WO2021088992A1 - 作为prmt5抑制剂的四氢异喹啉螺环化合物 - Google Patents

作为prmt5抑制剂的四氢异喹啉螺环化合物 Download PDF

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WO2021088992A1
WO2021088992A1 PCT/CN2020/127166 CN2020127166W WO2021088992A1 WO 2021088992 A1 WO2021088992 A1 WO 2021088992A1 CN 2020127166 W CN2020127166 W CN 2020127166W WO 2021088992 A1 WO2021088992 A1 WO 2021088992A1
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compound
carbonyl
azaspiro
dimethyl
nonane
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PCT/CN2020/127166
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English (en)
French (fr)
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刘磊
刘扬
赵春艳
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 CN202080077343.1A priority Critical patent/CN114728938B/zh
Publication of WO2021088992A1 publication Critical patent/WO2021088992A1/zh

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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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • 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
    • C07D471/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring

Definitions

  • This application relates to a tetrahydroisoquinoline spiro compound as a PRMT5 inhibitor, its preparation method, a pharmaceutical composition containing the compound, and its use in the treatment of PRMT5-mediated diseases.
  • DNA methylation, histone acetylation and methylation, non-coding RNA, and post-translational modifications are all epigenetic driving forces for cancer, and have nothing to do with changes in DNA sequence.
  • Arginine methylation is an important type of post-translational modification that affects cell growth and proliferation, apoptosis, angiogenesis, and metastasis by regulating transcription and post-transcriptional RNA processing.
  • methylarginine There are three types of methylarginine, ⁇ -NG-monomethylarginine (MMA), ⁇ -NG, N'G-asymmetric dimethylarginine (ADMA) and ⁇ -NG, N 'G-Symmetric dimethylarginine (SDMA).
  • This modification is catalyzed by the protein arginine methyltransferase (PRMT) family, which transfers methyl groups from S-adenosylmethionine (AdoMet) to the arginine side chains of histones and non-histone proteins.
  • PRMT protein arginine methyltransferase
  • AdoMet S-adenosylmethionine
  • PRMT5 is mainly a type II enzyme, which can catalyze the symmetric dimethylation of arginine.
  • PRMT5 was discovered for the first time in a two-hybrid experiment to detect proteins interacting with Janus tyrosine kinase (Jak2).
  • PRMT5 is a universal transcription repressor that forms a complex with other transcription factors, including BRG1 and hBRM, Blimp1 and Snail. PRMT5 participates in a variety of different cell biological processes by methylating a variety of substrates in the cytoplasm and nucleus, including histone H4 residue Arg3 (H4R3) and H3 residue Arg8 (H3R8). H4R3 methylation is related to transcriptional inhibition, whereas H3R8 methylation is considered to be related to both transcriptional activation and transcriptional inhibition.
  • PRMT5 In addition to direct induction of inhibitory histone markers, PRMT5's role in gene silencing is also mediated by the formation of multiple inhibitory protein complexes, including NuRD components, HDACs, MDB proteins and DNA methyltransferases. PRMT5 affects its substrate specificity by interacting with some binding proteins. The core component of this protein complex is MEP50. MEP50 is necessary for the enzymatic activity of PRMT5. Studies have found that PRMT5 can methylate proteins involved in RNA splicing, such as SmD3, which can be used to track the chemical activity of cellular biological PRMT5.
  • PRMT5 plays an important role in tumorigenesis. Studies have found that the expression of PRMT5 is up-regulated in a variety of tumors, including lymphoma, lung cancer, breast cancer and colorectal cancer. In addition, PRMT5 expression is increased in mantle cell lymphoma (MCL) patient samples, and PRMT5 knockout inhibits MCL cell proliferation, indicating that PRMT5 plays an important role in MCL. PRMT5 overexpression promotes cell proliferation. On the contrary, in melanoma, breast cancer and lung cancer cell lines, PRMT5 knockout inhibits the proliferation of these cells. Therefore, PRMT5 is a potential target for cancer treatment.
  • MCL mantle cell lymphoma
  • MTAP methylthioadenosine phosphorylase
  • This application relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof,
  • A is a 5-14 membered spirocyclic group optionally substituted by R 6;
  • R 1 and R 2 are independently selected from H, C 1-4 alkyl, halogen, C 1-4 alkoxy, or
  • R 1 , R 2 and the C atom to which they are connected together form a C 3-8 cycloalkyl or 3-8 membered heterocycloalkyl, the cycloalkyl or heterocycloalkyl is optionally substituted by halogen;
  • R 3 , R 4 , and R 5 are independently selected from H, halogen and C 1-4 alkyl;
  • n 1, 2, 3 or 4;
  • R 6 is independently selected from halogen, hydroxyl, cyano, amino, C 1-3 alkylamino, di(C 1-3 alkyl)amino, oxo, C 1-4 alkyl, C 1-4 alkoxy Group, C 1-4 alkoxycarbonyl, C 3-6 cycloalkyl and 3-6 membered heterocycloalkyl.
  • A is C 6-13 spirocycloalkyl or 6-13 membered spiroheterocycloalkyl, which is optionally substituted with R 6.
  • A is optionally substituted with R 6 among them
  • n, n', p, and q are independently selected from 1, 2, 3, and 4, and n+n'+p+q ⁇ 10;
  • W is selected from CH or N;
  • X and Y are independently selected from CH 2 , NH or O;
  • Z is selected from CH 2 , NH, O or a bond.
  • n, n', p, q are independently selected from 1, 2, and 3, and n+n'+p+q ⁇ 10.
  • n, n' are independently selected from 1, 2, and 3 and p, q are independently selected from 1 and 2.
  • W is N.
  • A is selected from optionally substituted with R 6
  • A is selected from optionally substituted with R 6
  • A is selected from
  • A is selected from
  • R 6 is independently selected from halogen, hydroxy, cyano, amino, C 1-3 alkylamino, oxo, C 1-4 alkyl, C 1-4 alkoxy, C 1- 4 Alkoxycarbonyl and C 3-6 cycloalkyl.
  • R 6 is independently selected from halogen, hydroxy, cyano, amino, oxo, C 1-4 alkyl, C 1-4 alkoxy, and C 1-4 alkoxycarbonyl.
  • R 6 is independently selected from fluoro, hydroxy, cyano, amino, oxo, methyl, methoxy, and methoxycarbonyl.
  • R 1 and R 2 are independently selected from H, C 1-4 alkyl, or R 1 , R 2 and the C atom to which they are attached together form a C 3-6 cycloalkyl group, and the C 3- The 6 cycloalkyl group is optionally substituted with halogen.
  • R 1 and R 2 are independently selected from H, methyl, ethyl, or R 1 , R 2 and the C atom to which they are attached together form a cyclopropyl or cyclobutyl, the cyclopropyl or Cyclobutyl is optionally substituted with fluorine.
  • R 1 and R 2 are methyl groups.
  • R 3 , R 4 , R 5 are independently selected from H, F, Cl, methyl, and ethyl.
  • R 3 , R 4 , and R 5 are H.
  • m is 1 or 2.
  • m is 1.
  • the compound of formula (I) of the present application or a pharmaceutically acceptable salt thereof is selected from a compound of formula (II) or a pharmaceutically acceptable salt thereof,
  • R 1 , R 2 , R 3 , R 4 , R 5 and m are as defined above.
  • the compound of formula (I) of the present application or a pharmaceutically acceptable salt thereof is selected from the compound of formula (III) or a pharmaceutically acceptable salt thereof,
  • R 1 , R 2 , R 3 , R 4 , R 5 and m are as defined above.
  • the compound of formula (I) of the present application or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the compound of formula (I) of the present application or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the present application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of formula (I) of the present application or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition of the present application further includes pharmaceutically acceptable excipients.
  • this application relates to a method for treating a disease mediated by PRMT5 in a mammal, including administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a mammal in need of such treatment, preferably a human, Or its pharmaceutical composition.
  • this application relates to a method for inhibiting PRMT5 activity in vivo or in vitro, the method comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof, Or its pharmaceutical composition.
  • this application relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a medicine for preventing or treating PRMT5-mediated diseases.
  • this application relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the prevention or treatment of PRMT5-mediated diseases.
  • the present application relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for preventing or treating PRMT5-mediated diseases.
  • the PRMT5-mediated disease is a proliferative disease, a metabolic disease or a blood disease, preferably a proliferative disease or a metabolic disease.
  • the proliferative disease is selected from cancer, autoimmune disease or inflammatory disease, preferably cancer.
  • the PRMT5-mediated disease is cancer.
  • 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.
  • stable means that when the optional range of substituents includes optional options that cannot be used to replace a specific group due to valence requirements, chemical stability, or other reasons, the optional range should be understood as the context according to the context Including those options suitable for substituting specific groups. For example, when considering the degree of optional substitution of a particular part, it should be understood that the number of substituents does not exceed the valence suitable for that part.
  • 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 document means that the part has an integer number of carbon atoms in the given range, that is, from an integer m to an integer n (inclusive).
  • 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, then each R has independent options.
  • the substituent can be bonded to any atom on the ring.
  • the structural unit It means that R 5 can be substituted at any position on the benzene ring.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • hydroxy refers to the -OH group.
  • cyano refers to the -CN group.
  • amino refers to the -NH 2 group.
  • 5-14 membered spirocyclic group refers to a cyclic group with at least two rings sharing one ring atom and forming a total of 5-14 ring atoms, in which all ring atoms can be carbon atoms or ring atoms It may contain at least one heteroatom selected from N, O or S.
  • the spirocyclic group may be saturated or partially saturated.
  • 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 - 6 alkyl refers to (e.g., methyl, ethyl, n-propyl, isopropyl, alkyl containing 1 to 6 carbon atoms, n-butyl, isobutyl, sec-butyl, Tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.).
  • the alkyl moiety (ie, alkyl) of alkoxy, alkylamino, and dialkylamino has the same definition as described above.
  • alkoxy refers to -O-alkyl
  • alkylamino refers to -NH-alkyl
  • dialkylamino refers to -N(alkyl) 2 .
  • cycloalkyl refers to a carbocyclic ring that is fully saturated and may exist as a monocyclic, bridged, or 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, diamond Alkyl, spiro[4.5]decane, etc.
  • Spirocycloalkyl refers to a cycloalkyl that exists as a spiro ring.
  • cycloalkenyl refers to a non-aromatic carbocyclic ring that is not 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 5- to 8-membered ring.
  • Non-limiting examples of cycloalkenyl include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, and the like.
  • heterocycloalkyl refers to a cyclic group that is fully saturated and may exist as a monocyclic, bridged, or spiro ring. Unless otherwise indicated, the heterocyclic ring is generally a 3 to 7 membered ring containing 1 to 3 heteroatoms (preferably 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, thiabutanyl, 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-dioxanyl, 1,4
  • the present application may refer to any compound or specific group of compounds defined herein by optional, preferred or suitable features or in other ways according to specific embodiments, the present application may also involve expressly excluding said optional, Any compound or specific group of compounds of a preferred or suitable feature or specific embodiment.
  • a measurable value e.g., a quantity or a period of time, etc.
  • the term "about” is intended to encompass reasonable variations in that value, for example, to allow for experimental error in measuring the value.
  • treatment means administering the compound or formulation described in this application to prevent, ameliorate or eliminate a disease or one or more symptoms related to the disease, and includes:
  • prevention is not absolute, but refers to where the administration of the compound or composition reduces the likelihood or severity of the condition, symptom, or disease state, and/or delays the onset of the condition, symptom, or disease state for a period of time Uses and results.
  • the terms "subject” and “patient” suitably refer to mammals, particularly humans.
  • terapéuticaally effective amount means (i) treatment or prevention of a particular disease, condition or disorder, (ii) reduction, amelioration or elimination of one or more symptoms of a particular disease, condition or disorder, or (iii) prevention or delay
  • the amount of the compound of the present application 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 this disclosure.
  • PRMT5-mediated disease refers to any disease, disorder, or other pathological condition in which PRMT5 is known to play a role. Therefore, in some embodiments, the application relates to treating or reducing the severity of one or more diseases in which PRMT5 is known to play a role.
  • 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.
  • 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. .
  • the compound of formula (I) is isolated as a pharmaceutically acceptable salt.
  • pharmaceutical composition refers to a mixture of one or more of the compounds of the application or their salts and pharmaceutically acceptable excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present application to the organism.
  • pharmaceutically acceptable excipients refers to those excipients that have no obvious stimulating effect on the organism and will not damage the biological activity and performance of the active compound.
  • Suitable auxiliary materials are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.
  • tautomer or "tautomeric form” refers to structural isomers of different energies that can interconvert via a low energy barrier.
  • proton tautomers also called proton transfer tautomers
  • proton migration such as keto-enol and imine-enamine isomerization.
  • a specific example of a proton tautomer is the imidazole moiety, in which protons can migrate between two ring nitrogens.
  • Valence tautomers include interconversion through the recombination of some bonding electrons.
  • 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 the atomic weight or mass number commonly found in nature.
  • isotopes that can be bound to 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 isotopically-labeled compounds of the present application can be prepared by the following procedures similar to those disclosed in the schemes and/or examples below, by replacing non-isotopically-labeled reagents with isotope-labeled reagents.
  • substitution with heavier isotopes can provide certain therapeutic advantages resulting from higher metabolic stability (for example, increased in vivo half-life or reduced dosage requirements), and therefore in certain situations
  • deuterium substitution can be partial or complete
  • partial deuterium substitution refers to the substitution of at least one hydrogen with at least one deuterium.
  • the compounds of the present application may be asymmetric, for example, have one or more stereoisomers.
  • all stereoisomers include, for example, enantiomers and diastereomers.
  • Enantiomers are characterized by the absolute configuration of their asymmetric centers, and are described by the R- and S-ordering rules of Cahn and Prelog, or by the way the molecule rotates the plane of polarized light, and is called right-handed or left-handed .
  • the compounds of the present application containing asymmetric carbon atoms can be isolated in an optically pure form or in a racemic form. The optically active pure form can be resolved from the racemic mixture or synthesized by using chiral raw materials or chiral reagents.
  • Non-limiting examples of stereoisomers include, but are not limited to:
  • Certain compounds of formula (I) may have one or more asymmetric centers and therefore may exist in multiple stereoisomeric configurations. Therefore, these compounds can be synthesized and/or separated as a mixture of enantiomers and/or as a single (pure) enantiomer, and in the case of two or more asymmetric centers, as a single non- Synthesis and/or separation of mixtures of enantiomers and/or diastereomers. It should be understood that this application includes all these enantiomers and diastereomers and all unfavorable mixtures thereof.
  • 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 present 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 method, dissolution method, granulation method, sugar-coated pill method, grinding method, emulsification method, freeze-drying method, etc.
  • the pharmaceutical composition is in 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 tabletting 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, and the like.
  • 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 100 mg/kg body weight, preferably 0.05 to 50 mg/kg body weight, more preferably 0.1 to 30 mg/kg body weight, in single or divided doses form.
  • the compounds of the present application 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 combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent replacement manners, preferred implementation manners include but are not limited to the embodiments of the present application.
  • the compound of general formula (I) of the present application can be prepared by those skilled in the art of organic synthesis through Route 1:
  • the compound M1 and M2 react in the presence of a base to form M3.
  • the compound M3 reacts to form M4 in the presence of a palladium catalyst/base/ethanol/CO.
  • the compound M5 is reacted with A-H in the presence of a condensing agent, and the amino protecting group is further removed to produce a compound of formula (I).
  • the route 1 can be further expressed as:
  • Figure 1 shows the ellipsoid of the compound m crystal.
  • Figure 2 shows the tumor growth curve of the in vivo drug efficacy experiment in mice using the Z-138 subcutaneous tumor model.
  • Figure 3 shows the mouse body weight change curve of the in vivo drug efficacy experiment in mice using the Z-138 subcutaneous tumor model.
  • reaction solution was reacted at room temperature for 4 hours. After the reaction is complete, the excess N,N-diisopropylethylamine and N,N-dimethylformamide are removed by rotary evaporation, then cooled in an ice bath, diluted with saturated brine (1L), and extracted with ethyl acetate (2 X 200 mL), the organic phases were combined and washed with 5% sodium carbonate aqueous solution (2 X 500 mL), and then washed with saturated brine (500 mL).
  • reaction solution was cooled to room temperature, concentrated under reduced pressure, adjusted to pH 10 with a 10% aqueous sodium hydroxide solution, and then extracted three times with ethyl acetate (400 mL), the organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure.
  • methyl(2-(3-bromophenyl)-2-methylpropyl)carbamate (g) (18.9g, 66.1mmol) was added to trifluoromethanesulfonic acid (TfOH) (240mL In), the reaction system was heated to 100°C and stirred for 16 hours. After the reaction was completed, the reaction solution was cooled to room temperature, slowly added to ice water (300 mL), and then extracted three times with dichloromethane (200 mL), the organic phase was dried and filtered with anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure.
  • TfOH trifluoromethanesulfonic acid
  • Ethyl-4,4-dimethyl-1-carbonyl-2-((3-carbonyl-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]iso Quinolin-1-yl) methyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylate (1-2) (750mg, 1.67mmol) was added to tetrahydrofuran (9.5mL), methanol To the mixed solution of (9.5 mL) and water (9.5 mL), sodium hydroxide (268 mg, 6.70 mmol) was added, heated to 70°C, and reacted for 16.0 hours.
  • the reaction system was cooled to room temperature, and Boc anhydride (1.09 g, 5.01 mmol) was added to react for 1.0 hour.
  • the reaction system was cooled to 0°C, the pH of the reaction solution was adjusted to 5.0 with 1N aqueous hydrochloric acid, and then extracted with ethyl acetate (50 mL) three times, the organic phase was dried and filtered with anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure .
  • Trifluoroacetic acid (1 mL) was added to a dichloromethane (2 mL) solution of Intermediate 4--1-PK1 (112 mg), and the reaction solution was stirred at room temperature for 1 hour. After the reaction is completed, it is cooled in an ice bath, and the pH value is adjusted to 9 by adding sodium carbonate aqueous solution.
  • reaction system was cooled to 0°C, the pH of the reaction solution was adjusted to 5.0 with 1N aqueous hydrochloric acid, and then extracted with ethyl acetate (50 mL) three times, the organic phase was dried and filtered with anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure .
  • the compound 5-4 was resolved by chiral chromatography column, resolution conditions:
  • peak 1 compound is tert-butyl (R)-3-((S)-1-hydroxy-2-(6-(2-hydroxy-7-azaspiro[3.5]nonane-7-carbonyl)-4 ,4-Dimethyl-1-carbonyl-3,4-dihydroisoquinoline-2(1H)-yl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (5-4-PK1)
  • peak 2 compound is tert-butyl (S)-3-((R)-1-hydroxy-2-(6-(2-hydroxy-7-azaspiro[3.5]nonane -7-carbonyl)-4,4-dimethyl-1-carbonyl-3,4-dihydroisoquinoline-2(1H)-yl)ethyl)-3,4-dihydroisoquinoline-2 (1H)-Carboxylic acid ester (5-4-PK2).
