WO2024083255A1 - 苄基或噻吩亚甲基取代的氨基喹唑啉衍生物及其作为sos1降解剂的用途 - Google Patents

苄基或噻吩亚甲基取代的氨基喹唑啉衍生物及其作为sos1降解剂的用途 Download PDF

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WO2024083255A1
WO2024083255A1 PCT/CN2023/125904 CN2023125904W WO2024083255A1 WO 2024083255 A1 WO2024083255 A1 WO 2024083255A1 CN 2023125904 W CN2023125904 W CN 2023125904W WO 2024083255 A1 WO2024083255 A1 WO 2024083255A1
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cancer
mmol
ethyl
amino
methoxy
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PCT/CN2023/125904
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French (fr)
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WO2024083255A8 (zh
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叶正清
冯焱
丁陈利
王俏
韦红
谢舒晨
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上海领泰生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the present invention belongs to the field of medical technology. Specifically, the present invention relates to an SOS1 degrading agent and its use as a therapeutic agent for preventing and/or treating cancer.
  • SOS1 (son of sevenless homolog 1) protein is a regulatory protein widely expressed in cells. As a key protein in the signaling pathway, SOS1 plays an important regulatory role in many signal transduction pathways in cells, such as participating in the regulation of Ras and Rac signaling pathways. SOS1 is composed of 1333 amino acids and contains a proline-rich domain (PxxP) at its C-terminus. This domain can bind to growth factor receptor-bound protein 2 (Grb2) in the Ras pathway to form a complex of Grb2 and SOS1, thereby bringing SOS1 to the vicinity of Ras protein on the cell membrane.
  • Prb2 growth factor receptor-bound protein 2
  • SOS1 catalyzes the binding of Ras to GTP, promotes the activation of Ras, and then activates multiple downstream signaling pathways, such as Ras-Raf-Mek-Erk and Ras-PI3K-AKT-mTOR.
  • PxxP can also bind to the SH3 (Src homology 3) domain of proteins such as E3B1 in the Rac pathway to form an EPS8-E3B1-SOS1 complex.
  • the EPS8-E3B1-SOS1 complex connects to actin filaments through EPS8, causing the conversion of GTP, thereby activating Rac and subsequently activating signaling pathways such as JNK and MAPK.
  • Ras mutant genes are considered to be the main oncogenes with a high incidence in human cancer. Studies have shown that 20-30% of tumor patients have Ras mutations, of which KRas mutations account for 85%, NRas and HRas account for 12% and 3%, respectively. However, due to the picomolar affinity of GTP for its binding site and the smooth surface of Ras protein, which lacks other suitable binding pockets, it is considered extremely challenging to directly act on Ras to inhibit its activity.
  • SOS1 Abnormal expression or mutation of SOS1 is also closely related to the occurrence of clinical diseases. Studies have shown that SOS1 mutations exist in NS patients and CFC patients. HGF1 is a rare autosomal dominant genetic disease, and its cause is also related to the mutation of the PxxP domain of SOS1. In addition, abnormal expression or mutation of SOS1 is also related to the occurrence of cancer.
  • WO2018172250A1, WO2020173935A1, WO2019201848A1, WO2020180768A1, WO2020180770A1, WO2019122129A1 and EP3558979A1 disclose several types of SOS1 inhibitors, but so far, there are no reports on SOS1 degraders.
  • Proteolysis Targeting Chimeria is a technology different from traditional small molecule inhibitors.
  • PROTAC is a heterogeneous bifunctional molecule, one end of which is a small molecule inhibitor that can recognize the target protein through a connecting chain, and the other end is an E3 ubiquitin ligase ligand that can recognize E3 ubiquitin ligase.
  • This bifunctional molecule recognizes the target protein and E3 ubiquitin ligase in vivo, brings the target protein and E3 ubiquitin ligase together to form a ternary complex, and after ubiquitination of the target protein, it degrades the target protein through the ubiquitin-proteasome pathway in vivo.
  • PROTAC Compared with traditional small molecule inhibitors, PROTAC only needs to bring the target protein and E3 ubiquitin ligase closer to degrade the substrate. This mode of action allows this technology to be applied to some undruggable targets. On the other hand, since the target protein can be released after being degraded and continue to participate in the degradation process of the next protein, this degradation with catalytic effect allows a smaller PROTAC drug dose to achieve efficient degradation. On the other hand, traditional small molecule inhibitors are prone to drug resistance, often because point mutations occur, causing the small molecule inhibitors to lose their inhibitory effect on the target, while PROTAC can directly degrade the target protein, which can avoid the drug resistance caused by point mutations to a certain extent. Therefore, compared with traditional small molecule inhibitors, the use of PROTAC technology for the development of new drug small molecules has high advantages and feasibility.
  • the present invention provides a new SOS1 protein degrader and its application.
  • the SOS1 protein degrader of the present invention can effectively bind to or inhibit the SOS1 target protein, and further, can effectively degrade the SOS1 protein.
  • the present invention solves the above technical problems through the following methods.
  • the present invention provides a compound of formula I, its stereoisomers, enantiomers, diastereomers, deuterated compounds, hydrates, solvates, prodrugs or pharmaceutically acceptable salts thereof: SLE I
  • L is L is connected to S through A or B;
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • L is E is
  • a and S in L are linked.
  • the compound represented by formula I is any of the following compounds:
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising G and a pharmaceutically acceptable carrier, diluent or excipient, wherein G is the above-mentioned compound, its stereoisomer, enantiomer, diastereomer, deuterated substance, hydrate, solvate, prodrug, or pharmaceutically acceptable salt thereof.
  • the present invention provides a method for degrading SOS1 protein, comprising contacting the above-mentioned compound, its stereoisomer, enantiomer, diastereomer, deuterated product, hydrate, solvate, metabolite, prodrug, or pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition with SOS1 protein.
  • the present invention provides a compound, a stereoisomer, an enantiomer, a diastereomer, a deuterated substance, a hydrate, a solvate, a metabolite, a prodrug, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use as a drug for treating or preventing a disease or condition mediated by SOS1.
  • the present invention provides the above-mentioned compound, its stereoisomer, enantiomer, diastereomer, deuterated product, hydrate, solvate, metabolite, prodrug, or pharmaceutically acceptable salt, or the above-mentioned pharmaceutical composition for use as a drug for treating or preventing diseases or disorders (cancer) caused by the interaction between SOS1 and Ras (such as KRAS) or SOS1 and Rac (such as KRAS).
  • diseases or disorders cancer
  • the present invention provides a compound, a stereoisomer, an enantiomer, a diastereomer, a deuterated compound, a hydrate, a solvate, a metabolite, a prodrug, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use in preparing a drug for treating or preventing a disease or condition mediated by SOS1 (e.g., cancer).
  • SOS1 e.g., cancer
  • the present invention provides the above-mentioned compound, its stereoisomers, enantiomers, diastereomers, deuterated compounds, hydrates, solvates, metabolites, prodrugs, or pharmaceutically acceptable salts, or the use of the above-mentioned pharmaceutical composition in the preparation of drugs for treating or preventing diseases or disorders (such as cancer) caused by the interaction between SOS1 and Ras (such as KRAS) or SOS1 and Rac (such as KRAS).
  • diseases or disorders such as cancer
  • SOS1 and Ras such as KRAS
  • SOS1 and Rac such as KRAS
  • the present invention provides the above-mentioned compound, its stereoisomer, enantiomer, diastereomer, deuterated product, hydrate, solvate, metabolite, prodrug, or pharmaceutically acceptable salt, or the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating or preventing cancer.
  • the cancer can be pancreatic cancer, lung cancer, colorectal cancer, cholangiocarcinoma, multiple myeloma, melanoma, uterine cancer, endometrial cancer, thyroid cancer, acute myeloid leukemia, bladder cancer, urothelial carcinoma, gastric cancer, cervical cancer, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, esophageal cancer, chronic lymphocytic leukemia, hepatocellular carcinoma, breast cancer, ovarian cancer, prostate cancer, glioblastoma, renal cancer or sarcoma.
  • the present invention provides a compound, a stereoisomer, an enantiomer, a diastereomer, a deuterated product, a hydrate, a solvate, a metabolite, a prodrug, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use in preparing a drug for treating or preventing pancreatic cancer, lung cancer, colorectal cancer, cholangiocarcinoma, multiple myeloma, melanoma, uterine cancer, endometrial cancer, thyroid cancer, acute myeloid leukemia, bladder cancer, urothelial carcinoma, gastric cancer, cervical cancer, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, esophageal cancer, chronic lymphocytic leukemia, hepatocellular carcinoma, breast cancer, ovarian cancer, prostate cancer, glioblastoma, renal cancer and sarcoma.
  • the present invention provides a compound, a stereoisomer, an enantiomer, a diastereomer, a deuterated product, a hydrate, a solvate, a metabolite, a prodrug, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use in preparing a drug for treating or preventing neurofibromatosis type 1 (NF1), Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), capillary malformation-arteriovenous malformation syndrome (CM-AVM), Costello syndrome (CS), cardiofacial-cutaneous syndrome (CFC), Leggs syndrome and hereditary gingival fibromatosis.
  • NF1 neurofibromatosis type 1
  • NS Noonan syndrome
  • NSML Noonan syndrome with multiple lentigines
  • CM-AVM capillary malformation-arteriovenous malformation syndrome
  • CS Costello syndrome
  • CFC cardiofacial-cutaneous syndrome
  • the SOS1-mediated disease or condition may be cancer, neurofibromatosis type 1 (NF1), Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), capillary malformation-arteriovenous malformation syndrome (CM-AVM), Costello syndrome (CS), cardiofacial-cutaneous syndrome (CFC), Leggs syndrome or hereditary gingival fibromatosis; wherein the cancer may be the cancer described in any embodiment of the present invention.
  • NF1 neurofibromatosis type 1
  • NS Noonan syndrome
  • NML Noonan syndrome with multiple lentigines
  • C-AVM capillary malformation-arteriovenous malformation syndrome
  • CS Costello syndrome
  • CFC cardiofacial-cutaneous syndrome
  • Leggs syndrome hereditary gingival fibromatosis
  • the present invention provides a method for treating or preventing a disease or condition mediated by SOS1, comprising administering to a patient in need thereof a therapeutically effective amount of the above-mentioned compound, its stereoisomer, enantiomer, diastereomer, deuterated substance, hydrate, solvate, metabolite, prodrug, or pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition.
  • the present invention provides a method for treating or preventing a disease or condition (such as cancer) regulated by the interaction between SOS1 and Ras (such as KRAS) or SOS1 and Rac (such as KRAS), comprising administering a therapeutically effective amount of the above-mentioned compound, or its stereoisomer, enantiomer, diastereomer, deuterated substance, hydrate, solvate, metabolite, prodrug, or pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition to a patient in need thereof.
  • a disease or condition such as cancer
  • SOS1 and Ras such as KRAS
  • SOS1 and Rac such as KRAS
  • the cancer may be selected from:
  • sarcoma angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma
  • myxoma rhabdomyoma, fibroma, lipoma, and teratoma group
  • bronchial carcinoma squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar carcinoma (bronchiolar carcinoma), bronchial adenoma, sarcoma, lymphoma, enchondroma, hamartoma, mesothelioma;
  • Gastrointestinal tract esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma);
  • kidney adenocarcinoma, Wilms' tumor, lymphoma, leukemia
  • bladder and urethra squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma
  • prostate adenocarcinoma, sarcoma
  • testis salivanal carcinoma, teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatoid tumor, lipoma
  • liver cancer hepatocellular carcinoma
  • cholangiocarcinoma hepatoblastoma
  • angiosarcoma hepatocellular adenoma
  • hemangioma hepatocellular adenoma
  • Biliary tract gallbladder cancer, ampullary cancer, bile duct cancer;
  • Bone osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, enchondroma (osteochondral exostosis), benign enchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor;
  • Nervous system skull (osteomas, hemangiomas, granulomas, xanthomas, osteitis malformation), meninges (meningiomas, meningosarcomas, gliomas), brain (astrocytomas, medulloblastomas, gliomas, ependymomas, germinal cell tumors (pinealomas), glioblastoma multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital tumors), spinal neurofibromas, meningiomas, gliomas, sarcomas);
  • Gynecology uterus (endometrial cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumor, Sertoli-Leydig cell tumor, germ cell tumor, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma)), fallopian tube (cancer);
  • Blood myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma);
  • Skin malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, nevus dysplasia, lipoma, hemangioma, dermatofibroma, keloid, psoriasis;
  • Adrenal gland neuroblastoma.
  • the cancer can be selected from pancreatic cancer, lung cancer, colorectal cancer, cholangiocarcinoma, multiple myeloma, melanoma, uterine cancer, endometrial cancer, thyroid cancer, acute myeloid leukemia, bladder cancer, urothelial carcinoma, gastric cancer, cervical cancer, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, esophageal cancer, chronic lymphocytic leukemia, hepatocellular carcinoma, breast cancer, ovarian cancer, prostate cancer, glioblastoma, renal cancer and sarcoma.
  • the present invention also provides a use of the above-mentioned compound, or its stereoisomer, enantiomer, diastereomer, deuterated product, hydrate, solvate, prodrug, or pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for treating and/or preventing a KRAS-mediated disease or condition.
  • the present invention also provides a method for treating and/or preventing a KRAS-mediated disease or condition, comprising administering to a patient in need thereof a therapeutically effective amount of the above-mentioned compound, or its stereoisomers, enantiomers, diastereomers, deuterated substances, hydrates, solvates, metabolites, prodrugs, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof.
  • the KRAS may be a mutant KRAS.
  • the mutant KRAS is preferably one or more of KRAS G12C, KRAS G12D, KRAS G13D and KRAS G12V.
  • “Pharmaceutical composition” means a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.
  • the present invention also provides pharmaceutically acceptable salts of the compounds of formula (I).
  • pharmaceutically acceptable salt refers to acid addition salts or base addition salts of relatively nontoxic compounds of the present invention.
  • the acid addition salts are salts formed by the compounds of formula (I) of the present invention and suitable inorganic or organic acids, which can be prepared in the final separation and purification process of the compounds, or can be prepared by reacting the purified compounds of formula (I) in their free alkali form with suitable organic or inorganic acids.
  • Representative acid addition salts include hydrochlorides, hydrobromides, sulfates, bisulfates, sulfites, acetates, oxalates, valerates, oleates, palmitates, stearates, laurates, borates, benzoates, lactates, phosphates, hydrogen phosphates, carbonates, bicarbonates, toluate, citrates, maleates, fumarates, succinates, benzoates, tartrates, methanesulfonates, p-toluenesulfonates, gluconates, lactobionates, and lauryl sulfonates, etc.
  • the base addition salt is a salt formed by the compound of formula (I) and a suitable inorganic base or organic base, including, for example, salts formed with alkali metals, alkaline earth metals, quaternary ammonium cations, such as sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, tetramethyl quaternary ammonium salts, tetraethyl quaternary ammonium salts, etc.; amine salts include salts formed with ammonia ( NH3 ), primary amines, secondary amines or tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, etc.
  • quaternary ammonium cations such as sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, tetramethyl quaternary ammonium salts, tetraethyl
  • the compounds of the present invention or their pharmaceutically acceptable salts can be administered to mammals, including humans, orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically (powders, ointments or drops), or intratumorally.
  • the compound of the present invention may be administered at a dosage of about 0.05-300 mg/kg body weight/day, preferably 10-300 mg/kg body weight/day, more preferably 10-200 mg/kg body weight/day.
  • the compounds of the present invention or their pharmaceutically acceptable salts can be formulated into solid dosage forms for oral administration, including but not limited to capsules, tablets, pills, powders, granules and the like.
  • the compound of formula (I) of the present invention is mixed as an active ingredient with at least one conventional inert excipient (or carrier), for example, sodium citrate or dicalcium phosphate, or with the following ingredients: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, etc.; (2) binders, such as hydroxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic, etc.; (3) humectants, such as glycerol, etc.; (4) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, etc.; (5) solubilizing
  • the solid dosage forms such as tablets, pills, capsules, pills and granules can be coated or microencapsulated with coating and shell materials such as enteric coatings and other materials known in the art. They can contain opacifiers, and the release of the active ingredient in such compositions can be delayed in a certain part of the digestive tract.
  • coating and shell materials such as enteric coatings and other materials known in the art. They can contain opacifiers, and the release of the active ingredient in such compositions can be delayed in a certain part of the digestive tract.
  • Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active ingredient can also be formed into microcapsules with one or more of the above-mentioned excipients.
  • the compounds of the present invention or their pharmaceutically acceptable salts can be formulated into liquid dosage forms for oral administration, including but not limited to pharmaceutically acceptable emulsions, solutions, suspensions, syrups and tinctures, etc.
  • the liquid dosage form may contain inert diluents such as water and other solvents, solubilizers and emulsifiers conventionally used in the art, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, etc.
  • inert diluents such as water and other solvents, solubilizers and emulsifiers conventionally used in the art, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-buty
  • liquid dosage form of the present invention may also contain conventional adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices, etc.
  • the suspending agent includes, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol, microcrystalline cellulose, aluminum methylate and agar, or a mixture of these substances.
  • the compounds of the present invention or their pharmaceutically acceptable salts can be formulated into dosage forms for parenteral injection, including but not limited to physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for re-dissolving into sterile injectable solutions or dispersions.
  • Suitable carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the compounds of the present invention or their pharmaceutically acceptable salts can also be formulated into dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays and inhalants, etc.
  • dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays and inhalants, etc.
  • active ingredients the compounds of formula (I) of the present invention or their pharmaceutically acceptable salts are mixed with physiologically acceptable carriers and optional preservatives, buffers, or propellants that may be needed under sterile conditions.
  • the present invention also provides a pharmaceutical composition, which contains the compound of formula (I) of the present invention or its pharmaceutically acceptable salt as an active ingredient, and a pharmaceutically acceptable carrier, excipient or diluent.
  • a pharmaceutically acceptable carrier excipient or diluent.
  • composition of the present invention can be formulated into conventional pharmaceutical preparations according to conventional preparation methods, such as tablets, pills, capsules, powders, granules, emulsions, suspensions, dispersions, solutions, syrups, elixirs, ointments, drops, suppositories, inhalants, sprays, etc.
  • the compounds of the present invention or their pharmaceutically acceptable salts can be administered alone or (if necessary) in combination with other pharmaceutically acceptable therapeutic agents, such as other anti-tumor drugs.
