WO2025242012A1 - 一类蛋白靶向降解嵌合体及其用途 - Google Patents

一类蛋白靶向降解嵌合体及其用途

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Publication number
WO2025242012A1
WO2025242012A1 PCT/CN2025/095596 CN2025095596W WO2025242012A1 WO 2025242012 A1 WO2025242012 A1 WO 2025242012A1 CN 2025095596 W CN2025095596 W CN 2025095596W WO 2025242012 A1 WO2025242012 A1 WO 2025242012A1
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mmol
piperidin
dioxopiperidin
esi
yield
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English (en)
French (fr)
Inventor
姜正羽
尤启冬
陈学涛
徐如俊
康文静
吴慧丹
吴婷婷
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China Pharmaceutical University
Ascentage Pharma Suzhou Co Ltd
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China Pharmaceutical University
Ascentage Pharma Suzhou Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/06—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • This invention belongs to the field of medicinal chemistry, specifically relating to a class of protein-targeted degradation chimeras and their uses.
  • B-cell lymphokine 6 plays a crucial role in humoral immune responses. It is a human gene of approximately 24 kb, encoding a protein of about 95 kDa. BCL6 is a member of the POZ/BTB/Zinc finger protein family and is primarily composed of three parts: 1) The N-terminal POZ/BTB domain: This is the main functional region responsible for transcriptional repression. When BCL6 exerts its transcriptional repression function, the BTB domain spontaneously forms a dimer. Three important transcriptional co-repressors, SMRT, BCOR, or NCOR, competitively bind to the BTB binding site, collectively exerting transcriptional repression.
  • the central region also known as the RD2 domain
  • the C-terminal zinc finger domain consists of six identical zinc finger structures, primarily binding to DNA and is a prerequisite for BCL6's transcriptional repression function.
  • BCL6 is a transcriptional repressor that recruits co-repressors through its BTB domain.
  • BCL6 protein is overexpressed in lymphoma, breast cancer, ovarian cancer, non-small cell lung cancer, and glioma, and is closely associated with poor patient prognosis. When BCL6 protein is highly expressed, the p53 tumor suppressor gene is almost absent. In GC responses, chromosomal translocations and point mutations of BCL6 lead to persistently high BCL6 protein expression, promoting malignant proliferation of B cells and resulting in B-cell lymphoma. Most non-Hodgkin lymphomas (NHL) originate from GC, with diffuse large B-cell lymphoma (DLBCL) being the most common subtype.
  • NHL non-Hodgkin lymphomas
  • BCL6 is considered a carcinogenic driver of DLBCL.
  • Numerous preclinical studies have also shown that blocking the interaction between the BCL6-BTB domain and its transcriptional co-repressors can inhibit GC formation and NHL cell proliferation, representing an effective and safe treatment strategy for NHL without toxic side effects or macrophage-driven inflammatory responses.
  • BCL6 is a highly promising target for cancer therapy, including but not limited to Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, T-cell non-Hodgkin's lymphoma, NK/T-cell non-Hodgkin's lymphoma, and diffuse large B-cell lymphoma.
  • BCL6 drugs in development, categorized into small molecule inhibitors (8 classes), protein-targeted degradation chimeras (5 classes), and molecular gels (5 classes).
  • BMS-986458 Only BMS's PROTAC drug (BMS-986458) has entered Phase I clinical trials. Therefore, developing novel small molecule degraders that balance druggability and safety, and can rapidly and efficiently degrade BCL6 remains urgent and necessary.
  • the purpose of this invention is to provide a class of BCL6 protein-targeted degradation chimeras and their uses.
  • L is the Linker connecting POI and E3L;
  • E3L is independently selected from:
  • R1 , R2 , R3 , R4 , R5 , R6, R7, R8, R9 , R10 , R11, R12 , R13 , R14 , R15 , R16 , R17, R18 , R19 , R20 , and R21 are independently selected from -H, halogen, -C1 to C3 alkyl, -C1 to C3 haloalkyl, and -C1 to C3 alkoxy.
  • R 22 is independently selected from -H, -C1 to -C3 alkyl groups
  • POIs are selected independently from:
  • R 23 is independently selected from -H and halogens
  • R24 and R25 are independently selected from -H and -C1 to C3 alkyl groups
  • L is selected independently from:
  • n 1, 2 or 3.
  • a compound or a pharmaceutically acceptable salt or solvate thereof showing one of the following chemical structures:
  • a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt or solvate thereof.
