYP2C9、CYP2D6和CYP3A4的IC 50值 Table 8 The IC 50 values of the compounds of the present invention on CYP2C9, CYP2D6 and CYP3A4
Figure PCTCN2021099653-appb-000083
Figure PCTCN2021099653-appb-000083
测试实施例9：化合物对MCF-7异种移植瘤的生长抑制实验Test Example 9: Growth inhibition experiment of compound on MCF-7 xenograft tumor
实验试剂：Experimental reagents:
人乳腺癌MCF-7细胞：ECACC-86012803Human breast cancer MCF-7 cells: ECACC-86012803
17β-***片：Innovative Research of America,Cat No.:SE-121,60-day release,0.18mg/pellet17β-estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.18mg/pellet
EMEM培养液：ATCC,Cat No.:30-2003EMEM culture medium: ATCC, Cat No.: 30-2003
胎牛血清：Hyclone；Cat No.:SV30087.03Fetal Bovine Serum: Hyclone; Cat No.: SV30087.03
Antibiotic-Antimycotic：Gibco,Cat No.:15240-062Antibiotic-Antimycotic: Gibco, Cat No.: 15240-062
0.25％胰酶-EDTA：Gibco,Cat No.:25200-0720.25% Pancreatin-EDTA: Gibco, Cat No.: 25200-072
DPBS：Corning,Cat No.:21-031-CVRDPBS: Corning,Cat No.:21-031-CVR
基质胶：Corning,Cat No.:354234Matrigel: Corning, Cat No.: 354234
实验方法：experimental method:
动物信息：Balb/c裸小鼠，雌性，6-8周，体重约18-22克，动物购自上海灵畅生物科技有限公司，将小鼠饲养在SPF级的环境中，每个笼位单独送排风，所有动物都可以自由获取标准认证的商业实验室饮食和自由饮水。Animal information: Balb/c nude mice, female, 6-8 weeks old, weighing about 18-22 grams, the animals were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were kept in an SPF environment, each cage With separate air supply and exhaust, all animals have free access to standard-certified commercial laboratory food and free drinking water.
细胞培养：人乳腺癌MCF-7细胞株体外培养，培养条件为EMEM(细胞培养液)中加入10％胎牛血清，1％Antibiotic-Antimycotic，37℃、5％CO 2孵箱。一周两次用0.25％胰酶-EDTA消化液进行常规消化处理传代。当细胞饱和度为80％-90％，数量达到要求时，收取细胞，计数。 Cell culture: Human breast cancer MCF-7 cell line is cultured in vitro, and the culture conditions are EMEM (cell culture medium) with 10% fetal bovine serum, 1% Antibiotic-Antimycotic, 37°C, 5% CO 2 incubator. Use 0.25% pancreatin-EDTA digestion solution twice a week for routine digestion and passage. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected and counted.
细胞接种：将0.2ml/(含1×10 7)MCF-7细胞悬液(DPBS加基质胶，体积比为1:1)皮下接种于每只 小鼠的右后背，并于细胞接种前两天皮下接种17β-***片。在接种细胞后第6天，依据肿瘤体积随机分组给药，分组当天为Day 0。 Cell inoculation: 0.2ml/(containing 1×10 7 ) MCF-7 cell suspension (DPBS plus Matrigel, volume ratio 1:1) was subcutaneously inoculated on the right back of each mouse, and before cell inoculation 17β-estradiol tablets were inoculated subcutaneously in two days. On the 6th day after cell inoculation, drugs were administered in random groups according to the tumor volume, and the day of grouping was Day 0.
给药：化合物的给药剂量为3mg/kg或10mg/kg，口服给药(PO)，每天一次给药(QD)x3周，PO，QDx3周。每组6只小鼠。Administration: The dosage of the compound is 3 mg/kg or 10 mg/kg, oral administration (PO), once a day (QD) x 3 weeks, PO, QD x 3 weeks. Each group has 6 mice.
肿瘤测量和实验指标：Tumor measurement and experimental indicators:
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a x b 2，a和b分别表示肿瘤的长径和短径。每周两次测量小鼠体重。 The diameter of the tumor was measured with a vernier caliper twice a week. The calculation formula of tumor volume is: V=0.5a x b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of the mice was measured twice a week.