  • the components to be combined can be administered simultaneously or sequentially, in the form of a single preparation or in the form of different preparations.
  • the combination can include not only a combination of the compounds of the present invention and one other active agent, but also a combination of the compounds of the present invention and two or more other active agents.
  • other pharmaceutically acceptable therapeutic agents that can be used together or in combination with the SOS1 degrading agent compound of formula (I) may be: EGFR and/or its mutant inhibitors, ErbB2 (Her2) and/or its mutant inhibitors, ALK and/or its mutant inhibitors, MEK and/or its mutant inhibitors, Kras and/or its mutant inhibitors, BCR-ABL and/or its mutant inhibitors, FGFR1/FGFR2/FGFR3 and/or its mutant inhibitors, ROS1 and/or its mutant inhibitors, c-MET and/or its mutant inhibitors, AXL and /or its mutant inhibitors, NTRK1 and/or its mutant inhibitors, RET and/or its mutant inhibitors, taxanes, platinum-containing compounds, antimetabolites, mitotic kinase inhibitors, immunotherapeutics, anti-angiogenic drugs, topoisomerase inhibitors, A-Raf/B-Raf/C-RAf and/or its mutant inhibitors, ERK and/or its mutant inhibitors,
  • other pharmaceutically acceptable therapeutic agents that can be used together or in combination with the SOS1 degrading agent compound of formula (I) may be: afatinib, erlotinib, gefitinib, lapatinib, cetuximab, panitumumab, osimertinib, olmutinib, EGF-816, trastuzumab, pertuzumab, crizotinib, alectinib, entrectinib, brigatinib, trametinib, cobimetinib , binimetinib, selumetinib, refametinib, imatinib, dasatinib, nilotinib, nintedanib, crizotinib, lorlatinib, ceritinib, merestinib, paclitaxel, nab-paclitaxel, docetaxel, cis
  • the reagents and raw materials used in the present invention are commercially available.
  • the positive and progressive effect of the present invention is that: the present invention proves through the SOS1 kinase activity test experiment that the compound of formula I of the present invention can effectively bind to the SOS1 target protein or produce an inhibitory effect, and further, can effectively degrade the SOS1 protein, thereby achieving the effect of preventing or treating diseases or symptoms related to SOS1 or caused by the interaction between SOS1 and Ras or SOS1 and Rac.
  • Step 1 Preparation of benzyl 4-(2-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy-d 2 )ethyl)piperidine-1-carboxylate
  • Step 2 Preparation of benzyl 4-(2-(piperidin-4-ylmethoxy-d 2 )ethyl)piperidine-1-carboxylate
  • Benzyl 4-(2-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy-d 2 )ethyl)piperidine-1-carboxylate (350 mg, 0.758 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was added and stirred at room temperature for 2 hours. The reaction solution was directly concentrated under reduced pressure to obtain benzyl 4-(2-(piperidin-4-ylmethoxy-d 2 )ethyl)piperidine-1-carboxylate, which was directly used in the next reaction without purification.
  • Step 3 Preparation of benzyl 4-(2-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)methoxy-( 2 2 )ethyl)piperidine-1-carboxylate
  • Step 4 Preparation of 1-(2-chloro-5-(4-((2-(piperidin-4-yl)ethoxy)methyl-d 2 )piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Example 1 (1R, 4R)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)-N-(3-(3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecane-9-yl)propyl)-N-methylcyclohexane-1-carboxamide
  • Step 1 Preparation of (E)-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-3-azaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester
  • Phosphorylacetic acid (1.27 g, 5.7 mmol) was dissolved in THF (50 mL), and NaH (342 mg, 8.55 mmol, 60%) was added at 0°C, and the mixture was stirred for 2 h. Then, tert-butyl 9-aldehyde-3-azaspiro[5.5]undecane-3-carboxylate (800 mg, 2.85 mmol) was added, and stirring was continued at room temperature for 2 h. Water (100 mL) was added to dilute the reaction solution, and the mixture was extracted with ethyl acetate (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 2 Preparation of tert-butyl 9-(3-ethoxy-3-oxopropyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 3 Preparation of tert-butyl 9-(3-hydroxypropyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 4 Preparation of tert-butyl 9-(3-oxopropyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 5 Preparation of tert-butyl 9-(3-(benzyl(methyl)amino)propyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 6 Preparation of tert-butyl 9-(3-(methylamino)propyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 7 Preparation of tert-butyl 9-(3-((1R,4R)-4-(4-(((R)-1-(3-((tert-butoxycarbonyl)amino)-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)-N-methylcyclohexane-1-carboxamido)propyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • tert-butyl 9-(3-(methylamino)propyl)-3-azaspiro[5.5]undecane-3-carboxylate 130 mg, 0.40 mmol was added and stirring was continued for 1 hour.
  • the reaction solution was diluted with water (40 mL) and extracted with ethyl acetate (40 mL x 3). The organic phase was washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • Step 8 Preparation of (1R,4R)-N-(3-(3-azaspiro[5.5]undec-9-yl)propyl)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)-N-methylcyclohexane-1-carboxamide
  • Step 9 Preparation of (1R,4R)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)-N-(3-(3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)propyl)-N-methylcyclohexane-1-carboxamide
  • Pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (73.6 mg, 0.17 mmol), DIEA (65.8 mg, 0.51 mmol) and (1R,4R)-N-(3-(3-azaspiro[5.5]undec-9-yl)propyl)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)-N-methylcyclohexane-1-carboxamide (120 mg, 0.17 mmol) were added to DMSO (10 mL), and the reaction solution was stirred at room temperature for 30 min.
  • Example 2 1-(2-chloro-5-(4-((2-(1-((1R,4R)-4-(7-methoxy-2-methyl-4-(((R)-1-(4-(2-((methylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)quinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)ethoxy)methyl)piperidin-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 1 Preparation of tert-butyl 4-(2-(pyridin-4-ylmethoxy)ethyl)piperidine-1-carboxylate
  • Step 2 Preparation of tert-butyl 4-(2-(piperidin-4-ylmethoxy)ethyl)piperidine-1-carboxylate
  • Step 3 Preparation of tert-butyl 4-(2-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)methoxy)ethyl)piperidine-1-carboxylate
  • Step 4 Preparation of 1-(2-chloro-5-(4-((2-(piperidin-4-yl)ethoxy)methyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • reaction solution was stirred at room temperature for 2 hours and concentrated to obtain 1-(2-chloro-5-(4-((2-(piperidin-4-yl)ethoxy)methyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione, which was directly used in the next step reaction.
  • Step 5 Preparation of tert-butyl (2-(5-((R)-1-((6-((1R,4R)-4-(4-(2-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)methoxy)ethyl)piperidine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)thiophen-3-yl)benzyl)(methyl)carboxylate
  • reaction solution was stirred at room temperature for 2 hours. Water (20 mL) was added to the reaction solution, and then extracted with ethyl acetate (20 mL x 2). The organic phases were combined and washed with saturated brine (30 mL x 3), and dried over anhydrous sodium sulfate.
  • Step 6 Preparation of 1-(2-chloro-5-(4-((2-(1-((1R,4R)-4-(7-methoxy-2-methyl-4-(((R)-1-(4-(2-((methylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)quinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)ethoxy)methyl)piperidin-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 1 Preparation of tert-butyl 4-(2-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methoxy)ethyl)piperidine-1-carboxylate
  • Step 2 Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(4-((2-(piperidin-4-yl)ethoxy)methyl)piperidin-1-yl)isoindoline-1,3-dione
  • Step 3 Preparation of tert-butyl (2-(5-((1R)-1-((6-((1R,4R)-4-(4-(2-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methoxy)ethyl)piperidine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)thiophen-3-yl)benzyl)(methyl)carboxylate
  • Step 4 Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(4-((2-(1-((1R,4R)-4-(7-methoxy-2-methyl-4-(((R)-1-(4-(2-((methylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)quinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)ethoxy)methyl)piperidin-1-yl)isoindoline-1,3-dione
  • Step 2 Preparation of tert-butyl 4-(4-(pyridin-4-yl)n-butyl)piperazine-1-carboxylate
  • Step 3 Preparation of tert-butyl 4-(4-(piperidin-4-yl)n-butyl)piperazine-1-carboxylate
  • Step 4 Preparation of tert-butyl 4-(4-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)n-butyl)piperazine-1-carboxylate
  • Step 5 Preparation of 1-(2-chloro-5-(4-(4-(piperazin-1-yl)n-butyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 6 Preparation of tert-butyl (2-(5-((R)-1-((6-((1R,4R)-4-(4-(4-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)n-butyl)piperazine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)thiophen-3-yl)benzyl)(methyl)carboxylate
  • the reaction solution was stirred at room temperature for 2 hours. Water (20 mL) was added to the reaction solution, and it was extracted with ethyl acetate (20 mL x 2). The organic phases were combined and washed with saturated brine (30 mL x 3), and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated.
  • Step 7 Preparation of 1-(2-chloro-5-(4-(4-(4-((1R,4R)-4-(7-methoxy-2-methyl-4-(((R)-1-(4-(2-((methylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)quinazolin-6-yl)cyclohexane-1-carbonyl)piperazin-1-yl)n-butyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Pd(dppf)Cl 2 (262 mg, 0.36 mmol) and sodium carbonate (2.01 g, 18.92 mmol) were added to a mixed solution of 2,6-bis(benzyloxy)-3-bromopyridine (2.80 g, 7.57 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxybenzofuran-2-yl)benzoic acid (1.87 g, 11.35 mmol) in dioxane/H 2 O (50 mL/10 ml).
  • Step 3 Preparation of benzyl 4-((3-(4-(2,6-dioxopiperidin-3-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperazine-1-carboxylate
  • Step 4 Preparation of 3-(4-(9-(piperazin-1-ylmethyl)-3-azaspiro[5.5]undecane-3-carbonyl)phenyl)piperidine-2,6-dione
  • reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to obtain 3-(4-(9-(piperazin-1-ylmethyl)-3-azaspiro[5.5]undecane-3-carbonyl)phenyl)piperidine-2,6-dione, which was directly used in the next step.
  • Step 5 Preparation of tert-butyl (2-(5-((1R)-1-((6-((1R,4R)-4-(4-((3-(4-(2,6-dioxopiperidin-3-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperazine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)thiophen-3-yl)benzyl)(methyl)carboxylate
  • Step 6 Preparation of 3-(4-(9-((4-((1R,4R)-4-(7-methoxy-2-methyl-4-(((R)-1-(4-(2-((methylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)quinazolin-6-yl)cyclohexane-1-carbonyl)piperazin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carbonyl)phenyl)piperidine-2,6-dione
  • Trifluoroacetic acid (1.0 mL) was added to a solution of tert-butyl (2-(5-((1R)-1-((6-((1R,4R)-4-(4-((3-(4-(2,6-dioxopiperidin-3-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperazine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)thiophen-3-yl)benzyl)(methyl)carboxylate (160 mg, 0.15 mmol) in dichloromethane (2.0 mL), and the mixture was stirred at 30° C.
  • Example 6 1-(2-chloro-5-(4-(2-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-chloro-6-((dimethylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)methoxy)ethyl)piperazine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 1 Preparation of tert-butyl 4-(2-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-chloro-6-((dimethylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)methoxy)ethyl)piperazine-1-carboxylate
  • reaction solution was diluted with water (50 mL), extracted with ethyl acetate (30 mL x 3), and the organic phase was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 2 Preparation of ((1R,4R)-4-(4-(((R)-1-(4-(2-chloro-6-((dimethylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexyl)(4-((2-(piperazin-1-yl)ethoxy)methyl)piperidin-1-yl)methanone
  • Step 3 Preparation of 1-(2-chloro-5-(4-(2-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-chloro-6-((dimethylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)methoxy)ethyl)piperazine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 3). The organic phase was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Example 7 1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperazin-1-yl)propyl)piperidin-1-yl)-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 4 Preparation of 3-((5-(4-(3-(benzoyloxy)propyl)piperidin-1-yl)-2-chlorophenyl)amino)propanoic acid
  • Benzoic acid 3-(1-(3-amino-4-chlorophenyl)piperidin-4-yl)propyl ester (900 mg, 2.42 mmol) and acrylic acid (1.74 g, 24.19 mmol) were added to H2O /AcOH (5/1, 12 mL), and the reaction solution was stirred at 110°C overnight under a nitrogen atmosphere. The reaction solution was directly concentrated under reduced pressure, and the crude product was purified by pre-HPLC (acetonitrile/formic acid water system) to obtain 3-((5-(4-(3-(benzoyloxy)propyl)piperidin-1-yl)-2-chlorophenyl)amino)propanoic acid.
  • pre-HPLC acetonitrile/formic acid water system
  • Step 5 Preparation of 3-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl)piperidin-4-yl)propyl benzoate
  • Step 6 Preparation of 1-(2-chloro-5-(4-(3-hydroxypropyl)piperidin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Benzoic acid 3-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl)piperidin-4-yl)propyl ester (500mg, 1.06mmol) was dissolved in methanol/concentrated hydrochloric acid (1:1, 8mL), and the reaction liquid was heated to 70°C and stirred overnight under nitrogen atmosphere.
  • Step 7 Preparation of 3-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl)piperidin-4-yl)propanal
  • Step 8 Preparation of tert-butyl 4-(3-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl)piperidin-4-yl)propyl)piperazine-1-carboxylate
  • Step 9 Preparation of 1-(2-chloro-5-(4-(3-(piperazin-1-yl)propyl)piperidin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 10 Preparation of tert-butyl (3-((R)-1-((6-((1R,4R)-4-(4-(3-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl)piperidin-4-yl)propyl)piperazine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)-5-(trifluoromethyl)phenyl)carboxylate
  • Step 11 Preparation of 1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperazin-1-yl)propyl)piperidin-1-yl)-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Example 8 1-(2-chloro-5-(4-(3-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-chloro-6-((dimethylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)oxy)propyl)piperazine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 1 Preparation of benzyl 4-(3-ethoxy-3-oxopropoxy)piperidine-1-carboxylate
  • Step 3 Preparation of 4-(3-oxopropoxy)piperidine-1-carboxylic acid benzyl ester
  • Step 4 Preparation of tert-butyl 4-(3-((1-((benzyloxy)carbonyl)piperidin-4-yl)oxy)propyl)piperazine-1-carboxylate
  • Benzyl 4-(3-oxopropoxy)piperidine-1-carboxylate (370 mg, 1.27 mmol) was dissolved in THF (10 mL), and tert-butyl piperazine-1-carboxylate (236 mg, 1.27 mmol) was added in sequence and stirred at room temperature for 10 min, sodium triacetoxyborohydride (808 mg, 3.81 mmol), and the reaction solution was stirred at room temperature for 2 hours.
  • Step 5 Preparation of tert-butyl 4-(3-(piperidin-4-yloxy)propyl)piperazine-1-carboxylate
  • Step 6 Preparation of tert-butyl 4-(3-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-chloro-6-((dimethylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)oxy)propyl)piperazine-1-carboxylate
  • Step 7 Preparation of ((1R,4R)-4-(4-(((R)-1-(4-(2-chloro-6-((dimethylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexyl)(4-(3-(piperazin-1-yl)propoxy)piperidin-1-yl)methanone
  • Step 8 Preparation of 1-(2-chloro-5-(4-(3-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-chloro-6-((dimethylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)oxy)propyl)piperazine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Example 9 3-((R)-1-((6-((1R,4R)-4-(4-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperazine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)-2-methylbenzyl cyanide
  • Step 1 Preparation of tert-butyl 9-((4-((benzyloxy)carbonyl)piperazin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 2 Preparation of tert-butyl 9-(piperazin-1-ylmethyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • reaction solution was cooled to room temperature, it was filtered through diatomaceous earth, and the filtrate was directly concentrated under reduced pressure to obtain 9-(piperazin-1-ylmethyl)-3-azaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester, which was directly used in the next step without purification.
  • Step 3 Preparation of (1R,4R)-4-(4-(((R)-1-(3-cyano-2-methylphenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carboxylic acid methyl ester
  • Step 4 Preparation of (1R,4R)-4-(4-(((R)-1-(3-cyano-2-methylphenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carboxylic acid
  • Step 5 Preparation of tert-butyl 9-((4-((1R,4R)-4-(4-(((R)-1-(3-cyano-2-methylphenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperazin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • reaction solution was diluted with water (50 mL), extracted with ethyl acetate (30 mL x 3), and the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 6 Preparation of 3-((R)-1-((6-((1R,4R)-4-(4-((3-azaspiro[5.5]undec-9-yl)methyl)piperazine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)-2-methylbenzonitrile
  • Step 7 Preparation of 3-((R)-1-((6-((1R,4R)-4-(4-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperazine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)-2-methylbenzonitrile
  • Example 10 1-(2-chloro-5-(4-((2-(1-((1R,4R)-4-(4-(((R)-1-(4-(2-chloro-6-((dimethylamino)methyl)phenyl)thiophen-2-yl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)ethoxy)methyl- d2 )piperidin-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Example 11 1-(5-(4-((2-(1-((1R,4R)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)ethoxy)methyl- d2 )piperidine-1-carbonyl)-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 1 Preparation of (1R,4R)-4-(4-(((R)-1-(3-((tert-butyloxycarbonyl)amino)-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carboxylic acid pentafluorophenyl ester
  • reaction solution was diluted with water (50 mL), extracted with dichloromethane (50 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 2 Preparation of tert-butyl (3-((R)-1-((6-((1R,4R)-4-(4-(2-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin- 4 -yl)methoxy-d2)ethyl)piperidine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)-5-(trifluoromethyl)phenyl)carboxylate
  • Step 3 Preparation of 1-(5-(4-((2-(1-((1R,4R)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperidin-4-yl)ethoxy)methyl- d2 )piperidine-1-carbonyl)-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Example 12 1-(2-chloro-5-(9-((4-((1R,4R)-4-(4-(((R)-1-(3-(difluoromethyl)-2-methylphenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperazin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 1 Preparation of methyl (1R,4R)-4-(4-(((R)-1-(3-(difluoromethyl)-2-methylphenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carboxylate
  • reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 3). The organic phase was washed with saturated aqueous sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 2 Preparation of (1R,4R)-4-(4-(((R)-1-(3-(difluoromethyl)-2-methylphenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carboxylic acid
  • Step 3 Preparation of 1-(2-chloro-5-(9-((4-((1R,4R)-4-(4-(((R)-1-(3-(difluoromethyl)-2-methylphenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperazin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • the mixture was diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 3). The organic phase was washed with saturated aqueous sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Example 13 1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperazin-1-yl)propoxy)piperidine-1-carbonyl)-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 1 Preparation of tert-butyl (3-((R)-1-((6-((1R,4R)-4-(4-(3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxy)propyl)piperazine-1-carbonyl)cyclohexyl)-7-methoxy-2-methylquinazolin-4-yl)amino)ethyl)-5-(trifluoromethyl)phenyl)carboxylate
  • Step 2 Preparation of 1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)cyclohexane-1-carbonyl)piperazin-1-yl)propoxy)piperidine-1-carbonyl)-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Test Example 1 KRAS-G12C/SOS1 binding assay
  • the following method is used to determine the degree of inhibition of KRAS-G12C/SOS1 binding by the preferred compounds of the present invention under in vitro conditions.