  • the disease is cancer.
  • the cancer is Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, T-cell non-Hodgkin lymphoma, NK/T-cell non-Hodgkin lymphoma, or diffuse large B-cell lymphoma.
  • the above pharmaceutical composition is used for the preparation of a medicament for treating a disease that is treated or alleviated by degrading BCL6 protein.
  • the disease is cancer.
  • the cancer is Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, T-cell non-Hodgkin lymphoma, NK/T-cell non-Hodgkin lymphoma, or diffuse large B-cell lymphoma.
  • a method of treating a disease comprising administering to an individual suffering from the disease a therapeutically effective amount of any of the above-mentioned compounds or a pharmaceutically acceptable salt or solvate thereof, or administering a therapeutically effective amount of the above-mentioned pharmaceutical composition; wherein the disease is a disease that is treated or alleviated by degrading BCL6 protein.
  • the disease is cancer.
  • the cancer is Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, T-cell non-Hodgkin lymphoma, NK/T-cell non-Hodgkin lymphoma, or diffuse large B-cell lymphoma.
  • the compounds provided by this invention, or their pharmaceutically acceptable salts or solvates are protein-targeting degradation chimeras with novel chemical structures. These compounds, or their pharmaceutically acceptable salts or solvates, exhibit excellent degradation activity against BCL6 protein and can be used to prepare BCL6 degrading agents. They have the potential to be developed into drugs for treating or alleviating diseases by degrading BCL6 protein. These diseases include cancers such as Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, T-cell non-Hodgkin lymphoma, NK/T-cell non-Hodgkin lymphoma, and diffuse large B-cell lymphoma.
  • Examples 1-3 were synthesized according to synthetic route 2.
  • Synthetic Route 2 Reagents and Conditions: (a) NaBH(OAc) 3 , DCE, rt, 3h; (b) H2 , Pd/C, EtOH, rt, 16h; (c) DIPEA, DMSO, 100°C, 3h; (d) TFA, DCM, rt, 1h; (e) DIPEA, NMP, 170°C, microwave, 3h.
  • Example 4 was synthesized according to synthesis route 3.
  • Synthetic Route 3 Reagents and Conditions: (a) Pd(OAc) 2 , Xantphos, Cs2CO3 , dioxane , 110°C, 10h; (b) LiOH, MeOH, H2O , 40°C, 10h; (c) EDCI, HOBt, DIPEA, DMF, 25°C, 10h; (d) TFA, DCM, rt, 1h; (e) DIPEA, NMP, 170°C, microwave, 3h.
  • Examples 5 and 6 were synthesized according to synthesis route 4.
  • Synthetic Route 4 Reagents and Conditions: (a) NaBH(OAc) 3 , DCE, rt, 12h; (b) TFA, DCM, rt, 3h; (c) DIPEA, DMSO, 100°C, 3h; (d) H2 , Pd/C, EtOH, rt, 16h; (e) DIPEA, NMP, microwave, 170°C, 3h.
  • Example 5 Following general synthesis method 5, using 34a (132 mg, 0.28 mmol) as the reactant, the target product, a yellow solid, was obtained in Example 5 (18 mg, yield: 9.1%).
  • Examples 7-9 were synthesized according to synthetic route 5.
  • Synthetic Route 5 Reagents and Conditions: (a) NaBH(OAc) 3 , DCE, rt, 3h; (b) H2 , Pd/C, EtOH, rt, 16h; (c) DIPEA, DMSO, 100°C, 3h; (d) TFA, DCM, rt, 1h; (e) DIPEA, DMSO, 100°C, 10h; (f) DIPEA, EDCI, HOBt, DMF, rt, 12h.
  • Examples 10-11 were synthesized according to synthetic route 6.
  • Synthetic Route 6 Reagents and Conditions: (a) ( PPh3 ) 3 RhCl, EtOH, 80°C, 18h; (b) KI, pyridine, 115°C, 12h; (c) TFA, DCM, rt, 3h; (d) AcOH, NaBH(OAc) 3 , DCE, rt, 3h; (e) TFA, DCM, rt, 3h; (f) EDCI, HOBt, DIPEA, DMF, rt, 12h.
  • Examples 12-16 were synthesized according to synthetic route 7.