化合物的抑瘤疗效用肿瘤生长抑制率TGI(％)来评价。The anti-tumor efficacy of the compound was evaluated by the tumor growth inhibition rate TGI (%).
TGI(％)＝[(1-(某处理组给药结束时平均瘤体积-该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]x100％。TGI(%)=[(1-(Average tumor volume at the end of a certain treatment group-Average tumor volume at the start of the treatment group)/(Average tumor volume at the end of the solvent control group-When the solvent control group starts treatment Average tumor volume)] x 100%.
实验结果如表9所示。The experimental results are shown in Table 9.
表9 MCF-7皮下瘤模型肿瘤体积Table 9 Tumor volume of MCF-7 subcutaneous tumor model
Figure PCTCN2021099653-appb-000084
Figure PCTCN2021099653-appb-000084
测试实施例10：化合物对T47D异种移植瘤的生长抑制实验Test Example 10: Growth inhibition experiment of compound on T47D xenograft tumor
实验试剂：Experimental reagents:
人乳腺癌T47D细胞：ATCC,HTB-133Human breast cancer T47D cells: ATCC, HTB-133
17β-***片：Innovative Research of America,Cat No.:SE-121,60-day release,0.36mg/pellet17β-estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.36mg/pellet
RPMI1640培养液：Gibco,Cat No.:22400-089RPMI1640 culture medium: Gibco, Cat No.: 22400-089
胎牛血清：Gbico；Cat No.:10099-141CFetal Bovine Serum: Gbico; Cat No.: 10099-141C
青链霉素(Pen Strep)：Gibco,Cat No.:15240-122Pen Strep: Gibco, Cat No.: 15240-122
0.25％胰酶-EDTA：Gibco,Cat No.:25200-0720.25% Pancreatin-EDTA: Gibco, Cat No.: 25200-072
D-PBS(无钙镁离子磷酸盐缓冲液)：Hyclone,Cat.No.:SH30256.01D-PBS (calcium-magnesium ion-free phosphate buffer): Hyclone, Cat. No.: SH30256.01
基质胶：Corning,Cat No.:356237Matrigel: Corning, Cat No.: 356237
牛胰岛素：上海翊圣，Cat No.:40107ES60Bovine insulin: Shanghai Yisheng, Cat No.: 40107ES60
实验方法：experimental method:
动物信息：NPG小鼠，雌性，6-8周，体重约18-22克，动物购自北京维通达生物技术有限公司，将小鼠饲养在SPF级的环境中，每个笼位单独送排风，所有动物都可以自由获取标准认证的商业实验室饮食和自由饮水。Animal information: NPG mice, female, 6-8 weeks old, weighing about 18-22 grams, the animals were purchased from Beijing Weitongda Biotechnology Co., Ltd. The mice were kept in an SPF environment, and each cage was sent out separately Wind, all animals have free access to standard certified commercial laboratory food and drinking water.
细胞培养：人乳腺癌T47D细胞株体外培养，培养条件为RPMI1640(细胞培养液)中加入10％胎牛血清，8μg/ml牛胰岛素，1％pen-strep，37℃、5％CO 2孵箱。一周一次用0.25％胰酶-EDTA消化液进行常规消化处理传代。当细胞汇合度为80％-90％，数量达到要求时，收取细胞，计数。 Cell culture: human breast cancer T47D cell line is cultured in vitro, and the culture conditions are RPMI1640 (cell culture medium) with 10% fetal bovine serum, 8μg/ml bovine insulin, 1% pen-strep, 37°C, 5% CO 2 incubator . Use 0.25% pancreatin-EDTA digestion solution for routine digestion and passage once a week. When the cell confluence is 80%-90% and the number reaches the requirement, the cells are collected and counted.
细胞接种：将0.1ml/(含1×10 7)T47D细胞的悬液(DPBS加基质胶，体积比为1:1)皮下接种于每只小鼠的右侧腰背部，并于细胞接种前两天皮下接种17β-***片。在接种细胞后第29天，依据肿瘤体积随机分组给药，分组当天为Day 0。 Cell inoculation: 0.1ml/(containing 1×10 7 ) T47D cell suspension (DPBS plus Matrigel, volume ratio 1:1) subcutaneously inoculated on the right back of each mouse, and before cell inoculation 17β-estradiol tablets were inoculated subcutaneously in two days. On the 29th day after cell inoculation, drugs were administered in random groups according to the tumor volume, and the day of grouping was Day 0.