  • This method uses a KRAS-G12C/SOS1 binding assay kit (Cisbio, 63ADK000CB16PEG) to determine the competitive inhibition of KRAS-G12C/SOS1 binding by the preferred compounds using homogeneous time-resolved fluorescence technology (HTRF).
  • HTRF homogeneous time-resolved fluorescence technology
  • the experimental process is as follows (for detailed methods, refer to the kit instructions): the compound of the present invention is first dissolved in DMSO at a concentration of 20mM, and the buffer in the kit is used for isocratic dilution so that the final concentration range of the test compound in the reaction system is 10000nM to 0.04nM, and the final concentration of DMSO is 0.5%.
  • 2 ⁇ L of the compound is incubated with 4 ⁇ L 1x Tag1-SOS1 and 4 ⁇ L 1x Tag2-KRAS-G12C (containing 10 ⁇ M guanosine triphosphate, GTP) at 25°C for 15min, and then 5 ⁇ L 1x Anti-Tag1-Tb3+ and 5 ⁇ L 1x Anti-Tag2-XL665 are added to the reaction system and incubated on ice for 3h. After the incubation, the fluorescence intensity of each well at an excitation wavelength of 337 nm was measured on the microplate reader EnVision (PerkinElmer, 2105) in HTRF mode, and the fluorescence intensity of each well at an emission wavelength of 620 nm and 665 nm was read.
  • 4 ⁇ L 1x Tag1-SOS1 and 4 ⁇ L 1x Tag2-KRAS-G12C containing 10 ⁇ M guanosine triphosphate, GTP
  • the inhibition rate of the compound at each concentration was calculated by comparing the fluorescence intensity ratio with that of the control group, and then the nonlinear curve fitting of the logarithmic concentration-inhibition rate was performed by GraphPad Prism 8 to obtain the IC50 value of the compound, as shown in Table 1.
  • the degradation effect of the compounds on SOS1 was investigated in human non-small cell lung cancer cells NCI-H358 (ATCC or Chinese Academy of Sciences Stem Cell Bank) with KRAS G12C mutation, human lung cancer cells A-427 (ATCC) with KRAS G12D mutation, human colorectal adenocarcinoma cells SW-620 (ATCC) with KRAS G12V mutation, and human colorectal adenocarcinoma epithelial cells DLD-1 (ATCC) with KRAS G13D mutation.
  • the specific method is:
  • 0.95 mL of cells were plated in each well of a 24-well cell culture plate to a cell density of 5 ⁇ 105 cells/well; the cell plate was placed in a 5% CO 2 incubator at 37°C overnight. Then 50 ⁇ L of the diluted compound solution was added to the corresponding wells with cells to make the final concentration of the compound in the range of 0.03 to 3000 nM. The final concentration of DMSO was 0.25%. After administration, the cell plate was placed in a 5% CO 2 incubator and cultured at 37°C for 24 hours. After removing the cell culture medium in the 24 wells, the cells were washed twice with 1 ⁇ PBS.
  • the NCI-H358 cells attached to the bottom of the cell culture plate were added with 180 ⁇ L RIPA (strong) lysis buffer (Biyuntian, P0013B), supplemented with 1mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail (Biyuntian, P1008) and protease phosphatase inhibitor cocktail (Biyuntian, P1045) for lysis.
  • RIPA strong lysis buffer
  • 1mM phenylmethylsulfonyl fluoride 1mM phenylmethylsulfonyl fluoride
  • protease inhibitor cocktail Biyuntian, P1008
  • protease phosphatase inhibitor cocktail Biyuntian, P1045
  • the cell lysate supernatant after centrifugation was frozen at -80°C for testing and used for WB method to detect the degradation level of SOS1 protein.
  • the total protein concentration in the cell lysate supernatant was determined using the BCA protein quantification kit (Tiangen, PA115-02).
  • PBS and 5 ⁇ SDS-PAGE protein loading buffer (Biyuntian, P0015L) were adjusted to 0.5 ⁇ g/ ⁇ L, 100°C water bath for 15min, then placed on ice for 5min, centrifuged at 14000g and 4°C for 1min, and mixed as WB loading sample.
  • Precast gel Keygen, KGMG010W15 was used for protein electrophoresis, with a loading volume of 10 ⁇ L (total protein 5 ⁇ g), Tris-MOPS-SDS electrophoresis buffer (Adamas, P1598253) and 120V constant voltage electrophoresis for 55min.
  • the protein on the gel strip was transferred to the PVDF membrane at a constant current of 250mA for 65min. After the transfer, the membrane was placed in 1 ⁇ QuickBlock blocking solution (Biyuntian, P0235) and incubated at room temperature for 30min.
  • the PVDF membrane was incubated with the primary antibody of SOS1 (Abcam, ab140621) at 4°C overnight, washed with TBST buffer (2.4g Tris, 8.8g NaCl, 1.5mL Tween 20, adjusted to pH 7.4, and fixed to 1L) for 30min (10min/time), incubated with the secondary antibody (Abcam, ab205718) at room temperature for 2h, washed with TBST buffer for 30min (10min/time), and finally incubated with Clarity Western ECL Substrate (BIO-RAD, 170-5061) for 5min for luminescence color development, and the chemiluminescence imaging system (Qinxiang, ChemiScope 6200Touch) was used for color development and protein map photography.
  • SOS1 Abcam, ab140621
  • TBST buffer 2.4g Tris, 8.8g NaCl, 1.5mL Tween 20, adjusted to pH 7.4, and fixed to 1L
  • the protein map was analyzed by the Qinxiang chemiluminescence analysis software for grayscale value.
  • the degradation rate was calculated by comparing with the grayscale correction value of the control group. Then, nonlinear curve fitting was performed using GraphPad Prism 8 with the logarithmic concentration-inhibition rate to obtain the DC 50 and D max values of the compound.
  • the inhibitory effect of the compounds on 3D cell proliferation was investigated in human non-small cell lung cancer cells NCI-H358 (ATCC or Chinese Academy of Sciences Stem Cell Bank) with KRAS G12C mutation, human metastatic pancreatic adenocarcinoma cells AsPC-1 (ATCC) with KRAS G12D mutation, human lung adenocarcinoma cells NCI-H441 (ATCC) with KRAS G12V mutation, and human colorectal adenocarcinoma epithelial cells DLD-1 (ATCC) with KRAS G13D mutation.
  • the specific method is:
  • DMSO control wells and culture medium control wells were also set up.
  • the DMSO control wells contained DMSO and cells, and the culture medium wells contained only culture medium.
  • the plate cover was covered, and the 384-well low-adsorption microplate was placed in a 5% carbon dioxide incubator and incubated at 37°C for 7 days. After 7 days, the microwell plate was removed, 40 ⁇ L of CTG reagent (Promega, G9683) was added to each well, and the chemiluminescence program was used to read the results on the microplate reader EnVision after incubation at room temperature for 30 minutes.
  • CTG reagent Promega, G9683
  • inhibition rate % (mean value of DMSO control group - single compound concentration reading) / (mean value of DMSO control group - mean value of culture medium control group) ⁇ 100, and then nonlinear curve fitting was performed using GraphPad Prism 8 with the logarithmic concentration-inhibition rate to obtain the IC50 value of the compound.
  • IC 50 ⁇ 15nM it is A; when 15nM ⁇ IC 50 ⁇ 30nM, it is B; when 30nM ⁇ IC 50 ⁇ 50nM, it is C; when 50nM ⁇ IC 50 ⁇ 100nM, it is D; when 100nM ⁇ IC 50 ⁇ 1000nM, it is E.
  • the compounds of the present invention can effectively bind to the SOS1 target protein or produce an inhibitory effect, and the compounds of the present invention can effectively and specifically degrade the SOS1 protein.

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Abstract

本发明公开了一种SOS1降解剂及其应用。本发明提供了一种如式(I)示的化合物、和/或其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药和/或其药学上可接受的盐。本发明的化合物可作为SOS1降解剂。

Description

苄基或噻吩亚甲基取代的氨基喹唑啉衍生物及其作为SOS1降解剂的用途
本申请要求申请日为2022年10月21日的中国专利申请2022112963993的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于医药技术领域，具体的，本发明涉及SOS1降解剂，及其作为预防和/或治疗癌症治疗剂的应用。
背景技术
SOS1(son of sevenless homolog 1)蛋白是一种在细胞中广泛表达的调控蛋白，作为信号通路中的关键蛋白，SOS1在细胞内许多信号转导通路中起着重要的调控作用，例如参与调控Ras和Rac信号通路。SOS1由1333个氨基酸组成，在其C端包含一个富含脯氨酸的结构域(PxxP),该结构域可与Ras通路中的生长因子受体结合蛋白2(growth factor receptor-bound protein 2,Grb2)相结合，形成Grb2和SOS1的复合物，从而将SOS1带至细胞膜Ras蛋白附近，SOS1催化Ras与GTP的结合，促进Ras的激活，进而激活多条下游信号通路，例如Ras-Raf-Mek-Erk、Ras-PI3K-AKT-mTOR。PxxP也可与Rac通路中的E3B1等蛋白的SH3(Src homology 3)结构域相结合，形成EPS8-E3B1-SOS1复合物，EPS8-E3B1-SOS1复合物通过EPS8连接肌动蛋白丝，引起GTP的转换，从而激活Rac，随后激活JNK和MAPK等信号通路。
Ras突变基因被认为是人类癌症具有高发生率的主要致癌基因，研究表明，20-30％的肿瘤患者都存在着Ras的突变，其中KRas的突变占了85％，NRas和HRas分别占12％和3％。然而由于GTP对其结合位点的皮摩尔亲和力，以及Ras蛋白表面光滑，缺乏其他合适的结合口袋，因此，直接作用于Ras抑制其活性被认为是极具挑战性的。
SOS1的异常表达或突变与临床疾病的发生也有着密切的关系。研究表明，在NS患者、CFC患者体内都存在SOS1的突变，HGF1是一种罕见的常染色体显性遗传病，其病因也与SOS1的PxxP结构域突变有关，此外，SOS1的异常表达或突变与癌症的发生也有关系。
WO2018172250A1、WO2020173935A1、WO2019201848A1、WO2020180768A1、WO2020180770A1、WO2019122129A1和EP3558979A1公开了几类SOS1抑制剂，但目前为止，未有SOS1降解剂的相关报道。蛋白降解靶向嵌合体(Proteolysis Targeting Chimeria，PROTAC)是一种不同于传统小分子抑制剂的技术，传统小分子抑制剂通常需要作用于靶蛋白的活性位点才能抑制其活性，而PROTAC为一种异质双功能分子，其一端为可识别靶蛋白的小分子抑制剂，通过连接链，另一端为可识别E3泛素连接酶的E3泛素连接酶配体，这种双功能分子在体内识别靶蛋白和E3泛素连接酶，将靶蛋白和E3泛素连接酶拉近，形成三元复合物，将靶蛋白泛素化后，在体内通过泛素－蛋白酶体途径将靶蛋白降解。相较于传统小分子抑制剂，一方面PROTAC只需要将靶蛋白与E3泛素连接酶拉近，使底物降解，这种作用模式使得这种技术可以应用于一些不可成药靶点；另一方面，由于靶蛋白在被降解后，PROTAC分子还可释放出来继续参与下一蛋白的降解过程，因此这种具有催化效果的降解作用，使得较少的PROTAC药物剂量就可以实现高效的降解；再一方面，传统的小分子抑制剂易产生耐药性常常是因为发生了点突变，使得小分子抑制剂失去了对靶点的抑制作用，而PROTAC可以直接将靶蛋白降解，在一定程度上能过避免点突变产生的耐药性。因此，相较于传统小分子抑制剂，运用PROTAC技术进行新药小分子研发具有很高的优势和可行性。
发明内容
为了解决现有技术中SOS1蛋白降解剂缺乏的技术问题，本发明提供了一种新的SOS1蛋白降解剂及其应用。本发明所述的SOS1蛋白降解剂能够有效的与SOS1靶蛋白结合或产生抑制效果，进一步地，能够有效地降解SOS1蛋白。
本发明是通过以下方法来解决上述技术问题的。
本发明提供了一种式I所示化合物、其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药或其药学上可接受的盐：
S-L-E
I
其中，S为
L为 L通过A或B与S相连；
E为
在本发明一优选实施方案中，所述式I所示化合物、其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药或其药学上可接受的盐里，某些基团具有如下定义，未提及的基团的定义如上任一方案所述(本段内容以下简称为“在本发明一优选实施方案中”)：
在本发明一优选实施方案中，S为L为
在本发明一优选实施方案中，S为
L为
在本发明一优选实施方案中，S为
L为
在本发明一优选实施方案中，S为
L为
在本发明一优选实施方案中，S为
L为
在本发明一优选实施方案中，S为
L为
在本发明一优选实施方案中，S为L为
在本发明一优选实施方案中，S为
L为
在本发明一优选实施方案中，S为L为
在本发明一优选实施方案中，S为
E为
在本发明一优选实施方案中，S为
E为
在本发明一优选实施方案中，S为
E为
在本发明一优选实施方案中，S为
E为
在本发明一优选实施方案中，S为
E为
在本发明一优选实施方案中，S为
E为
在本发明一优选实施方案中，S为
E为
在本发明一优选实施方案中，S为
E为
在本发明一优选实施方案中，S为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，L为E为
在本发明一优选实施方案中，S为L为 E为
在本发明一优选实施方案中，S为L为 E为
在本发明一优选实施方案中，S为L为 E为
在本发明一优选实施方案中，S为L为 E为
在本发明一优选实施方案中，S为L为 E为
在本发明一优选实施方案中，S为L为E为
在本发明一优选实施方案中，S为L为 E为
在本发明一优选实施方案中，S为L为E为
在本发明一优选实施方案中，S为L为 E为
在本发明一优选实施方案中，L中A与S相连。
在本发明一优选实施方案中，所述式I所示化合物为如下任一化合物：




本发明还提供一种药物组合物，其包含G和药学上可接受的载体、稀释剂或赋形剂，所述的G为上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药，或其药学上可接受的盐。