  • Synthetic Route 7 Reagents and Conditions: (a) DIPEA, DMSO, 100°C, 3h; (b) TFA, DCM, rt, 1h; (c) NaBH(OAc) 3 , DCE, rt, 5h; (d) TFA, DCM, rt, 1h; (e) DIPEA, EDCI, HOBt, DMF, rt, 12h.
  • Examples 17-18 were synthesized according to synthetic route 8.
  • Synthetic Route 8 Reagents and Conditions: (a ) Cs2CO3 , Xantphos, Pd2 (dba) 3 , dioxane, 100°C for 4h; ( b) Cs2CO3, PdCl2(dppf)CH2Cl2, dioxane, 100°C for 4h; (c) H2 , Pd / C , THF, 12h; (d) TFA, DCM, 3h; (e) NaBH(OAc) 3 , DCE, 6h; (f) TFA, DCM, 3h; (g) EDCI, HOBt, DIPEA, DMF, 12h.
  • Example 19 was synthesized according to synthetic route 9.
  • Synthetic Route 9 Reagents and Conditions: (a) K2CO3 , DMF, 60°C, 4h; (b ) H2 , Pd/C, EtOH, rt, 6h; (c) NaHCO3 , DMF, 80°C, 16h; (d) TFA, DCM, rt, 3h; (e) NaBH(OAc) 3 , DCE, rt, 5h; (f) TFA, DCM, rt, 2h; (g) DIPEA, EDCI, HOBt, rt, 12h.
  • Synthetic Method 13 55 (400 mg, 2.51 mmol), 47b (604 mg, 2.51 mmol), and potassium carbonate (695 mg, 5.03 mmol) were added to a round-bottom flask, followed by 10 mL of DMF solution. The mixture was stirred at 60 °C for 4 h, and the reaction was monitored by TLC until complete. After cooling to room temperature, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • Synthetic Method 14 57 (800 mg, 2.29 mmol), 57a (1.10 g, 5.72 mmol), and sodium bicarbonate (1.92 g, 22.90 mmol) were added to a round-bottom flask, followed by 15 mL of DMF solution. The mixture was stirred at 85 °C for 16 h, and the reaction was monitored by TLC until complete. After cooling to room temperature, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • Example 20 was synthesized according to synthesis route 10.
  • Synthetic Route 10 Reagents and Conditions: (a) K2CO3 , DMF, 60°C, 4h; (b ) H2 , Pd/C, EtOH, rt, 6h; (c) NaHCO3 , DMF, 80°C, 16h; (d) TFA, DCM, rt, 3h; (e) DIPEA, EDCI, HOBt, rt, 12h.
  • Example 21 was synthesized according to synthetic route 11.
  • Synthetic Route 11 Reagents and Conditions: (a) Cs2CO3 , Xantphos , Pd(OAc) 2 , dioxane, 110°C, 10h; (b) LiOH, MeOH, H2O , rt, 12h; (c) EDCI, HOBt, TEA, DMF, 12h; (d) TFA, DCM, 3h; (e) NaBH(OAc) 3 , DCE, 6h; (f) TFA, DCM, 3h; (g) EDCI, HOBt, DIPEA, DMF, 12h.
  • intermediate 63 (200 mg, 0.46 mmol) was added to a round-bottom flask containing a methanol:water mixture of 8 mL:2 mL, followed by lithium hydroxide (55 mg, 2.30 mmol). The reaction was carried out at 40 °C for 10 hours, and TLC was used to monitor the completeness of the reaction. After the reaction cooled to room temperature, dilute hydrochloric acid was added to adjust the pH of the solution to 5–6. The mixture was extracted with ethyl acetate, and the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • intermediate 64 (180 mg, 0.43 mmol) was added to a round-bottom flask containing 5 mL of LDM solution, followed by DIPEA (276 mg, 2.14 mmol), EDCI (123 mg, 0.64 mmol), and HOBt (87 mg, 0.64 mmol). The mixture was stirred at room temperature for half an hour, then 27a (83 mg, 0.51 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. The reaction was monitored by TLC until complete. Extraction was performed with ethyl acetate, and the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • Example 22 was synthesized according to synthetic route 12.
  • Synthetic Route 12 Reagents and Conditions: (a) DIPEA, DMSO, 100°C, 10h; (b) Dys-Martin oxidant, DMF, 100°C, 2h; (c) NaBH(OAc) 3 , DCE, 6h.
  • Example 23 was synthesized according to synthetic route 13.
