WO2024078370A1 - 具有取代苯基螺[吲哚啉-3,3'-吡咯烷]结构的小分子化合物 - Google Patents

具有取代苯基螺[吲哚啉-3,3'-吡咯烷]结构的小分子化合物 Download PDF

Info

Publication number
WO2024078370A1
WO2024078370A1 PCT/CN2023/123063 CN2023123063W WO2024078370A1 WO 2024078370 A1 WO2024078370 A1 WO 2024078370A1 CN 2023123063 W CN2023123063 W CN 2023123063W WO 2024078370 A1 WO2024078370 A1 WO 2024078370A1
Authority
WO
WIPO (PCT)
Prior art keywords
mmol
substituted
unsubstituted
chloro
alkyl
Prior art date
Application number
PCT/CN2023/123063
Other languages
English (en)
French (fr)
Inventor
赵玉军
李佳
周宇波
程靖
严子琴
吕细林
张世言
周飞龙
刘晨
林芷彤
姜凯龙
徐德华
胡小蓓
Original Assignee
中国科学院上海药物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海药物研究所 filed Critical 中国科学院上海药物研究所
Publication of WO2024078370A1 publication Critical patent/WO2024078370A1/zh

Links

Classifications

    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • 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
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/10—Spiro-condensed systems

Definitions

  • the present invention belongs to the field of drug synthesis, and specifically relates to a compound having a substituted phenyl spiro[indoline-3,3'-pyrrolidine] structure, its stereoisomers, enantiomers or pharmaceutically acceptable salts thereof, and a preparation method and use thereof.
  • the tumor suppressor p53 exerts antiproliferative effects in response to various stresses, including cell growth arrest, DNA repair, and apoptosis. Mice lacking p53 develop normally but are susceptible to multiple tumors.
  • the TP53 gene encoding the p53 protein is mutated or deleted in nearly 50% of human cancers, rendering cells unable to function as a p53 tumor suppressor. Although p53 remains wild-type in the remaining 50% of human cancers, its function is inhibited by a variety of inhibitors. Studies have shown that two proteins are essential for the regulation of p53—MDM2 and MDMX (also known as MDM4).
  • MDM2 and MDMX The embryonic lethality caused by knocking out the MDM2 and MDMX genes can be rescued by knocking out the TP53 gene in mice at the same time, clarifying the role of MDM2 and MDMX as major negative endogenous regulators of p53.
  • MDM2 and MDMX inhibit the gene transcription function of the p53 protein by interacting with the transcription activation domain (TAD) of the p53 protein through their amino-terminal p53 binding domains.
  • TAD transcription activation domain
  • MDM2 can promote the ubiquitination and proteasomal degradation of MDM2 itself, MDMX, and p53 proteins.
  • MDM2 can also promote the transport of p53 protein out of the nucleus, making it impossible for p53 to contact its target DNA, thereby reducing its transcriptional ability.
  • MDMX although it cannot act as an E3 ubiquitin ligase to degrade like MDM2, it can form a stable heterodimer with the carboxyl-terminal RING domain of MDM2 through the carboxyl-terminal RING domain to promote MDM2-mediated p53 ubiquitination.
  • RG7112 NCT00559533, NCT00623870, NCT01677780, NCT01164033, NCT01605526, NCT01143740 and NCT01635296
  • RG7388 Ding et al., J Med Chem 2013, 56(14), 5979-83
  • MI-77301 NCT01636479 and NCT01985191
  • AMG 232 NCT01723020 and NCT02016729
  • WK298 (Popowicz et al., Cell Cycle 2010, 9(6), 1104-11), ATSP-7041 (Chang et al., Proc Natl Acad Sci U S A 2013, 110(36), E3445-54), RO-5963 (Graves et al., Proc Natl Acad Sci U S A 2012, 109(29), 11788-93) and ALRN-6924 (Carvajal et al., Sci Transl Med 2018, 10(436)) are compounds reported in the literature that can simultaneously inhibit the MDM2/p53 and MDMX/p53 interactions. Small molecule inhibitors that block the MDM2/p53 and MDMX/p53 interactions have the potential to treat related diseases.
  • the object of the present invention is to provide a small molecule inhibitor that blocks the MDM2/p53 and/or MDMX/p53 interaction.
  • Ar is a substituted or unsubstituted phenyl group, wherein the substitution means that one or more hydrogen atoms on the phenyl group are replaced by a group selected from the group consisting of halogen, deuterium, cyano, hydroxyl, amino, nitro, substituted or unsubstituted C2-C4 alkynyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C2-C10 acyl, substituted or unsubstituted C1-C4 alkylcarbonyl;
  • R1 and R2 are each independently hydrogen, deuterium, halogen, cyano, substituted or unsubstituted C2-C4 alkynyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C4 alkoxy;
  • Y and Z are each independently hydrogen, -(CH 2 ) m -substituted or unsubstituted 6-10 membered aryl, -(CH 2 ) m -substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted C1-C6 alkyl, -(CH 2 ) m -substituted or unsubstituted C3-C8 cycloalkyl, -(CH 2 ) m -substituted or unsubstituted 5-13 membered heterocyclyl;
  • R 4 is substituted or unsubstituted C1-C8 alkyl, -(CH 2 ) m -substituted or unsubstituted C3-C8 cycloalkyl, -(CH 2 ) m -substituted or unsubstituted 4-13 membered heterocyclyl, -(CH 2 ) m -substituted or unsubstituted 6-10 membered aryl, -(CH 2 ) m -substituted or unsubstituted 5-12 membered heteroaryl, C2-C8 alkynyl, C2-C8 alkenyl;
  • n is independently 0, 1, 2, 3 or 4 at each occurrence
  • R 5 is substituted or unsubstituted C1-C8 alkyl, -(CH 2 ) m -substituted or unsubstituted C3-C8 cycloalkyl, -(CH 2 ) m -substituted or unsubstituted 4-12 membered heterocycloalkyl;
  • each of the substitutions described above independently refers to the substitution of one or more hydrogen atoms on the group with a group selected from the group consisting of halogen, deuterium, cyano, hydroxy, amino, nitro, C1-C4 alkylsulfonyl, C1-C4 alkylsulfonamidocarbonyl (C1-C4 alkylSO 2 NHCO-), carboxyl, -CONH 2 , C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 alkoxy, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkylethynyl, mono(C1-C4 alkyl)amino, di(C1-C4 alkyl)amino, C1-C4 alkylcarbonylamino, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, C1-C4 al
  • Ar is a substituted or unsubstituted phenyl group, wherein the substitution refers to that 1, 2 or 3 hydrogen atoms on the phenyl group are replaced by groups selected from the group consisting of halogen, C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 alkoxy.
  • Ar is a substituted or unsubstituted phenyl group, wherein the substitution refers to that 1, 2 or 3 hydrogen atoms on the phenyl group are replaced by groups selected from the group consisting of fluorine, chlorine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy.
  • R 1 and R 2 are each independently hydrogen, deuterium, halogen, cyano, C1-C4 alkyl. In another preferred embodiment, R 1 and R 2 are each independently hydrogen, deuterium, halogen. In another preferred embodiment, R 1 and R 2 are each independently hydrogen, Deuterium, chlorine, fluorine.
  • R 3 is Y is hydrogen or C1-C4 alkyl; Z is -(CH 2 ) m -substituted or unsubstituted phenyl, -(CH 2 ) m -substituted or unsubstituted 5-7 membered heteroaryl, -(CH 2 ) m -substituted or unsubstituted C6-C8 cycloalkyl, -(CH 2 ) m -substituted or unsubstituted 5-8 membered heterocyclyl.
  • Z is -(CH 2 ) m -substituted or unsubstituted phenyl, -(CH 2 ) m -substituted or unsubstituted 6 membered heteroaryl, -(CH 2 ) m -substituted or unsubstituted C5-C8 cycloalkyl, -(CH 2 ) m -substituted or unsubstituted 6-8 membered heterocyclyl.
  • the above substitution means that 1, 2 or 3 hydrogen atoms on the group are substituted by a group selected from the group consisting of hydroxyl, carboxyl, C1-C4 alkoxy, halogen, amino, deuterium, C1-C4 alkyl, -CONH 2 , C1-C4 alkylaminoCO-, cyano, C1-C4 alkyl substituted with carboxyl, C1-C4 alkyl-CO-O-C1-C4 alkylene-O-CO-.
  • m is 0, 1, 2 or 3.
  • R4 is hydrogen, substituted or unsubstituted C1-C6 alkyl, -( CH2 ) m -substituted or unsubstituted C3-C6 cycloalkyl, -( CH2 ) m -substituted or unsubstituted 4-6 membered heterocyclyl, -( CH2 ) m -substituted or unsubstituted phenyl, -( CH2 ) m -substituted or unsubstituted 5-7 membered heteroaryl, C3-C6 alkynyl, C3-C6 alkenyl.
  • m is 0, 1, 2 or 3.
  • substitution means that 1, 2 or 3 hydrogen atoms on the group are replaced by a group selected from the group consisting of halogen, deuterium, cyano, hydroxyl, C1-C4 alkoxycarbonyl, C1-C4 alkyl, C2-C4 alkynyl, C2-C4 alkenyl.
  • R 5 is substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-6 membered heterocycloalkyl; in another preferred embodiment, the above substitution means that 1, 2 or 3 hydrogen atoms on the group are replaced by a group selected from the following group: C1-C4 alkyl, C1-C4 alkylCO-, C1-C4 alkylCOO-.
  • R 5 is C1-C6 alkyl, C1-C4 alkyl substituted C3-C6 cycloalkyl, C1-C4 alkyl and/or C1-C4 alkyl CO-substituted 4-6 membered heterocycloalkyl.
  • R 5 is tert-butyl
  • the compound has a structure as shown in Formula II, III, IV, V, VI, VII or VIII:
  • each substituent is a corresponding group in a specific compound.
  • heterocyclic group and heteroaryl group each independently contain 1, 2, 3 or 4 heteroatoms selected from N and O.
  • the compound is selected from any one of compounds Nos. 1-97.
  • the compound provided by the present invention can be used as a small molecule inhibitor for inhibiting the protein-protein interaction of MDM2-p53 and MDMX-p53 proteins.
  • the second aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound as described in the first aspect, one or more of its enantiomers, diastereomers, racemates or pharmaceutically acceptable salts; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition optionally further comprises a pharmaceutically acceptable excipient, which is selected from the following group: adhesives, fillers, diluents, disintegrants, suspending agents, suspending aids, sustained (controlled) release agents, lyoprotectants, coating agents, enteric materials, lubricants, glidants, anti-adherents, sweeteners, flavoring agents, plasticizers, opacifiers, solubilizers, humectants, solvents, osmotic pressure regulators, colorants, pigments, surfactants, emulsifiers, water-soluble matrices, fat-soluble matrices, oily matrices, pore-forming agents, gelling agents, preservatives, buffers, chelating agents, antioxidants, or a combination thereof.
  • a pharmaceutically acceptable excipient which is selected from the following group: adhesives, fillers, diluents, disintegrants, suspending agents, suspending aids, sustained (controlled) release agents,
  • the third aspect of the present invention provides the use of the compound described in the first aspect, its enantiomers, diastereomers, racemates or pharmaceutically acceptable salts thereof for preparing small molecule inhibitors that block the MDM2/p53 and/or MDMX/p53 interaction; or for preparing drugs for treating diseases related to the activity or expression of MDM2 or MDMX protein.
  • the disease associated with the activity or expression of MDM2 or MDMX protein is selected from the group consisting of glioma, liposarcoma, skin melanoma, squamous cell carcinoma, retinoblastoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, liver cancer, soft tissue sarcoma, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, osteosarcoma and colon cancer.
  • the main advantages of the present invention include:
  • a novel class of small molecule compounds having a substituted phenylspiro[indoline-3,3'-pyrrolidine] structure and similar structures is provided.
  • the preparation method thereof has the advantages of mild reaction conditions, abundant and readily available raw materials, simple operation and post-treatment, and good enantioselectivity.
  • a small molecule inhibitor that simultaneously inhibits MDM2-p53 and MDMX-p53 protein-protein interactions.
  • This type of inhibitor has a strong proliferation inhibition ability against p53 wild-type and MDM2 overexpressing cells, and has good solubility, high bioavailability, and excellent metabolic properties. It is a potential anti-tumor drug.
  • FIG1 shows the absolute stereo configuration of JM085-CF2.
  • FIG. 2 shows the absolute stereo configuration of JN110.
  • FIG3 is a standard curve of compound concentration-peak area ratio.
  • FIG. 4 shows the results of a study on the mechanism of action of 18(JN122) in HCT116 cells.
  • FIG5 shows the results of the study on the mechanism of action of 18 (JN122) in various solid tumor cancer cell lines
  • FIG. 6 shows the results of a study on the mechanism of action of 18(JN122) in MOLM-13 cells.
  • FIG. 7 shows the results of a pharmacodynamic study of 18(JN122) in a MOLM-13 mouse xenograft tumor model.
  • the inventors of the present invention unexpectedly developed small molecule compounds with substituted phenyl spiro[indoline-3,3'-pyrrolidine] structures and similar structures, which can inhibit MDM2-p53 and MDMX-p53 protein-protein interactions and regulate p53-mediated gene expression in tumor cells, and can therefore be used to inhibit diseases related to MDM2-p53 and MDMX-p53 interactions, such as the prevention and treatment of cancer.
  • the inventors completed the present invention.
  • the halogen is F, Cl, Br or I.
  • C 1 -C 6 refers to having 1, 2, 3, 4, 5 or 6 carbon atoms
  • C 1 -C 4 refers to having 1, 2, 3 or 4 carbon atoms
  • 4-12 membered refers to having 4, 5, 6, 7, 8, 9, 10, 11 or 12 ring atoms, and so on.
  • alkyl refers to a saturated linear or branched hydrocarbon moiety.
  • C1 - C8 alkyl refers to a straight or branched alkyl group having 1 to 8 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl, etc.; preferably ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.
  • alkoxy refers to an -O-(alkyl) group.
  • C 1 -C 6 alkoxy refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy and butoxy.
  • alkenyl refers to a straight or branched hydrocarbon moiety containing at least one double bond.
  • C2 - C6 alkenyl refers to a straight or branched alkenyl group containing one double bond having 2 to 6 carbon atoms, including but not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl and hexenyl.
  • alkynyl refers to a straight or branched alkynyl group containing a triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl (2-methyl 3-butynyl, 2-pentynyl, 3-pentynyl) and hexynyl, etc.
  • cycloalkyl refers to a saturated monocyclic, bridged or spirocyclic hydrocarbon moiety.
  • C 3 -C 8 cycloalkyl refers to a monocyclic, bridged or spirocyclic alkyl group having 3 to 8 carbon atoms in the ring. Including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclodecyl etc.
  • bridged ring group includes, but is not limited to, bicyclo[1,1,1]pentyl, bicyclo[2,1,1]hexyl, bicyclo[2,2,1]heptyl, bicyclo[2,2,2]octyl, bicyclo[3,2,2]octyl etc.
  • aryl refers to a hydrocarbon moiety containing one or more aromatic rings.
  • C 6 -C 10 aryl refers to an aromatic ring group having 6 to 10 carbon atoms without heteroatoms in the ring, such as phenyl, naphthyl, etc.
  • heterocyclyl refers to a saturated or unsaturated, non-aromatic cyclic group containing at least one (such as 1, 2, 3 or 4) ring heteroatom (such as N, O or S), such as tetrahydropyridyl, pyrrolinyl, dihydropyridyl, dihydrofuranyl, dihydrothiophenyl, morpholinyl.
  • heteroaryl refers to an aromatic cyclic group containing at least one (such as 1, 2, 3 or 4) ring heteroatoms (such as N, O or S), for example furanyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, quinolyl, isoquinolyl, indolyl, pyrimidinyl, pyranyl.
  • the compound of the present invention has a structure as shown in general formula I:
  • the compound has a structure shown in the following formula I-1:
  • Ar is a substituted or unsubstituted phenyl group, wherein the substitution means that 1, 2, 3 or 4 hydrogen atoms on the phenyl group are replaced by a group selected from the group consisting of fluorine, chlorine, bromine, methoxy, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, halogenated C1-C4 alkyl (such as trifluoromethyl); R1 and R2 are each independently hydrogen, deuterium, fluorine, chlorine or bromine;
  • R3 is OH or Y is H;
  • Z is H, substituted or unsubstituted C1-C4 alkyl, -(CH 2 ) m -substituted or unsubstituted phenyl, -(CH 2 ) m -substituted or unsubstituted 5-7 membered heteroaryl, -(CH 2 ) m -substituted or unsubstituted C3-C8 cycloalkyl, or -(CH 2 ) m -substituted or unsubstituted 5-7 membered heterocyclyl; or Y, Z and N form a substituted or unsubstituted 5-7 membered heterocyclyl; the substitution refers to substitution by 1, 2, 3 or 4 groups selected from the group consisting of fluorine, chlorine, bromine, amino, hydroxyl, carboxyl, C1-C4 alkoxy, -CONH 2 , C1-C4 al
  • R 4 is substituted or unsubstituted C1-C4 alkyl, -(CH 2 ) m -substituted or unsubstituted phenyl, -(CH 2 ) m -substituted or unsubstituted substituted C3-C6 cycloalkyl; each m is independently 0, 1 or 2; the substitution refers to substitution by 1, 2, 4 or 3 groups selected from the group consisting of fluorine, chlorine, bromine, cyano, amino, hydroxyl, nitro, carboxyl, C1-C4 alkoxy, -CONH 2 , C1-C4 alkyl-CONH-, C1-C4 alkyl-NHCO-, C1-C4 alkyl, C1-C4 alkyl-S(O 2 )-C1-C4 alkylene-, C1-C4 alkylSO 2 -, 5-7 membered heteroaryl, C1-C4 alkyl substituted with carboxy
  • R 5 is substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 5-7 membered heterocyclyl; the substitution refers to substitution with one or more groups selected from the following group: C1-C4 alkyl, C1-C4 alkylcarbonyl.
  • the heteroaryl group in the present invention is selected from the group consisting of tetrazolyl, isoxazolyl, oxazolyl, pyridyl, imidazolyl and pyrazolyl.
  • heteroaryl group in the present invention is selected from:
  • heterocyclic group in the present invention is selected from:
  • the compounds of the present invention can be prepared through the following reaction routes.
  • Step 1 Aldehyde S1 and substituted 2-fluorophenylacetonitrile S2 are reacted in an appropriate solvent (e.g., methanol, ethanol, tetrahydrofuran, 2-
  • an appropriate solvent e.g., methanol, ethanol, tetrahydrofuran
  • 2- Step 2 S3 is mixed with the corresponding raw material S4-2 in an appropriate solvent (for example, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or N, N-dimethylformamide, etc.), and reacted at room temperature or at an appropriately elevated temperature (for example, 40-60°C) to obtain the intermediate S3.
  • an appropriate solvent for example, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or N, N-dimethylformamide, etc.
  • Step 2 S3 is mixed with the corresponding raw material S4-2 in an appropriate solvent (for example, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.), and silver fluoride and a base (triethylamine, N, N-diisopropylethylamine and DBU, etc.) are added to react at room temperature to obtain the intermediate S5-2.
  • Step 3 S5-2 is subjected to a hydrogenation reduction reaction to obtain the intermediate S6-2 (for example, Pd/C hydrogen hydrogenation reduction, Raney nickel hydrogen hydrogenation reduction, and Raney nickel hydrazine hydrate hydrogenation reduction, etc.).
  • an appropriate solvent for example, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.
  • silver fluoride and a base triethylamine, N, N-diisopropylethylamine and DBU, etc
  • Step 4 S6-2 and FmocCl are mixed in a suitable solvent (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile), a base (e.g., triethylamine, N,N-diisopropylethylamine, DBU, etc.) is added, and the reaction is carried out at room temperature to obtain the intermediate S7-2.
  • a suitable solvent e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile
  • a base e.g., triethylamine, N,N-diisopropylethylamine, DBU, etc.
  • S7-2 is dissolved in dichloromethane, trifluoroacetic acid is added, and the reaction is carried out at room temperature to obtain the intermediate S8-2.
  • Step 6 S8-2 is dissolved in a suitable solvent (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N,N-dimethylformamide, or acetonitrile), a base (e.g., triethylamine, N,N-diisopropylethylamine, DBU, etc.) and a condensation reagent (e.g., diphenylphosphinoyl chloride, CDI, PyBOP, HATU, EDCI, etc.) are added, and the reaction is carried out for half an hour, and an amine is added, and the reaction is carried out at room temperature to obtain the intermediate S9-2.
  • a suitable solvent e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N,N-dimethylformamide, or acetonitrile
  • a base e.g., triethylamine,
  • Step 7 S9-2 and R 4 CHO are dissolved in a suitable solvent (e.g., methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N,N-dimethylformamide, etc.), a reducing agent (sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride, etc.) is added, and the intermediate S10-2 is obtained by reductive amination reaction.
  • Step 8 S10-2 is dissolved in a suitable solvent (e.g., N,N-dimethylformamide, etc.), piperidine is added, and the reaction is carried out at room temperature to obtain the intermediate S11-2.
  • a suitable solvent e.g., methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N,N-dimethylformamide, etc.
  • a suitable solvent e.g., methanol, tetrahydrofuran, ethanol, 1,2-
  • Step 9 S11-2 is dissolved in a suitable solvent (e.g., acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene and benzene, etc.), a base (e.g., potassium carbonate, cesium carbonate and sodium carbonate, etc.) is added, and the reaction is carried out at an elevated temperature (e.g., 80-120°C) to obtain the final product I.
  • a suitable solvent e.g., acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene and benzene, etc.
  • a base e.g., potassium carbonate, cesium carbonate and sodium carbonate, etc.
  • Step 1 Aldehyde S1 and substituted 2-fluorophenylacetonitrile S2 are mixed in a suitable solvent (e.g., methanol, ethanol, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or N, N-dimethylformamide, etc.). Add a suitable base (sodium methoxide, ethanol Sodium, etc.), react at room temperature or at an appropriately elevated temperature (e.g., 40-60°C) to obtain intermediate S3.
  • a suitable solvent e.g., methanol, ethanol, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or N, N-dimethylformamide, etc.
  • a suitable base sodium methoxide, ethanol Sodium, etc.
  • Step 2 S3 is mixed with the corresponding raw material S4 in an appropriate solvent (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.), silver fluoride and a base (triethylamine, N,N-diisopropylethylamine and DBU, etc.) are added, and the intermediate S5 is reacted at room temperature.
  • Step 3 S5 is subjected to a hydrogenation reduction reaction to obtain intermediate S6 (e.g., Pd/C hydrogen hydrogenation reduction, Raney nickel hydrogen hydrogenation reduction, and Raney nickel hydrazine hydrate hydrogenation reduction, etc.).
  • an appropriate solvent e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.
  • silver fluoride and a base triethylamine, N,N-diisopropylethyl
  • Step 4 S6 is mixed with FmocCl in an appropriate solvent (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.), a base (triethylamine, N,N-diisopropylethylamine and DBU, etc.) is added, and the intermediate S7 is reacted at room temperature.
  • an appropriate solvent e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.
  • a base triethylamine, N,N-diisopropylethylamine and DBU, etc.
  • Step 6 S8 is dissolved in an appropriate solvent (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N, N-dimethylformamide or acetonitrile, etc.), a base (triethylamine, N, N-diisopropylethylamine and DBU, etc.) and a condensation reagent (diphenylphosphinoyl chloride, CDI, PyBOP, HATU and EDCI, etc.) are added, and an amine is added after reacting for half an hour, and the reaction is carried out at room temperature to obtain intermediate S9.
  • an appropriate solvent e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N, N-dimethylformamide or acetonitrile, etc.
  • a base triethylamine, N, N-diisopropylethylamine and DBU, etc.
  • Step 7 S9 and R 4 CHO are dissolved in an appropriate solvent (e.g., methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N, N-dimethylformamide, etc.), a reducing reagent (sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride, etc.) is added, and intermediate S10 is obtained by reductive amination reaction.
  • Step 8 S10 is dissolved in a suitable solvent (e.g., N,N-dimethylformamide, etc.), piperidine is added, and the reaction is carried out at room temperature to obtain the intermediate S11.
  • an appropriate solvent e.g., methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N, N-dimethylformamide, etc.
  • a reducing reagent sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride, etc.
  • Step 9 S11 is dissolved in a suitable solvent (e.g., acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene and benzene, etc.), a base (e.g., potassium carbonate, cesium carbonate and sodium carbonate, etc.) is added, and the reaction is carried out at an elevated temperature (e.g., 80-120°C) to obtain the final product II.
  • a suitable solvent e.g., acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene and benzene, etc.
  • a base e.g., potassium carbonate, cesium carbonate and sodium carbonate, etc.
  • Step 1 S9 is dissolved in a suitable solvent (e.g., N,N-dimethylformamide, etc.), piperidine is added, and the reaction is carried out at room temperature to obtain intermediate S12.
  • Step 2 S12 and p-methoxybenzaldehyde are dissolved in a suitable solvent (e.g., methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N,N-dimethylformamide, etc.), a reducing agent (sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride, etc.) is added, and intermediate S13 is obtained by reductive amination reaction.
  • a suitable solvent e.g., N,N-dimethylformamide, etc.
  • a suitable solvent e.g., methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N,N-dimethylformamide, etc.
  • Step 3 S13 is dissolved in a suitable solvent (e.g., acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene and benzene, etc.), a base (e.g., potassium carbonate, cesium carbonate and sodium carbonate, etc.) is added, and the reaction is carried out at an elevated temperature (e.g., 80-120°C) to obtain intermediate S14.
  • a suitable solvent e.g., acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene and benzene, etc.
  • a base e.g., potassium carbonate, cesium carbonate and sodium carbonate, etc.
  • Step 4 S14 and R 4 CHO are dissolved in a suitable solvent (for example, methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N,N-dimethylformamide, etc.), and a reducing agent (sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride, etc.) is added to obtain intermediate S15 through reductive amination reaction.
  • a suitable solvent for example, methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N,N-dimethylformamide, etc.
  • a reducing agent sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride, etc.
  • I, I-1 can be synthesized using the above three synthesis strategies.
  • Step 1 S3 is mixed with the corresponding raw material S4 in a suitable solvent (for example, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.), and after removing oxygen and replacing with nitrogen, cuprous acetate and R-(+)-1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) are added, and a base (triethylamine, N,N-diisopropylethylamine and DBU, etc.) is added dropwise.
  • a suitable solvent for example, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.
  • the bisphosphine ligand can also be R-(+)-1,1'-binaphthyl-2,2'-bis(4-methylphenyl)phosphine, R-(+)-1,1'-binaphthyl-2,2'-bis(3,5-dimethylphenyl)phosphine, R-(+)-1,1'-binaphthyl-2,2'-bis(4-iodophenyl)phosphine, R-(+)-1,1'-binaphthyl-2,2'-bis(4-methoxyphenyl)phosphine, (R)-(+)-2,2'-bis(diphenylphosphino)-5,5',6,6',7,7',8,8'-octa
  • Step 2 S16 is subjected to hydrogenation reduction reaction to obtain intermediate S17 (such as Pd/C hydrogenation reduction, Raney nickel hydrogenation reduction, and Raney nickel hydrazine hydrate hydrogenation reduction).
  • Step 3 S17 is mixed with FmocCl in a suitable solvent (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile), a base (e.g., triethylamine, N,N-diisopropylethylamine, and DBU) is added, and the intermediate S18 is obtained by reaction at room temperature.
  • a suitable solvent e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile
  • a base e.g., triethylamine, N,N-diisopropylethylamine, and DBU
  • Step 4 S18 and R 4 CHO are dissolved in a suitable solvent (e.g., methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane, and N,N-dimethylformamide), a reducing agent (e.g., sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride, etc.) is added, and the intermediate S19 is obtained by reductive amination reaction.
  • Step 5 S19 is dissolved in dichloromethane, trifluoroacetic acid is added, and the intermediate S20 is obtained by reaction at room temperature.
  • Step 6 S20 is dissolved in a suitable solvent (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N, N-dimethylformamide or acetonitrile, etc.), and a base (1-methylimidazole, triethylamine, N, N-diisopropylethylamine and DBU, etc.) and a condensation reagent (ethylsulfonyl chloride, diphenylphosphinoyl chloride, CDI, PyBOP, HATU and EDCI, etc.), add amine after half an hour of reaction, and react at room temperature to obtain intermediate S21.
  • a suitable solvent e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N, N-dimethylformamide or acetonitrile, etc.
  • a base 1-methylimidazole, triethylamine, N,
  • Step 7 S21 is dissolved in an appropriate solvent (for example: N, N-dimethylformamide, etc.), piperidine is added, and react at room temperature to obtain intermediate S22.
  • Step 8 S22 is dissolved in an appropriate solvent (for example: acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide, toluene and benzene, etc.), a base (for example: potassium carbonate, cesium carbonate and sodium carbonate, etc.) is added, and react at an elevated temperature (for example 80-120°C) to obtain the final product II, which is a single enantiomer or a mixture of enantiomers with ee>50%.
  • an appropriate solvent for example: N, N-dimethylformamide, etc.
  • a base for example: potassium carbonate, cesium carbonate and sodium carbonate, etc.
  • S23 to S29 are mixtures of optically active enantiomers with ee>20%
  • Step 1 S3 is mixed with the corresponding raw material S4-2 in a suitable solvent (for example, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.), and after removing oxygen and replacing with nitrogen, cuprous acetate and R-(+)-1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) are added, and a base (triethylamine, N,N-diisopropylethylamine and DBU, etc.) is added dropwise.
  • a suitable solvent for example, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile, etc.
  • the bisphosphine ligand can also be R-(+)-1,1'-binaphthyl-2,2'-bis(4-methylphenyl)phosphine, R-(+)-1,1'-binaphthyl-2,2'-bis(3,5-dimethylphenyl)phosphine, R-(+)-1,1'-binaphthyl-2,2'-bis(4-iodophenyl)phosphine, R-(+)-1,1'-binaphthyl-2,2'-bis(4-methoxyphenyl)phosphine, (R)-(+)-2,2'-bis(diphenylphosphino)-5,5',6,6',7,7',8,8'-octa
  • Step 2 S23 is subjected to hydrogenation reduction reaction to obtain intermediate S24 (such as Pd/C hydrogenation reduction, Raney nickel hydrogenation reduction, and Raney nickel hydrazine hydrate hydrogenation reduction).
  • Step 3 S24 is mixed with FmocCl in a suitable solvent (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile), a base (e.g., triethylamine, N,N-diisopropylethylamine, DBU, etc.) is added, and the reaction is carried out at room temperature to obtain the intermediate S25.
  • a suitable solvent e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or acetonitrile
  • a base e.g., triethylamine, N,N-diisopropylethylamine, DBU, etc.
  • Step 4 S25 and R 4 CHO are dissolved in a suitable solvent (e.g., methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N,N-dimethylformamide, etc.), and a reducing agent (sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride, etc.) is added to obtain intermediate S26 through a reductive amination reaction.
  • a suitable solvent e.g., methanol, tetrahydrofuran, ethanol, 1,2-dichloroethane and N,N-dimethylformamide, etc.
  • a reducing agent sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride, etc.
  • Step 6 S27 is dissolved in a suitable solvent (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N,N-dimethylformamide or acetonitrile, etc.), and a base (1-methylimidazole, triethylamine, N,N-diisopropylethylamine and DBU, etc.) and a condensation agent (ethylsulfonyl chloride, diphenylphosphinoyl chloride, CDI, PyBOP, HATU and EDCI, etc.) are added at zero degrees Celsius. After reacting for half an hour, an amine is added, and the reaction is carried out at room temperature to obtain intermediate S28.
  • a suitable solvent e.g., tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N,N-dimethylformamide or acetonitrile, etc.
  • a base 1-methylimi
  • Step 7 S28 is dissolved in a suitable solvent (e.g., N,N-dimethylformamide, etc.), piperidine is added, and the reaction is carried out at room temperature to obtain the intermediate S29.
  • Step 8 S29 is dissolved in a suitable solvent (e.g., acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene and benzene, etc.), a base (e.g., potassium carbonate, cesium carbonate and sodium carbonate, etc.) is added, and the reaction is carried out at an elevated temperature (e.g., 80-120° C.) to obtain the final product I, which is a single enantiomer or a mixture of enantiomers with ee>20%.
  • a suitable solvent e.g., N,N-dimethylformamide, etc.
  • a base e.g., potassium carbonate, cesium carbonate and sodium carbonate, etc.
  • Synthetic intermediate 1 4-((2′,3S,4′,5′-R)-6-chloro-4-((2,3-difluorophenyl)-1-(4-methoxybenzyl)-2-ylbenzyl neopentylspiro[indoline-3,3′-pyrrolidine]-5′carboxamide)-3-methoxybenzoic acid methyl ester (JM158)
  • Step 1 Synthesis of (Z)-2-(4-chloro-2-fluorophenyl)-3-(2,3-difluorophenyl)acrylonitrile (JM029)
  • 2,3-Difluorobenzaldehyde (2.9g, 20mmol) and 4-chloro-2-fluorobenzeneacetonitrile (3.4g, 20mmol) were weighed into a 250mL round-bottom flask, 150mL of methanol was added to dissolve the reactants, 4.8mL of 5N sodium methoxide methanol solution was added dropwise, and the reaction system was stirred overnight at 50°C. After the reaction was completed, the mixture was cooled to room temperature and filtered to obtain 5.8g of crude product with a yield of 99%.
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(2,3-difluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JM036)
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(2,3-difluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JM040)
  • JM036 (1.6 g, 8.5 mmol) was weighed into a 100 mL round-bottom flask, dissolved with tetrahydrofuran/EtOH (30 mL/10 mL), heated to 55 °C, 4 g of Raney nickel and 10 mL of hydrazine hydrate were added, reacted for 2 h, filtered, the solvent was dried, and purified by normal phase column to obtain 1.6 g of the target product with a yield of 24%.
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-(((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(2,3-difluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (JM048)
  • JM040 (1.4 g, 2.7 mmol) was weighed into a 50 mL single-mouth bottle, dissolved in dry tetrahydrofuran, and diisopropylethylamine (1.4 g, 10.8 mmol) and FmocCl (1.1 g, 4.1 mmol) were added, and the reaction was allowed to react overnight at room temperature.
  • the reactants were dried under reduced pressure, dissolved in 5 mL of dichloromethane, and 4 mL of trifluoroacetic acid was added, and the reaction was allowed to react overnight at room temperature.
  • the reaction solution was dried, saturated sodium bicarbonate solution was added, and the reaction solution was extracted three times with dichloromethane.
  • the organic phases were combined, dried, and purified by normal phase column to obtain 1.31 g of the target product with a yield of 73%.
  • Step 5 Synthesis of methyl 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(2,3-difluorophenyl)-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoate (JM148)
  • JM048 (515 mg, 0.8 mmol) was added to a 50 mL single-mouth bottle, dissolved in dry tetrahydrofuran, and diisopropylethylamine (504 mg, 3.9 mmol) was added. The mixture was stirred for 5 minutes, and diphenylphosphinoyl chloride (555 mg, 2.3 mmol) was added. After stirring for half an hour, methyl 4-amino-3-methoxybenzoate (562 mg, 3.1 mmol) was added. The mixture was reacted at room temperature overnight. Saturated sodium bicarbonate solution was added to the reaction solution, extracted with dichloromethane, and the organic phase was dried and purified by normal phase column.
  • the crude product was dissolved in DMF (2 mL), and piperidine (0.4 mL) was added. The mixture was reacted at room temperature for 15 minutes, washed with 1N HCl solution 3 times, washed with saturated sodium chloride solution 3 times, and the organic phase was dried and purified by normal phase column to obtain 285 mg of the target product with a yield of 73%.
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(2,3-difluorophenyl)-4-fluoro-1-(4-methoxybenzyl)-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid methyl ester (JM158)
  • Synthetic intermediate 2 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(3-chloro-2-fluorophenyl)-1-(4-methoxybenzyl)-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid methyl ester (YM155)
  • Step 1 Synthesis of methyl 4-((2R,3S,4S,5S)-4-(aminomethyl)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (YI045)
  • Step 2 Synthesis of methyl 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(3-chloro-2-fluorophenyl)-1-(4-methoxybenzyl)-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoate (YM155)
  • Step 1 Synthesis of enantiomers (2R,3S,4S,5S)-4-(aminomethyl)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester and enantiomers (2S,3R,4R,5R)-4-(aminomethyl)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YM159)
  • SM1 (5.45 g, 1.04 mmol) was weighed into a 100 mL round-bottom flask, and 2 g of Raney Nickel was added. The mixture was dissolved in tetrahydrofuran/ethanol and heated to 55° C. Then 10 mL of hydrazine hydrate was added and the mixture was reacted until no bubbling occurred. The filtrate was filtered and dried, and purified by normal phase column to obtain 1.9 g of the target compound with a yield of 36%.
  • Step 2 Synthesis of the epimer (2R, 3S, 4S, 5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-(((R)-2-methoxy-2-phenylacetamide)methyl)-5-neopentylpyrrolidine-2-carboxylic acid and the epimer (2S, 3R, 4R, 5R)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-(((R)-2-methoxy-2-phenylacetamide)methyl)-5-neopentylpyrrolidine-2-carboxylic acid (YN003)
  • YM159 (210 mg, 0.4 mmol) was dissolved in dichloromethane, (R)-(-)-alpha-methoxyphenylacetyl chloride (110 mg, 0.6 mmol) and triethylamine (162 mg, 1.6 mmol) were added, and the mixture was stirred at room temperature overnight. After the reaction, water was added, and the mixture was extracted with dichloromethane. The organic phase was dried and purified with a normal phase column to obtain a crude product. The crude product was dissolved in dichloromethane (5 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at room temperature overnight. After the reaction, water was added, and the mixture was extracted with dichloromethane. The organic phase was dried and purified with a normal phase column to obtain 226 mg of the target compound, with a yield of 91%.
  • Step 3 Synthesis of (2S, 3R, 4R, 5R)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-((R)-2-methoxy-2-phenylacetamide)methyl)-5-neopentyl-N-((S)-1-phenylethyl)pyrrolidine-2-carboxamide (YN015-CF1) and (2R, 3S, 4S, 5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-((R)-2-methoxy-2-phenylacetamide)methyl)-5-neopentyl-N-((S)-1-phenylethyl)pyrrolidine-2-carboxamide (YN015-CF2)
  • YN003 (226 mg, 0.36 mmol) was weighed into a bottle, dissolved in tetrahydrofuran, and diisopropylethylamine (232 mg, 1.8 mmol) and diphenylphosphinoyl chloride (255 mg, 1.08 mmol) were added. After stirring at room temperature for 30 min, (S)-1-phenylethylamine (176 mg, 1.46 mmol) was added and reacted at room temperature overnight. Water was added, extracted with dichloromethane, and the organic phase was spin-dried and purified with a normal phase column to obtain a mixture of 200 mg of the target compound with a yield of 77%. The above operation was repeated to obtain a total of 400 mg of the mixture.
  • Step 4 Synthesis of (2S,3R,4R,5R)-4-(aminomethyl)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (YN16-CF1)
  • YN015-CF1 (383 mg, 0.5 mmol) was dissolved in 10 mL of concentrated hydrochloric acid, and ethanol was added and refluxed for 18 h. After the reaction was completed, 187 mg of trifluoroacetate of YN16-CF1 was purified by HPLC with a yield of 75%.
  • Step 5 Synthesis of (2S,3R,4R,5R)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-(((4-methoxybenzyl)amino)methyl)-5-neopentylpyrrolidine-2-carboxylic acid (YN18-CF1)
  • Step 6 Synthesis of methyl 4-((2S,3R,4R,5R)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-(((4-methoxybenzyl)amino)methyl)-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoate (YN20-CF1)
  • YN18-CF1 (133 mg, 0.22 mmol) was weighed into a bottle, dissolved in tetrahydrofuran, and diisopropylethylamine (144 mg, 1.12 mmol) and diphenylphosphinoyl chloride (156 mg, 0.66 mmol) were added. After stirring at room temperature for 30 min, 3-methoxy-4-aminobenzoic acid methyl ester (163 mg, 0.9 mmol) was added and reacted at room temperature overnight. Water was added and extracted with dichloromethane. The organic phase was spin-dried and purified with a normal phase column to obtain the crude product of the target compound YN20-CF1, which was directly used in the next step.
  • Step 1 Synthesis of (2R,3S,4S,5S)-4-(aminomethyl)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (YN17-CF2)
  • step 4 of the synthetic method of YN16-CF1, YN015-CF2 was used as the raw material to obtain 203 mg of trifluoroacetate of YN17-CF2 with a yield of 81%.
  • Step 2 Synthesis of (2R,3S,4S,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-(((4-methoxybenzyl)amino)methyl)-5-neopentylpyrrolidine-2-carboxylic acid (YN19-CF2)
  • step 5 of the synthesis method of YN18-CF1, YN17-CF2 was used as the raw material to obtain 181 mg of trifluoroacetate of YN19-CF2 with a yield of 60%.
  • Step 3 Synthesis of methyl 4-((2R,3S,4S,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-(((4-methoxybenzyl)amino)methyl)-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoate (YN22-CF2)
  • step six of the synthetic method of YN20-CF1, YN19-CF2 was used as the raw material to obtain the crude product of YN22-CF2.
  • YM155 40 mg, 0.054 mmol
  • formaldehyde 41 mg, 0.5 mmol
  • sodium acetate borohydride 106 mg, 0.5 mmol
  • 0.1 mL acetic acid weighed into a 50 mL round bottom flask, dissolved in 1,2-dichloroethane, and left at room temperature overnight.
  • reaction steps refer to the final product 1, the target product 20.2 mg, yield 47%.
  • reaction steps refer to the final product 1, the target product 5 mg, yield 11%.
  • reaction steps refer to the final product 1, the target product 9.3 mg, yield 21%.
  • reaction steps refer to the final product 1, the target product 10.3 mg, yield 16%.
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (TA046)
  • reaction steps refer to step 2 of intermediate 1, and the target product 3.16 g is obtained with a yield of 33.5%.
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(2-fluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (TA048)
  • reaction steps refer to step 3 of intermediate 1, and the target product 0.67 g is obtained with a yield of 21%.
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-(((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(2-fluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (TA051)
  • reaction steps refer to step 4 of intermediate 1, to obtain 0.36 g of the target product, the yield is 40.6%.
  • Step 5 Synthesis of methyl 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(2-fluorophenyl)-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoate (JN015)
  • reaction steps refer to step 5 of intermediate 1, and the target product 56 mg is obtained with a yield of 25%.
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(2,3-difluorophenyl)-4-fluoro-1-(4-methoxybenzyl)-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid methyl ester (JN020)
  • Step 7 Synthesis of 4-((2'S,3S,4'R,5'R)-6-chloro-4'-(2,3-difluorophenyl)-2'-neopentylspiro[dihydroindole-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid (JN22)
  • Step 1 Synthesis of (Z)-2-(4-chloro-2-fluorophenyl)-3-(3-fluorophenyl)acrylonitrile (JM075)
  • reaction steps refer to step 1 of intermediate 1, and 5.3 g of the target product is obtained with a yield of 96%.
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JM083)
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-fluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JM087)
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-(((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-fluorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (JM093)
  • Step 5 4-(2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-1-ethyl-3-(3-fluorophenyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JM153)
  • JM093 (131 mg, 0.2 mmol) was added into a 50 mL single-mouth bottle, dissolved in dry tetrahydrofuran, and diisopropylethylamine (129 mg, 1.0 mmol) was added, stirred for 5 minutes, and diphenylphosphinoyl chloride (143 mg, 0.6 mmol) was added. After stirring for half an hour, methyl 4-amino-3-methoxybenzoate (147 mg, 4.0 mmol) was added, and the mixture was reacted at room temperature overnight. Saturated sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted with dichloromethane.
  • the organic phase was dried and purified with a normal phase column to obtain 116 mg of a crude product.
  • 116 mg of crude product, acetaldehyde (47 mg, 1.1 mmol), sodium acetate borohydride (234 mg, 1.1 mmol) and 0.1 mL of acetic acid were weighed into a 50 mL round-bottom flask, dissolved in 1,2-dichloroethane, left at room temperature overnight, added with saturated sodium bicarbonate solution, extracted with ethyl acetate, the organic phase was spin-dried, the crude product was dissolved in DMF (2 mL), piperidine (0.4 mL) was added, reacted at room temperature for 15 minutes, water was added, extracted with ethyl acetate, the organic phase was spin-dried and purified by normal phase column to obtain 32 mg of the target product with a yield of 38%.
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(3-fluorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JM159)
  • Step 1 Synthesis of (Z)-2-(4-chloro-2-fluorophenyl)-3-(2,3-dichlorophenyl)acrylonitrile (YI116)
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(2,3-dichlorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YI124-1)
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(2,3-dichlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YI124-2)
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-(((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(2,3-dichlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (YI126)
  • reaction steps refer to step 4 of intermediate 1, and the target product 1.08 g is obtained with a yield of 80%.
  • Step 5 4-(2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-1-ethyl-3-(2,3-dichlorophenyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JN012)
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(2,3-dichlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JN17)
  • Step 1 Synthesis of (Z)-2-(4-chloro-2-fluorophenyl)-3-(4-chlorophenyl)acrylonitrile (YI037)
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(4-chlorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YI040)
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(4-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YI051)
  • reaction steps refer to step 3 of intermediate 1, to obtain 584 mg of the target product, yield 37%.
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-((((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(4-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (YI054)
  • reaction steps refer to step 4 of intermediate 1, and the target product 722 mg is obtained with a yield of 95%.
  • Step 5 4-(2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-1-ethyl-3-(4-chlorophenyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JM155)
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(4-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JN01)
  • Step 1 Synthesis of (Z)-2-(4-chloro-2-fluorophenyl)-3-(2-chlorophenyl)acrylonitrile (YI091)
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(2-chlorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YI097-1)
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(2-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YI097-2)
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-(((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(2-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (YI100)
  • reaction steps refer to step 4 of intermediate 1, to obtain 1.12 g of the target product, yield 86%.
  • Step 5 4-(2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-1-ethyl-3-(2-chlorophenyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JN013)
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(2-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JN18)
  • reaction steps refer to step 1 of intermediate 1, and 2.7 g of the target product is obtained with a yield of 92%.
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YH156)
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YH160)
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-((((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (YI005)
  • Step 5 4-(2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-1-ethyl-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (YN54)
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (YN55)
  • Step 1 Synthesis of (Z)-2-(4-chloro-2-fluorophenyl)-3-(3-methylphenyl)acrylonitrile (JM005)
  • reaction steps refer to step 1 of intermediate 1, and the target product 5 g is obtained with a yield of 93%.
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-methylphenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JM010)
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-methylphenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JM014)
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-(((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-methylphenyl)-5-neopentylpyrrolidine-2-carboxylic acid (JM018)
  • Step 5 4-(2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-1-ethyl-3-(3-methylphenyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JM149)
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(3-methylphenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JM157)
  • Step 1 Synthesis of (Z)-2-(4-chloro-2-fluorophenyl)-3-(3-methoxyphenyl)acrylonitrile (JM030)
  • reaction steps refer to step 1 of intermediate 1, and the target product 5.5 g is obtained with a yield of 96%.
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-methoxyphenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JM037)
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-methoxyphenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JM041)
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-(((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-methoxyphenyl)-5-neopentylpyrrolidine-2-carboxylic acid (JM049)
  • Step 5 4-(2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-1-ethyl-3-(3-methoxyphenyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JM154)
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(3-methoxyphenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JM160)
  • Step 1 Synthesis of (Z)-2-(5-chloro-2-fluorophenyl)-3-(2,3-dichlorophenyl)acrylonitrile (YK090)
  • Step 2 Synthesis of (2R, 3S, 4R, 5S)-4-(5-chloro-2-fluorophenyl)-4-cyano-3-(2,3-dichlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YK093)
  • Step 3 Synthesis of (2R,3S,4S,5S)-4-(aminomethyl)-4-(5-chloro-2-fluorophenyl)-3-(2,3-dichlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (YK108)
  • reaction steps refer to step 3 of intermediate 1, to obtain 1.23 g of the target product, yield 32%.
  • Step 4 Synthesis of (2R,3S,4S,5S)-4-((((((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(5-chloro-2-fluorophenyl)-3-(2,3-dichlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (YK109)
  • reaction steps refer to step 4 of intermediate 1, to obtain the target product 1.08 g, yield 69%.
  • Step 5 4-(2R,3S,4S,5S)-4-(aminomethyl)-4-(5-chloro-2-fluorophenyl)-3-(2,3-dichlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JN014)
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-5-chloro-4'-(2,3-dichlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JN19)
  • Step 1 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JN110)
  • Step 2 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JN105)
  • reaction steps refer to step 3 of intermediate 1, to obtain the target product 1.5 mg, yield 47%.
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(((9H-fluoro-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JN107)
  • JN105 (2.6 g, 5.1 mmol) was weighed into a 100 mL single-mouth bottle, dissolved in dry dichloromethane, and diisopropylethylamine (2.6 g, 20.4 mmol) and FmocCl (2.0 g, 7.6 mmol) were added, and reacted at room temperature overnight. After drying, the target product was purified by normal phase column to obtain 3.2 g, with a yield of 86%.
  • Step 4 Synthesis of (2R,3R,4S,5S)-4-(((9H-fluoro-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxylic acid (JN113)
  • Step 5 Synthesis of methyl 4-(((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (JN115)
  • JN113 (1.0 g, 1.4 mmol) was added to a 100 mL single-mouth bottle, dissolved in dry dichloromethane, 1-methylimidazole (345 mg, 4.2 mmol) was added at zero degrees Celsius, stirred for 10 minutes, ethylsulfonyl chloride (361 mg, 2.8 mmol) was added, methyl 4-amino-3-methoxybenzoate (769 mg, 4.2 mmol) was added after stirring for half an hour, and reacted at room temperature for 2 hours. Saturated sodium bicarbonate solution was added to the reaction solution, extracted with dichloromethane, and the organic phase was dried and purified by normal phase column to obtain 1.47 g of crude product.
  • Step 6 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JN122)
  • Step 1 Synthesis of tert-butyl (E)-5-(1-methylcyclobutyl)-3-pentenoate (LCC123)
  • Step 2 Synthesis of tert-butyl (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-((1-methylcyclobutyl)methyl)pyrrolidine-2-carboxylate (LCC127)
  • Step 3 Synthesis of tert-butyl (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-((1-methylcyclobutyl)methyl)pyrrolidine-2-carboxylate (LCC132)
  • Step 4 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-((1-methylcyclobutyl)methyl)pyrrolidine-2-carboxylic acid tert-butyl ester (LCC134)
  • Step 5 Synthesis of (2R,3R,4S,5S)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-((1-methylcyclobutyl)methyl)pyrrolidine-2-carboxylic acid (LCC142)
  • reaction steps refer to step 4 of final product 18, to obtain 40 mg of the target product, yield 35%.
  • Step 6 Synthesis of methyl 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-((1-methylcyclobutyl)methyl)pyrrolidine-2-carboxamido)-3-methoxybenzoate (LCC150)
  • Step 7 Synthesis of 4-((2'S,3S,4'R,5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-((1-methylcyclobutyl)methyl)spiro[indole]-1-((1-methylcyclobutyl)methyl) ...-1-((1-methylcyclobutyl)methyl)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-((1-methylcyclobutyl)methyl)-1-((1-methylcyclobutyl)methyl)-6-chloro [3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (LCC153)
  • Step 1 Synthesis of (E)-5-(1-methylcyclopentyl)-3-pentenoic acid tert-butyl ester (LCC043)
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-((1-methylcyclopentyl)methyl)pyrrolidine-2-carboxylic acid tert-butyl ester (LCC044)
  • Step 3 Synthesis of tert-butyl (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-((1-methylcyclopentyl)methyl)pyrrolidine-2-carboxylate (LCC050)
  • Step 4 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-((1-methylcyclopentyl)methyl)pyrrolidine-2-carboxylic acid tert-butyl ester (LCC051)
  • Step 5 Synthesis of (2R,3R,4S,5S)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-((1-methylcyclopentyl)methyl)pyrrolidine-2-carboxylic acid (LCC055)
  • reaction steps refer to step 4 of final product 18, to obtain 63 mg of the target product, yield 35%.
  • Step 6 Synthesis of 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-((1-methylcyclopentyl)methyl)pyrrolidine-2-carboxamido)-3-methoxybenzoic acid methyl ester (LCC059)
  • Step 7 Synthesis of 4-((2'S,3S,4'R,5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-((1-methylcyclopentyl)methyl)spiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (LCC063)
  • Step 1 Synthesis of (E)-5-(1-methylcyclohexyl)-3-pentenoic acid tert-butyl ester (LCC110)
  • Step 2 Synthesis of tert-butyl (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-((1-methylcyclohexyl)methyl)pyrrolidine-2-carboxylate (LCC111)
  • Step 3 Synthesis of tert-butyl (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-((1-methylcyclohexyl)methyl)pyrrolidine-2-carboxylate (LCC113)
  • Step 4 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-((1-methylcyclohexyl)methyl)pyrrolidine-2-carboxylic acid tert-butyl ester (LCC115)
  • Step 5 Synthesis of (2R,3R,4S,5S)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-((1-methylcyclopentyl)methyl)pyrrolidine-2-carboxylic acid (LCC119)
  • reaction steps refer to step 4 of final product 18, to obtain the target product 119 mg, yield 62%.
  • Step 6 Synthesis of methyl 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-((1-methylcyclohexyl)methyl)pyrrolidine-2-carboxamido)-3-methoxybenzoate (LCC126)
  • Step 7 Synthesis of 4-((2'S,3S,4'R,5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-((1-methylcyclohexyl)methyl)spiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (LCC131)
  • Step 1 (E)-tert-butyl 2-((3,3-dimethylpentylidene)amino)acetate (TC070)
  • reaction steps refer to step 1 of final product 33, and the target product 5.1 g is obtained with a yield of 99%.
  • Step 2 Synthesis of tert-butyl (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-(2,2-dimethylbutyl)pyrrolidine-2-carboxylate (TC074)
  • Step 3 Synthesis of tert-butyl (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-(2,2-dimethylbutyl)pyrrolidine-2-carboxylate (TC076)
  • Step 4 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-(2,2-dimethylbutyl)pyrrolidine-2-carboxylic acid tert-butyl ester (TC078)
  • Step 5 Synthesis of (2R,3R,4S,5S)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-(2,2-dimethylbutyl)-1-ethylpyrrolidine-2-carboxylic acid (TC082)
  • reaction steps refer to step 4 of final product 18, to obtain the target product 145 mg, yield 72%.
  • Step 6 Synthesis of 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-(2,2-dimethylbutyl)-1-ethylpyrrolidine-2-carboxamide)-3-methoxybenzoate methyl ester (TC087)
  • Step 7 Synthesis of 4-((2'S,3S,4'R,5'R)-6-chloro-4'-(3-chlorophenyl)-2'-(2,2-dimethylbutyl)-1'-ethylspiro[indoline-3,3'-pyrrolidine]-5'-carboxyl)-3-methoxybenzoic acid (TC90)
  • reaction steps refer to step 1 of final product 33, and the target product 5.1 g is obtained with a yield of 99%.
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-(2-ethyl-2-methylbutyl)pyrrolidine-2-carboxylic acid tert-butyl ester (TC020)
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-(2-ethyl-2-methylbutyl)pyrrolidine-2-carboxylic acid tert-butyl ester (TC021)
  • Step 4 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-(2-ethyl-2-methylbutyl)pyrrolidine-2-carboxylic acid tert-butyl ester (TC022)
  • Step 5 Synthesis of (2R,3R,4S,5S)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-(2-ethyl-2-methylbutyl)pyrrolidine-2-carboxylic acid (TC024)
  • reaction steps refer to step 4 of final product 18, and the target product 210 mg is obtained with a yield of 45.6%.
  • Step 6 Synthesis of 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-(2-ethyl-2-methylbutyl)pyrrolidine-2-carboxamido)-3-methoxybenzoic acid methyl ester (TC025)
  • Step 7 Synthesis of 4-((2'S,3S,4'R,5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-(2-ethyl-2-methylbutyl)spiro[indoline-3,3'-pyrrolidine]-5'-amino)-3-methoxybenzoic acid (TC29)
  • Step 1 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)benzoate (JP12)
  • Step 2 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamido)benzoic acid (JP16)
  • Step 1 (2R, 3R, 4S, 5S)-4-((tert-butyloxycarbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ09)
  • JN105 60 mg, 0.12 mmol was weighed into a 50 mL single-necked bottle, dissolved in dry dichloromethane, and diisopropylethylamine (31 mg, 0.24 mmol) and (Boc) 2 O (39 mg, 0.18 mmol) were added, and the reaction was allowed to proceed overnight at room temperature.
  • the product was extracted with methane and washed twice with saturated sodium chloride.
  • the organic phase was dried with anhydrous sodium sulfate, and then purified by column chromatography to obtain 75 mg of the target product with a yield of 98%.
  • Step 2 (2R, 3R, 4S, 5S)-4-((tert-butyloxycarbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(2-hydroxyethyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ12)
  • the reactants were dissolved in 10 mL of tetrahydrofuran, 65 mg of tetrabutylammonium fluoride trihydrate was added, and the reaction was allowed to react at room temperature for two hours.
  • the reaction solution was extracted three times with dichloromethane, the organic phases were combined, dried with anhydrous sodium sulfate, and purified by normal phase column to obtain 55 mg of the target product with a yield of 85%.
  • Step 3 (2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(2-fluoroethyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ19)
  • JQ12 (56 mg, 0.09 mmol) was weighed into a 50 mL single-mouth bottle, dissolved in dry dichloromethane, and diethylaminosulfur trifluoride (28 mg, 0.18 mmol) was added at 0°C, and the reaction was allowed to react overnight at room temperature. After the reaction was completed, the mixture was extracted with dichloromethane, washed twice with saturated sodium chloride, and the organic phase was dried with anhydrous sodium sulfate, spin-dried, and purified by column to obtain 49 mg of crude product. The reactants were dissolved in 4 mL of dichloromethane, and 0.6 mL of trifluoroacetic acid was added, and the mixture was allowed to react overnight at room temperature.
  • Step 4 (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(2-fluoroethyl)-5-neopentylpyrrolidine-2-carboxylic acid (JQ38)
  • JQ19 (149 mg, 0.27 mmol) was weighed into a 50 mL single-mouth bottle, dissolved in dry dichloromethane, and diisopropylethylamine (140 mg, 1.1 mmol) and FmocCl (105 mg, 0.31 mmol) were added, and the reaction was allowed to react overnight at room temperature. After drying, the crude product was purified by normal phase column to obtain 130 mg. The reactants were dissolved in 3 mL of dichloromethane, and 3 mL of trifluoroacetic acid was added, and the reaction was allowed to react overnight at room temperature. Saturated sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with ethyl acetate.
  • Step 5 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(2-fluoroethyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JQ42)
  • reaction steps refer to the steps of final product 18 Step 5, 32 mg of the target product was obtained with a yield of 82%.
  • Step 6 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-(2-fluoroethyl)-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamide]-3-methoxybenzoic acid (JQ44)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-(oxetane-3-ylmethyl)pyrrolidine-2-carboxylic acid (JQ83)
  • reaction steps refer to step 4 of final product 18, to obtain 46 mg of the target product, yield 23%.
  • Step 2 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-(oxetane-3-ylmethyl)pyrrolidine-2-carboxamido)-3-methoxybenzoate (JQ94)
  • Step 3 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-2'-neopentyl-1'-(oxetane -3-ylmethyl)spiro[indole-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid (JQ97)
  • Step 1 Synthesis of methyl 2-(4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)phenyl)acetate (JP25)
  • Step 2 Synthesis of 2-(4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamide)phenyl)acetic acid (JP27)
  • Step 1 Synthesis of methyl 2-(4-aminophenyl)acetate-4-(((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)methyl)benzoate (JP14)
  • Step 2 Synthesis of 4-(((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamide)methyl)benzoic acid (JP20)
  • Step 1 Synthesis of benzyl (2R, 5S)-5-((tert-butyloxycarbonyl)amino)tetrahydro-2H-pyran-2-carboxylate (JP11)
  • JP11 43 mg, 0.13 mmol was dissolved in 3 mL of dichloromethane, and 3 mL of 4N hydrochloric acid 1,4-dioxane solution was added, and the reaction was allowed to proceed overnight at room temperature. The reaction solution was dried by vortexing to obtain 40 mg of crude product, which was directly used for the next step.
  • Step 3 Synthesis of benzyl (2S, 5R)-5-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)tetrahydro-2H-pyran-2-carboxylate (JP19)
  • Step 4 Synthesis of (2'S, 3S, 4'R, 5'R)-N-((3R, 6S)-6-carbamoyltetrahydro-2H-pyran-3-yl)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamide (JP23)
  • JP19 (33 mg, 0.05 mmol) was weighed into a bottle, dissolved in 2 mL DMF, potassium carbonate (29 mg, 0.2 mmol) was added, and stirred at 110 ° C overnight. After the reaction was completed, it was cooled to room temperature, water was added, and ethyl acetate was extracted. After the organic phase was dried by rotation, the crude product was dissolved in 5 mL 7N hydrochloric acid methanol solution and reacted at room temperature for three hours. The reaction solution was dried by rotation and purified by HPLC to obtain 16 mg of the target product with a yield of 46%.
  • Final product 65 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamido)bicyclo[2.2.2]octane-1-carboxylic acid (JP04)
  • Step 1 Synthesis of 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorobenzene 1-ethyl-5-neopentylpyrrolidine-2-carboxamide) bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (JP01)
  • Step 2 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamido)bicyclo[2.2.2]octane-1-carboxylic acid (JP04)
  • Step 1 Synthesis of (pivaloyloxy) 4-amino-3-methoxybenzoic acid methyl ester (JP58)
  • Reactant was dissolved in tetrahydrofuran (THF)/methanol (10mL/10mL) mixed solution, deoxygenated for 5 minutes, nitrogen exchanged, palladium/carbon 40mg was added, deoxygenated for 5 minutes, nitrogen exchanged, and reacted at room temperature overnight.
  • the reaction solution was spin-dried, column purified, and 236mg of target product was obtained, with a yield of 84%.
  • Step 2 Synthesis of (pivaloyloxy)methyl-4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (JP50)
  • Step 3 Synthesis of (pivaloyloxy)methyl-4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoate (JP65)
  • Step 1 Synthesis of methyl (isopropyloxycarbonyl)oxy)4-amino-3-methoxybenzoate (JP70)
  • Step 2 Synthesis of ((isopropoxycarbonyl)oxy)methyl-4-((2R,3R,4S,5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (JP72)
  • JN113 50 mg, 0.07 mmol was added to a 50 mL single-mouth bottle, dissolved in dry dichloromethane, and heated at zero degrees Celsius.
  • 1-methylimidazole 18 mg, 0.21 mmol
  • ethylsulfonyl chloride 18 mg, 0.14 mmol
  • JP70 88 mg, 0.21 mmol
  • Step 3 Synthesis of (isopropoxycarbonyl)oxy)methyl-4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoate (JP90)
  • JP72 was dissolved in 1 mL DMF, and 0.5 mL diethylamine was added. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, diethylamine was dried by spin drying, potassium carbonate (12 mg, 0.08 mmol) was added to the reaction solution, and the reaction was allowed to react at 80°C overnight. After the reaction was completed, water was added, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated sodium chloride solution three times, dried over anhydrous sodium sulfate, and purified by HPLC. The target product was 5 mg with a yield of 13%.
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentyl-N-(pyridin-4-yl)pyrrolidine-2-carboxamide (JP13)
  • Step 2 Synthesis of (2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentyl-N-(pyridin-4-yl)spiro[indole-3,3'-pyrrolidine]-5'-carboxamide (JP17)
  • Step 1 Synthesis of methyl (1R, 4r)-4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamido)cyclohexane-1-carboxylate (JP15)
  • Step 2 Synthesis of (1R, 4r)-4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamido)cyclohexane-1-carboxylic acid (JP21)
  • Step 1 Synthesis of methyl (1S, 4s)-4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamido)cyclohexane-1-carboxylate (JP24)
  • Step 2 Synthesis of (1S, 4s)-4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamido)cyclohexane-1-carboxylic acid (JP26)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-N-((1r, 4R)-4-hydroxycyclohexyl)-5-neopentylpyrrolidine-2-carboxamide (JP06-2)
  • Step 1 Synthesis of tert-butyl (1r, 4R)-4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamido)cyclohexyl)carbamate (JP30)
  • Step 2 Synthesis of (2'S, 3S, 4'R, 5'R)-N-((1r, 4R)-4-aminocyclohexyl)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide (JP33)
  • JP30 (35 mg, 0.05 mmol) was weighed into a bottle, dissolved with 2 mL of DMF, potassium carbonate (29 mg, 0.2 mmol) was added, and stirred at 110°C overnight. After the reaction was completed, it was cooled to room temperature, water was added, and ethyl acetate was extracted. After the organic phase was dried by rotation, the crude product was dissolved in 3 mL of dichloromethane, 3 mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for two hours. The reaction solution was dried by rotation, and HPLC was purified to obtain 23 mg of the target product with a yield of 68 %.
  • Step 1 Synthesis of tert-butyl (1s, 4S)-4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)cyclohexyl)carbamate (JP31)
  • Step 2 Synthesis of (2'S, 3S, 4'R, 5'R)-N-((1s, 4S)-4-aminocyclohexyl)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamide (JP34)
  • Step 1 Synthesis of (1r, 4R)-4-aminocyclohexyl (2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxylate (JP06-1)
  • Step 2 Synthesis of (1r, 4R)-4-aminocyclohexyl (2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentyl spiro[indoline-3,3'-pyrrolidine]-5'-carboxylate (JP08)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-isobutyl-5-neopentylpyrrolidine-2-carboxylic acid (JQ124)
  • the reactants were dissolved with 2 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added, and allowed to react at room temperature overnight.
  • the reaction solution was dried, saturated sodium bicarbonate solution was added, and the mixture was extracted three times with dichloromethane.
  • the organic phases were combined, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and then spin-dried.
  • the target product 60 mg was purified by normal phase column with a yield of 59%.
  • Step 2 Synthesis of 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-isobutyl-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JQ128)
  • Step 3 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-isobutyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid (JQ129)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(cyclopropylmethyl)-5-neopentylpyrrolidine-2-carboxylic acid (JQ48)
  • reaction steps refer to step 4 of final product 18, to obtain the target product 30 mg, yield 14%.
  • Step 2 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(cyclopropylmethyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (JQ51)
  • Step 3 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-(cyclopropylmethyl)-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamide]-3-methoxybenzoic acid (JQ53)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(cyclobutylmethyl)-5-neopentylpyrrolidine-2-carboxylic acid (JQ141)
  • reaction steps refer to step 1 of final product 75, to obtain the target product 90 mg, yield 58%.
  • Step 2 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(cyclobutylmethyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (JQ146)
  • Step 3 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-(cyclobutylmethyl)-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid (JQ148)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4- (4-Chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(cyclopentylmethyl)-5-neopentylpyrrolidine-2-carboxylic acid (JQ80)
  • reaction steps refer to step 4 of final product 18, to obtain 52 mg of the target product, yield 12%.
  • Step 2 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(cyclopentylmethyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (JQ86)
  • Step 3 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-(cyclopentylmethyl)-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid (JQ91)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(cyclohexylmethyl)-5-neopentylpyrrolidine-2-carboxylic acid (JQ81)
  • reaction steps refer to step 4 of final product 18, to obtain the target product 21 mg, yield 10%.
  • Step 2 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(cyclohexylmethyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (JQ92)
  • Step 3 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-(cyclohexylmethyl)-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid (JQ96)
  • reaction steps refer to step 6 of final product 10, and 2 mg of trifluoroacetate salt of the target product is obtained with a yield of 25%.
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-((tetrahydrofuran-3-yl)methyl)pyrrolidine-2-carboxylic acid (JQ157)
  • reaction steps refer to step 1 of final product 75, to obtain 64 mg of the target product, yield 40%.
  • Step 2 Synthesis of methyl 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-(((R or S)-tetrahydrofuran-3-yl)methyl)pyrrolidine-2-carboxamido)-3-methoxybenzoate (JQ159-1 and JQ159-2)
  • Step 3 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-2'-neopentyl-1'-(((R or S)-tetrahydrofuran-3-yl)methyl)spiro[indoline-3,3'-pyrrolidine]-5'-carboxamide]-3-methoxybenzoic acid (JR01 and JR02)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-((tetrahydro-2H-pyran-4-yl)methyl)pyrrolidine-2-carboxylic acid (JQ110)
  • reaction steps refer to step 1 of final product 75, to obtain 20 mg of the target product, yield 19%.
  • Step 2 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-((tetrahydro-2H-pyran-4-yl)methyl)pyrrolidine-2-carboxamido)-3-methoxybenzoate (JQ119)
  • Step 3 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-2'-neopentyl-1'-((tetrahydro-2H-pyran-4-yl)methyl)spiro[indole-3,3'-pyrrolidine]-5'-carboxamide]-3-methoxybenzoic acid (JQ122)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(furan-2-ylmethyl)-5-neopentylpyrrolidine-2-carboxylic acid (JQ142)
  • reaction steps refer to step 1 of final product 75, to obtain 37 mg of the target product, yield 25%.
  • Step 2 Synthesis of 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(furan-2-ylmethyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JQ147)
  • Step 3 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-(furan-2-ylmethyl)-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JQ149)
  • Step 1 Synthesis of tert-butyl (2R, 3R, 4S, 5S)-4-(((tert-butyloxycarbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-(2-oxoethyl)pyrrolidine-2-carboxylate (JQ46)
  • Dimethyl sulfoxide (141 mg, 1.8 mmol) was weighed into a 100 mL round-bottom flask, and ultra-dry dichloromethane was added.
  • Oxalyl chloride (115 mg, 0.9 mmol) was added dropwise at -78 °C.
  • a dichloromethane solution of JQ12 200 mg, 0.3 mmol was added dropwise to the reaction solution.
  • the mixture was stirred at -78 °C for 1 hour, and triethylamine (273 mg, 2.7 mmol) was added dropwise. The mixture was naturally warmed to room temperature and reacted for 3 hours.
  • Step 2 Synthesis of (2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(2,2-difluoroethyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ50)
  • reaction steps refer to step 3 of final product 49, to obtain 160 mg of the target product, yield 66%.
  • Step 3 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(2,2-difluoroethyl)-5-neopentylpyrrolidine-2-carboxylic acid (JQ55)
  • Step 4 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(2,2-difluoroethyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (JQ56)
  • Step 5 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-(2,2-difluoroethyl)-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid (JQ57)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-(prop-2-yn-1-yl)pyrrolidine-2-carboxylic acid (JQ155)
  • reaction steps refer to step 1 of final product 75, to obtain 80 mg of the target product, yield 63%.
  • Step 2 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-(prop-2-yn-1-yl)pyrrolidine-2-carboxamido)-3-methoxybenzoate (JQ156)
  • Step 3 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentyl-1-(prop-2-yn-1-yl)pyrrolidine-2-carboxamido)-3-methoxybenzoate (JQ158)
  • Step 1 Synthesis of (Z)-2-(4-chloro-2,3-difluorophenyl)-3-(3-chlorophenyl)acrylonitrile (JQ64)
  • Step 2 Synthesis of (2R, 3R, 4R, 5S)-4-(4-chloro-2,3-difluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ66)
  • Step 3 Synthesis of (2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2,3-difluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ100)
  • Step 4 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2,3-difluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ102)
  • Step 5 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2,3-difluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxylic acid (JQ111)
  • Step 6 Synthesis of 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2,3-difluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JQ117)
  • Step 7 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-7-fluoro-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamide]-3-methoxybenzoic acid (JQ120)
  • Step 1 Synthesis of (Z)-2-(4-chloro-2,5-difluorophenyl)-3-(3-chlorophenyl)acrylonitrile (JQ63)
  • Step 2 Synthesis of (2R, 3R, 4R, 5S)-4-(4-chloro-2,5-difluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ67)
  • Step 3 Synthesis of (2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2,5-difluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ101)
  • Step 4 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2,5-difluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JQ103)
  • Step 5 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2,5-difluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxylic acid (JQ112)
  • Step 6 Synthesis of 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2,5-difluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoic acid methyl ester (JQ118)
  • Step 7 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-5-fluoro-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid (JQ121)
  • reaction steps refer to step 1 of final product 33, and the target product 5.2 g is obtained with a yield of 99%.
  • Step 2 Synthesis of (2R,3R,4R,5S)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-4-cyano-5-(2,2,3-trimethylbutyl)pyrrolidine-2-carboxylic acid tert-butyl ester (TC129)
  • reaction steps refer to step 2 of intermediate 1, and the target product 3.1 g is obtained with a yield of 31%.
  • Step 3 Synthesis of (2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-(2,2,3-trimethyl Tert-Butyl)pyrrolidine-2-carboxylate (TC130)
  • Step 4 Synthesis of tert-butyl (2R,3R,4S,5S)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-(2,2,3-trimethylbutyl)pyrrolidine-2-carboxylate (TC132)
  • Step 5 Synthesis of (2R,3R,4S,5S)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-(2,2,3-trimethylbutyl)pyrrolidine-2-carboxylic acid (TC137)
  • reaction steps refer to step 4 of final product 18, and the target product 170 mg is obtained with a yield of 85%.
  • Step 6 Synthesis of 4-((2R,3R,4S,5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-ethyl-5-(2,2,3-trimethylbutyl)pyrrolidine-2-carboxamido)-3-methoxybenzoic acid methyl ester (TC140)
  • Step 7 Synthesis of 4-((2'S,3S,4'R,5'R)-6-chloro-4'-(3-chlorophenyl)-2'-(2,2-dimethylbutyl)-1'-ethylspiro[indoline-3,3'-pyrrolidine]-5'-carboxyl)-3-methoxybenzoic acid (TC145)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-1-allyl-4-((tert-butyloxycarbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JR58)
  • JQ09 400 mg, 0.66 mmol was weighed into a 35 mL sealed tube, dissolved in 5 mL ultra-dry DMF, and allyl bromide (799 mg, 6.6 mmol) and cesium carbonate (646 mg, 1.98 mmol) were added, and reacted at 90°C overnight. After the reaction, it was cooled to room temperature, water was added, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was spin-dried and purified with a normal phase column to obtain 246 mg of the target product with a yield of 57%.
  • Step 2 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-1-allyl-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JR59)
  • Step 3 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-1-allyl-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxylic acid (JR60)
  • Step 4 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-1-allyl-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoate (JR61)
  • Step 5 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-1'-allyl-6-chloro-4'-(3-chlorophenyl)-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid (JR62)
  • Step 1 Synthesis of (2R, 3R, 4S, 5S)-4-((tert-butyloxycarbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(3-hydroxypropyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JR46)
  • Step 2 Synthesis of (2R, 3R, 4S, 5S)-4-((tert-butyloxycarbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(3-fluoropropyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JR47)
  • JR46 200 mg, 0.35 mmol was weighed into a 50 mL single-mouth bottle, dissolved in dry dichloromethane, and diethylaminosulfur trifluoride (114 mg, 0.71 mmol) was added at 0°C, and reacted at room temperature overnight. After the reaction, dichloromethane was extracted, washed twice with saturated sodium chloride, and the organic phase was dried over anhydrous sodium sulfate, spin-dried, and purified by column to obtain 158 mg of the target product with a yield of 67%.
  • Step 3 Synthesis of (2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(3-fluoropropyl)-5-neopentylpyrrolidine-2-carboxylic acid tert-butyl ester (JR50)
  • reaction steps refer to step 2 of the final product 94, and the target product 60 mg is obtained with a yield of 28%.
  • Step 4 Synthesis of ((2R, 3R, 4S, 5S)-4-(9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(3-fluoropropyl)-5-neopentylpyrrolidine-2-carboxylic acid (JR63)
  • reaction steps refer to step 3 of final product 94, to obtain 34 mg of the target product, yield 61%.
  • Step 5 Synthesis of methyl 4-((2R, 3R, 4S, 5S)-4-(aminomethyl)-4-(4-chloro-2-fluorophenyl)-3-(3-chlorophenyl)-1-(3-fluoropropyl)-5-neopentylpyrrolidine-2-carboxamide)-3-methoxybenzoate (JR64)
  • Step 6 Synthesis of 4-((2'S, 3S, 4'R, 5'R)-6-chloro-4'-(3-chlorophenyl)-1-(3-fluoropropyl)-2'-neopentylspiro[indole-3,3'-pyrrolidine]-5'-carboxamide]-3-methoxybenzoic acid (JR55)
  • MDMX His-tagged MDMX (14-111, C17S) was expressed in E. coli and purified by Ni affinity column and then by Superdex75 molecular sieve. The purity of the obtained MDMX protein was greater than 95% and the protein concentration was 12.5 ⁇ M.
  • FAM-tagged PDI polypeptide [Cancer Res 2007, 67, 8810-8817] was used as a fluorescent labeled molecular probe, wherein the dissociation constant K d of the MDMX/FAM-PDI interaction was 2.1 nM.
  • the 96-well plate was purchased from Corning (black, #3694).
  • the multifunctional microplate reader was a product of TECAN, model: SPARK 10M.
  • the detection buffer was 10mM Tris (pH 7.5), 200mM NaCl (Sigma), 0.01% Tween-20 and 0.01% Trition X-100 (Sigma), and the experimental water was Millipore-Q pure water.
  • test compound was dissolved in DMSO to form a 20 mM standard stock solution.
  • standard stock solution of the test compound was diluted with DMSO in an EP tube to form a working sample solution.
  • concentration of the prepared working sample solution 25 times the highest sample concentration required on the test plate (25 ⁇ test compound solution).
  • the compound was diluted 3 times in an EP tube for later use.
  • Wells A1-A3 are used as blank control groups: add 100uL detection buffer.
  • Wells A4-A6 are used as negative signal reference groups: add 100 ⁇ L buffer containing only 5nM fluorescent labeled molecular probe.
  • Wells A7-A9 are used as positive reference groups: add 100 ⁇ L mixed solution containing 5nM fluorescent labeled molecular probe and 60nM MDMX protein.
  • the reaction plate was covered with aluminum foil, and the 96-well plate was placed on a 96-well plate shaker and incubated at room temperature for 1 hour.
  • the fluorescence polarization mP value at Ex485nm/Em530nm was read using a microplate reader.
  • the measured mP value was plotted against the compound concentration gradient.
  • the sample compound concentration corresponding to the median of the maximum and minimum mP values was the IC 50 value ([I] 50 ) of the compound binding to the protein.
  • Ki [I] 50 /([L] 50 / Kd +[P] 0 / Kd +1).
  • [L] 50 represents 50% of the concentration of the fluorescent labeled molecular probe in the above test system
  • [P] 0 represents the concentration of MDMX protein in the above test system
  • K d is the dissociation constant between the protein and the fluorescent labeled molecular probe.
  • Example 3 FP detection of Ki values of compounds and MDX2 protein
  • FAM-tagged PDI polypeptide [Cancer Res 2007, 67, 8810-8817] was used as a fluorescent labeled molecular probe, wherein the dissociation constant K d of the MDM2/FAM-PDI interaction was 0.7 nM.
  • the 96-well plate was purchased from Corning (black, #3694).
  • the multifunctional microplate reader was a product of TECAN, model: SPARK 10M.
  • the detection buffer 100mM potassium phosphate (pH 8.0), 100ug/mL Bovine-r-globulin (Sigma) and 0.01% Trition X-100 (Sigma), and the experimental water was Millipore-Q pure water.
  • test compound was dissolved in DMSO to form a 20 mM standard stock solution.
  • standard stock solution of the test compound was diluted with DMSO in an EP tube to form a working sample solution.
  • concentration of the prepared working sample solution 25 times the highest sample concentration required on the test plate (25 ⁇ test compound solution).
  • the compound was diluted 3 times in an EP tube for later use.
  • Wells A1-A3 are used as blank control groups: add 100 ⁇ L of detection buffer.
  • Wells A4-A6 are used as negative signal reference groups: add 100 ⁇ L of buffer containing only 2 nM fluorescently labeled molecular probe.
  • Wells A7-A9 are used as positive reference groups: add 100 ⁇ L of a mixed solution containing 2 nM fluorescently labeled molecular probe and 20 nM MDM2 protein.
  • the reaction plate was covered with aluminum foil, and the 96-well plate was placed on a 96-well plate shaker and incubated at room temperature for 0.5 h.
  • the fluorescence polarization mP value at Ex485nm/Em530nm was read using an ELISA reader.
  • the measured mP value was plotted against the compound concentration gradient.
  • the sample compound concentration corresponding to the median of the maximum and minimum mP values was the IC 50 value ([I] 50 ) of the compound binding to the protein.
  • Ki [I] 50 /([L] 50 / Kd +[P] 0 / Kd +1).
  • [L] 50 represents 50% of the concentration of the fluorescent labeled molecular probe in the above test system
  • [P] 0 represents the concentration of MDM2 protein in the above test system
  • K d is the dissociation constant between the protein and the fluorescent labeled molecular probe.
  • the Ki values of the example compounds for inhibiting the MDM2/p53 interaction were measured and are shown in Table 2.
  • the experimental data show that the compounds have very good activity in inhibiting the MDM2/p53 interaction.
  • Step 1 Synthesis of (2R,3S,4S,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-1-fluorophenyl)-5-neopentyl-4-((3-nitrobenzyl)amino)methyl)pyrrolidine-2-carboxylic acid (JM043-CF2)
  • YN17-CF2 [239 mg, 0.5 mmol, an intermediate compound in the reaction process of synthesizing intermediate 4 (YN22-CF2)] was dissolved in methanol, and m-nitrobenzaldehyde (154 mg, 1.0 mmol), sodium cyanoborohydride (129 mg, 2.0 mmol) and 1 mL of acetic acid were added, and stirred at room temperature overnight. After the reaction, HPLC purification was performed to obtain 224 mg of the trifluoroacetate of the target compound, with a yield of 73%.
  • Step 2 Synthesis of methyl 4-((2R,3S,4S,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-1-fluorophenyl)-5-neopentyl-4-(((3-nitrobenzyl)amino)methyl)pyrrolidine-2-carboxamide)-3-methoxybenzoate (JM079-CF2)
  • JM043-CF2 (78 mg, 0.13 mmol) was weighed into a bottle, dissolved in tetrahydrofuran, and diisopropylethylamine (84 mg, 0.65 mmol) and diphenylphosphinoyl chloride (93 mg, 0.39 mmol) were added. After stirring at room temperature for 30 min, 3-methoxy-4-aminobenzoic acid methyl ester (94 mg, 0.52 mmol) was added and reacted at room temperature overnight. Water was added and extracted with dichloromethane. The organic phase was spin-dried and purified with a normal phase column to obtain the crude product of the target compound JM079-CF2, which was directly used in the next step.
  • Step 3 Synthesis of 4-((2'S,3S,4'S,5'R)-6-chloro-4'-(3-chloro-2-fluorophenyl)-2'-neopentyl-1-(3-nitrobenzyl)spiro[indoline-3,3'-pyrrolidine]-5'-carboxamide)-3-methoxybenzoic acid (JM085-CF2)
  • JM079-CF2 The crude product of JM079-CF2 was dissolved in N,N-dimethylformamide, potassium carbonate (63 mg, 0.45 mmol) was added, and the mixture was stirred at 100°C overnight. After the reaction, HPLC purification was performed to obtain 47 mg of trifluoroacetate of the target compound JM085-CF2, with a two-step reaction yield of 49%.
  • the target compound was dissolved in ethyl acetate, washed twice with saturated sodium bicarbonate solution, and once with saturated brine to obtain the free form of JM085-CF2.
  • the diffraction data and results of the X-ray single crystal diffraction test compound are shown in Table 4:
  • the crystal data show that the absolute stereo configuration of JN110 is shown in Figure 2.
  • Single enantiomer JN110 can be obtained by CuOAc and (R)-BINAP catalytic system, and intermediate JN110 can be used to synthesize the target product JN122 with an ee value greater than 95%.
  • the compound will not undergo configurational changes, so the absolute stereo configuration of JN122 should be consistent with that of JN110.
  • Cell growth inhibition assay Dissolve the sample to be tested in 100% dimethyl sulfoxide to prepare a 20mM stock solution of the compound. Dilute the compound with 100% dimethyl sulfoxide to the highest concentration required for the experiment (1mM or 10mM).
  • the cell density of each well was as follows: about 3000 per well for HCT116 and RKO, about 5000 per well for U2-OS, and about 8000 per well for JEG-3, and the total volume of each well was 150 ⁇ L.
  • two control groups were set up: (1) a control group with cells and culture medium but no compound added; (2) a group with only complete culture medium added, no cells and no compound.
  • 15 ⁇ L of CCK-8 reagent was added to each well and then incubated at 37°C for 2-3 hours.
  • the absorbance value at a wavelength of 450 nm was read using a TECAN microplate reader.
  • cell growth inhibition rate [absorption value of the experimental group - absorption value of only adding complete medium (no cell and no compound group)] / [absorption value of the group with cells but no compound - absorption value of only adding complete medium (no cell and no compound group)] ⁇ 100%.
  • the above data were processed using GraphPad Prism 7.0 software, and the IC50 value was the compound concentration corresponding to 50% cell growth inhibition rate.
  • Example 6 Comparison of the solubility of compounds 2 (YN11), 14 (YN55) and reference compounds RG7388 and YM30 in 5% DMSO/H 2 O
  • Compound sample concentration test The sample to be tested was dissolved in 50 ⁇ L DMSO, 950 ⁇ L water was added, vortexed for 5 min, ultrasonicated for 15 min, centrifuged at 14000 rpm for 30 min, 90 ⁇ L was added with 10 ⁇ L internal standard solution, and 20 ⁇ L was injected into UPLC liquid chromatograph. After measuring the peak area of the test sample and the peak area of the internal standard and calculating the ratio, the concentration of the test sample in 5% DMSO/H 2 O saturated solution was obtained by substituting into the standard curve equation.
  • Example 7 Comparison of the pharmacokinetic properties of YM34, 2 (YN11) and 14 (YN55)
  • ICR mice (species: SPF grade, source: animals transferred from the animal reserve bank of the experimental institution, Experimental Animal Management Department of Shanghai Institute of Family Planning Science) were given blood samples through the submandibular vein or other appropriate methods at different time points (IV group: 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 8h and 24h after administration, PO group: 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h and 24h after administration, 0.03ml of blood was collected at each time point), K2-EDTA anticoagulation, blood samples were centrifuged within 1h after collection to obtain plasma (centrifugation conditions: 6800g, 6 minutes, 2-8°C), and the samples to be tested were stored in a refrigerator at -80°C for use before analysis.
  • the concentration of the test samples was detected by LC-MS and the relevant parameters were calculated.
  • the solvent conditions used in this experiment were: 5% DMSO, 10% Cremophor and 85% PBS.
  • the intravenous dose was 5 mg/kg and the oral dose was 15 mg/kg.
  • Three male mice were used for each of the intravenous and oral experiments.
  • mice Using the above method, the pharmacokinetic parameters of YM34, 2 (YN11) and 14 (YN55) in mice were experimentally determined, and the data are shown in Tables 7-9.
  • the oral bioavailability of YM34 is 17%.
  • the oral bioavailability of YN11 and YN55 is 34.34% and 30.31%, respectively, which is twice that of YM34.
  • the maximum blood concentration (C max ) of YM34 is 2177.88 ng/mL, while that of YN11 and YN55 are 4089.43 ng/mL and 4151.35 ng/mL, respectively.
  • the maximum blood concentration parameters of YN11 and YN55 are 1 times higher than those of YM34.
  • the half-life (T 1/2 ) of YM34 is 3.18 hours, while that of YN11 and YN55 is 12.61 hours and 7.42 hours, respectively.
  • the half-life of YN11 and YN55 is 2 times and 1 time longer than that of YM34, respectively.
  • the experimental data show that the pharmacokinetic properties of 2 (YN11), 14 (YN55) and YM34 are better than those of YN11, YN55 has better oral absorption, better in vivo exposure, higher plasma drug concentration, and longer in vivo residence time, indicating that such drugs (including YN11 and YN55) can bind to drug targets longer and more fully in animals, and their concentrations in animals are higher, which is more conducive to achieving better disease treatment effects.
  • HCT-116 cells were seeded into 6-well plates and treated with inhibitors for 24 or 48 hours. Cells were collected by centrifugation and lysed with RIPA lysis buffer (Beyotime, product number P0013B) containing PMSF (Beyotime, product number ST506) according to the operating manual. The lysate was centrifuged at 13000g for 30 minutes at 4°C, and the supernatant was transferred to a transparent EP tube for subsequent analysis. Proteins were standardized to 20-40 ⁇ g/lane and separated using 8% or 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose (NC) membranes and blocked with 5% skim milk dissolved in TBST buffer for 1 hour.
  • NC nitrocellulose
  • p53 antibody was purchased from Millipore (product number #OP43), MDM4, GAPDH, ⁇ -Actin, and BAX antibodies were purchased from Proteintech (product numbers #17914-1-AP, 10494-1-AP, 66009-1-Ig, and 50599-2-Ig, respectively), and p21, MDM2, PARP, PUMA, and CL-PARP antibodies were purchased from Cell Signaling Technology (product numbers 2947, 86934, 9542, 12450, and 5625, respectively).
  • JN122 dose-dependently increased the expression of p53 and its target proteins p21 and MDM2 in HCT116 cells, indicating that p53/MDM2 interaction was inhibited and p53 stability was increased.
  • JN122 at 39.6nM could significantly upregulate the concentrations of p53, p21 and MDM2
  • RG7388 at 156nM and Nutlin-3a at 2.5 ⁇ M could achieve similar effects, indicating that JN122 was more effective than RG7388 and Nutlin-3a.
  • JN122, Nutlin-3a and RG7388 could significantly upregulate the expression of PUMA and BAX, indicating the activation of apoptotic pathways.
  • JN122 can be observed to cleave PARP (a biomarker of apoptosis), and treatment of HCT116 cells with the inhibitor for 48 hours may lead to a decrease in the concentrations of MDM2, MDM4, and BAX due to cell death.
  • JN122 can efficiently activate the intracellular p53 function, exhibit anti-cancer activity, and its activity is better than RG7388.
  • RKO colon cancer
  • H460 large cell lung cancer
  • U2-OS osteosarcoma
  • MSTO-211H mesothelioma
  • HepG2 liver cancer
  • A549 non-small cell lung cancer
  • Hela cervical cancer
  • HeLa is a p53 wild-type cell
  • p53 protein cannot be detected due to the continuous degradation of E6 oncoprotein.
  • the experimental steps are basically the same as before, except that HCT-116 cells are replaced with the above cells.
  • the expression level of MDM4 was high in RKO, H460 and U2-OS cells, which are considered to be MDM4 overexpressing cancer cell lines; the expression level of MDM2 was high in MSTO-211H and HepG2, while the expression level of MDM2 was relatively low in A549 cell line; no stably expressed p53 was detected in Hela cells, and the expression levels of MDM2 and MDM4 were also low.
  • all cells were treated with 0.6 ⁇ M JN122, which is the concentration of JN122 in HCT-116 cells. JN122 exhibited a strong p53 activation effect.
  • JN122 promoted a significant increase in the concentrations of p53, p21 and MDM2, and the promoting effect of JN122 was better than that of RG7388. However, no similar phenomenon was observed in Hela cells.
  • JN122 can activate p53 and upregulate the expression level of its target protein, showing stronger activity than RG7388.
  • the data show that JN122's activation of p53 activity depends on the tumor cells being able to express a certain concentration of wild-type p53.
  • HCT-116 cells were replaced with MOLM-13 (human acute myeloid leukemia) as a p53 wild-type cell line.
  • MOLM-13 human acute myeloid leukemia
  • JN122 increases the expression of p53 and its target proteins p21 and MDM2 in a dose-dependent manner, and decreases the expression of MDM4 in a dose-dependent manner, which may be due to the increase in the E3 ubiquitin ligase activity of MDM2.
  • 37.5nM of JN122 can significantly promote the cleavage of apoptosis markers PARP and Caspase-3, while RG7388 can achieve a similar effect only at 150nM.
  • JN122 can induce p53 activation in MOLM-13 cells and promote cell cycle arrest and apoptosis.
  • the activity of JN122 in activating p53 in MOLM-13 is about four times stronger than that of RG7388.
  • Example 9 In vivo efficacy of 18(JN122) in the MOLM-13 xenograft tumor model in mice
  • mice Female NOD.CB17-PrkdcscidIl2rgtm1/Bcgen (B-NDG) mice were purchased from Biocytogen Biotech Co., Ltd., Haimen, China. Number of animals in each group: 20 in the solvent control group (Vehicle) and 10 in the drug-treated group. Mice were intravenously injected with 200 ⁇ L of MOLM-13 cells (2 ⁇ 10 4 cells) suspended in PBS.
  • the JN122 25 mg/kg group was gavaged once a day for 21 days; the JN122 50 mg/kg group was gavaged once a day for 21 days; the JN122 100 mg/kg group was gavaged once a day for 21 days; the RG7388 50 mg/kg group was gavaged once a day for 21 days; the solvent control group was gavaged once a day with an equal amount of 5% DMSO + 10% EL + 85% saline for 21 days. The survival rate of mice was monitored every day.
  • the in vivo efficacy of JN122 was evaluated in the MOLM-13 xenograft tumor mouse model.
  • the median survival of mice in the solvent control group was 20 days (survival period 18 to 26 days); oral administration of 100 mg/kg of JN122 once a day extended the median survival of mice to 31 days (survival period 26 to 33 days, p ⁇ 0.001); oral administration of 25 mg/kg of JN122 once a day, the median survival of mice was 25.5 days (survival period 20 to 27 days, p ⁇ 0.01); oral administration of 50 mg/kg of JN122 once a day, the median survival of mice was 25.5 days (survival period 17 to 28 days, p ⁇ 0.001); oral administration of 50 mg/kg of RG7388 once a day, the median survival of mice was 27.5 days (survival period 26 to 30 days, p ⁇ 0.001). Therefore, consistent with RG7388, 18(JN122) exhibited potent anti-leukemia efficacy in
  • JN122 has very good anti-tumor efficacy in the human tumor mouse transplant tumor model.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