本发明提供了一种降解SOS1蛋白的方法，包括使上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物与SOS1蛋白接触。
本发明提供了一种上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物用作治疗或预防SOS1介导的疾病或病症的药物使用。
本发明提供了一种上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物用作治疗或预防由SOS1与Ras(例如KRAS)或者SOS1与Rac(例如KRAS)相互作用所引起的疾病或病症(癌症)的药物的应用。
本发明提供了一种上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物在制备治疗或预防SOS1介导的疾病或病症的药物(例如癌症)中的应用。
本发明提供了上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物在制备治疗或预防由SOS1与Ras(例如KRAS)或者SOS1与Rac(例如KRAS)相互作用所引起的疾病或病症(例如癌症)的药物的应用。
本发明提供了一种上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物在制备治疗或预防癌症药物中的应用。
本发明任一实施方案中，所述的癌症可为胰腺癌、肺癌、结肠直肠癌、胆管上皮癌、多发性骨髓瘤、黑色素瘤、子宫癌、子宫内膜癌、甲状腺癌、急性髓性白血病、膀胱癌、尿路上皮癌、胃癌、子宫颈癌、头颈部鳞状细胞癌、弥漫性大B细胞淋巴瘤、食道癌、慢性淋巴细胞性白血病、肝细胞癌、乳腺癌、卵巢癌、前列腺癌、成胶质细胞瘤、肾癌或肉瘤。
本发明提供了一种上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物在制备治疗或预防胰腺癌、肺癌、结肠直肠癌、胆管上皮癌、多发性骨髓瘤、黑色素瘤、子宫癌、子宫内膜癌、甲状腺癌、急性髓性白血病、膀胱癌、尿路上皮癌、胃癌、子宫颈癌、头颈部鳞状细胞癌、弥漫性大B细胞淋巴瘤、食道癌、慢性淋巴细胞性白血病、肝细胞癌、乳腺癌、卵巢癌、前列腺癌、成胶质细胞瘤、肾癌及肉瘤药物的应用。
本发明提供了一种上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物在制备治疗或预防1型神经纤维瘤病(NF1)、努南综合征(NS)、伴有多雀斑的努南综合征(NSML)、毛细血管畸形-动静脉畸形综合征(CM-AVM)、科斯特洛综合征(CS)、心-面-皮肤综合症(CFC)、莱格斯综合征和遗传性牙龈纤维瘤病药物的应用。
本发明任一实施方案中，所述的SOS1介导的疾病或病症可为癌症、1型神经纤维瘤病(NF1)、努南综合征(NS)、伴有多雀斑的努南综合征(NSML)、毛细血管畸形-动静脉畸形综合征(CM-AVM)、科斯特洛综合征(CS)、心-面-皮肤综合症(CFC)、莱格斯综合征或遗传性牙龈纤维瘤病；其中，所述的癌症可为本发明任一方案所述的癌症。
本发明提供一种治疗或预防由SOS1介导的疾病或病症的方法，包括向有需要的患者施用治疗有效量的上述化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物。
本发明提供一种治疗或预防由SOS1与Ras(例如KRAS)或者SOS1与Rac(例如KRAS)相互作用调控的疾病或病症(例如癌症)的方法，包括向有需要的患者施用治疗有效量的上述化合物，或其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或上述药物组合物。
本发明某些实施方案中，所述的癌症可选自：
心脏:肉瘤(血管肉瘤、纤维肉瘤、横纹肌肉瘤、脂肪肉瘤)、黏液瘤、横纹肌瘤、纤维瘤、脂肪瘤和畸胎瘤组；
肺:支气管癌(鳞状细胞癌、未分化小细胞癌、未分化大细胞癌、腺癌)、肺泡癌(细支气管癌)、支气管腺瘤、肉瘤、淋巴瘤、软骨瘤错构瘤、间皮瘤；
胃肠道食管(鳞状细胞癌、腺癌、平滑肌肉瘤、淋巴瘤)、胃(癌、淋巴瘤、平滑肌肉瘤)、胰腺(导管腺癌、胰岛素瘤、胰高血糖素瘤、胃泌素瘤、类癌肿瘤、血管活性肠肽瘤)、小肠(腺癌、淋巴瘤、类癌肿瘤、卡波西氏肉瘤、平滑肌瘤、血管瘤、脂肪瘤、神经纤维瘤、纤维瘤)、大肠(腺癌、管状腺瘤、绒毛腺瘤、错构瘤、平滑肌瘤)；
泌尿生殖系统:肾(腺癌、肾母细胞瘤、淋巴瘤、白血病)、膀胱和尿道(鳞状细胞癌、移行细胞癌、腺癌)、前列腺(腺癌、肉瘤)、睾丸(精原细胞瘤、畸胎瘤、胚胎癌、畸胎瘤、绒毛膜癌、肉瘤、间质细胞癌、纤维瘤、纤维腺瘤、腺瘤样瘤、脂肪瘤)；
肝:肝癌(肝细胞癌)、胆管癌、肝母细胞瘤、血管肉瘤、肝细胞腺瘤、血管瘤；
胆道:胆囊癌、壶腹癌、胆管癌；
骨:骨肉瘤(骨肉瘤)、纤维肉瘤、恶性纤维组织细胞瘤、软骨肉瘤、尤因氏肉瘤、恶性淋巴瘤(网状细胞肉瘤)、多发性骨髓瘤、恶性巨细胞瘤脊索瘤、软骨瘤(骨软骨外生骨病)、良性软骨瘤、成软骨瘤、软骨黏液纤维瘤、骨样骨瘤和巨细胞瘤；
神经系统:颅骨(骨瘤、血管瘤、肉芽肿、黄色瘤、畸形骨炎)、脑膜(脑膜瘤、脑膜肉瘤、胶质瘤)、大脑(星形细胞瘤、髓母细胞瘤、胶质瘤、室管膜瘤、生发细胞瘤(松果体瘤)、多形胶质母细胞瘤、少突胶质母细胞瘤、神经鞘瘤、视网膜母细胞瘤、先天性肿瘤)、脊髓神经纤维瘤、脑膜瘤、胶质瘤、肉瘤)；
妇科:子宫(子宫内膜癌(浆液性囊腺癌、粘液性囊腺癌、未分类癌)、颗粒-鞘膜细胞瘤、支持间质细胞瘤、无生殖细胞瘤、恶性畸胎瘤)、外阴(鳞状细胞癌、上皮内癌、腺癌、纤维肉瘤、黑素瘤)、阴道(透明细胞癌、鳞状细胞癌、葡萄状肉瘤(胚胎型横纹肌肉瘤))、输卵管(癌)；
血液学:血液(髓系白血病(急性和慢性)、急性淋巴母细胞白血病、慢性淋巴细胞白血病、骨髓增生性疾病、多发性骨髓瘤、骨髓增生异常综合征)、霍奇金病、非霍奇金淋巴瘤(恶性淋巴瘤)；
皮肤:恶性黑色素瘤、基底细胞癌、鳞状细胞癌、卡波西肉瘤、痣发育不良痣、脂肪瘤、血管瘤、皮肤纤维瘤、瘢痕疙瘩、银屑病；
肾上腺:成神经细胞瘤。
本发明某些实施方案中，所述的癌症可选自胰腺癌、肺癌、结肠直肠癌、胆管上皮癌、多发性骨髓瘤、黑色素瘤、子宫癌、子宫内膜癌、甲状腺癌、急性髓性白血病、膀胱癌、尿路上皮癌、胃癌、子宫颈癌、头颈部鳞状细胞癌、弥漫性大B细胞淋巴瘤、食道癌、慢性淋巴细胞性白血病、肝细胞癌、乳腺癌、卵巢癌、前列腺癌、成胶质细胞瘤、肾癌及肉瘤。
本发明还提供了一种上述化合物，或其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药，或其药学上可接受的盐或前述的药物组合物在制备用于治疗和/或预防KRAS介导的疾病或病症的药物中的应用。
本发明还提供了一种治疗和/或预防KRAS介导的疾病或病症的方法，包括向有需要的患者施用治疗有效量的上述化合物，或其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或其药物组合物。
本发明某些实施方案中，所述的KRAS可为突变型KRAS。所述突变型KRAS优选为KRAS G12C、KRAS G12D、KRAS G13D和KRAS G12V中的一种或多种。
详细说明：除非有相反陈述、下列用在说明书和权利要求书中的术语具有下述含义。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物、以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药、利于活性成分的吸收进而发挥生物活性。
本发明还提供式(I)化合物药学上可接受的盐。术语“药学上可接受的盐”是指相对无毒的本发明化合物的酸加成盐或碱加成盐。所述酸加成盐为本发明式(I)化合物与合适的无机酸或者有机酸形成的盐，这些盐可在化合物最后的分离和提纯过程中制备，或者可用纯化的式(I)化合物以其游离碱形式与适宜的有机酸或无机酸进行反应来制备。代表性酸加成盐包括盐酸盐、氢溴酸盐、硫酸盐、硫酸氢盐、亚硫酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月硅酸盐、硼酸盐、苯甲酸盐、乳酸盐、磷酸盐、磷酸氢盐、碳酸盐、碳酸氢盐、甲苯甲酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、苯甲酸盐、酒石酸盐、甲磺酸盐、对甲苯磺酸盐、葡萄糖酸盐、乳糖酸盐和月桂基磺酸盐等。所述碱加成盐为式(I)化合物与合适的无机碱或者有机碱形成的盐，包括例如与碱金属、碱土金属、季铵阳离子形成的盐，例如钠盐、锂盐、钾盐、钙盐、镁盐、四甲基季铵盐、四乙基季铵盐等；胺盐包括与氨(NH3)、伯胺、仲胺或叔胺形成的盐，如甲胺盐、二甲胺盐、三甲胺盐、三乙胺盐、乙胺盐等。
本发明的化合物或其药学上可接受的盐可给药于哺乳动物包括人，可口服、直肠、肠胃外(静脉内、肌肉内或皮下)、局部给药(粉剂、软膏剂或滴剂)、或瘤内给药。
本发明化合物的给药剂量可以大约为0.05-300mg/kg体重/天，优选10-300mg/kg体重/天，更优选10-200mg/kg体重/天。
本发明化合物或其药学上可接受的盐可以配制为用于口服给药的固体剂型，包括但不限于胶囊剂、片剂、丸剂、散剂和颗粒剂等。在这些固体剂型中，本发明式(I)化合物作为活性成分与至少一种常规惰性赋形剂(或载体)混合，例如与柠檬酸钠或磷酸二钙，或与下述成分混合：(1)填料或增容剂，如淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸等；(2)粘合剂，如羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶等；(3)保湿剂，如甘油等；(4)崩解剂，如琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐和碳酸钠等；(5)缓溶剂，如石蜡等；(6)吸收加速剂，如季铵化合物等；(7)润湿剂如鲸蜡醇和单硬脂酸甘油酯等；(8)吸附剂，如高岭土等；和(9)润滑剂，如滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠等，或其混合物。胶囊剂、片剂和丸剂中也可包含缓冲剂。
所述固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材料例如肠溶衣和其他本领域公知的材料进行包衣或微囊化。它们可包含不透明剂，并且这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时、活性成分也可与上述赋形剂中的一种或多种形成微胶囊形式。
本发明化合物或其药学上可接受的盐可以配制为用于口服给药的液体剂型，包括但不限于药学上可接受的乳液、溶液、悬浮液、糖浆和酊剂等。除了作为活性成分的式(I)化合物或其药学上可接受的盐外，液体剂型可包含本领域中常规采用的惰性稀释剂如水和其他溶剂、增溶剂和乳化剂，如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油类，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油等或这些物质的混合物等。除了这些惰性稀释剂外，本发明液体剂型也可包含常规助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料等。
所述悬浮剂包括如乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇只、微晶纤维素、甲醇铝和琼脂等或这些物质的混合物。
本发明化合物或其药学上可接受的盐可以配制为用于肠胃外注射的剂型包括但不限于生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，以及用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明化合物或其药学上可接受的盐也可以配制为用于局部给药的剂型包括如软膏剂、散剂、栓剂、滴剂、喷射剂和吸入剂等。作为活性成分的本发明式(I)化合物或其药学上可接受的盐在无菌条件下和生理上可接受的载体及任选的防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明还提供药物组合物，它含有本发明式(I)化合物或其药学上可接受的盐作为活性成分，以及药学上可接受载体、赋形剂或稀释剂。在制备药物组合物时，通常是将本发明式(I)化合物或其药学上可接受的盐与药学上可接受载体、赋形剂或稀释剂混合。
可以按常规制备方法将所述本发明组合物配制为常规药物制剂。例如片剂、丸剂、胶囊剂、散剂、颗粒剂、乳液剂、混浮剂、分散液、溶液剂、糖浆剂、酏剂、软膏剂、滴剂、栓剂、吸入剂、喷射剂等。
本发明所述的化合物或其药学上可接受的盐可以单独给药，或者(如果需要)与其他药学上可接受的治疗剂联合给药，如与其他抗肿瘤药物。待组合的各成分可同时或顺序的给予，以单一制剂形式或以不同制剂的形式给予。所述组合不仅可包括本发明化合物和一种其他活性剂的组合，也可包括本发明化合物和两种或更多种其他活性剂的组合。
本发明中，可与SOS1降解剂式(I)化合物一起或组合使用的其他药学上可接受的治疗剂可为：EGFR和/或其突变体抑制剂、ErbB2(Her2)和/或其突变体抑制剂、ALK和/或其突变体抑制剂、MEK和/或其突变体抑制剂、Kras和/或其突变体抑制剂、BCR-ABL和/或其突变体抑制剂、FGFR1/FGFR2/FGFR3和/或其突变体抑制剂、ROS1和/或其突变体抑制剂、c-MET和/或其突变体抑制剂、AXL和/或其突变体抑制剂、NTRK1和/或其突变体抑制剂、RET和/或其突变体抑制剂、紫杉烷、含铂化合物、抗代谢物、有丝分裂激酶抑制剂、免疫治疗剂、抗血管生成药物、拓扑异构酶抑制剂、A-Raf/B-Raf/C-RAf和/或其突变体抑制剂、ERK和/或其突变体抑制剂、细胞凋亡抑制剂、mTOR抑制剂、外遗传调控剂、IGF1/2和/或IGF1-R抑制剂、Ras GEF和/或其突变体抑制剂、PI3K和/或其突变体抑制剂。
本发明中，可与SOS1降解剂式(I)化合物一起或组合使用的其他药学上可接受的治疗剂可为：阿法替尼(afatinib)、厄洛替尼(erlotinib)、吉非替尼(gefitinib)、拉帕替尼(lapatinib)、西妥昔单抗、帕尼单抗、奥希替尼、奥莫替尼(olmutinib)、EGF-816、、曲妥珠单抗(trastuzumab)、帕妥珠单抗(pertuzumab)、唑替尼(crizotinib)、阿雷替尼(alectinib)、恩曲替尼(entrectinib)、布吉替尼(brigatinib)、曲美替尼(trametinib)、考比替尼(cobimetinib)、贝美替尼(binimetinib)、司美替尼(selumetinib)、瑞法替尼(refametinib)、伊马替尼(imatinib)、达沙替尼(dasatinib)、尼罗替尼(nilotinib)、尼达尼布(nintedanib)、克唑替尼、劳拉替尼(lorlatinib)、色瑞替尼(ceritinib)、美乐替尼(merestinib)、紫杉醇(paclitaxel)、nab-紫杉醇、多西他赛(docetaxel)、顺铂(cisplatin)、卡铂(carboplatin)、奥沙利铂(oxaliplatin)、5-氟尿嘧啶、卡培他滨(capecitabine)、氟尿苷、阿糖胞苷(cytarabine)、吉西他滨(gemcitabine)、曲氟尿苷(trifluridine)及替比嘧啶(tipiracil)的组合(＝TAS102)、帕博西尼(palbociclib)、瑞博西尼(ribociclib)、阿贝西尼(abemaciclib)、伊匹单抗(ipilimumab)、纳武单抗(nivolumab)、帕博利珠单抗(pembrolizumab)、阿替珠单抗(atezolizumab)、阿维鲁单抗(avelumab)、德瓦鲁单抗(durvalumab)、匹利珠单抗(pidilizumab)、PDR-001(＝斯帕珠单抗(spartalizumab))、贝伐珠单抗(bevacizumab)、伊立替康(irinotecan)、脂质体伊立替康、托泊替康(topotecan)、乌利替尼(ulixertinib)、雷帕霉素、替西罗莫司(temsirolimus)、依维莫司(everolimus)、利达莫司(ridaforolimus)、JQ-1、GSK525762、OTX 015(＝MK8628)、CPI 0610、TEN-010(＝RO6870810)、珍妥珠单抗(xentuzumab)(WO2010/066868中的抗体60833)或MEDI-573(＝杜昔妥单抗(dusigitumab))。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明通过SOS1激酶活性测试实验证明本发明所述式Ⅰ化合物能够有效的与SOS1靶蛋白结合或产生抑制效果，进一步地，能够有效地降解SOS1蛋白，从而达到预防或治疗与SOS1相关或与SOS1与Ras或者SOS1与Rac相互作用所引起的疾病或病症的效果。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
中间体1：1-(2-氯-5-(4-((2-(哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮
步骤1：4-(2-((1-(叔-丁氧羰基)哌啶-4-基)甲氧基-d2)乙基)哌啶-1-羧酸苄酯的制备
将4-(羟基甲基-d2)哌啶-1-羧酸叔丁酯(600mg,2.76mmol)用无水THF(20mL)溶解，0℃下加入NaH(60％,133mg,3.31mmol)并搅拌1小时，加入4-(2-(甲苯磺酰)乙基)哌啶-1-羧酸苄酯(1.73g,4.14mmol)，反应液升温至65℃并搅拌过夜。向反应液中缓慢加入水(30mL)，用乙酸乙酯(50mL x 3)萃取，有机相用饱和食盐水(100mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。粗品经硅胶柱层析(乙酸乙酯:石油醚＝1:3)纯化得到4-(2-((1-(叔-丁氧羰基)哌啶-4-基)甲氧基-d2)乙基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝463.2.
步骤2：4-(2-(哌啶-4-基甲氧基-d2)乙基)哌啶-1-羧酸苄酯的制备
将4-(2-((1-(叔-丁氧羰基)哌啶-4-基)甲氧基-d2)乙基)哌啶-1-羧酸苄酯(350mg,0.758mmol)用二氯甲烷(10mL)溶解，然后加入三氟乙酸(2mL)并室温搅拌2小时，反应液直接减压浓缩得到4-(2-(哌啶-4-基甲氧基-d2)乙基)哌啶-1-羧酸苄酯，无需纯化直接用于下步反应。
LC-MS:(ESI,m/z):[M+H]+＝363.1.
步骤3：4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基-d2)乙基)哌啶-1-羧酸苄酯的制备
将4-(2-(哌啶-4-基甲氧基-d2)乙基)哌啶-1-羧酸苄酯(350mg,0.758mmol)用二甲亚砜(5mL)溶解，加入4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(330mg,0.758mmol)和DIEA(985mg,7.58mmol)，反应液室温搅拌2小时。加水(50mL)稀释，用二氯甲烷(30mL x 3)萃取，经无水硫酸钠干燥，过滤，减压浓缩，粗品将硅胶柱层析(乙酸乙酯)纯化得到4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基-d2)乙基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝613.2.
步骤4：1-(2-氯-5-(4-((2-(哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基-d2)乙基)哌啶-1-羧酸苄酯(300mg,0.489mmol)加入到三氟乙酸(5mL)中，反应液升温至90℃并搅拌3小时，直接减压浓缩，残余物用二氯甲烷(30mL)溶解，然后缓慢加入饱和碳酸氢钠溶液直至溶液pH＝8，分离有机相，水相用二氯甲烷/甲醇溶液(10/1,40mL x 5)萃取，合并有机相，经无水硫酸钠干燥，过滤，减压浓缩，得到1-(2-氯-5-(4-((2-(哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮，无需纯化直接用于下步反应。
LC-MS:(ESI,m/z):[M+H]+＝479.1.
实施例1：(1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)-N-(3-(3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)丙基)-N-甲基环己烷-1-甲酰胺
步骤1：(E)-9-(3-乙氧基-3-氧代丙-1-烯-1-基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将磷酰基乙酸(1.27g,5.7mmol)溶于THF(50mL)中，0℃下加入NaH(342mg,8.55mmol,60％)，搅拌2h，然后加入9-醛基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(800mg,2.85mmol)，继续室温搅拌2h，反应液中加水(100mL)稀释，乙酸乙酯(100mL x 2)萃取，无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(乙酸乙酯:石油醚＝1:10)纯化得到(E)-9-(3-乙氧基-3-氧代丙-1-烯-1-基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
1H NMR(400MHz,DMSO-d6)δ6.86(dd,J＝15.8,6.9Hz,1H),5.80(dd,J＝15.8,1.1Hz,1H),4.15-4.02(m,2H),3.30-3.22(m,4H),2.23-2.05(m,1H),1.72-1.60(m,2H),1.57-1.49(m,2H),1.46-1.34(m,13H),1.32(s,1H),1.28(s,1H),1.25-1.19(m,5H).
步骤2：9-(3-乙氧基-3-氧代丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将(E)-9-(3-乙氧基-3-氧代丙-1-烯-1-基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(740mg,2.1mmol)溶于EtOH(30mL)中，氢气气氛下加入Pd/C(100mg)，反应液室温搅拌1h，硅藻土过滤，滤液直接减压浓缩得到9-(3-乙氧基-3-氧代丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
1H NMR(400MHz,DMSO-d6)δ4.05(dq,J＝14.2,7.2Hz,2H),3.30-3.20(m,4H),2.27(t,J＝7.7Hz,2H),1.66-1.55(m,2H),1.51-1.41(m,4H),1.40-1.32(m,11H),1.25-1.12(m,6H),1.08-0.97(m,4H).