  • Example 8 2-((S)-2-hydroxy-2-((R)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-4,4-dimethyl Base-6-(2,7-diazaspiro[3.5]nonane-7-carbonyl)-3,4-dihydroisoquinolin-1(2H)-one hydrochloride (compound 7) and 2- ((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-4,4-dimethyl-6-(2 Preparation of ,7-diazaspiro[3.5]nonane-7-carbonyl)-3,4-dihydroisoquinolin-1(2H)-one hydrochloride (compound 8)
  • Compound 7-1 was resolved by chiral resolution (the resolution conditions were the same as those in Example 7), and two compounds with a single configuration were obtained.
  • the compound of peak 1 was tert-butyl (R)-3-((S)-2-( 6-(2-(tert-Butoxycarbonyl)-2,7-diazaspiro[3.5]nonane-7-carbonyl)-4,4-dimethyl-1-carbonyl-3,4-di Hydroisoquinoline-2(1H)-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-1-PK1) (45mg), peak 2
  • the compound is tert-butyl (S)-3-((R)-2-(6-(2-(tert-butoxycarbonyl)-2,7-diazaspiro[3.5]nonane-7- (Carbonyl)-4,4-Dimethyl-1-carbonyl-3,4-dihydr
  • the compound 9-1 was subjected to chiral resolution (the resolution conditions were the same as in Example 4), and two compounds with a single configuration were obtained.
  • the compound of peak 1 was tert-butyl (R)-3-((S)-2-( 4,4-Dimethyl-1-carbonyl-6-(2-carbonyl-7-azaspiro[3.5]nonane-7-carbonyl)-3,4-dihydroisoquinoline-2(1H)- Yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (9-1-PK1)(24mg), peak 2 compound is tert-butyl(S)- 3-((R)-2-(4,4-Dimethyl-1-carbonyl-6-(2-carbonyl-7-azaspiro[3.5]nonane-7-carbonyl)-3,4-di Hydroisoquinoline-2(1H)-yl)-1-hydroxyethyl)-3,4-
  • Diffusion method (dichloromethane-methyl tert-butyl ether system) was used to obtain a single crystal sample of compound m.
  • the obtained sample was subjected to X-ray crystal diffraction analysis at the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences to determine the absolute structure of compound m. type.
  • the test results are shown in Table 1 and Figure 1.
  • the reaction system was cooled to room temperature, Boc anhydride (430 mg, 2 mmol) was added, and the reaction was carried out at room temperature for 1.0 hour. After the reaction was complete, the reaction system was cooled to 0°C, the pH of the reaction solution was adjusted to 5.0 with 1N aqueous hydrochloric acid, and extracted with ethyl acetate (50 mL) three times, the organic phase was dried and filtered with anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure.
  • 2-methoxy-7-azaspiro[3.5]nonane hydrochloride (12-4) 230 mg, 1.2 mmol was added, and the reaction was carried out at room temperature for 1.5 hours. After the reaction was completed, saturated brine (50 mL) was added, and the mixture was extracted twice with ethyl acetate (20 mL). The organic phases were combined and washed with saturated brine (50 mL) three times, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure.
  • Example 13 replace 2-cyano-7-azaspiro[3.5]nonane-7-carboxylic acid tert-butyl ester (13-1) with 6,6-difluoro-2-aza Spiro[3.3]heptane-2-carboxylic acid tert-butyl ester to obtain the target product 6-(6,6-difluoro-2-azaspiro[3.3]heptane-2-carbonyl)-2-((R) -2-Hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-4,4-dimethyl-3,4-dihydroisoquine Lin-1(2H)-one hydrochloride (Compound 20).
  • reaction solution was cooled to room temperature, slowly added to ice water (1L), extracted with ethyl acetate (1L) three times, the organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was reduced Pressure concentration.
  • the reaction system was cooled to 0°C, the pH of the reaction solution was adjusted to 8.0 with potassium carbonate, extracted three times with ethyl acetate (200 mL), the organic phase was washed three times with saturated brine, dried and filtered with anhydrous sodium sulfate, and the filtrate Concentrated under reduced pressure to obtain the target intermediate 1-(3-bromophenyl)-3-oxocyclobutane-1-carbonitrile (37-3) (10 g, yield: 74.0%), and its properties: yellow liquid.
  • reaction system was reduced to 0°C, slowly poured into an ice-water mixture (500 mL), adjusted to pH 8 with solid sodium bicarbonate, extracted three times with dichloromethane (200 mL), and the organic phase was washed with saturated brine Three times, dry and filter with anhydrous sodium sulfate, and concentrate the filtrate under reduced pressure.
  • reaction solution was concentrated, water (200 mL) and ethyl acetate (100 mL) were added to the crude product, the pH was adjusted to 8 with solid sodium carbonate, extracted three times with ethyl acetate (200 mL), and washed with saturated sodium chloride solution Three times, dry and filter with anhydrous sodium sulfate, and concentrate the filtrate under reduced pressure to obtain the target intermediate (1-(3-bromophenyl)-3,3-difluorocyclobutyl)methylamine (37-5) (crude product, 6.6) g, yield: 100%).
  • reaction system was cooled to room temperature, di-tert-butyl dicarbonate (O(Boc) 2 ) (595 mg, 2.73 mmol) was added, and the reaction was carried out at room temperature for 3.5 hours.
  • the reaction solution was reduced to 0°C, adjusted to pH 5.0 with 1N aqueous hydrochloric acid, and then extracted three times with ethyl acetate (50 mL), the organic phase was dried and filtered with anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure.
  • Example 13 replace 2-cyano-7-azaspiro[3.5]nonane-7-carboxylic acid tert-butyl ester (13-1) with 9-oxo-3-azaspiro[ 5.5] Undecane-3-carboxylic acid tert-butyl ester to obtain the target product 3-(2-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquine (Aline-3-yl) ethyl)-4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinoline-6-carbonyl)-3-azaspiro[5.5] Undecane-9-one hydrochloride (Compound 52).
  • Example 13 replace 2-cyano-7-azaspiro[3.5]nonane-7-carboxylic acid tert-butyl ester (13-1) with 2-oxo-1,7-diazepine Heterospiro[3.5]nonane-7-carboxylic acid tert-butyl ester to obtain the target compound 2-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinoline -3-yl)ethyl)-4,4-dimethyl-6-(2-oxo-1,7-diazaspiro[3.5]nonane-7-carbonyl)-3,4-dihydro Isoquinolin-1(2H)-one (Compound 55).
  • the crude product 1-methyl-1,7-diazaspiro[3.5]nonane-7-carboxylic acid tert-butyl ester (56-2) (100mg, 0.42mmol) was dissolved in dichloromethane (4.0mL ) Solution, then add trifluoroacetic acid (2.0 mL), and the reaction solution was stirred at room temperature for 1 hour. After the reaction was completed, it was concentrated under reduced pressure to obtain the target crude product 1-methyl-1,7-diazaspiro[3.5]nonane trifluoroacetate (56-3) (121 mg, yield: 86%).
  • Test example 1 PRMT5 enzymatic activity inhibition experiment
  • PRMT5/MEP50 protein was purchased from BPS bioscience company (U.S.); Histone H4 Peptide substrate was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; Anti-Histone H4 (symmetric di methyl R3) antibody-ChIP Grade was purchased from Ai Bokang (U.S.); S-(5-Adenosyl)-L-methionine chloride dihydrochloride was purchased from Sigma (U.S.); 384-well plates, AlphaScreen Streptavidin Donor beads and AlphaScreen Protein A Acceptor beads were purchased from PerkinElmer Instruments Limited company (United States).
  • Enzyme activity detection Use Echo to drive the compound into a 384-well plate to make the final concentration of the compound 0-1000 nM (initial concentration 1000 nM, 3-fold dilution, 10 points), and DMSO content 0.5%.
  • Prepare 6X detection reagent containing AlphaScreen Protein A Acceptor beads and Histone H4 (symmetric dimethyl R3) antibody add 5 ⁇ L to each well, and incubate for 60 minutes at room temperature.
  • Prepare 6X detection reagent containing AlphaScreen Streptavidin Donor beads add 5 ⁇ L to each well, and incubate at room temperature for 60 minutes.
  • Envision detection signal value The test results are shown in Table 2.
  • Test Example 2 Inhibitory activity of the compound on tumor cell proliferation
  • Z-138 cells were purchased from ATCC (U.S.); IMDM medium and penicillin-streptomycin were purchased from Sigma (U.S.); horse serum was purchased from Hyclone (U.S.); 96-well plates were purchased from Corning ( United States); Cell-Titer Glo reagent was purchased from Promega (United States).
  • Cell culture Z-138 cells were cultured in IMDM medium containing 10% horse serum + 1% penicillin-streptomycin at 37°C and 5% CO 2 . Cells in the logarithmic growth phase can be used in experiments.
  • Cell proliferation activity detection Cell-Titer Glo reagent was used to detect the proliferation inhibitory activity of the compound on Z-138 cells. Adjust the cell concentration, inoculate a 96-well plate (500/well) with 180 ⁇ L per well, and equilibrate for 10-15 minutes at 37°C and 5% CO 2. Add 20 ⁇ L of the cell culture medium containing the compound to each well to make the final concentration of the compound reach 0-300 nM (initial concentration 300 nM, 3-fold dilution, 10 points), and the DMSO content is 0.1%. The cell plate was incubated at 37°C and 5% CO 2 for 8 days.
  • the medium was changed: 100 ⁇ L of supernatant was slowly aspirated, and 100 ⁇ L of fresh culture medium containing the compound was added to keep the compound concentration unchanged.
  • Cell-Titer Glo reagent was used to detect cell viability.
  • Test Example 3 The compound's inhibitory activity experiment on Z-138 cells SDMA
  • Z-138 cells were purchased from ATCC (U.S.); IMDM medium and penicillin-streptomycin were purchased from Sigma (U.S.); Horse serum was purchased from Hyclone (U.S.); Hoechst antibody was purchased from Invitrogen (U.S.) ); Alexa Fluor 488 goat anti-rabbit IgG antibody was purchased from Santa Company; Anti-dimethyl-Arginine symmetric (SYM11) antibody was purchased from Merck Company (USA).
  • Cell culture Z-138 cells were cultured in IMDM medium containing 10% horse serum + 1% penicillin-streptomycin at 37°C and 5% CO 2 . Cells in the logarithmic growth phase can be used in experiments.
  • Immunofluorescence detection Use immunofluorescence to detect the effect of compounds on SDMA in Z-138 cells. Adjust the cell concentration to 1*10 5 /mL, inoculate a 384-well plate (4000/well) with 40 ⁇ L per well, and equilibrate for 10-15 minutes at 37°C and 5% CO 2. Using Echo, the compound was injected into a 384-well plate, so that the final concentration of the compound was 0-300 nM (initial concentration 300 nM, 3-fold dilution, 10 points), and the DMSO content was 0.1%. The cell plate was incubated at 37°C and 5% CO 2 for 2 days.
  • human liver cells were purchased from Biopredic; mouse liver cells were purchased from BioIVT; acetonitrile and methanol were purchased from Merck; AOPI stains were purchased from Nexcelom; dexamethasone was purchased from NIFDC; DMSO was purchased from Beijing Solei Bao Technology Co., Ltd.; DPBS (10x), GlutaMAX TM -1 (100x) and human recombinant insulin were purchased from Gibco by Life Technologies; fetal bovine serum was purchased from Corning; formic acid was purchased from DIKMAPURE; Isotonic Percoll was purchased from GE Healthcare; Alprazolam was purchased from Supelco; caffeine was purchased from ChromaDex.inc; HEPES, tolbutamide and Williams' Medium E were purchased from Sigma.
  • the specific preparation information of the hepatocyte resuscitation solution is shown in Table 3 below.
  • the hepatocytes were quickly placed in a 37°C water bath and gently shaken until all ice crystals were dispersed, sprayed with 70% ethanol and transferred to a biological safety cabinet.
  • hepatocyte tubule containing 50 mL of resuscitation medium, and centrifuge it at 100 g for 10 minutes. After centrifugation, aspirate the recovery medium and add sufficient incubation medium to obtain a cell suspension with a cell density of about 1.5 ⁇ 10 6 cells/mL.
  • Use Cellometer Vision to count liver cells and determine the density of viable cells. The survival rate of liver cells must be greater than 75%.
  • Use the incubation medium to dilute the hepatocyte suspension to a viable cell density of 0.5 ⁇ 10 6 viable cells/mL.
  • mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.; DMSO, HP- ⁇ -CD (hydroxypropyl- ⁇ -cyclodextrin), MC (methyl cellulose), acetonitrile were purchased From Merck (USA).
  • mice 6 female CB17-SCID mice (20-30g, 4-6 weeks) were randomly divided into 2 groups with 3 mice in each group.
  • the first group was given compound 12 by tail vein injection at a dose of 2 mg/kg and the vehicle was an aqueous solution of 5% DMSO + 95% 10% HP- ⁇ -CD.
  • the second group was given compound 12 orally at a dose of 10 mg/kg and the vehicle was 0.5 %MC aqueous solution. Feed and water normally before the animal experiment.
  • Mice in each group were subjected to intravenous blood sampling at 0.083 (intravenous injection group only), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h before and after administration.
  • the collected whole blood samples were placed in a K 2 EDTA anticoagulation tube, and after centrifugation for 5 min (4000 rpm, 4° C.), plasma was taken for testing.
  • Z138 cells were purchased from ATCC; IMDM culture medium, penicillin and 0.25% pancreatin-EDTA were purchased from Gibco; horse serum was purchased from Hyclone.
  • mice CB17-SCID mice, female, 5-6 weeks old, weighing about 13-20 grams, the animals were purchased from Shanghai Lingchang Biological Technology Co., Ltd. The mice were kept in an SPF-level environment, and each cage was independent When sending and exhausting air, all animals have free access to standard certified commercial laboratory food and drinking water.
  • Cell culture Human mantle cell lymphoma Z-138 cell line is cultured in vitro, and the culture conditions are IMDM (cell culture medium) with 10% horse serum, 1% penicillin solution, 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 85%-90% and the number reaches the requirement, the cells are collected and counted.
  • IMDM cell culture medium
  • pancreatin-EDTA digestion solution twice a week for routine digestion and passage.
  • PBS 0.1ml/(containing 1 ⁇ 10 7 ) Z-138 cell suspension
  • Matrigel 1:1
  • PBS phosphate buffered saline solution without calcium and magnesium ions
  • Matrigel matrigel.
  • the dose of compound 12 is 25 mg/kg, PO, twice a day (BID) x 3 weeks, or 50 mg/kg, PO, once a day (QD) x 3 weeks.
  • the dosage of compound 13 is 50 mg/kg, PO, twice a day (BID) x 3 weeks. Each group has 6 mice.
  • the tumor diameter was measured with vernier calipers twice a week.
  • the body weight of the mice was measured twice a week.
  • the anti-tumor efficacy of the compound is 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 beginning of the treatment group)/(Average tumor volume at the end of the solvent control group-when the solvent control group starts Average tumor volume)] x 100%.
  • mice became ill or died during the experiment.
  • compound 12 at 25 mg/kg twice a day and compound 13 at 50 mg/kg twice a day have a significant inhibitory effect on tumor growth and shrink tumors.
  • the effect shows good anti-tumor efficacy.
  • Compound 12 and compound 13 did not significantly affect the body weight of the mice at the tried doses, nor did they cause any death of the mice, and the mice could tolerate them.