给药：阳性药Fulvestrant(
Figure PCTCN2021099653-appb-000085
氟维司群注射液，阿斯利康)的给药剂量为250mg/kg，皮下注射给药(SC)，每周一次给药(QW)x4周，化合物的给药剂量为3mg/kg或10mg/kg，口服给药(PO)，每天一次给药(QD)x4周，PO，QDx4周。每组6只小鼠。 Administration: Positive drug Fulvestrant (
Figure PCTCN2021099653-appb-000085
Fulvestrant injection, AstraZeneca) at a dosage of 250mg/kg, subcutaneous injection (SC), once a week (QW) x 4 weeks, the compound dosage is 3mg/kg or 10mg /kg, oral administration (PO), once a day (QD) x 4 weeks, PO, QD x 4 weeks. Each group has 6 mice.
肿瘤测量和实验指标：Tumor measurement and experimental indicators:
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a x b 2，a和b分别表示肿瘤的长径和短径。每周两次测量小鼠体重。 The diameter of the tumor was measured with a vernier caliper twice a week. The calculation formula of tumor volume is: V=0.5a x b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of the mice was measured twice a week.
化合物的抑瘤疗效用肿瘤生长抑制率TGI(％)来评价。The anti-tumor efficacy of the compound was evaluated by the tumor growth inhibition rate TGI (%).
TGI(％)＝[(1-(某处理组给药结束时平均瘤体积-该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]x100％。TGI(%)=[(1-(Average tumor volume at the end of a certain treatment group-Average tumor volume at the start of the treatment group)/(Average tumor volume at the end of the solvent control group-When the solvent control group starts treatment Average tumor volume)] x 100%.
实验结果如表10所示。The experimental results are shown in Table 10.
表10 T-47D皮下瘤模型肿瘤体积Table 10 Tumor volume of T-47D subcutaneous tumor model
Figure PCTCN2021099653-appb-000086
Figure PCTCN2021099653-appb-000086
测试实施例11：Langendorff离体心脏灌流实验Test Example 11: Langendorff isolated heart perfusion experiment
实验试剂：Experimental reagents:
Glucose(葡萄糖),General-reagent,目录号:G2410AGlucose, General-reagent, catalog number: G2410A
EDTA,Sinopharm,目录号:10009617EDTA, Sinopharm, catalog number: 10009617
Heparin sodium salt(肝素钠),Sinopharm,目录号:63007131Heparin sodium salt, Sinopharm, catalog number: 63007131
Sodium pentobarbital(戊巴比妥钠),Solarbio,目录号:P8410Sodium pentobarbital (sodium pentobarbital), Solarbio, catalog number: P8410
受试化合物：实施例4化合物、实施例8化合物、GDC-9545(CN107108611A表1化合物340，按专利方法制备)、AZD-9833(CN109843888A实例17化合物，按专利方法制备)。Test compounds: Example 4 compound, Example 8 compound, GDC-9545 (CN107108611A Table 1 compound 340, prepared according to the patent method), AZD-9833 (CN109843888A Example 17 compound, prepared according to the patent method).