本发明公开了一种具有取代苯基螺[吲哚啉-3,3'-吡咯烷]结构的小分子化合物，结构如通式I所示，各取代基的定义如说明书和权利要求书所述。本发明化合物，能够抑制MDM2-p53、MDMX-p53蛋白蛋白相互作用，作为MDM2-p53、MDMX-p53蛋白蛋白相互作用小分子抑制剂，用于制备预防和/或治疗与MDM2、MDMX相关疾病，尤其是肿瘤的药物。

Description

具有取代苯基螺[吲哚啉-3,3′-吡咯烷]结构的小分子化合物 技术领域
本发明属于药物合成领域，具体涉及一类具有取代苯基螺[吲哚啉-3,3'-吡咯烷]结构的化合物，其立体异构体、对映体或其药学上可接受的盐，其制备方法和用途。
技术背景
肿瘤抑制因子p53在应对各种应激时发挥抗增殖作用，包括细胞生长停滞，DNA修复和细胞凋亡等。缺乏p53的小鼠虽然正常发育，但是容易诱发多种肿瘤。编码p53蛋白的TP53基因在近50％的人类癌症中发生突变或缺失，使得细胞无法发挥p53肿瘤抑制因子的功能。尽管p53在剩下的50％的人类癌症中仍保持野生型状态，但其功能受到多种抑制因子的抑制。研究表明：有两种蛋白质对p53的调控至关重要——MDM2和MDMX(也称为MDM4)。通过同时敲除小鼠的TP53基因可以挽救因敲除MDM2和MDMX基因导致的胚胎致死，阐明了MDM2和MDMX作为p53主要负内源性调节因子的作用。体外研究显示：MDM2和MDMX通过它们的氨基末端p53结合结构域与p53蛋白的转录激活结构域(TAD)作用，来抑制p53蛋白的基因转录功能。此外，MDM2可以促进MDM2自身、MDMX和p53蛋白泛素化和蛋白酶体降解。相反地，p53特异性地与MDM2P2启动子结合并激活其转录，从而形成一个自动调节反馈环——MDM2-p53反馈环。MDM2还可以促进p53蛋白转运到细胞核外，使p53无法接触其靶向DNA，从而降低其转录能力。而对于MDMX，虽然它不能像MDM2一样作为E3泛素连接酶发挥降解作用，但是它可以通过羧基末端RING结构域与MDM2的羧基末端RING结构域作用形成稳定的异二聚体，以促进MDM2介导的p53泛素化。
研究表明：与正常细胞相比，癌细胞中的MDM2和MDMX致癌因子水平异常升高，p53介导的基因转录功能被抑制，p53水平降低，这些特征与肿瘤细胞的过度生长密切相关。
RG7112(NCT00559533,NCT00623870,NCT01677780,NCT01164033,NCT01605526,NCT01143740和NCT01635296)，RG7388(Ding et al.,J Med Chem 2013,56(14),5979-83)，MI-77301(NCT01636479和NCT01985191)和AMG 232(NCT01723020和NCT02016729)等文献中报道的化合物可以选择性阻断MDM2/p53相互作用。SJ-172550(Reed et al.,J Biol Chem 2010,285(14),10786-96；Bista et al.,PLoS One 2012,7(6),e37518)，CTX-1(Karan et al.,Mol Cancer Ther 2016,15(4),574-582)和K-178(Uesato et al.,Bioorg Med Chem 2016,24(8),1919-26)等文献中报道的化合物可以选择性阻断MDMX/p53相互作用。WK298(Popowicz et al.,Cell Cycle 2010,9(6),1104-11)，ATSP-7041(Chang et al.,Proc Natl Acad Sci U S A 2013,110(36),E3445-54)，RO-5963(Graves et al.,Proc Natl Acad Sci U S A 2012,109(29),11788-93)和ALRN-6924(Carvajal et al.,Sci Transl Med 2018,10(436))等文献报道的化合物可以同时抑制MDM2/p53和MDMX/p53相互作用。小分子抑制剂阻断MDM2/p53和MDMX/p53相互作用，具有治疗相关疾病的潜力。
发明内容
本发明的目的在于提供一种阻断MDM2/p53和/或MDMX/p53相互作用的小分子抑制剂。
本发明的第一方面，提供一种如式(I)所述的化合物、其对映异构体、非对映异构体、消 旋体或其药学上可接受的盐，
式中，Ar为取代或未取代的苯基，其中，所述的取代是指苯基上的一个或多个氢原子被选自下组的基团取代：卤素、氘、氰基、羟基、氨基、硝基、取代或未取代的C2-C4炔基、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C2-C10酰基、取代或未取代的C1-C4烷基羰基；
R1和R2各自独立地为氢、氘、卤素、氰基、取代或未取代的C2-C4炔基、取代或未取代的C1-C4烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C4烷氧基；
R3为
Y、Z各自独立地为氢、-(CH2)m-取代或未取代的6-10元芳基、-(CH2)m-取代或未取代的5-12元杂芳基、取代或未取代的C1-C6烷基、-(CH2)m-取代或未取代的C3-C8环烷基、-(CH2)m-取代或未取代的5-13元杂环基；
R4为取代或未取代的C1-C8烷基、-(CH2)m-取代或未取代的C3-C8环烷基、-(CH2)m-取代或未取代的4-13元杂环基、-(CH2)m-取代或未取代的6-10元芳基、-(CH2)m-取代或未取代的5-12元杂芳基、C2-C8炔烃基、C2-C8烯基；
m在各出现处各自独立地为0、1、2、3或4；
R5为取代或未取代的C1-C8烷基、-(CH2)m-取代或未取代的C3-C8环烷基、-(CH2)m-取代或未取代的4-12元杂环烷基；
除非另有定义，以上所述的各取代各自独立地是指基团上的一个或多个氢原子被选自下组的基团取代：卤素、氘、氰基、羟基、氨基、硝基、C1-C4烷基磺酰基、C1-C4烷基磺酰胺基羰基(C1-C4烷基SO2NHCO-)、羧基、-CONH2、C1-C4烷基、C3-C6环烷基、C1-C4烷氧基、C2-C4烯基、C2-C4炔基、C1-C4烷基乙炔基、单(C1-C4烷基)氨基、二(C1-C4烷基)氨基、C1-C4烷基羰基氨基、C1-C4烷氧基羰基氨基、C1-C4烷氧基羰基、C1-C4烷基SO2-、C1-C4烷基-S(O2)-C1-C4亚烷基-、羧基取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷基-S-、C2-C10酰基、C1-C4烷基羰基、C1-C4烷基氨基羰基、5-12元杂芳基、5-12元杂芳基羰基、C1-C4烷基5-12元杂芳基羰基、C1-C4烷基-CO-O-C1-C4亚烷基-O-CO-。
在另一优选例中，Ar为取代或未取代的苯基，其中，所述的取代是指苯基上的1、2或3个氢原子被选自下组的基团取代：卤素、C1-C4烷基、C3-C6环烷基、C1-C4烷氧基。在另一优选例中，Ar为取代或未取代的苯基，其中，所述的取代是指苯基上的1、2或3个氢原子被选自下组的基团取代：氟、氯、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基。
在另一优选例中，R1和R2各自独立地为氢、氘、卤素、氰基、C1-C4烷基。在另一优选例中，R1和R2各自独立地为氢、氘、卤素。在另一优选例中，R1和R2各自独立地为氢、 氘、氯、氟。
在另一优选例中，R3为Y为氢、C1-C4烷基；Z为-(CH2)m-取代或未取代的苯基、-(CH2)m-取代或未取代的5-7元杂芳基、-(CH2)m-取代或未取代的C6-C8环烷基、-(CH2)m-取代或未取代的5-8元杂环基。在另一优选例中，Z为-(CH2)m-取代或未取代的苯基、-(CH2)m-取代或未取代的6元杂芳基、-(CH2)m-取代或未取代的C5-C8环烷基、-(CH2)m-取代或未取代的6-8元杂环基。在另一优选例中，上述取代是指基团上的1、2或3个氢原子被选自下组的基团取代：羟基、羧基、C1-C4烷氧基、卤素、氨基、氘、C1-C4烷基、-CONH2、C1-C4烷基氨基CO-、氰基、羧基取代的C1-C4烷基、、C1-C4烷基-CO-O-C1-C4亚烷基-O-CO-。在另一优选例中，m为0、1、2或3。
在另一优选例中，R4为氢、取代或未取代的C1-C6烷基、-(CH2)m-取代或未取代的C3-C6环烷基、-(CH2)m-取代或未取代的4-6元杂环基、-(CH2)m-取代或未取代的苯基、-(CH2)m-取代或未取代的5-7元杂芳基、C3-C6炔基、C3-C6烯基。在另一优选例中，m为0、1、2或3。在另一优选例中，上述取代是指基团上的1、2或3个氢原子被选自下组的基团取代：卤素、氘、氰基、羟基、C1-C4烷氧基羰基、C1-C4烷基、C2-C4炔基、C2-C4烯基。
在另一优选例中，R5为取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的4-6元杂环烷基；在另一优选例中，上述取代是指基团上的1、2或3个氢原子被选自下组的基团取代：C1-C4烷基、C1-C4烷基CO-、C1-C4烷基COO-。
在另一优选例中，R5为C1-C6烷基、C1-C4烷基取代的C3-C6环烷基、C1-C4烷基和/或C1-C4烷基CO-取代的4-6元杂环烷基。在另一优选例中，R5为叔丁基、
在另一优选例中，所述化合物具有如式II、III、IV、V、VI、VII或VIII所示的结构：