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Abstract

本申请公开了式(I)所示作为PRMT5抑制剂的四氢异喹啉螺环化合物、其制备方法、含有该化合物的药物组合物、以及其在治疗PRMT5介导的疾病中的用途，其中，A为任选被R 6取代的5-14元螺环基。

Description

作为PRMT5抑制剂的四氢异喹啉螺环化合物 技术领域
本申请涉及作为PRMT5抑制剂的四氢异喹啉螺环化合物、其制备方法、含有该化合物的药物组合物、以及其在治疗PRMT5介导的疾病中的用途。
背景技术
表观遗传学改变是驱动和维持肿瘤恶性化表型的关键介质。DNA甲基化，组蛋白乙酰化和甲基化，非编码RNA，翻译后修饰的变化都是癌症发生的表观遗传驱动力，而与DNA序列的变化无关。精氨酸甲基化是一类重要的翻译后修饰，通过调节转录和转录后RNA处理影响细胞生长和增殖，凋亡，血管生成和转移。存在三种类型的甲基精氨酸，ω-NG-单甲基精氨酸(MMA)，ω-NG，N’G-不对称二甲基精氨酸(ADMA)和ω-NG，N’G-对称二甲基精氨酸(SDMA)。这种修饰是由蛋白质精氨酸甲基转移酶(PRMT)家族催化的，从S-腺苷甲硫氨酸(AdoMet)把甲基转移到组蛋白和非组蛋白的精氨酸侧链。在人类基因组中注释了九个PRMT基因，基于产生的甲基精氨酸类型分为I型(PRMT1,2,3,4,6和8)，II型(PRMT5和PRMT9)，和III型酶(PRMT7)。PRMT5主要是II型酶，可催化精氨酸的对称二甲基化。PRMT5是在检测与Janus酪氨酸激酶(Jak2)相互作用蛋白的双杂交实验中被首次发现的。
PRMT5是一种通用的转录抑制因子，与其他转录因子形成复合物，包括BRG1和hBRM，Blimp1以及Snail。PRMT5通过对多种细胞质和细胞核中的底物的甲基化，包括组蛋白H4残基Arg3(H4R3)和H3残基Arg8(H3R8)而参与多种不同的细胞生物学过程。H4R3甲基化与转录抑制有关，相反H3R8甲基化被视为既与转录激活又和转录抑制有关。PRMT5除了直接诱导抑制性组蛋白标记外，该酶在基因沉默中的作用还通过形成多抑制蛋白复合物来介导，包括NuRD组分，HDACs，MDB蛋白和DNA甲基转移酶。PRMT5通过与一些结合蛋白的相互作用进而影响其底物特异性。这种蛋白质复合物中的核心成分是MEP50。MEP50对于PRMT5的酶学活性是必须的。研究发现，PRMT5可以甲基化参与RNA剪接的蛋白，比如SmD3，可用于跟踪细胞生物PRMT5的化学活性。
PRMT5在肿瘤发生中起重要作用。研究发现PRMT5在多种肿瘤中的表达上调，包括淋巴瘤，肺癌，乳腺癌和结直肠癌。此外，PRMT5表达在套细胞淋巴瘤(MCL)病人样本中增高，并且PRMT5敲除抑制MCL细胞增殖，表明PRMT5在MCL中起重要作用。PRMT5过表达促进细胞增生，相反，在黑色素瘤，乳腺癌和肺癌细胞系中，PRMT5敲除抑制这些细胞的增殖。因此，PRMT5是癌症治疗的潜在靶点。
甲基硫腺苷磷酸化酶(MTAP)的丧失赋予了细胞对PRMT5及其结合蛋白WDR77的选择性依赖。MTAP由于与通常缺失的肿瘤抑制基因CDKN2A靠近而经常丢失。携带MTAP缺失的细胞的胞内甲硫基腺苷(MTA，被MTAP裂解的代谢物)浓度增加。此外，MTA特异性抑制PRMT5的酶活性。与MTAP表达的细胞相比，MTA或PRMT5小分子抑制剂显著抑制MTAP缺失的癌细胞系的细胞活力。
因此，本领域需要开发能够抑制PRMT5的活性并治疗各种PRMT5相关疾病的小分子。
发明内容
本申请涉及式(I)化合物或其药学上可接受的盐，
Figure PCTCN2020127166-appb-000001
其中，
A为任选被R 6取代的5-14元螺环基；
R 1、R 2独立地选自H、C 1-4烷基、卤素、C 1-4烷氧基，或
R 1、R 2与其连接的C原子共同形成C 3-8环烷基或3-8元杂环烷基，所述环烷基或杂环烷基任选地被卤素取代；
R 3、R 4、R 5独立地选自H、卤素和C 1-4烷基；
m为1、2、3或4；
R 6独立地选自卤素、羟基、氰基、氨基、C 1-3烷基氨基、二(C 1-3烷基)氨基、氧代、C 1-4烷基、C 1-4烷氧基、C 1-4烷氧基羰基、C 3-6环烷基和3-6元杂环烷基。
在一些实施方案中，A为C 6-13螺环烷基或6-13元螺杂环烷基，所述螺环烷基或螺杂环烷基任选被R 6取代的。
在一些实施方案中，A为任选被R 6取代的
Figure PCTCN2020127166-appb-000002
其中
n、n’、p、q独立地选自1、2、3和4，且n+n’+p+q≤10；
W选自CH或N；
X、Y独立地选自CH 2、NH或O；
Z选自CH 2、NH、O或键。
在一些实施方案中，n、n’、p、q独立地选自1、2和3，且n+n’+p+q≤10。
在一些实施方案中，n、n’独立地选自1、2和3，p、q独立地选自1和2。
在一些实施方案中，W为N。
在一些实施方案中，A选自任选被R 6取代的
Figure PCTCN2020127166-appb-000003
Figure PCTCN2020127166-appb-000004
Figure PCTCN2020127166-appb-000005
在一些实施方案中，A选自任选被R 6取代的
Figure PCTCN2020127166-appb-000006
Figure PCTCN2020127166-appb-000007
在一些实施方案中，A选自
Figure PCTCN2020127166-appb-000008
Figure PCTCN2020127166-appb-000009
在一些实施方案中，A选自
Figure PCTCN2020127166-appb-000010
Figure PCTCN2020127166-appb-000011
在一些实施方案中，R 6独立地选自卤素、羟基、氰基、氨基、C 1-3烷基氨基、氧代、C 1-4烷基、C 1-4烷氧基、C 1-4烷氧基羰基和C 3-6环烷基。
在一些实施方案中，R 6独立地选自卤素、羟基、氰基、氨基、氧代、C 1-4烷基、C 1-4烷氧基和C 1-4烷氧基羰基。
在一些实施方案中，R 6独立地选自氟、羟基、氰基、氨基、氧代、甲基、甲氧基和甲氧基羰基。
在一些实施方案中，R 1、R 2独立地选自H、C 1-4烷基，或R 1、R 2与其连接的C原子共同形成C 3-6环烷基，所述C 3-6环烷基任选地被卤素取代。
在一些实施方案中，R 1、R 2独立地选自H、甲基、乙基，或R 1、R 2与其连接的C原子共同形成环丙基或环丁基，所述环丙基或环丁基任选地被氟取代。
在一些实施方案中，R 1、R 2为甲基。
在一些实施方案中，R 3、R 4、R 5独立地选自H、F、Cl、甲基和乙基。
在一些实施方案中，R 3、R 4、R 5为H。
在一些实施方案中，m为1或2。
在一些实施方案中，m为1。
在一些实施方案中，本申请的式(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，
Figure PCTCN2020127166-appb-000012
其中A、R 1、R 2、R 3、R 4、R 5和m如上定义。
在一些实施方案中，本申请的式(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，
Figure PCTCN2020127166-appb-000013
其中A、R 1、R 2、R 3、R 4、R 5和m如上定义。
在一些实施方案中，本申请的式(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：
Figure PCTCN2020127166-appb-000014
Figure PCTCN2020127166-appb-000015
Figure PCTCN2020127166-appb-000016
在一些实施方案中，本申请的式(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：
Figure PCTCN2020127166-appb-000017
Figure PCTCN2020127166-appb-000018
另一方面，本申请涉及药物组合物，其包含本申请的式(I)化合物或其药学上可接受的 盐。在一些实施方案中，本申请的药物组合物还包括药学上可接受的辅料。
另一方面，本申请涉及治疗哺乳动物由PRMT5介导的疾病的方法，包括对需要该治疗的哺乳动物，优选人类，给予治疗有效量的式(I)化合物或其药学上可接受的盐、或其药物组合物。
另一方面，本申请涉及一种在体内或体外抑制PRMT5活性的方法，所述方法包括向有此需要的受试者施用治疗有效量的式(I)化合物或其药学上可接受的盐、或其药物组合物。
另一方面，本申请涉及式(I)化合物或其药学上可接受的盐、或其药物组合物在制备预防或者治疗PRMT5介导的疾病的药物中的用途。
另一方面，本申请涉及式(I)化合物或其药学上可接受的盐、或其药物组合物在预防或者治疗PRMT5介导的疾病中的用途。
另一方面，本申请涉及预防或者治疗PRMT5介导的疾病的式(I)化合物或其药学上可接受的盐、或其药物组合物。
在一些实施方案中，所述PRMT5介导的疾病为增殖性疾病、代谢疾病或血液疾病，优选增殖性疾病或代谢疾病。
在一些实施方案中，所述增殖性疾病选自癌症、自身免疫性疾病或炎性疾病，优选癌症。
在一些实施方案中，所述PRMT5介导的疾病为癌症。
定义
除非另有说明，本申请中所用的下列术语具有下列含义。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。
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。所述“稳定的”是指当取代基的可选范围包含因价数要求、化学稳定性或其他原因而不能用于取代特定基团的可选项时，该可选范围根据上下文应被理解为包括适合取代特定基团的那些可选项。例如，当考虑特定部分的可选取代程度时，应该理解的是，取代基的数目不超过适合于该部分的化合价。
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。
本文中的C m-n，是该部分具有给定范围即整数m至整数n中的整数个碳原子(包括端值)。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。
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。
当其中一个变量选自键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表键时表示该结构实际上是A-Z。
当一个取代基的键交叉连接到一个环上的两个原子时，这种取代基可以与这个环上的任 意原子相键合。例如，结构单元
Figure PCTCN2020127166-appb-000019
表示R 5可在苯环上的任意一个位置发生取代。
术语“卤”或“卤素”是指氟、氯、溴和碘。
术语“羟基”指-OH基团。
术语“氰基”指-CN基团。
术语“氨基”指-NH 2基团。
术语“5-14元螺环基”是指一类至少有两个环共享一个环原子形成的环原子总数为5-14的环状基团，其中环原子可全部为碳原子，或环原子可含有至少一个选自N、O或S的杂原子。所述螺环基可以是饱和的或部分饱和的。
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- 6烷基”指含有1至6个碳原子的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、1-甲基丁基、2-甲基丁基、3-甲基丁基、新戊基、己基、2-甲基戊基等)。类似地，烷氧基、烷基氨基和二烷基氨基的烷基部分(即烷基)具有上述相同定义。
术语“烷氧基”指-O-烷基。
术语“烷基氨基”指-NH-烷基。
术语“二烷基氨基”指-N(烷基) 2。
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、螺[4.5]癸烷等。螺环烷基指以螺环存在的环烷基。
术语“环烯基”是指不完全饱和的并且可以以呈单环、桥环或螺环存在的非芳族碳环。除非另有指示，该碳环通常为5至8元环。环烯基的非限制性实例包括但不限于环戊烯基、环戊二烯基、环己烯基、环己二烯基、环庚烯基、环庚二烯基等。
术语“杂环烷基”是指完全饱和的并且可以以单环、桥环或螺环存在的环状基团。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(优选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。优选为具有5或6个环原子的单环杂环烷基。螺杂环烷基指以螺环存在的杂环烷基。
尽管本申请可以通过任选的、优选的或合适的特征或以其他方式根据特定实施方案涉及本文中定义的任何化合物或特定的化合物组，但是本申请还可以涉及明确排除所述任选的、优选的或合适的特征或特定实施方案的任何化合物或特定的化合物组。
当在本文中与可度量值(例如，一个数量或一段时间等)结合使用时，术语“约”意在涵盖该值的合理变化，例如，以允许测量所述值时出现实验误差。
术语“治疗”意为将本申请所述化合物或制剂进行给药以预防、改善或消除疾病或与所 述疾病相关的一个或多个症状，且包括：
(i)预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时；
(ii)抑制疾病或疾病状态，即遏制其发展；
(iii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
应当理解，术语“预防”不是绝对的，而是指其中化合物或组合物的施用减轻病况、症状或疾病状态的可能性或严重性，和/或将病况、症状或疾病状态的发作延迟一段时间的用途和结果。
如本文单独使用或与另一个或多个术语结合使用时，术语“受试者”和“患者”适当地是指哺乳动物，特别是人类。
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申请化合物的用量。构成“治疗有效量”的本申请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。
术语“PRMT5介导的疾病”是指其中已知PRMT5起作用的任何疾病、病症或其他病理状况。因此，在一些实施方案中，本申请涉及治疗或减轻其中已知PRMT5起作用的一种或多种疾病的严重性。
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。
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。在一些实施方案中，式(I)化合物以药学上可接受的盐被分离。
术语“药物组合物”是指一种或多种本申请的化合物或其盐与药学上可接受的辅料组成的混合物。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。
术语“药学上可接受的辅料”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等。
词语“包括(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising应理解为开放的、非排他性的意义，即“包括但不限于”。
本申请的化合物和中间体还可以以不同的互变异构体形式存在，并且所有这样的形式包含于本申请的范围内。术语“互变异构体”或“互变异构体形式”是指可经由低能垒互变的不同能量的结构异构体。例如，质子互变异构体(也称为质子转移互变异构体)包括经由质子迁移的互变，如酮-烯醇及亚胺-烯胺异构化。质子互变异构体的具体实例是咪唑部分，其中质子可在两个环氮间迁移。价互变异构体包括通过一些成键电子的重组的互变。
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。
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。
此外，用较重同位素(诸如氘(即 2H))取代可以提供某些由更高的代谢稳定性产生的治疗优点(例如增加的体内半衰期或降低的剂量需求)，并且因此在某些情形下可能是优选的，其中氘取代可以是部分或完全的，部分氘取代是指至少一个氢被至少一个氘取代。
本申请化合物可以是不对称的，例如，具有一个或多个立体异构体。除非另有说明，所有立体异构体都包括，如对映异构体和非对映异构体。对映异构体的特征在于其不对称中心的绝对构型，并由Cahn和Prelog的R-和S-排序规则描述，或由分子旋转偏振光平面的方式描述，并称为右旋或左旋。本申请的含有不对称碳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。立体异构体的非限制性实例包括但不限于：
Figure PCTCN2020127166-appb-000020
某些式(I)化合物可以具有一个或多个不对称中心，因此可以以多种立体异构构型存在。因此，这些化合物可以作为对映异构体的混合物和/或作为单个(纯)对映异构体合成和/或分离，并且在两个或更多个不对称中心的情况下，作为单一非对映异构体和/或非对映异构体的混合物合成和/或分离。应理解，本申请包括所有这些对映异构体和非对映异构体及其所有不利的混合物。
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。
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。
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、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
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。
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。
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。
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100mg/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。
本申请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申请的实施例。
本申请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申请的化学变化及其所需的试剂和物料。为了获得本申请的化合物，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。
本领域合成路线规划中的一个重要考量因素是为反应性官能团(如本申请中的氨基)选择合适的保护基，例如，可参考Greene's Protective Groups in Organic Synthesis(4th Ed).Hoboken,New Jersey:John Wiley&Sons,Inc.本申请引用的所有参考文献整体上并入本申请。
在一些实施方案中，本申请通式(I)的化合物可以由有机合成领域技术人员通过路线1来制备：
Figure PCTCN2020127166-appb-000021
路线1
化合物M1与M2反应生成M3，M3在乙醇/CO存在下反应生成M4，M4在碱的存在下水解酯基，并在Boc酸酐的存在下生成M5，M5与A-H反应，并进一步脱去氨基保护基生成式(I)化合物，其中所述R 1、R 2、R 3、R 4、R 5、A和m如上文式(I)中所定义。
在一些实施方案中，所述化合物M1与M2在碱存在下反应生成M3。
在一些实施方案中，所述化合物M3在钯催化剂/碱/乙醇/CO存在下反应生成M4。
在一些实施方案中，所述化合物M5在缩合剂的存在下与A-H反应，并进一步脱去氨基 保护基生成式(I)化合物。
在一些实施方案中，所述路线1可进一步表示为：
Figure PCTCN2020127166-appb-000022
路线1
化合物M1与M2在NaH/DMF存在下反应生成M3，M3在[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物/碱/乙醇/CO存在下反应生成M4，M4在碱的存在下水解酯基，在Boc酸酐的存在下生成M5，M5在缩合剂的存在下与A-H反应，并进一步脱去氨基保护基生成式(I)化合物。
附图说明
图1所示为化合物m晶体的椭球图。
图2所示为采用Z-138皮下瘤模型进行的小鼠体内药效实验的肿瘤生长曲线。
图3所示为采用Z-138皮下瘤模型进行的小鼠体内药效实验的小鼠体重变化曲线。
具体实施方式
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。
实施例1、叔丁基3-(环氧乙烷-2基)-3,4-二氢异喹啉-2(1H)-羧酸酯(中间体1)的制备
Figure PCTCN2020127166-appb-000023
化合物中间体b：
Figure PCTCN2020127166-appb-000024
室温下将(S)-2-(叔丁氧羰基)-1,2,3,4-四氢异喹啉-3-羧酸(a)(55g，200mmol)，二甲羟胺盐酸盐(29.4g，300mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)(91g，240mmol)加入到1L单口瓶中，再加入无水N,N-二甲基甲酰胺(500mL)，氮气保护后冰浴冷却，然后滴加N,N-二异丙基乙胺(DIEA)(104mL，600mmol)。反应液在室温反应4小时。反应完全后旋蒸除去过量的N,N-二异丙基乙胺和N,N-二甲基甲酰胺，然后冰浴冷却，用饱和食盐水(1L)稀释，用乙酸乙酯萃取(2 X 200mL)，有机相合并后用5％碳酸钠水溶液洗涤(2 X 500mL)，然后用饱和食盐水洗涤(500mL)。无水硫酸钠干燥过滤，滤液减压浓缩后柱层析(洗脱剂梯度：石油醚/乙酸乙酯＝2/1)纯化，得到目标中间体叔丁基(S)-3-(甲氧基(甲基)氨基甲酰)-3,4-二氢异喹啉-2(1H)-羧酸酯(b)(62g,收率：97％),性状:无色油状物。
LCMS:Rt:1.76min；MS m/z(ESI):321.3[M+H]。
Chiral-HPLC:Rt:3.159
化合物中间体c：
Figure PCTCN2020127166-appb-000025
室温下将叔丁基(S)-3-(甲氧基(甲基)氨基甲酰)-3,4-二氢异喹啉-2(1H)-羧酸酯(b)(20g，62.5mmol)称量到500mL三口瓶中，加入无水四氢呋喃(200mL)中，冷却至-70℃，缓慢滴加DIBAL-H甲苯溶液(1.5M，83mL，125mmol)，反应液在-70℃搅拌1小时。反应完全后在-70℃缓慢加入饱和氯化铵溶液(100mL)淬灭，然后加入0.5N盐酸水溶液稀释(200mL)。分层后有机相用饱和氯化钠水溶液洗涤(2 X 200mL)，然后用无水硫酸钠干燥过滤，滤液减压浓缩，柱层析(洗脱剂梯度：石油醚/乙酸乙酯＝8/1)纯化，得中间体叔丁基(S)-3-甲酰基-3,4-二氢异喹啉-2(1H)-羧酸酯(c)(15g,收率:92％),性状:黄色油状物。
LCMS:Rt:1.93min；MS m/z(ESI):206.1[M-56+H]。
Chiral-HPLC:Rt:2.018
化合物中间体1：
Figure PCTCN2020127166-appb-000026
室温下称取氢化钠(2.1g，54mmol)于三口瓶中，氮气保护，加入无水DMSO(30mL)，在80℃搅拌45分钟。冷却至室温，加入无水四氢呋喃(20mL)稀释。继续冷却至0℃，加入三甲基碘化硫(11.3g，55.5mmol)的无水DMSO(30mL)溶液。搅拌10分钟后加入叔丁基(S)-3-甲酰基-3,4-二氢异喹啉-2(1H)-羧酸酯(c)(5g,19.2mmol)的四氢呋喃溶液(20mL)，缓慢升至室温过夜。反应完全后冰浴冷却，加入饱和氯化铵溶液淬灭(10mL)，用饱和食盐水稀释(300mL)，乙酸乙酯萃取(2 X 60mL)，有机相合并后无水硫酸钠干燥过滤，滤液减压浓缩，反相柱纯化(H 2O-MeCN-50-100)，分离组分浓缩后用乙酸乙酯萃取，无水硫酸钠干燥过滤，滤液减压浓缩，得到叔丁基3-(环氧乙烷-2基)-3,4-二氢异喹啉-2(1H)-羧酸酯(中间体1)(2.3g，收率：42％)性状:黄色油状物。
LCMS:Rt:1.10min；MS m/z(ESI):220.0[M-56+H]。
实施例2、6-溴-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮(中间体2)的制备
Figure PCTCN2020127166-appb-000027
化合物中间体e：
Figure PCTCN2020127166-appb-000028
室温下将2-(3-溴苯基)乙腈(d)(20.0g，102mmol)，加入到超干四氢呋喃中(400mL)，-78℃下缓慢滴加双(三甲基硅基)氨基钠(NaHMDS)(127mL，251mmol)。滴加完毕后，在-78℃下反应0.5小时。然后缓慢滴加碘甲烷(16.8mL，270mmol)并在-78℃下反应2小时。反应完全后升温至-10℃，缓慢加入饱和氯化铵(150mL)淬灭，然后用乙酸乙酯(200mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：石油醚:乙酸乙酯＝40:1)纯化，得到目标中间体2-(3-溴苯基)-2-甲基丙腈(e)(22.5g,收率：98％),性状:黄色油状物。
化合物中间体f：
Figure PCTCN2020127166-appb-000029
室温下将2-(3-溴苯基)-2-甲基丙腈(e)(29.0g，129.5mmol)加入到超干四氢呋喃中(300mL)，0℃下缓慢滴加硼烷-四氢呋喃(389mL，385.5mmol)。滴加完毕后加热至70℃，反应3.0小时。反应完全后降温至0℃下缓慢加入甲醇(150mL)再滴加浓盐酸(35mL)，加热至70℃，反应2.0小时。将反应液冷却至室温，减压浓缩，用10％氢氧化钠水溶液调pH至10，然后用乙酸乙酯(400mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：二氯甲烷:甲醇＝30:1，0.5％三乙胺)纯化，得到目标中间体2-(3-溴苯基)-2-甲基丙烷-1-胺(f)(24.2g,收率：82％),性状:黄色油状物。
LCMS:Rt:0.742min；MS m/z(ESI):228.0和230.0[M+H]。
化合物中间体g：
Figure PCTCN2020127166-appb-000030
室温下将2-(3-溴苯基)-2-甲基丙烷-1-胺(f)(24.0g，105.3mmol)和三乙胺(31.9g，315.9mmol)加入到二氯甲烷(320mL)中，冷却至0℃，缓慢滴加氯甲酸甲酯(14.9g，157.9mmol)。滴加完毕后，室温反应1.0小时。反应完全后冷却至0℃，加入水(300mL)淬灭，然后用二氯甲烷(200mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：石油醚:乙酸乙酯＝5:1)纯化，得到目标中间体甲基(2-(3-溴苯基)-2- 甲基丙基)氨基甲酸酯(g)(18.9g,收率：63.5％),性状:黄色油状物。
LCMS:Rt:1.827min；MS m/z(ESI):327.0和329.0[M+H+CH 3CN]。
化合物中间体2：
Figure PCTCN2020127166-appb-000031
室温下将甲基(2-(3-溴苯基)-2-甲基丙基)氨基甲酸酯(g)(18.9g，66.1mmol)，加入到三氟甲磺酸(TfOH)(240mL)中，反应体系加热至100℃搅拌反应16小时。反应完全后将反应液冷却至室温，缓慢加入到冰水(300mL)中，然后用二氯甲烷(200mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物用反相色谱柱(洗脱剂梯度：乙腈/水＝34％,含0.1％三氟乙酸)纯化，得到目标中间体6-溴-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮(中间体2)(9.1g,收率：54.2％),性状:白色固体。
LCMS:Rt:1.505min；MS m/z(ESI):254.0和256.0[M+H]。
1H NMR(400MHz,CD 3OD)：δ7.84(d,J＝8.4Hz,1H),7.59(d,J＝2.0Hz,1H),7.52(dd,J＝8.4,2.0Hz,1H),3.28(s,2H),1.34(s,6H).