步骤3：9-(3-羟基丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将9-(3-乙氧基-3-氧代丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(700mg,1.98mmol)加入到THF(30mL)中，0℃逐滴加入LiBH4(174mg,7.92mmol)的THF溶液，反应液加热到80℃并搅拌16h。反应液加水(40mL)稀释，乙酸乙酯(40mL x 3)萃取，有机相用饱和食盐水(40mL)洗，无水硫酸钠干燥，减压浓缩，粗品经硅胶柱层析(PE:EA＝4:1)纯化得到9-(3-羟基丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
1H NMR(400MHz,DMSO-d6)δ4.32(t,J＝5.1Hz,1H),3.37-3.32(m,2H),3.29-3.15(m,4H),1.69-1.62(m,2H),1.54-1.32(m,15H),1.26-1.10(m,6H),1.09-0.98(m,3H).
步骤4：9-(3-氧代丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将9-(3-羟基丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(400mg,1.29mmol)和PCC(660mg,2.58mmol)加入到EA(20mL)中，室温搅拌2h，硅藻土过滤，滤液直接减压浓缩，粗品经硅胶柱层析(PE:EA＝5:1)纯化得到9-(3-氧代丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
1H NMR(400MHz,DMSO-d6)δ9.66(s,1H),3.30-3.19(m,4H),2.46-2.35(m,2H),1.66-1.57(m,2H),1.49-1.42(m,4H),1.42-1.33(m,11H),1.22-1.17(m,3H),1.06-0.99(m,4H).
步骤5：9-(3-(苄基(甲基)氨基)丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将9-(3-氧代丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(200mg,0.65mmol)，N-甲基-1-苯基甲胺(95mg,0.78mmol)和STAB(413.4mg,1.95mmol)加入到THF(30mL)中，反应液在60℃条件下搅拌2h。反应液加水(40mL)稀释，用乙酸乙酯(40mL x 3)萃取，有机相用饱和食盐水(40mL)洗涤，无水硫酸钠干燥，减压浓缩，粗品经硅胶柱层析(PE:EA＝10:1)纯化得到9-(3-(苄基(甲基)氨基)丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝415.3.
步骤6：9-(3-(甲基氨基)丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将9-(3-(苄基(甲基)氨基)丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(180mg,0.43mmol)加入到MeOH(20mL)中，氢气气氛下，加入Pd(OH)2/C(60mg),室温搅拌2h，硅藻土过滤，滤液直接减压浓缩，得到9-(3-(甲基氨基)丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝325.3.
步骤7：9-(3-((1R,4R)-4-(4-(((R)-1-(3-((叔-丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑林-6-基)-N-甲基环己烷-1-羧酰氨基)丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将(1R,4R)-4-(4-(((R)-1-(3-((叔-丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸(200mg,0.33mmol)，HATU(152mg,0.40mmol)和DIEA(128mg,0.99mmol)加入DMF(30mL)中，室温搅拌10min，再加入9-(3-(甲基氨基)丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(130mg,0.40mmol)，继续搅拌1小时。反应液用水(40mL)稀释，乙酸乙酯(40mL x 3)萃取，有机相用饱和食盐水(40mL)洗涤，无水硫酸钠干燥，减压浓缩，粗品经硅胶柱层析(PE:EA＝10:1)纯化得到9-(3-((1R,4R)-4-(4-(((R)-1-(3-((叔-丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑林-6-基)-N-甲基环己烷-1-羧酰氨基)丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝909.5.
步骤8：(1R,4R)-N-(3-(3-氮杂螺[5.5]十一烷-9-基)丙基)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)-N-甲基环己烷-1-甲酰胺的制备
将9-(3-((1R,4R)-4-(4-(((R)-1-(3-((叔-丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑林-6-基)-N-甲基环己烷-1-羧酰氨基)丙基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(170mg,0.19mmol)加入到HCl/二氧六环(4M,5mL)溶液中，室温搅拌2h，直接减压浓缩得到(1R,4R)-N-(3-(3-氮杂螺[5.5]十一烷-9-基)丙基)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)-N-甲基环己烷-1-甲酰胺，直接用于下步反应。
步骤9：(1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)-N-(3-(3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)丙基)-N-甲基环己烷-1-甲酰胺的制备
将4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(73.6mg,0.17mmol)，DIEA(65.8mg,0.51mmol)和(1R,4R)-N-(3-(3-氮杂螺[5.5]十一烷-9-基)丙基)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)-N-甲基环己烷-1-甲酰胺(120mg,0.17mmol)加入到DMSO(10mL)中，反应液室温搅拌30min，用水(20mL)稀释，乙酸乙酯(20mL x 3)萃取，有机相用饱和食盐水(20mL)洗涤，无水硫酸钠干燥，减压浓缩，粗品使用Prep-HPLC纯化得到(1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)-N-(3-(3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)丙基)-N-甲基环己烷-1-甲酰胺。
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),8.12(s,1H),8.05(d,J＝6.8Hz,1H),7.63(d,J＝8.2Hz,1H),7.54(s,1H),7.38(d,J＝5.5Hz,1H),6.99(s,1H),6.88(s,1H),6.85(s,1H),6.70(s,1H),5.66-5.40(m,3H),3.89(s,3H),3.79-3.70(m,1H),3.66-3.46(m,4H),3.28-3.22(m,2H),3.10-2.86(m,4H),2.90-2.55(m,5H),2.36(s,3H),1.9-1.75(m,4H),1.72-1.39(m,14H),1.35-0.96(m,9H).
实施例2：1-(2-氯-5-(4-((2-(1-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮
步骤1：4-(2-(吡啶-4-基甲氧基)乙基)哌啶-1-羧酸叔丁酯的制备
将4-(2-羟乙基)哌啶-1-甲酸叔丁酯(2.00g,8.72mmol)溶于DMF(25mL)溶液中，冷却至0-5℃。分批加入NaH(1.74g,43.6mmol,60％)，然后室温搅拌30分钟，将混合物在0-5℃搅拌30分钟，然后加入4-(溴甲基)吡啶氢溴酸盐(2.64g,10.46mmol)并在室温搅拌3h。混合物用NH4Cl(100mL)水溶液稀释并用乙酸乙酯(100mL x 3)萃取。将合并的有机相用食盐水(100mL)洗涤，经硫酸钠干燥，过滤并减压浓缩，所得粗品经硅胶柱层析(PE:EtOAc＝50:1～1:1)纯化，得到4-(2-(吡啶-4-基甲氧基)乙基)哌啶-1-羧酸叔丁酯。
步骤2：4-(2-(哌啶-4-基甲氧基)乙基)哌啶-1-羧酸叔丁酯的制备
向4-(2-(吡啶-4-基甲氧基)乙基)哌啶-1-羧酸叔丁酯(1.2g,3.74mmol)的i-PrOH/H2O(20mL/20mL)溶液中加入Pd/C(150mg)，在氢气球保护下65℃下反应过夜。反应液经硅藻土过滤，滤液浓缩，得到4-(2-(哌啶-4-基甲氧基)乙基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝327.2.
步骤3：4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基)乙基)哌啶-1-羧酸叔丁酯的制备
向4-(2-(哌啶-4-基甲氧基)乙基)哌啶-1-羧酸叔丁酯(200mg,0.61mmol)的DMSO(2mL)溶液中加入4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(280mg,1.64mmol)和DIEA(237mg,1.83mmol)，反应液在室温下搅拌2小时。反应液中加入水(20mL)，用乙酸乙酯(20mL x 2)萃取，合并有机相并用饱和盐水(30mL x 3)洗涤，经无水硫酸钠干燥。过滤，滤液浓缩并通过硅胶柱层析(DCM:MeOH＝100:1～10:1)纯化，得到4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基)乙基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+Na]+＝599.2.
步骤4：1-(2-氯-5-(4-((2-(哌啶-4-基)乙氧基)甲基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基)乙基)哌啶-1-羧酸叔丁酯(250mg,粗品)溶于DCM(4.5mL)中，滴加TFA(1.5mL)。将反应液在室温搅拌2小时并浓缩得到1-(2-氯-5-(4-((2-(哌啶-4-基)乙氧基)甲基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮，直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝477.1.
步骤5：(2-(5-((R)-1-((6-((1R,4R)-4-(4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基)乙基)哌啶-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯的制备
将1-(2-氯-5-(4-((2-(哌啶-4-基)乙氧基)甲基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(150mg,0.27mmol)溶于DMSO(4mL)中，加入(1R,4R)-4-(4-(((R)-1-(4-(2-(((叔-丁氧基羰基)(甲基)氨基))甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-甲酸五氟苯酯(232mg,0.28mmol)和DIEA(105mg,0.81mmol)。将反应液在室温搅拌2小时。反应液中加入水(20mL)，然后用乙酸乙酯(20mL x 2)萃取，合并有机相并用饱和盐水(30mL x 3)洗涤，经无水硫酸钠干燥。过滤，滤液浓缩，所得粗品经硅胶柱层析(DCM:MeOH＝100:1～5:1)纯化，得到(2-(5-((R)-1-((6-((1R,4R)-4-(4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基)乙基)哌啶-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[(M-Boc)/2+H]+＝502.2.
步骤6：1-(2-氯-5-(4-((2-(1-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
向(2-(5-((R)-1-((6-((1R,4R)-4-(4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基)乙基)哌啶-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯(130mg,粗品)的DCM(3mL)溶液中加TFA(1mL)。将反应液室温搅拌2小时，反应液直接减压浓缩，所得粗品经pre-HPLC纯化得到1-(2-氯-5-(4-((2-(1-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M/2+H]+＝502.2.
1H NMR(400MHz,CD3OD)δ7.99(s,1H),7.67–7.59(m,1H),7.55–7.26(m,6H),7.22–7.11(m,2H),7.01(s,1H),6.06(q,J＝6.5Hz,1H),4.66–4.49(m,2H),4.08–3.92(m,4H),3.79–3.68(m,4H),3.49(t,J＝6.1Hz,2H),3.18–3.01(m,3H),2.91–2.48(m,8H),2.21(s,3H),2.01–1.49(m,20H),1.38–1.11(m,6H).
实施例3：2-(2,6-二氧代哌啶-3-基)-5-(4-((2-(1-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基)哌啶-1-基)异吲哚啉-1,3-二酮
步骤1：4-(2-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)哌啶-4-基)甲氧基)乙基)哌啶-1-羧酸叔丁酯的制备
将4-(2-(哌啶-4-基甲氧基)乙基)哌啶-1-甲酸叔丁酯(200mg,0.61mmol)溶于DMSO(4mL)中，加入2-(2,6-二氧代哌啶)-3-基)-5-氟异二氢吲哚-1,3-二酮(186mg,0.67mmol)和DIEA(237mg,1.83mmol)。将混合物在70℃反应16小时。冷却至室温，反应液中加入水(20mL)，用乙酸乙酯(20mL x 2)萃取。合并有机相并用饱和氯化钠水溶液(30mL x 3)洗涤，无水硫酸钠干燥。过滤，滤液浓缩，所得粗品经硅胶柱层析(DCM:MeOH＝100:1～20:1)纯化，得到4-(2-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)哌啶-4-基)甲氧基)乙基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M-Boc+H]+＝483.1.
步骤2：2-(2,6-二氧代哌啶-3-基)-5-(4-((2-(哌啶-4-基)乙氧基)甲基)哌啶-1-基)异吲哚啉-1,3-二酮的制备
将4-(2-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)哌啶-4-基)甲氧基)乙基)哌啶-1-羧酸叔丁酯(240mg,粗品)溶于DCM(4.5mL)，加入TFA(1.5mL)，所得反应液在室温搅拌2小时。反应结束，将反应液浓缩得到2-(2,6-二氧代哌啶-3-基)-5-(4-((2-(哌啶-4-基)乙氧基)甲基)哌啶-1-基)异吲哚啉-1,3-二酮。
LC-MS:(ESI,m/z):[M+H]+＝483.2.
步骤3：(2-(5-((1R)-1-((6-((1R,4R)-4-(4-(2-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)哌啶-4-基)甲氧基)乙基)哌啶-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯的制备
向2-(2,6-二氧代哌啶-3-基)-5-(4-((2-(哌啶-4-基)乙氧基)甲基)哌啶-1-基)异吲哚啉-1,3-二酮(150mg,0.31mmol)的DMSO(4mL)溶液中加入(1R,4R)-4-(4-(((R)-1-(4-(2-(((叔丁氧基羰基)(甲基)氨基))甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸五氟苯酯(285mg,0.35mmol)和DIEA(136mg,1.05mmol)。将混合物在室温搅拌2小时。反应液中加入水(20mL)，用乙酸乙酯(20mL x 2)萃取，合并有机相并用饱和盐水(30mL x 3)洗涤，经无水硫酸钠干燥。过滤，滤液浓缩，所得粗品经硅胶柱层析(DCM:MeOH＝100:1～5:1)纯化，得到(2-(5-((1R)-1-((6-((1R,4R)-4-(4-(2-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)哌啶-4-基)甲氧基)乙基)哌啶-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M/2+H]+＝555.3.
步骤4：2-(2,6-二氧代哌啶-3-基)-5-(4-((2-(1-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基)哌啶-1-基)异吲哚啉-1,3-二酮的制备
将(2-(5-((1R)-1-((6-((1R,4R)-4-(4-(2-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)哌啶-4-基)甲氧基)乙基)哌啶-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯(180mg,粗品)溶于DCM(1mL)中，加入TFA(0.5mL)。将混合物在室温搅拌2小时，浓缩，所得粗品经pre-HPLC纯化得到2-(2,6-二氧代哌啶-3-基)-5-(4-((2-(1-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基)哌啶-1-基)异吲哚啉-1,3-二酮。
LC-MS:(ESI,m/z):[M+H]+＝1009.3.
1H NMR(400MHz,CD3OD)δ8.12(s,1H),7.67–7.60(m,1H),7.57–7.52(m,1H),7.46–7.38(m,3H),7.32(t,J＝2.2Hz,1H),7.27(s,1H),7.22–7.16(m,2H),7.03(s,1H),6.11(q,J＝6.8Hz,1H),4.64–4.46(m,4H),4.21(s,2H),4.05(d,J＝13.2Hz,2H),3.97(s,3H),3.59–3.54(m,2H),3.23–2.93(m,6H),2.83–2.65(m,4H),2.59–2.50(m,6H),1.94–1.80(m,10H),1.73–1.66(m,4H),1.57–1.54(m,2H),1.41–1.33(m,8H).
实施例4：1-(2-氯-5-(4-(4-(4-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)正丁基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮
步骤1：4-(吡啶-4-基)丁醛的制备
将4-(吡啶-4-基)丁烷-1-醇(2.50g,16.55mmol)溶于DCM(50mL)中，冷却至0-10℃，分批加入戴斯-马丁氧化剂(8.42g,19.86mmol)。室温反应3小时，加入硫代硫酸钠水溶液和碳酸氢钠水溶液淬灭，用DCM(30mL x 2)萃取。有机相用饱和NaCl溶液洗涤，无水Na2SO4干燥，过滤，滤液浓缩，所得粗品经硅胶柱层析(DCM:MeOH＝50:1)纯化得到4-(吡啶-4-基)丁醛。
1H NMR(400MHz,CDCl3)δ9.79(s,1H),8.52(d,J＝5.6Hz,2H),7.13(d,J＝5.6Hz,2H),2.66(t,J＝7.2Hz,2H),2.50(t,J＝7.2Hz,2H),2.03–1.94(m,2H).
步骤2：4-(4-(吡啶-4-基)正丁基)哌嗪-1-羧酸叔丁酯的制备
向4-(吡啶-4-基)丁醛(1.60g,10.73mmol)和哌嗪-1-羧酸叔丁酯(3.00g,16.10mmol)的1,2-二氯乙烷/甲醇(28mL/14mL)的混合液中加入乙酸(3mL)。将混合物在30℃下搅拌1小时。0℃下加入NaBH(OAc)3(4.55g,21.48mmol)，室温反应16小时。反应完毕，反应液中加入H2O(10mL)，用乙酸乙酯(30ml x 3)萃取。将合并的有机相用无水硫酸钠干燥，过滤并在真空下浓缩。粗品将硅胶柱层析(DCM:MeOH＝20:1)纯化，得到4-(4-(吡啶-4-基)正丁基)哌嗪-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝320.2.
步骤3：4-(4-(哌啶-4-基)正丁基)哌嗪-1-羧酸叔丁酯的制备
向4-(4-(吡啶-4-基)正丁基)哌嗪-1-羧酸叔丁酯(1.10g,3.44mmol)的i-PrOH/H2O(20mL/20mL)溶液中加入Pd/C(150mg)在氢气球保护下75℃下反应16小时。反应液经硅藻土过滤，滤液浓缩得到4-(4-(哌啶-4-基)正丁基)哌嗪-1-羧酸叔丁酯，直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝326.3.
步骤4：4-(4-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)正丁基)哌嗪-1-羧酸叔丁酯的制备
向4-(4-(哌啶-4-基)正丁基)哌嗪-1-羧酸叔丁酯(300mg,0.92mmol)的DMSO(6mL)溶液中加入4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(401mg,0.92mmol)和DIEA(238mg,1.84mmol)，反应液在室温下搅拌2小时。反应液中加入水(20mL)，用乙酸乙酯(20mL x 2)萃取，合并有机相并用饱和盐水(30mL x 3)洗涤，经无水硫酸钠干燥，过滤，滤液浓缩，所得粗品经硅胶柱层析(DCM:MeOH＝100:1～10:1)纯化，得到4-(4-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)正丁基)哌嗪-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝576.0.
步骤5：1-(2-氯-5-(4-(4-(哌嗪-1-基)正丁基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将4-(4-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)正丁基)哌嗪-1-羧酸叔丁酯(150mg,0.315mmol)溶于DCM(3mL)中，然后滴加TFA(1.5mL)。将反应液在室温搅拌1小时并浓缩得到1-(2-氯-5-(4-(4-(哌嗪-1-基)正丁基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮，直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝476.0.