各基团的定义如前所述。
在另一优选例中，各取代基为具体化合物中对应的基团。
在另一优选例中，上述杂环基、杂芳基各自独立地含有1、2、3或4个选自N、O的杂原子。
在另一优选例中，所述化合物选自序号1-97任一的化合物。
本发明提供的化合物，能够用作抑制MDM2-p53、MDMX-p53蛋白蛋白相互作用的小分子抑制剂。
本发明的第二方面，提供一种药物组合物，包括如第一方面所述的化合物，其对映异构体、非对映异构体、消旋体或药学上可接受的盐中的一种或多种；和药学上可接受的载体。
在另一优选例中，所述药物组合物任选的还包含药学上可接受的辅料，所述辅料选自下组：粘合剂、填充剂、稀释剂、崩解剂、混悬剂、助悬剂、缓(控)释剂、冻干保护剂、包衣剂、肠溶材料、润滑剂、助流剂、抗粘剂、甜味剂、风味剂、增塑剂、遮光剂、增溶剂、保湿剂、溶剂、渗透压调节剂、着色剂、色素、表面活性剂、乳化剂、水溶性基质、脂溶性基质、油脂性基质、致孔剂、凝胶剂、防腐剂、缓冲剂、螯合剂、抗氧剂，或其组合。
本发明的第三方面，提供第一方面所述的化合物、其对映异构体、非对映异构体、消旋体或其药学上可接受的盐的用途，用于制备阻断MDM2/p53和/或MDMX/p53相互作用的小分子抑制剂；或用于制备治疗与MDM2或MDMX蛋白的活性或表达量相关的疾病的药物。
在另一优选例中，所述与MDM2或MDMX蛋白的活性或表达量相关的疾病选自下组：神经胶质瘤、脂肪肉瘤、皮肤黑色素瘤、鳞状上皮细胞癌、视网膜母细胞癌、乳腺癌、食道癌、肺癌、卵巢癌、胃癌、膀胱癌、肝癌、软组织肉瘤、慢性淋巴白血病、急性髓性白血病、淋巴瘤、骨肉瘤和结肠癌。
与现有技术相比，本发明的主要优点包括：
(1)提供了一类结构新颖的具有取代苯基螺[吲哚啉-3,3'-吡咯烷]结构及其类似结构的小分子化合物，其制备方法具有反应条件温和、原料丰富易得、操作及后处理简单、对映选择性好等优点。
(2)提供了一种同时抑制MDM2-p53、MDMX-p53蛋白蛋白相互作用的小分子抑制剂，该类抑制剂对于p53野生型、MDM2过表达细胞具有很强的增殖抑制能力，且其溶解度好、生物利用度高、代谢性质优良，是一类潜在的抗肿瘤药物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述 的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅，在此不再一一累述。
附图说明
图1示出JM085-CF2的绝对立体构型。
图2示出JN110的绝对立体构型。
图3为化合物浓度-峰面积比值标准曲线。
图4示出18(JN122)在HCT116细胞中的作用机制研究结果。
图5示出18(JN122)在多种实体瘤癌细胞系中的作用机制研究结果
图6示出18(JN122)在MOLM-13细胞中的作用机制研究结果。
图7示出18(JN122)在MOLM-13小鼠异种移植瘤模型中的药效学研究结果。
具体实施方式
本发明的发明人经过长期而深入的研究，意外地研发出具有取代苯基螺[吲哚啉-3,3'-吡咯烷]结构及其类似结构的小分子化合物，其能够抑制MDM2-p53、MDMX-p53蛋白蛋白相互作用，调控肿瘤细胞内p53介导的基因表达，因此可以用于抑制MDM2-p53、MDMX-p53相互作用相关的疾病，如癌症的预防和治疗。基于上述发现，发明人完成了本发明。
术语
在本发明中，所述卤素为F、Cl、Br或I。
在本发明中，除非特别指出，所用术语具有本领域技术人员公知的一般含义。
在本发明中，术语“C1-C6”是指具有1、2、3、4、5或6个碳原子，“C1-C4”是指具有1、2、3或4个碳原子，依此类推。“4-12元”是指具有4、5、6、7、8、9、10、11或12个环原子，依此类推。
在本发明中，术语“烷基”表示饱和的线性或支链烃部分，例如术语“C1-C8烷基”是指具有1至8个碳原子的直链或支链烷基，非限制性地包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、戊基和已基等；优选乙基、正丙基、异丙基、正丁基、异丁基、仲丁基和叔丁基。
在本发明中，术语“烷氧基”表示-O-(烷基)基团。例如术语“C1-C6烷氧基”是指具有1至6个碳原子的直链或支链烷氧基，非限制性地包括甲氧基、乙氧基、正丙氧基、异丙氧基和丁氧基等。
在本发明中，术语“烯基”表示包含至少一个双键的直链或支链烃基部分，例如术语“C2-C6烯基”是指具有2至6个碳原子的含有一个双键的直链或支链烯基，非限制性地包括乙烯基、丙烯基、丁烯基、异丁烯基、戊烯基和己烯基等。
在本发明中，术语“炔基”是指含有一个三键的直链或支链炔基，非限制性地包括乙炔基、丙炔基、丁炔基、异丁炔基、戊炔基(2-甲基3-丁炔基、2-戊炔基、3-戊炔基)和己炔基等。
在本发明中，术语“环烷基”表示饱和的单环、桥环或螺环的环状烃基部分，例如术语“C3-C8环烷基”是指在环上具有3至8个碳原子的单环、桥环或螺环的环状烷基，非限制性地 包括环丙基、环丁基、环戊基、环己基、环庚基、环辛基和环癸基等。术语“桥环基”非限制性包括双环[1,1,1]戊烷基、双环[2,1,1]己烷基、双环[2,2,1]庚烷基、双环[2,2,2]辛烷基、双环[3,2,2]辛烷基等。
在本发明中，术语“芳基”表示包含一个或多个芳环的烃基部分。例如术语“C6-C10芳基”是指在环上不含杂原子的具有6至10个碳原子的芳香族环基，如苯基、萘基等。
在本发明中，术语“杂环基”表示包含至少一个(如1、2、3或4个)环杂原子(例如N，O或S)的饱和或不饱和的、非芳香性的环状基团，例如四氢吡啶基、吡咯啉基、二氢吡啶基、二氢呋喃基、二氢噻吩基、吗啉基。
在本发明中，术语“杂芳基”表示包含至少一个(如1、2、3或4个)环杂原子(例如N，O或S)的芳香性的环状基团，例如呋喃基、吡咯基、噻吩基、噁唑基、咪唑基、噻唑基、吡啶基、喹啉基、异喹啉基、吲哚基、嘧啶基、吡喃基。
化合物
本发明的化合物，结构如通式I所示：
在另一优选例中，所述的化合物具有如下式I-1所示的结构：
在另一优选例中，Ar为取代或未取代的苯基，所述取代是指苯基上的1、2、3或4个氢原子被选自下组的基团取代：氟、氯、溴、甲氧基、甲基、乙基、正丙基、异丙基、正丁基、异丁基、卤代C1-C4烷基(如三氟甲基)；R1和R2各自独立地为氢、氘、氟、氯或溴；
R3为OH或Y为H；Z为H、取代或未取代的C1-C4烷基、-(CH2)m-取代或未取代的苯基、-(CH2)m-取代或未取代的5-7元杂芳基、-(CH2)m-取代或未取代的C3-C8环烷基、或-(CH2)m-取代或未取代的5-7元杂环基；或Y、Z与N形成取代或未取代的5-7元杂环基；所述取代是指被选自下组的1、2、3或4个基团取代：氟、氯、溴、氨基、羟基、羧基、C1-C4烷氧基、-CONH2、C1-C4烷基-CONH-、C1-C4烷基-NHCO-、C1-C4烷基、C1-C4烷基-S(O2)-C1-C4亚烷基-、单(C1-C4烷基)氨基、二(C1-C4烷基)氨基、C1-C4烷基SO2NHCO-、羧基取代的C1-C4烷基、羟基取代的C1-C4烷基；各m独立地为0、1或2；
R4为取代或未取代的C1-C4烷基、-(CH2)m-取代或未取代的苯基、-(CH2)m-取代或未取 代的C3-C6环烷基；各m独立地为0、1或2；所述取代是指被选自下组的1、2、4或3个基团取代：氟、氯、溴、氰基、氨基、羟基、硝基、羧基、C1-C4烷氧基、-CONH2、C1-C4烷基-CONH-、C1-C4烷基-NHCO-、C1-C4烷基、C1-C4烷基-S(O2)-C1-C4亚烷基-、C1-C4烷基SO2-、5-7元杂芳基、羧基取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷基SO2NHCO-；
R5为取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的5-7元杂环基；所述取代是指被选自下组的一个或多个基团取代：C1-C4烷基、C1-C4烷基羰基。
在另一优选例中，本发明中的杂芳基选自：四唑基、异恶唑基、恶唑基、吡啶基、咪唑基、吡唑基。
在另一优选例中，本发明中的杂芳基选自：
在另一优选例中，本发明中的杂环基选自：
制备方法
本发明的化合物，可以通过下述反应路线制备。
路线一：制备化合物(I)的路线
步骤一：醛S1与取代的2-氟苯乙腈S2在适当溶剂(例如：甲醇、乙醇、四氢呋喃、2- 甲基四氢呋喃、二氯甲烷、或者N、N-二甲基甲酰胺等)中混合。加入适当碱(甲醇钠、乙醇钠等)，在室温或适当升高温度条件下(例如40-60℃)条件下，反应得到中间体S3。步骤二：S3与相应原料S4-2在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、或者乙腈等)混合，加入氟化银和碱(三乙胺、N,N-二异丙基乙胺和DBU等)，在室温条件下,反应得到中间体S5-2。步骤三：S5-2通过氢化还原反应得到中间体S6-2(例如Pd/C氢气氢化还原，雷尼镍氢气氢化还原以及雷尼镍水合肼氢化还原等)。步骤四：S6-2与FmocCl在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、或者乙腈等)中混合，加入碱(三乙胺、N,N-二异丙基乙胺和DBU等)，在室温条件下,反应得到中间体S7-2。步骤五：S7-2在二氯甲烷中溶解，加入三氟乙酸，在室温条件下,反应得到中间体S8-2。步骤六：S8-2在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、N、N-二甲基甲酰胺或者乙腈等)中溶解，加入碱(三乙胺、N,N-二异丙基乙胺和DBU等)和缩合试剂(二苯基次磷酰氯、CDI、PyBOP、HATU和EDCI等)，反应半小时后加入胺，在室温条件下,反应得到中间体S9-2。步骤七：S9-2和R4CHO在合适的溶剂中溶解(例如：甲醇、四氢呋喃、乙醇、1，2-二氯乙烷和N,N-二甲基甲酰胺等)，加入还原试剂(硼氢化钠、醋酸硼氢化钠、氰基硼氢化钠等)，通过还原胺化反应得到中间体S10-2。步骤八：S10-2在适当溶剂(例如：N,N-二甲基甲酰胺等)中溶解，加入哌啶，在室温条件下,反应得到中间体S11-2。步骤九：S11-2在适当溶剂(例如：乙腈、二甲基亚砜、N,N-二甲基甲酰胺、甲苯和苯等)中溶解，加入碱(例如：碳酸钾、碳酸铯和碳酸钠等)，在升高温度条件下(例如80-120℃)，反应得到终产物I。
路线二：制备化合物(II)的路线
步骤一：醛S1与取代的2-氟苯乙腈S2在适当溶剂(例如：甲醇、乙醇、四氢呋喃、2-甲基四氢呋喃、二氯甲烷、或者N、N-二甲基甲酰胺等)中混合。加入适当碱(甲醇钠、乙醇 钠等)，在室温或适当升高温度条件下(例如40-60℃)条件下，反应得到中间体S3。步骤二：S3与相应原料S4在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、或者乙腈等)混合，加入氟化银和碱(三乙胺、N,N-二异丙基乙胺和DBU等)，在室温条件下,反应得到中间体S5。步骤三：S5通过氢化还原反应得到中间体S6(例如Pd/C氢气氢化还原，雷尼镍氢气氢化还原以及雷尼镍水合肼氢化还原等)。步骤四：S6与FmocCl在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、或者乙腈等)中混合，加入碱(三乙胺、N,N-二异丙基乙胺和DBU等)，在室温条件下,反应得到中间体S7。步骤五：S7在二氯甲烷中溶解，加入三氟乙酸，在室温条件下,反应得到中间体S8。步骤六：S8在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、N、N-二甲基甲酰胺或者乙腈等)中溶解，加入碱(三乙胺、N,N-二异丙基乙胺和DBU等)和缩合试剂(二苯基次磷酰氯、CDI、PyBOP、HATU和EDCI等)，反应半小时后加入胺，在室温条件下,反应得到中间体S9。步骤七：S9和R4CHO在合适的溶剂中溶解(例如：甲醇、四氢呋喃、乙醇、1，2-二氯乙烷和N,N-二甲基甲酰胺等)，加入还原试剂(硼氢化钠、醋酸硼氢化钠、氰基硼氢化钠等)，通过还原胺化反应得到中间体S10。步骤八：S10在适当溶剂(例如：N,N-二甲基甲酰胺等)中溶解，加入哌啶，在室温条件下,反应得到中间体S11。步骤九：S11在适当溶剂(例如：乙腈、二甲基亚砜、N,N-二甲基甲酰胺、甲苯和苯等)中溶解，加入碱(例如：碳酸钾、碳酸铯和碳酸钠等)，在升高温度条件下(例如80-120℃)，反应得到终产物II。
路线三：制备化合物(II)的路线
步骤一：S9在适当溶剂(例如：N,N-二甲基甲酰胺等)中溶解，加入哌啶，在室温条件下,反应得到中间体S12。步骤二：S12和对甲氧基苯甲醛在合适的溶剂中溶解(例如：甲醇、四氢呋喃、乙醇、1，2-二氯乙烷和N,N-二甲基甲酰胺等)，加入还原试剂(硼氢化钠、醋酸硼氢化钠、氰基硼氢化钠等)，通过还原胺化反应得到中间体S13。步骤三：S13在适当溶剂(例如：乙腈、二甲基亚砜、N,N-二甲基甲酰胺、甲苯和苯等)中溶解，加入碱(例如：碳酸钾、碳酸铯和碳酸钠等)，在升高温度条件下(例如80-120℃)，反应得到中间体S14。步骤四：S14和R4CHO在合适的溶剂中溶解(例如：甲醇、四氢呋喃、乙醇、1，2-二氯乙烷和N,N-二甲基甲酰胺等)，加入还原试剂(硼氢化钠、醋酸硼氢化钠、氰基硼氢化钠等)，通过还原胺化反应得到中间体S15。步骤五：S15在三氟乙酸中溶解，在升高温度的条件下，反应得到终产物II。
I，I-1可以利用上述三种合成策略合成制备。
路线四：采用手性催化的合成方法，制备具有光学活性的化合物(II)
步骤一：S3与相应原料S4在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、或者乙腈等)混合，除氧气，氮气置换后，加入醋酸亚铜和R-(+)-1,1'-联萘-2,2'-双二苯膦(BINAP)，滴加碱(三乙胺、N,N-二异丙基乙胺和DBU等)，在室温条件下,反应得到中间体S16，S16为单一对映体或者ee>50％的对映体混合物。类似的，双膦配体还可以为R-(+)-1,1'-联萘-2,2'-双二(4-甲基苯)膦，R-(+)-1,1'-联萘-2,2'-双二(3,5-二甲基苯)膦，R-(+)-1,1'-联萘-2,2'-双二(4-碘苯)膦,R-(+)-1,1'-联萘-2,2'-双二(4-甲氧基苯)膦,(R)-(+)-2,2'-双(二苯磷基)-5,5',6,6',7,7',8,8'-八氢-1,1'-联萘等。步骤二：S16通过氢化还原反应得到中间体S17(例如Pd/C氢气氢化还原，雷尼镍氢气氢化还原以及雷尼镍水合肼氢化还原等)。步骤三：S17与FmocCl在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、或者乙腈等)中混合，加入碱(三乙胺、N,N-二异丙基乙胺和DBU等)，在室温条件下,反应得到中间体S18。步骤四：S18和R4CHO在合适的溶剂中溶解(例如：甲醇、四氢呋喃、乙醇、1，2-二氯乙烷和N,N-二甲基甲酰胺等)，加入还原试剂(硼氢化钠、醋酸硼氢化钠、氰基硼氢化钠等)，通过还原胺化反应得到中间体S19。步骤五：S19在二氯甲烷中溶解，加入三氟乙酸，在室温条件下,反应得到中间体S20。步骤六：S20在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、N、N-二甲基甲酰胺或者乙腈等)中溶解，零摄氏度下加入碱(1-甲基咪唑、三乙胺、N,N-二异丙基乙胺和DBU等)和缩合试剂(乙基磺酰氯、二苯基次磷酰氯、CDI、PyBOP、HATU和 EDCI等)，反应半小时后加入胺，在室温条件下,反应得到中间体S21。步骤七：S21在适当溶剂(例如：N,N-二甲基甲酰胺等)中溶解，加入哌啶，在室温条件下,反应得到中间体S22。步骤八：S22在适当溶剂(例如：乙腈、二甲基亚砜、N,N-二甲基甲酰胺、甲苯和苯等)中溶解，加入碱(例如：碳酸钾、碳酸铯和碳酸钠等)，在升高温度条件下(例如80-120℃)，反应得到终产物II，终产物II为单一对映体或者ee>50％的对映体混合物。
路线五：采用手性催化的合成方法，制备具有光学活性的化合物(I)
S23到S29为ee>20％具有光学活性对映体混合物
步骤一：S3与相应原料S4-2在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、或者乙腈等)混合，除氧气，氮气置换后，加入醋酸亚铜和R-(+)-1,1'-联萘-2,2'-双二苯膦(BINAP)，滴加碱(三乙胺、N,N-二异丙基乙胺和DBU等)，在室温条件下,反应得到中间体S23，S23为单一对映体或者ee>20％的对映体混合物。类似的，双膦配体还可以为R-(+)-1,1'-联萘-2,2'-双二(4-甲基苯)膦，R-(+)-1,1'-联萘-2,2'-双二(3,5-二甲基苯)膦，R-(+)-1,1'-联萘-2,2'-双二(4-碘苯)膦,R-(+)-1,1'-联萘-2,2'-双二(4-甲氧基苯)膦,(R)-(+)-2,2'-双(二苯磷基)-5,5',6,6',7,7',8,8'-八氢-1,1'-联萘等。步骤二：S23通过氢化还原反应得到中间体S24(例如Pd/C氢气氢化还原，雷尼镍氢气氢化还原以及雷尼镍水合肼氢化还原等)。步骤三：S24与FmocCl在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、或者乙腈等)中混合，加入碱(三乙胺、N,N-二异丙基乙胺和DBU等)，在室温条件下,反应得到中间体S25。步骤四： S25和R4CHO在合适的溶剂中溶解(例如：甲醇、四氢呋喃、乙醇、1，2-二氯乙烷和N,N-二甲基甲酰胺等)，加入还原试剂(硼氢化钠、醋酸硼氢化钠、氰基硼氢化钠等)，通过还原胺化反应得到中间体S26。步骤五：S26在二氯甲烷中溶解，加入三氟乙酸，在室温条件下,反应得到中间体S27。步骤六：S27在适当溶剂(例如：四氢呋喃、2-甲基四氢呋喃、二氯甲烷、N、N-二甲基甲酰胺或者乙腈等)中溶解，零摄氏度下加入碱(1-甲基咪唑、三乙胺、N,N-二异丙基乙胺和DBU等)和缩合试剂(乙基磺酰氯、二苯基次磷酰氯、CDI、PyBOP、HATU和EDCI等)，反应半小时后加入胺，在室温条件下,反应得到中间体S28。步骤七：S28在适当溶剂(例如：N,N-二甲基甲酰胺等)中溶解，加入哌啶，在室温条件下,反应得到中间体S29。步骤八：S29在适当溶剂(例如：乙腈、二甲基亚砜、N,N-二甲基甲酰胺、甲苯和苯等)中溶解，加入碱(例如：碳酸钾、碳酸铯和碳酸钠等)，在升高温度条件下(例如80-120℃)，反应得到终产物I，终产物I为单一对映体或者ee>20％的对映体混合物。
实施例1：化合物的合成
合成中间体1：4-((2′,3S,4′,5′-R)-6-氯-4-((2,3-二氟苯基)-1-(4-甲氧基苄基)-2基苄新戊螺[吲哚啉-3,3′-吡咯烷]-5′羧酰胺)-3-甲氧苯甲酸甲酯(JM158)
步骤一：合成(Z)-2-(4-氯-2-氟苯基)-3-(2,3-二氟苯基)丙烯腈(JM029)
将2,3-二氟苯甲醛(2.9g,20mmol)和4-氯-2-氟苯乙腈(3.4g,20mmol)称入250mL圆底烧瓶中，加150mL甲醇溶解反应物，滴加5N甲醇钠的甲醇溶液4.8mL，反应体系在50℃条件下搅拌过夜。反应结束后，冷至室温，过滤得粗产品5.8g,产率99％。1H NMR(500MHz,Chloroform-d)δ8.00(dd,J＝8.0,6.2Hz,1H),7.78(s,1H),7.56(t,J＝8.4Hz,1H),7.35–7.16(m,4H).
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(2,3-二氟苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(JM036)
将JM029(5.7g,20mmol)和AgF(2.54g,20mmol)称入100mL圆底烧瓶中，加30mL超干二氯甲烷溶解，滴加三乙胺4.4mL和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(4.4g,20mmol)，室温反应24h。反应结束后，向反应液中加入饱和氯化铵，用二氯甲烷萃取(30mL×3次)，合并有机相，用饱和氯化钠洗两次，用无水硫酸钠干燥，旋干有机相过柱纯化得目标产物6.8g，产率67％。1H NMR(500MHz,Chloroform-d)δ7.45(dd,J＝7.9,6.0Hz,1H),7.35(t, J＝8.5Hz,1H),7.19–7.02(m,4H),4.68(dd,J＝7.5,1.9Hz,1H),4.16(d,J＝7.5Hz,1H),4.08(d,J＝8.9Hz,1H),1.62(ddd,J＝14.2,9.1,1.7Hz,1H),1.38(s,9H),1.28(dd,J＝14.3,0.9Hz,1H),0.89(s,9H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(2,3-二氟苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(JM040)
将JM036(1.6g,8.5mmol)称入100mL圆底烧瓶中，用四氢呋喃/EtOH(30mL/10mL)溶解，加热到55℃，加入雷尼镍4g，水合肼10mL，反应2h，过滤，转干溶剂，过正相柱纯化得目标产物1.6g，产率24％。1H NMR(500MHz,Chloroform-d)δ7.49(t,J＝6.7Hz,1H),7.09(t,J＝8.6Hz,1H),7.06–6.94(m,4H),4.20–4.13(m,3H),3.29(dd,J＝13.1,1.5Hz,1H),3.19(d,J＝13.2Hz,1H),1.50–1.44(m,2H),1.25(s,9H),0.94(s,9H).
步骤四：合成(2R,3R,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(2,3-二氟苯基)-5-新戊基吡咯烷-2-羧酸(JM048)
JM040(1.4g,2.7mmol)称入50mL单口瓶中，用干燥的四氢呋喃溶解，加入二异丙基乙基胺(1.4g,10.8mmol)和FmocCl(1.1g,4.1mmol)，室温反应过夜。减压旋干反应物，用5mL二氯甲烷溶解反应物，加入4mL三氟乙酸，室温过夜。转干反应液，加入饱和碳酸氢钠溶液，用二氯甲烷萃取三次，合并有机相，转干后正相柱纯化得目标产物1.31g，产率73％。
步骤五：合成甲基4-((2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(2,3-二氟苯基)-5-新戊基吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸酯(JM148)
JM048(515mg,0.8mmol)加入50mL单口瓶中，用干燥的四氢呋喃溶解，加入二异丙基乙基胺(504mg,3.9mmol)，搅拌5分钟，加入二苯基次磷酰氯(555mg,2.3mmol)，搅拌半小时后加入4-氨基-3-甲氧基苯甲酸甲酯(562mg,3.1mmol)，室温反应过夜。向反应液中加入饱和碳酸氢钠溶液，二氯甲烷萃取，有机相转干后正相柱纯化，得到的粗品用DMF(2mL)溶解，加入哌啶(0.4mL)，室温反应15分钟，1N HCl溶液洗3次，饱和氯化钠溶液洗3次，有机相旋干后过正相柱纯化得目标产物285mg，产率73％。1H NMR(400MHz,Methanol-d4)δ8.26(d,J＝8.5Hz,1H),7.61–7.53(m,2H),7.47–7.36(m,1H),7.34(d,J＝8.5Hz,1H),7.32–7.05(m,4H),4.65(d,J＝10.3Hz,1H),4.46(d,J＝10.9Hz,1H),3.99(d,J＝12.4Hz,1H),3.92(s,3H),3.86(s,3H),3.78(dd,J＝14.3,3.2Hz,1H),3.56(d,J＝14.5Hz,1H),1.73(d,J＝13.7Hz,1H),1.69–1.57(m,1H),1.23(s,9H).
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(2,3-二氟苯基)-4-氟-1-(4-甲氧基苄基)-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸甲酯(JM158)
烘干一个50mL单口瓶，加入JM148(285mg,0.46mmol)和4-甲氧基苯甲醛(252mg,1.85mmol)，用5mL甲醇溶解反应物，加入氰基硼氢化钠(117mg,1.85mmol)和0.1mL乙酸，室温反应过夜，旋干反应物，加饱和碳酸氢钠，乙酸乙酯萃取，饱和食盐水洗涤，无水硫酸钠干燥，旋转蒸发仪浓缩。将反应得到的粗品加入烘干的50ml单口瓶中，用5mL DMF溶解，加入碳酸钾(197mg,1.42mmol)，110℃搅拌过夜，反应结束后冷至室温，加水，乙酸乙酯萃取，饱和食盐水洗涤，无水硫酸钠干燥，有机相旋干后正相柱纯化，得目标产物186mg，产率72％。1H NMR(400MHz,Chloroform-d)δ10.50(s,1H),8.48(d,J＝8.4Hz,1H),7.65(d,J＝8.5Hz,1H),7.55(s,1H),7.05(d,J＝8.1Hz,2H),7.03–6.96(m,1H),6.98–6.90(m,2H),6.84–6.73(m,3H),6.65(d,J＝7.9Hz,1H),6.34(s,1H),4.39(d,J＝9.1Hz,1H),4.07(d,J＝14.7Hz, 1H),4.01–3.94(m,2H),3.94–3.85(m,6H),3.81(s,3H),3.27(d,J＝10.2Hz,1H),3.23(d,J＝9.5Hz,1H),3.09(d,J＝10.1Hz,1H),1.46(d,J＝14.4Hz,1H),1.20–1.08(m,1H),0.96(s,9H).
合成中间体2：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯-2-氟苯基)-1-(4-甲氧基苄基)-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸甲酯(YM155)
步骤一：合成甲基4-((2R,3S,4S,5S)-4-(氨基甲基)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-5-新戊基吡咯烷-2-羧酰胺)-3-甲氧基苯甲酸酯(YI045)
将甲基4-((2R,3S,4R,5S)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-氰基-5-新戊基吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸酯(2.5g,3.96mmol)溶于四氢呋喃/乙醇(10/10mL)，加入雷尼镍2.5g，升至55℃，加入水合肼10mL，反应至不产生气体。过滤，滤液减压蒸干后用正相柱纯化得目标化合物637mg，产率：25％。1H NMR(500MHz,Methanol-d4)δ8.24(d,J＝8.2Hz,1H),7.59–7.50(m,2H),7.50–7.43(m,1H),7.40(t,J＝8.6Hz,1H),7.36–7.26(m,2H),7.26–7.13(m,2H),4.59(d,J＝10.2Hz,1H),4.41(d,J＝11.1Hz,1H),3.97(d,J＝10.3Hz,1H),3.91(s,3H),3.85(s,3H),3.78(dd,J＝14.4,3.2Hz,1H),3.52(d,J＝14.4Hz,1H),1.72(d,J＝13.6Hz,1H),1.60(dd,J＝13.8,11.3Hz,1H),1.23(s,9H).13C NMR(126MHz,Methanol-d4)δ172.68,168.02,163.96(d,JC-F＝249.5Hz),161.84(TFA,q,JC-F＝36.5Hz),158.22(d,JC-F＝248.2Hz),149.58,136.71(d,JC-F＝11.3Hz),132.46,131.36,130.89(d,JC-F＝5.0Hz),130.48,127.23(d,JC-F＝1.3Hz),126.76,126.01(d,JC-F＝10.1Hz),125.95,123.89,123.12(d,JC-F＝11.3Hz),122.73(d,JC-F＝20.2Hz),119.34,118.88(d,JC-F＝29.0Hz),117.50(TFA,q,JC-F＝289.8Hz),111.83,63.89,63.55,58.24(d,JC-F＝3.8Hz),56.34,52.62,43.20,38.24(d,JC-F＝7.6Hz),32.14,30.68.ESI-MS理论计算值C32H3635Cl2F2N3O4[M+H]+＝634.2,实验测得：634.1。
步骤二：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯-2-氟苯基)-1-(4-甲氧基苄基)-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸甲酯(YM155)
YI045(322mg,0.51mmol)，4-甲氧基苯甲醛(136mg,1mmol)，氰基硼氢化钠(126mg,2mmol)，乙酸0.1mL，碳酸钾(207mg,1.5mmol),反应步骤参见中间体1的步骤六，得目标产物363mg，产率93％。1H NMR(500MHz,Chloroform-d)δ10.47(s,1H),8.46(d,J＝8.6Hz,1H),7.62(dd,J＝8.5,1.9Hz,1H),7.52(d,J＝1.9Hz,1H),7.30–7.20(m,1H),7.03(d,J＝8.0Hz,1H),6.95–6.87(m,3H),6.86(d,J＝8.6Hz,1H),6.79–6.73(m,2H),6.62(dd,J＝7.9,1.8Hz,1H),6.31(d,J＝1.8Hz,1H),4.34(d,J＝9.1Hz,1H),4.03(d,J＝14.9Hz,1H),3.96–3.90(m,2H),3.87(s,3H),3.86(s,3H),3.78(s,3H),3.23(d,J＝10.1Hz,1H),3.18(d,J＝9.4Hz,1H),3.02(dd,J＝10.0,1.3Hz,1H),1.42(dd,J＝14.2,1.3Hz,1H),1.15–1.06(m,1H),0.93(s,9H).
合成中间体3：合成甲基4-((2S,3R,4R,5R)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((4-甲氧基苄基)氨基)甲基)-5-新戊基吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸酯(YN20-CF1)
步骤一：合成对映体(2R,3S,4S,5S)-4-(氨基甲基)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯和对映体(2S,3R,4R,5R)-4-(氨基甲基)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯(YM159)
将SM1(5.45g,1.04mmol)称入100mL圆底烧瓶，加入雷尼镍(Raney Nickel)2g，用四氢呋喃/乙醇溶解，升至55℃，加水合肼10mL，反应至不冒泡，过滤旋干滤液，正相柱纯化得目标化合物1.9g，产率：36％。1H NMR(500MHz,Methanol-d4)δ7.54–7.42(m,2H),7.34(dd,J＝8.7,2.2Hz,1H),7.31–7.22(m,3H),4.72(d,J＝10.3Hz,1H),4.67(d,J＝9.9Hz,1H),4.30(d,J＝9.9Hz,1H),3.66–3.60(m,1H),3.60–3.52(m,1H),1.77(dd,J＝14.4,10.4Hz,1H),1.67–1.54(m,1H),1.31(s,9H),1.08(s,9H).13C NMR(126MHz,Methanol-d4)δ170.86,163.01(TFA,q,JC-F＝35.3Hz),162.47(d,JC-F＝250.7Hz),157.99(d,JC-F＝248.2Hz),137.10(d,JC-F＝11.3Hz),131.82,130.70(d,JC-F＝5.0Hz),129.59,127.05(d,JC-F＝3.8Hz),126.50(d,JC-F＝3.8Hz),124.95(d,JC-F＝13.9Hz),123.09(d,JC-F＝10.1Hz),122.71(d,JC-F＝18.9Hz),119.01(d,JC-F＝30.2Hz),118.00(TFA,q,JC-F＝292.3Hz),84.70,63.16(d,JC-F＝3.8Hz),61.59,55.58(d,JC-F＝5.0Hz),51.79,42.42,39.29(d,JC-F＝6.3Hz),31.83,30.01,27.88.LRMS(ESI)calculated for C27H35 35Cl2F2N2O2[M+H]+＝527.2,obtained:527.2。
步骤二：合成差向异构体(2R,3S,4S,5S)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((R)-2-甲氧基-2-苯基乙酰胺)甲基)-5-新戊基吡咯烷-2-羧酸和差向异构体(2S,3R,4R,5R)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((R)-2-甲氧基-2-苯基乙酰胺)甲基)-5-新戊基吡咯烷-2-羧酸(YN003)
将YM159(210mg,0.4mmol)溶于二氯甲烷，加入(R)-(-)-alpha-甲氧基苯乙酰氯(110mg，0.6mmol)和三乙胺(162mg，1.6mmol)，室温搅拌过夜。反应结束后加水，用二氯甲烷萃取，有机相旋干后用正相柱纯化得粗品。粗品溶于二氯甲烷(5mL)，加入三氟醋酸(2mL)，室温过夜。反应结束后加水，用二氯甲烷萃取，有机相旋干后用正相柱纯化得目标化合物226mg，产率：91％。
步骤三：合成(2S,3R,4R,5R)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((R)-2-甲氧基-2-苯基乙酰胺)甲基)-5-新戊基-N-((S)-1-苯乙基)吡咯烷-2-甲酰胺(YN015-CF1)和(2R,3S,4S,5S)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((R)-2-甲氧基-2-苯基乙酰胺)甲基)-5-新戊基-N-((S)-1-苯乙基)吡咯烷-2-甲酰胺(YN015-CF2)
将YN003(226mg，0.36mmol)称入瓶中，用四氢呋喃溶解，加入二异丙基乙基胺(232mg，1.8mmol)和二苯基次磷酰氯(255mg，1.08mmol)，室温搅拌30min后加入(S)-1-苯乙基胺(176mg，1.46mmol)，室温反应过夜。加水，用二氯甲烷萃取，有机相旋干后用正相柱纯化得目标化合物的混合物200mg，产率：77％。重复上述操作，累计混合物400mg。将混合物用反相HPLC纯化，分离得YN015-CF1,172mg，1H NMR(500MHz,Methanol-d4)δ7.73(t,J＝7.0Hz,1H),7.67–7.54(m,1H),7.38(t,J＝8.0Hz,1H),7.31–7.19(m,5H),7.17–7.08(m,3H),7.04–6.97(m,2H),6.94(t,J＝8.8Hz,1H),6.89–6.82(m,2H),6.75(dd,J＝8.5,2.6Hz,1H),5.22–4.96(m,2H),4.88(dd,J＝10.4,5.7Hz,1H),4.52(d,J＝9.4Hz,1H),4.45(s,1H),4.18(d,J＝15.3Hz,1H),3.74(dd,J＝15.2,2.5Hz,1H),3.06(s,3H),1.82(dd,J＝15.3,9.5Hz,1H),1.46–1.35(m,4H),0.82(s,9H).YN015-CF2,172mg，1H NMR(500MHz,Methanol-d4)δ7.65(t,J＝7.2Hz,1H),7.56(t,J＝7.5Hz,1H),7.40–7.18(m,10H),7.17–6.97(m,4H),5.03(d,J＝10.0Hz,1H),4.97(q,J＝6.8Hz,1H),4.85–4.62(m,2H),4.52(s,1H),4.17–3.95(m,1H),3.85(d,J＝15.3Hz,1H),3.10(s,3H),1.97(dd,J＝15.2,9.9Hz,1H),1.60(d,J＝15.0Hz,1H),1.23(d,J＝7.0Hz,3H),0.95(s,9H).重复上述操作，累计混合物YN05-CF1 383mg，YN05-CF2 383mg。
步骤四：合成(2S,3R,4R,5R)-4-(氨基甲基)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-5-新戊基吡咯烷-2-羧酸(YN16-CF1)
将YN015-CF1(383mg，0.5mmol)用浓盐酸10mL溶解，加入乙醇，回流18h，反应结束后，用HPLC纯化得YN16-CF1的三氟醋酸盐187mg，产率75％。1H NMR(500MHz,Methanol-d4)δ7.55–7.48(m,1H),7.47–7.41(m,1H),7.40–7.36(m,1H),7.36–7.20(m,3H),5.07(d,J＝11.1Hz,1H),4.97(d,J＝10.9Hz,1H),4.29(d,J＝11.1Hz,1H),3.82(d,J＝14.2Hz,1H),3.72–3.63(m,1H),2.09–1.94(m,1H),1.84–1.70(m,1H),1.13(s,9H).
步骤五：合成(2S,3R,4R,5R)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((4-甲氧基苄基)氨基)甲基)-5-新戊基吡咯烷-2-羧酸(YN18-CF1)
将YN16-CF1(187mg，0.4mmol)溶于甲醇，加入对甲氧基苯甲醛(109mg，0.8mmol)、氰基硼氢化钠(101mg，1.6mmol)和醋酸1mL，室温搅拌过夜。反应结束HPLC纯化得目标化合物的三氟醋酸盐133mg，产率：47％。
步骤六：合成甲基4-((2S,3R,4R,5R)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((4-甲氧基苄基)氨基)甲基)-5-新戊基吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸酯(YN20-CF1)
将YN18-CF1(133mg，0.22mmol)称入瓶中，用四氢呋喃溶解，加入二异丙基乙基胺(144mg，1.12mmol)和二苯基次磷酰氯(156mg，0.66mmol)，室温搅拌30min后加入3-甲氧基-4-氨基苯甲酸甲酯(163mg，0.9mmol)，室温反应过夜。加水，用二氯甲烷萃取，有机相旋干后用正相柱纯化得目标化合物YN20-CF1的粗品，直接用于下一步。
合成中间体4：合成甲基4-((2R,3S,4S,5S)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((4-甲氧基苄基)氨基)甲基)-5-新戊基吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸酯(YN22-CF2)
步骤一：合成(2R,3S,4S,5S)-4-(氨基甲基)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-5-新戊基吡咯烷-2-羧酸(YN17-CF2)
参照合成中间体3，步骤四YN16-CF1的合成方法，以YN015-CF2为原料，得到YN17-CF2的三氟醋酸盐203mg，产率81％。1H NMR(500MHz,Methanol-d4)δ7.55–7.48(m,1H),7.47–7.41(m,1H),7.40–7.36(m,1H),7.36–7.20(m,3H),5.01(d,J＝11.1Hz,1H),4.95(d,J＝10.9Hz,1H),4.28(d,J＝11.1Hz,1H),3.81(d,J＝14.2Hz,1H),3.71–3.61(m,1H),2.09–1.94(m,1H),1.84–1.70(m,1H),1.12(s,9H).
步骤二：合成(2R,3S,4S,5S)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((4-甲氧基苄基)氨基)甲基)-5-新戊基吡咯烷-2-羧酸(YN19-CF2)
参照合成中间体3，步骤五YN18-CF1的合成方法，以YN17-CF2为原料，得到YN19-CF2的三氟醋酸盐181mg，产率60％。
步骤三：合成甲基4-((2R,3S,4S,5S)-3-(3-氯-2-氟苯基)-4-(4-氯-2-氟苯基)-4-(((4-甲氧基苄基)氨基)甲基)-5-新戊基吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸酯(YN22-CF2)
参照合成中间体3，步骤六YN20-CF1的合成方法，以YN19-CF2为原料，得到YN22-CF2的粗品。
终产物1：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯-2-氟苯基)-1'-甲基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(YM157)
将YM155(40mg,0.054mmol)，甲醛(41mg,0.5mmol)，醋酸硼氢化钠(106mg,0.5mmol)和0.1mL乙酸称入50mL圆底烧瓶中，用1，2-二氯乙烷溶解，室温过夜。加水，用乙酸乙酯萃取，有机相旋干，加1ml三氟乙酸，50℃搅拌1h，加饱和碳酸氢钠溶液，用乙酸乙酯萃取，有机相旋干，加氢氧化锂一水合物(8mg,0.2mmol)，用四氢呋喃/H2O(v/v＝2mL/mL)溶解，室温搅拌过夜，HPLC纯化得目标化合物12.4mg，产率38％。1H NMR(500MHz,Methanol-d4)δ8.26(d,J＝8.4Hz,1H),7.65(dd,J＝8.4,1.8Hz,1H),7.61(d,J＝1.7Hz,1H),7.51–7.43(m,1H),7.43–7.30(m,2H),7.19(t,J＝8.0Hz,1H),6.73(dd,J＝8.1,1.9Hz,1H),6.49(d,J＝1.8Hz,1H),5.19–5.05(m,1H),4.68–4.48(m,1H),4.30–4.04(m,1H),3.87(s,3H),3.75(d,J＝10.9Hz,1H),3.57(d,J＝11.0Hz,1H),3.06(s,3H),2.01–1.71(m,2H),0.82(s,9H).ESI-MS理论计算值C32H35 35Cl2FN3O4[M+H]+＝614.2,实验测得：614.2。
终产物2：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯-2-氟苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(YN11)
YM155(50mg,0.068mmol)，乙醛(30mg,0.68mmol)，醋酸硼氢化钠(144mg,0.68mmol)，乙酸0.1mL，三氟乙酸1mL，氢氧化锂一水合物(14mg,0.34mmol)，反应步骤参见终产物1，得目标产物20.2mg，产率47％。1H NMR(500MHz,Methanol-d4)δ8.20(d,J＝8.4Hz,1H),7.65(d,J＝8.9Hz,1H),7.61(s,1H),7.45(t,J＝7.5Hz,1H),7.37(s,1H),7.32(d,J＝8.1Hz,1H),7.19(t,J＝7.9Hz,1H),6.72(dd,J＝8.1,1.9Hz,1H),6.50(d,J＝1.8Hz,1H),4.57–4.31(m,1H),4.26–4.04(m,1H),3.86(s,3H),3.76(d,J＝11.1Hz,1H),3.61(dd,J＝11.1,6.9Hz,1H),3.51(d,J＝10.7Hz,1H),3.31–3.16(m,2H),2.01–1.61(m,2H),1.40(t,J＝7.0Hz,3H),0.83(s,9H).ESI-MS理论计算值C33H37 35Cl2FN3O4[M+H]+＝628.2,实验测得：628.7。
终产物3：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯-2-氟苯基)-1'-丙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(YN51)
YM155(50mg,0.068mmol)，丙醛(39mg,0.68mmol)，醋酸硼氢化钠(144mg,0.68mmol)，乙酸0.1mL，三氟乙酸1mL，氢氧化锂一水合物(14mg,0.34mmol)，反应步骤参见终产物1，得目标产物5mg，产率11％。1H NMR(400MHz,Methanol-d4)δ8.27(d,J＝8.4Hz,1H),7.65(d,J＝8.4Hz,1H),7.63(s,1H),7.39(t,J＝7.4Hz,1H),7.32(t,J＝7.5Hz,1H),7.23(d,J＝7.8Hz,1H),7.15(t,J＝7.9Hz,1H),6.69(dd,J＝8.0,1.9Hz,1H),6.46(d,J＝1.9Hz,1H),4.90–4.43(m,1H),4.42–4.18(m,1H),3.90(s,3H),3.89–3.85(m,1H),3.67(d,J＝10.8Hz,1H),3.56–3.36(m,2H),3.11–2.79(m,1H),2.02–1.89(m,1H),1.86–1.46(m,3H),1.03(t,J＝7.3Hz,3H),0.92(s,9H).ESI-MS理论计算值C34H39 35Cl2FN3O4[M+H]+＝642.2,实验测得：642.3。
终产物4：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯-2-氟苯基)-1'-丁基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(YN52)
YM155(50mg,0.068mmol)，丁醛(49mg,0.68mmol)，醋酸硼氢化钠(144mg,0.68mmol)，乙酸0.1mL，三氟乙酸1mL，氢氧化锂一水合物(14mg,0.34mmol)，反应步骤参见终产物1，得目标产物9.3mg，产率21％。1H NMR(500MHz,Methanol-d4)δ8.24(d,J＝8.4Hz,1H),7.66(dd,J＝8.4,1.8Hz,1H),7.63(d,J＝1.7Hz,1H),7.41(t,J＝7.4Hz,1H),7.34(t,J＝5.7Hz,1H),7.27(d,J＝8.1Hz,1H),7.17(t,J＝7.9Hz,1H),6.70(dd,J＝8.1,1.9Hz,1H),6.47(d,J＝1.9Hz,1H),4.89–4.57(m,1H),4.44–4.18(m,1H),4.08–3.95(m,1H),3.89(s,3H),3.71(d,J＝10.4Hz,1H),3.56–3.40(m,2H),3.15–2.83(m,1H),2.01–1.87(m,1H),1.82–1.71(m,1H),1.71–1.54(m,2H),1.52–1.41(m,2H),0.95(t,J＝7.3Hz,3H),0.89(s,9H).ESI-MS理论计算值C35H41 35Cl2FN3O4[M+H]+＝656.2,实验测得：656.3。
终产物5：4-((2'S,3S,4'S,5'R)-6-氯-4'-(2,3-二氟苯基)-1'-甲基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN05-2)
JM158(80mg,0.11mmol)，甲醛(90mg,1.1mmol)，醋酸硼氢化钠(236mg,1.1mmol)，乙酸0.1mL，三氟乙酸1mL，氢氧化锂一水合物(14mg,0.34mmol)，反应步骤参见终产物1，得目标产物17.2mg，产率26％。1H NMR(400MHz,Methanol-d4)δ8.23(d,J＝8.4Hz,1H),7.64(d,J＝8.7Hz,1H),7.60(s,1H),7.39(d,J＝8.1Hz,1H),7.30–7.10(m,3H),6.72(d,J＝8.1Hz,1H),6.49(s,1H),5.15(d,J＝10.2Hz,1H),4.63(d,J＝10.0Hz,1H),4.23–4.06(m,1H),3.87(s,3H),3.76(d,J＝11.0Hz,1H),3.61(d,J＝11.0Hz,1H),3.07(s,3H),2.06–1.77(m,2H),0.80(s,9H).ESI-MS理论计算值C32H35 35ClF2N3O4[M+H]+＝598.2,实验测得：598.2。
终产物6：4-((2'S,3S,4'S,5'R)-6-氯-4'-(2,3-二氟苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JM151)
JM158(60mg,0.08mmol)，乙醛(36mg,0.8mmol)，醋酸硼氢化钠(160mg,0.8mmol)，乙酸0.1mL，三氟乙酸1mL，氢氧化锂一水合物(13mg,0.3mmol)，反应步骤参见终产物1， 得目标产物8.6mg，产率18％。1H NMR(400MHz,Methanol-d4)δ8.20(d,J＝8.4Hz,1H),7.66(d,J＝8.6Hz,1H),7.61(s,1H),7.31(d,J＝8.2Hz,1H),7.29–7.09(m,3H),6.72(d,J＝8.1Hz,1H),6.49(s,1H),5.21–4.90(m,1H),4.59–4.31(m,1H),4.23–4.03(m,1H),3.86(s,3H),3.75(d,J＝11.0Hz,1H),3.67–3.58(m,1H),3.55(d,J＝11.4Hz,1H),3.33–3.05(m,1H),2.02–1.63(m,2H),1.40(t,J＝7.0Hz,3H),0.85(s,9H).ESI-MS理论计算值C33H37 35ClF2N3O4[M+H]+＝612.2,实验测得：612.2。
终产物7：4-((2'S,3S,4'S,5'R)-6-氯-4'-(2,3-二氟苯基)-1'-丙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JM156)
JM158(58mg,0.08mmol)，丙醛(47mg,0.8mmol)，醋酸硼氢化钠(170mg,0.8mmol)，乙酸0.1mL，三氟乙酸1mL，氢氧化锂一水合物(9mg,0.2mmol)，反应步骤参见终产物1，得目标产物4.1mg，产率8％。1H NMR(400MHz,Methanol-d4)δ8.24(d,J＝8.4Hz,1H),7.66(d,J＝8.8Hz,1H),7.62(s,1H),7.28(d,J＝8.2Hz,1H),7.24–7.05(m,3H),6.71(d,J＝8.0Hz,1H),6.49(s,1H),4.90–4.53(m,1H),4.48–4.20(m,1H),4.16–3.97(m,1H),3.88(s,3H),3.72(d,J＝11.0Hz,1H),3.63–3.50(m,1H),3.50–3.38(m,1H),3.12–2.89(m,1H),2.02–1.90(m,1H),1.89–1.47(m,3H),1.03(t,J＝7.4Hz,3H),0.90(s,9H).ESI-MS理论计算值C34H39 35ClF2N3O4[M+H]+＝626.2,实验测得：626.3。
终产物8：4-((2'S,3S,4'S,5'R)-6-氯-4'-(2,3-二氟苯基)-1'-丁基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN06)
JM158(70mg,0.1mmol)，丁醛(73mg,1.0mmol)，醋酸硼氢化钠(212mg,1.0mmol)，乙酸0.1mL，三氟乙酸1mL，氢氧化锂一水合物(21mg,0.5mmol)，反应步骤参见终产物1，得目标产物10.3mg，产率16％。1H NMR(400MHz,Methanol-d4)δ8.22(d,J＝8.3Hz,1H),7.65(d,J＝8.9Hz,1H),7.62(s,1H),7.29(d,J＝8.2Hz,1H),7.24–7.11(m,3H),6.71(d,J＝8.1Hz,1H),6.48(s,1H),4.93–4.66(m,1H),4.54–4.24(m,1H),4.13–3.96(m,1H),3.88(s,3H),3.73(d,J＝11.0Hz,1H),3.61–3.42(m,2H),3.20–2.91(m,1H),1.97(d,J＝16.3Hz,1H),1.88–1.73(m,2H),1.72–1.55(m,2H),1.54–1.37(m,2H),0.94(t,J＝7.6Hz,3H),0.87(s,9H).ESI-MS理论计算值C35H41 35ClF2N3O4[M+H]+＝640.2,实验测得：640.2。
终产物9：4-((2'S,3S,4'S,5'R)-6-氯-4'-(2-氟苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN22)
步骤一：合成(E)-2-(4-氯-2-氟苯基)-3-(2-氟苯基)丙烯腈(TA042)
2-氟苯甲醛(2.5g,20mmol)，4-氯-2-氟苯乙腈(3.4g,20mmol)，5N甲醇钠的甲醇溶液4.8mL，反应步骤参见中间体1的步骤一，得目标产物5.3g,产率96％。1H NMR(500MHz,Chloroform-d)δ8.27(td,J＝7.7,1.6Hz,1H),7.82(s,1H),7.56(t,J＝8.3Hz,1H),7.51–7.42(m,1H),7.29(t,J＝7.6Hz,1H),7.25(dd,J＝8.4,2.0Hz,1H),7.22(dd,J＝10.8,2.1Hz,1H),7.19–7.13(m,1H).
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(2-氟苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(TA046)
TA042(5.3g,19.3mmol)，AgF(2.54g,20mmol)，三乙胺4.4mL和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(4.4g,20mmol)，反应步骤参见中间体1的步骤二，得目标产物3.16g，产率33.5％。1H NMR(400MHz,Chloroform-d)δ7.69(t,J＝7.5Hz,1H),7.34(t,J＝8.5Hz,1H),7.29–7.12(m,3H),7.08(d,J＝8.6Hz,1H),6.88(t,J＝9.0Hz,1H),4.68(d,J＝7.6Hz,1H),4.22(d,J＝7.6Hz,1H),4.12(d,J＝8.9Hz,1H),1.70–1.58(m,1H),1.37(s,9H),1.30(d,J＝14.4Hz,1H),0.91(s,9H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(2-氟苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(TA048)
TA046(3.16g,6.47mmol)，雷尼镍8.0g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物0.67g，产率21％。1H NMR(400MHz,Methanol-d4)δ7.48(t,J＝7.7Hz,1H),7.34–7.29(m,1H),7.27–7.13(m,4H),7.01(t,J＝9.6Hz,1H),4.37–4.19(m,3H),3.38(d,J＝14.1Hz,1H),3.24(d,J＝14.1Hz,1H),1.57–1.41(m,3H),1.33(s,9H),1.00(s,9H).
步骤四：合成(2R,3R,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(2-氟苯基)-5-新戊基吡咯烷-2-羧酸(TA051)
TA048(0.67g,1.4mmol)，FmocCl(0.52g,2.0mmol)，二异丙基乙基胺(0.7g,5.4mmol)和三氟乙酸4mL，反应步骤参见中间体1的步骤四，得目标产物0.36g，产率40.6％。
步骤五：合成甲基4-((2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(2-氟苯基)-5-新戊基吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸酯(JN015)
TA051(239mg,0.4mmol)，4-氨基-3-甲氧基苯甲酸甲酯(268mg,1.5mmol)，二苯基次磷酰氯(264mg,1.1mmol)，二异丙基乙基胺(239mg,1.9mmol)和哌啶0.4mL，反应步骤参见中间体1的步骤五，得目标产物56mg，产率25％。1H NMR(500MHz,Methanol-d4):δ8.27(d,J＝8.4Hz,1H),7.62–7.56(m,2H),7.43–7.34(m,2H),7.34–7.27(m,2H),7.23(t,J＝7.5Hz,1H),7.17(dd,J＝13.1,2.2Hz,1H),7.07(dd,J＝10.9,8.2Hz,1H),4.63(d,J＝10.4Hz,1H),4.42(d,J＝11.2Hz,1H),3.96–3.89(m,4H),3.88(s,3H),3.74(dd,J＝14.4,3.2Hz,1H),3.56(d,J＝ 13.7Hz,1H),1.71(d,J＝13.7Hz,1H),1.59(dd,J＝13.8,11.3Hz,1H),1.23(s,9H).ESI-MS理论计算值C32H37ClF2N3O4[M+H]+＝600.2，实验测得：600.2。
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(2,3-二氟苯基)-4-氟-1-(4-甲氧基苄基)-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸甲酯(JN020)
JN015(56mg,0.1mmol)，4-甲氧基苯甲醛(64mg,0.5mmol)，氰基硼氢化钠(32mg,0.5mmol)，乙酸0.1mL，碳酸钾(42mg,0.3mmol)，反应步骤参见中间体1的步骤六，得目标产物53mg，产率76％。1H NMR(500MHz,Chloroform-d)δ8.51(d,J＝8.5Hz,1H),7.65(dd,J＝8.5,1.7Hz,1H),7.55(d,J＝1.7Hz,1H),7.21(dd,J＝9.1,3.7Hz,1H),7.11–7.06(m,2H),7.04(t,J＝7.4Hz,1H),6.96(dd,J＝11.3,8.2Hz,1H),6.92(d,J＝8.4Hz,2H),6.78(d,J＝8.5Hz,2H),6.64(dd,J＝7.9,1.8Hz,1H),6.29(d,J＝1.7Hz,1H),4.40(d,J＝9.2Hz,1H),4.10(d,J＝15.1Hz,1H),4.01(d,J＝9.1Hz,1H),3.90(s,3H),3.89(s,3H),3.89–3.85(m,1H),3.81(s,3H),3.29(d,J＝10.0Hz,1H),3.22(d,J＝9.6Hz,1H),3.13(d,J＝9.9Hz,1H),1.47(d,J＝14.2Hz,1H),1.14(dd,J＝14.4,9.7Hz,1H),0.97(s,9H).
步骤七：合成4-((2'S,3S,4'R,5'R)-6-氯-4'-(2,3-二氟苯基)-2'-新戊基螺[二氢吲哚-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JN22)
JN020(53mg,0.08mmol)，乙醛(34mg,0.8mmol)，醋酸硼氢化钠(162mg,0.8mmol)，乙酸0.1mL，三氟乙酸1mL，氢氧化锂一水合物(15mg,0.4mmol)，反应步骤参见终产物1，得目标产物10.3mg，产率21％。1H NMR(400MHz,Methanol-d4)δ8.18(d,J＝8.3Hz,1H),7.65(d,J＝8.5Hz,1H),7.60(s,1H),7.51–7.27(m,3H),7.22(t,J＝7.7Hz,1H),7.10(t,J＝9.7Hz,1H),6.71(d,J＝8.0Hz,1H),6.48(s,1H),5.22–4.91(m,1H),4.56–4.34(m,1H),4.28–4.03(m,1H),3.83(s,3H),3.77(d,J＝11.1Hz,1H),3.69–3.49(m,2H),3.40–3.33(m,1H),2.02–1.67(m,2H),1.42(t,J＝7.2Hz,3H),0.81(s,9H).ESI-MS理论计算值C33H38 35ClFN3O4[M+H]+＝594.3,实验测得：594.3。
终产物10：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氟苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JM159)
步骤一：合成(Z)-2-(4-氯-2-氟苯基)-3-(3-氟苯基)丙烯腈(JM075)
间氟苯甲醛(2.5g,20mmol)，4-氯-2-氟苯乙腈(3.4g,20mmol)，5N甲醇钠的甲醇溶液4.8mL，反应步骤参见中间体1的步骤一，得目标产物5.3g,产率96％。1H NMR(400MHz,Chloroform-d)δ7.66(d,J＝7.9Hz,1H),7.62(dt,J＝9.9,2.1Hz,1H),7.59–7.53(m,2H),7.47(td,J＝8.1,5.8Hz,1H),7.31–7.22(m,2H),7.19(td,J＝8.3,2.2Hz,1H)..
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-氟苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(JM083)
JM075(5.3g,19mmol)，AgF(2.4g,19mmol)，三乙胺3.2mL和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(4.9g,23mmol)，反应步骤参见中间体1的步骤二，得目标产物2.4g，产率26％。1H NMR(500MHz,Chloroform-d)δ7.34(t,J＝8.5Hz,1H),7.25–7.18(m,1H),7.18(dd,J＝12.4.2.1Hz,1H),7.11(dd,J＝8.5,2.2Hz,1H),6.94(dd,J＝7.8,2.6Hz,1H),6.91(dt,J＝10.0,1.5Hz,1H),4.24(d,J＝7.8Hz,1H),4.16(d,J＝7.7Hz,1H),4.05(d,J＝9.0Hz,1H),1.60(ddd,J＝14.3,9.2,0.7Hz,1H),1.37(s,9H),1.28(dd,J＝14.1,1.2Hz,1H),0.88(s,9H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氟苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(JM087)
JM083(2.2g,4.5mmol)，雷尼镍2.7g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物594mg，产率27％。1H NMR(500MHz,Chloroform-d)δ7.16(td,J＝7.9,6.1Hz,1H),7.10–6.99(m,2H),6.93–6.82(m,3H),4.27(d,J＝8.7Hz,1H),4.11(d,J＝9.1Hz,1H),3.91(dd,J＝8.6,2.1Hz,1H),3.25(d,J＝13.3Hz,1H),3.04(d,J＝13.3Hz,1H),1.48–1.38(m,2H),1.28(s,9H),0.88(s,9H).
步骤四：合成(2R,3R,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氟苯基)-5-新戊基吡咯烷-2-羧酸(JM093)
JM087(494mg,1.0mmol)，FmocCl(389mg,1.5mmol)，二异丙基乙基胺(516mg,4.0mmol)和三氟乙酸5mL，反应步骤参见中间体1的步骤四，得目标产物396mg，产率60％。
步骤五：4-(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-1-乙基-3-(3-氟苯基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JM153)
JM093(131mg,0.2mmol)加入50mL单口瓶中，用干燥的四氢呋喃溶解，加入二异丙基乙基胺(129mg,1.0mmol)，搅拌5分钟，加入二苯基次磷酰氯(143mg,0.6mmol)，搅拌半小时后加入4-氨基-3-甲氧基苯甲酸甲酯(147mg,4.0mmol)，室温反应过夜，向反应液中加入饱和碳酸氢钠溶液，二氯甲烷萃取，有机相转干后正相柱纯化，得到粗品116mg。将粗品116mg，乙醛(47mg,1.1mmol)，醋酸硼氢化钠(234mg,1.1mmol)和0.1mL乙酸称入50mL圆底烧瓶中，用1，2-二氯乙烷溶解，室温过夜，加饱和碳酸氢钠溶液，用乙酸乙酯萃取，有机相旋干，得到的粗品用DMF(2mL)溶解，加入哌啶(0.4mL)，室温反应15分钟，加水，乙酸乙酯萃取，有机相旋干后过正相柱纯化，得目标产物32mg，产率38％。1H NMR(400MHz,Methanol-d4)δ8.26(dd,J＝8.5,2.2Hz,1H),7.68–7.56(m,2H),7.51(t,J＝7.6Hz,1H),7.39–7.30(m,2H),7.27(d,J＝13.6Hz,1H),7.06(t,J＝7.6Hz,1H),6.96(d,J＝10.4Hz,1H),6.90(d,J＝7.8Hz,1H),4.42(d,J＝8.8Hz,1H),4.36(d,J＝9.2Hz,1H),4.09(d,J＝9.2Hz,1H),3.93(s,3H),3.88(s,3H),3.58–3.37(m,2H),3.29–3.20(m,1H),3.09–2.93(m,1H),2.00(dd,J＝15.4,9.0Hz,1H),1.45(d,J＝15.0Hz,1H),1.24(t,J＝6.1Hz,3H),1.02(s,9H).
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氟苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JM159)
将JM153(32mg,0.05mmol)称入瓶中，用2mL DMF溶解，加入碳酸钾(29mg,0.2mmol)，110℃搅拌过夜，反应结束后冷至室温，加水，乙酸乙酯萃取，有机相旋干后，粗品溶于10mL水/四氢呋喃/MeOH(V/V/V＝1/1/1)混合溶剂中，加入氢氧化锂一水合物(21mg,0.5mmol)，室 温过夜。旋干反应液，HPLC纯化目标产物13.4mg,产率45％。1H NMR(400MHz,Methanol-d4)δ8.18(d,J＝8.3Hz,1H),7.63(d,J＝8.5Hz,1H),7.58(s,1H),7.42–7.23(m,2H),7.06(d,J＝9.4Hz,1H),7.05–6.95(m,2H),6.73(d,J＝8.0Hz,1H),6.45(s,1H),5.25–4.91(m,1H),4.32–4.01(m,2H),3.82(s,3H),3.77–3.60(m,2H),3.58–3.48(m,1H),3.32–3.13(m,1H),2.01–1.90(m,1H),1.89–1.59(m,1H),1.41(t,J＝7.1Hz,3H),0.86(s,9H).ESI-MS理论计算值C33H38 35ClFN3O4[M+H]+＝594.2,实验测得：594.5。
终产物11：4-((2'S,3S,4'S,5'R)-6-氯-4'-(2,3-二氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN17)
步骤一：合成(Z)-2-(4-氯-2-氟苯基)-3-(2,3-二氯苯基)丙烯腈(YI116)
2,3-二氯苯甲醛(3.5g,20mmol)，4-氯-2-氟苯乙腈(3.4g,20mmol)，5N甲醇钠的甲醇溶液4.8mL，反应步骤参见中间体1的步骤一，得目标产物6.23g,产率95％。1H NMR(500MHz,Chloroform-d)δ7.98–7.89(m,2H),7.62–7.54(m,2H),7.39–7.32(m,1H),7.28(dd,J＝2.1,0.9Hz,1H),7.23(d,J＝2.1Hz,1H).
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(2,3-二氯苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(YI124-1)
YI116(6.23g,19mmol)，AgF(2.41g,19mmol)，三乙胺4.24mL和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(4g,19mmol)，反应步骤参见中间体1的步骤二，得目标产物4.57g，产率45％。1H NMR(500MHz,Chloroform-d)δ7.72(dd,J＝7.9,1.6Hz,1H),7.39(dd,J＝8.0,1.6Hz,1H),7.38–7.29(m,2H),7.14(dd,J＝12.3,2.1Hz,1H),7.12–7.08(m,1H),5.02(d,J＝6.4Hz,1H),4.09(d,J＝8.9Hz,1H),4.03(d,J＝6.3Hz,1H),1.74–1.61(m,1H),1.42(s,9H),1.29(dd,J＝14.3,1.0Hz,1H),0.91(s,9H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(2,3-二氯苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(YI124-2)
YI124-1(4.57g,8.5mmol)，雷尼镍4.5g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物1.04g，产率23％。1H NMR(500MHz,Chloroform-d)δ7.87(dd,J＝7.9,1.6Hz,1H),7.34(dd,J＝7.9,1.4Hz,1H),7.22(t,J＝7.9Hz,1H),7.11(t,J＝8.6Hz,1H),7.05(dd,J＝8.7,2.2Hz,1H),6.97(dd,J＝12.9,2.2Hz,1H),4.44(d,J＝8.9Hz,1H),4.20(dd,J＝9.1,1.8Hz,1H),4.01(d,J＝8.9Hz,1H),3.36–3.29(m,2H),1.60–1.47(m,2H),1.26(s,9H),1.00(s,9H).
步骤四：合成(2R,3R,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(2,3-二氯苯基)-5-新戊基吡咯烷-2-羧酸(YI126)
YI124-2(1.04g,1.91mmol)，FmocCl(740mg,2.87mmol)，二异丙基乙基胺(986mg,7.62mmol)和三氟乙酸4mL，反应步骤参见中间体1的步骤四，得目标产物1.08g，产率80％。
步骤五：4-(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-1-乙基-3-(2,3-二氯苯基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JN012)
YI126(200mg,0.29mmol)，二异丙基乙基胺(188mg,1.45mmol)，二苯基次磷酰氯(207mg,0.87mmol)，4-氨基-3-甲氧基苯甲酸甲酯(209mg,1.15mmol)，乙醛(39mg,0.9mmol)，醋酸硼氢化钠(191mg,0.9mmol)，乙酸(0.1mL)，哌啶(0.3mL)，反应步骤参见终产物10的步骤五，得目标产物10mg，产率8％。1H NMR(400MHz,Methanol-d4)δ8.27(d,J＝8.3Hz,1H),7.70–7.63(m,2H),7.63(s,1H),7.52(d,J＝8.0Hz,1H),7.46–7.39(m,2H),7.29(d,J＝8.7Hz,1H),7.18(d,J＝13.5Hz,1H),4.62(d,J＝10.7Hz,1H),4.57(d,J＝8.1Hz,1H),4.14(d,J＝9.2Hz,1H),3.98(s,3H),3.89(s,3H),3.65–3.55(m,2H),3.41(d,J＝14.5Hz,1H),3.19–3.06(m,1H),2.16–2.04(m,1H),1.52(d,J＝14.8Hz,1H),1.25(t,J＝6.0Hz,3H).
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(2,3-二氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN17)
JN012(10mg,0.02mmol)，碳酸钾(12mg,0.08mmol)，氢氧化锂一水合物(8mg,0.2mmol)，反应步骤参见终产物10的步骤六，得到目标产物3.4mg,产率27％。1H NMR(500MHz,Methanol-d4)δ8.37–8.21(m,1H),7.67(dd,J＝8.4,1.7Hz,1H),7.63(s,1H),7.61–7.53(m,1H),7.49(d,J＝7.8Hz,1H),7.40(t,J＝7.7Hz,1H),7.33–7.18(m,1H),6.68(dd,J＝8.1,1.9Hz,1H),6.45(d,J＝1.8Hz,1H),4.82–4.62(m,1H),4.6–4.2(m,1H),4.20–3.93(m,1H),3.89(s,3H),3.71–3.60(m,1H),3.60–3.46(m,2H),3.14–2.85(m,1H),2.02–1.48(m,2H),1.34(t,J＝6.3Hz,3H),0.93(s,9H).ESI-MS理论计算值C33H37 35Cl3N3O4[M+H]+＝644.2,实验测得：644.7。
终产物12：4-((2'S,3S,4'S,5'R)-6-氯-4'-(4-氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN01)
步骤一：合成(Z)-2-(4-氯-2-氟苯基)-3-(4-氯苯基)丙烯腈(YI037)
对氯苯甲醛(1.4g,10mmol)，4-氯-2-氟苯乙腈(1.7g,10mmol)，5N甲醇钠的甲醇溶液3mL，反应步骤参见中间体1的步骤一，得目标产物2.78g,产率95％。1H NMR(500MHz,Chloroform-d)δ7.85–7.78(m,2H),7.57–7.50(m,2H),7.48–7.42(m,2H),7.25–7.18(m,2H).
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(4-氯苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(YI040)
YI037(2.7g,9.1mmol)，AgF(1.16g,9.1mmol)，三乙胺2.1mL和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(1.94g,9.1mmol)，反应步骤参见中间体1的步骤二，得目标产物1.57g，产率34％。1H NMR(500MHz,Chloroform-d)δ7.32(t,J＝8.5Hz,1H),7.26–7.21(m,2H),7.18(dd,J＝12.3,2.1Hz,1H),7.11(dd,J＝8.7,2.4Hz,3H),4.23(d,J＝7.9Hz,1H),4.13(d,J＝7.8Hz,1H),4.06(dd,J＝9.2,1.2Hz,1H),1.60(ddd,J＝14.4,9.2,1.1Hz,1H),1.37(s,9H),1.29(dd,J＝14.3,1.2Hz,1H),0.89(s,9H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(4-氯苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(YI051)
YI040(1.57g,3.1mmol)，雷尼镍1.57g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物584mg，产率37％。
步骤四：合成(2R,3R,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(4-氯苯基)-5-新戊基吡咯烷-2-羧酸(YI054)
YI051(584mg,1.15mmol)，FmocCl(385mg,1.49mmol)，二异丙基乙基胺(592mg,4.6mmol)和三氟乙酸4mL，反应步骤参见中间体1的步骤四，得目标产物722mg，产率95％。
步骤五：4-(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-1-乙基-3-(4-氯苯基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JM155)
YI054(300mg,0.44mmol)，二异丙基乙基胺(284mg,2.2mmol)，二苯基次磷酰氯(313mg,1.32mmol)，4-氨基-3-甲氧基苯甲酸甲酯(322mg,1.78mmol)，乙醛(40mg,0.9mmol)，醋酸硼氢化钠(191mg,0.9mmol)，乙酸(0.1mL)，哌啶(0.4mL)，反应步骤参见终产物10的步骤五，得目标产物20mg，产率8％。1H NMR(400MHz,Methanol-d4)δ8.26(d,J＝8.3Hz,1H),7.68–7.57(m,2H),7.49(t,J＝9.0Hz,1H),7.39–7.30(m,3H),7.26(d,J＝13.6Hz,1H),7.11(d,J＝8.0Hz,2H),4.42(d,J＝8.8Hz,1H),4.32(d,J＝9.1Hz,1H),4.03(d,J＝9.2Hz,1H),3.94(s,3H),3.88(s,3H),3.52–3.38(m,2H),3.26(d,J＝14.6Hz,1H),3.05–2.93(m,1H),2.00(dd,J＝15.2,9.2Hz,1H),1.45(d,J＝15.1Hz,1H),1.25(t,J＝7.0Hz,3H),1.03(s,9H).
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(4-氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN01)
JM155(21mg,0.03mmol)，碳酸钾(19mg,0.13mmol)，氢氧化锂一水合物(13mg,0.3mmol)，反应步骤参见终产物10的步骤六，得到目标产物6.6mg,产率36％。1H NMR(400MHz,Methanol-d4)δ8.22(d,J＝8.4Hz,1H),7.65(d,J＝8.6Hz,1H),7.60(s,1H),7.41–7.29(m,3H),7.23(d,J＝8.2Hz,2H),6.73(d,J＝8.0Hz,1H),6.46(s,1H),5.09–4.89(m,1H),4.40–4.15(m,1H),4.13–3.98(m,1H),3.84(s,3H),3.76–3.61(m,2H),3.60–3.45(m,1H),3.29–3.12(m,1H),2.14–1.91(m,1H),1.89–1.62(m,1H),1.43(t,J＝7.1Hz,3H),0.90(s,9H).ESI-MS理论计算值C33H38 35Cl2N3O4[M+H]+＝610.2,实验测得：610.2。
终产物13：4-((2'S,3S,4'S,5'R)-6-氯-4'-(2-氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN18)
步骤一：合成(Z)-2-(4-氯-2-氟苯基)-3-(2-氯苯基)丙烯腈(YI091)
2-氯苯甲醛(2.82g,20mmol),4-氯-2-氟苯乙腈(3.4g,20mmol),5N甲醇钠的甲醇溶液4.8mL，反应步骤参见中间体1的步骤一，得目标产物5.46g,产率93％。1H NMR(500MHz,Chloroform-d)δ8.11(dd,J＝5.5,3.8Hz,1H),7.94(s,1H),7.57(t,J＝8.3Hz,1H),7.52–7.46(m,1H),7.44–7.37(m,2H),7.29–7.20(m,2H).
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(2-氯苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(YI097-1)
YI091(5.46g,18.6mmol)，AgF(2.36g,18.6mmol)，三乙胺4mL和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(3.97g,18.6mmol)，反应步骤参见中间体1的步骤二，得目标产物4g，产率43％。1H NMR(500MHz,Chloroform-d)δ7.80(dd,J＝8.1,1.4Hz,1H),7.37(ddd,J＝8.2,6.8,1.8Hz,1H),7.32(t,J＝8.5Hz,1H),7.25–7.16(m,2H),7.12(dd,J＝12.2,2.1Hz,1H),7.09–7.03(m,1H),4.92(d,J＝6.7Hz,1H),4.18–4.10(m,1H),4.08(d,J＝6.7Hz,1H),1.72–1.57(m,1H),1.40(s,9H),1.30(dd,J＝14.4,1.1Hz,1H),0.92(s,9H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(2-氯苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(YI097-2)
YI097-1(4g,7.91mmol)，雷尼镍4g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物1g，产率24％。1H NMR(500MHz,Chloroform-d)δ7.80(dd,J＝7.9,1.6Hz,1H),7.31–7.20(m,2H),7.17–7.10(m,2H),7.05(dd,J＝8.6,2.3Hz,1H),6.95(dd,J＝12.8,2.2Hz,1H),4.33(d,J＝8.9Hz,1H),4.19(dd,J＝9.6,1.2Hz,1H),4.03(d,J＝8.9Hz,1H),3.41–3.29(m,2H),1.57(dd,J＝13.8,1.3Hz,1H),1.51(d,J＝9.7Hz,1H),1.25(s,9H),1.01(s,9H).
步骤四：合成(2R,3R,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(2-氯苯基)-5-新戊基吡咯烷-2-羧酸(YI100)
YI097-2(980mg,1.93mmol)，二异丙基乙基胺(996mg,7.72mmol)，FmocCl(745mg,2.89mmol)，三氟乙酸4mL，反应步骤参见中间体1的步骤四，得目标产物1.12g，产率86％。
步骤五：4-(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-1-乙基-3-(2-氯苯基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JN013)
YI100(200mg,0.3mmol)，二异丙基乙基胺(194mg,1.5mmol)，二苯基次磷酰氯(214mg,0.9mmol)，4-氨基-3-甲氧基苯甲酸甲酯(215mg,1.2mmol)，乙醛(44mg,1.0mmol)，醋酸硼氢化钠(212mg,1.0mmol)，乙酸(0.1mL)，哌啶(0.3mL)，反应步骤参见终产物10的步骤五，得目标产物15mg，产率17％。1H NMR(400MHz,Methanol-d4)δ8.26(d,J＝8.3Hz,1H),7.67(d,J＝8.3Hz,1H),7.65–7.57(m,2H),7.44(t,J＝7.5Hz,1H),7.41–7.28(m,2H),7.26(d,J＝ 8.7Hz,1H),7.16(d,J＝13.4Hz,1H),4.63(d,J＝9.9Hz,1H),4.55(d,J＝8.1Hz,1H),4.18(d,J＝9.1Hz,1H),3.96(s,3H),3.88(s,3H),3.63(d,J＝14.6Hz,1H),3.60–3.49(m,1H),3.39(d,J＝14.5Hz,1H),3.20–3.04(m,1H),2.07(dd,J＝14.8,10.3Hz,1H),1.51(d,J＝14.9Hz,1H),1.24(t,J＝7.0Hz,3H),1.14(s,9H).
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(2-氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN18)
JN013(15mg,0.03mmol)，碳酸钾(17mg,0.12mmol)，氢氧化锂一水合物(13mg,0.3mmol)，反应步骤参见终产物10的步骤六，得到目标产物9mg,产率50％。1H NMR(500MHz,Methanol-d4)δ8.22(d,J＝8.4Hz,1H),7.70–7.63(m,2H),7.61(s,1H),7.44(t,J＝7.6Hz,1H),7.35(d,J＝7.8Hz,1H),7.33–7.23(m,2H),6.67(dd,J＝8.1,1.9Hz,1H),6.45(d,J＝1.9Hz,1H),4.92–4.50(m,1H),4.42–4.01(m,1H),3.96–3.83(m,1H),3.84(s,3H),3.69(d,J＝10.1Hz,1H),3.66–3.49(m,2H),3.27–3.11(m,1H),2.02–1.86(m,1H),1.83–1.60(m,1H),1.39(t,J＝7.0Hz,3H),0.87(s,9H).ESI-MS理论计算值C33H38 35Cl2N3O4[M+H]+＝610.2,实验测得：610.6。
终产物14：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(YN55)
步骤一：合成(Z)-2-(4-氯-2-氟苯基)-3-(3-氯苯基)丙烯腈(YH132)
间氯苯甲醛(1.4g,10mmol)，4-氯-2-氟苯乙腈(1.7g,10mmol)，5N甲醇钠的甲醇溶液3mL，反应步骤参见中间体1的步骤一，得目标产物2.7g,产率92％。1H NMR(500MHz,Chloroform-d)δ7.84–7.80(m,1H),7.78(dt,J＝2.6,1.3Hz,1H),7.58–7.49(m,2H),7.46–7.38(m,2H),7.24(dd,J＝2.0,0.7Hz,1H),7.22(dd,J＝10.9,2.1Hz,1H).
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(YH156)
YH132(2.7g,9.3mmol)，AgF(1.17g,9.3mmol)，三乙胺2.1mL和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(1.97g,9.3mmol)，反应步骤参见中间体1的步骤二，得目标产物1.42g，产率30％。
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(YH160)
YH156(1.42g,2.8mmol)雷尼镍1.4g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物117mg，产率8％。1H NMR(500MHz,Chloroform-d)δ7.25–7.01(m,6H),6.97(dd, J＝7.5,1.7Hz,1H),4.28(d,J＝8.7Hz,1H),4.11(d,J＝9.1Hz,1H),3.87(dd,J＝8.8,2.1Hz,1H),3.25(d,J＝13.3Hz,1H),3.07(d,J＝13.3Hz,1H),1.56–1.35(m,2H),1.30(s,9H),0.91(s,9H).
步骤四：合成(2R,3R,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸(YI005)
YH160(236mg,0.46mmol)，FmocCl(180mg,0.7mmol)，二异丙基乙基胺(239mg,1.8mmol)和三氟乙酸2mL，反应步骤参见中间体1的步骤四，得目标产物219mg，产率70％。
步骤五：4-(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-1-乙基-3-(3-氯苯基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(YN54)
YI05(200mg,0.3mmol)，二异丙基乙基胺(184mg,1.5mmol)，二苯基次磷酰氯(214mg,0.9mmol)，4-氨基-3-甲氧基苯甲酸甲酯(218mg,1.2mmol)，乙醛(20mg,0.46mmol)，醋酸硼氢化钠(98mg,0.46mmol)，乙酸(0.1mL)，哌啶(0.2mL)，反应步骤参见终产物10的步骤五，得目标产物12mg，产率6％。1H NMR(400MHz,Methanol-d4):δ8.33–8.21(m,1H),7.60–7.43(m,3H),7.36–7.28(m,3H),7.25(d,J＝13.6Hz,1H),7.20–7.00(m,2H),4.46(d,J＝8.9Hz,1H),4.37(d,J＝9.2Hz,1H),4.06(d,J＝9.3Hz,1H),3.90(s,3H),3.83(s,3H),3.55–3.40(m,2H),3.39–3.27(m,1H),3.13–2.98(m,1H),2.07(dd,J＝15.1,9.1Hz,1H),1.49(d,J＝14.7Hz,1H),1.25(t,J＝6.9Hz,3H),1.05(s,9H).13C NMR(126MHz,Methanol-d4):δ173.78,167.87,162.63(d,J＝249.7Hz),149.60,138.87,136.18(d,J＝12.1Hz),135.53,132.41,132.02(d,J＝4.9Hz),131.25,130.16,129.06,128.40,126.79,126.71,124.88(d,J＝8.9Hz),123.82,119.76,118.99(d,J＝30.2Hz),111.87,69.98,68.25,60.93,58.34,56.38,52.63,46.75,41.09(d,J＝5.8Hz),38.82,32.01,30.34,15.69.ESI-MS理论计算值C34H41Cl2FN3O4[M+H]+＝644.2；实测值:644.3.
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(YN55)
YN54(12mg,0.02mmol)，碳酸钾(8mg,0.06mmol)，氢氧化锂一水合物(4mg,0.1mmol)，反应步骤参见终产物10的步骤六，得到目标产物4mg,产率33％。1H NMR(500MHz,Methanol-d4)δ8.25(d,J＝8.3Hz,1H),7.64(dd,J＝8.4,1.7Hz,1H),7.60(s,1H),7.42–7.20(m,4H),7.10(d,J＝7.5Hz,1H),6.71(dd,J＝8.1,1.9Hz,1H),6.43(d,J＝1.9Hz,1H),4.89–4.65(m,1H),4.40–3.92(m,2H),3.86(s,3H),3.72–3.55(m,2H),3.55–3.40(m,1H),3.21–2.82(m,1H),2.16–1.84(m,1H),1.76–1.44(m,1H),1.37(t,J＝6.2Hz,3H),0.92(s,9H).13C NMR(126MHz,CDCl3):δ173.15,171.53,152.87,147.95,139.46,134.20,134.11,132.37,129.53,129.04,128.19,127.35,126.93,124.56,124.32,123.26,118.80,118.41,111.12,109.96,70.59,68.14,62.93,61.26,55.71,48.59,44.87,37.78,30.75,29.93,14.97.ESI-MS理论计算值C33H38 35Cl2N3O4[M+H]+＝610.2,实验测得：610.2。
终产物15：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-甲基苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JM157)
步骤一：合成(Z)-2-(4-氯-2-氟苯基)-3-(3-甲基苯基)丙烯腈(JM005)
间甲基苯甲醛(2.4g,20mmol)，4-氯-2-氟苯乙腈(3.4g,20mmol)，5N甲醇钠的甲醇溶液4.8mL，反应步骤参见中间体1的步骤一，得目标产物5g,产率93％。1H NMR(500MHz,Chloroform-d)δ7.72(dd,J＝7.7,1.7Hz,1H),7.67(s,1H),7.56–7.49(m,2H),7.37(t,J＝7.7Hz,1H),7.29(dd,J＝7.5,1.1Hz,1H),7.24(ddd,J＝8.3,2.0,0.7Hz,1H),7.21(dd,J＝10.7,2.0Hz,1H),2.42(s,3H).
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-甲基苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(JM010)
JM005(5g,20mmol)，AgF(2.54g,20mmol)，三乙胺4.5mL和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(4.4g,20mmol)，反应步骤参见中间体1的步骤二，得目标产物2.8g，产率29％。1H NMR(500MHz,Chloroform-d)δ7.33(t,J＝8.5Hz,1H),7.17(dd,J＝12.3,2.1Hz,1H),7.12(t,J＝7.6Hz,1H),7.08(dd,J＝8.5,2.2Hz,1H),7.04(d,J＝7.6Hz,1H),7.01(s,1H),6.93(d,J＝7.9Hz,1H),4.27(d,J＝7.6Hz,1H),4.14(d,J＝7.6Hz,1H),4.06(d,J＝9.0Hz,1H),2.27(s,3H),1.61(dd,J＝14.4,9.1Hz,1H),1.37(s,9H),1.30(dd,J＝14.4,1.2Hz,1H),0.89(s,9H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-甲基苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(JM014)
JM010(2.6g,5.4mmol)，雷尼镍3.2g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物418mg，产率16％。1H NMR(500MHz,Chloroform-d)δ7.11(t,J＝8.6Hz,1H),7.05–7.01(m,2H),6.99(dd,J＝13.0,2.3Hz,1H),6.93(d,J＝7.6Hz,1H),6.88(s,1H),6.83(d,J＝7.8Hz,1H),4.26(d,J＝8.6Hz,1H),4.02(dd,J＝8.2,2.3Hz,1H),3.89(dd,J＝8.6,1.7Hz,1H),3.19(d,J＝13.6Hz,1H),3.05(d,J＝13.6Hz,1H),2.19(s,3H),1.42–1.31(m,2H),1.25(s,9H),0.85(s,9H).
步骤四：合成(2R,3R,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-甲基苯基)-5-新戊基吡咯烷-2-羧酸(JM018)
JM014(1.0g,2.0mmol)，FmocCl(793mg,1.5mmol)，二异丙基乙基胺(1.0g,8.2mmol)和三氟乙酸5mL，反应步骤参见中间体1的步骤四，得目标产物1.1g，产率85％。
步骤五：4-(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-1-乙基-3-(3-甲基苯基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JM149)
JM018(200mg,0.3mmol)，二异丙基乙基胺(206mg,1.6mmol)，二苯基次磷酰氯(228mg, 0.96mmol)，4-氨基-3-甲氧基苯甲酸甲酯(226mg,1.25mmol)，乙醛(44mg,1.0mmol)，醋酸硼氢化钠(212mg,1.0mmol)，乙酸(0.1mL)，哌啶(0.2mL)，反应步骤参见终产物10的步骤五，得目标产物37mg，产率20％。1H NMR(400MHz,Methanol-d4):δ8.25(d,J＝8.2Hz,1H),7.66–7.55(m,2H),7.49(t,J＝9.0Hz,1H),7.40–7.19(m,3H),7.14(d,J＝7.7Hz,1H),7.01–6.89(m,2H),4.44(d,J＝9.1Hz,1H),4.40(d,J＝8.0Hz,1H),4.12(d,J＝8.9Hz,1H),3.91(s,3H),3.88(s,3H),3.57–3.37(m,2H),3.30–3.24(m,1H),3.09–3.00(m,1H),2.28(s,3H),1.98(dd,J＝15.9,9.0Hz,1H),1.45(d,J＝15.1Hz,1H),1.26(t,J＝7.1Hz,3H),0.99(s,9H).ESI-MS理论计算值C35H44ClFN3O4[M+H]+＝624.3,实验测得：624.3.
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-甲基苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JM157)
JM149(26mg,0.04mmol)，碳酸钾(24mg,0.17mmol)，氢氧化锂一水合物(17mg,0.4mmol)，反应步骤参见终产物10的步骤六，得到目标产物9.4mg,产率40％。1H NMR(400MHz,Methanol-d4)δ8.19(d,J＝8.4Hz,1H),7.63(d,J＝8.5Hz,1H),7.56(s,1H),7.35(d,J＝8.0Hz,1H),7.21(t,J＝7.7Hz,1H),7.14(d,J＝7.7Hz,1H),7.10–6.98(m,1H),6.72(d,J＝8.0Hz,1H),6.44(s,1H),5.22–5.01(m,1H),4.46–3.96(m,2H),3.77(s,3H),3.72–3.52(m,3H),3.53–3.29(m,1H),2.28(s,3H),2.15–1.94(m,1H),1.90–1.64(m,1H),1.45(t,J＝7.1Hz,3H),0.85(s,9H).ESI-MS理论计算值C34H41 35ClN3O4[M+H]+＝590.3,实验测得：590.3。
终产物16：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-甲氧基苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JM160)
步骤一：合成(Z)-2-(4-氯-2-氟苯基)-3-(3-甲氧基苯基)丙烯腈(JM030)
间甲氧基苯甲醛(2.7g,20mmol)，4-氯-2-氟苯乙腈(3.4g,20mmol)，5N甲醇钠的甲醇溶液4.8mL，反应步骤参见中间体1的步骤一，得目标产物5.5g,产率96％。1H NMR(500MHz,Chloroform-d)δ7.55–7.50(m,2H),7.49(t,J＝2.0Hz,1H),7.39(dt,J＝8.0,1.7Hz,1H),7.37(t,J＝7.7Hz,1H),7.25–7.21(m,1H),7.19(dd,J＝10.7,1.9Hz,1H),7.02(ddd,J＝7.6,2.5,1.4Hz,1H),3.87(s,3H).
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-甲氧基苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(JM037)
JM030(5.4g,20mmol)，AgF(2.54g,20mmol)，三乙胺4.5mL和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(4.4g,20mmol)，反应步骤参见中间体1的步骤二，得目标产物3.9g，产率39％。1H NMR(500MHz,Chloroform-d)δ7.34(t,J＝8.5Hz,1H),7.16(dd,J＝9.8,2.6Hz, 1H),7.15–7.11(m,1H),7.08(dd,J＝8.4,2.1Hz,1H),6.80–6.76(m,1H),6.75–6.71(m,2H),4.27(d,J＝7.7Hz,1H),4.14(d,J＝7.8Hz,1H),4.05(d,J＝9.0Hz,1H),3.71(s,3H),1.67–1.56(m,1H),1.37(s,9H),1.31–1.27(m,1H),0.88(s,9H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-甲氧基苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(JM041)
JM037(5.3g,9.8mmol)，雷尼镍4.6g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物1.3g，产率33％。1H NMR(500MHz,Chloroform-d)δ7.07(t,J＝8.6Hz,1H),7.04–6.91(m,3H),6.66–6.57(m,3H),4.23(d,J＝8.6Hz,1H),4.00(dd,J＝7.0,1.8Hz,1H),3.89(dd,J＝8.6,2.0Hz,1H),3.15(d,J＝13.4Hz,1H),2.99(d,J＝13.4Hz,1H),1.35–1.26(m,2H),1.22(s,9H),0.80(s,9H).
步骤四：合成(2R,3R,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-甲氧基苯基)-5-新戊基吡咯烷-2-羧酸(JM049)
JM041(1.0g,2.0mmol)，FmocCl(774mg,3.0mmol)，二异丙基乙基胺(1.0g,8.0mmol)和三氟乙酸4mL，反应步骤参见中间体1的步骤四，得目标产物1.3g，产率94％。
步骤五：4-(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-1-乙基-3-(3-甲氧基苯基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JM154)
JM049(200mg,0.3mmol)，二异丙基乙基胺(194mg,1.5mmol)，二苯基次磷酰氯(214mg,0.9mmol)，4-氨基-3-甲氧基苯甲酸甲酯(221mg,1.2mmol)，乙醛(44mg,1.0mmol)，醋酸硼氢化钠(212mg,1.0mmol)，乙酸(0.1mL)，哌啶(0.2mL)，反应步骤参见终产物10的步骤五，得目标产物41mg，产率26％。1H NMR(400MHz,Methanol-d4)δ8.25(d,J＝8.2Hz,1H),7.66–7.56(m,2H),7.50(t,J＝9.0Hz,1H),7.32(d,J＝9.1Hz,1H),7.31–7.20(m,2H),6.88(d,J＝8.4Hz,1H),6.83(d,J＝7.7Hz,1H),6.56(s,1H),4.53–4.34(m,2H),4.15(d,J＝8.8Hz,1H),3.91(s,3H),3.87(s,3H),3.67(s,3H),3.48(d,J＝14.4Hz,1H),3.45–3.36(m,1H),3.26(d,J＝14.5Hz,1H),3.09–2.96(m,1H),1.97(dd,J＝15.3,8.4Hz,1H),1.45(d,J＝15.1Hz,1H),1.25(t,J＝6.8Hz,3H),0.98(s,9H).
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-甲氧基苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JM160)
JM154(39mg,0.06mmol)，碳酸钾(34mg,0.25mmol)，氢氧化锂一水合物(25mg,0.6mmol)，反应步骤参见终产物10的步骤六，得到目标产物18.2mg,产率50％。1H NMR(400MHz,Methanol-d4)δ8.20(d,J＝8.4Hz,1H),7.63(d,J＝8.5Hz,1H),7.56(s,1H),7.35(d,J＝8.0Hz,1H),7.25(t,J＝8.0Hz,1H),6.96–6.82(m,2H),6.78–6.65(m,2H),6.45(s,1H),5.19–5.02(m,1H),4.45–3.97(m,2H),3.79(s,3H),3.66(s,3H),3.65–3.49(m,3H),3.31–3.16(m,1H),2.18–1.94(m,1H),1.92–1.66(m,1H),1.43(t,J＝7.1Hz,3H),0.85(s,9H).ESI-MS理论计算值C34H41 35ClN3O5[M+H]+＝606.2,实验测得：606.2。
终产物17：4-((2'S,3S,4'S,5'R)-5-氯-4'-(2,3-二氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN19)
步骤一：合成(Z)-2-(5-氯-2-氟苯基)-3-(2,3-二氯苯基)丙烯腈(YK090)
5-氯-2-氟苯乙腈(3.4g,20mmol)，2,3-二氯苯甲醛(3.5g,20mmol)，5N甲醇钠的甲醇溶液4.8mL，反应步骤参见中间体1的步骤一，得目标产物6.1g,产率93％。1H NMR(500MHz,Chloroform-d)δ7.98–7.89(m,2H),7.59(ddd,J＝14.3,7.4,2.0Hz,2H),7.42–7.32(m,2H),7.15(dd,J＝10.5,8.8Hz,1H).
步骤二：合成(2R，3S，4R，5S)-4-(5-氯-2-氟苯基)-4-氰基-3-(2,3-二氯苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯(YK093)
YK090(6.1g,18.65mmol)，AgF(2.37g,18.65mmol)，三乙胺4.15mL，(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(3.97g,19mmol)，反应步骤参见中间体1的步骤二，得目标产物3.72g，产率37％。1H NMR(500MHz,Chloroform-d)δ7.72(dd,J＝8.0,1.6Hz,1H),7.45–7.38(m,2H),7.36–7.28(m,2H),7.05(dd,J＝12.0,8.8Hz,1H),5.05(dd,J＝6.3,1.0Hz,1H),4.08(d,J＝8.8Hz,1H),4.02(d,J＝6.3Hz,1H),1.65(ddd,J＝14.5,9.0,1.2Hz,1H),1.42(s,9H),1.31(dd,J＝14.5,1.1Hz,1H),0.90(s,9H).
步骤三：合成(2R,3S,4S,5S)-4-(氨基甲基)-4-(5-氯-2-氟苯基)-3-(2,3-二氯苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(YK108)
YK093(3.72g,6.8mmol)，雷尼镍3g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物1.23g，产率32％。
步骤四：合成(2R,3S,4S,5S)-4-((((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(5-氯-2-氟苯基)-3-(2，3-二氯苯基)-5-新戊基吡咯烷-2-羧酸(YK109)
YK108(1.2g,2.2mmol)，二异丙基乙基胺(1.2g,8.8mmol，FmocCl(854mg,3.3mmol)，三氟乙酸3mL，反应步骤参见中间体1的步骤四，得目标产物1.08g，产率69％。
步骤五：4-(2R,3S,4S,5S)-4-(氨基甲基)-4-(5-氯-2-氟苯基)-3-(2,3-二氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JN014)
YK109(200mg,0.3mmol)，二异丙基乙基胺(188mg,1.5mmol)，二苯基次磷酰氯(207mg,0.9mmol)，4-氨基-3-甲氧基苯甲酸甲酯(208mg,1.2mmol)，乙醛(44mg,1.0mmol)，醋酸硼氢化钠(212mg,1.0mmol)，乙酸(0.1mL)，哌啶(0.2mL)，反应步骤参见终产物10的步骤五，得目标产物10mg，产率10％。1H NMR(400MHz,Methanol-d4)δ8.27(d,J＝8.2Hz,1H),7.72–7.57(m,3H),7.56–7.47(m,2H),7.46–7.37(m,2H),7.07(t,J＝11.2Hz,1H),4.71–4.52(m,2H),4.13(d,J＝9.4Hz,1H),3.98(s,3H),3.89(s,3H),3.71–3.54(m,2H),3.41(d,J＝14.7Hz,1H),3.19–3.06(m,1H),2.20–2.04(m,1H),1.55(d,J＝15.1Hz,1H),1.24(t,J＝5.9Hz,3H), 1.21(s,9H).ESI-MS理论计算值C34H40Cl3FN3O4[M+H]+＝678.2,实验测得：678.2.
步骤六：合成4-((2'S,3S,4'S,5'R)-5-氯-4'-(2,3-二氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN19)
JN014(10mg,0.02mmol)，碳酸钾(12mg,0.08mmol)，氢氧化锂一水合物(8mg,0.2mmol)，反应步骤参见终产物10的步骤六，得到目标产物5mg,产率38％。1H NMR(500MHz,Methanol-d4)δ8.27(d,J＝8.0Hz,1H),7.68(dd,J＝8.4,1.8Hz,1H),7.65–7.56(m,2H),7.50(d,J＝8.0Hz,1H),7.42(d,J＝8.3Hz,1H),7.40(s,1H),7.06(dd,J＝8.4,2.1Hz,1H),6.54(d,J＝8.3Hz,1H),4.89–4.66(m,1H),4.6–4.2(m,1H),4.23–4.02(m,1H),3.89(s,3H),3.67(d,J＝10.8Hz,1H),3.62–3.47(m,2H),3.15–2.92(m,1H),2.01–1.83(m,1H),1.74–1.52(m,1H),1.36(t,J＝6.8Hz,3H),0.93(s,9H).ESI-MS理论计算值C33H37 35Cl3N3O4[M+H]+＝644.2,实验测得：644.2。
终产物18：4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN122)
步骤一：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(JN110)
将YH132(2.4g,8.3mmol)和(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(2.12g,10.0mmol)称入250mL圆底烧瓶中，加入80ml 2-甲基四氢呋喃溶解，反应溶液除氧气，氮气置换，加入醋酸亚铜(103mg,0.83mmol)和R-(+)-1,1'-联萘-2,2'-双二苯膦(569mg,0.91mmol)，反应溶液除氧气，氮气置换，滴加三乙胺(839mg,8.3mmol)，反应溶液除氧气，氮气置换，室温反应24h。反应结束后，反应液用10％醋酸铵洗三次，饱和氯化钠洗两次，有机相用无水硫酸钠干燥，旋干后过柱纯化得目标产物3.2g，产率76％,ee值97％(手性柱，DaicelIG，流动相为乙腈和水,10％乙腈至100％乙腈)，比旋光[α]20＝60.1°(c＝1g/100mL in CHCl3)。1H NMR(500MHz,Chloroform-d)δ7.35(t,J＝8.5Hz,1H),7.26–7.23(m,1H),7.23–7.20(m,1H),7.20–7.17(m,1H),7.16(t,J＝1.9Hz,1H),7.12(dd,J＝8.5,2.2Hz,1H),7.09(d,J＝7.6Hz,1H),4.24(d,J＝7.5Hz,1H),4.13(dd,J＝7.7,1.1Hz,1H),4.04(d,J＝9.0Hz,1H),1.63(dd,J＝15.3,9.7Hz,1H),1.38(s,9H),1.28(d,J＝14.9Hz,1H),0.89(s,9H).
步骤二：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊吡咯烷-2-羧酸叔丁酯(JN105)
JN110(3.2g,2.8mmol)雷尼镍2.5g，水合肼8mL，反应步骤参见中间体1的步骤三，得目标产物1.5mg，产率47％。
步骤三：合成(2R,3R,4S,5S)-4-(((9H-氟-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸叔丁基酯(JN107)
JN105(2.6g,5.1mmol)称入100mL单口瓶中，用干燥的二氯甲烷溶解，加入二异丙基乙基胺(2.6g,20.4mmol)和FmocCl(2.0g,7.6mmol)，室温反应过夜。转干后正相柱纯化得目标产物3.2g，产率86％。
步骤四：合成(2R,3R,4S,5S)-4-(((9H-氟-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-羧酸(JN113)
JN107(3.2g,4.4mmol)，乙醛(968mg,22mmol)，醋酸硼氢化钠(4.7g,22mmol)和10mL乙酸称入100mL圆底烧瓶中，用1，2-二氯乙烷溶解，室温过夜，加饱和碳酸氢钠溶液，用乙酸乙酯萃取，有机相旋干后过正相柱纯化，得粗品3.2g。用10mL二氯甲烷溶解反应物，加入8mL三氟乙酸，室温过夜。转干反应液，加入饱和碳酸氢钠溶液，用二氯甲烷萃取三次，合并有机相，转干后正相柱纯化得目标产物2.1g，产率70％。
步骤五：合成4-(((2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-羧酰胺)-3-甲氧基苯甲酸甲酯(JN115)
JN113(1.0g,1.4mmol)加入100mL单口瓶中，用干燥的二氯甲烷溶解，零摄氏度下加入1-甲基咪唑(345mg,4.2mmol)，搅拌10分钟，加入乙基磺酰氯(361mg,2.8mmol)，搅拌半小时后加入4-氨基-3-甲氧基苯甲酸甲酯(769mg,4.2mmol)，室温反应2小时，向反应液中加入饱和碳酸氢钠溶液，二氯甲烷萃取，有机相转干后正相柱纯化，得到粗品1.47g。将粗品1.47g用DMF(10mL)溶解，加入哌啶(2mL)，室温反应15分钟，用1N盐酸洗三次，饱和食盐水洗三次，有机相旋干后过正相柱纯化，得目标产物800mg，产率89％。1H NMR(400MHz,Chloroform-d)δ8.43(d,J＝8.5Hz,1H),7.62(d,J＝8.5Hz,1H),7.50(s,1H),7.35–7.05(m,6H),7.02(s,1H),4.39–4.18(m,3H),3.86(s,6H),3.39–3.19(m,2H),3.00(d,J＝14.1Hz,1H),2.90(dd,J＝13.7,6.6Hz,1H),1.82(dd,J＝15.2,7.2Hz,1H),1.38(d,J＝15.1Hz,1H),1.21(d,J＝6.4Hz,3H),0.83(s,9H).
步骤六：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺环[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(JN122)
JN115(831mg,1.3mmol)，碳酸钾(711mg,5.2mmol)，氢氧化锂一水合物(242mg,5.8mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐780mg,产率83％，ee值96.6％(手性柱，DaicelIG,流动相为乙腈和水，10％乙腈至100％乙腈)，比旋光[α]20＝-6.1°(c＝0.85g/100mL in CH3OH)。1H NMR(400MHz,Methanol-d4)δ8.17(d,J＝8.4Hz,1H),7.63(d,J＝8.5Hz,1H),7.57(s,1H),7.45–7.21(m,4H),7.14(d,J＝7.4Hz,1H),6.74(d,J＝8.1Hz,1H),6.46(s,1H),5.22–5.05(m,1H),4.37–4.05(m,2H),3.82(s,3H),3.71(d,J＝11.3Hz,2H),3.56(d,J＝11.1Hz,1H),3.42–3.34(m,1H),2.18–1.94(m,1H),1.96–1.75(m,1H),1.44(t,J＝7.1Hz,3H),0.84(s,9H).ESI-MS理论计算值C33H38 35Cl2N3O4[M+H]+＝610.2,实验测得：610.2。
终产物33：4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-((1-甲基环丁基)甲基)螺[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(LCC153)
步骤一：合成(E)-5-(1-甲基环丁基)-3-戊烯酸叔丁酯(LCC123)
LCC122(250mg,2.3mmol)溶于二氯甲烷，滴加甘氨酸叔丁酯(293g,2.3mmol)，室温反应18h，硫酸钠干燥，过滤后旋干有机相，得目标产物550mg，产率99％。
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-4-氰基-5-((1-甲基环丁基)甲基)吡咯烷-2-甲酸叔丁酯(LCC127)
YH123(474mg,1.64mmol)，AgF(310mg,2.44mmol)，三乙胺263mg，LCC123(550mg,2.5mmol)，反应步骤参见中间体1的步骤二，得目标产物150mg，产率30％。1H NMR(500MHz,Chloroform-d):δ7.35(t,J＝8.5Hz,1H),7.25–7.16(m,4H),7.13–7.06(m,2H),4.21(d,J＝7.7Hz,1H),4.11(d,J＝7.7Hz,1H),3.98(dd,J＝9.7,1.9Hz,1H),1.95–1.77(m,4H),1.76–1.65(m,2H),1.59(t,J＝7.4Hz,2H),1.36(s,9H),1.15(s,3H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-((1-甲基环丁基)甲基)吡咯烷-2-甲酸叔丁酯(LCC132)
LCC127(140mg,0.3mmol)，雷尼镍1g，水合肼1mL，反应步骤参见中间体1的步骤三，得目标产物70mg，产率50％。
步骤四：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-((1-甲基环丁基)甲基)吡咯烷-2-甲酸叔丁酯(LCC134)
LCC132(58mg,0.11mmol)，二异丙基乙基胺(57mg,0.44mmol，Fmoc-Cl(42mg,0.16mmol)，反应步骤参见终产物18的步骤三，得目标产物75mg，产率92％。
步骤五：合成(2R,3R,4S,5S)-4-(((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-((1-甲基环丁基)甲基)吡咯烷-2-羧酸(LCC142)
LCC134(75mg,0.1mmol)，乙醛(44mg,1.0mmol),醋酸硼氢化钠(212mg,1.0mmol),乙酸(1.0ml),三氟乙酸1mL，反应步骤参见终产物18的步骤四，得目标产物40mg，产率35％。
步骤六：合成4-((2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-((1-甲基环丁基)甲基)吡咯烷-2-甲酰氨基)-3-甲氧基苯甲酸甲酯(LCC150)
LCC142(40mg,0.05mmol)，N-甲基咪唑(31mg,0.2mmol)，乙基磺酰氯(32mg,0.2mmol)，4-氨基-3-甲氧基苯甲酸甲酯(32mg,0.2mmol)，哌啶(1mL)，反应步骤参见终产物18的步骤五，得目标产物12mg，产率10％。
步骤七：合成4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-((1-甲基环丁基)甲基)螺[吲 哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(LCC153)
LCC150(12mg,0.02mmol)，碳酸钾(10mg,0.07mmol)，氢氧化锂一水合物(60mg,0.2mmol)，反应步骤参见终产物10的步骤六，得到目标产物3mg,产率17％。1H NMR(400MHz,Methanol-d4)δ8.22(d,J＝8.2Hz,1H),7.63(d,J＝8.4Hz,1H),7.57(s,1H),7.47–7.18(m,4H),7.09(d,J＝7.7Hz,1H),6.70(d,J＝7.9Hz,1H),6.38(s,1H),4.53–3.69(m,7H),3.62(s,2H),3.12(s,1H),2.20–1.96(m,2H),1.94–1.79(m,2H),1.76–1.52(m,3H),1.38(s,3H),1.22(s,3H).ESI-MS理论计算值C34H3835Cl2N3O4[M+H]+＝622.2,实验测得：622.3。
终产物34：4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-((1-甲基环戊基)甲基)螺[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(LCC63)
步骤一：合成(E)-5-(1-甲基环戊基)-3-戊烯酸叔丁酯(LCC043)
LCC041(340mg,2.7mmol)，甘氨酸叔丁酯(353mg,2.7mmol)，反应步骤参见终产物33的步骤一，得目标产物618mg,产率99％。
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-4-氰基-5-((1-甲基环戊基)甲基)吡咯烷-2-甲酸叔丁酯(LCC044)
YH132(501mg,1.72mmol)，AgF(328mg,2.58mmol)，三乙胺218mg，LCC043(618mg,2.58mmol)，反应步骤参见中间体1的步骤二，得目标产物234mg，产率30％。1H NMR(400MHz,Chloroform-d)δ7.35(t,J＝8.5Hz,1H),7.25–7.20(m,2H),7.20–7.15(m,2H),7.11(t,J＝9.6Hz,2H),4.24(d,J＝7.8Hz,1H),4.13(d,J＝7.6Hz,1H),4.05(d,J＝9.1Hz,1H),1.77–1.55(m,7H),1.40–1.36(m,10H),1.21–1.08(m,2H),0.94(s,3H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-((1-甲基环戊基)甲基)吡咯烷-2-甲酸叔丁酯(LCC050)
LCC044(234mg,0.4mmol)，雷尼镍1g，水合肼1mL，反应步骤参见中间体1的步骤三，得目标产物99mg，产率45％。
步骤四：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-((1-甲基环戊基)甲基)吡咯烷-2-甲酸叔丁酯(LCC051)
LCC050(99mg,0.19mmol)，二异丙基乙基胺(96mg,0.7mmol，Fmoc-Cl(72mg,0.3mmol)，反应步骤参见终产物18的步骤三，得目标产物130mg，产率78％。
步骤五：合成(2R,3R,4S,5S)-4-(((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-((1-甲基环戊基)甲基)吡咯烷-2-羧酸(LCC055)
LCC051(124mg,0.15mmol)，乙醛(76mg,1.7mmol),醋酸硼氢化钠(365mg,1.7mmol),乙酸(1ml),三氟乙酸2mL，反应步骤参见终产物18的步骤四，得目标产物63mg，产率35％。
步骤六：合成4-((2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-((1-甲基环戊基)甲基)吡咯烷-2-甲酰氨基)-3-甲氧基苯甲酸甲酯(LCC059)
LCC055(63mg,0.08mmol)，N-甲基咪唑(22mg,0.3mmol)，乙基磺酰氯(23mg,0.2mmol)，4-氨基-3-甲氧基苯甲酸甲酯(47mg,0.3mmol)，哌啶(1mL)，反应步骤参见终产物18的步骤五，得目标产物30mg，产率28％。1H NMR(500MHz,MeOH-d4):δ8.26(d,J＝8.2Hz,1H),7.62(d,J＝8.3Hz,2H),7.54(d,J＝8.5Hz,1H),7.39–7.24(m,4H),7.15(d,J＝2.0Hz,1H),7.06(d,J＝7.2Hz,1H),4.39(d,J＝8.9Hz,1H),4.33(d,J＝9.3Hz,1H),4.01(d,J＝9.3Hz,1H),3.96(s,3H),3.89(s,3H),3.51–3.39(m,2H),3.02(dq,J＝13.4,6.8Hz,1H),2.13–2.02(m,1H),1.80–1.41(m,9H),1.32(dt,J＝14.7,9.2Hz,2H),1.25(t,J＝7.0Hz,3H),1.13(s,3H).
步骤七：合成4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-((1-甲基环戊基)甲基)螺[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(LCC063)
LCC059(20mg,0.03mmol)，碳酸钾(17mg,0.1mmol)，氢氧化锂一水合物(100mg,0.2mmol)，反应步骤参见终产物10的步骤六，得到目标产物12mg,产率40％。1H NMR(500MHz,Methanol-d4)δ8.20(d,J＝8.5Hz,1H),7.70–7.53(m,2H),7.45–7.22(m,4H),7.12(d,J＝7.6Hz,1H),6.80–6.66(m,1H),6.43(d,J＝1.9Hz,1H),4.38–3.97(m,2H),3.83(s,3H),3.77–3.62(m,2H),3.56(s,1H),3.25(s,1H),2.11(s,1H),1.91(s,1H),1.72–1.50(m,3H),1.50–1.32(m,6H),1.15(s,2H),0.97(s,3H).ESI-MS理论计算值C35H40 35Cl2N3O4[M+H]+＝636.2,实验测得：636.4。
终产物35：4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-((1-甲基环己基)甲基)螺[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(LCC131)
步骤一：合成(E)-5-(1-甲基环己基)-3-戊烯酸叔丁酯(LCC110)
脂肪醛LCC108(1.88g,13mmol)，甘氨酸叔丁酯(1.76g,13mmol)，反应步骤参见终产物33的步骤一，得目标产物3.27g,产率99％。
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-4-氰基-5-((1-甲基环己基)甲基)吡咯烷-2-甲酸叔丁酯(LCC111)
YH132(2.5g,8.6mmol)，AgF(1.64g,13mmol)，三乙胺1.4g，LCC110(3.27g,13mmol)， 反应步骤参见中间体1的步骤二，得目标产物1.3g，产率30％。1H NMR(400MHz,Chloroform-d4)δ7.37(t,J＝8.5Hz,1H),7.25–7.22(m,1H),7.22–7.15(m,3H),7.15–7.08(m,2H),4.23(d,J＝7.6Hz,1H),4.13(d,J＝7.6Hz,1H),4.04(d,J＝8.8Hz,1H),1.66–1.57(m,2H),1.40–1.37(m,10H),1.37–1.18(m,8H),0.86(s,3H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-((1-甲基环己基)甲基)吡咯烷-2-甲酸叔丁酯(LCC113)
LCC111(1.3g,2.4mmol)，雷尼镍2g，水合肼5mL，反应步骤参见中间体1的步骤三，得目标产物655mg，产率50％。
步骤四：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-((1-甲基环己基)甲基)吡咯烷-2-甲酸叔丁酯(LCC115)
LCC113(912mg,1.67mmol)，二异丙基乙基胺(860mg,6.7mmol，Fmoc-Cl(645mg,2.5mmol)，反应步骤参见终产物18的步骤三，得目标产物1g，产率78％。
步骤五：合成(2R,3R,4S,5S)-4-(((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-((1-甲基环戊基)甲基)吡咯烷-2-羧酸(LCC119)
LCC115(200mg,0.3mmol)，乙醛(114mg,2.6mmol),醋酸硼氢化钠(551mg,2.6mmol),乙酸(1.5ml),三氟乙酸3mL，反应步骤参见终产物18的步骤四，得目标产物119mg，产率62％。
步骤六：合成4-((2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-((1-甲基环己基)甲基)吡咯烷-2-甲酰氨基)-3-甲氧基苯甲酸甲酯(LCC126)
LCC119(119mg,0.16mmol)，N-甲基咪唑(39mg,0.5mmol)，乙基磺酰氯(42mg,0.3mmol)，4-氨基-3-甲氧基苯甲酸甲酯(87mg,0.5mmol)，哌啶(2mL)，反应步骤参见终产物18的步骤五，得目标产物109mg，产率30％。1H NMR(400MHz,Methanol-d4)δ8.26(d,J＝8.8Hz,1H),7.66–7.48(m,3H),7.39–7.24(m,5H),7.19–7.02(m,2H),4.42(d,J＝8.2Hz,1H),4.34(d,J＝9.1Hz,1H),4.05(d,J＝9.2Hz,1H),3.95(s,3H),3.88(s,3H),3.55–3.39(m,2H),3.08–2.96(m,1H),1.97–1.81(m,1H),1.60–1.33(m,8H),1.33–1.14(m,6H),1.10(s,3H).
步骤七：合成4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-((1-甲基环己基)甲基)螺[吲哚啉-3,3'-吡咯烷]-5'-羧酰胺)-3-甲氧基苯甲酸(LCC131)
LCC126(85mg,0.13mmol)，碳酸钾(69mg,0.5mmol)，氢氧化锂一水合物(130mg,0.2mmol)，反应步骤参见终产物10的步骤六，得到目标产物24mg,产率39％。1H NMR(400MHz,Methanol-d4)δ8.17(d,J＝8.4Hz,1H),7.67–7.52(m,2H),7.41–7.24(m,4H),7.14(d,J＝7.5Hz,1H),6.79–6.68(m,1H),6.46(s,1H),5.07(s,1H),4.44–4.05(m,2H),3.81(s,3H),3.77–3.69(m,2H),3.64–3.54(m,1H),3.35(s,1H),1.94(s,2H),1.57–1.38(m,6H),1.37–1.11(m,6H),1.09–0.76(m,4H).ESI-MS理论计算值C36H42 35Cl2N3O4[M+H]+＝650.3,实验测得：650.4。
终产物37：4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-2'-(2,2-二甲基丁基)-1'-乙基螺[吲哚啉-3,3'-吡咯烷]-5'-羧基)-3-甲氧基苯甲酸(TC90)
步骤一：(E)-2-((3,3-二甲基戊亚基)氨基)乙酸叔丁酯(TC070)
TC069(2.5,21.9mmol)，甘氨酸叔丁酯(3.2g,22.9mmol)，反应步骤参见终产物33的步骤一，得目标产物5.1g，产率99％。
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-4-氰基-5-(2,2-二甲基丁基)吡咯烷-2-甲酸叔丁酯(TC074)
TC070(5.1g,22.5mmol)，YH132(6.1g,20.8mmol)，AgF(2.54g,20mmol)，三乙胺4.5mL，反应步骤参见中间体1的步骤二，得目标产物3.2g，产率30％。1H NMR(500MHz,Chloroform-d)δ7.36(t,J＝8.5Hz,1H),7.26(d,J＝1.7Hz,1H),7.25–7.22(m,1H),7.22–7.19(m,1H),7.19–7.16(m,1H),7.13(dd,J＝8.5,2.2Hz,1H),7.10(dt,J＝7.5,1.6Hz,1H),4.24(d,J＝7.6Hz,1H),4.14(d,J＝7.6Hz,1H),4.02(d,J＝8.9Hz,1H),1.60(dd,J＝14.5,9.0Hz,2H),1.38(s,9H),1.26–1.19(m,2H),0.84(s,3H),0.82(s,3H),0.67(t,J＝7.5Hz,3H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-(2,2-二甲基丁基)吡咯烷-2-甲酸叔丁酯(TC076)
TC074(3.2g,6.2mmol)，雷尼镍3g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物1.7g，产率52％。1H NMR(500MHz,Methanol-d4)δ7.29–7.23(m,5H),7.15(s,1H),7.06–6.99(m,1H),4.34(d,J＝8.5Hz,1H),4.13(d,J＝8.4Hz,1H),4.03(d,J＝8.2Hz,1H),3.25(d,J＝14.0Hz,1H),3.05(d,J＝14.1Hz,1H),1.48–1.41(m,2H),1.34(s,9H),1.30–1.26(m,2H),0.84(s,3H),0.82(s,3H),0.71(t,J＝7.4Hz,3H).
步骤四：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-(2,2-二甲基丁基)吡咯烷-2-甲酸叔丁酯(TC078)
TC076(1.7g,3.24mmol)，二异丙基乙基胺(1.68g,13mmol)，Fmoc-Cl(1.27g,4.9mmol)，反应步骤参见终产物18的步骤三，得目标产物2.3g，产率95％。
步骤五：合成(2R,3R,4S,5S)-4-(((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-(2,2-二甲基丁基)-1-乙基吡咯烷-2-羧酸(TC082)
TC078(200mg,0.269mmol)，乙醛(120mg,2.7mmol)，三乙酰氧基硼氢化钠(570mg,2.7mmol)，乙酸3mL，三氟乙酸2mL，反应步骤参见终产物18的步骤四，得目标产物145mg，产率72％。
步骤六：合成4-((2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-(2,2-二甲基丁基)-1-乙基吡咯烷-2-羧酰胺)-3-甲氧基苯甲酸酯甲酯(TC087)