  • Synthetic Route 13 Reagents and Conditions: (a ) Na2CO3 , Pd(PPh) 3Cl2 , dioxane , 110°C, 4h; (b) Pd/C, EtOH, rt, 16h; (c) LiOH, MeOH, H2O , rt, 16h; (d) BuLi, THF, DMF, -78°C, 6h; (e) K2CO3 , DMF, rt, 5h; ( f ) NaBH3CN , DIPEA, DCE; (g) TFA, DCM, 3h; (h) Dimethylchlorosilane, ACN, 6h; (i) Pd/C, H2 , rt, 12h; Boc2O , EtOH, THF, rt, 3h; ( j ) Tf2O , TEA, DCM, rt, 3h; (k) K2CO3 ,DMF,70°C,4
  • Example 24 was synthesized according to synthetic route 14.
  • Synthetic Route 14 Reagents and Conditions: (a) N,N-dimethyl-bromoacetamide, Cs2CO3 , DMF , rt, 3h; (b) Fe, NH4Cl , EtOH, H2O , 80°C, 3h; (c) DIPEA, DMSO, 100°C, 3h; (d) DIPEA, DMSO, 100°C, 10h; (e) DIPEA, EDCI, HOBt, rt, 12h.
  • BCL6 protein degradation assay OCI-LY1 cells (Zhejiang Meisen Cell Technology Co., Ltd.) were seeded in 6-well plates, and different concentrations of the compound were added. Twelve hours after drug administration, cells were collected by centrifugation, mixed with medium-efficiency RIPA lysis buffer (Beyotime Biotechnology), and centrifuged again. The supernatant was collected, and protein concentration was determined by the BCA method. Protein samples were mixed with protein loading buffer (Beyotime Biotechnology) and heated dry at 100°C for 10 min for sample preparation. The samples were then added to a 12% polyacrylamide gel SDS-PAGE. Electrophoresis was performed at 60V until the marker left the stacking gel, followed by continued electrophoresis at 120V.
  • the membrane was transferred to a wet transfer buffer containing 10% methanol for 90 min.
  • the transferred PVDF membrane was then cut into desired bands, blocked with milk for 2 hours, and anti-BCL6 antibody (abcam) and ⁇ -Actin antibody (Proteintech) diluted with milk were added to the corresponding bands, respectively, and incubated overnight at 4°C.
  • the primary antibody was washed away with TBST, and the membrane was incubated with secondary antibody at room temperature for 45 min.
  • the secondary antibody solution was then washed away again with TBST, and the membrane was scanned using an Odyssey Infrared Imaging System (LI-COR, Lincoln, Iowa, USA).
  • DC 50 refers to the concentration of the BCL6 degrading agent required to achieve 50% degradation of BCL6 protein.
  • the calculation process is as follows: Starting from 1000 nM, the degrading agent was serially diluted 5-fold to nine concentrations, and the degradation of BCL6 protein at each concentration was detected by Western blotting. Grayscale analysis was performed using ImageJ software to calculate the remaining amount of BCL6. The DC 50 value was obtained by fitting the logarithm (Log(C)) of the remaining protein amount and concentration using Graphpad 8.0 software.
  • Cell antiproliferative activity assay Cells in logarithmic growth phase were seeded at a density of 5000 cells per well in 96-well plates (Coring, 3799) with 100 ⁇ L of RPMI 1640 medium (Adamas) containing 20% FBS and incubated overnight at 37°C in a 5% CO2 incubator. The next day, 100 ⁇ L of different concentrations of drug solution prepared in the medium were added, with three replicates for each concentration (denoted as RLU test ), and control and blank wells were also included. The control wells contained cells, culture medium, and the same concentration of drug solution (denoted as RLU control ), while the blank wells contained culture medium (denoted as RLU blank ).