步骤6：(2-(5-((R)-1-((6-((1R,4R)-4-(4-(4-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)正丁基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯的制备
将1-(2-氯-5-(4-(4-(哌嗪-1-基)正丁基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(280mg,粗品)溶于DMSO(4mL)中，加入(1R,4R)-4-(4-((R)-1-(4-(2-((叔丁氧羰基)(甲基)氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸酯(255mg,0.315mmol)和DIEA(203mg,1.575mmol)。将反应液在室温搅拌2小时。反应液中加入水(20mL)，用乙酸乙酯(20mL x 2)萃取，合并有机相并用饱和盐水(30mL x 3)洗涤，经无水硫酸钠干燥。过滤，滤液浓缩，所得粗品经硅胶柱层析(DCM:MeOH＝100:1～5:1)纯化，得到(2-(5-((R)-1-((6-((1R,4R)-4-(4-(4-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)正丁基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝1102.5.
步骤7：1-(2-氯-5-(4-(4-(4-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)正丁基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
向(2-(5-((R)-1-((6-((1R,4R)-4-(4-(4-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)正丁基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯(140mg,0.13mmol)的二氯甲烷(2.0mL)溶液中加入三氟乙酸(1.0mL)，于30℃下搅拌1小时。反应完成后，减压浓缩。粗品用pre-HPLC纯化，得到1-(2-氯-5-(4-(4-(4-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)正丁基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝1002.3.
1H NMR(400MHz,CDCl3)δ7.54–7.49(m,2H),7.41–7.29(m,6H),7.22–7.12(m,3H),6.50-6.23(m,1H),6.09–6.03(m,1H),4.75–4.52(s,1H),3.91(s,3H),3.85(s,2H),3.78–3.45(m,8H),3.08–2.31(m,24H),2.00–1.83(m,4H),1.59–1.42(m,5H),1.37–0.89(m,8H).
实施例5：3-(4-(9-((4-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)哌啶-2,6-二酮
步骤1：4-(2,6-二(苄氧基)吡啶-3-基)苯甲酸的制备
向2,6-双(苄氧基)-3-溴吡啶(2.80g,7.57mmol)和4-(4,4,5,5-四甲基-1,3,2-二氧苯并呋喃-2-基)苯甲酸(1.87g,11.35mmol)的二氧六环/H2O(50mL/10ml)的混合溶液中加入Pd(dppf)Cl2(262mg,0.36mmol)和碳酸钠(2.01g,18.92mmol)。混合物在氮气保护下80℃反应16小时，冷却至室温，加入1N HCl水溶液调节pH＝2，乙酸乙酯(30mL x 3)萃取，合并有机相并用无水硫酸钠干燥后减压浓缩。所得粗品经硅胶柱层析(DCM:MeOH＝100:1～2:1)纯化，得到4-(2,6-二(苄氧基)吡啶-3-基)苯甲酸。
LC-MS:(ESI,m/z):[M+H]+＝412.1.
步骤2：4-(2,6-二氧代哌啶-3-基)苯甲酸的制备
向4-(2,6-二(苄氧基)吡啶-3-基)苯甲酸(0.50g,1.22mmol)的i-PrOH(20mL)溶液中加入Pd/C(0.2mg)，在氢气球保护下40℃下反应16小时。反应液经硅藻土过滤，滤液浓缩得4-(2,6-二氧代哌啶-3-基)苯甲酸，直接用于下一步反应。
LC-MS:(ESI,m/z):[M-H]-＝231.9.
步骤3：4-((3-(4-(2,6-二氧代哌啶-3-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羧酸苄酯的制备
将4-(2,6-二氧代哌啶-3-基)苯甲酸(200m g,0.86mmol)和HATU(400mg,1.03mmol)溶于DMF(6mL)中，搅拌3分钟，依次加入DIEA(220mg,0.72mmol)和4-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羧酸苄酯(320mg,0.86mmol)，反应液在25℃下搅拌1小时。反应液加水(30mL)稀释，用乙酸乙酯(10mL x 2)萃取。有机相用盐水洗涤，用无水硫酸钠干燥，过滤，滤液浓缩。所得粗品经硅胶柱层析(DCM:MeOH＝30:1)纯化得到4-((3-(4-(2,6-二氧代哌啶-3-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝601.1.
步骤4：3-(4-(9-(哌嗪-1-基甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)哌啶-2,6-二酮的制备
向4-((3-(4-(2,6-二氧代哌啶-3-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羧酸苄酯(230mg,0.38mmol)的i-PrOH(5mL)溶液中加入Pd/C(0.2mg)，在氢气球保护下室温反应2小时。反应液经硅藻土过滤，滤液浓缩得到3-(4-(9-(哌嗪-1-基甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)哌啶-2,6-二酮，直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝467.0.
步骤5：(2-(5-((1R)-1-((6-((1R,4R)-4-(4-((3-(4-(2,6-二氧代哌啶-3-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯的制备
向3-(4-(9-(哌嗪-1-基甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)哌啶-2,6-二酮(250mg,粗品)的DMSO(2mL)溶液中加入(1R,4R)-4-(4-((R)-1-(4-(2-((叔丁氧羰基)(甲基)氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸五氟苯酯(308mg,0.38mmol)和DIEA(245mg,1.90mmol)，反应液在室温下搅拌2小时。反应液中加入水(20mL)，然后用乙酸乙酯(20mL x 2)萃取，合并有机相并用饱和盐水(30mL x 3)洗涤，经无水硫酸钠干燥。过滤，滤液浓缩，所得粗品经pre-HPLC纯化得到(2-(5-((1R)-1-((6-((1R,4R)-4-(4-((3-(4-(2,6-二氧代哌啶-3-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M/2+H]+＝547.4.
步骤6：3-(4-(9-((4-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)哌啶-2,6-二酮的制备
向(2-(5-((1R)-1-((6-((1R,4R)-4-(4-((3-(4-(2,6-二氧代哌啶-3-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)噻吩-3-基)苄基)(甲基)羧酸叔丁酯(160mg,0.15mmol)的二氯甲烷(2.0mL)溶液中加入三氟乙酸(1.0mL)，于30℃下搅拌1小时。反应完成后，减压浓缩。粗品用pre-HPLC纯化，得到3-(4-(9-((4-((1R,4R)-4-(7-甲氧基-2-甲基-4-(((R)-1-(4-(2-((甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)哌啶-2,6-二酮。
LC-MS:(ESI,m/z):[M/2+H]+＝497.4.
1H NMR(400MHz,CD3OD)δ7.42–7.39(m,3H),7.32–7.27(m,6H),7.24–7.12(m,2H),7.11(s,1H),6.06–5.98(m,1H),5.66(d,J＝8.0Hz,1H),3.92(s,3H),3.83–3.79(m,1H),3.75–3.68(m,3H),3.65–3.59(m,2H),3.56–3.48(m,2H),3.42–3.26(m,2H),3.14–3.02(m,1H),2.78–2.12(m,17H),2.05–1.70(m,14H),1.54–1.10(m,13H).
实施例6：1-(2-氯-5-(4-(2-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)甲氧基)乙基)哌嗪-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮
步骤1：4-(2-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)甲氧基)乙基)哌嗪-1-羧酸叔丁酯的制备
将(1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸(145mg,0.24mmol)用DMF(5mL)溶解，然后加入4-(2-(哌啶-4-基甲氧基)乙基)哌嗪-1-羧酸叔丁酯(80mg,0.24mmol)，HATU(102mg,0.31mmol)和DIEA(93mg,0.72mmol)，反应液室温搅拌过夜。反应液加水(50mL)稀释，乙酸乙酯(30mL x 3)萃取，有机相用饱和氯化钠溶液(50mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(甲醇:二氯甲烷＝1:9)纯化得到4-(2-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)甲氧基)乙基)哌嗪-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝902.5.
步骤2：((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己基)(4-((2-(哌嗪-1-基)乙氧基)甲基)哌啶-1-基)甲酮的制备
4-(2-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)甲氧基)乙基)哌嗪-1-羧酸叔丁酯(120mg,0.13mmol)用二氯甲烷(5mL)溶解，然后加入三氟乙酸(1mL)并室温搅拌2小时，反应液直接减压浓缩得到((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己基)(4-((2-(哌嗪-1-基)乙氧基)甲基)哌啶-1-基)甲酮，无需纯化直接用于下步反应。
LC-MS:(ESI,m/z):[M+H]+＝802.4.
步骤3：1-(2-氯-5-(4-(2-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)甲氧基)乙基)哌嗪-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己基)(4-((2-(哌嗪-1-基)乙氧基)甲基)哌啶-1-基)甲酮(120mg,0.13mmol)用二甲亚砜(5mL)溶解，加入4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(57mg,0.13mmol)和DIEA(168mg,1.3mmol)，反应液室温搅拌2小时。反应液中加水(50mL)稀释，用乙酸乙酯(30mL x 3)萃取，有机相用饱和氯化钠溶液(50mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用pre-HPLC纯化得到1-(2-氯-5-(4-(2-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)甲氧基)乙基)哌嗪-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝1052.4.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),8.31(d,J＝8.3Hz,1H),8.05(s,1H),7.63(d,J＝8.2Hz,1H),7.55(s,1H),7.45-7.31(m,4H),7.24(s,1H),7.02(s,1H),6.96(s,1H),6.04-5.88(m,1H),4.44-4.34(m,1H),3.97-3.86(m,4H),3.78-3.71(m,1H),3.66-3.54(m,3H),3.50-3.45(m,2H),3.27-3.20(m,2H),3.15-2.87(m,5H),2.81-2.56(m,6H),2.45-2.37(m,5H),2.01(s,6H),1.93-1.42(m,17H),1.13-0.91(m,2H).
实施例7：1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)丙基)哌啶-1-基)-2-氯苯基)二氢嘧啶-2,4(1H,3H)-二酮
步骤1：3-(1-(4-氯-3-硝基苯基)哌啶-4-基)丙-1-醇的制备
将1-氯-4-氟-2-硝基苯(3.0g,17.04mmol)用DMSO(30mL)溶解，然后加入3-(哌啶-4-基)丙-1-醇(3.66g，25.57mmol)和DIEA(6.59g，51.12mmol)并升温至50℃搅拌过夜。反应液中加入水(200mL)，乙酸乙酯(100mL x 3)萃取，有机相用饱和氯化钠水溶液洗，无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(石油醚:乙酸乙酯＝1:4)纯化得到3-(1-(4-氯-3-硝基苯基)哌啶-4-基)丙-1-醇。
LC-MS:(ESI,m/z):[M+H]+＝299.1.
步骤2：苯甲酸3-(1-(4-氯-3-硝基苯基)哌啶-4-基)丙酯的制备
3-(1-(4-氯-3-硝基苯基)哌啶-4-基)丙-1-醇(200mg,0.67mmol)用四氢呋喃(10mL)溶解，然后依次加入苯甲酰氯(122mg,0.8mmol)和三乙胺(203mg,0.8mmol)，反应液加热至70℃搅拌过夜。反应液中加入水(50mL)，乙酸乙酯(50mL x 3)萃取，有机相用饱和氯化钠水溶液洗，无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(石油醚:乙酸乙酯＝1:9)纯化得到苯甲酸3-(1-(4-氯-3-硝基苯基)哌啶-4-基)丙酯。
LC-MS:(ESI,m/z):[M+H]+＝403.2.
步骤3：苯甲酸3-(1-(3-氨基-4-氯苯基)哌啶-4-基)丙酯的制备
将苯甲酸3-(1-(4-氯-3-硝基苯基)哌啶-4-基)丙酯(1.0g,2.49mmol)用EtOH(10mL)溶解，然后加入水合肼(2mL)和Raney Ni(固液浑浊液,2mL)并室温搅拌1小时，过滤，滤液减压浓缩得到苯甲酸3-(1-(3-氨基-4-氯苯基)哌啶-4-基)丙酯。无需纯化直接用于下步反应。
LC-MS:(ESI,m/z):[M+H]+＝373.1.
步骤4：3-((5-(4-(3-(苯甲酰氧)丙基)哌啶-1-基)-2-氯苯基)氨基)丙酸的制备
将苯甲酸3-(1-(3-氨基-4-氯苯基)哌啶-4-基)丙酯(900mg,2.42mmol)和丙烯酸(1.74g,24.19mmol)加入到H2O/AcOH(5/1,12mL)中，反应液在氮气氛围下110℃搅拌过夜，反应液直接减压浓缩，粗品经pre-HPLC(乙腈/甲酸水体系)纯化得到3-((5-(4-(3-(苯甲酰氧)丙基)哌啶-1-基)-2-氯苯基)氨基)丙酸。
LC-MS:(ESI,m/z):[M+H]+＝445.2.
步骤5：苯甲酸3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙酯的制备
将3-((5-(4-(3-(苯甲酰氧)丙基)哌啶-1-基)-2-氯苯基)氨基)丙酸(850mg,1.91mmol)用AcOH(8mL)溶解，然后加入尿素(1.19g,19.14mmol)，反应液升温至110℃搅拌过夜。反应液中加入水(80mL)，乙酸乙酯(50mL x 3)萃取，合并有机相，饱和氯化钠水溶液洗，无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(PE:EA＝2:1)纯化得到苯甲酸3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙酯。
LC-MS:(ESI,m/z):[M+H]+＝470.1.
步骤6：1-(2-氯-5-(4-(3-羟基丙基)哌啶-1-基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
苯甲酸3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙酯(500mg,1.06mmol)用甲醇/浓盐酸(1:1,8mL)溶解，反应液氮气氛围下加热至70℃搅拌过夜。直接减压浓缩，粗品经pre-HPLC(乙腈/甲酸水体系)纯化得到1-(2-氯-5-(4-(3-羟基丙基)哌啶-1-基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝366.1.
步骤7：3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙醛的制备
1-(2-氯-5-(4-(3-羟基丙基)哌啶-1-基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(200mg,0.55mmol)用二氯甲烷(10mL)溶解，加入Dess-Martin(466mg,1.10mmol)并室温搅拌过夜，反应液直接减压浓缩，粗品经硅胶柱层析(二氯甲烷:甲醇＝10:1)纯化得到3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙醛。
LC-MS:(ESI,m/z):[M+H]+＝364.0.
步骤8：4-(3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙基)哌嗪-1-羧酸叔丁酯的制备
将3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙醛(110mg,0.30mmol)和哌嗪-1-羧酸叔丁酯(84mg,0.45mmol)用THF(10mL)溶解，反应液室温搅拌1小时，加入三乙酰氧基硼氢化钠(95mg,0.45mmol)并室温搅拌过夜。反应液中加水(50mL)，乙酸乙酯(50mL x 3)萃取，合并有机相，饱和氯化钠水溶液洗，无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(PE:EA＝2:1)纯化得到4-(3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙基)哌嗪-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝534.2.
步骤9：1-(2-氯-5-(4-(3-(哌嗪-1-基)丙基)哌啶-1-基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将4-(3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙基)哌嗪-1-羧酸叔丁酯(120mg,0.225mmol)用DCM/TFA(1:1,8mL)溶解并室温搅拌1小时，反应液减压浓缩得到1-(2-氯-5-(4-(3-(哌嗪-1-基)丙基)哌啶-1-基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。无需纯化直接用于下步反应。
LC-MS:(ESI,m/z):[M+H]+＝434.2.
步骤10：(3-((R)-1-((6-((1R,4R)-4-(4-(3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-5-(三氟甲基)苯基)羧酸叔丁酯的制备
将1-(2-氯-5-(4-(3-(哌嗪-1-基)丙基)哌啶-1-基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(90mg,0.21mmol)和(1R,4R)-4-(4-(((R)-1-(3-((叔-丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸(130mg,0.21mmol)用DMF(5mL)溶解，然后加入DIEA(55mg,0.42mmol)和HATU(120mg,0.32mmol)并室温搅拌1小时，反应液加水(50mL)稀释，然后用二氯甲烷(50mL x 3)萃取，无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(二氯甲烷:甲醇＝10:1)纯化得到(3-((R)-1-((6-((1R,4R)-4-(4-(3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-5-(三氟甲基)苯基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝1018.4.
步骤11：1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)丙基)哌啶-1-基)-2-氯苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将(3-((R)-1-((6-((1R,4R)-4-(4-(3-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯基)哌啶-4-基)丙基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-5-(三氟甲基)苯基)羧酸叔丁酯(100mg,0.10mmol)用DCM/TFA(1/1,8mL)溶解并室温搅拌1小时，反应液直接减压浓缩，粗品使用pre-HPLC纯化得到1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)丙基)哌啶-1-基)-2-氯苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝918.4.
1H NMR(400MHz,DMSO-d6)δ10.39(s,1H),8.20-8.00(m,2H),7.30(d,J＝8.8Hz,1H),7.03(s,1H),6.99(s,1H),6.94-6.86(m,2H),6.85(s,1H),6.70(s,1H),5.63-5.46(m,3H),3.88(s,3H),3.75-3.60(m,3H),3.60-3.53(m,1H),3.52-3.40(m,4H),2.98-2.92(m,1H),2.80-2.60(m,6H),2.35-2.28(m,8H),1.89-1.43(m,16H),1.29-1.11(m,4H).
实施例8：1-(2-氯-5-(4-(3-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)氧)丙基)哌嗪-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮
步骤1：4-(3-乙氧基-3-氧代丙氧基)哌啶-1-羧酸苄酯的制备
将4-氰基哌啶-1-羧酸苄酯(5.0g,21.28mmol)用乙腈(100mL)溶解，然后加入丙烯酸甲酯(4.256g,42.56mmol)和碳酸铯(13.87g,42.56mmol)，反应液常温搅拌过夜。减压浓缩，残留物加水(100mL)，然后用乙酸乙酯(100mL x 3)萃取，有机相用饱和食盐水(150mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。粗品经硅胶柱层析(乙酸乙酯:石油醚＝1:4)纯化得到4-(3-乙氧基-3-氧代丙氧基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝336.1.
步骤2：4-(3-羟基丙氧基)哌啶-1-羧酸苄酯的制备
将4-(3-乙氧基-3-氧代丙氧基)哌啶-1-羧酸苄酯(1.5g,4.47mmol)溶解于无水四氢呋喃(50mL)中，氮气氛围下，向溶液中缓慢加入硼烷四氢呋喃溶液(45mL,45mmol)，反应液升温至50℃并搅拌3小时。冷却至室温，缓慢滴加甲醇至无气泡产生，直接减压浓缩，粗品经硅胶柱层析(甲醇:二氯甲烷＝1:9)纯化得到4-(3-羟基丙氧基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝294.1.