TC082(145mg,0.18mmol)，4-氨基-3-甲氧基苯甲酸甲酯(100mg,0.53mmol)，N-甲基咪 唑(45mg,0.53mmol)，乙基磺酰氯(46mg,0.354mmol)，哌啶(0.2mL)，反应步骤参见终产物18的步骤五，得目标产物92.5mg，产率78％。1H NMR(500MHz,Methanol-d4)δ8.27(d,J＝8.8Hz 1H),7.62(t,J＝7.5Hz,2H),7.58–7.50(m,1H),7.41–7.23(m,4H),7.15(s,1H),7.07(d,J＝7.1Hz,1H),4.40(d,J＝8.9Hz,1H),4.32(d,J＝9.2Hz,1H),4.03(d,J＝9.1Hz,1H),3.96(s,3H),3.89(s,3H),3.49–3.38(m,2H),3.29(d,J＝16.3Hz,1H),3.05–2.95(m,1H),1.95(dd,J＝15.1,9.1Hz,1H),1.45(d,J＝15.0Hz,1H),1.33(tt,J＝7.3,4.1Hz,2H),1.24(t,J＝7.0Hz,3H),1.06(s,3H),0.93(s,3H),0.80(t,J＝7.5Hz,3H).
步骤七：合成4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-2'-(2,2-二甲基丁基)-1'-乙基螺[吲哚啉-3,3'-吡咯烷]-5'-羧基)-3-甲氧基苯甲酸(TC90)
TC087(84mg,0.127mmol)，碳酸钾(70mg,0.51mmol)，氢氧化锂一水合物(120mg,2.86mmol)，反应步骤参见终产物10的步骤六，得到目标产物36.2mg,产率46％。1H NMR(500MHz,Methanol-d4)δ8.20(d,J＝8.4Hz,1H),7.63(dd,J＝8.4,1.8Hz,1H),7.58(s,1H),7.49–7.21(m,4H),7.13(d,J＝7.5Hz,1H),6.73(dd,J＝8.0,1.9Hz,1H),6.46(d,J＝1.9Hz,1H),4.31–4.07(m,2H),3.83(s,3H),3.69(d,J＝10.8Hz,2H),3.50(s,1H),3.31–3.19(m,2H),2.06–1.69(m,2H),1.42(t,J＝7.1Hz,3H),1.34–1.06(m,2H),0.89(s,3H),0.72(t,J＝7.4Hz,6H).ESI-MS理论计算值C34H39 35Cl2N3O4[M+H]+＝624.23,实验测得：624.3。
终产物38：4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-(2-乙基-2-甲基丁基)螺[吲哚啉-3,3'-吡咯烷]-5'-氨基)-3-甲氧基苯甲酸(TC29)
步骤一：(E)-2-((3-乙基-3-甲基戊亚基)氨基)乙酸叔丁酯(TC019)
TC018(2.9,22.7mmol)，甘氨酸叔丁酯(3.5g,25mmol)，反应步骤参见终产物33的步骤一，得目标产物5.1g，产率99％。
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-4-氰基-5-(2-乙基-2-甲基丁基)吡咯烷-2-甲酸叔丁酯(TC020)
YH132(5.6g,19.2mmol)，TC019(5.4g,22.4mmol)，AgF(2.5g,19.2mmol)，三乙胺4.3mL，反应步骤参见中间体1的步骤二，得目标产物3.2g，产率31.2％。1H NMR(400MHz,Chloroform-d)δ7.38(t,J＝8.5Hz,1H),7.24-7.21(m,1H),7.22–7.18(m,1H),7.19–7.15(m,2H),7.13(dd,J＝8.4,2.2Hz,1H),7.10(dt,J＝7.1,2.6Hz,1H),4.23(d,J＝7.6Hz,1H),4.15(d,J＝7.5Hz,1H),4.00(d,J＝8.9Hz,1H),1.59(dd,J＝14.7,8.9Hz,2H),1.38(s,9H),1.32–1.12(m,4H),0.79(s,3H),0.66(t,J＝7.5Hz,3H),0.59(t,J＝7.5Hz,3H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-(2-乙基-2-甲基丁基)吡咯烷-2-甲酸叔丁酯(TC021)
TC020(3.2g,6.0mmol)，雷尼镍3g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物1.1g，产率34％。1H NMR(400MHz,Methanol-d4):δ7.38–7.27(m,4H),7.20(t,J＝8.8Hz,1H),7.08(s,1H),6.98(d,J＝7.8Hz,1H),4.34(d,J＝7.5Hz,1H),4.24(t,J＝5.2Hz,1H),4.15(t,J＝7.6Hz,1H),3.40(d,J＝13.8Hz,1H),3.05(d,J＝13.8Hz,1H),1.94(s,2H),1.40(s,9H),1.35–1.22(m,4H),0.86(s,3H),0.72(t,J＝7.5Hz,3H),0.63(t,J＝7.5Hz,3H).
步骤四：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-(2-乙基-2-甲基丁基)吡咯烷-2-甲酸叔丁酯(TC022)
TC021(340mg,0.63mmol)，二异丙基乙基胺(326mg,2.52mmol)，Fmoc-Cl(243mg,0.94mmol)，反应步骤参见终产物18的步骤三，得目标产物380mg，产率79％。
步骤五：合成(2R,3R,4S,5S)-4-(((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-(2-乙基-2-甲基丁基)吡咯烷-2-羧酸(TC024)
TC023(380g,0.486mmol)，乙醛(214mg,4.9mmol)，三乙酰氧基硼氢化钠(1g,4.9mmol)，乙酸3mL，三氟乙酸3mL，反应步骤参见终产物18的步骤四，得目标产物210mg，产率45.6％。
步骤六：合成4-((2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-(2-乙基-2-甲基丁基)吡咯烷-2-甲酰氨基)-3-甲氧基苯甲酸甲酯(TC025)
TC024(100mg,0.132mmol)，4-氨基-3-甲氧基苯甲酸甲酯(75mg,0.395mmol)，N-甲基咪唑(35mg,0.395mmol)，乙基磺酰氯(35mg,0.264mmol)，哌啶(0.2mL)，反应步骤参见终产物18的步骤五，得目标产物85mg，产率99％。1H NMR(500MHz,Methanol-d4)δ8.26(d,J＝8.8Hz,1H),7.61(td,J＝4.6,2.2Hz,2H),7.55(s,1H),7.37–7.24(m,4H),7.17(s,1H),7.09(d,J＝6.9Hz,1H),4.36(d,J＝9.4Hz,2H),4.11(d,J＝9.1Hz,1H),3.94(s,3H),3.88(s,3H),3.48–3.38(m,2H),3.03–2.91(m,1H),1.91–1.83(m,1H),1.47(d,J＝15.1Hz,1H),1.41–1.27(m,4H),1.25(t,J＝7.0Hz,3H),1.18(q,J＝14.2,7.3Hz,1H),1.00(s,3H),0.83(t,J＝7.4Hz,3H),0.59(t,J＝7.4Hz,3H).
步骤七：合成4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-(2-乙基-2-甲基丁基)螺[吲哚啉-3,3'-吡咯烷]-5'-氨基)-3-甲氧基苯甲酸(TC29)
TC025(85mg,0.127mmol)，碳酸钾(70mg,0.51mmol)，氢氧化锂一水合物(120mg,2.86mmol)，反应步骤参见终产物10的步骤六，得到目标产物58mg,产率71.6％。1H NMR(400MHz,Methanol-d4)δ8.23(d,J＝8.4Hz,1H),7.63(dd,J＝8.4,1.8Hz,1H),7.59(s,1H),7.37–7.23(m,4H),7.11(d,J＝7.1Hz,1H),6.71(dd,J＝8.0,1.9Hz,1H),6.44(d,J＝1.8Hz,1H),4.05(s,2H),3.85(s,3H),3.66(d,J＝10.7Hz,2H),3.52(d,J＝11.4Hz,1H),3.25–3.05(m,1H),2.02–1.84(m,1H),1.73(s,1H),1.38(t,J＝7.0Hz,3H),1.34–1.24(m,4H),1.22–1.01(m,1H),0.86(s,3H),0.77(t,J＝7.4Hz,3H),0.56(t,J＝7.4Hz,3H).ESI-MS理论计算值C35H42 35Cl2N3O4[M+H]+＝638.25,实验测得：638.0。
终产物43：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)苯甲酸(JP16)
步骤一：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)苯甲酸甲酯(JP12)
JN113(40mg,0.06mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(15mg,0.11mmol)，4-氨基苯甲酸甲酯(26mg,0.17mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物50mg，产率99％。1H NMR(500MHz,Methanol-d4)δ7.96(d,J＝8.4Hz,2H),7.70(d,J＝8.7Hz,2H),7.42–7.25(m,5H),7.13–6.99(m,2H),4.70–4.64(m,1H),4.63(d,J＝7.3Hz,1H),4.56(d,J＝7.2Hz,1H),3.85(s,3H),3.68(d,J＝14.2Hz,1H),3.21–3.11(m,1H),3.09–2.90(m,2H),1.70(dd,J＝15.5,4.7Hz,1H),1.54(dd,J＝15.5,3.1Hz,1H),1.29(t,J＝7.1Hz,3H),0.78(s,9H).ESI-MS理论计算值C33H39Cl2FN3O3[M+H]+＝614.2，实验测得：614.2。
步骤二：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)苯甲酸(JP16)
JP12(45mg,0.08mmol)，碳酸钾(42mg,0.32mmol)，氢氧化锂一水合物(17mg,0.4mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐27mg,产率49％。1H NMR(400MHz,Methanol-d4)δ7.99(d,J＝8.9Hz,2H),7.68(d,J＝8.7Hz,2H),7.41(d,J＝8.1Hz,1H),7.32–7.24(m,3H),7.21–7.10(m,1H),6.75(dd,J＝8.1,1.9Hz,1H),6.47(d,J＝1.8Hz,1H),5.05–4.93(m,1H),4.34(d,J＝9.8Hz,1H),4.25–4.09(m,1H),3.73(d,J＝11.1Hz,1H),3.70–3.60(m,1H),3.58–3.40(m,2H),2.03(d,J＝16.0Hz,1H),1.94(dd,J＝15.6,4.2Hz,1H),1.43(t,J＝7.1Hz,3H),0.77(s,9H).ESI-MS理论计算值C32H36Cl2N3O3[M+H]+＝580.2，实验测得：580.2。终产物49：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(2-氟乙基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JQ44)
步骤一：(2R，3R，4S，5S)-4-((叔丁氧羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ09)
JN105(60mg,0.12mmol)称入50mL单口瓶中，用干燥的二氯甲烷溶解，加入二异丙基乙基胺(31mg,0.24mmol)和(Boc)2O(39mg,0.18mmol)，室温反应过夜。反应结束后，二氯 甲烷萃取，饱和氯化钠洗两次，有机相用无水硫酸钠干燥，旋干后过柱纯化得目标产物75mg，产率98％。1H NMR(400MHz,Chloroform-d)δ7.46(d,J＝7.0Hz,1H),7.20–6.89(m,5H),6.77(d,J＝7.7Hz,1H),4.23(d,J＝7.6Hz,1H),4.19(d,J＝9.0Hz,1H),4.09(d,J＝7.6Hz,1H),3.27(d,J＝12.7Hz,1H),3.22–3.13(m,1H),1.38(s,9H),1.37–1.34(m,1H),1.15(d,J＝14.1Hz,1H),0.85(s,9H).
步骤二：(2R，3R，4S，5S)-4-((叔丁氧羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(2-羟乙基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ12)
JQ09(75mg,0.12mmol)，叔丁基二甲基硅氧基乙醛(64mg,0.37mmol)，醋酸硼氢化钠(77mg,0.36mmol)和1mL乙酸称入50mL圆底烧瓶中，用1，2-二氯乙烷溶解，室温过夜，加饱和碳酸氢钠溶液，用乙酸乙酯萃取，有机相旋干后过正相柱纯化，得粗品84mg。用10mL四氢呋喃溶解反应物，加入65mg四丁基氟化铵三水合物，室温反应两小时。反应液用二氯甲烷萃取三次，合并有机相，用无水硫酸钠干燥，转干后正相柱纯化得目标产物55mg，产率85％。1H NMR(400MHz,Chloroform-d)δ7.72(d,J＝6.2Hz,1H),7.20–6.97(m,5H),6.75(d,J＝7.8Hz,1H),4.51–4.22(m,3H),3.90(t,J＝10.6Hz,1H),3.78–3.64(m,1H),3.42(d,J＝13.2Hz,1H),3.27–3.16(m,1H),3.12–2.93(m,2H),1.52(dd,J＝15.9,4.2Hz,1H),1.45(s,9H),1.41–1.35(m,1H),0.66(s,9H).
步骤三：(2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(2-氟乙基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ19)
JQ12(56mg,0.09mmol)称入50mL单口瓶中，用干燥的二氯甲烷溶解，0℃下加入二乙胺基三氟化硫(28mg,0.18mmol)，室温反应过夜。反应结束后，二氯甲烷萃取，饱和氯化钠洗两次，有机相用无水硫酸钠干燥，旋干后过柱纯化得粗品49mg。用4mL二氯甲烷溶解反应物，加入0.6mL三氟乙酸，室温过夜。向反应液中加入饱和碳酸氢钠溶液，用乙酸乙酯萃取三次，合并有机相，用饱和氯化钠溶液洗一次，转干后正相柱纯化得粗品42mg，直接投下一步。
步骤四：(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(2-氟乙基)-5-新戊基吡咯烷-2-羧酸(JQ38)
JQ19(149mg,0.27mmol)称入50mL单口瓶中，用干燥的二氯甲烷溶解，加入二异丙基乙基胺(140mg,1.1mmol)和FmocCl(105mg,0.31mmol)，室温反应过夜。转干后正相柱纯化得粗品130mg。用3mL二氯甲烷溶解反应物，加入3mL三氟乙酸，室温过夜。向反应液中加入饱和碳酸氢钠溶液，用乙酸乙酯萃取三次，合并有机相，用饱和氯化钠溶液洗一次，转干后正相柱纯化得目标产物80mg，产率66％。1H NMR(400MHz,Chloroform-d)δ7.78(d,J＝7.5Hz,2H),7.66(d,J＝7.5Hz,2H),7.46–7.38(m,2H),7.38–7.31(m,2H),7.17–7.09(m,3H),7.06–7.00(m,3H),6.74(d,J＝7.8Hz,1H),4.69–4.44(m,4H),4.41–4.19(m,4H),3.50(d,J＝13.3Hz,1H),3.46–3.23(m,2H),3.14(dd,J＝13.1,7.0Hz,1H),1.56–1.36(m,2H),0.67(s,9H).
步骤五：4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(2-氟乙基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ42)
JQ38(40mg,0.05mmol)，1-甲基咪唑(13mg,0.16mmol)，乙基磺酰氯(13mg,0.1mmol)，4-氨基-3-甲氧基苯甲酸甲酯(29mg,0.16mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步 骤五，得目标产物32mg，产率82％。1H NMR(400MHz,Methanol-d4)δ8.23(d,J＝8.8Hz,1H),7.65–7.55(m,2H),7.46(t,J＝8.8Hz,1H),7.36–7.24(m,4H),7.11(d,J＝2.1Hz,1H),7.05(dt,J＝6.6,1.9Hz,1H),4.82–4.57(m,2H),4.54(d,J＝8.2Hz,1H),4.45(d,J＝9.0Hz,1H),4.11(d,J＝8.1Hz,1H),3.92(s,3H),3.87(s,3H),3.72–3.55(m,1H),3.49(d,J＝14.5Hz,1H),3.45–3.32(m,1H),3.21(dd,J＝14.8,2.3Hz,1H),1.99–1.87(m,1H),1.50(dd,J＝15.3,1.8Hz,1H),1.00(s,9H).ESI-MS理论计算值C34H40Cl2F2N3O4[M+H]+＝662.2，实验测得：662.2。
步骤六：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(2-氟乙基)-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JQ44)
JQ42(32mg,0.05mmol)，碳酸钾(28mg,0.2mmol)，氢氧化锂一水合物(21mg,0.5mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐20.7mg,产率56％。1H NMR(400MHz,Methanol-d4)δ8.34(d,J＝8.9Hz,1H),7.69–7.59(m,2H),7.30(d,J＝8.1Hz,1H),7.26–7.19(m,2H),7.18–7.15(m,1H),7.04(dt,J＝6.5,2.0Hz,1H),6.85(dd,J＝8.1,1.9Hz,1H),6.60(d,J＝1.8Hz,1H),4.66(dt,J＝47.5,4.7Hz,2H),4.31(d,J＝9.4Hz,1H),3.93(s,3H),3.84(d,J＝8.8Hz,2H),3.81–3.65(m,1H),3.54(d,J＝11.0Hz,1H),3.40(d,J＝11.0Hz,1H),3.10–2.90(m,1H),1.95(dd,J＝15.4,9.6Hz,1H),1.24(d,J＝15.4Hz,1H),0.99(s,9H).ESI-MS理论计算值C33H37Cl2FN3O4[M+H]+＝628.2，实验测得：628.2。
终产物55：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-2'-新戊基-1'-(氧杂环丁烷-3-基甲基)螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ97)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-(氧杂环丁烷-3-基甲基)吡咯烷-2-羧酸(JQ83)
JN107(200mg,0.27mmol)，氧杂环丁烷-3-甲醛(118mg,1.37mmol)，醋酸硼氢化钠(291mg,1.37mmol)，乙酸1mL，三氟乙酸3mL，反应步骤参见终产物18的步骤四，得到目标产物46mg，产率23％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-(氧杂环丁烷-3-基甲基)吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸甲酯(JQ94)
JQ83(46mg,0.06mmol)，1-甲基咪唑(15mg,0.18mmol)，乙基磺酰氯(16mg,0.12mmol)，4-氨基-3-甲氧基苯甲酸甲酯(34mg,0.18mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物26mg，产率54％。1H NMR(400MHz,Methanol-d4)δ8.26(d,J＝8.4Hz,1H),7.67–7.52(m,3H),7.39–7.23(m,4H),7.19(s,1H),7.07(d,J＝7.3Hz,1H),4.79–4.73(m,1H),4.63–4.56(m,1H),4.56–4.51(m,1H),4.47–4.37(m,2H),4.31(d,J＝9.8Hz,1H),4.01(d,J＝9.9Hz,1H),3.95(s,3H),3.88(s,3H),3.49–3.42(m,1H),3.37(dd,J＝14.5,2.7Hz,1H),3.30–3.24(m,2H),2.09(dd,J＝15.2,9.0Hz,1H),1.49(d,J＝15.0Hz,1H),1.06(s,9H),0.79(d,J＝13.6Hz,1H).ESI-MS理论计算值C36H43Cl2FN3O5[M+H]+＝686.3，实验测得：686.3。
步骤三：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-2'-新戊基-1'-(氧杂环丁烷 -3-基甲基)螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ97)
JQ94(26mg,0.04mmol)，碳酸钾(21mg,0.16mmol)，氢氧化锂一水合物(9mg,0.2mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐8.5mg,产率28％。1H NMR(500MHz,Methanol-d4)δ8.34(d,J＝8.8Hz,1H),7.67–7.61(m,2H),7.28–7.16(m,4H),7.04(dt,J＝7.4,1.6Hz,1H),6.72(dd,J＝8.0,1.9Hz,1H),6.46(d,J＝1.9Hz,1H),4.81(dd,J＝7.8,6.1Hz,1H),4.60(d,J＝6.6Hz,2H),4.45–4.38(m,1H),4.23–4.11(m,1H),3.94(s,3H),3.90–3.83(m,1H),3.83–3.71(m,2H),3.52(d,J＝10.7Hz,1H),3.41(d,J＝10.7Hz,1H),3.28–3.21(m,1H),3.02–2.90(m,1H),2.05(dd,J＝15.3,8.7Hz,1H),1.35(d,J＝15.5Hz,1H),1.03(s,9H).ESI-MS理论计算值C35H40Cl2N3O5[M+H]+＝652.2，实验测得：652.2。
终产物56：2-(4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺)-3-甲氧基苯基)乙酸(JP27)
步骤一：合成2-(4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)苯基)乙酸甲酯(JP25)
JN113(40mg,0.06mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(15mg,0.11mmol)，2-(4-氨基苯基)乙酸甲酯(35mg,0.17mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物36mg，产率88％。1H NMR(500MHz,Methanol-d4)δ7.50(d,J＝8.3Hz,2H),7.40–7.27(m,5H),7.22(d,J＝8.4Hz,2H),7.07(s,1H),7.06–7.00(m,1H),4.68–4.61(m,1H),4.61–4.51(m,2H),3.68(d,J＝14.3Hz,1H),3.65(s,3H),3.60(s,2H),3.19–3.07(m,1H),3.01(d,J＝14.2Hz,1H),2.99–2.90(m,1H),1.68(dd,J＝15.5,4.6Hz,1H),1.54(dd,J＝15.5,3.2Hz,1H),1.30(t,J＝7.1Hz,3H),0.78(s,9H).ESI-MS理论计算值C34H41Cl2FN3O3[M+H]+＝628.3，实验测得：628.3。
步骤二：合成2-(4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺)苯基)乙酸(JP27)
JP25(33mg,0.05mmol)，碳酸钾(30mg,0.22mmol)，氢氧化锂一水合物(12mg,0.27mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐9mg,产率24％。1H NMR(400MHz,Methanol-d4)δ7.49(d,J＝8.2Hz,2H),7.41(d,J＝8.0Hz,1H),7.33–7.22(m,5H),7.17–7.09(m,1H),6.75(dd,J＝8.0,1.9Hz,1H),6.47(d,J＝1.8Hz,1H),4.90–4.77(m,1H),4.42–4.12(m,2H),3.71(d,J＝11.0Hz,1H),3.68–3.63(m,1H),3.58(s,2H),3.55–3.42(m,2H),2.10–1.85(m,2H),1.45(t,J＝7.1Hz,3H),0.78(s,9H).ESI-MS理论计算值C33H38Cl2N3O3[M+H]+＝594.2，实验测得：594.1。
终产物57：4-(((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺)甲基)-3-甲氧基苯甲酸(JP20)
步骤一：合成2-(4-氨基苯基)乙酸甲酯-4-(((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)甲基)苯甲酸甲酯(JP14)
JN113(40mg,0.06mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(15mg,0.11mmol)，4-(氨基甲基)苯甲酸甲酯(35mg,0.17mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物43mg，产率99％。1H NMR(500MHz,Methanol-d4)δ7.87(d,J＝8.2Hz,2H),7.38–7.24(m,5H),7.21(d,J＝8.1Hz,2H),7.07–6.98(m,2H),4.63–4.54(m,1H),4.52–4.33(m,4H),3.87(s,3H),3.68(d,J＝14.2Hz,1H),3.19–3.01(m,2H),2.98–2.88(m,1H),1.66(dd,J＝15.6,4.5Hz,1H),1.53(dd,J＝15.7,3.2Hz,1H),1.21(t,J＝7.2Hz,3H),0.76(s,9H).ESI-MS理论计算值C34H41Cl2FN3O3[M+H]+＝628.3，实验测得：628.3。
步骤二：合成4-(((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺)甲基)苯甲酸(JP20)
JP14(43mg,0.07mmol)，碳酸钾(39mg,0.28mmol)，氢氧化锂一水合物(15mg,0.35mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐25mg,产率51％。1H NMR(400MHz,Methanol-d4)δ7.82(d,J＝8.2Hz,2H),7.41–7.26(m,3H),7.23(d,J＝1.9Hz,1H),7.13(dt,J＝7.6,1.5Hz,1H),7.03(d,J＝7.9Hz,2H),6.72(dd,J＝8.0,1.9Hz,1H),6.44(d,J＝1.8Hz,1H),4.79(d,J＝10.8Hz,1H),4.58(d,J＝15.3Hz,1H),4.28(d,J＝15.3Hz,1H),4.23–4.03(m,2H),3.77–3.60(m,2H),3.57(d,J＝11.0Hz,1H),3.51–3.35(m,1H),2.14–1.82(m,2H),1.35(t,J＝7.1Hz,3H),0.76(s,9H).ESI-MS理论计算值C33H38Cl2N3O3[M+H]+＝594.2，实验测得：594.3。
终产物61：(2'S，3S，4'R，5'R)-N-((3R，6S)-6-氨基甲酰基四氢-2H-吡喃-3-基)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺(JP23)
步骤一：合成苄基(2R，5S)-5-((叔丁氧羰基)氨基)四氢-2H-吡喃-2-羧酸酯(JP11)
(2R，5S)-5-((叔丁氧羰基)氨基)四氢-2H-吡喃-2-羧酸(30mg，0.12mmol)，苄溴(31mg，0.18mmol)和碳酸钾(50mg，0.36mmol)溶于10mL超干乙腈中，80℃反应过 夜。反应结束后，旋干反应液，加水，乙酸乙酯萃取三次，合并有机相，饱和氯化钠溶液洗一次，无水硫酸钠干燥，旋干后过柱纯化得目标产物44mg，产率99％。1H NMR(500MHz,Chloroform-d)δ7.41–7.28(m,5H),5.18(s,2H),4.48(d,J＝8.3Hz,1H),4.16(dd,J＝11.1,4.4Hz,1H),3.96(d,J＝10.3Hz,1H),3.78–3.45(m,1H),3.24–2.96(m,1H),2.20–1.99(m,2H),1.84–1.65(m,1H),1.42(s,9H).
步骤二：合成苄基(2R，5S)-5-氨基四氢-2H-吡喃-2-羧酸盐酸盐(JP18)
JP11(43mg，0.13mmol)溶于3mL二氯甲烷中，加入3mL 4N的盐酸1，4-二氧六环溶液，室温反应过夜。旋干反应液，得到粗品40mg，直接投下一步。1H NMR(500MHz,Methanol-d4)δ7.42–7.26(m,5H),5.31–5.12(m,2H),4.27–4.07(m,2H),3.51(dd,J＝11.4,9.2Hz,1H),3.31–3.21(m,1H),2.26–2.09(m,2H),1.85–1.67(m,2H).
步骤三：合成苄基(2S，5R)-5-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)四氢-2H-吡喃-2-羧酸酯(JP19)
JN113(35mg,0.05mmol)，1-甲基咪唑(13mg,0.15mmol)，乙基磺酰氯(13mg,0.1mmol)，JP18(40mg,0.15mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物34mg，产率91％。1H NMR(400MHz,Methanol-d4)δ8.43(d,J＝7.7Hz,1H),7.46–7.22(m,9H),7.01(d,J＝2.2Hz,1H),6.97(dt,J＝6.5,2.0Hz,1H),5.24–5.09(m,2H),4.56(d,J＝3.9Hz,1H),4.46(d,J＝6.3Hz,1H),4.38(d,J＝7.3Hz,1H),4.02(dd,J＝10.6,2.5Hz,1H),3.95–3.77(m,2H),3.66(d,J＝14.2Hz,1H),3.17–2.92(m,3H),2.91–2.75(m,1H),2.17–1.97(m,2H),1.80–1.45(m,4H),1.25(t,J＝7.1Hz,3H),0.75(s,9H).ESI-MS理论计算值C38H47Cl2FN3O4[M+H]+＝698.3，实验测得：698.3。
步骤四：合成(2'S，3S，4'R，5'R)-N-((3R，6S)-6-氨基甲酰基四氢-2H-吡喃-3-基)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺(JP23)
将JP19(33mg,0.05mmol)称入瓶中，用2mL DMF溶解，加入碳酸钾(29mg,0.2mmol)，110℃搅拌过夜，反应结束后冷至室温，加水，乙酸乙酯萃取，有机相旋干后，粗品溶于5mL7N的盐酸甲醇溶液中，室温反应三小时。旋干反应液，HPLC纯化得目标产物16mg,产率46％。1H NMR(400MHz,Methanol-d4)δ7.37(d,J＝8.1Hz,1H),7.34–7.25(m,2H),7.19(d,J＝2.0Hz,1H),7.08(dt,J＝6.9,1.8Hz,1H),6.73(dd,J＝8.1,1.9Hz,1H),6.45(d,J＝1.9Hz,1H),4.64(d,J＝10.2Hz,1H),4.36–4.00(m,2H),3.93–3.82(m,1H),3.78–3.55(m,4H),3.54–3.37(m,2H),2.88(t,J＝10.7Hz,1H),2.23–1.80(m,4H),1.61–1.46(m,2H),1.40(t,J＝7.1Hz,3H),0.76(s,9H).ESI-MS理论计算值C31H41Cl2N4O3[M+H]+＝587.3，实验测得：587.3。
终产物65：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)双环[2.2.2]辛烷-1-羧酸(JP04)
步骤一：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯 基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)双环[2.2.2]辛烷-1-羧酸甲酯(JP01)
JN113(60mg,0.1mmol)，1-甲基咪唑(25mg,0.3mmol)，乙基磺酰氯(26mg,0.2mmol)，4-氨基双环[2.2.2]辛烷-1-羧酸甲酯(46mg,0.25mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物58mg，产率76％。1H NMR(500MHz,Methanol-d4)δ7.53–7.24(m,5H),7.08(s,1H),7.04(d,J＝7.2Hz,1H),4.60–4.37(m,3H),3.65(d,J＝14.4Hz,1H),3.60(s,3H),3.24–3.03(m,3H),1.96–1.80(m,12H),1.76(dd,J＝15.9,5.1Hz,1H),1.57(dd,J＝15.8,3.1Hz,1H),1.29(t,J＝7.1Hz,3H),0.79(s,9H).
步骤二：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)双环[2.2.2]辛烷-1-羧酸(JP04)
JP01(48mg,0.06mmol)，碳酸钾(31mg,0.22mmol)，氢氧化锂一水合物(12mg,0.28mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐25.2mg,产率63％。1H NMR(400MHz,Methanol-d4)δ7.36(d,J＝8.1Hz,1H),7.33–7.24(m,2H),7.20(d,J＝2.2Hz,1H),7.07(dt,J＝6.9,1.5Hz,1H),6.73(dd,J＝8.0,1.9Hz,1H),6.45(d,J＝1.8Hz,1H),4.76–4.52(m,1H),4.39–3.84(m,2H),3.67(d,J＝11.1Hz,1H),3.61(dd,J＝12.9,6.9Hz,1H),3.55–3.36(m,2H),2.03–1.66(m,14H),1.42(t,J＝7.1Hz,3H),0.83–0.60(m,9H).ESI-MS理论计算值C34H44Cl2N3O3[M+H]+＝612.3，实验测得：612.4。
终产物66：(新戊酰氧基)甲基-4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸酯(JP65)
步骤一：合成(新戊酰氧基)4-氨基-3-甲氧基苯甲酸甲酯(JP58)
4-((苄氧基)羰基)氨基)-3-甲氧基苯甲酸(300mg，1.0mmol)，特戊酸氯甲酯(227mg，1.5mmol)和碳酸钾(414mg，3.0mmol)溶于10mL DMF中，室温反应1小时。反应结束后，加水，乙酸乙酯萃取三次，合并有机相，饱和氯化钠溶液洗三次，无水硫酸钠干燥，旋干后过柱纯化，得粗品396mg。将反应物溶于四氢呋喃/甲醇(10mL/10mL)混合溶液中，除氧气5分钟，氮气置换，加入钯/碳40mg，除氧气5分钟，氮气置换，室温反应过夜。旋干反应液，过柱纯化，得目标产物236mg，产率84％。1H NMR(400MHz,Methanol-d4)δ7.49(dd,J＝8.3,1.8Hz,1H),7.41(d,J＝1.9Hz,1H),6.68(d,J＝8.2Hz,1H),5.92(s,2H),3.88(s,3H),1.20(s,9H).
步骤二：合成(新戊酰氧基)甲基-4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸酯(JP50)
JN113(50mg,0.07mmol)，1-甲基咪唑(18mg,0.21mmol)，乙基磺酰氯(18mg,0.14mmol)，JP58(60mg,0.21mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物43mg，产率80％。1H NMR(400MHz,Methanol-d4)δ8.31(d,J＝8.8Hz,1H),7.70–7.60(m,2H),7.53(t,J＝8.7Hz,1H),7.39–7.30(m,3H),7.29–7.23(m,1H),7.14(s,1H),7.05(d,J＝7.1Hz,1H),5.97(s,2H),4.43(d,J＝9.2Hz,1H),4.31(d,J＝9.4Hz,1H),3.97(s,3H)，3.99–3.95(m,1H),3.56–3.40(m,2H),3.29–3.22(m,1H),3.08–2.96(m,1H),2.07–1.97(m,1H),1.45(d,J＝14.9Hz,1H),1.24(t,J＝7.1Hz,3H),1.21(s,9H),1.06(s,9H).ESI-MS理论计算值C39H49Cl2FN3O6[M+H]+＝744.3，实验测得：744.3。
步骤三：合成(新戊酰氧基)甲基-4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸酯(JP65)
JP62(48mg，0.06mmol)和碳酸钾(36mg，0.26mmol)溶于1.5mL DMF中，110℃反应8小时。反应结束后，HPLC纯化，得到目标产物的三氟乙酸盐10mg,产率20％。1H NMR(400MHz,Methanol-d4)δ8.40(d,J＝8.9Hz,1H),7.73–7.57(m,2H),7.25–6.95(m,5H),6.63(dd,J＝8.0,1.9Hz,1H),6.36(d,J＝1.8Hz,1H),5.97(s,2H),4.70–4.52(m,1H),4.04(d,J＝9.4Hz,1H),3.98(s,3H),3.71–3.63(m,2H),3.52–3.37(m,2H),3.27(d,J＝10.4Hz,1H),2.65–2.49(m,1H),1.94(dd,J＝15.2,10.3Hz,1H),1.21(s,9H),1.18(t,J＝7.0Hz,3H),1.03(s,9H).ESI-MS理论计算值C39H48Cl2N3O6[M+H]+＝724.3，实验测得：724.4。
终产物67：(异丁酰氧基)甲基4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺)-3-甲氧基苯甲酸酯(JP90)
步骤一：合成(异丙氧羰基)氧基)4-氨基-3-甲氧基苯甲酸甲酯(JP70)
4-((苄氧基)羰基)氨基)-3-甲氧基苯甲酸(269mg，0.9mmol)，氯甲基异丙基碳酸二酯(203mg，1.34mmol)，碳酸钾(373mg，2.7mmol)，钯/碳36mg，反应步骤参见终产物66的步骤一，得到目标产物217mg，产率82％。1H NMR(400MHz,Chloroform-d)δ7.59(dd,J＝8.2,1.8Hz,1H),7.46(d,J＝1.7Hz,1H),6.75(d,J＝8.2Hz,1H),5.95(s,2H),4.91(hept,J＝6.3Hz,1H),3.88(s,3H),1.29(d,J＝6.3Hz,6H).
步骤二：合成((异丙氧羰基)氧基)甲基-4-((2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸酯(JP72)
JN113(50mg,0.07mmol)加入50mL单口瓶中，用干燥的二氯甲烷溶解，零摄氏度下加 入1-甲基咪唑(18mg,0.21mmol)，搅拌10分钟，加入乙基磺酰氯(18mg,0.14mmol)，搅拌半小时后加入JP70(88mg,0.21mmol)，室温反应2小时，有机相转干后正相柱纯化，得到粗品102mg，直接投下一步。
步骤三：合成(异丙氧羰基)氧基)甲基-4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺)-3-甲氧基苯甲酸酯(JP90)
JP72溶于1mL DMF中，加入0.5mL二乙胺，室温反应1小时。反应结束后，旋干二乙胺，向反应液中加入碳酸钾(12mg，0.08mmol)，80℃反应过夜。反应完成后，加水，乙酸乙酯萃取三次，合并有机相，饱和氯化钠溶液洗三次，无水硫酸钠干燥后HPLC纯化，的目标产物5mg，产率13％。1H NMR(400MHz,Methanol-d4)δ8.40(d,J＝8.4Hz,1H),7.71–7.60(m,2H),7.26–7.08(m,4H),7.04(d,J＝5.7Hz,1H),6.63(dd,J＝7.8,1.5Hz,1H),6.37(d,J＝1.9Hz,1H),5.95(s,2H),4.96–4.91(m,1H),4.17–4.03(m,1H),3.97(s,3H),3.81–3.59(m,2H),3.56–3.37(m,2H),3.31–3.22(m,1H),2.68–2.44(m,1H),2.01–1.87(m,1H),1.29(d,J＝6.2Hz,6H),1.25–1.14(m,3H),1.02(s,9H).ESI-MS理论计算值C38H46Cl2N3O7[M+H]+＝726.3，实验测得：726.3。
终产物68：(2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基-N-(吡啶-4-基)螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺(JP17)
步骤一：合成(2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基-N-(吡啶-4-基)吡咯烷-2-甲酰胺(JP13)
JN113(40mg,0.06mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(15mg,0.11mmol)，4-氨基吡啶(16mg,0.17mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物40mg，产率99％。1H NMR(500MHz,Methanol-d4)δ8.64(d,J＝7.1Hz,2H),8.30–8.20(m,2H),7.41–7.14(m,5H),7.12–6.99(m,2H),4.78(d,J＝7.0Hz,1H),4.71–4.64(m,1H),4.54(dd,J＝7.3,2.5Hz,1H),3.68(d,J＝14.1Hz,1H),3.22–3.08(m,1H),2.94–2.86(m,2H),1.68(dd,J＝15.6,4.5Hz,1H),1.51(dd,J＝15.6,3.1Hz,1H),1.26(t,J＝7.2Hz,3H),0.78(s,9H).ESI-MS理论计算值C30H36Cl2FN4O[M+H]+＝557.2，实验测得：557.0。
步骤二：合成(2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基-N-(吡啶-4-基)螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺(JP17)
JP13(34mg,0.06mmol)，碳酸钾(35mg,0.25mmol)，反应步骤参见终产物66的步骤三，得到目标产物的三氟乙酸盐17mg,产率44％。1H NMR(400MHz,Methanol-d4)δ9.89–7.98(m,4H),7.35(d,J＝8.0Hz,1H),7.29–7.20(m,3H),7.18–7.10(m,1H),6.73(dd,J＝8.0,1.9Hz,1H),6.46(d,J＝1.8Hz,1H),4.20(d,J＝9.5Hz,1H),4.14–4.01(m,1H),3.66(d,J＝11.0Hz,1H),3.53–3.37(m,2H),3.36–3.31(m,2H),1.86(d,J＝4.1Hz,2H),1.32(t,J＝7.1Hz,3H),0.79(s,9H).ESI-MS理论计算值C30H35Cl2N4O[M+H]+＝537.2，实验测得：537.2。
终产物69：(1R，4r)-4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基 螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)环己烷-1-甲酸(JP21)
步骤一：合成甲基(1R，4r)-4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺基)环己烷-1-甲酸酯(JP15)
JN113(40mg,0.06mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(15mg,0.11mmol)，反式-4-氨基环己烷-1-甲酸甲酯盐酸盐(33mg,0.17mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物50mg，产率99％。1H NMR(500MHz,Methanol-d4)δ8.46(d,J＝7.9Hz,1H),7.40–7.26(m,4H),7.03(s,1H),7.01–6.93(m,1H),4.57(d,J＝3.8Hz,1H),4.50(d,J＝7.5Hz,1H),4.41(d,J＝7.5Hz,1H),3.74–3.57(m,5H),3.22–3.09(m,1H),3.04(d,J＝14.2Hz,1H),2.98–2.88(m,1H),2.26(tt,J＝12.1,3.4Hz,1H),2.09–1.97(m,2H),1.97–1.88(m,1H),1.77–1.68(m,1H),1.66(d,J＝4.6Hz,1H),1.62–1.37(m,3H),1.35–1.30(m,1H),1.27(t,J＝7.2Hz,3H),1.13(qd,J＝12.7,3.7Hz,1H),0.76(s,9H).ESI-MS理论计算值C33H45Cl2FN3O3[M+H]+＝620.3，实验测得：620.2。
步骤二：合成(1R，4r)-4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)环己烷-1-甲酸(JP21)
JP15(44mg,0.07mmol)，碳酸钾(41mg,0.29mmol)，氢氧化锂一水合物(15mg,0.35mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐24mg,产率49％。1H NMR(400MHz,Methanol-d4)δ7.38(d,J＝8.1Hz,1H),7.33–7.24(m,2H),7.20(d,J＝2.0Hz,1H),7.08(dt,J＝6.9,1.9Hz,1H),6.73(dd,J＝8.0,1.9Hz,1H),6.46(d,J＝1.8Hz,1H),4.67(d,J＝10.2Hz,1H),4.34–3.99(m,2H),3.78–3.58(m,3H),3.54–3.36(m,2H),2.20(tt,J＝12.0,3.5Hz,1H),2.11–1.96(m,3H),1.96–1.75(m,2H),1.60(d,J＝10.5Hz,1H),1.56–1.43(m,2H),1.43(t,J＝7.5Hz,3H),1.34–1.19(m,1H),1.06(qd,J＝12.7,3.8Hz,1H),0.75(s,9H).ESI-MS理论计算值C32H42Cl2N3O3[M+H]+＝586.3，实验测得：586.3。
终产物70：(1S，4s)-4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)环己烷-1-甲酸(JP26)
步骤一：合成甲基(1S，4s)-4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺基)环己烷-1-甲酸酯(JP24)
JN113(40mg,0.06mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(15mg,0.11mmol)，顺式-4-氨基环己烷-1-甲酸甲酯盐酸盐(33mg,0.17mmol)，哌啶(1.5mL)，反应步骤参见终产 物18的步骤五，得目标产物42mg，产率99％。1H NMR(500MHz,Methanol-d4)δ7.45–7.27(m,5H),7.08–6.98(m,2H),4.56(t,J＝3.9Hz,1H),4.51–4.28(m,2H),3.85–3.76(m,1H),3.70–3.66(m,1H),3.65(s,3H),3.21–3.05(m,2H),3.03–2.90(m,1H),2.56–2.45(m,1H),1.97–1.87(m,1H),1.80–1.65(m,4H),1.63–1.48(m,4H),1.46–1.36(m,1H),1.28(t,J＝7.1Hz,3H),0.78(s,9H).ESI-MS理论计算值C33H45Cl2FN3O3[M+H]+＝620.3，实验测得：620.3。
步骤二：合成(1S，4s)-4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)环己烷-1-甲酸(JP26)
JP15(39mg,0.07mmol)，碳酸钾(36mg,0.26mmol)，氢氧化锂一水合物(14mg,0.33mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐15mg,产率33％。1H NMR(400MHz,Methanol-d4)δ7.37(d,J＝8.1Hz,1H),7.33–7.25(m,2H),7.19(d,J＝2.2Hz,1H),7.13–7.05(m,1H),6.73(dd,J＝8.0,1.9Hz,1H),6.45(d,J＝1.8Hz,1H),4.79–4.57(m,1H),4.41–3.93(m,2H),3.89–3.76(m,1H),3.69(d,J＝11.0Hz,1H),3.63(dd,J＝12.8,6.9Hz,1H),3.56–3.37(m,2H),2.57–2.30(m,1H),2.09–1.80(m,3H),1.78–1.48(m,5H),1.41(t,J＝7.1Hz,3H),1.38–1.27(m,2H),0.76(s,9H).ESI-MS理论计算值C32H42Cl2N3O3[M+H]+＝586.3，实验测得：586.3。
终产物71：(2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-N-((1r，4R)-4-羟基环己基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺(JP09)
步骤一：合成(2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-N-((1r，4R)-4-羟基环己基)-5-新戊基吡咯烷-2-甲酰胺(JP06-2)
JN113(60mg,0.1mmol)，1-甲基咪唑(25mg,0.3mmol)，乙基磺酰氯(26mg,0.2mmol)，反式-4-氨基环己烷-1-醇(26mg,0.22mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得JP06-2 29mg，产率42％。1H NMR(500MHz,Methanol-d4)δ7.53–7.17(m,5H),7.01(s,1H),6.97(d,J＝6.6Hz,1H),4.66–4.44(m,3H),4.37(d,J＝7.4Hz,1H),3.80–3.60(m,2H),3.25–3.05(m,1H),2.98(d,J＝14.7Hz,1H),2.93–2.79(m,1H),2.13(d,J＝12.7Hz,1H),2.09–1.97(m,2H),1.77(d,J＝13.3Hz,1H),1.73–1.57(m,2H),1.52(dd,J＝15.6,3.4Hz,1H),1.47–1.15(m,6H),0.76(s,9H).ESI-MS理论计算值C31H43Cl2FN3O2[M+H]+＝578.3，实验测得：578.3。步骤二：合成(2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-N-((1r，4R)-4-羟基环己基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺(JP09)
JP06-2(27mg,0.05mmol)，碳酸钾(26mg,0.2mmol)，反应步骤参见终产物66的步骤三，得到目标产物的三氟乙酸盐6.2mg,产率18％。1H NMR(400MHz,Methanol-d4)δ7.38–7.23(m,3H),7.18(d,J＝1.6Hz,1H),7.12–7.00(m,1H),6.72(dd,J＝8.0,1.9Hz,1H),6.44(d,J＝1.8Hz,1H),5.75–5.42(m,2H),4.70–4.39(m,1H),4.29–3.87(m,3H),3.63(d,J＝10.6Hz,1H),3.58–3.51(m,1H),3.51–3.40(m,1H),2.36(dd,J＝16.3,4.1Hz,1H),2.25–1.50(m,8H),1.50–1.42(m,1H),1.38(t,J＝7.1Hz,3H),0.79(s,9H).ESI-MS理论计算值C31H42Cl2N3O2 [M+H]+＝558.3，实验测得：558.3。
终产物72：(2'S，3S，4'R，5'R)-N-((1r，4R)-4-氨基环己基)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺(JP33)
步骤一：合成(1r，4R)-4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺基)环己基)氨基甲酸叔丁酯(JP30)
JN113(40mg,0.06mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(15mg,0.11mmol)，反式-4-氨基环己基氨基甲酸叔丁酯(36mg,0.17mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物35mg，产率78％。1H NMR(400MHz,Methanol-d4)δ7.42–7.22(m,5H),7.00(s,1H),6.97(dt,J＝6.4,2.0Hz,1H),4.57(d,J＝3.9Hz,1H),4.53–4.43(m,1H),4.35(d,J＝7.3Hz,1H),3.73–3.54(m,2H),3.30–3.18(m,1H),3.16–3.04(m,1H),2.98(d,J＝14.2Hz,1H),2.93–2.80(m,1H),2.04–1.78(m,3H),1.74–1.67(m,1H),1.64(dd,J＝15.7,4.5Hz,1H),1.52(dd,J＝15.7,3.3Hz,1H),1.42(s,9H),1.36–1.10(m,7H),0.75(s,9H).ESI-MS理论计算值C36H52Cl2FN4O3[M+H]+＝677.3，实验测得：677.4。
步骤二：合成(2'S，3S，4'R，5'R)-N-((1r，4R)-4-氨基环己基)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺(JP33)
将JP30(35mg,0.05mmol)称入瓶中，用2mL DMF溶解，加入碳酸钾(29mg,0.2mmol)，110℃搅拌过夜，反应结束后冷至室温，加水，乙酸乙酯萃取，有机相旋干后，粗品溶于3mL二氯甲烷中，加入3mL三氟乙酸，室温反应两小时。旋干反应液，HPLC纯化得目标产物23mg,产率68％。1H NMR(500MHz,Methanol-d4)δ7.37(d,J＝8.1Hz,1H),7.32–7.24(m,2H),7.21(t,J＝2.2Hz,1H),7.10(dt,J＝6.8,1.9Hz,1H),6.72(dd,J＝8.1,1.9Hz,1H),6.45(d,J＝1.8Hz,1H),4.76(d,J＝10.3Hz,1H),4.27–4.03(m,2H),3.79–3.56(m,3H),3.47(d,J＝11.4Hz,2H),3.04(tt,J＝11.9,4.0Hz,1H),2.16–1.83(m,5H),1.65(d,J＝12.8Hz,1H),1.56–1.43(m,4H),1.39(t,J＝7.2Hz,3H),1.18(qd,J＝12.9,3.6Hz,1H),0.75(s,9H).ESI-MS理论计算值C31H43Cl2N4O[M+H]+＝557.3，实验测得：557.3。
终产物73：(2'S，3S，4'R，5'R)-N-((1s，4S)-4-氨基环己基)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺(JP34)
步骤一：合成(1s，4S)-4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)环己基)氨基甲酸叔丁酯(JP31)
JN113(40mg,0.06mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(15mg,0.11mmol)，顺式-4-氨基环己基氨基甲酸叔丁酯(36mg,0.17mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物44mg，产率98％。1H NMR(400MHz,Methanol-d4)δ7.40–7.25(m,5H),7.05(s,1H),7.04–6.99(m,1H),4.57(t,J＝3.6Hz,1H),4.53–4.39(m,2H),3.87–3.72(m,1H),3.66(d,J＝14.2Hz,1H),3.58–3.42(m,1H),3.17–3.09(m,1H),3.05(d,J＝14.3Hz,1H),2.99–2.87(m,1H),1.80–1.46(m,10H),1.43(s,9H),1.29(t,J＝7.1Hz,3H),0.77(s,9H).ESI-MS理论计算值C36H52Cl2FN4O3[M+H]+＝677.3，实验测得：677.2。
步骤二：合成(2'S，3S，4'R，5'R)-N-((1s，4S)-4-氨基环己基)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺(JP34)
JP31(44mg,0.07mmol)，碳酸钾(36mg,0.26mmol)，三氟乙酸(3mL)，反应步骤参见终产物72的步骤二，得到目标产物的三氟乙酸盐30mg,产率64％。1H NMR(500MHz,Methanol-d4)δ7.38(d,J＝8.0Hz,1H),7.35–7.20(m,3H),7.13(dt,J＝7.3,1.4Hz,1H),6.72(dd,J＝8.1,1.9Hz,1H),6.44(d,J＝1.9Hz,1H),5.01(d,J＝11.0Hz,1H),4.31–4.05(m,2H),3.97(dd,J＝8.1,4.7Hz,1H),3.69(d,J＝11.0Hz,1H),3.65(dd,J＝13.0,7.0Hz,1H),3.60–3.34(m,2H),3.25–3.15(m,1H),2.15–1.49(m,9H),1.43(t,J＝7.1Hz,3H),1.39–1.23(m,1H),0.77(s,9H).ESI-MS理论计算值C31H43Cl2N4O[M+H]+＝557.3，实验测得：557.3。
终产物74：(1r，4R)-4-氨基环己基(2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-羧酸盐(JP08)
步骤一：合成(1r，4R)-4-氨基环己基(2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-羧酸酯(JP06-1)
JN113(60mg,0.1mmol)，1-甲基咪唑(25mg,0.3mmol)，乙基磺酰氯(26mg,0.2mmol)，反式-4-氨基环己烷-1-醇(26mg,0.22mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得JP06-1 42mg，产率61％。1H NMR(500MHz,Methanol-d4)δ7.46–7.19(m,5H),7.09–6.89(m,2H),4.57(t,J＝4.2Hz,1H),4.49(d,J＝7.7Hz,1H),4.43(t,J＝8.0Hz,1H),3.79–3.55(m,2H),3.55–3.40(m,1H),3.20–3.09(m,1H),3.05(d,J＝13.4Hz,1H),2.93(dt,J＝12.7,6.3Hz,1H),2.02–1.81(m,3H),1.81–1.59(m,2H),1.55(dd,J＝15.7,3.2Hz,1H),1.39–1.30(m,3H),1.27(t,J＝6.9Hz,3H),1.20–1.08(m,1H),0.76(s,9H).ESI-MS理论计算值C31H43Cl2FN3O2[M+H]+＝578.3，实验测得：578.3。
步骤二：合成(1r，4R)-4-氨基环己基(2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-羧酸盐(JP08)
JP06-1(40mg,0.07mmol)，碳酸钾(39mg,0.28mmol)，反应步骤参见终产物66的步骤三，得到目标产物的三氟乙酸盐16.5mg,产率35％。1H NMR(400MHz,Methanol-d4)δ7.37(d,J＝8.1Hz,1H),7.33–7.23(m,2H),7.20(t,J＝1.7Hz,1H),7.08(dt,J＝6.8,1.9Hz,1H),6.73(dd,J＝8.0,1.9Hz,1H),6.45(d,J＝1.9Hz,1H),4.76–4.56(m,1H),4.44–3.95(m,2H),3.68(d,J＝ 11.1Hz,1H),3.66–3.55(m,2H),3.54–3.35(m,3H),2.11–1.86(m,4H),1.81(d,J＝12.9Hz,1H),1.55(d,J＝11.3Hz,1H),1.40(t,J＝7.2Hz,3H),1.37–1.17(m,3H),1.08(qd,J＝12.7,3.4Hz,1H),0.76(s,9H).ESI-MS理论计算值C31H42Cl2N3O2[M+H]+＝558.3，实验测得：558.3。
终产物75：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-异丁基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ129)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-异丁基-5-新戊基吡咯烷-2-羧酸(JQ124)
JN107(100mg,0.14mmol)，异丁醛(98mg,1.4mmol)，醋酸硼氢化钠(297mg,1.4mmol)和1mL乙酸称入50mL圆底烧瓶中，用4mL 1，2-二氯乙烷溶解，室温反应过夜，加饱和碳酸氢钠溶液，乙酸乙酯萃取，有机相旋干后溶于20mL醋酸，加入氰基硼氢化钠(44mg，0.7mmol)，室温反应过夜，向反应液中加入饱和碳酸氢钠溶液，用乙酸乙酯萃取三次，合并有机相，饱和氯化钠溶液洗一次，无水硫酸钠干燥后旋干，正相柱纯化得粗品94mg。用2mL二氯甲烷溶解反应物，加入2mL三氟乙酸，室温过夜。转干反应液，加入饱和碳酸氢钠溶液，用二氯甲烷萃取三次，合并有机相，饱和氯化钠溶液洗一次，无水硫酸钠干燥后旋干，正相柱纯化得目标产物60mg，产率59％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-异丁基-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ128)
JQ124(49mg,0.07mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(19mg,0.14mmol)，4-氨基-3-甲氧基苯甲酸甲酯(37mg,0.2mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物39mg，产率72％。1H NMR(400MHz,Methanol-d4)δ8.18(d,J＝8.5Hz,1H),7.64–7.54(m,2H),7.43(t,J＝8.9Hz,1H),7.38–7.27(m,4H),7.16–7.02(m,2H),4.54–4.44(m,2H),4.40(d,J＝8.2Hz,1H),3.87(s,6H),3.59(d,J＝14.3Hz,1H),3.21(d,J＝14.3Hz,1H),3.02(dd,J＝12.7,5.0Hz,1H),2.65(dd,J＝12.5,8.7Hz,1H),1.97(dd,J＝14.2,7.5Hz,1H),1.78(dd,J＝15.5,5.9Hz,1H),1.53(d,J＝15.4Hz,1H),1.04(d,J＝6.7Hz,3H),0.95(d,J＝6.6Hz,3H),0.88(s,9H).ESI-MS理论计算值C36H45Cl2FN3O4[M+H]+＝672.3，实验测得：672.2。
步骤三：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-异丁基-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ129)
JQ128(39mg,0.06mmol)，碳酸钾(33mg,0.24mmol)，氢氧化锂一水合物(13mg,0.3mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐25.2mg，产率56％。1H NMR(400MHz,Methanol-d4)δ8.23(d,J＝8.4Hz,1H),7.63(dd,J＝8.4,1.7Hz,1H),7.57(d,J＝1.7Hz,1H),7.39–7.30(m,3H),7.27(t,J＝7.8Hz,1H),7.11(dt,J＝7.6,1.5Hz,1H),6.76(dd,J＝8.1,1.9Hz,1H),6.48(d,J＝1.9Hz,1H),4.52–4.14(m,1H),4.12–3.95(m,1H),3.81(s,3H),3.68(d,J＝10.9Hz,1H),3.58(d,J＝11.0Hz,1H),3.47–3.32(m,1H),3.08–2.83(m,1H),2.28–1.94(m,2H),1.82–1.42(m,1H),1.11(d,J＝6.7Hz,3H),1.09(d,J＝7.1Hz, 3H),0.93(s,9H).ESI-MS理论计算值C35H42Cl2N3O4[M+H]+＝638.3，实验测得：638.3。
终产物76：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(环丙基甲基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JQ53)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(环丙基甲基)-5-新戊基吡咯烷-2-羧酸(JQ48)
JN107(200mg,0.27mmol)，环丙烷甲醛(96mg,1.37mmol)，醋酸硼氢化钠(290mg,1.37mmol)，乙酸1mL，三氟乙酸3mL，反应步骤参见终产物18的步骤四，得到目标产物30mg，产率14％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(环丙基甲基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ51)
JQ48(25mg,0.03mmol)，1-甲基咪唑(8mg,0.09mmol)，乙基磺酰氯(8mg,0.06mmol)，4-氨基-3-甲氧基苯甲酸甲酯(18mg,0.09mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物23mg，产率98％。1H NMR(400MHz,Methanol-d4)δ8.28(dd,J＝9.0,1.4Hz,1H),7.65–7.58(m,2H),7.54(t,J＝8.8Hz,1H),7.34(dd,J＝9.1,2.0Hz,1H),7.33–7.28(m,2H),7.25(dd,J＝13.6,2.2Hz,1H),7.11(t,J＝2.2Hz,1H),7.06(dt,J＝6.7,1.9Hz,1H),4.47(d,J＝9.2Hz,1H),4.34(d,J＝9.8Hz,1H),3.99(d,J＝9.8Hz,1H),3.94(s,3H),3.87(s,3H),3.63(dd,J＝12.8,5.4Hz,1H),3.44(d,J＝14.5Hz,1H),3.24(dd,J＝14.4,2.7Hz,1H),2.52(dd,J＝12.8,8.1Hz,1H),2.00(dd,J＝14.8,9.0Hz,1H),1.45(d,J＝14.9Hz,1H),1.06(s,9H),1.04–0.94(m,1H),0.56–0.41(m,2H),0.40–0.19(m,2H).ESI-MS理论计算值C36H43Cl2FN3O4[M+H]+＝670.3，实验测得：670.3。
步骤三：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(环丙基甲基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JQ53)
JQ51(23mg,0.03mmol)，碳酸钾(18mg,0.14mmol)，氢氧化锂一水合物(8mg,0.17mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐11mg，产率44％。1H NMR(400MHz,Methanol-d4)δ8.23(d,J＝8.4Hz,1H),7.63(dd,J＝8.4,1.7Hz,1H),7.58(d,J＝1.7Hz,1H),7.41–7.24(m,4H),7.14(d,J＝7.5Hz,1H),6.73(dd,J＝8.0,1.9Hz,1H),6.45(d,J＝1.8Hz,1H),4.36–4.12(m,1H),4.12–3.95(m,1H),3.83(s,3H),3.72–3.51(m,2H),3.27–3.01(m,1H),2.15–1.88(m,1H),1.75–1.55(m,1H),1.25–1.09(m,1H),0.90(s,9H),0.75(dd,J＝12.4,5.3Hz,1H),0.68–0.60(m,1H),0.60–0.50(m,1H),0.42(dt,J＝9.8,5.0Hz,1H).ESI-MS理论计算值C35H40Cl2N3O4[M+H]+＝636.2，实验测得：636.2。
终产物77：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(环丁基甲基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ148)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(环丁基甲基)-5-新戊基吡咯烷-2-羧酸(JQ141)
JN107(146mg,0.2mmol)，环丁烷甲醛(84mg,1.0mmol)，醋酸硼氢化钠(212mg,1.0mmol)，乙酸1mL，氰基硼氢化钠(126mg,2.0mmol)，三氟乙酸3mL，反应步骤参见终产物75的步骤一，得到目标产物90mg，产率58％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(环丁基甲基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ146)
JQ141(45mg,0.06mmol)，1-甲基咪唑(15mg,0.18mmol)，乙基磺酰氯(16mg,0.12mmol)，4-氨基-3-甲氧基苯甲酸甲酯(31mg,0.18mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物39mg，产率81％。1H NMR(400MHz,Methanol-d4)δ8.34–8.18(m,1H),7.65–7.57(m,2H),7.49(t,J＝9.0Hz,1H),7.36–7.29(m,3H),7.26(dd,J＝13.5,2.3Hz,1H),7.11(s,1H),7.06(dt,J＝6.3,2.0Hz,1H),4.43(d,J＝8.5Hz,1H),4.30(d,J＝9.4Hz,1H),4.06(d,J＝9.2Hz,1H),3.93(s,3H),3.87(s,3H),3.47(d,J＝14.5Hz,1H),3.40(dd,J＝12.4,6.7Hz,1H),3.30–3.24(m,1H),2.94(dd,J＝12.4,7.7Hz,1H),2.74–2.55(m,1H),2.17–1.71(m,7H),1.46(d,J＝15.0Hz,1H),1.02(s,9H).ESI-MS理论计算值C37H45Cl2FN3O4[M+H]+＝684.3，实验测得：684.3。
步骤三：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(环丁基甲基)-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ148)
JQ146(39mg,0.06mmol)，碳酸钾(32mg,0.24mmol)，氢氧化锂一水合物(13mg,0.3mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐22.4mg，产率49％。1H NMR(400MHz,Methanol-d4)δ8.24(d,J＝8.4Hz,1H),7.64(dd,J＝8.4,1.7Hz,1H),7.58(d,J＝1.7Hz,1H),7.39–7.23(m,4H),7.12(d,J＝7.5Hz,1H),6.74(dd,J＝8.0,1.9Hz,1H),6.47(d,J＝1.9Hz,1H),4.94–4.64(m,1H),4.37–4.14(m,1H),3.96(d,J＝9.8Hz,1H),3.82(s,3H),3.74–3.51(m,3H),3.21–2.98(m,1H),2.83–2.65(m,1H),2.36–2.02(m,3H),2.01–1.76(m,4H),1.73–1.43(m,1H),0.96(s,9H).ESI-MS理论计算值C36H42Cl2N3O4[M+H]+＝650.3，实验测得：650.3。
终产物78：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(环戊基甲基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ91)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4- (4-氯-2-氟苯基)-3-(3-氯苯基)-1-(环戊基甲基)-5-新戊基吡咯烷-2-羧酸(JQ80)
JN107(400mg,0.55mmol)，环戊烷甲醛(268mg,2.74mmol)，醋酸硼氢化钠(581mg,2.74mmol)，乙酸2mL，三氟乙酸2mL，反应步骤参见终产物18的步骤四，得到目标产物52mg，产率12％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(环戊基甲基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ86)
JQ80(52mg,0.07mmol)，1-甲基咪唑(17mg,0.2mmol)，乙基磺酰氯(17mg,0.13mmol)，4-氨基-3-甲氧基苯甲酸甲酯(36mg,0.2mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物34mg，产率64％。1H NMR(400MHz,Methanol-d4)δ8.19(d,J＝8.2Hz,1H),7.65–7.58(m,2H),7.43(t,J＝8.9Hz,1H),7.38–7.24(m,4H),7.15–7.04(m,2H),4.47(d,J＝8.7Hz,1H),4.29(d,J＝8.4Hz,1H),3.90(s,3H),3.88(s,3H),3.55(d,J＝14.4Hz,1H),3.26–3.17(m,2H),2.79(dd,J＝12.4,8.6Hz,1H),2.33–2.18(m,1H),1.96–1.90(m,1H),1.84(dd,J＝15.3,7.0Hz,1H),1.79–1.54(m,5H),1.50(dd,J＝15.3,2.2Hz,1H),1.42–1.19(m,3H),0.92(s,9H).ESI-MS理论计算值C38H47Cl2FN3O4[M+H]+＝698.3，实验测得：698.3。
步骤三：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(环戊基甲基)-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ91)
JQ86(34mg,0.05mmol)，碳酸钾(27mg,0.2mmol)，氢氧化锂一水合物(11mg,0.25mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐12.7mg，产率33％。1H NMR(500MHz,Methanol-d4)δ8.28(s,1H),7.64(dt,J＝8.3,1.9Hz,1H),7.59(s,1H),7.46–7.16(m,5H),7.09(d,J＝7.6Hz,1H),6.70(dd,J＝8.0,2.0Hz,1H),6.43(d,J＝2.0Hz,1H),4.26–3.73(m,6H),3.73–3.35(m,1H),3.22–2.57(m,1H),2.44–1.23(m,11H),0.97(s,9H).ESI-MS理论计算值C37H44Cl2N3O4[M+H]+＝664.3，实验测得：664.3。
终产物79：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(环己基甲基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ96)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(环己基甲基)-5-新戊基吡咯烷-2-羧酸(JQ81)
JN107(200mg,0.27mmol)，环己烷甲醛(153mg,1.37mmol)，醋酸硼氢化钠(290mg,1.37mmol)，乙酸1mL，三氟乙酸2mL，反应步骤参见终产物18的步骤四，得到目标产物21mg，产率10％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(环己基甲基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ92)
JQ81(21mg,0.03mmol)，1-甲基咪唑(8mg,0.09mmol)，乙基磺酰氯(8mg,0.06mmol)，4-氨基-3-甲氧基苯甲酸甲酯(15mg,0.08mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物6mg，产率24％。1H NMR(400MHz,Methanol-d4)δ8.19(d,J＝8.3Hz,1H), 7.69–7.57(m,2H),7.48(t,J＝7.9Hz,1H),7.39–7.28(m,4H),7.15(s,1H),7.09(d,J＝6.2Hz,1H),4.50–4.30(m,3H),3.90(s,3H),3.89(s,3H),3.54(d,J＝14.3Hz,1H),3.21(d,J＝14.3Hz,1H),3.02(dd,J＝12.6,5.3Hz,1H),2.70(dd,J＝12.5,8.0Hz,1H),1.91–1.74(m,3H),1.73–1.61(m,3H),1.51(d,J＝15.3Hz,1H),1.45–1.13(m,4H),1.09–0.94(m,2H),0.90(s,9H).ESI-MS理论计算值C39H49Cl2FN3O4[M+H]+＝712.3，实验测得：712.3。
步骤三：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(环己基甲基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ96)
JQ96(6mg,0.01mmol)，碳酸钾(5mg,0.04mmol)，氢氧化锂一水合物(2mg,0.05mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐2mg，产率25％。1H NMR(500MHz,Methanol-d4)δ8.36(s,1H),7.71–7.63(m,2H),7.41–7.00(m,5H),6.69(d,J＝8.1Hz,1H),6.42(d,J＝2.2Hz,1H),3.93(s,3H),3.90–3.45(m,7H),2.28–2.18(m,1H),1.87–1.77(m,3H),1.77–1.65(m,3H),1.41–1.01(m,6H),0.98(s,9H).ESI-MS理论计算值C38H46Cl2N3O4[M+H]+＝678.3，实验测得：678.2。
终产物80及81：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-2'-新戊基-1'-(((R或S)-四氢呋喃-3-基)甲基)螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JR01及JR02)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-((四氢呋喃-3-基)甲基)吡咯烷-2-羧酸(JQ157)
JN107(146mg,0.2mmol)，四氢呋喃-3-甲醛(100mg,1.0mmol)，醋酸硼氢化钠(212mg,1.0mmol)，乙酸1mL，氰基硼氢化钠(126mg,2.0mmol)，三氟乙酸3mL，反应步骤参见终产物75的步骤一，得到目标产物64mg，产率40％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-(((R或S)-四氢呋喃-3-基)甲基)吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸甲酯(JQ159-1及JQ159-2)
JQ157(64mg,0.08mmol)，1-甲基咪唑(20mg,0.24mmol)，乙基磺酰氯(21mg,0.16mmol)，4-氨基-3-甲氧基苯甲酸甲酯(44mg,0.24mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，反应结束后，HPLC纯化，得到JQ159-1 20mg，产率31％。1H NMR(400MHz,Methanol-d4)δ8.23(d,J＝8.3Hz,1H),7.62(d,J＝1.7Hz,1H),7.60(d,J＝1.8Hz,1H),7.55(t,J＝8.9Hz,1H),7.41–7.25(m,4H),7.20(d,J＝2.1Hz,1H),7.09(d,J＝7.1Hz,1H),4.50–4.37(m,2H),4.15(d,J＝9.3Hz,1H),3.94(s,3H),3.91(dd,J＝8.3,4.8Hz,1H),3.88(s,3H),3.77–3.63(m,3H),3.55–3.43(m,1H),3.35(d,J＝15.8Hz,1H),3.28(d,J＝8.1Hz,1H),2.93(dd,J＝12.3,6.7Hz,1H),2.62–2.45(m,1H),2.22–2.11(m,1H),1.95(dd,J＝15.2,8.2Hz,1H),1.67(dq,J＝12.1,7.5Hz,1H),1.48(dd,J＝15.2,1.7Hz,1H),0.98(s,9H).ESI-MS理论计算值C37H45Cl2FN3O5[M+H]+＝700.3，实验测得：700.3。
得到JQ159-2 24mg，产率37％。1H NMR(400MHz,Methanol-d4)δ8.24(d,J＝8.9Hz,1H), 7.65–7.60(m,2H),7.55(t,J＝9.0Hz,1H),7.39–7.27(m,4H),7.20(t,J＝2.2Hz,1H),7.09(dt,J＝6.8,2.0Hz,1H),4.50–4.36(m,2H),4.21(d,J＝8.9Hz,1H),3.93(s,3H),3.91–3.89(m,1H),3.88(s,3H),3.84(dd,J＝8.3,5.2Hz,1H),3.77–3.68(m,1H),3.55(dd,J＝8.5,5.7Hz,1H),3.52–3.45(m,1H),3.36–3.32(m,1H),3.30–3.22(m,1H),2.92(dd,J＝12.5,7.8Hz,1H),2.58(hept,J＝6.7Hz,1H),2.02–1.93(m,1H),1.89(dd,J＝15.3,7.6Hz,1H),1.81–1.69(m,1H),1.50(dd,J＝15.0,1.9Hz,1H),0.97(s,9H).ESI-MS理论计算值C37H45Cl2FN3O5[M+H]+＝700.3，实验测得：700.3。
步骤三：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-2'-新戊基-1'-(((R或S)-四氢呋喃-3-基)甲基)螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JR01及JR02)
JQ159-1(20mg,0.03mmol)，碳酸钾(17mg,0.12mmol)，氢氧化锂一水合物(7mg,0.15mmol),反应步骤参见终产物10的步骤六，得到JR01的三氟乙酸盐12.1mg，产率25％。1H NMR(400MHz,Methanol-d4)δ8.31(d,J＝8.2Hz,1H),7.69–7.59(m,2H),7.33–7.20(m,4H),7.08(dt,J＝7.4,1.6Hz,1H),6.80(dt,J＝8.0,1.7Hz,1H),6.56–6.51(m,1H),4.64–4.39(m,1H),4.07–3.80(m,7H),3.76–3.65(m,2H),3.58(d,J＝10.9Hz,1H),3.48(d,J＝10.9Hz,1H),3.36(d,J＝11.4Hz,1H),2.83(d,J＝8.9Hz,1H),2.62–2.43(m,1H),2.28–2.11(m,1H),2.02(dd,J＝15.5,8.6Hz,1H),1.75–1.62(m,1H),1.36(d,J＝15.6Hz,1H),0.97(s,9H).ESI-MS理论计算值C36H42Cl2N3O5[M+H]+＝666.3，实验测得：666.2。
JQ159-2(24mg,0.03mmol)，碳酸钾(19mg,0.14mmol)，氢氧化锂一水合物(7mg,0.15mmol)，反应步骤参见终产物10的步骤六，得到JR02的三氟乙酸盐13mg，产率56％。1H NMR(400MHz,Methanol-d4)δ8.30(d,J＝8.3Hz,1H),7.68–7.60(m,2H),7.36–7.17(m,4H),7.07(dt,J＝7.4,1.6Hz,1H),6.80(dd,J＝8.1,1.9Hz,1H),6.54(d,J＝1.9Hz,1H),4.69–4.36(m,1H),4.06–3.97(m,1H),3.96–3.82(m,6H),3.73(q,J＝7.7Hz,1H),3.63–3.54(m,2H),3.48(d,J＝10.9Hz,1H),3.39–3.32(m,1H),3.01–2.81(m,1H),2.66–2.48(m,1H),2.12–1.93(m,2H),1.93–1.78(m,1H),1.41(d,J＝15.7Hz,1H),0.96(s,9H).ESI-MS理论计算值C36H42Cl2N3O5[M+H]+＝666.3，实验测得：666.2。
终产物82：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-2'-新戊基-1'-((四氢-2H-吡喃-4-基)甲基)螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JQ122)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-((四氢-2H-吡喃-4-基)甲基)吡咯烷-2-羧酸(JQ110)
JN107(100mg,0.14mmol)，四氢-2H-吡喃-4-甲醛(78mg,0.68mmol)，醋酸硼氢化钠(144mg,0.68mmol)，乙酸1mL，氰基硼氢化钠(89mg,1.4mmol)，三氟乙酸2mL，反应步骤参见终产物75的步骤一，得到目标产物20mg，产率19％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-((四氢-2H-吡喃-4-基)甲基)吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸甲酯(JQ119)
JQ110(19mg,0.03mmol)，1-甲基咪唑(8mg,0.09mmol)，乙基磺酰氯(8mg,0.06mmol)，4-氨基-3-甲氧基苯甲酸甲酯(16mg,0.09mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物17mg，产率69％。1H NMR(400MHz,Methanol-d4)δ8.19(d,J＝8.3Hz,1H),7.68–7.59(m,2H),7.49(t,J＝7.9,6.2Hz,1H),7.39–7.25(m,4H),7.20(s,1H),7.09(d,J＝6.8Hz,1H),4.51–4.39(m,2H),4.34(d,J＝8.4Hz,1H),3.97(d,J＝11.4Hz,1H),3.90(s,3H),3.89(s,3H),3.87–3.84(m,1H),3.54(d,J＝14.4Hz,1H),3.45(t,J＝11.8Hz,1H),3.27(d,J＝14.3Hz,1H),3.10(dd,J＝13.1,5.4Hz,1H),2.76(dd,J＝12.7,7.9Hz,1H),1.98–1.86(m,1H),1.85–1.64(m,3H),1.52(d,J＝15.2Hz,1H),1.43–1.12(m,3H),0.91(s,9H).ESI-MS理论计算值C38H47Cl2FN3O5[M+H]+＝714.3，实验测得：714.3。
步骤三：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-2'-新戊基-1'-((四氢-2H-吡喃-4-基)甲基)螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JQ122)
JQ119(17mg,0.02mmol)，碳酸钾(14mg,0.1mmol)，氢氧化锂一水合物(5mg,0.1mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐9.5mg，产率60％。1H NMR(400MHz,Methanol-d4)δ8.28(d,J＝8.4Hz,1H),7.64(d,J＝8.9Hz,1H),7.61(s,1H),7.37–7.20(m,4H),7.07(d,J＝7.3Hz,1H),6.76(d,J＝8.1Hz,1H),6.49(s,1H),4.71–4.34(m,1H),4.25–3.89(m,4H),3.87(s,3H),3.60(d,J＝10.9Hz,1H),3.52–3.38(m,2H),3.23(t,J＝12.0Hz,1H),2.92–2.55(m,1H),2.16–1.84(m,3H),1.76(d,J＝12.9Hz,1H),1.60–1.25(m,3H),0.95(s,9H).ESI-MS理论计算值C37H44Cl2N3O5[M+H]+＝680.3，实验测得：680.3。
终产物83：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(呋喃-2-基甲基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺)-3-甲氧基苯甲酸(JQ149)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(呋喃-2-基甲基)-5-新戊基吡咯烷-2-羧酸(JQ142)
JN107(146mg,0.2mmol)，呋喃-2-甲醛(96mg,1.0mmol)，醋酸硼氢化钠(212mg,1.0mmol)，乙酸1mL，氰基硼氢化钠(126mg,2.0mmol)，三氟乙酸3mL，反应步骤参见终产物75的步骤一，得到目标产物37mg，产率25％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(呋喃-2-基甲基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ147)
JQ142(37mg,0.05mmol)，1-甲基咪唑(13mg,0.15mmol)，乙基磺酰氯(13mg,0.1mmol)，4-氨基-3-甲氧基苯甲酸甲酯(27mg,0.15mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物40mg，产率99％。1H NMR(400MHz,Methanol-d4)δ8.12(d,J＝8.4Hz,1H),7.61–7.45(m,3H),7.39–7.23(m,5H),7.12(s,1H),7.06(dd,J＝5.4,3.2Hz,1H),6.39(d,J＝3.2Hz,1H),6.24(dd,J＝3.2,1.9Hz,1H),4.63–4.48(m,2H),4.33(d,J＝9.4Hz,1H),4.13(d,J＝14.0Hz,1H),4.04(d,J＝9.3Hz,1H),3.92(s,3H),3.86(s,3H),3.50(d,J＝14.5Hz,1H),3.34(d,J＝13.3Hz,1H),2.11(dd,J＝15.2,8.8Hz,1H),1.55(d,J＝15.1Hz,1H),1.09(s,9H). ESI-MS理论计算值C37H41Cl2FN3O5[M+H]+＝696.2，实验测得：696.2。
步骤三：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(呋喃-2-基甲基)-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺)-3-甲氧基苯甲酸(JQ149)
JQ147(40mg,0.06mmol)，碳酸钾(32mg,0.24mmol)，氢氧化锂一水合物(13mg,0.3mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐25.4mg，产率55％。1H NMR(400MHz,Methanol-d4)δ8.16(d,J＝8.3Hz,1H),7.62–7.53(m,2H),7.35(d,J＝1.8Hz,1H),7.32(d,J＝8.1Hz,1H),7.28–7.20(m,2H),7.17(t,J＝2.0Hz,1H),7.10–7.03(m,1H),6.85(dd,J＝8.1,1.9Hz,1H),6.61(d,J＝1.9Hz,1H),6.49(d,J＝3.2Hz,1H),6.27(dd,J＝3.3,1.9Hz,1H),4.61(d,J＝13.8Hz,1H),4.38(d,J＝9.9Hz,1H),3.93(s,3H),3.93–3.89(m,2H),3.86(d,J＝10.0Hz,1H),3.61(d,J＝11.0Hz,1H),3.48(d,J＝11.0Hz,1H),2.10(dd,J＝15.5,8.9Hz,1H),1.38(d,J＝15.4Hz,1H),1.04(s,9H).ESI-MS理论计算值C36H38Cl2N3O5[M+H]+＝662.2，实验测得：662.2。
终产物84：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(2,2-二氟乙基)-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ57)
步骤一：合成(2R，3R，4S，5S)-4-(((叔丁氧羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-(2-氧乙基)吡咯烷-2-甲酸叔丁酯(JQ46)
二甲基亚砜(141mg，1.8mmol)称入一个100mL圆底烧瓶中，加入超干二氯甲烷，-78℃下滴加草酰氯(115mg，0.9mmol)，搅拌15分钟后，向反应液中滴加JQ12(200mg，0.3mmol)的二氯甲烷溶液，-78℃搅拌1小时，滴加三乙胺(273mg，2.7mmol)，自然升温至室温，反应3小时。反应结束后，加水，乙酸乙酯萃取三次，合并有机相，饱和氯化钠溶液洗一次，无水硫酸钠干燥后旋干，得粗品220mg，直接投下一步。
步骤二：合成(2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(2,2-二氟乙基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ50)
JQ46(264mg,0.42mmol)，二乙胺基三氟化硫(203mg,1.26mmol)，三氟乙酸1mL，反应步骤参见终产物49的步骤三，得目标产物160mg，产率66％。
步骤三：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(2,2-二氟乙基)-5-新戊基吡咯烷-2-羧酸(JQ55)
JQ50(170mg,0.61mmol)，二异丙基乙基胺(315mg,2.44mmol)，FmocCl(238mg,0.92mmol)，三氟乙酸4mL，反应步骤参见终产物49的步骤四，得目标产物108mg，产率50％。
步骤四：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(2,2-二氟乙基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ56)
JQ55(50mg,0.06mmol)，1-甲基咪唑(17mg,0.2mmol)，乙基磺酰氯(16mg,0.12mmol)，4-氨基-3-甲氧基苯甲酸甲酯(36mg,0.2mmol)，哌啶(2mL)，反应步骤参见终产物18的步骤五，得目标产物40mg，产率83％。1H NMR(400MHz,Methanol-d4)δ8.22(d,J＝8.8Hz,1H),7.64–7.54(m,2H),7.48(t,J＝8.8Hz,1H),7.37–7.30(m,3H),7.28(dd,J＝13.5,2.2Hz,1H),7.13(s,1H),7.06(d,J＝6.6Hz,1H),6.08(tt,J＝55.5,3.9Hz,1H),4.54(d,J＝8.4Hz,1H),4.42(d,J＝9.2Hz,1H),4.08(d,J＝9.1Hz,1H),3.91(s,3H),3.86(s,3H),3.84–3.76(m,1H),3.55–3.35(m,2H),3.24(dd,J＝14.5,2.3Hz,1H),1.95(dd,J＝15.5,8.7Hz,1H),1.55(d,J＝15.3Hz,1H),1.02(s,9H).ESI-MS理论计算值C34H39Cl2F3N3O4[M+H]+＝680.2，实验测得：680.2。
步骤五：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-(2,2-二氟乙基)-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ57)
JQ56(40mg,0.06mmol)，碳酸钾(34mg,0.24mmol)，氢氧化锂一水合物(13mg,0.3mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐28.2mg，产率62％。1H NMR(400MHz,Methanol-d4)δ8.34(d,J＝8.8Hz,1H),7.71–7.60(m,2H),7.29(d,J＝8.1Hz,1H),7.25–7.18(m,2H),7.15(d,J＝2.1Hz,1H),7.09–6.98(m,1H),6.84(dd,J＝8.1,1.9Hz,1H),6.61(d,J＝1.8Hz,1H),6.02(tt,J＝55.6,3.9Hz,1H),4.24(d,J＝9.1Hz,1H),3.94(s,3H),3.88–3.68(m,3H),3.48(d,J＝10.9Hz,1H),3.37(d,J＝11.0Hz,1H),3.03(qd,J＝14.7,4.1Hz,1H),1.87(dd,J＝15.5,9.3Hz,1H),1.24(d,J＝15.5Hz,1H),0.99(s,9H).ESI-MS理论计算值C33H36Cl2F2N3O4[M+H]+＝646.2，实验测得：646.2。
终产物85：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-2'-新戊基-1'-(丙-2-炔-1-基)螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ158)
步骤一：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-(丙-2-炔-1-基)吡咯烷-2-羧酸(JQ155)
JN107(132mg,0.18mmol)，丙炔醛(101mg,1.8mmol)，醋酸硼氢化钠(382mg,1.8mmol)，乙酸2mL，氰基硼氢化钠(114mg,1.8mmol)，三氟乙酸2mL，反应步骤参见终产物75的步骤一，得到目标产物80mg，产率63％。
步骤二：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-(丙-2-炔-1-基)吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸甲酯(JQ156)
JQ155(49mg,0.07mmol)，1-甲基咪唑(18mg,0.21mmol)，乙基磺酰氯(18mg,0.14mmol)，4-氨基-3-甲氧基苯甲酸甲酯(38mg,0.21mmol)，哌啶(2mL)，反应步骤参见终产物18的步骤五，得目标产物40mg，产率74％。1H NMR(400MHz,Methanol-d4)δ8.26(d,J＝8.9Hz,1H),7.65–7.53(m,3H),7.38–7.31(m,3H),7.28(dd,J＝13.6,2.2Hz,1H),7.18(s,1H),7.16–7.07(m,1H),4.51(d,J＝9.3Hz,1H),4.41(d,J＝8.1Hz,1H),4.16–4.03(m,2H),3.98–3.93(m, 1H),3.92(s,3H),3.87(s,3H),3.49(d,J＝14.6Hz,1H),3.39–3.32(m,1H),2.81(t,J＝2.2Hz,1H),1.96(dd,J＝15.3,8.4Hz,1H),1.50(dd,J＝15.3,1.8Hz,1H),1.03(s,9H).ESI-MS理论计算值C35H39Cl2FN3O4[M+H]+＝654.2，实验测得：654.2。
步骤三：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基-1-(丙-2-炔-1-基)吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸甲酯(JQ158)
JQ156(40mg,0.06mmol)，碳酸钾(34mg,0.24mmol)，氢氧化锂一水合物(13mg,0.3mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐13.7mg，产率31％。1H NMR(400MHz,Methanol-d4)δ8.31(d,J＝8.8Hz,1H),7.66–7.60(m,2H),7.33(d,J＝8.1Hz,1H),7.26–7.19(m,2H),7.17(s,1H),7.11–7.02(m,1H),6.84(dd,J＝8.1,1.9Hz,1H),6.59(d,J＝1.9Hz,1H),4.47(d,J＝10.1Hz,1H),4.01(dd,J＝17.0,2.5Hz,1H),3.94(s,3H),3.89–3.82(m,2H),3.77(dd,J＝16.9,2.4Hz,1H),3.65(d,J＝11.2Hz,1H),3.43(d,J＝11.1Hz,1H),2.82(t,J＝2.3Hz,1H),1.91(dd,J＝16.0,8.0Hz,1H),1.34(dd,J＝15.5,1.5Hz,1H),0.97(s,9H).ESI-MS理论计算值C34H36Cl2N3O4[M+H]+＝620.2，实验测得：620.2。
终产物86：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-7-氟-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JQ120)
步骤一：合成(Z)-2-(4-氯-2,3-二氟苯基)-3-(3-氯苯基)丙烯腈(JQ64)
3-氯苯甲醛(1.5g,10.7mmol)，2-(4-氯-2,3-二氟苯基)乙腈(2.0g,10.7mmol)，5N甲醇钠的甲醇溶液2.6mL，反应步骤参见中间体1的步骤一，得目标产物3.2g,产率97％。1H NMR(400MHz,Chloroform-d)δ7.83(dt,J＝7.4,2.2Hz,1H),7.81(t,J＝1.6Hz,1H),7.55(s,1H),7.50–7.41(m,2H),7.38–7.27(m,2H).
步骤二：合成(2R，3R，4R，5S)-4-(4-氯-2,3-二氟苯基)-3-(3-氯苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ66)
(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(1.9g,9.0mmol)，JQ64(2.3g,7.5mmol)，醋酸亚铜(92mg,0.75mmol)，R-(+)-1,1'-联萘-2,2'-双二苯膦(514mg,0.83mmol)，三乙胺(758mg,7.5mmol),反应步骤参见终产物18的步骤一，得目标产物3.5g，产率90％。1H NMR(400MHz,Chloroform-d)δ7.29–7.22(m,2H),7.21–7.12(m,3H),7.10(dt,J＝7.3,1.6Hz,1H),4.24(d,J＝7.5Hz,1H),4.11(d,J＝7.6Hz,1H),4.04(d,J＝9.1Hz,1H),1.64(dd,J＝14.4,9.2Hz,1H),1.39(s,9H),1.29(dd,J＝14.4,1.2Hz,1H),0.91(s,9H).
步骤三：合成(2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2,3-二氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ100)
JQ66(3.5g,6.7mmol)，雷尼镍5g，水合肼10mL，反应步骤参见中间体1的步骤三，得 目标产物1.4g，产率40％。1H NMR(400MHz,Chloroform-d)δ7.25–7.14(m,3H),7.13–7.08(m,1H),7.00–6.94(m,1H),6.88–6.77(m,1H),4.31(dd,J＝8.0,1.7Hz,1H),4.15(dd,J＝9.8,3.9Hz,1H),4.00(d,J＝8.4Hz,1H),3.33(dd,J＝13.9,6.0Hz,1H),3.04(dd,J＝13.6,3.9Hz,1H),1.54–1.45(m,1H),1.35(s,9H),0.96(d,J＝9.7Hz,1H),0.92(s,9H).
步骤四：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2,3-二氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ102)
JQ100(1.2g,2.3mmol)，二异丙基乙基胺(1.2g,9.0mmol)，FmocCl(880mg,3.4mmol)，反应步骤参见终产物18的步骤三，得目标产物1.8g，产率99％。
步骤五；合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2,3-二氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-羧酸(JQ111)
JQ102(153mg,0.2mmol)，乙醛(44mg,1.0mmol)，醋酸硼氢化钠(212mg,1.0mmol)，乙酸(1mL)，三氟乙酸(3mL)，反应步骤参见终产物18的步骤四，得目标产物96mg，产率67％。
步骤六：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2,3-二氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ117)
JQ111(53mg,0.07mmol)，1-甲基咪唑(19mg,0.22mmol)，乙基磺酰氯(20mg,0.15mmol)，4-氨基-3-甲氧基苯甲酸甲酯(40mg,0.22mmol)，哌啶(2mL)，反应步骤参见终产物18的步骤五，得目标产物67mg，产率99％。1H NMR(400MHz,Methanol-d4)δ8.26(d,J＝8.4Hz,1H),7.69–7.54(m,2H),7.42(t,J＝8.0Hz,1H),7.38–7.28(m,3H),7.16(s,1H),7.05(d,J＝7.1Hz,1H),4.44(d,J＝9.2Hz,1H),4.34(d,J＝9.2Hz,1H),4.02–3.96(m,1H),3.95(s,3H),3.88(s,3H),3.55–3.40(m,2H),3.37–3.32(m,1H),3.11–2.96(m,1H),2.07(d,J＝10.0Hz,1H),1.46(d,J＝15.0Hz,1H),1.24(t,J＝7.0Hz,3H),1.06(s,9H).ESI-MS理论计算值C34H40Cl2F2N3O4[M+H]+＝662.2，实验测得：662.3。
步骤七：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-7-氟-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JQ120)
JQ117(67mg,0.1mmol)，碳酸钾(56mg,0.4mmol)，氢氧化锂一水合物(17mg,0.4mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐42.6mg，产率57％。1H NMR(400MHz,Methanol-d4)δ8.16(d,J＝8.3Hz,1H),7.62(d,J＝8.5Hz,1H),7.57(s,1H),7.36–7.24(m,3H),7.21(d,J＝8.2Hz,1H),7.13(d,J＝7.4Hz,1H),6.79(t,J＝7.2Hz,1H),5.16–4.98(m,1H),4.34–4.05(m,2H),3.83(s,3H),3.77(d,J＝11.1Hz,1H),3.67(dd,J＝13.0,7.1Hz,1H),3.60(d,J＝11.1Hz,1H),3.31–3.16(m,1H),2.04(dd,J＝16.3,5.9Hz,1H),1.92–1.69(m,1H),1.41(t,J＝7.1Hz,3H),0.84(s,9H).ESI-MS理论计算值C33H37Cl2FN3O4[M+H]+＝628.2，实验测得：628.2。
终产物87：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-5-氟-2'-新戊基螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ121)
步骤一：合成(Z)-2-(4-氯-2,5-二氟苯基)-3-(3-氯苯基)丙烯腈(JQ63)
3-氯苯甲醛(2.5g,18.0mmol)，2-(4-氯-2,5-二氟苯基)乙腈(3.4g,18.0mmol)，5N甲醇钠的甲醇溶液4.3mL，反应步骤参见中间体1的步骤一，得目标产物4.3g,产率80％。1H NMR(400MHz,Chloroform-d)δ7.83(dt,J＝6.8,1.7Hz,1H),7.80(d,J＝1.7Hz,1H),7.56(s,1H),7.49–7.38(m,3H),7.32–7.27(m,1H).
步骤二：合成(2R，3R，4R，5S)-4-(4-氯-2,5-二氟苯基)-3-(3-氯苯基)-4-氰基-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ67)
(E)-2-(((3,3-二甲基亚丁基)氨基)乙酸叔丁酯(2.1g,9.7mmol)，JQ63(2.5g,8.1mmol)，醋酸亚铜(100mg,0.81mmol)，R-(+)-1,1'-联萘-2,2'-双二苯膦(555mg,0.89mmol)，三乙胺(818mg,8.1mmol),反应步骤参见终产物18的步骤一，得目标产物4.0g，产率95％。1H NMR(400MHz,Chloroform-d)δ7.30–7.20(m,5H),7.17(t,J＝1.9Hz,1H),7.10(dt,J＝7.3,1.6Hz,1H),4.22(d,J＝7.6Hz,1H),4.13(d,J＝7.6Hz,1H),4.02(d,J＝9.0Hz,1H),1.62(dd,J＝14.3,9.1Hz,1H),1.38(s,9H),1.29(dd,J＝14.3,1.2Hz,1H),0.90(s,9H).
步骤三：合成(2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2,5-二氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ101)
JQ67(4.0g,7.6mmol)，雷尼镍5g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物1.9g，产率48％。1H NMR(400MHz,Chloroform-d)δ7.30–7.24(m,1H),7.23–7.07(m,3H),7.02(dd,J＝11.1,6.9Hz,1H),6.96(dd,J＝7.6,1.5Hz,1H),4.27(d,J＝8.8Hz,1H),4.07(dd,J＝8.4,2.1Hz,1H),3.83(dd,J＝8.8,2.0Hz,1H),3.22(d,J＝13.1Hz,1H),3.05(d,J＝13.1Hz,1H),1.50–1.38(m,2H),1.30(s,9H),0.93(s,9H).
步骤四：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2,5-二氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JQ103)
JQ101(1.7g,3.2mmol)，二异丙基乙基胺(1.7g,12.8mmol)，FmocCl(1.2g,4.8mmol)，反应步骤参见终产物18的步骤三，得目标产物2.5g，产率99％。
步骤五；合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2,5-二氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-羧酸(JQ112)
JQ102(160mg,0.21mmol)，乙醛(46mg,1.05mmol)，醋酸硼氢化钠(223mg,1.05mmol)，乙酸(1mL)，三氟乙酸(3mL)，反应步骤参见终产物18的步骤四，得目标产物115mg，产率76％。
步骤六：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2,5-二氟苯基)-3-(3-氯苯基)-1-乙基-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JQ118)
JQ112(67mg,0.09mmol)，1-甲基咪唑(23mg,0.27mmol)，乙基磺酰氯(24mg,0.18mmol)，4-氨基-3-甲氧基苯甲酸甲酯(51mg,0.27mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物57mg，产率81％。1H NMR(400MHz,Methanol-d4)δ8.24(dd,J＝8.9,1.8Hz,1H),7.63–7.55(m,2H),7.49(t,J＝9.3Hz,1H),7.38(dd,J＝12.7,6.5Hz,1H),7.35–7.26(m,2H),7.18(s,1H),7.08(d,J＝6.5Hz,1H),4.41(d,J＝8.9Hz,1H),4.31(d,J＝9.2Hz,1H),3.99(d,J＝9.3Hz,1H),3.93(s,3H),3.87(s,3H),3.54–3.38(m,2H),3.30–3.22(m,1H),3.09–2.94(m,1H),2.04(dd,J＝13.3,5.6Hz,1H),1.47(d,J＝15.0Hz,1H),1.24(t,J＝7.0Hz,3H),1.07(s,9H).ESI-MS理论计算值C34H40Cl2F2N3O4[M+H]+＝662.2，实验测得：662.2。
步骤七：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-5-氟-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JQ121)
JQ118(57mg,0.09mmol)，碳酸钾(50mg,0.36mmol)，氢氧化锂一水合物(17mg,0.4mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐45.4mg，产率68％。1H NMR(400MHz,Methanol-d4)δ8.17(d,J＝8.3Hz,1H),7.62(d,J＝8.5Hz,1H),7.57(s,1H),7.40(d,J＝9.0Hz,1H),7.35(s,1H),7.33–7.24(m,2H),7.15(d,J＝7.0Hz,1H),6.54(d,J＝5.9Hz,1H),5.06–4.97(m,1H),4.27–4.04(m,2H),3.83(s,3H),3.74–3.62(m,2H),3.55(d,J＝11.0Hz,1H),3.32–3.21(m,1H),2.03(dd,J＝14.7,6.9Hz,1H),1.93–1.69(m,1H),1.40(t,J＝7.1Hz,3H),0.87(s,9H).ESI-MS理论计算值C33H37Cl2FN3O4[M+H]+＝628.2，实验测得：628.2。
终产物93：4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-1'-乙基-2'-(2,2,3-三甲基丁基)螺[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(TC145)
步骤一：(E)-2-((3,3,4-三甲基亚戊基)氨基)乙酸叔丁酯(TC128)
TC127(2.8,22.1mmol)，甘氨酸叔丁酯(3.2g,24.5mmol)，反应步骤参见终产物33的步骤一，得目标产物5.2g，产率99％。
步骤二：合成(2R,3R,4R,5S)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-4-氰基-5-(2,2,3-三甲基丁基)吡咯烷-2-甲酸叔丁酯(TC129)
TC128(5.2g,21.5mmol)，YH132(5.4g,18.4mmol)，AgF(2.4g,18.4mmol)，三乙胺4.25mL，反应步骤参见中间体1的步骤二，得目标产物3.1g，产率31％。1H NMR(500MHz,Chloroform-d)δ7.37(t,J＝8.5Hz,1H),7.26–7.22(m,1H),7.22–7.17(m,2H),7.16(t,J＝1.9Hz,1H),7.13(dd,J＝8.5,1.7Hz,1H),7.10(dt,J＝7.4,1.6Hz,1H),4.24(d,J＝7.6Hz,1H),4.15(d,J＝7.6,Hz,1H),4.04(d,J＝8.9Hz,1H),1.67–1.58(m,1H),1.46(h,J＝6.7Hz,1H),1.38(s,9H),1.37–1.32(m,1H),0.81(d,J＝6.4Hz,6H),0.77(s,3H),0.61(d,J＝6.8Hz,3H).
步骤三：合成(2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-(2,2,3-三甲基 丁基)吡咯烷-2-甲酸叔丁酯(TC130)
TC129(3.1g,5.8mmol)，雷尼镍3g，水合肼10mL，反应步骤参见中间体1的步骤三，得目标产物1.6g，产率43％。1H NMR(400MHz,Methanol-d4)δ7.37–7.26(m,4H),7.21(t,J＝8.7Hz,1H),7.09(s,1H),6.99(d,J＝5.4Hz,1H),4.34(d,J＝7.7Hz,1H),4.27(t,J＝5.2Hz,1H),4.13(d,J＝7.8Hz,1H),3.38(d,J＝13.9Hz,1H),3.07(d,J＝13.8Hz,1H),1.45–1.41(m,2H),1.39(s,9H),1.35–1.25(m,1H),0.91–0.82(m,9H),0.67(d,J＝6.7Hz,3H).
步骤四：合成(2R,3R,4S,5S)-4-(((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-(2,2,3-三甲基丁基)吡咯烷-2-甲酸叔丁酯(TC132)
TC130(1.4g,2.6mmol)，二异丙基乙基胺(1.32g,10.2mmol)，Fmoc-Cl(1.0g,3.8mmol)，反应步骤参见终产物18的步骤三，得目标产物1.6g，产率82％。
步骤五：合成(2R,3R,4S,5S)-4-(((((9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-(2,2,3-三甲基丁基)吡咯烷-2-羧酸(TC137)
TC132(200mg,0.26mmol)，乙醛(117mg,2.64mmol)，三乙酰氧基硼氢化钠(560mg,2.64mmol)，乙酸3mL，三氟乙酸4mL，反应步骤参见终产物18的步骤四，得目标产物170mg，产率85％。
步骤六：合成4-((2R,3R,4S,5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-乙基-5-(2,2,3-三甲基丁基)吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸甲酯(TC140)
TC137(170mg,0.23mmol)，4-氨基-3-甲氧基苯甲酸甲酯(119mg,0.66mmol)，N-甲基咪唑(54mg,0.66mmol)，乙基磺酰氯(57mg,0.44mmol)，哌啶(0.2mL)，反应步骤参见终产物18的步骤五，得目标产物120mg，产率81％。1H NMR(500MHz,Methanol-d4)δ8.27(d,J＝8.9Hz,1H),7.66–7.47(m,3H),7.39–7.24(m,4H),7.19(s,1H),7.12(d,J＝6.7Hz,1H),4.33(t,J＝8.3Hz,2H),4.08(d,J＝9.2Hz,1H),3.95(s,3H),3.88(s,3H),3.51–3.35(m,2H),3.35–3.32(m,1H),3.09–2.91(m,1H),1.95(q,J＝15.1,8.9Hz,1H),1.57–1.40(m,2H),1.24(t,J＝7.0Hz,3H),1.12(s,3H),0.90(d,J＝6.8Hz,3H),0.81(d,J＝6.8Hz,3H),0.79(s,3H).
步骤七：合成4-((2'S,3S,4'R,5'R)-6-氯-4'-(3-氯苯基)-2'-(2,2-二甲基丁基)-1'-乙基螺[吲哚啉-3,3'-吡咯烷]-5'-羧基)-3-甲氧基苯甲酸(TC145)
TC140(103mg,0.15mmol)，碳酸钾(85mg,0.62mmol)，氢氧化锂一水合物(120mg,2.86mmol)，反应步骤参见终产物10的步骤六，得到目标产物23mg,产率41％。1H NMR(500MHz,Methanol-d4)δ8.21(d,J＝8.4Hz,1H),7.63(dd,J＝8.4,1.8Hz,1H),7.58(s,1H),7.42–7.21(m,4H),7.13(d,J＝7.5Hz,1H),6.72(dd,J＝8.0,1.9Hz,1H),6.45(d,J＝1.9Hz,1H),4.14(s,2H),3.84(s,3H),3.73–3.61(m,2H),3.53(s,1H),3.30(s,2H),2.02-1.81(m,2H),1.54–1.28(m,4H),0.97(s,3H),0.83(d,J＝6.7Hz,3H),0.71(d,J＝6.8Hz,3H),0.60(s,3H).ESI-MS理论计算值C35H42 35Cl2N3O4[M+H]+＝638.25,实验测得：638.2。
终产物94：4-((2'S，3S，4'R，5'R)-1'-烯丙基-6-氯-4'-(3-氯苯基)-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JR62)
步骤一：合成(2R，3R，4S，5S)-1-烯丙基-4-((叔丁氧羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JR58)
将JQ09(400mg,0.66mmol)称入35mL封管中，用5mL超干DMF溶解，加入烯丙基溴(799mg,6.6mmol)和碳酸铯(646mg,1.98mmol)，90℃反应过夜。反应结束后冷至室温，加水，乙酸乙酯萃取，饱和食盐水洗涤，无水硫酸钠干燥，有机相旋干后正相柱纯化，得目标产物246mg，产率57％。
步骤二：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-1-烯丙基-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JR59)
将JR58(280mg,0.43mmol)用15mL二氯甲烷溶解，加入1mL三氟乙酸，室温反应过夜。向反应液中加入饱和碳酸氢钠溶液，用乙酸乙酯萃取三次，合并有机相，用饱和氯化钠溶液洗一次后旋干有机相。用20mL二氯甲烷溶解反应物，加入二异丙基乙基胺(222mg,1.7mmol)和FmocCl(167mg,0.65mmol)，室温反应过夜。转干后正相柱纯化得粗品110mg，产率33％。
步骤三：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-1-烯丙基-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-羧酸(JR60)
将JR59(110mg,0.14mmol)溶于3mL二氯甲烷中，加入3mL三氟乙酸，室温反应过夜。向反应液中加入饱和碳酸氢钠溶液，用乙酸乙酯萃取三次，合并有机相，用饱和氯化钠溶液洗一次，转干后正相柱纯化得目标产物83mg，产率83％。1H NMR(400MHz,Chloroform-d)δ7.78(d,J＝7.5Hz,2H),7.59(d,J＝7.2Hz,2H),7.41(t,J＝7.4Hz,2H),7.37–7.29(m,2H),7.23–7.08(m,4H),7.05(t,J＝7.9Hz,1H),6.97(s,1H),6.76(d,J＝7.8Hz,1H),6.03–5.79(m,1H),5.46–5.30(m,2H),4.57(d,J＝7.8Hz,1H),4.50–4.29(m,4H),4.27–4.17(m,1H),3.84–3.68(m,2H),3.52(d,J＝13.9Hz,1H),3.27(d,J＝13.6Hz,1H),1.60(dd,J＝15.9,4.6Hz,1H),1.46(d,J＝15.1Hz,1H),0.68(s,9H).
步骤四：合成4-((2R，3R，4S，5S)-1-烯丙基-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-5-新戊基吡咯烷-2-甲酰胺基)-3-甲氧基苯甲酸甲酯(JR61)
JR60(40mg,0.06mmol)，1-甲基咪唑(14mg,0.17mmol)，乙基磺酰氯(16mg,0.12mmol)，4-氨基-3-甲氧基苯甲酸甲酯(31mg,0.17mmol)，哌啶(1.5mL)，反应步骤参见终产物18的步骤五，得目标产物19mg，产率41％。1H NMR(400MHz,Methanol-d4)δ8.23(d,J＝8.9Hz,1H),7.64–7.57(m,2H),7.52(t,J＝8.8Hz,1H),7.39–7.30(m,3H),7.27(dd,J＝13.5,2.2Hz,1H),7.14–7.10(m,1H),7.06(dt,J＝6.4,2.0Hz,1H),6.07–5.89(m,1H),5.37(d,J＝17.1,1H),5.16 (d,J＝10.0Hz,1H),4.46(d,J＝8.6Hz,1H),4.26(d,J＝9.4Hz,1H),4.09(dd,J＝13.3,5.7Hz,1H),4.03(d,J＝9.4Hz,1H),3.93(s,3H),3.87(s,3H),3.55(dd,J＝13.3,7.7Hz,1H),3.47(d,J＝14.5Hz,1H),3.30–3.26(m,1H),2.02(dd,J＝15.1,8.9Hz,1H),1.49(d,J＝15.0Hz,1H),1.05(s,9H).ESI-MS理论计算值C35H41 35Cl2FN3O4[M+H]+＝656.2,实验测得：656.2。
步骤五：合成4-((2'S，3S，4'R，5'R)-1'-烯丙基-6-氯-4'-(3-氯苯基)-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺基)-3-甲氧基苯甲酸(JR62)
JR61(20mg,0.03mmol)，碳酸钾(17mg,0.12mmol)，氢氧化锂一水合物(7mg,0.15mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐7mg,产率32％。1H NMR(400MHz,Methanol-d4)δ8.28(d,J＝9.1Hz,1H),7.69–7.59(m,2H),7.28–7.00(m,5H),6.70(dd,J＝7.9,1.9Hz,1H),6.43(d,J＝1.9Hz,1H),6.04–5.89(m,1H),5.55–5.43(m,1H),5.34–5.19(m,1H),4.38–4.23(m,1H),4.21–3.67(m,7H),3.55(d,J＝10.7Hz,1H),3.40(d,J＝10.6Hz,1H),2.03–1.90(m,1H),1.54–1.30(m,1H),0.98(s,9H).ESI-MS理论计算值C34H38 35Cl2N3O4[M+H]+＝622.2,实验测得：622.2。
终产物95：4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1-(3-氟丙基)-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JR55)
步骤一：合成(2R，3R，4S，5S)-4-((叔丁氧羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(3-羟丙基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JR46)
JQ09(300mg,0.49mmol)，3-[(叔丁基二甲硅烷基)氧基]-1-丙醛(278mg,1.48mmol)，醋酸硼氢化钠(314mg,1.48mmol)，乙酸(1mL)和1N四丁基氟化铵四氢呋喃溶液(0.9mL,0.88mmol)。反应步骤参见终产物49的步骤二，得目标产物207mg，产率63％。
步骤二：合成(2R，3R，4S，5S)-4-((叔丁氧羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(3-氟丙基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JR47)
JR46(200mg,0.35mmol)称入50mL单口瓶中，用干燥的二氯甲烷溶解，0℃下加入二乙胺基三氟化硫(114mg,0.71mmol)，室温反应过夜。反应结束后，二氯甲烷萃取，饱和氯化钠洗两次，有机相用无水硫酸钠干燥，旋干后过柱纯化得目标产物158mg，产率67％。
步骤三：合成(2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(3-氟丙基)-5-新戊基吡咯烷-2-羧酸叔丁酯(JR50)
JR46(158mg,0.27mmol)，三氟乙酸(1mL)，二异丙基乙基胺(139mg,1.1mmol)和FmocCl(105mg,0.4mmol)，反应步骤参见终产物94的步骤二，得目标产物60mg，产率28％。
步骤四：合成((2R，3R，4S，5S)-4-(9H-芴-9-基)甲氧基)羰基)氨基)甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(3-氟丙基)-5-新戊基吡咯烷-2-羧酸(JR63)
JR50(60mg,0.08mmol)，三氟乙酸(3mL)，反应步骤参见终产物94的步骤三，得目标产物34mg，产率61％。
步骤五：合成4-((2R，3R，4S，5S)-4-(氨基甲基)-4-(4-氯-2-氟苯基)-3-(3-氯苯基)-1-(3-氟丙基)-5-新戊基吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸甲酯(JR64)
JR63(47mg,0.06mmol)，1-甲基咪唑(15mg,0.18mmol)，乙基磺酰氯(17mg,0.13mmol)，4-氨基-3-甲氧基苯甲酸甲酯(33mg,0.18mmol)，哌啶(2mL)，反应步骤参见终产物18的步骤五，得目标产物29mg，产率62％。1H NMR(400MHz,Methanol-d4)δ8.21(d,J＝8.8Hz,1H),7.66–7.56(m,2H),7.48(t,J＝8.8Hz,1H),7.39–7.23(m,4H),7.16–7.11(m,1H),7.05(dt,J＝6.8,1.8Hz,1H),4.70–4.58(m,1H),4.57–4.46(m,2H),4.43(d,J＝8.9Hz,1H),4.12(d,J＝8.9Hz,1H),3.93(s,3H),3.88(s,3H),3.58–3.44(m,2H),3.24(d,J＝14.8Hz,1H),3.15–3.04(m,1H),2.13–1.88(m,3H),1.50(dd,J＝15.3,2.0Hz,1H),1.00(s,9H).ESI-MS理论计算值C35H42 35Cl2F2N3O4[M+H]+＝676.3,实验测得：676.2。
步骤六：合成4-((2'S，3S，4'R，5'R)-6-氯-4'-(3-氯苯基)-1-(3-氟丙基)-2'-新戊基螺[吲哚-3,3'-吡咯烷]-5'-甲酰胺]-3-甲氧基苯甲酸(JR55)
JR64(29mg,0.04mmol)，碳酸钾(23mg,0.16mmol)，氢氧化锂一水合物(9mg,0.2mmol)，反应步骤参见终产物10的步骤六，得到目标产物的三氟乙酸盐17.6mg,产率59％。1H NMR(500MHz,Methanol-d4)δ8.29(d,J＝8.4Hz,1H),7.70–7.59(m,2H),7.35–7.20(m,4H),7.07(d,J＝7.4Hz,1H),6.75(dd,J＝8.0,1.9Hz,1H),6.48(d,J＝1.9Hz,1H),4.72–4.42(m,3H),4.07–3.82(m,5H),3.69–3.61(m,1H),3.59(d,J＝10.8Hz,1H),3.45(d,J＝10.6Hz,1H),3.05–2.85(m,1H),2.23–1.94(m,3H),1.55–1.40(m,1H),0.96(s,9H).ESI-MS理论计算值C34H39 35Cl2FN3O4[M+H]+＝642.2,实验测得：642.2。
选用相应的原料，可以合成下述表1中的各个化合物：
表1