实施例3、2-(2-羟基-2-(1,2,3,4-四氢异喹啉-3-基)乙基)-6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物1)的制备
Figure PCTCN2020127166-appb-000032
化合物1-1：
Figure PCTCN2020127166-appb-000033
室温下将6-溴-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮(中间体2)(1.07g，4.22mmol)，加入到超干N,N-二甲基甲酰胺(60mL)中，氮气保护，0℃下分批次加入氢化钠(0.253g,6.33mmol)。加完后室温搅拌反应0.5小时，然后将叔丁基3-(环氧乙烷-2基)-3,4-二氢异喹啉-2(1H)-羧酸酯(中间体1)(2.9g，10.54mmol)加入。加热至40℃，反应16.0小时。反应完全后冷却至0℃，缓慢加入饱和氯化铵溶液(60mL)淬灭，然后用乙酸乙酯(100mL)萃取三次，有机相合并后用饱和食盐水洗涤两次，无水硫酸钠干燥过滤，滤液减压浓缩经反相色谱柱(洗脱剂梯度：乙腈/水＝62％,含0.1％三氟乙酸)纯化，得到粗产品目标中间体1-((6-溴-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮(1-1)(2.02g)。
LCMS:Rt:1.919min；MS m/z(ESI):455.0和457.0[M+H]。
化合物1-2：
Figure PCTCN2020127166-appb-000034
室温下将1-((6-溴-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮(1-1)(2.02g，4.44mmol)，[1,1'-双(二苯基膦)二茂铁]二氯化钯(163mg，0.22mmol)和醋酸钾(1.3g，13.32mmol)加入到无水乙醇(40mL)中，CO置换3次，加热至70℃反应16.0小时。反应完全后冷却至室温，将反应液减压浓缩，加入饱和食盐水(100mL)，然后用乙酸乙酯(100mL)萃取两次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物经正相色谱柱(洗脱剂梯度：石油醚：乙酸乙酯＝2:1,)纯化，得到目标中间体乙基-4,4-二甲基-1-羰基-2-((3-羰基-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-1,2,3,4-四氢异喹啉-6-羧酸酯(1-2)(753mg,收率：38％)。
LCMS:Rt:1.847min；MS m/z(ESI):449.0[M+H]。
化合物1-3：
Figure PCTCN2020127166-appb-000035
室温下将乙基-4,4-二甲基-1-羰基-2-((3-羰基-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-1,2,3,4-四氢异喹啉-6-羧酸酯(1-2)(750mg，1.67mmol)加入到四氢呋喃(9.5mL)、甲醇(9.5mL)和水(9.5mL)的混合溶液中，然后加入氢氧化钠(268mg，6.70mmol)，加热至70℃，反应16.0小时。反应完全后，将反应体系降温至室温，加入Boc酸酐(1.09g，5.01mmol)反应1.0小时。反应完全后，将反应体系降温至0℃，用1N的盐酸水溶液调节反应液的pH至5.0，后用乙酸乙酯(50mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物经反相色谱柱(洗脱剂梯度：乙腈/水＝52％)得到目标中间体2-(2-(2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-羰基-1,2,3,4-四氢异喹啉-6-羧酸(1-3)(772mg,收率：93％)。
LCMS:Rt:1.851min；MS m/z(ESI):495[M+H]。
化合物1-4：
Figure PCTCN2020127166-appb-000036
室温下将2-(2-(2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-羰基-1,2,3,4-四氢异喹啉-6-羧酸(1-3)(180mg,0.3mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(231mg,0.6mmol)和N,N-二异丙基乙胺(157mg,1.22mmol)加入到超干N,N-二甲基甲酰胺(2.5mL)中，然后加入7-氮杂螺环[3.5]壬-2-醇盐酸盐(77mg，0.36mmol)，室温反应1.0小时。反应完全后加入水(25mL)，然后用二氯甲烷(20mL)萃取三 次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：石油醚:乙酸乙酯＝1:1)纯化，得到目标中间体3-(1-羟基-2-(6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(1-4)(96mg,收率：46.4％)。
LCMS:Rt:1.68min；MS m/z(ESI):618.3。
化合物1：
Figure PCTCN2020127166-appb-000037
向中间体3-(1-羟基-2-(6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(1-4)(50mg)的二氯甲烷(2mL)溶液中加入三氟乙酸(TFA)(1mL)，反应液在室温下搅拌1小时。反应完毕后减压浓缩,经高效液相色谱纯化，洗脱剂梯度：
Figure PCTCN2020127166-appb-000038
得终产物2-(2-羟基-2-(1,2,3,4-四氢异喹啉-3-基)乙基)-6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物1)(11mg,收率:28％)。HPLC显示该化合物包含两组峰，手性HPLC分析显示该化合物包含4个异构体。
1H NMR(400MHz,DMSO-d6)：δ9.31-9.72(m,1H),9.04(s,1H),7.89-7.93(m,1H),7.36(s,1H),7.21-7.31(m,5H),4.08-4.40(m,4H),3.85-4.07(m,2H),3.45-3.59(m,6H),3.33-3.43(m,1H),3.02-3.29(m,4H),2.15(s,2H),1.47-1.54(m,4H),1.42(s,2H),1.29-1.32(m,6H).
HPLC：Rt:6.62min 37.598％,Rt:6.70min 62.402％,@214nm，
Rt:6.62min 37.331％,Rt:6.70min 62.669％,@254nm
LCMS:Rt:1.28min；MS m/z(ESI):518.4[M+H]。
实施例4、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐和2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐混合物(化合物2)的制备
Figure PCTCN2020127166-appb-000039
化合物2-1：
Figure PCTCN2020127166-appb-000040
室温下将2-(2-(2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-羰基-1,2,3,4-四氢异喹啉-6-羧酸(1-3)(150mg,0.304mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(231mg,0.608mmol)和N,N-二异丙基乙胺(157mg,1.22mmol)加入到超干N,N-二甲基甲酰胺(2.5mL)，然后加入1,7-二氮杂螺[3.5]壬烷-1-羧酸叔丁基酯(82.0mg,0.364mmol)，室温反应1.0小时。反应完全后加入饱和食盐水(30mL)，用乙酸乙酯(30mL)萃取两次，有机相用饱和食盐水洗涤两次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：石油醚:乙酸乙酯＝1:2)纯化，得到目标中间体叔丁基3-(2-(6-(1-(叔丁氧基羰基)-1,7-二氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(2-1)(195mg,收率：91.5％)。
LCMS:Rt:1.851min；MS m/z(ESI):703.0[M+H]。
化合物2-1-PK1和化合物2-1-PK2：
Figure PCTCN2020127166-appb-000041
室温下将叔丁基3-(2-(6-(1-(叔丁氧基羰基)-1,7-二氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(2-1)(195mg)经手性色谱柱(SFC)拆分，拆分条件：
仪器 Waters SFC150
流动相 55％EtOH in CO 2
色谱柱 MIC
进样量 3mL
流速 70g/min
循环进样时间 4.5min
溶样浓度 140mg/40mL
得到一对对映异构体2-1-PK1(主峰，81mg)；另一对对映异构体2-1-PK2(39mg)。
Chiral-HPLC：2-1-PK1,Rt:1.987min；2-1-PK2,Rt:3.642min.
化合物2：
Figure PCTCN2020127166-appb-000042
在0℃下向中间体2-1-PK1(81mg)的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完全后减压浓缩，残余物用高效液相制备色谱法(色谱条件同实施例3)纯化，得到目标化合物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐和2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐混合物(化合物2)(2.31mg,收率:4％)。
1H NMR(400MHz,CD 3OD)：δ8.11-8.01(m,1H),7.48(d,J＝1.2Hz,1H),7.40(d,J＝7.6Hz,1H),7.34-7.19(m,4H),5.69(s,1H),4.52-4.30(m,3H),4.21(s,1H),4.02-3.86(m,2H),3.71-3.56(m,4H),3.48(s,1H),3.32-3.28(m,4H)，3.07(d,J＝6.4Hz,2H),2.49-2.91(m,2H),2.85-2.11(m,2H),1.40(d,J＝7.2Hz,6H).
HPLC：97.881％@214nm，97.094％@254nm
LCMS:Rt:0.436min；MS m/z(ESI):503.2[M+H]。
实施例5、2-((R)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐和2-((S)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐混合物(化合物3)的制备
Figure PCTCN2020127166-appb-000043
化合物3：
Figure PCTCN2020127166-appb-000044
在0℃下向中间体2-1-PK2(39mg)的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完全后减压浓缩，残余物用高效液相制备色谱法(色谱条 件同实施例3)纯化，得到目标化合物2-((R)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐和2-((S)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐混合物(化合物3)(6.08mg,收率:22％)。
1H NMR(400MHz,CD 3OD)：δ8.05(t,J＝7.2Hz,14.8Hz,1H),7.47(d,J＝1.2Hz,1H),7.40(d,J＝7.6Hz,1H),7.34-7.20(m,4H),5.69(s,1H),4.42(s,2H),4.21(s,1H),4.15-4.04(m,2H),3.99-3.80(m,2H),3.72-3.43(m,6H),3.27-3.18(m,2H),3.14-2.95(m,2H),2.50-2.35(m,2H),2.30-2.12(m,2H),1.39(s,6H).
HPLC：99.405％@214nm，99.108％@254nm
LCMS:Rt:0.594min；MS m/z(ESI):503.2[M+H]。
实施例6、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(6-氮杂螺[2.5]辛烷-6-羰基)-3,4-二氢异喹啉-1(2H)-酮和2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(6-氮杂螺[2.5]辛烷-6-羰基)-3,4-二氢异喹啉-1(2H)-酮混合物(化合物4)的制备
Figure PCTCN2020127166-appb-000045
化合物4-1：
Figure PCTCN2020127166-appb-000046
室温下将2-(2-(2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-羰基-1,2,3,4-四氢异喹啉-6-羧酸(1-3)(150mg,0.3mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(231mg,0.6mmol)和N,N-二异丙基乙胺(157mg,1.216mmol)加入到超干N,N-二甲基甲酰胺(2.5mL)，然后加入6-氮杂螺环[2.5]辛烷盐酸盐(54mg，0.36mmol)，室温反应1.0小时。反应完全后加入水(25mL)，然后用二氯甲烷(200mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：石油醚:乙酸乙酯＝1:1)纯化，得到目标中间体叔丁基3-(2-(4,4-二甲基-1-羰基-6-(6-氮杂螺[2.5]辛烷-6-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(4-1)(140mg,收率：83.5％)。
化合物4-1-PK1和4-1-PK-2：
Figure PCTCN2020127166-appb-000047
室温下将叔丁基3-(2-(4,4-二甲基-1-羰基-6-(6-氮杂螺[2.5]辛烷-6-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(4-1)经手性柱拆分(拆分条件同实施例4)，得两对对映体4-1-PK1(主峰)以及4-1-PK2。
LCMS:Rt:1.90min；MS m/z(ESI):588.3[M+H]。
Chiral-HPLC：4-1-PK1，Rt：1.86min.
化合物4：
Figure PCTCN2020127166-appb-000048
向中间体4-1-PK1(112mg)的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完毕后冰浴冷却，加入碳酸钠水溶液调pH值到9。乙酸乙酯萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥过滤，滤液减压浓缩后，得终产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(6-氮杂螺[2.5]辛烷-6-羰基)-3,4-二氢异喹啉-1(2H)-酮和2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(6-氮杂螺[2.5]辛烷-6-羰基)-3,4-二氢异喹啉-1(2H)-酮的混合物(化合物4)(25mg,收率:27％)。
1H NMR(400MHz,CD 3OD)：δ8.04(d,J＝7.6Hz,1H),7.44(d,J＝1.2Hz,1H)7.38(dd,J＝8Hz,1.2Hz,1H),7.10-7.16(m,3H),7.05-7.07(m,1H),4.01-4.12(m,3H),3.94(dd,J＝12.8Hz,4Hz,1H),3.80(s,2H),3.55-3.69(m,2H),3.41(s,2H),2.81-3.02(m,3H),1.51(s,2H),1.33-1.39(m,8H),0.41(d,J＝8.8Hz,4H)。
HPLC：95.959％@214nm，96.363％@254nm
LCMS:Rt:0.86min；MS m/z(ESI):488.3[M+H]。
实施例7、2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物5)和2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物6)的制备
Figure PCTCN2020127166-appb-000049
化合物5-1-PK1：
Figure PCTCN2020127166-appb-000050
室温下将6-溴-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮(中间体2)(1.1g，4.34mmol)，加入到超干N,N-二甲基甲酰胺(60mL)中，0℃下分批次加入氢化钠(0.26g,6.5mmol)，加完后室温搅拌反应0.5小时，然后加入叔丁基3-(环氧乙烷-2-基)-3,4-二氢异喹啉-2(1H)羧酸酯(中间体1)(1.8g，6.54mmol)。反应在40℃加热16.0小时。HPLC以及LCMS显示有两组产物峰，分别为两对对映异构体。反应完全后，缓慢加入冰水(60mL)淬灭，用乙酸乙酯(100mL)萃取两次，有机相用饱和食盐水洗涤两次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物加入乙酸乙酯(20mL)，超声固体析出，过滤收集滤饼，经乙酸乙酯重结晶两次得其中一对对映异构体，(1R,10aS)-1-((6-溴-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮和(1S,10aR)-1-((6-溴-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮的混合物(5-1-PK1)，为HPLC中两组峰的主峰(425mg，收率：23％)。
LCMS:Rt:1.92min；MS m/z(ESI):455.0和457.0[M+H].
化合物5-2：
Figure PCTCN2020127166-appb-000051
室温下将化合物5-1-PK1(425mg，0.94mmol)，[1,1'-双(二苯基膦)二茂铁]二氯化钯(40mg，0.05mmol)和醋酸钾(196mg，1.88mmol)加入到无水乙醇(10mL)中，CO置换3次，加热至70℃反应16.0小时。反应完全后，反应液冷却至室温，减压浓缩，加入饱和食盐水(50mL)，然后用乙酸乙酯(50mL)萃取三次，有机相合并后用无水硫酸钠干燥过滤，滤液减压浓缩，得一对对映异构体，4,4-二甲基-1-羰基-2-(((1R,10aS)-3-羰基-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-1,2,3,4-四氢异喹啉-6-羧酸乙酯和4,4-二甲基-1-羰基-2-(((1R,10aS)-3-羰基-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-1,2,3,4-四氢异喹啉-6-羧酸乙酯的混合物(5-2)(580mg)。
LCMS:Rt:1.895min；MS m/z(ESI):449.0[M+H]。
化合物5-3：
Figure PCTCN2020127166-appb-000052
室温下将化合物5-2(580mg，1.30mmol)加入到四氢呋喃(7mL)、甲醇(7mL)和水(7mL)的混合溶液中，加入氢氧化钠(207mg，5.19mmol)，70℃反应16.0小时。反应完全后，将反应体系降温至室温，加入Boc酸酐(1.13g，5.19mmol)反应1.0小时。反应完全后，将反应体系降温至0℃，用1N的盐酸水溶液调节反应液的pH至5.0，后用乙酸乙酯(50mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物经反相色谱柱(洗脱剂梯度：乙腈/水＝52％)得到目标中间体，2-((R)-2-((S)-2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-羰基-1,2,3,4-四氢异喹啉-6-羧酸和2-((S)-2-((R)-2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-羰基-1,2,3,4-四氢异喹啉-6-羧酸的混合物(5-3)(363mg,两步收率：79％)。
LCMS:Rt:1.799min；MS m/z(ESI):495.2[M+H]。
化合物5-4：
Figure PCTCN2020127166-appb-000053
室温下将化合物5-3(180mg,0.3mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(231mg,0.6mmol)和N,N-二异丙基乙胺(157mg,1.216mmol)加入到超干N,N-二甲基甲酰胺(2.5mL)，然后加入7-氮杂螺环[3.5]壬-2-醇盐酸盐(77mg，0.36mmol)，室温反应1.0小时。反应完全后加入水(25mL)，用二氯甲烷(20mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：石油醚:乙酸乙酯＝1:1)纯化，得到目标中间体，叔丁基(R)-3-((S)-1-羟基-2-(6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯和叔丁基(S)-3-((R)-1-羟基-2-(6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯的混合物(5-4)(90mg,收率：43.5％)。
LCMS:Rt:1.68min；MS m/z(ESI):618.3[M+H]。
化合物5-4-PK1和5-4-PK2：
Figure PCTCN2020127166-appb-000054
将化合物5-4经手性色谱柱拆分，拆分条件：
仪器 Waters SFC150
流动相 55％EtOH in CO 2
色谱柱 WHELK
进样量 7mL
流速 80g/min
循环进样时间 5min
溶样浓度 50mg/40mL
得两个单一构型化合物。其中峰1化合物为叔丁基(R)-3-((S)-1-羟基-2-(6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(5-4-PK1)，峰2化合物为叔丁基(S)-3-((R)-1-羟基-2-(6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(5-4-PK2)。
LCMS:Rt:1.68min；MS m/z(ESI):618.3[M+H]。
Chiral-HPLC：5-4-PK1，Rt：2.79min；5-4-PK2，Rt：3.76min.