步骤3：4-(3-氧代丙氧基)哌啶-1-羧酸苄酯的制备
将4-(3-羟基丙氧基)哌啶-1-羧酸苄酯(500mg,1.70mmol)用乙腈(15mL)溶解，加入IBX(952mg,3.40mmol)，反应液加热回流2小时。直接减压浓缩，粗品经硅胶柱层析(甲醇:二氯甲烷＝1:9)纯化得到4-(3-氧代丙氧基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝292.1.
步骤4：4-(3-((1-((苄氧基)羰基)哌啶-4-基)氧)丙基)哌嗪-1-羧酸叔丁酯的制备
将4-(3-氧代丙氧基)哌啶-1-羧酸苄酯(370mg,1.27mmol)用THF(10mL)溶解，依次加入哌嗪-1-羧酸叔丁酯(236mg,1.27mmol)并室温搅拌10min，三乙酰氧基硼氢化钠(808mg，3.81mmol)，反应液室温搅拌2小时。用水(50mL)稀释，二氯甲烷(30mL x 3)萃取，经无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(甲醇:二氯甲烷＝1:9)纯化得到4-(3-((1-((苄氧基)羰基)哌啶-4-基)氧)丙基)哌嗪-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝462.2.
步骤5：4-(3-(哌啶-4-基氧)丙基)哌嗪-1-羧酸叔丁酯的制备
将4-(3-((1-((苄氧基)羰基)哌啶-4-基)氧)丙基)哌嗪-1-羧酸叔丁酯(250mg,0.54mmol)用乙酸乙酯(10mL)溶解，加入Pd/C(10％,75mg)，反应体系用氢气置换3次，然后在氢气氛围下加热到70℃并搅拌过夜。过滤，滤饼用乙酸乙酯(5mL x 3)淋洗，滤液直接减压浓缩得到4-(3-(哌啶-4-基氧)丙基)哌嗪-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝328.3.
步骤6：4-(3-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)氧)丙基)哌嗪-1-羧酸叔丁酯的制备
将4-(3-(哌啶-4-基氧)丙基)哌嗪-1-羧酸叔丁酯(110mg,0.34mmol)用DMF(5mL)溶解，分别加入(1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸(202mg,0.34mmol)，HATU(144mg,0.44mmol)和DIEA(132mg,1.02mmol)，反应液室温搅拌过夜。用水(50mL)稀释，二氯甲烷(30mL x 3)萃取，经无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(甲醇:二氯甲烷＝1:9)纯化得到4-(3-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)氧)丙基)哌嗪-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝902.4.
步骤7：((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己基)(4-(3-(哌嗪-1-基)丙氧基)哌啶-1-基)甲酮的制备
将4-(3-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)氧)丙基)哌嗪-1-羧酸叔丁酯(90mg,0.10mmol)用二氯甲烷(5mL)溶解，加入三氟乙酸(1mL)并室温搅拌2小时，反应液直接减压浓缩得到((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己基)(4-(3-(哌嗪-1-基)丙氧基)哌啶-1-基)甲酮，无需纯化直接用于下步反应；
LC-MS:(ESI,m/z):[M+H]+＝802.4.
步骤8：1-(2-氯-5-(4-(3-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)氧)丙基)哌嗪-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己基)(4-(3-(哌嗪-1-基)丙氧基)哌啶-1-基)甲酮(90mg,0.10mmol)用二甲亚砜(5mL)溶解，加入4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(44mg,0.10mmol)和DIEA(129mg,1.0mmol)，反应液室温搅拌2小时。反应液加水(50mL)稀释，用二氯甲烷(30mL x 3)萃取，经无水硫酸钠干燥，过滤，减压浓缩，粗品经pre-HPLC纯化得到1-(2-氯-5-(4-(3-((1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)氧)丙基)哌嗪-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝1054.5.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),8.31(d,J＝8.2Hz,1H),8.05(s,1H),7.65(s,1H),7.63(s,1H),7.56(s,1H),7.46-7.31(m,4H),7.24(s,1H),7.02(s,1H),6.96(s,1H),6.05-5.86(m,1H),3.89(s,3H),3.87-3.80(m,1H),3.79-3.56(m,6H),3.52-3.42(m,4H),3.16-3.06(m,3H),2.98-2.89(m,1H),2.77-2.60(m,5H),2.44-2.32(m,8H),2.01(s,6H),1.89-1.75(m,6H),1.74-1.68(m,3H),1.68-1.48(m,6H),1.44-1.26(m,2H).
实施例9：3-((R)-1-((6-((1R,4R)-4-(4-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-2-甲基苯甲氰
步骤1：9-((4-((苄氧基)羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将9-醛基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(300mg,1.06mmol)和哌嗪-1-羧酸苄酯(282mg,1.27mmol)用四氢呋喃(10mL)溶解，搅拌下加入三乙酰氧基硼氢化钠(674mg,3.18mmol)并室温搅拌过夜。反应液加水(50mL)，用二氯甲烷(30mL x 3)萃取，经无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(乙酸乙酯:石油醚＝1:1)纯化得到9-((4-((苄氧基)羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝486.3.
步骤2：9-(哌嗪-1-基甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将9-((4-((苄氧基)羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(200mg,0.41mmol)用乙酸乙酯(15mL)溶解，加入氢氧化钯炭(20％,100mg)，所得混合物在氢气氛围下升温至70℃并搅拌过夜。反应液冷却至室温后，用硅藻土过滤，滤液直接减压浓缩得到9-(哌嗪-1-基甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯，无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝352.4.
步骤3：(1R,4R)-4-(4-(((R)-1-(3-氰基-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸甲酯的制备
将(1R,4R)-4-(4-羟基-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸甲酯(200mg,0.61mmol)，BOP(344mg,0.78mmol)和DBU(232mg,1.52mmol)用DMF(5mL)溶解，室温搅拌30分钟后，加入(R)-3-(1-氨基乙基)-2-甲基苯甲氰(154mg,0.78mmol)，反应液升温至70℃并搅拌过夜。冷却至室温，反应液加水(50mL)稀释，用乙酸乙酯(30mL x 3)萃取，有机相用饱和食盐水(50mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(乙酸乙酯:石油醚＝9:1)纯化得到(1R,4R)-4-(4-(((R)-1-(3-氰基-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸甲酯。
LC-MS:(ESI,m/z):[M+H]+＝473.2.
步骤4：(1R,4R)-4-(4-(((R)-1-(3-氰基-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸的制备
将(1R,4R)-4-(4-(((R)-1-(3-氰基-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸甲酯(130mg,0.27mmol)溶解于四氢呋喃/水(10mL/5mL)混合液中，加入氢氧化锂(53mg,2.2mmol)，反应液升温至50℃并搅拌过夜。冷却至室温，反应液加水(30mL)稀释，用饱和柠檬酸钠水溶液调节混合液的pH值至中性，再用乙酸乙酯(30mL x 3)萃取，有机相用饱和食盐水(50mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，得到(1R,4R)-4-(4-(((R)-1-(3-氰基-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸。
LC-MS:(ESI,m/z):[M+H]+＝459.2.
步骤5：9-((4-((1R,4R)-4-(4-(((R)-1-(3-氰基-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将(1R,4R)-4-(4-(((R)-1-(3-氰基-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸(120mg,0.26mmol)和9-(哌嗪-1-基甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(120mg,0.34mmol)用DMF(5mL)溶解，然后加入HATU(148mg,0.39mmol)和N,N-二异丙基乙胺(100mg,0.78mmol)，反应液室温搅拌2小时。反应液加水(50mL)稀释，乙酸乙酯(30mL x 3)萃取，有机相用饱和食盐水(50mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(甲醇:二氯甲烷＝1:9)纯化得到9-((4-((1R,4R)-4-(4-(((R)-1-(3-氰基-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝792.4.
步骤6：3-((R)-1-((6-((1R,4R)-4-(4-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-2-甲基苯甲氰的制备
将9-((4-((1R,4R)-4-(4-(((R)-1-(3-氰基-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(160mg,0.20mmol)用二氯甲烷(5mL)溶解，三氟乙酸(2mL)加入并室温搅拌2小时，反应液直接减压浓缩得到3-((R)-1-((6-((1R,4R)-4-(4-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-2-甲基苯甲氰，无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝692.4.
步骤7：3-((R)-1-((6-((1R,4R)-4-(4-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-2-甲基苯甲氰的制备
将3-((R)-1-((6-((1R,4R)-4-(4-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-2-甲基苯甲氰(160mg,TFA盐,0.20mmol)用二甲亚砜(3mL)溶解，搅拌下加入4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(95mg,0.22mmol)和N,N-二异丙基乙胺(77mg,0.60mmol)。反应液室温搅拌过夜，反应液加水(50mL)，过滤，滤饼经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到3-((R)-1-((6-((1R,4R)-4-(4-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-2-甲基苯甲氰。
LC-MS:(ESI,m/z):[M+H]+＝942.5.
1H NMR(400MHz,DMSO-d6)δ10.52(s,1H),8.25(s,1H),7.83(d,J＝8.0Hz,1H),7.71-7.60(m,2H),7.55(s,1H),7.39(t,J＝7.7Hz,2H),7.02(s,1H),5.78-5.68(m,1H),3.91(s,3H),3.81-3.38(m,8H),3.3-3.25(m,2H),3.01-2.93(m,1H),2.80-2.63(m,7H),2.48-2.21(m,7H),1.90-1.77(m,4H),1.75-1.20(m,18H),1.16-0.92(m,4H).
实施例10：1-(2-氯-5-(4-((2-(1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮
将1-(2-氯-5-(4-((2-(哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(190mg,0.397mmol)用DMF(5mL)溶解，分别加入(1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸(85mg,0.143mmol)，DIEA(156mg,1.2mmol)和PyBOP(250mg,0.48mmol)，反应液在N2保护下60℃搅拌16h，反应液加水(40mL)稀释，用乙酸乙酯(40mL x 4)萃取，饱和食盐水洗，无水硫酸钠干燥，减压浓缩，粗品经pre-HPLC纯化得到1-(2-氯-5-(4-((2-(1-((1R,4R)-4-(4-(((R)-1-(4-(2-氯-6-((二甲基氨基)甲基)苯基)噻吩-2-基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝1053.4.
1H NMR(400MHz,MeOD)δ8.01(s,1H),7.64(s,1H),7.62(s,1H),7.52(s,1H),7.44-7.35(m,3H),7.30(t,J＝7.8Hz,1H),7.09(d,J＝1.1Hz,1H),7.01(s,1H),6.95(s,1H),6.06(d,J＝6.8Hz,1H),4.68-4.47(m,2H),4.10-4.00(m,1H),3.95(s,3H),3.82-3.60(m,4H),3.49(t,J＝6.2Hz,2H),3.27(s,2H),3.17-3.02(m,3H),2.91-2.80(m,3H),2.75-2.67(m,1H),2.66-2.55(m,1H),2.52(s,3H),2.05(s,6H),2.00-1.58(m,19H),1.58-1.49(m,2H),1.32-1.29(m,2H).
实施例11：1-(5-(4-((2-(1-((1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)-2-氯苯基)二氢嘧啶-2,4(1H,3H)-二酮
步骤1：(1R,4R)-4-(4-(((R)-1-(3-((叔-丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸五氟苯酯的制备
将(1R,4R)-4-(4-(((R)-1-(3-((叔-丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸(200mg,0.33mmol)用DMF(5mL)溶解，加入2,3,4,5,6-五氟苯酚(74mg,0.40mmol)和DCC(82mg,0.40mmol)，反应液升温至70℃并搅拌2h。反应液用水(50mL)稀释，二氯甲烷(50mL x 3)萃取，有机相用无水硫酸钠干燥，过滤，减压浓缩。粗品经硅胶柱层析(乙酸乙酯:石油醚＝1:1)纯化得到(1R,4R)-4-(4-(((R)-1-(3-((叔-丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸五氟苯酯。
LC-MS:(ESI,m/z):[M+H]+＝769.4.
步骤2：(3-((R)-1-((6-((1R,4R)-4-(4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基-d2)乙基)哌啶-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-5-(三氟甲基)苯基)羧酸叔丁酯的制备
将(1R,4R)-4-(4-(((R)-1-(3-((叔-丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸五氟苯酯(200mg,0.26mmol)用二甲亚砜(5mL)溶解，加入1-(2-氯-5-(4-((2-(哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(144mg,0.30mmol)和DIEA(117mg,0.90mmol)，反应液室温搅拌2小时。反应液加水(50mL)稀释，用二氯甲烷(30mL x 3)萃取，经无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(甲醇:二氯甲烷＝1:9)纯化得到(3-((R)-1-((6-((1R,4R)-4-(4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基-d2)乙基)哌啶-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-5-(三氟甲基)苯基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝1063.4.
步骤3：1-(5-(4-((2-(1-((1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)-2-氯苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将(3-((R)-1-((6-((1R,4R)-4-(4-(2-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)甲氧基-d2)乙基)哌啶-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-5-(三氟甲基)苯基)羧酸叔丁酯(150mg,0.14mmol)溶于DCM(5mL)中，加入TFA(1mL)，室温搅拌1小时，减压浓缩后，加入MeOH/DCM(1/9,10mL)溶解，再加入稀释一倍的饱和碳酸氢钠水溶液调节pH至中性，搅拌10分钟后，用MeOH/DCM(1/9,30mL x 3)萃取，无水硫酸钠干燥，过滤，减压浓缩，粗品经pre-HPLC纯化得到1-(5-(4-((2-(1-((1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌啶-4-基)乙氧基)甲基-d2)哌啶-1-羰基)-2-氯苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝963.2
1H NMR(400MHz,DMSO-d6)δ10.52(s,1H),8.12(d,J＝8.1Hz,1H),8.06(s,1H),7.63(d,J＝8.2Hz,1H),7.54(d,J＝2.0Hz,1H),7.38(dd,J＝8.2,2.0Hz,1H),6.99(s,1H),6.88(s,1H),6.85(s,1H),6.70(s,1H),5.62-5.51(m,3H),4.54-4.30(m,2H),3.96-3.83(m,4H),3.80-3.51(m,3H),3.40(t,J＝6.4Hz,2H),3.11-2.89(m,3H),2.80-2.59(m,4H),2.36(s,3H),1.92-1.37(m,20H),1.20-0.89(m,4H).
实施例12：1-(2-氯-5-(9-((4-((1R,4R)-4-(4-(((R)-1-(3-(二氟甲基)-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮
步骤1：(1R,4R)-4-(4-(((R)-1-(3-(二氟甲基)-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸甲酯的制备
将(1R,4R)-4-(4-羟基-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸甲酯(400mg,1.212mmol)用DMF(5mL)溶解，加入BOP(696mg,1.576mmol)及DBU(461mg,3.03mmol)，室温搅拌0.5小时后，加入(R)-1-(3-(二氟甲基)-2-甲基苯基)乙基-1-胺(244mg,1.33mmol)，反应液升温至70℃搅拌过夜。反应液用水(50mL)稀释，乙酸乙酯(30mL x 3)萃取，有机相用饱和氯化钠水溶液(50mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品经硅胶柱层析(乙酸乙酯:石油醚＝9:1)纯化得到(1R,4R)-4-(4-(((R)-1-(3-(二氟甲基)-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸甲酯。
LC-MS:(ESI,m/z):[M+H]+＝498.2.
步骤2：(1R,4R)-4-(4-(((R)-1-(3-(二氟甲基)-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸的制备
将(1R,4R)-4-(4-(((R)-1-(3-(二氟甲基)-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸甲酯(178mg,0.36mmol)用THF(5mL)溶解，加入LiOH(44mg,1.83mmol)，反应液升温至50℃搅拌2小时。放映业加水(50mL)稀释，用柠檬酸调节pH至中性，乙酸乙酯(30mL x 3)萃取，有机相用饱和氯化钠水溶液(50mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩得到(1R,4R)-4-(4-(((R)-1-(3-(二氟甲基)-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸，无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝484.2.
步骤3：1-(2-氯-5-(9-((4-((1R,4R)-4-(4-(((R)-1-(3-(二氟甲基)-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将(1R,4R)-4-(4-(((R)-1-(3-(二氟甲基)-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸(128mg，0.265mmol)用DMF(5mL)溶解，分别加入1-(2-氯-5-(9-(哌嗪-1-基甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(133mg，0.265mmol)，DIEA(103mg，0.795mmol)和HATU(141mg，0.307mmol)，反应液室温搅拌2小时。用水(50mL)稀释，乙酸乙酯(30mL x 3)萃取，有机相用饱和氯化钠水溶液(50mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品经Prep-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到1-(2-氯-5-(9-((4-((1R,4R)-4-(4-(((R)-1-(3-(二氟甲基)-2-甲基苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝967.3.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),8.23(d,J＝7.3Hz,1H),8.08(s,1H),7.68(d,J＝8.0Hz,1H),7.63(d,J＝8.2Hz,1H),7.55(d,J＝1.9Hz,1H),7.41-7.35(m,2H),7.35-7.04(m,2H),6.97(s,1H),5.82-5.72(m,1H),3.87(s,3H),3.80-3.69(m,1H),3.66-3.54(m,3H),3.54-3.42(m,4H),3.30-3.20(m,2H),3.00-2.89(m,1H),2.78-2.70(m,2H),2.58-2.54(m,3H),2.38-2.21(m,7H),2.15-2.07(m,2H),1.91-1.77(m,4H),1.74-1.24(m,17H),1.17-0.95(m,4H).
实施例13：1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)丙氧基)哌啶-1-羰基)-2-氯苯基)二氢嘧啶-2,4(1H,3H)-二酮
步骤1：(3-((R)-1-((6-((1R,4R)-4-(4-(3-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)氧)丙基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-5-(三氟甲基)苯基)羧酸叔丁酯的制备
将1-(2-氯-5-(4-(3-(哌嗪-1-基)丙氧基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(90mg,0.13mmol)，HATU(59mg.0.16mmol)和DIEA(50mg,0.39mmol)加入到(1R,4R)-4-(4-(((R)-1-(3-((叔丁氧羰基)氨基)-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羧酸(80mg,0.16mmol)的DMF(5mL)溶液中，室温搅拌2小时，加入水(50mL)搅拌，过滤，滤饼水洗3次，然后用二氯甲烷(20mL)溶解，无水硫酸钠干燥，减压浓缩，得到粗品(3-((R)-1-((6-((1R,4R)-4-(4-(3-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)氧)丙基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-5-(三氟甲基)苯基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝1062.5.