实施例2：FP检测化合物与MDMX蛋白的Ki值
His标记的MDMX(14-111,C17S)在E.coli中表达，先用Ni亲和柱纯化再用Superdex75分子筛纯化，所得MDMX蛋白纯度大于95％，蛋白浓度为12.5μM。采用FAM标记的PDI多肽(FAM-PDI)[Cancer Res 2007,67,8810-8817]作为荧光标记分子探针，其中MDMX/FAM-PDI相互作用的解离常数Kd为2.1nM。
96-孔板购自Corning公司(黑色，#3694)。多功能酶标仪为TECAN公司产品，型号：SPARK 10M。检测缓冲液：10mM Tris(pH 7.5)、200mM NaCl(Sigma)、0.01％Tween-20和0.01％Trition X-100(Sigma)，实验用水为Millipore-Q纯水。
首先待测试化合物用DMSO溶解成20mM的标准母液。随后，在EP管中用DMSO将测试化合物的标准母液稀释成工作样品溶液，所制备的工作样品溶液浓度＝测试板上所需最高样品浓度的25倍(25×测试化合溶液)，在EP管中3倍梯度稀释化合物，备用。
分别在96孔板的B1-D1至B12-D12孔加入梯度稀释的待测化合物A的25×测试化合溶液4μL，在E1-G1至E12-G12孔加入梯度稀释的待测化合物B的25×测试化合溶液4μL。最后向上述每孔中加入96μL含5.2nM FAM-PDI和62.5nM MDMX蛋白的检测缓冲液。
A1-A3孔作为空白对照组：加入100uL检测缓冲液。A4-A6孔作为阴性信号参照组：加入100μL只含5nM荧光标记分子探针的缓冲液。A7-A9孔作为阳性参照组：加入100μL含5nM荧光标记分子探针和60nM MDMX蛋白的混合溶液。
在上述反应板覆盖上铝箔纸，并将96孔板放置于96孔板摇床上室温孵育1h后，用酶标仪读取在Ex485nm/Em530nm时的荧光偏振mP值。所测得mP值对化合物浓度梯度做曲线，mP极大值和极小值的中值对应的样本化合物浓度，即为化合物与蛋白的结合的IC50值([I]50)。
根据此IC50值([I]50)，利用公式计算化合物与蛋白的结合率常数
Ki：Ki＝[I]50/([L]50/Kd+[P]0/Kd+1)。
其中[L]50表示上述测试体系内荧光标记分子探针浓度的50％；[P]0表示上述测试体系内MDMX蛋白浓度，Kd是蛋白和荧光标记分子探针的解离常数。
利用上述方法，测得实施例化合物抑制MDMX/p53相互作用的Ki值如下表2所示。实验数据表明，化合物具有抑制MDMX/p53相互作用的活性。
实施例3：FP检测化合物与MDX2蛋白的Ki值
His标记的MDM2(1-118)在E.coli中表达，先用Ni亲和柱纯化再用Superdex75分子筛纯化，所得MDMX蛋白纯度大于95％，蛋白浓度为151μM。采用FAM标记的PDI多肽(FAM-PDI)[Cancer Res 2007,67,8810-8817]作为荧光标记分子探针，其中MDM2/FAM-PDI相互作用的解离常数Kd为0.7nM。
96-孔板购自Corning公司(黑色，#3694)。多功能酶标仪为TECAN公司产品，型号：SPARK 10M。检测缓冲液：100mM potassium phosphate(pH 8.0)、100ug/mL Bovine-r-globulin(Sigma)和0.01％Trition X-100(Sigma)，实验用水为Millipore-Q纯水。
首先待测试化合物用DMSO溶解成20mM的标准母液。随后，在EP管中用DMSO将测试化合物的标准母液稀释成工作样品溶液，所制备的工作样品溶液浓度＝测试板上所需最高样品浓度的25倍(25×测试化合溶液)，在EP管中3倍梯度稀释化合物，备用。
分别在96孔板的B1-D1至B12-D12孔加入梯度稀释的待测化合物A的25×测试化合溶液4μL，在E1-G1至E12-G12孔加入梯度稀释的待测化合物B的25×测试化合溶液4μL。最后向上述每孔中加入96μL含2.08nM FAM-PDI和20.8nM MDM2蛋白的检测缓冲液。
A1-A3孔作为空白对照组：加入100μL检测缓冲液。A4-A6孔作为阴性信号参照组：加入100μL只含2nM荧光标记分子探针的缓冲液。A7-A9孔作为阳性参照组：加入100μL含2nM荧光标记分子探针和20nM MDM2蛋白的混合溶液。
在上述反应板覆盖上铝箔纸，并将96孔板放置于96孔板摇床上室温孵育0.5h后，用酶标仪读取在Ex485nm/Em530nm时的荧光偏振mP值。所测得mP值对化合物浓度梯度做曲线，mP极大值和极小值的中值对应的样本化合物浓度，即为化合物与蛋白的结合的IC50值([I]50)。
根据此IC50值([I]50)，利用公式计算化合物与蛋白的结合率常数
Ki：Ki＝[I]50/([L]50/Kd+[P]0/Kd+1)。
其中[L]50表示上述测试体系内荧光标记分子探针浓度的50％；[P]0表示上述测试体系内MDM2蛋白浓度，Kd是蛋白和荧光标记分子探针的解离常数。
利用上述方法，测得实施例化合物抑制MDM2/p53相互作用的Ki值如表2所示。实验数据表明，化合物抑制MDM2/p53相互作用的活性非常好。
表2：实施例化合物抑制MDMX/p53和MDM2/p53相互作用活性