离体心脏的制备：Preparation of the isolated heart:
成年Hartley豚鼠，体重250～300g，戊巴比妥钠(40mg/kg，i.p.)腹腔麻醉5～10分钟，麻醉前进行肝素化处理(500U/kg，i.p.)。迅速打开胸腔，将含有部分上腔静脉和较长主动脉弓的心脏立即从豚鼠胸腔分离，并将其浸入预冷的台式液中(台式液配制(mM)：131NaCl、4.0KCl、1.8CaCl 2、1.2MgSO 4、11.1葡萄糖、24.9NaHCO 3、1.2KH 2PO 4(pH 7.4))，迅速将豚鼠离体心脏主动脉套入langendorff灌流装置的灌流针并固定，用36.5±1℃ 95％O 2-5％CO 2饱和的台式液(pH 7.35±0.05)进行逆向灌流，流速控制在15～20ml/min，保持恒流灌流。记录电极的两个位点分别放置在心室外膜的两侧，产生双极反式心电图。 Adult Hartley guinea pigs, weighing 250-300g, were anesthetized intraperitoneally with sodium pentobarbital (40mg/kg, ip) for 5-10 minutes, and were treated with heparinization (500U/kg, ip) before anesthesia. The chest cavity was opened quickly, and the heart containing part of the superior vena cava and the longer aortic arch was immediately separated from the chest cavity of the guinea pig, and immersed in the pre-cooled bench-top solution (mM): 131NaCl, 4.0KCl, 1.8CaCl 2 , 1.2 MgSO 4 , 11.1 glucose, 24.9NaHCO 3 , 1.2KH 2 PO 4 (pH 7.4)), quickly insert the isolated guinea pig heart aorta into the perfusion needle of the Langendorff perfusion device and fix it with 36.5±1℃ 95% O 2- 5% CO 2 saturated bench-top solution (pH 7.35±0.05) is used for reverse perfusion, the flow rate is controlled at 15-20ml/min, and the perfusion is maintained at a constant flow. The two points of the recording electrode are placed on both sides of the ventricular membrane to generate a bipolar trans-ECG.
电信号记录：Electric signal record:
ECG(双极心室外膜心电图)通过心外膜上的一对表面电极记录，一个电极放置在右心室靠近房室环处，另一个电极放置在左心室表面，调节其位置以能够获得一个较大清晰的T波，无关电极放置在主动脉根部。稳定30分钟后，整个实验过程各记录电极的位置都不再调节。ECG (bipolar epicardial electrocardiogram) is recorded by a pair of surface electrodes on the epicardium. One electrode is placed on the right ventricle close to the atrioventricular ring, and the other electrode is placed on the surface of the left ventricle. Adjust its position to obtain a better comparison. Large and clear T waves, irrelevant electrodes are placed at the aortic root. After stabilizing for 30 minutes, the positions of the recording electrodes were no longer adjusted during the entire experiment.
所有的信号通过放大器放大并数字化处理后记录，采样速率为1kHz保存在硬盘里以脱机分析。所有的数据都由PowerLab离体心脏***实时记录。All signals are amplified by an amplifier and digitally processed and recorded. The sampling rate is 1kHz and saved in the hard disk for offline analysis. All data are recorded in real time by the PowerLab Isolated Heart System.
实验方案：Experimental program:
在稳定30分钟后(稳定30分钟后的结果作为溶剂阴性对照组结果)，所有符合实验条件的心脏用含化合物的灌流液进行灌流。同样的实验步骤重复三遍，使每一个心脏都经下列递增浓度(1.1、3.3、10μM)的灌流液灌流。灌流结束后用正常台式液清洗20分钟。在窦性心率下采集数据，用于评价受试化合物的作用。After 30 minutes of stabilization (the result after 30 minutes of stabilization was used as the result of the solvent negative control group), all the hearts that met the experimental conditions were perfused with the compound-containing perfusate. The same experimental procedure was repeated three times, so that each heart was perfused with the following increasing concentrations (1.1, 3.3, 10μM) of perfusate. After the perfusion, clean with normal bench-top fluid for 20 minutes. Data are collected at sinus heart rate to evaluate the effects of test compounds.
参数分析：Parameter analysis:
ECG结果显示了心脏的整体电活动性，下列参数均来自实验记录的ECG分析：RR间期、PR间期、QRS波群、QT间期和QTc(Bazett QTc＝QT/RR)。The ECG results show the overall electrical activity of the heart. The following parameters are from the ECG analysis recorded in the experiment: RR interval, PR interval, QRS complex, QT interval and QTc (Bazett QTc=QT/RR).
实验结果如表11所示。The experimental results are shown in Table 11.
表11 Langendorff试验心率变化结果Table 11 Heart rate change results of Langendorff test
Figure PCTCN2021099653-appb-000087
Figure PCTCN2021099653-appb-000087
注：*表示有显著性差异。Note: * indicates a significant difference.