步骤2：1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)丙氧基)哌啶-1-羰基)-2-氯苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将(3-((R)-1-((6-((1R,4R)-4-(4-(3-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)氧)丙基)哌嗪-1-羰基)环己基)-7-甲氧基-2-甲基喹唑啉-4-基)氨基)乙基)-5-(三氟甲基)苯基)羧酸叔丁酯(130mg，0.12mmol)用二氯甲烷(5mL)溶解，加入三氟乙酸(1mL)，然后反应液室温搅拌2小时，减压浓缩，粗品使用反相制备纯化得到1-(5-(4-(3-(4-((1R,4R)-4-(4-(((R)-1-(3-氨基-5-(三氟甲基)苯基)乙基)氨基)-7-甲氧基-2-甲基喹唑啉-6-基)环己烷-1-羰基)哌嗪-1-基)丙氧基)哌啶-1-羰基)-2-氯苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝962.5.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),8.17–7.98(m,2H),7.67–7.50(m,2H),7.47–7.34(m,1H),6.99(s,1H),6.88(s,1H),6.85(s,1H),6.70(s,1H),5.64–5.46(m,3H),3.88(s,4H),3.74(s,1H),3.62(d,J＝5.7Hz,1H),3.56–3.39(m,8H),3.18(s,1H),3.00-2.89(m,1H),2.80–2.70(m,2H),2.68-2.59(m,1H),2.40–2.25(m,9H),1.94–1.35(m,18H).
Ⅱ生物活性测试实施例
测试实施例1：KRAS-G12C/SOS1结合试验
以下方法用于测定本发明优选化合物在体外条件下对KRAS-G12C/SOS1结合抑制程度。本方法使用KRAS-G12C/SOS1结合试验试剂盒(Cisbio,63ADK000CB16PEG)，以均相时间分辨荧光技术(HTRF)测定优选化合物对KRAS-G12C/SOS1结合竞争抑制测定。
实验流程如下(详细方法参考试剂盒说明书)：将本发明中的化合物先溶解于DMSO中，浓度为20mM，用试剂盒中的缓冲液进行等梯度稀释，使受试化合物在反应体系中的终浓度范围为10000nM～0.04nM，DMSO的终浓度为0.5％。将2μL化合物与4μL 1x Tag1-SOS1、4μL 1xTag2-KRAS-G12C(含10μM三磷酸鸟苷,GTP)在25℃下孵育15min，随后向反应体系中加入5μL 1x Anti-Tag1-Tb3+以及5μL 1x Anti-Tag2-XL665在冰上下孵育3h。孵育结束后，在酶标仪EnVision(PerkinElmer,2105)上以HTRF模式测定各孔在激发波长为337nm，读取各孔在发射波长为620nm和665nm的荧光强度，使用公式Ratio＝(665nm/620nm)×104算出Ratio值。通过与对照组荧光强度比值进行对比，计算化合物在各浓度下的抑制率，进而通过GraphPad Prism 8以对数浓度-抑制率进行非线性曲线拟合，得出化合物的IC50值，见表1。
测试实施例2：SOS1降解试验
在KRAS G12C突变的人非小细胞肺癌细胞NCI-H358(ATCC或中国科学院干细胞库)、KRAS G12D突变的人肺癌细胞A-427(ATCC)、KRAS G12V突变的人结直肠腺癌细胞SW-620(ATCC)和KRAS G13D突变的人结直肠腺癌上皮细胞DLD-1(ATCC)上考察化合物对SOS1的降解作用。具体方法为：
在24孔细胞培养板每孔中铺0.95mL细胞，使细胞密度为5×105个/孔；细胞板置于5％CO2培养箱中37℃培养过夜。然后在相应铺有细胞的孔中加入50μL稀释后的化合物溶液，使化合物的终浓度在0.03～3000nM范围内。DMSO终浓度为0.25％。给药后的细胞板置于5％CO2培养箱中37℃培养24h后，移除24孔中细胞培养液后，用1×PBS清洗2次，细胞培养板底部贴壁的NCI-H358细胞加入180μL RIPA(强)裂解液(碧云天,P0013B)，补充1mM苯甲基磺酰氟、蛋白酶抑制剂混合物(碧云天,P1008)以及蛋白酶磷酸酶抑制剂混合物(碧云天,P1045)裂解，冰上静置30min后，将每孔中的蛋白裂解液分别转移至1.5mL离心管中，15000g、4℃离心20min。离心后的细胞裂解上清液冻存于-80℃待测，用于WB方法检测SOS1蛋白降解水平。细胞裂解上清液中总蛋白浓度使用BCA蛋白质定量试剂盒(天根,PA115-02)测定。
测试实施例3：人SOS1蛋白免疫印迹试验
根据BCA检测细胞裂解液中总蛋白的浓度，用PBS和5×SDS-PAGE蛋白上样缓冲液(碧云天,P0015L)调整至0.5μg/μL，100℃水浴15min，然后置于冰上冰浴5min后，14000g、4℃离心1min，混匀作为WB的上样样品。用预制胶(凯基,KGMG010W15)做蛋白电泳，上样量为10μL(总蛋白5μg)，Tris-MOPS-SDS电泳液(Adamas,P1598253)后120V 55min恒压电泳，电泳结束后，恒流250mA 65min把胶条上的蛋白转至PVDF膜上。转膜结束后将膜放入1×QuickBlock封闭液(碧云天,P0235)中室温孵育30min。封闭结束后，PVDF膜分别进行SOS1一抗(Abcam,ab140621)孵育4℃过夜，TBST缓冲液(2.4g Tris,8.8g NaCl,1.5mL Tween 20,调pH至7.4，定容至1L)洗涤膜30min(10min/次)，二抗(Abcam,ab205718)室温孵育2h，TBST缓冲液洗涤膜30min(10min/次)，最后用Clarity Western ECL Substrate(BIO-RAD,170-5061)孵育5min发光显色，化学发光成像系统(勤翔,ChemiScope 6200Touch)进行显色和蛋白图谱拍照。蛋白图谱通过勤翔化学发光分析软件进行灰度值分析。使用公式：灰度校正值＝(目的蛋白灰度值/对应内参灰度值)×103，计算出各样品的灰度校正值。再与对照组灰度校正值对比计算降解率。进而通过GraphPad Prism 8以对数浓度-抑制率进行非线性曲线拟合，得出化合物的DC50和Dmax值。
测试实施例4：3D细胞增殖抑制试验
在KRAS G12C突变的人非小细胞肺癌细胞NCI-H358(ATCC或中国科学院干细胞库)、KRAS G12D突变的人转移胰腺腺癌细胞AsPC-1(ATCC)、KRAS G12V突变的人肺腺癌细胞NCI-H441(ATCC)和KRAS G13D突变的人结直肠腺癌上皮细胞DLD-1(ATCC)上考察化合物对3D细胞增殖的抑制作用。具体方法为：
在384孔低吸附微孔板(Corning,3657)相应微孔中用Echo声波移液系统(Labcyte,Echo 550)加入200nL化合物梯度稀释溶液，使受试化合物在反应体系中的终浓度范围为20000nM～0.008nM。然后将40μL一定密度的细胞加入对应的微孔板中。除受试化合物测试孔外，同时设置DMSO对照孔和培养基对照孔，DMSO对照孔中含有DMSO和细胞，培养基孔中只含有培养基。加样完成后盖上板盖，将测384孔低吸附微孔板放入5％二氧化碳培养箱中，37℃孵育7天。7天后取出微孔板，每孔加入40μL CTG试剂(Promega,G9683)，室温孵育30分钟后在酶标仪EnVision上采用化学发光程序读数。通过细胞增殖抑制百分比计算公式：抑制率％＝(DMSO对照组平均值-化合物单浓度读值)/(DMSO对照组平均值-培养基对照组平均值)×100，计算化合物在各浓度时的抑制率，进而通过GraphPad Prism 8以对数浓度-抑制率进行非线性曲线拟合，得出化合物的IC50值。
实验结果如表1所示：
表1


“-”表示未检测；
当DC50≤50nM为A，当50nM<DC50≤100nM时为B，当100nM<DC50≤500nM为C，当500nM<DC50≤1000nM为D；1000nM<DC50≤10μM为E；
当80≤Dmax(％)≤100时为A，当50≤Dmax(％)<80时为B，当30≤Dmax(％)<50时为C，当Dmax(％)<30时为D；
当IC50≤15nM为A，当15nM<IC50≤30nM时为B，当30nM<IC50≤50nM为C，当50nM<IC50≤100nM为D，当100nM<IC50≤1000nM为E。
实验结论：本发明化合物能够有效的与SOS1靶蛋白结合或产生抑制效果，本发明所述化合物能够有效的特异性地降解SOS1蛋白。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (11)

  1. 一种式I所示化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药，或其药学上可接受的盐：
    S-L-E
    I
    其中，S为
    L为 L通过A或B与S相连；
    E为
  2. 如权利要求1所述的式I所示化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药，或其药学上可接受的盐，其特征在于，所述式I所示化合物满足如下条件中的一种或多种：
    (1)S为L为
    (2)S为L为
    (3)S为L为
    (4)S为L为
    (5)S为L为
    (6)S为L为
    (7)S为L为
    (8)S为L为
    (9)S为L为
    (10)S为E为
    (11)S为E为
    (12)S为E为
    (13)S为E为
    (14)S为E为
    (15)S为E为
    (16)S为E为
    (17)S为E为
    (18)S为E为
    (19)S为E为
    (20)L为E为
    (21)L为E为
    (22)L为E为
    (23)L为E为
    (24)L为E为
    (25)L为E为
    (26)L为E为
    (27)L为E为
    (28)L为E为
    (29)L为E为
    (30)L为E为
    (31)L为E为
    (32)L为E为
    (33)L为E为
    (34)L为E为
    和
    (35)L为E为
  3. 如权利要求1所述的式I所示化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药，或其药学上可接受的盐，其特征在于，所述式I所示化合物满足如下条件中的一种或多种：
    (1)S为L为 E为
    (2)S为L为
    (3)S为L为 E为
    (4)S为L为 E为
    (5)S为L为 E为
    (6)S为L为 E为
    (7)S为L为 E为
    (8)S为L为E为
    和
    (9)S为L为 E为
  4. 如权利要求3所述的式I所示化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药，或其药学上可接受的盐，其特征在于，L中A与S相连。
  5. 如权利要求1所述的式I所示化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药，或其药学上可接受的盐，其特征在于，所述式I所示化合物为如下任一化合物：




  6. 一种药物组合物，其包含G和药学上可接受的载体、稀释剂或赋形剂，所述的G为权利要求1-5至少一项所述的化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药，或其药学上可接受的盐。
  7. 一种如权利要求1-5至少一项所述的化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或如权利要求6所述的药物组合物在制备治疗或预防SOS1介导的疾病或病症、或由SOS1与Ras或者SOS1与Rac相互作用所引起的疾病或病症、或癌症的药物中的应用；较佳地，所述的SOS1介导的疾病或病症为癌症、1型神经纤维瘤病(NF1)、努南综合征(NS)、伴有多雀斑的努南综合征(NSML)、毛细血管畸形-动静脉畸形综合征(CM-AVM)、科斯特洛综合征(CS)、心-面-皮肤综合症(CFC)、莱格斯综合征或遗传性牙龈纤维瘤病；较佳地，所述的SOS1与Ras或者SOS1与Rac相互作用所引起的疾病或病症为癌症。
  8. 如权利要求7所述的应用，其特征在于，所述的癌症选自：
    心脏:肉瘤(血管肉瘤、纤维肉瘤、横纹肌肉瘤、脂肪肉瘤)、黏液瘤、横纹肌瘤、纤维瘤、脂肪瘤和畸胎瘤组；
    肺:支气管癌(鳞状细胞癌、未分化小细胞癌、未分化大细胞癌、腺癌)、肺泡癌(细支气管癌)、支气管腺瘤、肉瘤、淋巴瘤、软骨瘤错构瘤、间皮瘤；
    胃肠道食管(鳞状细胞癌、腺癌、平滑肌肉瘤、淋巴瘤)、胃(癌、淋巴瘤、平滑肌肉瘤)、胰腺(导管腺癌、胰岛素瘤、胰高血糖素瘤、胃泌素瘤、类癌肿瘤、血管活性肠肽瘤)、小肠(腺癌、淋巴瘤、类癌肿瘤、卡波西氏肉瘤、平滑肌瘤、血管瘤、脂肪瘤、神经纤维瘤、纤维瘤)、大肠(腺癌、管状腺瘤、绒毛腺瘤、错构瘤、平滑肌瘤)；
    泌尿生殖系统:肾(腺癌、肾母细胞瘤、淋巴瘤、白血病)、膀胱和尿道(鳞状细胞癌、移行细胞癌、腺癌)、前列腺(腺癌、肉瘤)、睾丸(精原细胞瘤、畸胎瘤、胚胎癌、畸胎瘤、绒毛膜癌、肉瘤、间质细胞癌、纤维瘤、纤维腺瘤、腺瘤样瘤、脂肪瘤)；
    肝:肝癌(肝细胞癌)、胆管癌、肝母细胞瘤、血管肉瘤、肝细胞腺瘤、血管瘤；
    胆道:胆囊癌、壶腹癌、胆管癌；
    骨:骨肉瘤(骨肉瘤)、纤维肉瘤、恶性纤维组织细胞瘤、软骨肉瘤、尤因氏肉瘤、恶性淋巴瘤(网状细胞肉瘤)、多发性骨髓瘤、恶性巨细胞瘤脊索瘤、软骨瘤(骨软骨外生骨病)、良性软骨瘤、成软骨瘤、软骨黏液纤维瘤、骨样骨瘤和巨细胞瘤；
    神经系统:颅骨(骨瘤、血管瘤、肉芽肿、黄色瘤、畸形骨炎)、脑膜(脑膜瘤、脑膜肉瘤、胶质瘤)、大脑(星形细胞瘤、髓母细胞瘤、胶质瘤、室管膜瘤、生发细胞瘤(松果体瘤)、多形胶质母细胞瘤、少突胶质母细胞瘤、神经鞘瘤、视网膜母细胞瘤、先天性肿瘤、脊髓神经纤维瘤、脑膜瘤、胶质瘤、肉瘤)；
    妇科:子宫(子宫内膜癌(浆液性囊腺癌、粘液性囊腺癌、未分类癌)、颗粒-鞘膜细胞瘤、支持间质细胞瘤、无生殖细胞瘤、恶性畸胎瘤)、外阴(鳞状细胞癌、上皮内癌、腺癌、纤维肉瘤、黑素瘤)、阴道(透明细胞癌、鳞状细胞癌、葡萄状肉瘤(胚胎型横纹肌肉瘤))、输卵管(癌)；
    血液学:血液(髓系白血病(急性和慢性)、急性淋巴母细胞白血病、慢性淋巴细胞白血病、骨髓增生性疾病、多发性骨髓瘤、骨髓增生异常综合征)、霍奇金病、非霍奇金淋巴瘤(恶性淋巴瘤)；
    皮肤:恶性黑色素瘤、基底细胞癌、鳞状细胞癌、卡波西肉瘤、痣发育不良痣、脂肪瘤、血管瘤、皮肤纤维瘤、瘢痕疙瘩、银屑病；
    肾上腺:成神经细胞瘤。
  9. 如权利要求8所述的应用，其特征在于，所述的癌症为胰腺癌、肺癌、结肠直肠癌、胆管上皮癌、多发性骨髓瘤、黑色素瘤、子宫癌、子宫内膜癌、甲状腺癌、急性髓性白血病、膀胱癌、尿路上皮癌、胃癌、子宫颈癌、头颈部鳞状细胞癌、弥漫性大B细胞淋巴瘤、食道癌、慢性淋巴细胞性白血病、肝细胞癌、乳腺癌、卵巢癌、前列腺癌、成胶质细胞瘤、肾癌或肉瘤。
  10. 一种如权利要求1-5至少一项所述的化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或如权利要求6所述的药物组合物在制备治疗或预防1型神经纤维瘤病(NF1)、努南综合征(NS)、伴有多雀斑的努南综合征(NSML)、毛细血管畸形-动静脉畸形综合征(CM-AVM)、科斯特洛综合征(CS)、心-面-皮肤综合症(CFC)、莱格斯综合征和遗传性牙龈纤维瘤病药物的应用。
  11. 一种如权利要求1-5至少一项所述的化合物，其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药，或其药学上可接受的盐，或如权利要求6所述的药物组合物在制备用于治疗和/或预防KRAS介导的疾病或病症的药物中的应用，所述的KRAS优选为突变型KRAS，所述突变型KRAS优选为KRAS G12C、KRAS G12D、KRAS G13D和KRAS G12V中的一种或多种。
PCT/CN2023/125904 2022-10-21 2023-10-23 苄基或噻吩亚甲基取代的氨基喹唑啉衍生物及其作为sos1降解剂的用途 WO2024083255A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022061348A1 (en) * 2020-09-16 2022-03-24 Biotheryx, Inc. Sos1 protein degraders, pharmaceutical compositions thereof, and their therapeutic applications
CN115043817A (zh) * 2021-03-09 2022-09-13 苏州泽璟生物制药股份有限公司 Sos1蛋白水解调节剂及其制备方法和应用
WO2022266248A1 (en) * 2021-06-16 2022-12-22 Biotheryx, Inc. Sos1 protein degraders, pharmaceutical compositions thereof, and their therapeutic applications
CN116768861A (zh) * 2022-06-23 2023-09-19 北京福元医药股份有限公司 Sos1蛋白降解靶向嵌合体及其组合物、制剂和用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022061348A1 (en) * 2020-09-16 2022-03-24 Biotheryx, Inc. Sos1 protein degraders, pharmaceutical compositions thereof, and their therapeutic applications
CN115043817A (zh) * 2021-03-09 2022-09-13 苏州泽璟生物制药股份有限公司 Sos1蛋白水解调节剂及其制备方法和应用
WO2022188819A1 (zh) * 2021-03-09 2022-09-15 苏州泽璟生物制药股份有限公司 Sos1蛋白水解调节剂及其制备方法和应用
WO2022266248A1 (en) * 2021-06-16 2022-12-22 Biotheryx, Inc. Sos1 protein degraders, pharmaceutical compositions thereof, and their therapeutic applications
CN116768861A (zh) * 2022-06-23 2023-09-19 北京福元医药股份有限公司 Sos1蛋白降解靶向嵌合体及其组合物、制剂和用途

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