*表示Ki在100,000nM和10,000nM之间；**表示Ki在10,000nM和100nM之间；***表示Ki在100nM和10nM之间；****表示Ki<10nM。
实验结果表明：化合物具有结合MDM2蛋白和MDM4蛋白的亲和力。部分化合物的亲和力特别强，其结合力常数Ki在nM区间。
实施例4：测定化合物的绝对立体构型
(1)合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯-2-氟苯基)-2'-新戊基-1-(3-硝基苄基)螺环[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺)-3-甲氧基苯甲酸(JM085-CF2)
步骤一：合成(2R,3S,4S,5S)-3-(3-氯-2-氟苯基)-4-(4-氯-1-氟苯基)-5-新戊基-4-((3-硝基苄基)氨基)甲基)吡咯烷-2-羧酸(JM043-CF2)
将YN17-CF2[239mg，0.5mmol，合成中间体4(YN22-CF2)的反应过程中的中间化合物]溶于甲醇，加入间硝基苯甲醛(154mg，1.0mmol)、氰基硼氢化钠(129mg，2.0mmol)和醋酸1mL，室温搅拌过夜。反应结束HPLC纯化得目标化合物的三氟醋酸盐224mg，产率：73％。
步骤二：合成甲基4-((2R,3S,4S,5S)-3-(3-氯-2-氟苯基)-4-(4-氯-1-氟苯基)-5-新戊基-4-(((3-硝基苄基)氨基)甲基)吡咯烷-2-甲酰胺)-3-甲氧基苯甲酸酯(JM079-CF2)
将JM043-CF2(78mg，0.13mmol)称入瓶中，用四氢呋喃溶解，加入二异丙基乙基胺(84mg，0.65mmol)和二苯基次磷酰氯(93mg，0.39mmol)，室温搅拌30min后加入3-甲氧基-4-氨基苯甲酸甲酯(94mg，0.52mmol)，室温反应过夜。加水，用二氯甲烷萃取，有机相旋干后用正相柱纯化得目标化合物JM079-CF2的粗品，直接用于下一步。
步骤三：合成4-((2'S,3S,4'S,5'R)-6-氯-4'-(3-氯-2-氟苯基)-2'-新戊基-1-(3-硝基苄基)螺环[吲哚啉-3,3'-吡咯烷]-5'-甲酰胺)-3-甲氧基苯甲酸(JM085-CF2)
将JM079-CF2的粗品溶于N,N-二甲基甲酰胺，加入碳酸钾(63mg，0.45mmol)，100℃搅拌过夜。反应结束后HPLC纯化得目标化合物JM085-CF2的三氟醋酸盐47mg，两步反应产率49％。将目标化合物用乙酸乙酯溶解，饱和碳酸氢钠溶液洗两次，饱和食盐水洗一次，得到游离形式的JM085-CF2。1H NMR(400MHz,Methanol-d4)δ8.18(d,J＝8.4Hz,1H),8.12(dd,J＝8.3,2.2Hz,1H),7.92(d,J＝2.1Hz,1H),7.59(dd,J＝8.4,1.8Hz,1H),7.54–7.47(m, 2H),7.46–7.42(m,1H),7.40(d,J＝8.1Hz,1H),7.38–7.29(m,2H),7.11(t,J＝7.9Hz,1H),6.79(dd,J＝8.0,1.8Hz,1H),6.57(d,J＝1.9Hz,1H),5.17(d,J＝10.6Hz,1H),4.39(d,J＝10.6Hz,1H),4.32(d,J＝15.5Hz,1H),4.26–4.13(m,2H),3.86(s,3H),3.72(s,3H),3.60(d,J＝10.9Hz,1H),3.38(d,J＝10.9Hz,1H),1.81(dd,J＝15.4,8.3Hz,1H),1.70(d,J＝15.1Hz,1H),0.95(s,9H).ESI-MS理论计算值C39H40 35Cl2FN4O6[M+H]+＝749.2,实验测得：749.2。
(2)单晶衍射测试：将23mg JM085-CF2称入10mL玻璃样品瓶中，加入1.5mL的二氯甲烷中，滴加1.5mL的正己烷，铝箔封口后用针扎3个小孔，室温静置2天长出单晶，X-ray单晶衍射测试化合物衍射数据和结果如表3所示：晶体数据表明JM085-CF2的绝对立体构型如图1所示。
表3 X-ray单晶衍射测试化合物衍射数据
结论：由于JM085-CF2的合成路线和与中间体2(YM155)的合成路线类似，因此JM085-CF2的立体化学与中间体2(YM155)的立体化学一致。中间体2(YM157)是合成终产物1(YM157)，2(YN11)，3(YN51)，4(YN52)的原料，因此可推断化合物1、2、3、4螺环母 核的立体化学与JM085-CF2螺环母核的立体化学完全一致。
X-ray单晶衍射测试化合物衍射数据和结果如表4所示：晶体数据表明JN110的绝对立体构型如图2所示。
表4 X-ray单晶衍射测试化合物衍射数据
结论：通过CuOAc和(R)-BINAP催化体系可得到单一对映体JN110，中间体JN110可以合成ee值大于95％的目标产物JN122。中间体JN110至终产物JN122的合成路线中，化合物不会产生构型变化，因此，JN122的绝对立体构型应与JN110一致。
实施例5：细胞生长抑制活性实验
细胞生长抑制实验(Cell growth inhibition assay)：用100％二甲基亚砜溶解待测样品，配制20mM的化合物母液。用100％二甲基亚砜稀释化合物至实验所需的最高浓度(1mM或者10mM)。
首先，在96孔平底透明细胞培养板的B1-G1孔中加入145μL细胞完全培养基，B2-G12孔中分别加入100μL完全培养基。然后，在96孔平底透明细胞培养板的B1-D1和E1-G1孔中分别加入5μL 1mM或者10mM化合物溶液，依次按3倍梯度稀释至96孔平底透明细胞培养板的B12-D12和E12-G12。最后，向各孔中加入50μL待测细胞溶液，每孔的细胞密度分别如下：HCT116和RKO为3000左右每孔，U2-OS为5000左右每孔，JEG-3为8000左右每孔，每孔总体积为150μL。在实验中，除所测化合物外，另设置两对照组分别是：(1)加细胞和培养基，但是不加化合物对照组；(2)仅加完全培养基，无细胞无化合物组。将96孔板置于含5％二氧化碳的37℃细胞培养箱中孵育4天后，每孔加入15μL CCK-8试剂，然后于37℃孵育2-3小时。用TECAN酶标仪读取其在450nm波长下的吸收值。
不同浓度化合物对细胞活性的影响用以下公式计算：细胞生长抑制率＝[实验组吸收值-仅加完全培养基(无细胞无化合物组)吸收值]/[加细胞不加化合物组吸收值-仅加完全培养基(无细胞无化合物组)吸收值]×100％。利用GraphPad Prism 7.0软件处理以上数据，所取IC50值为对细胞生长抑制率达50％时所对应的化合物浓度。
利用上述方法，测试了部分化合物和文献化合物YM30、Nutlin-3a、RG7388的细胞活性，数据如表5所示。
表5化合物细胞增殖抑制活性