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Abstract

公开了一类蛋白靶向降解嵌合体及其用途，该类蛋白靶向降解嵌合体化学结构新颖，且对BCL6蛋白具有优异的降解作用，可以用于制备BCL6降解剂药物，具有开发成治疗通过降解BCL6蛋白而得到治疗或缓解的疾病的药物的前景，所述通过降解BCL6蛋白而得到治疗或缓解的疾病包括霍奇金淋巴瘤、B细胞源性非霍奇金淋巴瘤、T细胞源性非霍奇金淋巴瘤、NK/T细胞源性非霍奇金淋巴瘤和弥漫性大B细胞淋巴瘤等癌症。

Description

一类蛋白靶向降解嵌合体及其用途 技术领域
本发明属于药物化学领域，具体涉及一类蛋白靶向降解嵌合体及其用途。
背景技术
B细胞淋巴因子6(BCL6)在体液免疫反应中扮演重要角色，是一个约为24kb的人类基因，编码大约95kD的BCL6蛋白。BCL6蛋白是POZ/BTB/Zinc finger蛋白家族的成员之一，主要由三个部分组成：1)、氨基端的POZ/BTB结构域：这是发挥转录抑制作用的主要功能区域。BCL6发挥转录抑制作用时，BTB结构域会自发形成二聚体，三个重要的转录共抑制因子SMRT、BCOR或NCOR通过竞争性结合方式结合到BTB结合位点，共同发挥转录抑制作用。这个过程参与了生发中心(GC)形成早期的转录调控。2)、中部区域(也称为RD2结构域)包含三个PEST结构域，其主要作用是招募MTA3或CTBP1等辅助因子，起到维持蛋白稳定的作用。3)、羧基端的锌指结构域由6个相同的锌指结构组成，主要发挥结合DNA的作用，是BCL6发挥转录抑制作用的前提条件。
BCL6是一种转录抑制因子，通过其BTB结构域招募共抑制因子。BCL6蛋白在淋巴瘤、乳腺癌、卵巢癌、非小细胞肺癌及胶质瘤中过度表达，并与患者预后不良密切相关。当BCL6蛋白高表达时，p53抑癌基因几乎是缺失的。在GC反应中，BCL6的染色体易位和点突变会导致BCL6蛋白持续高表达，促使B细胞发生恶性增殖，进而导致B细胞淋巴瘤的发生。大多数非霍奇金淋巴瘤(NHL)起源于GC，其中弥漫大B细胞淋巴瘤(DLBCL)是最常见的亚型，BCL6被认为是DLBCL的致癌驱动因子。许多临床前研究也表明，阻断BCL6-BTB结构域与其转录共抑制因子之间的相互作用可以抑制GC形成和NHL细胞增殖，这是治疗NHL的有效策略，并且具有良好的安全性，不会引起毒副作用和由巨噬细胞驱动的炎症反应。
BCL6是极具潜力的癌症治疗靶标，包括但不限于霍奇金淋巴瘤、B细胞源性非霍奇金淋巴瘤、T细胞源性非霍奇金淋巴瘤、NK/T细胞源性非霍奇金淋巴瘤或弥漫性大B细胞淋巴瘤。目前在研的BCL6药物约18种，分别为小分子抑制剂(8类)、蛋白靶向降解嵌合体(5类)和分子胶(5类)，仅BMS的PROTAC药物(BMS-986458)进入Ⅰ期临床研究。因此，开发结构新颖、兼顾良好成药性与安全性、能够快速高效地降解BCL6的小分子降解剂仍然十分迫切与必要。
发明内容
本发明的目的提供一类BCL6蛋白靶向降解嵌合体及其用途。
本发明上述目的通过如下技术方案实现：
一种化学结构如式Ⅰ所示的化合物或其药学上可接受的盐、溶剂化物：
POI-L-E3L
Ⅰ
其中：
L为连接POI和E3L的Linker；
E3L独立地选自：
其中：
R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21独立地选自-H、卤素、-C1～C3烷基、-C1～C3卤代烷基、-C1～C3烷氧基；
R22独立地选自-H、-C1～C3烷基；
POI独立地选自：
其中：
R23独立地选自-H、卤素；
R24、R25独立地选自-H、-C1～C3烷基；
L独立地选自：
其中：
n＝1、2或3。
一种如下化学结构之一所示的化合物或其药学上可接受的盐、溶剂化物：


一种药物组合物，含有上述化合物或其药学上可接受的盐、溶剂化物。
上述化合物或其药学上可接受的盐、溶剂化物用于制备BCL6蛋白降解剂的用途。