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。In the foregoing, the embodiments of the present invention have been described. However, the present invention is not limited to the above-mentioned embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (13)

  1. 式(I)所示化合物或其药学上可接受的盐：The compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
    Figure PCTCN2021099653-appb-100001
    Figure PCTCN2021099653-appb-100001
    其中：in:
    X 1、X 2、X 3、X 4独立地选自CR 7或N； X 1 , X 2 , X 3 , and X 4 are independently selected from CR 7 or N;
    R 7选自H、F、Cl、Br、I、OH、CN、C 1-C 6烷基、C 3-C 6环烷基、3-6元杂环基、C 1-C 6烷氧基、C 3-C 6环烷基氧基或3-6元杂环基氧基； R 7 is selected from H, F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclyl, C 1 -C 6 alkoxy Group, C 3 -C 6 cycloalkyloxy or 3-6 membered heterocyclyloxy;
    Het选自
    Figure PCTCN2021099653-appb-100002
    Het is selected from
    Figure PCTCN2021099653-appb-100002
    R 1、R 2、R 9独立地选自氢、OH、F、Cl、Br、I、C 1-C 6烷基或C 2-C 8烯基； R 1 , R 2 , and R 9 are independently selected from hydrogen, OH, F, Cl, Br, I, C 1 -C 6 alkyl or C 2 -C 8 alkenyl;
    或者，R 1、R 2与其连接的碳原子共同形成C 3-C 6环烷基或3-6元杂环烷基，所述C 3-C 6环烷基或3-6元杂环烷基任选被R a取代； Alternatively, R 1 , R 2 and the carbon atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, the C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group group is optionally substituted with R a;
    R 5选自C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基，所述C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基任选被1个或多个选自如下的基团取代：氘、F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3； R 5 is selected from C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, the C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected from one or more of the following Substitution of groups: deuterium, F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
    R 6选自H、C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基，所述C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3； R 6 is selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, said C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected by one or more Substitution from the following groups: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
    或者，R 1或R 2之一与R 6及其各自连接的C和N共同形成3-6元杂环基； Alternatively, one of R 1 or R 2 and R 6 and the C and N respectively connected to form a 3-6 membered heterocyclic group;
    R 3选自H、CN、COOH、C(O)OR b、OC(O)R b、CONH 2、C(O)NR bR c、SO 2R b、SO 2NR bR c、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基、C 3-C 6杂环基、C 6-C 10芳基或C 5-C 10杂芳基，所述C 1-C 6烷基、C 3-C 6环烷基、C 3-C 6杂环基、C 6-C 10芳基或C 5-C 10杂芳基任选被1个或多个R d取代； R 3 is selected from H, CN, COOH, C(O)OR b , OC(O)R b , CONH 2 , C(O)NR b R c , SO 2 R b , SO 2 NR b R c , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, C 3 -C 6 heterocyclyl, C 6 -C 10 aryl or C 5 -C 10 heteroaryl, The C 1 -C 6 alkyl group, C 3 -C 6 cycloalkyl group, C 3 -C 6 heterocyclic group, C 6 -C 10 aryl group or C 5 -C 10 heteroaryl group are optionally selected by 1 or Multiple R d substitutions;
    R 4选自H、F、Cl、Br、I、OH、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基或C 3-C 6杂环基，所述C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基或C 3-C 6杂环基任选被一个或多个选自如下的基团取代：F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3； R 4 is selected from H, F, Cl, Br, I, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 heterocyclyl , The C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group, C 3 -C 6 cycloalkyl group or C 3 -C 6 heterocyclic group is optionally selected from one or more groups as follows Replacement: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
    环Q选自C 6-C 10芳基或5-10元杂芳基，所述C 6-C 10芳基或5-10元杂芳基任选被1个或多个R 8取代； Ring Q is selected from a C 6 -C 10 aryl group or a 5-10 membered heteroaryl group, the C 6 -C 10 aryl group or a 5-10 membered heteroaryl group is optionally substituted by one or more R 8 ;