*表示IC50在100,000nM和10,000nM之间；**表示IC50在10,000nM和100nM之间；***表示IC50在100nM和10nM之间；****表示IC50<10nM；n.c.表示未进行测试。
上述实验结果表明，在HCT-116、RKO、U2-OS等肿瘤细胞模型中，部分实施例化合物例如YN55,JN122，JQ44，JQ97，JR01，JR02，JQ122，JQ149，JQ57，JQ158等与RG7388及YM30相比，细胞增殖抑制活性10倍以上的提高，具有明显的活性优势。
实施例6：比较化合物2(YN11)、14(YN55)和文献化合物RG7388、YM30在5％DMSO/H2O中的溶解度
化合物溶解度实验：
(1)内标液的制备：将3-甲氧基-4-氨基苯甲酸甲酯溶解于乙腈：水＝1：1体系中，配制为合适浓度的内标液(RG7388-1.4mM，YM30-5.5mM,YN55-5.5mM,YN11-11.2mM)。
(2)标准曲线的制备：设置6-7个浓度梯度，将待测样品用乙腈:水＝1:1体系两倍梯度稀释，每个浓度取90μL加入10μL内标液，再取20μL进样UPLC液相色谱仪。测得测试样品峰面积与内标物(Internal Standard,IS)峰面积并求比值后，与测试样品的浓度作标准曲线，结果如图3所示。
(3)化合物样品浓度测试：待测样品溶于50μLDMSO中，加入950μL水，涡旋5min，超声15min，14000rpm离心30min，取90μL加入10μL内标液，再取20μL进样UPLC液相色谱仪。测得测试样品峰面积与内标物峰面积并求比值后，代入标准曲线方程，求得测试样品在5％DMSO/H2O饱和溶液中的浓度。
利用上述方法，测得的RG7388,YM30,14(YN55),2(YN11)溶解度数据如表6所示。
表6化合物溶解度测试结果
实验数据表明，化合物YN55、YN11在5％DMSO/H2O中的饱和浓度明显高于文献化合物RG7388、YM30。在检测体系中，YN55、YN11溶解度是RG7388的溶解度的40倍以上，是YM30的两倍以上。YN55、YN11的良好溶解性，有利于化合物提高体内药代动力学和药效学性质。
实施例7：比较YM34、2(YN11)和14(YN55)的药代动力学性质
小鼠体内的药代动力学实验：ICR小鼠(种属：SPF级，来源：动物转移自实验机构动物储备库，上海市计划生育科学研究所实验动物经营部)静脉注射或口服受试化合物后，于不同时间点经颌下静脉或其它合适方式采血(IV组为给药后0.083h,0.25h,0.5h,1h,2h,4h,8h和24h，PO组为给药后0.25h,0.5h,1h,2h,4h,6h,8h和24h，每个时间点采血0.03ml)，K2-EDTA抗凝，血液样本采集后1h内离心得血浆(离心条件：6800g，6分钟，2-8℃)，待测样品在分析前存放于-80℃冰箱内备用。通过LC-MS检测受试样品的浓度后计算相关参数。本实验采用的溶媒条件为：5％DMSO、10％Cremophor和85％PBS。静脉给药剂量为5mg/kg，口服给药剂量为15mg/kg。静脉和口服实验各使用3只雄鼠。
利用上述方法，实验测定了YM34、2(YN11)和14(YN55)在小鼠体内的药代动力学动力学参数，数据如表7-9所示。
表7 YM34在ICR小鼠体内的药动学参数
表8 YN11在ICR小鼠体内的药动学参数
表9 YN55在ICR小鼠体内的药动学参数
实验数据表明，相同剂量口服条件下，2(YN11)和14(YN55)的药代动力学性质，比YM34显著提高：
(1)YM34的体内暴露量(AUC(0-t))为14423.25h*ng/mL，YN11和YN55的体内暴露量分别为68548.41h*ng/mL和58429.33h*ng/mL，后两者分别有4-5倍的提高。
(2)YM34的口服生物利用度为17％。YN11和YN55的口服生物利用度分别为34.34％和30.31，是YM34的2倍
(3)YM34的最大血药浓度(Cmax)是2177.88ng/mL，YN11和YN55的的最大血药浓度分别为4089.43ng/mL和4151.35ng/mL。YN11、YN55的最大血药浓度参数比YM34提高了1倍。
(4)YM34的半衰期(T1/2)是3.18小时，YN11和YN55的半衰期分别为12.61小时和7.42小时。YN11、YN55的半衰期比YM34分别提高了2倍和1倍。
综上所述，实验数据表明，比较2(YN11)、14(YN55)与YM34药代动力学性质，YN11、 YN55的口服吸收更好，体内暴露量更好，血浆药物浓度更高，体内滞留时间更长，说明此类药物(包括YN11、YN55)在动物体内能够与药靶更长时间、更充分结合，且其动物体内浓度更高，更有利于达到更好的疾病治疗效果。
实施例8：蛋白质印记分析(Western-Blot Assay)验证18(JN122)的作用机制
(1)18(JN122)在HCT116细胞中的作用机制研究,HCT116(结肠癌)为p53野生型且MDM2过表达的细胞系。
蛋白质印记分析(Western-Blot Assay)实验：将HCT-116细胞接种到6孔板中，并用抑制剂处理24小时或48小时，离心收集细胞，并根据操作手册用含有PMSF(Beyotime，产品编号ST506)的RIPA裂解缓冲液(Beyotime，产品编号P0013B)裂解细胞。将裂解物在4℃下以13000g离心30分钟，将上清液转移到透明的EP管中用于随后的分析。将蛋白质标准化至20～40μg/lane，用8％或12％的SDS-PAGE凝胶进行分离。将蛋白质转移到硝酸纤维素(NC)膜上，并用溶解在TBST缓冲液中的5％脱脂牛奶封闭1小时。随后，将样品与相应的抗体在4℃下孵育，并振荡过夜。用TBST缓冲液洗涤NC膜10分钟，重复两次，然后将其与IgG-HRP二抗在室温下孵育2小时，再次将膜洗涤三次。最后，使用增强化学发光检测试剂(Thermo Scientific，产品编号34577)对蛋白质进行可视化。p53抗体购自Millipore(产品编号#OP43)，MDM4、GAPDH、β-Actin和BAX抗体购自Proteintech(产品编号分别为#17914-1-AP、10494-1-AP、66009-1-Ig和50599-2-Ig)，p21、MDM2、PARP、PUMA和CL-PARP抗体购自Cell Signaling Technology(产品编号分别为2947、86934、9542、12450和5625)。
如图4所示，与RG7388和Nutlin-3a一致，JN122剂量依赖性地增加p53及其靶标蛋白p21和MDM2的表达，表明HCT116细胞中p53/MDM2相互作用受到抑制，p53稳定性增加。用抑制剂处理HCT116细胞24小时，JN122在39.6nM时即可明显上调p53、p21和MDM2的浓度，而RG7388在156nM，Nutlin-3a在2.5μM时才能达到类似的效果，这表明JN122比RG7388和Nutlin-3a效应更强。用抑制剂处理HCT116细胞48小时，JN122、Nutlin-3a和RG7388可明显上调PUMA和BAX的表达，表明凋亡途径的激活。JN122在5～10μM的浓度下，可以观察到PARP(细胞凋亡的生物标志物)切割，用抑制剂处理HCT116细胞48小时，可能由于细胞死亡导致MDM2、MDM4和BAX的浓度降低。
上述实验数据表明:在HCT-116细胞中，JN122可以高效激活细胞内p53功能,展现抗癌活性,且其活性优于RG7388。
(2)18(JN122)在多种实体瘤细胞系中的作用机制研究
RKO(结肠癌)、H460(大细胞肺癌)、U2-OS(骨肉瘤)、MSTO-211H(间皮瘤)、HepG2(肝癌)、A549(非小细胞肺癌)和Hela(宫颈癌)细胞均为p53野生型细胞，而SW480(结肠癌)中的p53发生突变。尽管HeLa为p53野生型细胞，但由于E6癌蛋白的不断降解，无法检测到p53蛋白。试验步骤基本同前，不同之处在于将HCT-116细胞替换为上述细胞。
如图5中A所示，RKO、H460和U2-OS细胞中MDM4的表达水平较高，被认为是MDM4过表达癌细胞系；MSTO-211H和HepG2中MDM2表达水平较高，而A549细胞系中MDM2表达水平相对较低；Hela细胞中没有检测到稳定表达的p53且MDM2和MDM4表达水平也较低。为了便于比较，所有细胞均用0.6μM的JN122处理，在HCT-116细胞中该浓度的 JN122表现出很强的p53激活效应。
如图5中B和5中C所示，在H460、A549、U2-OS、HepG2、MSTO-211H和RKO细胞中，JN122促进p53、p21和MDM2浓度明显增加，并且JN122的促进效果优于RG7388。但是，在Hela细胞中没有观察到类似现象。
上述实验数据表明:在H460、A549、U2-OS、HepG2、MSTO-211H和RKO六种细胞系中，JN122可以激活p53并上调其靶蛋白表达水平，表现出比RG7388更强的活性。同时数据表明，JN122激活p53活性依赖于肿瘤细胞能够表达一定浓度的野生型p53。
(3)18(JN122)在血液瘤MOLM-13细胞中的作用机制研究
试验步骤基本同前，不同之处在于将HCT-116细胞替换为MOLM-13(人急性骨髓白血病)为p53野生型细胞系。
图6中WB结果显示，JN122剂量依赖性地增加p53及其靶标蛋白p21和MDM2的表达，剂量依赖性地降低MDM4表达，这可能是由于MDM2的E3泛素连接酶活性增加。37.5nM的JN122即可明显促进细胞凋亡标志物PARP和Caspase-3的裂解，而RG7388在150nM时才可达到类似效果。
上述实验数据表明:JN122可以诱导MOLM-13细胞内p53激活，并促进细胞周期阻滞和细胞凋亡。同时，JN122激活MOLM-13中p53的活性强于RG7388四倍左右。
实施例9：18(JN122)在小鼠MOLM-13异种移植瘤模型中的体内药效
小鼠体内抗肿瘤药效实验：雌性NOD.CB17-PrkdcscidIl2rgtm1/Bcgen(B-NDG)小鼠，购自中国海门百奥赛图生物技术有限公司。每组动物数：溶剂对照组(Vehicle)20只，给药组10只。小鼠静脉注射200μL悬浮于PBS中的MOLM-13细胞(2×104cells)。在第6天，JN122 25mg/kg组每天灌胃给药一次，持续21天；JN122 50mg/kg组每天灌胃给药一次，持续21天；JN122 100mg/kg组每天灌胃给药一次，持续21天；RG7388 50mg/kg组每天灌胃给药一次，持续21天；溶剂对照组每天等量灌胃一次5％DMSO+10％EL+85％生理盐水，持续21天。每天监测小鼠存活率。
在MOLM-13异种移植瘤小鼠模型中评估JN122的体内药效，如图7所示，溶剂对照组(Vehicle)小鼠的中位生存期为20天(生存期18至26天)；每天一次口服给药100mg/kg的JN122，小鼠的中位生存期延长至31天(生存期26至33天，p<0.001)；每天一次口服给药25mg/kg的JN122，小鼠的中位生存期为25.5天(生存期20至27天，p<0.01)；每天一次口服给药50mg/kg的JN122，小鼠的中位生存期为25.5天(生存期17至28天，p<0.001)；每天一次口服给药50mg/kg的RG7388，小鼠的中位生存期为27.5天(生存期26至30天，p<0.001)。因此，与RG7388一致，18(JN122)在MOLM-13小鼠模型上展现出强有力的抗白血病疗效，可明显延长荷瘤小鼠的中位生存期。
上述实验数据表明:在人体肿瘤小鼠移植瘤模型中，JN122具有非常好的抗肿瘤药效。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种如式I所示的化合物、其对映异构体、非对映异构体、消旋体或其药学上可接受的盐，
    式中，Ar为取代或未取代的苯基，其中，所述的取代是指苯基上的一个或多个氢原子被选自下组的基团取代：卤素、氘、氰基、羟基、氨基、硝基、取代或未取代的C2-C4炔基、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C4烷氧基；
    R1和R2各自独立地为氢、氘、卤素、氰基、取代或未取代的C2-C4炔基、取代或未取代的C1-C4烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C4烷氧基；
    R3为
    Y、Z各自独立地为氢、-(CH2)m-取代或未取代的6-10元芳基、-(CH2)m-取代或未取代的5-12元杂芳基、取代或未取代的C1-C6烷基、-(CH2)m-取代或未取代的C3-C8环烷基、-(CH2)m-取代或未取代的5-13元杂环基；
    R4为取代或未取代的C1-C8烷基、-(CH2)m-取代或未取代的C3-C8环烷基、-(CH2)m-取代或未取代的4-13元杂环基、-(CH2)m-取代或未取代的6-10元芳基、-(CH2)m-取代或未取代的5-12元杂芳基、C2-C8炔烃基、C2-C8烯基；
    m在各出现处各自独立地为0、1、2、3或4；
    R5为取代或未取代的C1-C8烷基、取代或未取代的C3-C8环烷基、取代或未取代的4-12元杂环烷基；
    除非另有定义，以上所述的各取代各自独立地是指基团上的一个或多个氢原子被选自下组的基团取代：卤素、氘、氰基、羟基、氨基、硝基、C1-C4烷基磺酰基、C1-C4烷基磺酰胺基羰基(C1-C4烷基SO2NHCO-)、羧基、-CONH2、C1-C4烷基、C3-C6环烷基、C1-C4烷氧基、C2-C4烯基、C2-C4炔基、C1-C4烷基乙炔基、单(C1-C4烷基)氨基、二(C1-C4烷基)氨基、C1-C4烷基羰基氨基、C1-C4烷基COO-、C1-C4烷氧基羰基氨基、C1-C4烷氧基羰基、C1-C4烷基SO2-、C1-C4烷基-S(O2)-C1-C4亚烷基-、羧基取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷基-S-、C2-C10酰基、C1-C4烷基羰基、C1-C4烷基氨基羰基、C1-C4卤代烷基氨基羰基、5-12元杂芳基、5-12元杂芳基羰基、C1-C4烷基5-12元杂芳基羰基、C1-C4烷基-CO-O-C1-C4亚烷基-O-CO-。
  2. 一种如式I所示的化合物、其对映异构体、非对映异构体、消旋体或其药学上可接受的盐，
    式中，Ar为取代或未取代的苯基，其中，所述的取代是指苯基上的一个或多个氢原子被选自下组的基团取代：卤素、氘、氰基、羟基、氨基、硝基、取代或未取代的C2-C4炔基、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C4烷氧基；
    R1和R2各自独立地为氢、氘、卤素、氰基、取代或未取代的C2-C4炔基、取代或未取代的C1-C4烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C4烷氧基；
    R3为
    Y、Z各自独立地为氢、-(CH2)m-取代或未取代的6-10元芳基、-(CH2)m-取代或未取代的5-12元杂芳基、取代或未取代的C1-C6烷基、-(CH2)m-取代或未取代的C3-C8环烷基、-(CH2)m-取代或未取代的5-13元杂环基；
    R4为取代或未取代的C1-C8烷基、-(CH2)m-取代或未取代的C3-C8环烷基、-(CH2)m-取代或未取代的4-13元杂环基、-(CH2)m-取代或未取代的6-10元芳基、-(CH2)m-取代或未取代的5-12元杂芳基；
    m在各出现处各自独立地为0、1、2、3或4；
    R5为取代或未取代的C1-C8烷基、取代或未取代的C3-C8环烷基、取代或未取代的4-12元杂环烷基；
    除非另有定义，以上所述的各取代各自独立地是指基团上的一个或多个氢原子被选自下组的基团取代：卤素、氘、氰基、羟基、氨基、硝基、C2-C4炔基、C1-C4烷基磺酰基、C1-C4烷基磺酰胺基羰基(C1-C4烷基SO2NHCO-)、羧基、-CONH2、C1-C4烷基、C3-C6环烷基、C1-C4烷氧基、C2-C4烯基、C1-C4烷基乙炔基、单(C1-C4烷基)氨基、二(C1-C4烷基)氨基、C1-C4烷基羰基氨基、C1-C4烷基COO-、C1-C4烷氧基羰基氨基、C1-C4烷氧基羰基、C1-C4烷基SO2-、C1-C4烷基-S(O2)-C1-C4亚烷基-、羧基取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷基-S-、C2-C10酰基、C1-C4烷基羰基、C1-C4烷基氨基羰基、C1-C4卤代烷基氨基羰基、5-12元杂芳基、5-12元杂芳基羰基、C1-C4烷基5-12元杂芳基羰基。
  3. 如权利要求1或2所述的化合物，其特征在于，所述化合物具有如式II所示的结构：
    式中，Ar、R1、R2、R3、R4的定义如权利要求1所述。
  4. 如权利要求1或2所述的化合物，其特征在于，所述化合物具有如式IV所示的结构：
    式中，Ar、R1、R2、R4的定义如权利要求1所述。
  5. 如权利要求1或2所述的化合物，其特征在于，所述化合物具有如式VI所示的结构：
    式中，Ar、R1、R2、R4的定义如权利要求1所述。
  6. 如权利要求1或2所述的化合物，其特征在于，所述化合物具有如式VII或式VIII所示的结构：
    式中，Ar、R1、R2、R4的定义如权利要求1或2所述。
  7. 如权利要求1或2所述的化合物，其特征在于，所述化合物选自下组：