化合物5：
Figure PCTCN2020127166-appb-000055
室温下，向化合物叔丁基(R)-3-((S)-1-羟基-2-(6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(5-4-PK1)(24mg)的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完毕后减压浓缩,经高效液相色谱纯化(色谱条件同实施例3)得终产物2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物5)(5mg,收率:27％)。
1H NMR(400MHz,CD 3OD)：δ8.03(d,J＝8Hz,1H),7.43(s,1H),7.35(d,J＝8Hz,1H),7.21-7.31(m,4H),4.21-4.50(m,4H),3.98(dd,J＝14Hz,4.8Hz,1H),3.58-3.70(m,6H),3.28-3.35(m,4H),2.29-2.31(m,2H),1.68-1.72(m,4H),1.54(s,2H),1.39(d,J＝8Hz,6H).
HPLC：98.233％@214nm，98.402％@254nm
LCMS:Rt:0.74min；MS m/z(ESI):518.5[M+H]。
化合物6：
Figure PCTCN2020127166-appb-000056
向化合物叔丁基(S)-3-((R)-1-羟基-2-(6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(5-4-PK2)(21mg)的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完毕后减 压浓缩，经高效液相色谱纯化(色谱条件同实施例3)得目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(2-羟基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物6)(4mg,收率:27％)。
1H NMR(400MHz,CD 3OD)：δ8.03(d,J＝8Hz,1H),7.43(s,1H),7.35(d,J＝8Hz,1H),7.21-7.31(m,4H),4.31-4.50(m,2H),4.28-4.30(m,2H),3.98(dd,J＝14Hz,4.8Hz,1H),3.58-3.70(m,6H),3.28-3.32(m,4H),2.29-2.31(m,2H),1.67-1.74(m,4H),1.54(s,2H),1.39(d,J＝8Hz,6H).
HPLC：99.168％@214nm，99.232％@254nm
LCMS:Rt:0.74min；MS m/z(ESI):518.5[M+H]。
实施例8、2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物7)和2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物8)的制备
Figure PCTCN2020127166-appb-000057
化合物7-1：
Figure PCTCN2020127166-appb-000058
室温下将化合物5-3(140mg,0.3mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(231mg,0.6mmol)和N,N-二异丙基乙胺(157mg,1.216mmol)加入到超干N,N-二甲基甲酰胺(2.5mL)，加入2,7-二氮杂螺[3.5]壬烷-2-羧酸叔丁酯(77mg，0.36mmol)，室温反应1.0小时。反应完全后加入水(25mL)，用二氯甲烷(20mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：石油醚:乙酸乙酯＝1:1)纯化，得到目标中间体，叔丁基(R)-3-((S)-2-(6-(2-(叔丁氧基羰基)-2,7-二氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯和叔丁基(S)-3-((R)-2-(6-(2-(叔丁氧基羰基)-2,7-二氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯的混合物(7-1)(166mg,收率：83.4％)。
LCMS:Rt:1.88min；MS m/z(ESI):703.4[M+H]。
化合物7-1-PK1和7-1-PK2：
Figure PCTCN2020127166-appb-000059
将化合物7-1经手性拆分(拆分条件同实施例7)，得到两个单一构型的化合物，其中峰1化合物为叔丁基(R)-3-((S)-2-(6-(2-(叔-丁氧基羰基)-2,7-二氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(7-1-PK1)(45mg)，峰2化合物为叔丁基(S)-3-((R)-2-(6-(2-(叔-丁氧基羰基)-2,7-二氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(7-1-PK2)(107mg)。
LCMS:Rt:1.88min；MS m/z(ESI):703.4[M+H]。
Chiral-HPLC：7-1-PK1,Rt:3.13min；7-1-PK2，Rt:4.05min.
化合物7：
Figure PCTCN2020127166-appb-000060
向化合物叔丁基(R)-3-((S)-2-(6-(2-(叔-丁氧基羰基)-2,7-二氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(7-1-PK1)(24mg)的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完毕后减压浓缩,经高效液相色谱(色谱条件同实施例3)纯化得终产物2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物7)(5mg,收率:27％)。
1H NMR(400MHz,CD 3OD)：δ8.04(d,J＝7.6Hz,1H),7.45(s,1H),7.37(d,J＝7.6Hz,1H),7.21-7.31(m,4H),4.35-4.50(m,3H),3.92-4.01(m,5H),3.57-3.73(m,6H),3.30-3.36(m,4H),1.87-1.99(m,4H),1.39-1.41(d,J＝7.6Hz,6H).
HPLC：99.704％@214nm，99.917％@254nm
LCMS:Rt:0.42min；MS m/z(ESI):503.3[M+H]。
化合物8：
Figure PCTCN2020127166-appb-000061
向化合物叔丁基(S)-3-((R)-2-(6-(2-(叔-丁氧基羰基)-2,7-二氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-羰基-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯 (7-1-PK2)(21mg)的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完毕后减压浓缩，经高效液相色谱(色谱条件同实施例3)纯化得终产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物8)(4mg,收率:27％)。
1H NMR(400MHz,CD 3OD)：δ8.04(d,J＝7.6Hz,1H),7.45(s,1H),7.37(d,J＝7.6Hz,1H),7.21-7.31(m,4H),4.35-4.50(m,3H),3.92-4.01(m,5H),3.57-3.73(m,6H),3.30-3.36(m,4H),1.87-1.99(m,4H),1.39-1.41(d,J＝7.6Hz,6H).
HPLC：96.311％@214nm，99.784％@254nm
LCMS:Rt:0.52min；MS m/z(ESI):503.3[M+H]。
实施例9、2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物9)和2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物10)的制备
Figure PCTCN2020127166-appb-000062
化合物9-1：
Figure PCTCN2020127166-appb-000063
室温下将化合物5-3(135mg,0.3mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(231mg,0.6mmol)和N,N-二异丙基乙胺(157mg,1.216mmol)加入到超干N,N-二甲基甲酰胺(2.5mL)，加入7-氮杂螺环[3.5]壬-2-酮盐酸盐(77mg，0.36mmol)，室温反应1.0小时。反应完全后加入水(25mL)，然后用二氯甲烷(20mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：石油醚:乙酸乙酯＝1:1)纯化，得到目标中间体，叔丁基(R)-3-((S)-2-(4,4-二甲基-1-羰基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯和叔丁基(S)-3-((R)-2-(4,4-二甲基-1-羰基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯的混合物(9-1)(153mg,收率：91.0％)。
LCMS:Rt:1.54min；MS m/z(ESI):616.3[M+H]。
化合物9-1-PK1和9-1-PK2：
Figure PCTCN2020127166-appb-000064
将化合物9-1经手性拆分(拆分条件同实施例4)，得到两个单一构型的化合物，其中峰1化合物为叔丁基(R)-3-((S)-2-(4,4-二甲基-1-羰基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(9-1-PK1)(24mg)，峰2化合物为叔丁基(S)-3-((R)-2-(4,4-二甲基-1-羰基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(9-1-PK2)(88mg)。
LCMS:Rt:1.54min；MS m/z(ESI):616.3[M+H]。
Chiral-HPLC：9-1-PK1,Rt：3.12min；9-1-PK2,Rt：3.99min.
化合物9：
Figure PCTCN2020127166-appb-000065
向中间体叔丁基(R)-3-((S)-2-(4,4-二甲基-1-羰基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(9-1-PK1)(24mg)的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完毕后减压浓缩,经高效液相色谱(色谱条件同实施例3)纯化得终产物2-((S)-2-羟基-2-((R)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物9)(5mg,收率:27％)。
1H NMR(400MHz,DMSO-d6)：δ9.90(d,J＝8Hz,1H),9.08(d,J＝8Hz,1H),7.92(d,J＝7.6Hz,1H),7.40(s,1H),7.33(dd,J＝8Hz,1.2Hz,1H),7.21-7.28(m,4H),5.89(s,1H),4.32-4.38(m,2H),4.21-4.24(m,1H),3.97-4.02(m,2H),3.57-3.60(m,6H),3.17-3.37(m,5H),2.84-2.89(m,4H),1.64-1.76(m,2H),1.32(d,J＝12.8Hz,6H).
HPLC：98.504％@214nm，98.008％@254nm
LCMS:Rt:1.28min；MS m/z(ESI):516.3[M+H]。
化合物10:
Figure PCTCN2020127166-appb-000066
向化合物叔丁基(S)-3-((R)-2-(4,4-二甲基-1-羰基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯(9-1-PK2)(21mg) 的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完毕后减压浓缩，经高效液相色谱(色谱条件同实施例3)纯化得终产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-羰基-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物10)(4mg,收率:27％)。
1H NMR(400MHz,DMSO-d6)：δ9.90(d,J＝8Hz,1H),9.08(d,J＝8Hz,1H),7.92(d,J＝7.6Hz,1H),7.40(s,1H),7.33(dd,J＝8Hz,1.2Hz,1H),7.21-7.28(m,4H),5.89(s,1H),4.32-4.38(m,2H),4.21-4.24(m,1H),3.97-4.02(m,2H),3.57-3.60(m,6H),3.17-3.37(m,5H),2.84-2.89(m,4H),1.64-1.76(m,2H),1.32(d,J＝12.8Hz,6H).
HPLC：98.682％@214nm，98.990％@254nm
LCMS:Rt:1.28min；MS m/z(ESI):516.3[M+H]。
实施例10、2-((R)-2-((S)-2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-氧代-1,2,3,4-四氢异喹啉-6-羧酸(中间体3)的合成
Figure PCTCN2020127166-appb-000067
化合物h：
Figure PCTCN2020127166-appb-000068
将甲基三苯基溴化磷(238g，0.67mol)分散到无水四氢呋喃(1.5L)中，氮气保护下冷却至-70℃，缓慢滴加二(三甲基硅基)氨基钠(334mL，0.67mol)，控制温度低于-50℃，滴加完毕后缓慢升至室温，搅拌2小时。重新冷却至-70℃，缓慢滴加叔丁基(S)-3-甲酰基-3,4-二氢异喹啉-2(1H)-羧酸酯(c)的四氢呋喃溶液，控制温度低于-50℃，滴加完毕后缓慢升至室温过夜。TLC检测反应完毕后，冷却至0℃，加入饱和氯化铵溶液淬灭，缓慢加入1N盐酸水溶液调节pH到3-4，加入乙酸乙酯(500mL)萃取。有机相用饱和食盐水洗涤，无水硫酸钠干燥后过滤，滤液减压浓缩。残余物加入混合溶剂(乙酸乙酯/石油醚＝1/4)重结晶，过滤除去析出的三苯基氧化膦，滤液硅胶柱层析(乙酸乙酯/石油醚＝1/8)得目标产物(S)-3-乙烯基-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(h)(85g，98％)。
Chiral-HPLC:Rt:1.883
化合物i：
Figure PCTCN2020127166-appb-000069
将(S)-3-乙烯基-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(h)(25.9g，0.1mol)溶于乙酸乙酯/乙腈(500mL/500mL)溶液中，冷却至0℃，在10分钟内加入高碘酸钠(32.1g，0.15mol) 与三氯化钌水合物(1.6g，7.7mmol)的水溶液，反应液在0℃搅拌10分钟。TLC检测反应完毕，加入硫代硫酸钠饱和溶液(150mL)淬灭，搅拌30分钟后分层，有机相用饱和食盐水洗涤(200mL X 2)，无水硫酸钠干燥，过滤后滤液减压浓缩，残余物经硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/2-1/1)，得产物(S)-3-((S)-1,2-二羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(i)(极性较小的产物，14g，收率48％)。加大洗脱剂极性(乙酸乙酯/石油醚＝2/1-1/0)，得产物(S)-3-((R)-1,2-二羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(i-1)(极性较大的产物，8g，收率28％)。
化合物j：
Figure PCTCN2020127166-appb-000070
将(S)-3-((S)-1,2-二羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(i)(14g，0.047mol)溶于二氯甲烷(150mL)中，加入三乙胺(9.90mL，0.072mol)后在搅拌状态下分批次加入对甲苯磺酰氯(10.0g，0.052mol)，反应液在40℃搅拌过夜。将反应液冷却至室温，用饱和食盐水洗涤(100mL X 2)，有机相用无水硫酸钠干燥。过滤后滤液减压浓缩，残余物经硅胶柱层析(乙酸乙酯/石油醚＝1/4)得目标产物(S)-3-((S)-1-羟基-2-(甲苯磺酰氧代)乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(j)(12.6g，收率59％)。
化合物k：
Figure PCTCN2020127166-appb-000071
将(S)-3-((S)-1-羟基-2-(甲苯磺酰氧代)乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(j)(12.6g，28.2mmol)溶于N,N-二甲基甲酰胺(150mL)中，氮气保护下分批次加入氢化钠(1.70g，42.3mmol)，反应液在40℃搅拌1小时。TLC检测反应完全。冷却至0℃，滴加饱和食盐水淬灭，反应液直接反相柱纯化(水/乙腈＝50/10)，柱层析所得溶液用乙酸乙酯萃取(200mL X 3)，有机相经无水硫酸钠干燥后过滤，滤液减压浓缩得到中间体(S)-3-((S)-环氧乙烷-2-基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(k)(6.5g，收率72％)。
化合物m：
Figure PCTCN2020127166-appb-000072
室温下将6-溴-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮(中间体2)(1.07g，4.22mmol)，加入到超干N,N-二甲基甲酰胺(40mL)中，氮气保护，0℃下分批次加入氢化钠(0.253g,6.33mmol)加完后40℃搅拌反应2小时，然后将(S)-3-((S)-环氧乙烷-2-基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(k)(2.9g，10.54mmol)加入。加热至40℃，反应16.0小时。反应完全后将反应液冷却至0℃，缓慢倒入饱和氯化铵溶液(200mL)淬灭，过滤，滤饼用甲醇打浆过滤得 到粗品(1R,10aS)-1-((6-溴-4,4-二甲基-1-氧代-3,4-二氢异喹啉-2(1H)-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮(m)(350mg，收率18％)。
LCMS:Rt:1.738min；MS m/z(ESI):455.1,457.1[M+H]。
采用扩散法(二氯甲烷-甲基叔丁基醚体系)得到化合物m的单晶样品，所得样品在中国科学院上海有机化学研究所进行X-射线晶体衍射分析，用以确定化合物m的绝对构型。测试结果如表1和图1所示。
表1 化合物m的样品和晶体数据、结构精修
Figure PCTCN2020127166-appb-000073
由X-射线晶体衍射结果可以确认化合物m的绝对构型如下：
Figure PCTCN2020127166-appb-000074
化合物n：
Figure PCTCN2020127166-appb-000075
室温下将(1R,10aS)-1-((6-溴-4,4-二甲基-1-氧代-3,4-二氢异喹啉-2(1H)-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮(m)(1.84g，4mmol)，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(170mg，0.2mmol)和醋酸钾(1.3g，12mmol)加入到无水乙醇(40mL)中，CO置换3次，加热至70℃反应3小时。反应完全后冷却至室温，将反应液减压浓缩，加入饱和食盐水(100mL)，然后用乙酸乙酯(100mL)萃取两次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物经正相色谱柱(洗脱剂梯度：石油醚/乙酸乙酯＝1/1)纯化，得到4,4-二甲基-1-氧代-2-(((1R，10aS)-3-氧代-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-1,2,3,4-四氢异喹啉-6-羧酸乙酯(n)(粗品1.3g,收率：72％)。
LCMS:Rt:1.892min；MS m/z(ESI):449.2[M+H]。
中间体3：
Figure PCTCN2020127166-appb-000076
室温下将4,4-二甲基-1-氧代-2-(((1R，10aS)-3-氧代-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-1,2,3,4-四氢异喹啉-6-羧酸乙酯(n)(750mg，1.67mmol)加入到四氢呋喃(9.5mL)，甲醇(9.5mL)和水(9.5mL)的混合溶剂中，然后加入氢氧化钠(268mg，6.70mmol)，加热至70℃，反应16.0小时。将反应体系降至室温，加入Boc酸酐(430mg，2mmol)，室温反应1.0小时。反应完全后，将反应体系降温至0℃，用1N的盐酸水溶液调节反应液的pH至5.0，用乙酸乙酯(50mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。残余物经反相色谱柱纯化(洗脱剂：52％乙腈水溶液)得到目标产物2-((R)-2-((S)-2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-氧代-1,2,3,4-四氢异喹啉-6-羧酸(中间体3)(430mg,收率：87％)。
LCMS:Rt:1.093min；MS m/z(ESI):495.4[M+H]。
实施例11、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物12)的制备
Figure PCTCN2020127166-appb-000077
化合物12-2：
Figure PCTCN2020127166-appb-000078
室温下将2-氧代-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(12-1)(1.0g,4.18mmol)加入到甲醇中(10.0mL)，0℃下加入硼氢化钠(474mg,12.5mmol)，室温反应1.5小时。加入氯化铵水溶液(20.0mL)淬灭反应，用乙酸乙酯(50.0mL)萃取2次。有机相合并，用饱和食盐水(50mL)洗涤1次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(石油醚:乙酸乙酯＝1:1)纯化，得到目标中间体2-羟基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(12-2)(970mg,收率：96％)。
LCMS:Rt:1.446min；MS m/z(ESI):142.1[M+H-Boc].
化合物12-3：
Figure PCTCN2020127166-appb-000079
室温下将2-羟基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(12-2)(300mg,1.245mmol)加入到超干N,N-二甲基甲酰胺中(3.0mL)，然后加入氢化钠(200mg,4.98mmol)室温反应0.5小时，将碘甲烷(1.06mg,7.47mmol)加入，继续反应1.5小时。反应完全后加入水(50mL)，然后用乙酸乙酯(40mL)萃取3次。有机相合并，用饱和食盐水(50mL)洗涤3次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(石油醚:乙酸乙酯＝5:1)纯化，得到目标中间体2-甲氧基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(12-3)(313mg,收率：85％)。
化合物12-4：
Figure PCTCN2020127166-appb-000080
室温下将2-甲氧基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(12-3)(313mg，1.23mmol)加入到4N盐酸/二氧六环溶液中(3.0mL)，室温反应1.5小时，将反应液减压浓缩得中间体2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)(200mg,收率：85％)。
LCMS:Rt:0.571min；MS m/z(ESI):156.5[M+H].