    R 8选自F、Cl、Br、I、OH、CN、COOH、C(O)OR b、OC(O)R b、CONH 2、C(O)NR bR c、NR cC(O)R b、SO 2R b、SO 2NR b、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基、3-6元杂环基、C 6-C 10芳基或5-10元杂芳基，所述C 3-C 6环烷基、3-6元杂环基、C 6-C 10芳基或5-10元杂芳基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、OH、CN、C 1-C 6烷基和C 1-C 6烷氧基； R 8 is selected from F, Cl, Br, I, OH, CN, COOH, C(O)OR b , OC(O)R b , CONH 2 , C(O)NR b R c , NR c C(O) R b , SO 2 R b , SO 2 NR b , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclic group, C 6- C 10 aryl group or 5-10 membered heteroaryl group, the C 3 -C 6 cycloalkyl group, 3-6 membered heterocyclic group, C 6 -C 10 aryl group or 5-10 membered heteroaryl group is optionally Substitution with one or more groups selected from: F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl and C 1 -C 6 alkoxy;
    R a选自卤素、OH、CN、C 1-6烷基、C 1-6烷氧基、C 3- 6环烷基或3-6元杂环基，所述C 1-6烷基、C 1-6烷氧基、C 3- 6环烷基或3-6元杂环基任选被卤素取代； R a is selected from halo, OH, CN, C 1-6 alkyl, C 1-6 alkoxy, C 3 - 6 cycloalkyl or 3-6 membered heterocyclyl, a C 1-6 alkyl group, C 1-6 alkoxy, C 3 - 6 cycloalkyl or 3-6 membered heterocyclyl group optionally substituted by halogen;
    R b独立选自C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基、3-6元杂环基、C 6-C 10芳基或5-10元杂芳基； R b is independently selected from C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclyl, C 6 -C 10 aryl or 5-10 membered heteroaryl;
    R c独立选自H、C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基、3-6元杂环基、C 6-C 10芳基或5-10元杂芳基； R c is independently selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclic group, C 6 -C 10 aryl or 5-10 membered heteroaryl;
    R d独立选自F、Cl、Br、I、OH、CN、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基或3-6元杂环基，所述C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基或3-6元杂环基任选被1个或多个选自F、Cl、Br、I或OH的基团取代； R d is independently selected from F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or 3-6 membered heterocyclic group , The C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group, C 3 -C 6 cycloalkyl group or 3-6 membered heterocyclic group is optionally selected from one or more of F, Cl, Group substitution of Br, I or OH;
    n为0、1或2；n is 0, 1 or 2;
    m为1、2、3或4；m is 1, 2, 3 or 4;
    p为1或2；p is 1 or 2;
    条件是：(1)当n为0或1时，R 1不为H、F或甲基；(2)当m为1或2时，R 9不为H。 The conditions are: (1) when n is 0 or 1, R 1 is not H, F or methyl; (2) when m is 1 or 2, R 9 is not H.
  2. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其中所述式(I)所示化合物或其药学上可接受的盐选自式(Ia)化合物或其药学上可接受的盐：The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (Ia) or Pharmaceutically acceptable salt:
    Figure PCTCN2021099653-appb-100003
    Figure PCTCN2021099653-appb-100003
  3. 根据权利要求1或2所述的式(I)所示化合物或其药学上可接受的盐，其中所述Het选自
    Figure PCTCN2021099653-appb-100004
    所述R 1、R 2、R 5、n如权利要求1所定义。     The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the Het is selected from
    Figure PCTCN2021099653-appb-100004
    The R 1 , R 2 , R 5 , and n are as defined in claim 1.
  4. 根据权利要求3所述的式(I)所示化合物或其药学上可接受的盐，其中所述Het选自
    Figure PCTCN2021099653-appb-100005
    其中R 1、R 2与其连接的碳原子共同形成C 3-C 6环烷基或3-6元杂环烷基，所述C 3-C 6环烷基或3-6元杂环烷基任选被R a取代，所述R a、R 5和n如权利要求1所定义。     The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 3, wherein the Het is selected from
    Figure PCTCN2021099653-appb-100005
    Wherein R 1 , R 2 and the carbon atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, the C 3 -C 6 cycloalkyl group or a 3-6 membered heterocycloalkyl group optionally substituted with R a, said R a, R 5 and n are as defined in claim 1.
  5. 根据权利要求1-4任一项所述的式(I)所示化合物或其药学上可接受的盐，其中所述Het选自
    Figure PCTCN2021099653-appb-100006
    其中R 1或R 2与R 6以及各自连接的C和N一起形成3-6元杂环基。     The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the Het is selected from
    Figure PCTCN2021099653-appb-100006
    Wherein R 1 or R 2 and R 6 and the C and N respectively connected together form a 3-6 membered heterocyclic group.