  8. 一种药物组合物，其特征在于，包括：如权利要求1-7任一项所述的化合物，其对映异构体、非对映异构体、消旋体或药学上可接受的盐中的一种或多种；和药学上可接受的载体。
  9. 如权利要求1-8任一项所述的化合物、其对映异构体、非对映异构体、消旋体或其药学上可接受的盐或权利要求8所述的药物组合物的用途，其特征在于，用于制备阻断MDM2/p53和/或MDMX/p53相互作用的小分子抑制剂；或用于制备治疗与MDM2或MDMX蛋白的活性或表达量相关的疾病的药物。
  10. 如权利要求9所述的用途，其特征在于，所述与MDM2或MDMX蛋白的活性或表达量相关的疾病选自下组：神经胶质瘤、脂肪肉瘤、皮肤黑色素瘤、鳞状上皮细胞癌、视网膜母细胞癌、乳腺癌、食道癌、肺癌、卵巢癌、胃癌、膀胱癌、肝癌、软组织肉瘤、慢性淋巴白血病、急性髓性白血病、淋巴瘤、骨肉瘤和结肠癌。
  11. 一种权利要求1或2所述的化合物的制备方法，其特征在于，所述制备方法包括以下步骤：

    醛S1与取代的2-氟苯乙腈S2反应得到中间体S3；
    S3与S4-2反应得到中间体S5-2；
    S5-2通过氢化还原反应得到中间体S6-2；
    S6-2与FmocCl反应得到中间体S7-2；
    S7-2脱去叔丁基得到中间体S8-2；
    S8-2经缩合反应得到中间体S9-2；
    S9-2和R4CHO通过还原胺化反应得到中间体S10-2；
    S10-2脱保护得到中间体S11-2；
    S11-2在碱存在下反应得到式I所示的化合物，
    或者所述制备方法包括以下步骤：

    S3与S4-2通过不对称催化反应得到具有光学活性的手性中间体S23；
    S23通过氢化还原反应得到中间体S24；
    S24与FmocCl反应得到中间体S25；
    S25和R4CHO通过还原胺化反应得到中间体S26；
    S26脱去叔丁基应得到中间体S27；
    S27经缩合反应得到中间体S28；
    S28脱保护得到中间体S29；
    S29在碱存在下反应得到式I所示的化合物，为具有光学活性的手性化合物，
    各式中，Ar、R1、R2、R3、R4、R5的定义如前所述，
    或者所述制备方法包括以下步骤：
    S11-2经还原胺化反应得到中间体S40；
    S40在碱存在下反应得到中间体S41；
    S41脱保护得到式I所示的化合物。
    各式中，Ar、R1、R2、R3、R4、R5的定义如前所述；
    或者所述制备方法包括以下步骤：
    S9-2脱保护得到中间体S12-2；
    S12-2经还原胺化反应得到中间体S13-2；
    S13-2在碱存在下反应得到中间体S14-2；
    S14-2和具有醛基官能团化合物R4CHO通过还原胺化反应得到中间体S41；
    S41脱保护得到式I所示的化合物。
    各式中，Ar、R1、R2、R3、R4、R5的定义如前所述。
PCT/CN2023/123063 2022-10-13 2023-10-02 具有取代苯基螺[吲哚啉-3,3'-吡咯烷]结构的小分子化合物 WO2024078370A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202211255892.0 2022-10-13
CN202211255892 2022-10-13
CN202311004867.X 2023-08-10
CN202311004867 2023-08-10

Publications (1)

Publication Number Publication Date
WO2024078370A1 true WO2024078370A1 (zh) 2024-04-18

Family

ID=90668770

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/123063 WO2024078370A1 (zh) 2022-10-13 2023-10-02 具有取代苯基螺[吲哚啉-3,3'-吡咯烷]结构的小分子化合物

Country Status (2)

Country Link
CN (1) CN118047784A (zh)
WO (1) WO2024078370A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103298818A (zh) * 2010-11-12 2013-09-11 密歇根大学董事会 螺-吲哚酮mdm2拮抗剂
CN112912143A (zh) * 2018-10-08 2021-06-04 美国密歇根州立大学试剂中心 小分子mdm2蛋白降解剂
CN113387957A (zh) * 2021-06-09 2021-09-14 江苏亚尧生物科技有限公司 螺环吲哚酮-吡咯烷碳酸酯化合物和其组合物、制备方法及用途
CN113788818A (zh) * 2016-04-06 2021-12-14 密执安大学评议会 Mdm2蛋白质降解剂

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103298818A (zh) * 2010-11-12 2013-09-11 密歇根大学董事会 螺-吲哚酮mdm2拮抗剂
CN113788818A (zh) * 2016-04-06 2021-12-14 密执安大学评议会 Mdm2蛋白质降解剂
CN112912143A (zh) * 2018-10-08 2021-06-04 美国密歇根州立大学试剂中心 小分子mdm2蛋白降解剂
CN113387957A (zh) * 2021-06-09 2021-09-14 江苏亚尧生物科技有限公司 螺环吲哚酮-吡咯烷碳酸酯化合物和其组合物、制备方法及用途

Also Published As

Publication number Publication date
CN118047784A (zh) 2024-05-17

Similar Documents

Publication Publication Date Title
CN113365988B (zh) Shp2抑制剂及其应用
WO2021063346A1 (zh) Kras g12c抑制剂及其应用
TWI832962B (zh) 雜環化合物及其用途
CN108026050B (zh) 作为RORγ调节剂的新型化合物
JP6097289B2 (ja) セリン／スレオニンキナーゼインヒビターとしてのキナゾリン化合物
TWI768557B (zh) 作為ｈｄａｃ抑制劑之3-烷基-4-醯胺基-雙環[4,5,0]羥肟酸
JP6795588B2 (ja) ブロモドメイン阻害薬としての使用のためのピリジノンジカルボキサミド
CN110139864B (zh) 作为αV整联蛋白抑制剂的吡咯酰胺
CN104884460B (zh) 2‑取代的头孢烯化合物
WO2021000885A1 (zh) 喹唑啉酮类衍生物、其制备方法及其在医药上的应用
KR101944686B1 (ko) 페닐-3-아자-바이사이클로[3.1.0]헥스-3일-메타논 및 약제로서의 이의 용도
TW201726606A (zh) 作為免疫調節劑之化合物
CN112300069A (zh) 一种选择性钠通道调节剂及其制备和应用
JP6954921B2 (ja) ブロモドメイン阻害薬としてのピリジル誘導体
CN102753522A (zh) 取代的吡咯烷-2-甲酰胺类
TW201706257A (zh) 化合物
TW201427971A (zh) 二氫吡唑ｇｐｒ４０調節劑
TW202123942A (zh) Ｂｒｄ９雙官能基降解劑及其使用方法
TW201625538A (zh) 新穎吡啶衍生物
WO2008069242A1 (ja) 新規２環性複素環化合物
CN108602807B (zh) 杂环磺酰胺衍生物及含有其的药物
TW201625585A (zh) 吡咯啶ｇｐｒ４０調節劑
CN105263924B (zh) Cxcr7受体调节剂
JP2023540350A (ja) 中環状または大環状ベンジル置換複素環誘導体およびオレキシン－２受容体アゴニストとしてのそれらの使用
TW202019946A (zh) 具有ｂｅｔ蛋白質分解誘導作用的醯胺化合物及其作為醫藥的用途