化合物12-5：
Figure PCTCN2020127166-appb-000081
室温下将2-((R)-2-((S)-2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-氧代-1,2,3,4-四氢异喹啉-6-羧酸(中间体3)(500mg,1.01mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(570mg,1.5mmol)和N,N-二异丙基乙胺(520mg,4mmol)加入到超干N,N-二甲基甲酰胺(10mL)。然后加入2-甲氧基-7-氮杂螺环[3.5]壬烷盐酸盐(12-4)(230mg,1.2mmol)，室温反应1.5小时。反应完全后加入饱和食盐水(50mL)，用 乙酸乙酯(20mL)萃取2次，有机相合并后用饱和食盐水(50mL)洗涤3次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂：石油醚/乙酸乙酯＝1/4)纯化，得到目标中间体(S)-3-((R)-1-羟基-2-(6-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-氧代-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(12-5)(560mg,收率：88％)。
LCMS:Rt:1.734min；MS m/z(ESI):632.4[M+H]。
化合物12：
Figure PCTCN2020127166-appb-000082
室温下将(S)-3-((R)-1-羟基-2-(6-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-氧代-3,4-二氢异喹啉-2(1H)-基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(12-5)(560mg,0.88mmol)溶于4N盐酸/二氧六环溶液(5mL)，反应液在室温下搅拌1小时。反应完全后减压浓缩，残余物用高效液相制备色谱法(洗脱剂梯度如下表)纯化：
Figure PCTCN2020127166-appb-000083
得到目标化合物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物12)(361mg,收率:73％)。 1H NMR(400MHz,CD 3OD):δ8.04(d,J＝7.9Hz,1H),7.43(s,1H),7.36(d,J＝7.9Hz,1H),7.33-7.20(m,4H),4.49-4.28(m,3H),3.99-3.95(m,2H),3.70-3.56(m,6H),3.30-3.24(m,4H),3.21(s,3H),2.26-2.24(m,2H),1.72-1.54(m,6H),1.38(d,J＝7.6Hz,6H).
LCMS:Rt:1.370min；MS m/z(ESI):532.4[M+H]。
实施例12、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(6-氮杂螺[2.5]辛烷-6-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物11)的制备
Figure PCTCN2020127166-appb-000084
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为6-氮杂 螺环[2.5]辛烷盐酸盐，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(6-氮杂螺[2.5]辛烷-6-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物11)。
1H NMR(400MHz,CD 3OD):δ8.04(d,J＝7.9Hz,1H),7.45(d,J＝1.6Hz,1H),7.38(dd,J＝7.9Hz,1.6Hz,1H),7.33-7.18(m,4H),4.51-4.36(m,2H),4.35-4.27(m,1H),3.97(dd,J＝14.1Hz,4.8Hz,1H),3.81-3.79(m,2H),3.67-3.56(m,6H),3.40(s,2H),3.31-3.27(m,2H),1.51-1.49(m,2H),1.39(d,J＝7.7Hz,6H),0.45-0.37(m,4H).
LCMS:Rt:1.040min；MS m/z(ESI):488.4[M+H]。
实施例13、7-(2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-1-氧代-1,2,3,4-四氢异喹啉-6-羰基)-7-氮杂螺[3.5]壬烷-2-甲腈(化合物13)的制备
Figure PCTCN2020127166-appb-000085
化合物13-2：
Figure PCTCN2020127166-appb-000086
室温下将2-氰基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(13-1)(600mg,2.4mmol)溶于二氯甲烷(2mL)，缓慢加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完全后将反应液减压浓缩，得中间体7-氮杂螺[3.5]壬烷-2-甲腈三氟乙酸盐(13-2)(粗品，590mg,收率：100％)。直接用于下一步。
化合物13-3：
Figure PCTCN2020127166-appb-000087
室温下将2-((R)-2-((S)-2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-4,4-二甲基-1-氧代-1,2,3,4-四氢异喹啉-6-羧酸(中间体3)(990mg，2mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.14g，3mmol)和N,N-二异丙基乙胺(520mg，4mmol)加入到超干N,N-二甲基甲酰胺(10mL)。然后加7-氮杂螺[3.5]壬烷-2-甲腈三氟乙酸盐(13-2)(590mg,2.4mmol)和N,N-二异丙基乙胺(520mg,4mmol)的混合物，室温反应1.5小时。反应完全后加入饱和食盐水(50mL)，用乙酸乙酯(20mL)萃取2次，有机相合并后用饱和食盐水洗涤3次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂：石油醚/乙酸乙酯＝1/4)纯化，得到目标中间体(S)-3-((R)-2-(6-(2-氰基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-氧代-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(13-3)(890mg,收率：71％)。
LCMS:Rt:1.632min；MS m/z(ESI):627.3[M+H]。
化合物13：
Figure PCTCN2020127166-appb-000088
室温下将(S)-3-((R)-2-(6-(2-氰基-7-氮杂螺[3.5]壬烷-7-羰基)-4,4-二甲基-1-氧代-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(13-3)(890mg,1.42mmol)溶于二氯甲烷(5mL)，缓慢加入三氟乙酸(2mL)，反应液在室温下搅拌1小时。反应完全后冷却到0℃，加水稀释后，在冰浴下缓慢滴加饱和碳酸钠水溶液调节pH到10，用乙酸乙酯萃取(50mL X 2)后合并有机相，用饱和食盐水洗涤(50mL X 3)，用无水硫酸钠干燥后过滤，滤液减压浓缩，残余物加少量水后冻干，得到目标化合物7-(2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-1-氧代-1,2,3,4-四氢异喹啉-6-羰基)-7-氮杂螺[3.5]壬烷-2-甲腈(化合物13)(567mg,收率:75％)。
1H NMR(400MHz,CD 3OD):δ8.02(d,J＝7.9Hz,1H),7.42(d,J＝1.5Hz,1H),7.35(dd,J＝7.9Hz,1.5Hz,1H),7.14-7.10(m,3H)，7.05-7.03(m,1H)，4.11-3.97(m,3H),3.97-3.88(m,1H),3.69-3.54(m,5H),3.31-3.29(m,3H),2.93-2.91(m,3H),2.36-2.33(m,2H),2.20-2.18(m,2H),1.77-1.74(m,2H),1.65-1.61(m,2H),1.37(d,J＝2.1Hz,6H).
LCMS:Rt:1.318min；MS m/z(ESI):527.3[M+H]。
实施例14、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-氮杂螺[4.5]癸烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物15)的制备
Figure PCTCN2020127166-appb-000089
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为2-氮杂螺环[4.5]癸烷盐酸盐，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-氮杂螺[4.5]癸烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物15)。
1H NMR(400MHz,CD 3OD)：δ8.06-8.02(m,1H),7.54(s,1H),7.53-7.45(m,1H),7.31-7.21(m,4H),4.49-4.31(m,3H),4.02-3.95(m,1H),3.68-3.56(m,5H),3.49-3.43(m,2H),3.30-3.22(m,3H),1.86-1.76(m,2H),1.58-1.49(m,6H),1.47-1.30(m,10H).
LCMS:Rt:1.465min；MS m/z(ESI):516.2[M+H]。
实施例15、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物16)的制备
Figure PCTCN2020127166-appb-000090
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为7-氮杂螺[3.5]壬烷盐酸盐，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物16)。
1H NMR(400MHz,CD 3OD)：δ8.03(d,J＝8.0Hz,1H),7.42(d,J＝1.6Hz,1H),7.36-7.34(m,1H),7.31-7.21(m,4H),4.45-4.29(m,3H),4.00-3.95(m,1H),3.67-3.59(m,6H),3.30-3.27(m,4H),1.95-1.82(m,6H),1.70(s,2H),1.56(s,2H),1.39(d,J＝7.2Hz,6H).
LCMS:Rt:1.455min；MS m/z(ESI):502.2[M+H]。
实施例16、6-(4,4-二氟-6-氮杂螺[2.5]辛烷-6-羰基)-2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物17)的制备
Figure PCTCN2020127166-appb-000091
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为4,4-二氟-6-氮杂螺[2.5]辛烷盐酸盐，得到目标产物6-(4,4-二氟-6-氮杂螺[2.5]辛烷-6-羰基)-2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物17)。
1H NMR(400MHz,CD 3OD)：δ8.06(d,J＝7.6Hz,1H),7.48(s,1H),7.40(dd,J＝7.6Hz,1.6Hz,1H),7.31-7.25(m,3H),7.22(d,J＝7.2Hz,1H),4.50-4.37(m,2H),4.33-4.29(m,1H),4.05-3.96(m,2H),3.88(s,1H),3.68-3.50(m,5H),3.48(s,1H),3.28-3.25(m,2H),1.73-1.63(m,2H),1.39(d,J＝4.4Hz,6H),0.95-0.90(m,2H),0.57(s,2H).
LCMS:Rt:1.455min；MS m/z(ESI):524.1[M+H]。
实施例17、6-(1,1-二氟-6-氮杂螺[2.5]辛烷-6-羰基)-2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物18)的制备
Figure PCTCN2020127166-appb-000092
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为1,1-二氟-6-氮杂螺环[2.5]辛烷盐酸盐，得到目标产物6-(1,1-二氟-6-氮杂螺[2.5]辛烷-6-羰基)-2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物18)。
1H NMR(400MHz,CD 3OD)：δ8.05(d,J＝8.0Hz,1H),7.48(d,J＝1.6Hz,1H),7.41-7.39(m,1H),7.31-7.21(m,4H),4.49-4.41(m,2H),4.37-4.31(m,1H),4.00-3.95(m,1H),3.80(s,2H),3.67-3.57(m,4H),3.31-3.30(m,2H),3.29-3.27(m,2H),1.71-1.68(m,4H),1.40-1.39(m,6H),1.26(t,J＝8.4Hz,2H).
LCMS:Rt:1.409min；MS m/z(ESI):524.3[M+H]。
实施例18、6-(1,1-二氟-5-氮杂螺[2.4]庚烷-5-羰基)-2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物19)的制备
Figure PCTCN2020127166-appb-000093
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为1,1-二 氟-5-氮杂螺环[2.4]庚烷盐酸盐，得到目标产物6-(1,1-二氟-5-氮杂螺[2.4]庚烷-5-羰基)-2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物19)。
1H NMR(400MHz,CD 3OD)：δ8.04(d,J＝8.0Hz,1H),7.61-7.54(m,1H),7.52-7.47(m,1H),7.28-7.18(m,4H),4.43-4.27(m,3H),4.00-3.95(m,1H),3.87-3.71(m,2H),3.67-3.47(m,6H),3.25-3.22(m,2H),2.25-2.00(m,2H),1.58-1.43(m,2H),1.39(d,J＝6.8Hz,6H).
LCMS:Rt:1.060min；MS m/z(ESI):510.2[M+H].
实施例19、6-(6,6-二氟-2-氮杂螺[3.3]庚烷-2-羰基)-2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物20)的制备
Figure PCTCN2020127166-appb-000094
参考实施例13的合成方法，将2-氰基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(13-1)替换为6,6-二氟-2-氮杂螺[3.3]庚烷-2-羧酸叔丁酯，得到目标产物6-(6,6-二氟-2-氮杂螺[3.3]庚烷-2-羰基)-2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物20)。
1H NMR(400MHz,CD 3OD)：δ8.03(d,J＝8.0Hz,1H),7.68(d,J＝1.6Hz,1H),7.58(dd,J＝8.0Hz,1.6Hz,1H),7.31-7.25(m,3H),7.22(d,J＝7.2Hz,1H),4.50-4.34(m,4H),4.33-4.29(m,1H),4.26(s,2H),3.98(dd,J＝14.0Hz,4.8Hz,1H),3.67-3.57(m,4H),3.29-3.27(m,2H),2.83(t,J＝11.6Hz,4H),1.40(d,J＝8.4Hz,6H).
LCMS:Rt:1.381min；MS m/z(ESI):510.2[M+H]。
实施例20、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(3-氮杂螺[5.5]十一烷-3-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物21)的制备
Figure PCTCN2020127166-appb-000095
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为3-氮杂螺[5.5]十一烷盐酸盐，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(3-氮杂螺[5.5]十一烷-3-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物21)。
1H NMR(400MHz,CD 3OD)：δ8.04(d,J＝7.6Hz,1H),7.43(d,J＝1.2Hz,1H),7.37-7.35(m,1H),7.31-7.21(m,4H),4.49-4.34(m,3H),4.01-3.98(m,1H),3.74-3.56(m,6H),3.34-3.33(m,2H),3.30-3.27(m,2H),1.47-1.28(m,20H).
LCMS:Rt:1.523min；MS m/z(ESI):530.5[M+H]。
实施例21、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-氧代-6-氮杂螺[3.4]辛烷-6-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物22)的制备
Figure PCTCN2020127166-appb-000096
参考实施例13的合成方法，将2-氰基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(13-1)替换为2-氧代-6-氮杂螺[3.4]辛烷-6-羧酸叔丁酯，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-氧代-6-氮杂螺[3.4]辛烷-6-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物22)。
1H NMR(400MHz,DMSO-d 6)：δ9.71(s,1H),9.03-9.01(m,1H),7.91(d,J＝8.0Hz,1H),7.52(d,J＝4.8Hz,1H),7.47(d,J＝8.0Hz,1H),7.27-7.23(m,4H),4.55-4.49(m,3H),4.25-4.21(m,1H),3.98(dd,J＝14.0Hz,6.0Hz,1H),3.68(s,1H),3.59-3.33(m,7H),3.25-3.16(m,3H),3.08-2.93(m,3H),2.12-2.03(m,2H),1.32(d,J＝11.2Hz,6H).
LCMS:Rt:1.217min；MS m/z(ESI):502.4[M+H]。
实施例22、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-氧杂-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物34)的制备
Figure PCTCN2020127166-appb-000097
参考实施例13的合成方法，将2-氰基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(13-1)替换为2-氧杂-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-氧杂-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物34)。
1H NMR(400MHz,CD 3OD)：δ8.03(d,J＝8.0Hz,1H),7.42(d,J＝1.2Hz,1H),7.37-7.35(m,1H),7.14-7.09(m,3H),7.05-7.03(m,1H),4.49-4.45(m,4H),4.09-4.01(m,3H),4.01-3.91(m,1H),3.69-3.64(m,3H),3.61-3.54(m,2H),3.34-3.30(m,2H),2.97-2.89(m,3H),1.98(s,2H),1.85(s,2H),1.37(d,J＝1.6Hz,6H).
LCMS:Rt:0.834min；MS m/z(ESI):504.4[M+H]。
实施例23、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1-氧杂-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物35)的制备
Figure PCTCN2020127166-appb-000098
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为1-氧杂-7-氮杂螺[3.5]壬烷半草酸盐，得到中间体(S)-3-((R)-2-(4,4-二甲基-1-氧代-6-(1-氧杂-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(35-1)。
Figure PCTCN2020127166-appb-000099
化合物35：
向(S)-3-((R)-2-(4,4-二甲基-1-氧代-6-(1-氧杂-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-2(1H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(35-1)(35.0mg,0.061mmol)的二氯甲烷(2mL)溶液中加入溴化锌(69.0mg,0.307mmol)，30℃反应3.0小时。反应完毕后加入碳酸氢钠水溶液(10mL),用乙酸乙酯(20mL)萃取三次。有机相合并，用饱和食盐水(30mL)洗涤1次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物用高效液相制备色谱法(洗脱剂梯度：
Figure PCTCN2020127166-appb-000100
)纯化，得到目标化合物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1-氧杂-7-氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物35)(9.17mg,收率:30％)。 1H NMR(400MHz,DMSO-d6)：δ7.93-7.92(m,1H),7.39(s,1H),7.34(d,J＝8.0Hz,1H),7.09-7.07(m,3H),7.01(d,J＝4.8Hz,1H),5.49-5.31(m,1H),4.92(d,J＝5.6Hz,1H),4.46-4.45(m,1H),4.04(s,1H),3.95-3.92(m,2H),3.86-3.70(m,3H),3.57-3.40(m,5H),2.81-2.67(m,3H),2.42-2.22(m,2H),2.21-2.08(m,5H),1.30-1.23(m,6H).
LCMS:Rt:1.221min；MS m/z(ESI):504.4[M+H]。
实施例24、3,3-二氟-2'-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6'-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-2',3'-二氢-1'氢-螺环[环丁烷-1,4'-异喹啉]-1'-酮盐酸盐(化合物37)的制备
Figure PCTCN2020127166-appb-000101
化合物37-2：
Figure PCTCN2020127166-appb-000102
0℃下将2-(3-溴苯基)乙腈(37-1)(30g，153mmol)加入到N,N-二甲基甲酰胺(600mL)中，然后分批加入氢化钠(60％)(14g,352mmol)，反应0.5小时。再滴加1,3-二溴-2,2-二甲氧基丙烷(60g,229mmol)，升温到60℃反应16小时。反应完全后，将反应液冷却至室温，缓慢加到冰水(1L)中，用乙酸乙酯(1L)萃取三次，有机相用饱和食盐水洗涤两次，无水硫酸钠干燥过滤，滤液减压浓缩。粗品用正相色谱柱(洗脱剂梯度：石油醚/乙酸乙酯＝10/1)纯化，得到目标中间体1-(3-溴苯基)-3,3-二甲氧基环丁烷-1-甲腈(37-2)(16g,收率：35.3％)。
化合物37-3：
Figure PCTCN2020127166-appb-000103
0℃下将1-(3-溴苯基)-3,3-二甲氧基环丁烷-1-甲腈(37-2)(16g，54mmol)加入到四氢呋喃(160mL)和水(80mL)的混合溶液中，然后滴加浓盐酸(80mL)，室温下反应16小时。反应完全后，将反应体系降温至0℃，用碳酸钾调节反应液的pH至8.0，用乙酸乙酯(200mL)萃取三次，有机相用饱和食盐水洗涤三次，无水硫酸钠干燥过滤，滤液减压浓缩，得到目标中间体1-(3-溴苯基)-3-氧代环丁烷-1-甲腈(37-3)(10g,收率：74.0％),性状:黄色液体。 1H NMR(400MHz,DMSO-d 6):δ7.82(t,J＝1.9Hz,1H),7.66-7.59(m,2H),7.45(t,J＝7.9Hz,1H),4.12-4.04(m,2H),3.97-3.89(m,2H).
化合物37-4：
Figure PCTCN2020127166-appb-000104
0℃下将1-(3-溴苯基)-3-氧代环丁烷-1-甲腈(37-3)(10g,40mmol)加入到超干二氯甲烷(40mL),然后加入双(2-甲氧基乙基)氨基硫三氟化物(BAST)(18.4g,84mmol)，氮气保护下加热至30℃，反应48小时。反应完全后，将反应体系降至0℃,缓慢倒入冰水混合物(500mL)中,用固体碳酸氢钠调节pH至8,用二氯甲烷(200mL)萃取三次，有机相用饱和食盐水洗涤三次，无水硫酸钠干燥过滤，滤液减压浓缩。粗品用正相色谱柱(洗脱剂梯度：石油醚/乙酸乙酯＝10/1)纯化，得到目标中间体1-(3-溴苯基)-3,3-二氟环丁烷-1-甲腈(37-4)(5.89g,收率：54.1％)。
1H NMR(400MHz,DMSO-d 6)：δ7.76(t,J＝1.8Hz,1H),7.63(ddd,J＝7.9,1.8,1.0Hz,1H),7.54(ddd,J＝7.8,1.8,1.0Hz,1H),7.45(t,J＝7.9Hz,1H),3.58-3.52(m,2H),3.49-3.36(m,2H).