  6. 根据权利要求1或2所述的式(I)所示化合物或其药学上可接受的盐，其中所述Het选自
    Figure PCTCN2021099653-appb-100007
    所述R 5和p如权利要求1所定义。     The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the Het is selected from
    Figure PCTCN2021099653-appb-100007
    The R 5 and p are as defined in claim 1.
  7. 根据权利要求6所述的式(I)所示化合物或其药学上可接受的盐，其中所述Het选自
    Figure PCTCN2021099653-appb-100008
    进一步优选自
    Figure PCTCN2021099653-appb-100009
    其中所述R 5如权利要求1所定义。     The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 6, wherein the Het is selected from
    Figure PCTCN2021099653-appb-100008
    More preferably from
    Figure PCTCN2021099653-appb-100009
    Wherein said R 5 is as defined in claim 1.
  8. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其中所述式(I)化合物或其药学上可接受的盐选自式(II)化合物或其药学上可接受的盐：The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (II) or a pharmaceutically acceptable salt thereof Acceptable salt:
    Figure PCTCN2021099653-appb-100010
    Figure PCTCN2021099653-appb-100010
    其中：in:
    Y选自NR 10、O或S； Y is selected from NR 10 , O or S;
    R 10选自H、C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基，所述C 1-C 6烷基、C 2-C 8烯基、C 2-C 4炔基、C 3-C 6环烷基或3-6元杂环烷基任选被1个或多个选自如下的基团取代：F、Cl、Br、I、CN、OH、OCH 3和SO 2CH 3； R 10 is selected from H, C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl, said C 1 -C 6 alkyl, C 2 -C 8 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl or 3-6 membered heterocycloalkyl are optionally selected by one or more Substitution from the following groups: F, Cl, Br, I, CN, OH, OCH 3 and SO 2 CH 3 ;
    X 1、X 2、X 3、X 4、Het、R 3、R 4和R 6如权利要求1所定义； X 1 , X 2 , X 3 , X 4 , Het, R 3 , R 4 and R 6 are as defined in claim 1;
  9. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其中所述式(I)化合物或其药学上可接受的盐选自式(III)化合物或其药学上可接受的盐：The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (III) or a pharmaceutically acceptable salt thereof Acceptable salt:
    Figure PCTCN2021099653-appb-100011
    Figure PCTCN2021099653-appb-100011
    其中X 1、X 2、X 3、X 4、Het、R 3、R 4和R 6如权利要求1所定义。 Wherein X 1 , X 2 , X 3 , X 4 , Het, R 3 , R 4 and R 6 are as defined in claim 1.
  10. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其中所述式(I)化合物或其药学上可接受的盐选自式(IV)化合物或其药学上可接受的盐：The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IV) or a pharmaceutically acceptable salt thereof Acceptable salt:
    Figure PCTCN2021099653-appb-100012
    Figure PCTCN2021099653-appb-100012
    Figure PCTCN2021099653-appb-100013
    Figure PCTCN2021099653-appb-100013
    其中X 1、X 2、X 3、X 4、Het、R 3、R 4、R 6和R 8如权利要求1所定义。 Wherein X 1 , X 2 , X 3 , X 4 , Het, R 3 , R 4 , R 6 and R 8 are as defined in claim 1.
  11. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其中所述化合物选自如下结构之一：The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from one of the following structures:
    Figure PCTCN2021099653-appb-100014
    Figure PCTCN2021099653-appb-100014
    Figure PCTCN2021099653-appb-100015
    Figure PCTCN2021099653-appb-100015
    Figure PCTCN2021099653-appb-100016
    Figure PCTCN2021099653-appb-100016
  12. 一种药物组合物，所述组合物包含权利要求1至11任一项的化合物或其药学上可接受的盐，以及药学上可接受的载体或赋形剂。A pharmaceutical composition comprising the compound of any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
  13. 一种预防或治疗***受体相关性疾病的方法，所述方法包括向有此需要的对象施用预防或治疗有效剂量的权利要求1至11任一项的化合物或其药学上可接受的盐或者权利要求12所述的药物组合物，其中所述***受体相关性疾病优选为肿瘤。A method for preventing or treating estrogen receptor-related diseases, the method comprising administering to a subject in need thereof a preventive or therapeutically effective dose of the compound of any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof Or the pharmaceutical composition of claim 12, wherein the estrogen receptor-related disease is preferably a tumor.
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