化合物37-5：
Figure PCTCN2020127166-appb-000105
0℃下将1-(3-溴苯基)-3,3-二氟环丁烷-1-甲腈(37-4)(6.29g,23.1mmol)加入到超干四氢呋喃(100mL)，氮气保护，滴加1N的硼烷四氢呋喃溶液(69.3mL,69.3mmol)，室温下反应1小时，然后加热至70℃反应3小时。将反应体系降温至0℃，滴加甲醇(30mL)，浓盐酸(5.9mL,71mmol)然后将反应升温至70℃反应3h。反应完成后，将反应液浓缩，向粗品中加入水(200mL)和乙酸乙酯(100mL)，用固体碳酸钠调节pH至8，用乙酸乙酯(200mL)萃取三次，饱和氯化钠溶液洗涤三次，无水硫酸钠干燥过滤，滤液减压浓缩，得到目标中间体(1-(3-溴苯基)-3,3-二氟环丁基)甲胺(37-5)(粗品，6.6g,收率：100％)。
LCMS:Rt:0.780min；MS m/z(ESI):276.0,278.0[M+H]。
化合物37-6：
Figure PCTCN2020127166-appb-000106
0℃下将三光气(BTC)(3.4g,11.6mmol)加入到超干二氯甲烷(100mL)中，先后加入(1-(3-溴苯基)-3,3-二氟环丁基)甲胺(37-5)(6.4g，23.1mmol)和三乙胺(2.3g，23.1mmol)。反应0.5小时后，滴加甲醇(30mL)和三乙胺(11.6g，115.5mmol)。反应完成后，向反应体系加水(50mL)分液，用二氯甲烷(70mL)萃取3次，饱和氯化钠溶液洗涤3次，无水硫酸钠干燥过滤，滤液减压浓缩，残余物用正相色谱柱(洗脱剂梯度：石油醚/乙酸乙酯＝10/1)纯化，得到目标中间体((1-(3-溴苯基)-3,3-二氟环丁基)甲基)氨基甲酸甲酯(37-6)(5.2g,收率：67.3％)。
LCMS:Rt:1.530min；MS m/z(ESI):334.1,336.1[M+H]。
化合物37-7：
Figure PCTCN2020127166-appb-000107
室温下将((1-(3-溴苯基)-3,3-二氟环丁基)甲基)氨基甲酸甲酯(37-6)(4.7g,14mmol)加入到伊顿试剂(75mL)中，氮气保护，125℃下反应3小时。将反应液冷却至室温，缓慢倒入碎冰中，用乙酸乙酯(200mL)萃取3次，饱和氯化钠溶液洗涤3次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：石油醚/乙酸乙酯＝1/1)纯化，得到目标中间体6'-溴-3,3-二氟-2',3'-二氢-1'H-螺[环丁烷-1,4'-异喹啉]-1'-酮(37-7)(1g,收率：23.6％)。LCMS:Rt:1.291min；MS m/z(ESI):302.1,304.1[M+H]。
化合物37-8：
Figure PCTCN2020127166-appb-000108
室温下将6'-溴-3,3-二氟-2',3'-二氢-1'H-螺[环丁烷-1,4'-异喹啉]-1'-酮(37-7)(950mg，3.14mmol)、(S)-3-((S)-环氧乙烷-2-基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(k)(1g，3.77mmol)以及碳酸铯(2.04g，6.29mmol)加到N-甲基吡咯烷酮(20mL)中，氩气保护，85℃下反应16小时。反应完成后直接用反相色谱柱(洗脱剂梯度：乙腈/纯水＝2/1)纯化，得到目标中间体(1R,10aS)-1-((6'-溴-3,3-二氟-1'-羰基-1'H-螺[环丁烷-1,4'-异喹啉]-2'(3'H)-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮(37-8)(1.3g,收率：82.3％)。
LCMS:Rt:1.898min；MS m/z(ESI):503.0,505.0[M+H]。
化合物37-9：
Figure PCTCN2020127166-appb-000109
室温下将(1R,10aS)-1-((6'-溴-3,3-二氟-1'-羰基-1'H-螺[环丁烷-1,4'-异喹啉]-2'(3'H)-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮(37-8)(600mg，1.19mmol)，[1,1'-双(二苯基膦)二茂铁]二氯化钯(44mg，0.06mmol)和醋酸钾(350mg，3.57mmol)加入到无水乙醇(18.0mL)中，CO置换3次，加热至70℃反应2.0小时。反应完全后冷却至室温，减压浓缩。残余物经正相色谱柱(洗脱剂梯度：石油醚/乙酸乙酯＝5/1)纯化，得到目标中间体3,3-二氟-1'-羰基-2'-(((1R,10aS)-3-羰基-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-2',3'-二氢-1'H-螺[环丁烷-1,4'-异喹啉]-6'-羧酸乙酯(37-9)(453mg,收率：76％)。
LCMS:Rt:1.883min；MS m/z(ESI):497.1[M+H]。
化合物37-10：
Figure PCTCN2020127166-appb-000110
室温下将3,3-二氟-1'-羰基-2'-(((1R,10aS)-3-羰基-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-2',3'-二氢-1'H-螺[环丁烷-1,4'-异喹啉]-6'-羧酸乙酯(37-9)(453mg，0.91mmol)加入到甲醇(5.0mL)、四氢呋喃(5.0mL)和水(5.0mL)的混合溶液中，加入氢氧化钠(146mg，3.65mmol)，加热至70℃，反应16小时。反应完全后，将反应体系降温至室温，加入二碳酸二叔丁酯(O(Boc) 2)(595mg，2.73mmol)，室温反应3.5小时。将反应液降至0℃，用1N的盐酸水溶液调节pH至5.0，后用乙酸乙酯(50mL)萃取三次，有机相用无水硫酸钠干燥过滤，滤液减压浓缩。得到目标中间体2'-((R)-2-((S)-2-(叔-丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-3,3-二氟-1'-氧代-2',3'-二氢-1'H-螺[环丁烷-1,4'-异喹啉]-6'-羧酸(37-10) (350mg,收率：71％)。
LCMS:Rt:1.857min；MS m/z(ESI):543.1[M+H]。
化合物37-11：
Figure PCTCN2020127166-appb-000111
室温下将2'-((R)-2-((S)-2-(叔-丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-3,3-二氟-1'-氧代-2',3'-二氢-1'H-螺[环丁烷-1,4'-异喹啉]-6'-羧酸(37-10)(70mg,0.13mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(98mg,0.26mmol)和N,N-二乙基乙胺(52.2mg,0.52mmol)加入到超干N,N-二甲基甲酰胺(5.0mL)中，然后加入2-甲氧基-7-氮杂螺环[3.5]壬烷盐酸盐(12-4)(29.6mg,0.16mmol)，室温反应1.5小时。反应完全后加入饱和食盐水(50mL)，用乙酸乙酯(50mL)萃取两次，有机相用饱和食盐水洗涤(50mL)两次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(洗脱剂梯度：乙酸乙酯＝100％)纯化，得到目标中间体(S)-3-((R)-2-(3,3-二氟-6'-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-1'-氧代-1'H-螺[环丁烷-1,4'-异喹啉]-2'(3'H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(37-11)(65mg,收率：74％)。
LCMS:Rt:1.994min；MS m/z(ESI):680.4[M+H]。
化合物37：
Figure PCTCN2020127166-appb-000112
在0℃下向(S)-3-((R)-2-(3,3-二氟-6'-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-1'-氧代-1'H-螺[环丁烷-1,4'-异喹啉]-2'(3'H)-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(37-11)的二氯甲烷(2mL)溶液中加入三氟乙酸(1mL)，反应液在室温下搅拌1小时。反应完全后减压浓缩，残余物用高效液相制备色谱法(洗脱剂梯度参考实施例11)纯化，得到目标化合物3,3-二氟-2'-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6'-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-2',3'-二氢-1'氢-螺环[环丁烷-1,4'-异喹啉]-1'-酮盐酸盐(化合物37)(24.28mg,收率:41％)。
1H NMR(400MHz,CD 3OD)：δ8.08(d,J＝8Hz,1H),7.57(s,1H),7.43(d,J＝8Hz,1H),7.31-7.20(m,4H),4.44-4.37(m,2H),4.30-4.28(m,1H),3.98-3.90(m,4H),3.70-3.65(m,3H),3.60-3.58(m,1H),3.33-3.32(m,1H),3.29-3.25(m,3H),3.21(s,3H),2.99-2.90(m,4H),2.26-2.25(m,2H),1.72-1.69(m,4H),1.55(s,2H).
LCMS:Rt:0.974min；MS m/z(ESI):580.2[M+H]。
实施例25、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,6-二氮杂螺[3.3]庚烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物39)的制备
Figure PCTCN2020127166-appb-000113
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为2,6-二氮杂螺[3.3]庚烷-2-羧酸叔丁酯半草酸盐，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,6-二氮杂螺[3.3]庚烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物39)。
1H NMR(400MHz,CD 3OD)：δ8.07-8.03(m,1H),7.67(s,1H),7.58(d,J＝8.0Hz,1H),7.31-7.21(m,4H),5.56(s,2H),4.49-4.29(m,10H),4.03-3.96(m,1H),3.68-3.57(m,4H),3.29-3.25(m,1H),1.41(d,J＝7.2Hz,6H).
LCMS:Rt:0.356min；MS m/z(ESI):475.3[M+H]。
实施例26、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,7-二氮杂螺[3.5]壬烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物40)的制备
Figure PCTCN2020127166-appb-000114
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为2,7-二氮杂螺[3.5]壬烷-7-羧酸叔丁酯盐酸盐，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,7-二氮杂螺[3.5]壬烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物40)。
1H NMR(400MHz,CD 3OD)：δ8.04(d,J＝7.6Hz,1H),7.70(s,1H),7.62(d,J＝7.6Hz,1H),7.31-7.21(m,4H),4.49-4.34(m,3H),4.18(s,2H),4.01-3.97(m,3H),3.68-3.57(m,4H),3.30-3.29(m,2H),3.27-3.19(m,4H),2.07(t,J＝5.6Hz,4H),1.40(d,J＝8.4Hz,6H).
LCMS:Rt:1.102min；MS m/z(ESI):503.4[M+H]。
实施例27、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(9-羟基-3-氮杂螺[5.5]十一烷-3-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物51)的制备
Figure PCTCN2020127166-appb-000115
化合物51-2：
Figure PCTCN2020127166-appb-000116
室温下将9-氧代-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(51-1)(400mg,1.5mmol)加入到甲醇中(10.0mL)，0℃下加入硼氢化钠(171mg,4.5mmol)，室温反应1.5小时。加入氯化铵水溶液(10.0mL)淬灭反应，用乙酸乙酯(50.0mL)萃取2次。有机相合并，用饱和食 盐水(50mL)洗涤1次，无水硫酸钠干燥过滤，滤液减压浓缩。残余物用正相色谱柱(石油醚:乙酸乙酯＝2:1)纯化，得到9-羟基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(51-2)(305mg,收率：76％)。
LCMS:Rt:1.570min；MS m/z(ESI):270.0[M+H].
化合物51-3：
Figure PCTCN2020127166-appb-000117
室温下将9-羟基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(51-2)(305mg)加入到4N盐酸/二氧六环溶液中(5.0mL)，室温反应1.5小时，反应液减压浓缩得粗品中间体9-羟基-3-氮杂螺[5.5]十一烷盐酸盐(51-3)(155mg,收率：66％)。
其余步骤参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为9-羟基-3-氮杂螺[5.5]十一烷盐酸盐(51-3)，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(9-羟基-3-氮杂螺[5.5]十一烷-3-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物51)。
1H NMR(400MHz,CD 3OD)：δ8.03(d,J＝8.0Hz,1H),7.44(s,1H),7.36(d,J＝8.0Hz,1H),7.31-7.25(m,3H),7.23-7.21(m,1H),4.50-4.37(m,2H),4.34-4.30(m,1H),3.98(dd,J＝14.0Hz,4.8Hz,1H),3.75-3.71(m,2H),3.67-3.57(m,5H),3.35-3.33(m,2H),3.28-3.23(m,2H),1.76-1.74(m,4H),1.65-1.62(m,1H),1.49-1.46(m,3H),1.39(d,J＝7.6Hz,6H),1.32-1.24(m,4H).
LCMS:Rt:1.370min；MS m/z(ESI):546.1[M+H]。
实施例28、3-(2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-1-氧代-1,2,3,4-四氢异喹啉-6-羰基)-3-氮杂螺[5.5]十一烷-9-酮盐酸盐(化合物52)的制备
Figure PCTCN2020127166-appb-000118
参考实施例13的合成方法，将2-氰基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(13-1)替换为9-氧代-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯，得到目标产物3-(2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-1-氧代-1,2,3,4-四氢异喹啉-6-羰基)-3-氮杂螺[5.5]十一烷-9-酮盐酸盐(化合物52)。
1H NMR(400MHz,DMSO-d 6)：δ9.52-9.46(m,1H),9.02-8.95(m,1H),7.92(d,J＝8.0Hz,1H),7.40(s,1H),7.34(dd,J＝8.0Hz,1.2Hz,1H),7.29-7.23(m,4H),5.88(s,1H),4.40-4.23(m,3H),3.97(dd,J＝13.6Hz,6.0Hz,1H),3.65-3.55(m,5H),3.41-3.31(m,1H),3.27(s,2H),3.18-3.16(m,2H),2.27-2.24(m,4H),1.75-1.71(m,4H),1.61(s,2H),1.45(s,2H),1.32(d,J＝10.4Hz,6H).LCMS:Rt:1.387min；MS m/z(ESI):544.1[M+H]。
实施例29、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,7-二氮杂螺[4.4]壬烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物53)的制备
Figure PCTCN2020127166-appb-000119
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为2,7-二氮杂螺[4.4]壬烷-2-羧酸叔丁酯半草酸盐，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2,7-二氮杂螺[4.4]壬烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物53)。
1H NMR(400MHz,CD 3OD)：δ8.04(d,J＝7.6Hz,1H),7.60-7.51(m,2H),7.31-7.21(m,4H),4.49-4.34(m,3H),4.01-3.98(m,1H),3.75-3.58(m,7H),3.52-3.40(m,2H),3.30-3.26(m,1H),2.66(s,4H),2.18-2.03(m,4H),1.40(d,J＝8.4Hz,6H).
LCMS:Rt:0.385min；MS m/z(ESI):503.4[M+H]。
实施例30、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(8-氧杂-2-氮杂螺[4.5]癸烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物54)的制备
Figure PCTCN2020127166-appb-000120
参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为8-氧杂-2-氮杂螺[4.5]癸烷，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(8-氧杂-2-氮杂螺[4.5]癸烷-2-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物54)。
1H NMR(400MHz,CD 3OD)：δ8.08-8.00(m,1H),7.55(s,1H),7.48(d,J＝7.2Hz,1H),7.30-7.21(m,4H),4.51-4.35(m,2H),4.32-4.31(m,1H),4.00-3.96(m,1H),3.77-3.64(m,9H),3.61-3.48(m,3H),3.29-3.12(m,2H),1.96(t,J＝7.2Hz,1H),1.85(t,J＝6.8Hz,1H),1.67-1.64(m,2H),1.58-1.50(m,2H),1.40(d,J＝7.2Hz,6H).
LCMS:Rt:1.226min；MS m/z(ESI):518.4[M+H]。
实施例31、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-氧代-1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物55)的制备
Figure PCTCN2020127166-appb-000121
参考实施例13的合成方法，将2-氰基-7-氮杂螺[3.5]壬烷-7-羧酸叔丁酯(13-1)替换为2-氧代-1,7-二氮杂螺[3.5]壬烷-7-羧酸叔丁酯，得到目标化合物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(2-氧代-1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物55)。
1H NMR(400MHz,DMSO-d 6)：δ8.36(s,1H),7.92(d,J＝8.0Hz,1H),7.38(s,1H),7.33(d,J＝8.0Hz,1H),7.09(s,3H),7.02-7.00(m,1H),4.92(d,J＝5.2Hz,1H),3.96-3.80(m,4H),3.63-3.40(m,6H),3.29-3.19(m,1H),2.81-2.65(m,5H),2.33-2.23(m,1H),1.80-1.61(m,4H),1.30(d,J＝ 2.0Hz,6H).
LCMS:Rt:1.130min；MS m/z(ESI):517.4[M+H]。
实施例32、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1-甲基-1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物56)的制备
Figure PCTCN2020127166-appb-000122
化合物56-2：
Figure PCTCN2020127166-appb-000123
室温下将1,7-二氮杂螺[3.5]壬烷-7-羧酸叔丁酯(56-1)(230mg，1.0mmol)溶于甲醇(10.0mL)，加入冰醋酸(0.2mL)，随后依次加入氰基硼氢化钠(320mg，5.0mmol)和甲醛水溶液(1.0mL)。反应在室温下搅拌2小时。反应完全后用饱和碳酸钠水溶液调节反应液的pH至9.0，用二氯甲烷(30mL)萃取2次。合并有机相，用饱和食盐水(30mL)洗涤2次，无水硫酸钠干燥过滤，滤液减压浓缩，得目标粗品1-甲基-1,7-二氮杂螺[3.5]壬烷-7-羧酸叔丁酯(56-2)(210mg,收率：79％)。
LCMS:Rt:0.496min；MS m/z(ESI):241.1[M+H]。
化合物56-3：
Figure PCTCN2020127166-appb-000124
室温下将粗产品1-甲基-1,7-二氮杂螺[3.5]壬烷-7-羧酸叔丁酯(56-2)(100mg,0.42mmol)溶于二氯甲烷(4.0mL)溶液中,然后加入三氟乙酸(2.0mL)，反应液在室温下搅拌1小时。反应完全后减压浓缩，得目标粗产品1-甲基-1,7-二氮杂螺[3.5]壬烷三氟乙酸盐(56-3)(121mg,收率：86％)。
其余步骤参考实施例13的合成方法，将7-氮杂螺[3.5]壬烷-2-甲腈三氟乙酸盐(13-2)替换为1-甲基-1,7-二氮杂螺[3.5]壬烷三氟乙酸盐(56-3)，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-4,4-二甲基-6-(1-甲基-1,7-二氮杂螺[3.5]壬烷-7-羰基)-3,4-二氢异喹啉-1(2H)-酮(化合物56)。
1H NMR(400MHz,CD 3OD)：δ8.03(d,J＝8.0Hz,1H),7.45(d,J＝1.6Hz,1H),7.38(dd,J＝8.0Hz,1.6Hz,1H),7.14-7.10(m,3H),7.06-7.04(m,1H),4.64-4.56(m,1H),4.09-3.98(m,3H),3.93(dd,J＝14.0Hz,3.6Hz,1H),3.69-3.54(m,4H),3.18-3.12(m,3H),2.98-2.85(m,4H),2.25(s,3H),2.17-1.91(m,3H),1.83-1.57(m,3H),1.38(d,J＝2.0Hz,6H).
LCMS:Rt:0.640min；MS m/z(ESI):517.3[M+H]。
实施例33、2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(9-甲氧基-3-氮杂螺[5.5]十一 烷-3-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物57)的制备
Figure PCTCN2020127166-appb-000125
化合物57-2：
Figure PCTCN2020127166-appb-000126
将9-羟基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(51-2)(540mg,2mmol)溶于N,N-二甲基甲酰胺(10mL)，冷却至0℃,分批加入氢化钠(320mg,8mmol)，然后0℃反应1.0小时。加入碘甲烷(568mg，4mmol)后室温搅拌过夜。反应完全后加入饱和氯化铵溶液淬灭，用饱和食盐水(50mL)稀释，乙酸乙酯(20mL)萃取两次，有机相用饱和食盐水洗涤两次，无水硫酸钠干燥过滤，滤液减压浓缩得到目标中间体9-甲氧基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(57-2)(粗品，560mg,收率：100％)。
化合物57-3：
Figure PCTCN2020127166-appb-000127
将9-甲氧基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(57-2)(560mg,2mmol)溶于二氧六环(1mL)，冷却至0℃,加入4N盐酸/二氧六环溶液(4mL)，0℃反应1.0小时。反应完全后将反应液减压浓缩，残余物用甲基叔丁基醚打浆，过滤得到目标中间体9-甲氧基-3-氮杂螺[5.5]十一烷盐酸盐(57-3)(192mg,收率：44％)。
LCMS:Rt:0.415min；MS m/z(ESI):184.2[M+H]。
其余步骤参考实施例11的合成方法，将2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(12-4)替换为9-甲氧基-3-氮杂螺[5.5]十一烷盐酸盐(57-3)，得到目标产物2-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-6-(9-甲氧基-3-氮杂螺[5.5]十一烷-3-羰基)-4,4-二甲基-3,4-二氢异喹啉-1(2H)-酮盐酸盐(化合物57)。
1H NMR(400MHz,CD 3OD)：δ8.03(d,J＝8.0Hz,1H),7.44(s,1H),7.36(d,J＝8.0Hz,1H),7.34-7.16(m,4H),4.54-4.35(m,2H),4.32(s,1H),4.04-3.90(m,1H),3.80-3.52(m,6H),3.43-3.32(m,4H),3.30-3.18(m,4H),1.88-1.65(m,4H),1.61(s,1H),1.57-1.34(m,11H),1.34-1.19(m,2H).LCMS:Rt:1.381min；MS m/z(ESI):560.4[M+H]。
参考上述实施例11、13的合成方法，本申请还合成了以下化合物。
Figure PCTCN2020127166-appb-000128
Figure PCTCN2020127166-appb-000129
Figure PCTCN2020127166-appb-000130
测试例1：PRMT5酶学活性抑制实验
材料：PRMT5/MEP50蛋白购于BPS bioscience公司(美国)；Histone H4 Peptide底物购于生工生物工程(上海)股份有限公司；Anti-Histone H4(symmetric di methyl R3)antibody-ChIP Grade购于艾博抗公司(美国)；S-(5-Adenosyl)-L-methionine chloride dihydrochloride购于西格玛公司(美国)；384孔板、AlphaScreen Streptavidin Donor beads和AlphaScreen Protein A Acceptor beads购于珀金埃尔默仪器有限公司(美国)。
酶学活性检测：利用Echo将化合物打入384孔板中，使化合物终浓度为0-1000nM(起始浓度1000nM，3倍稀释，10个点)，DMSO含量为0.5％。每孔加入10μL 2X PRMT5/MEP50溶液，常温孵育30分钟。每孔加入10μL 2X PRMT5/MEP50底物溶液启动反应，常温孵育60分钟。准备含AlphaScreen Protein A Acceptor beads和Histone H4(symmetric dimethyl R3)抗体的6X检测试剂，每孔加入5μL，常温孵育60分钟。准备含AlphaScreen Streptavidin Donor beads的6X检测试剂，每孔加入5μL，常温孵育60分钟。Envision检测信号值。测试结果见表2。
测试例2：化合物对肿瘤细胞增殖的抑制活性
材料与细胞：Z-138细胞购于ATCC(美国)；IMDM培养基和青霉素-链霉素购于西格玛公司(美国)；马血清购于Hyclone公司(美国)；96孔板购于康宁公司(美国)；Cell-Titer Glo试剂购于普洛麦格公司(美国)。
细胞培养：Z-138细胞用含10％马血清+1％青霉素-链霉素的IMDM培养液于37℃、5％CO 2条件下培养。处于对数生长期细胞方可用于实验。
细胞增殖活性检测：利用Cell-Titer Glo试剂检测化合物对Z-138细胞的增殖抑制活性。调整细胞浓度，每孔180μL接种96孔板(500/孔)，置于37℃、5％CO 2条件下平衡10-15分钟。每孔加入20μL含化合物的细胞培养液，使化合物终浓度达到0-300nM(起始浓度300nM，3倍稀释，10个点)，DMSO含量为0.1％。细胞板置于37℃、5％CO 2条件下孵育8天。其中第四天换液：缓慢吸去100μL上清，并补充100μL含有化合物的新鲜培养液，保持化合物浓度不变。通过Cell-Titer Glo试剂检测细胞活性。
测试结果见表2。
测试例3：化合物对Z-138细胞SDMA的抑制活性实验
材料与细胞：Z-138细胞购于ATCC(美国)；IMDM培养基和青霉素-链霉素购于西格玛公司(美国)；马血清购于Hyclone公司(美国)；Hoechst抗体购于invitrogen公司(美国)；Alexa Fluor 488goat anti-rabbit IgG抗体购于Santa公司；Anti-dimethyl-Arginine symmetric(SYM11)抗体购于Merck公司(美国)。
细胞培养：Z-138细胞用含10％马血清+1％青霉素-链霉素的IMDM培养液于37℃、5％CO 2条件下培养。处于对数生长期细胞方可用于实验。
免疫荧光检测：利用免疫荧光检测化合物对Z-138细胞中SDMA的影响。调整细胞浓度为1*10 5/mL，每孔40μL接种384孔板(4000/孔)，置于37℃、5％CO 2条件下平衡10-15分钟。利用Echo将化合物打入384孔板中，使化合物终浓度为0-300nM(起始浓度300nM，3倍 稀释，10个点)，DMSO含量为0.1％。细胞板置于37℃、5％CO 2条件下孵育2天。每孔加入40μL 8％多聚甲醛，常温孵育30分钟。弃上清，PBS洗板，每孔加入40μL 0.5％PBST，常温孵育60分钟。弃上清，0.05％PBST洗板，每孔加入40μL封闭液，常温孵育60分钟。弃上清，每孔加入20μL一抗，4℃过夜。弃上清，0.05％PBST洗板，每孔加入20μL二抗，常温孵育60分钟。弃上清，0.05％PBST洗板，ImageXpress Nano检测荧光强度。测试结果见表2。
表2
Figure PCTCN2020127166-appb-000131
注：NA代表未测试。
测试例4：肝细胞代谢稳定性测试
实验材料：人肝细胞购自Biopredic公司；小鼠肝细胞购自BioIVT公司；乙腈和甲醇购自Merck公司；AOPI染色剂购自Nexcelom公司；***购自NIFDC公司；DMSO购自北京索莱宝科技有限公司；DPBS(10x)、GlutaMAX TM-1(100x)和人重组胰岛素购自Gibco by Life  Technologies；胎牛血清购自Corning公司；甲酸购自DIKMAPURE公司；Isotonic Percoll购自GE Healthcare公司；阿普***购自Supelco公司；咖啡因购自ChromaDex.inc；HEPES、甲苯磺丁脲和Williams’Medium E购自Sigma公司。
实验准备：
将受试物粉末用DMSO配制高浓度储备液，使用前用乙腈稀释到100μM的工作液，受试物终浓度为1μM。
肝细胞复苏液的具体制备信息见下表3。混合49.5mL Williams’E Medium和0.5mL GlutaMAX作为孵育液。将肝细胞复苏液和孵育液于使用前置于37℃水浴中至少预热15分钟。取一管超低温保存的肝细胞，确保肝细胞在复苏之前仍处于低温冰冻状态。将肝细胞迅速置于37℃水浴中并轻摇直至所有冰晶全部分散，喷洒70％乙醇后转移至生物安全柜中。将肝细胞小管的内容物倾入盛有50mL复苏培养基的离心管中，将其于100g离心10分钟。离心后，吸出复苏培养基并加入足量孵育培养基得到细胞密度约1.5×10 6个细胞/mL的细胞混悬液。用Cellometer Vision对肝细胞进行计数及确定活细胞密度，肝细胞成活率必须大于75％。利用孵育培养基稀释肝细胞混悬液至活细胞密度为0.5×10 6个活细胞/mL。
表3 肝细胞复苏液制备
Figure PCTCN2020127166-appb-000132
实验方法：
转移247.5μL活细胞(人肝细胞或鼠肝细胞)的混悬液或培养基到96孔深孔板，将深孔板置于涡旋上孵箱中预热10分钟。所有样品均采用双平行孵育。每孔加入2.5μL 100μM受试物进行反应起始，将深孔板放回孵箱涡旋器上。孵育样品，分别于0、15、30、60、90和120分钟，取25μL混悬液，加入125μL含内标的乙腈(100nM阿普***，200nM咖啡因，100nM甲苯磺丁脲)终止反应。涡旋10分钟，于3220g、4℃条件离心30分钟，离心结束后转移100μL上清液到进样板，加入150μL纯水混匀，用于LC-MS/MS分析。
所有的数据计算均通过Microsoft Excel软件进行。通过提取离子图谱检测峰面积。通过对母药消除百分比的自然对数与时间进行线性拟合，检测母药的体外半衰期(t 1/2)。
体外半衰期(t 1/2)通过斜率计算：
in vitro t 1/2＝0.693/k
实验结果如表4所示。
表4 肝细胞代谢稳定性测试结果
化合物 人t 1/2(min) 鼠t 1/2(min)
10 98.24 36.36
11 50.80 20.77
12 89.51 25.22
13 186.98 66.06
16 52.25 19.98
18 67.03 24.68
35 889.57 71.53
54 419.95 128.87
57 90.63 76.44
测试例5：小鼠药代动力学实验
实验材料：CB17-SCID小鼠购自北京维通利华实验动物技术有限公司；DMSO、HP-β-CD(羟丙基-β-环糊精)、MC(甲基纤维素)、乙腈购自Merck公司(美国)。
实验方法：雌性CB17-SCID小鼠6只(20-30g，4-6周)，随机分成2组，每组3只。第1组尾静脉注射给予化合物12，剂量为2mg/kg，溶媒为5％DMSO+95％10％HP-β-CD的水溶液，第2组口服给予化合物12，剂量10mg/kg，溶媒为0.5％MC水溶液。动物实验前正常喂食喂水。每组小鼠于给药前及给药后0.083(仅静脉注射组)、0.25、0.5、1、2、4、6、8和24h进行静脉采血。收集的全血样品置于K 2EDTA抗凝管中，离心5min后(4000rpm，4℃)取血浆待测。
取小鼠血浆样品10μL，加入150μL乙腈溶剂(其中含内标化合物)沉淀蛋白，涡旋0.5min后，离心(4700rpm，4℃)15min，上清液用含0.05％(v/v)FA的水稀释2倍，进样3μL于LC-MS/MS***(AB Sciex Triple Quad 6500+)进行定量检测。在测定样品浓度时随行CB17-SCID小鼠血浆标准曲线(线性范围：0.5-1000ng/mL)和质控样品。对10x稀释样品，取2μL样品加入18μL的空白血浆，涡旋0.5min后，加入300μL乙腈溶剂(其中含内标化合物)沉淀蛋白，其余处理步骤同不稀释样品。
按照上述相同的操作，不同的是将其中的化合物12替换为化合物13，进行所述药代动力学实验。
药代动力学测试结果如表5所示。
表5 小鼠药代动力学测试结果
Figure PCTCN2020127166-appb-000133
测试例6：小鼠体内药效实验
实验材料：Z138细胞购自ATCC；IMDM培养液、青链霉素和0.25％胰酶-EDTA购自Gibco 公司；马血清购自Hyclone公司。
动物信息：CB17-SCID小鼠，雌性，5-6周，体重约13-20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。
实验方法：
细胞培养：人套细胞淋巴瘤Z-138细胞株体外培养，培养条件为IMDM(细胞培养液)中加入10％马血清，1％青链霉素溶液，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85％-90％，数量达到要求时，收取细胞，计数。
细胞接种：将0.1ml/(含1×10 7)Z-138细胞悬液(PBS:Matrigel＝1：1)皮下接种于每只小鼠的右后背。在接种后第18天，测量肿瘤平均体积达到约130mm 3时，依据肿瘤体积和动物体重采用随机分层分组方法开始分组给药。PBS为无钙镁离子的磷酸缓冲盐溶液，Matrigel是基质胶。
给药：化合物12的给药剂量为25mg/kg，PO，每天两次给药(BID)x 3周，或50mg/kg，PO，每天一次给药(QD)x 3周。化合物13的给药剂量为50mg/kg，PO，每天两次给药(BID)x 3周。每组6只小鼠。
肿瘤测量和实验指标：
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。
化合物的抑瘤疗效用肿瘤生长抑制率TGI(％)来评价：
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％。
实验结果如表6、图2和图3所示。实验过程中无小鼠发病或死亡。在小鼠皮下移植瘤Z-138模型中，化合物12在25mg/kg一天两次给药时和化合物13在50mg/kg一天两次给药时对肿瘤生长都具有显著抑制作用，并且具有缩小肿瘤的效果，显示了良好的抗肿瘤药效。化合物12和化合物13在所尝试剂量下未显著影响小鼠体重，也未引起任何小鼠死亡，小鼠可以耐受。
表6 Z-138皮下肿瘤模型肿瘤体积
Figure PCTCN2020127166-appb-000134
以上所述，仅是本发明的较佳实施例，并非对本发明作任何形式上的限制，任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，依据本发明的技术实质，对以上实施例所作的任何简单的修改、等同替换与改进等，均仍属于本发明技术方案的保护范围之内。

Claims (27)

  1. 式(I)化合物或其药学上可接受的盐，
    Figure PCTCN2020127166-appb-100001
    其中，
    A为任选被R 6取代的5-14元螺环基；
    R 1、R 2独立地选自H、C 1-4烷基、卤素、C 1-4烷氧基，或
    R 1、R 2与其连接的C原子共同形成C 3-8环烷基或3-8元杂环烷基，所述环烷基或杂环烷基任选地被卤素取代；
    R 3、R 4、R 5独立地选自H、卤素和C 1-4烷基；
    m为1、2、3或4；
    R 6独立地选自卤素、羟基、氰基、氨基、C 1-3烷基氨基、二(C 1-3烷基)氨基、氧代、C 1-4烷基、C 1-4烷氧基、C 1-4烷氧基羰基、C 3-6环烷基和3-6元杂环烷基。
  2. 权利要求1所述的化合物，其中，A为C 6-13螺环烷基或6-13元螺杂环烷基，所述螺环烷基或螺杂环烷基任选被R 6取代的。
  3. 权利要求1所述的化合物，其中，A为任选被R 6取代的
    Figure PCTCN2020127166-appb-100002
    其中
    n、n’、p、q独立地选自1、2、3和4，且n+n’+p+q≤10；
    W选自CH或N；
    X、Y独立地选自CH 2、NH或O；
    Z选自CH 2、NH、O或键。
  4. 权利要求3所述的化合物，其中，n、n’、p、q独立地选自1、2和3，且n+n’+p+q≤10。
  5. 权利要求4所述的化合物，其中，n、n’独立地选自1、2和3，p、q独立地选自1和2。
  6. 权利要求3-5任一项所述的化合物，其中，W为N。
  7. 权利要求1-6任一项所述的化合物，其中，A选自任选被R 6取代的
    Figure PCTCN2020127166-appb-100003
    Figure PCTCN2020127166-appb-100004
    Figure PCTCN2020127166-appb-100005
  8. 权利要求7所述的化合物，其中，A选自任选被R 6取代的
    Figure PCTCN2020127166-appb-100006
    Figure PCTCN2020127166-appb-100007
  9. 权利要求1-8任一项所述的化合物，其中，R 6独立地选自卤素、羟基、氰基、氨基、C 1-3烷基氨基、氧代、C 1-4烷基、C 1-4烷氧基、C 1-4烷氧基羰基和C 3-6环烷基。
  10. 权利要求9所述的化合物，其中，R 6独立地选自卤素、羟基、氰基、氨基、氧代、C 1-4烷基、C 1-4烷氧基和C 1-4烷氧基羰基。
  11. 权利要求10所述的化合物，其中，R 6独立地选自氟、羟基、氰基、氨基、氧代、甲基、甲氧基和甲氧基羰基。
  12. 权利要求1-11任一项所述的化合物，其中，A选自
    Figure PCTCN2020127166-appb-100008
    Figure PCTCN2020127166-appb-100009
    Figure PCTCN2020127166-appb-100010
  13. 权利要求12所述的化合物，其中，A选自
    Figure PCTCN2020127166-appb-100011
    Figure PCTCN2020127166-appb-100012
  14. 权利要求1-13任一项所述的化合物，其中，R 1、R 2独立地选自H、C 1-4烷基，或R 1、R 2与其连接的C原子共同形成C 3-6环烷基，所述C 3-6环烷基任选地被卤素取代。
  15. 权利要求14所述的化合物，其中，R 1、R 2独立地选自H、甲基、乙基，或R 1、R 2与其连接的C原子共同形成环丙基或环丁基，所述环丙基或环丁基任选地被氟取代。
  16. 权利要求15所述的化合物，其中，R 1、R 2为甲基。
  17. 权利要求1-16任一项所述的化合物，其中，R 3、R 4、R 5独立地选自H、F、Cl、甲基和乙基。
  18. 权利要求17所述的化合物，其中，R 3、R 4、R 5为H。
  19. 权利要求1-18任一项所述的化合物，其中，m为1或2。
  20. 权利要求19所述的化合物，其中，m为1。
  21. 权利要求1-20任一项所述的化合物，其中，式(I)化合物选自式(II)化合物
    Figure PCTCN2020127166-appb-100013
  22. 权利要求1-20任一项所述的化合物，其中，式(I)化合物选自式(III)化合物
    Figure PCTCN2020127166-appb-100014
  23. 权利要求1所述的式(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：
    Figure PCTCN2020127166-appb-100015
    Figure PCTCN2020127166-appb-100016
  24. 权利要求1所述的式(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：
    Figure PCTCN2020127166-appb-100017
    Figure PCTCN2020127166-appb-100018
  25. 药物组合物，其包含本申请的权利要求1-24任一项所述化合物或其药学上可接受的盐。
  26. 权利要求1-24任一项所述化合物或其药学上可接受的盐、或权利要求25所述药物组合物在制备预防或者治疗PRMT5介导的疾病的药物中的用途。
  27. 一种治疗哺乳动物由PRMT5介导的疾病的方法，包括对需要该治疗的哺乳动物给予治疗有效量的权利要求1-24任一项所述化合物或其药学上可接受的盐或权利要求25所述药物组合物。
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WO2017153515A1 (en) * 2016-03-09 2017-09-14 Ctxt Pty Limited Tetrahydroisoquinolines as prmt5-inhibitors
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WO2014100695A1 (en) * 2012-12-21 2014-06-26 Epizyme, Inc. Prmt5 inhibitors and uses thereof
WO2014100764A2 (en) * 2012-12-21 2014-06-26 Epizyme, Inc. Methods of inhibiting prmt5
WO2017153515A1 (en) * 2016-03-09 2017-09-14 Ctxt Pty Limited Tetrahydroisoquinolines as prmt5-inhibitors
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