上述化合物或其药学上可接受的盐、溶剂化物用于制备治疗疾病的药物的用途，所述疾病为通过降解BCL6蛋白而得到治疗或缓解的疾病。
优选地，所述疾病为癌症。
更优选地，所述癌症为霍奇金淋巴瘤、B细胞源性非霍奇金淋巴瘤、T细胞源性非霍奇金淋巴瘤、NK/T细胞源性非霍奇金淋巴瘤或弥漫性大B细胞淋巴瘤。
上述药物组合物用于制备治疗疾病的药物的用途，所述疾病为通过降解BCL6蛋白而得到治疗或缓解的疾病。
优选地，所述疾病为癌症。
更优选地，所述癌症为霍奇金淋巴瘤、B细胞源性非霍奇金淋巴瘤、T细胞源性非霍奇金淋巴瘤、NK/T细胞源性非霍奇金淋巴瘤或弥漫性大B细胞淋巴瘤。
一种治疗疾病的方法，向患有该疾病的个体施用治疗有效量的上述任一化合物或其药学上可接受的盐、溶剂化物，或施用治疗有效量的上述药物组合物；其中，所述疾病为通过降解BCL6蛋白而得到治疗或缓解的疾病。
优选地，所述疾病为癌症。
更优选地，所述癌症为霍奇金淋巴瘤、B细胞源性非霍奇金淋巴瘤、T细胞源性非霍奇金淋巴瘤、NK/T细胞源性非霍奇金淋巴瘤或弥漫性大B细胞淋巴瘤。
有益效果：
本发明提供的化合物或其药学上可接受的盐、溶剂化物为一种蛋白靶向降解嵌合体，其化学结构新颖；该类化合物或其药学上可接受的盐、溶剂化物对BCL6蛋白具有优异的降解作用，可以用于制备BCL6降解剂药物，具有开发成治疗通过降解BCL6蛋白而得到治疗或缓解的疾病的药物的前景，所述通过降解BCL6蛋白而得到治疗或缓解的疾病包括霍奇金淋巴瘤、B细胞源性非霍奇金淋巴瘤、T细胞源性非霍奇金淋巴瘤、NK/T细胞源性非霍奇金淋巴瘤和弥漫性大B细胞淋巴瘤等癌症。
具体实施方式
下面结合实施例具体介绍本发明实质性内容，但并不以此限定本发明的保护范围。
合成路线1：
中间体11按照合成路线1合成。
合成路线1.试剂和条件:(a)1,2-二溴乙烷,K2CO3,DMF,rt,3h；(b)BBr3,DCM,0℃,4h；(c)Cs2CO3,CH3CN,60℃,1h；(d)KNO3,H2SO4,0℃,4h；(e)重氮基乙酸乙酯,DBU,EtOH,DMF,rt,16h；(f)NaOH,H2O,115℃,24h；(g)2-溴-N-甲基乙酰胺,Cs2CO3,DMF,rt,3h；(h)Fe,NH4Cl,EtOH,H2O,80℃,3h；(i)DIPEA,DMSO,100℃,3h.
中间体11按照合成路线1合成。
1-(2-溴乙基)-7-甲氧基吲哚啉-2，3-二酮(2)
1-(2-bromoethyl)-7-methoxyindoline-2,3-dione(2)
将1a(5.00g,28.22mmol)，1,2-二溴乙烷1b(10.6g,56.45mmol)，碳酸钾(7.80g,56.45mmol)加入到圆底烧瓶中，加入35mL的DMF溶液，室温搅拌3h后TLC监测反应完全结束。加入350mL的水搅拌半小时，抽滤，水洗，干燥得红色固体中间体2(6.80g，产率：84.8％)。1H NMR(300MHz,DMSO-d6)δ7.45(d,J＝8.1Hz,1H),7.16(dt,J＝15.4,7.4Hz,2H),4.20(t,J＝6.9Hz,2H),3.91(s,3H),3.66(t,J＝6.8Hz,2H)。ESI-MS:m/z:[M+H]+283.98。
1-(2-溴乙基)-7-羟基吲哚啉-2，3-二酮(3)
1-(2-bromoethyl)-7-hydroxyindoline-2,3-dione(3)
将中间体2(0.30g，1.06mmol)至加入含有6mL二氯甲烷溶液的圆底烧瓶中，冰浴30min，再缓慢滴加三溴化硼(0.79g，3.17mmol)，1h后撤去冰浴，室温反应2h，TLC监测反应完全结束。将反应冰浴半小时，缓慢滴加无水甲醇，待反应液无白色烟雾产生停止滴加，低压浓缩，残留物经过柱层析(洗脱体系为石油醚：乙酸乙酯＝4：1，v/v)纯化得中间体3，为红色固体(0.16g，产率：56.1％)。1H NMR(300MHz,DMSO-d6)δ10.48(s,1H),7.17(dd,J＝8.0,1.3Hz,1H),7.09(dd,J＝7.4,1.3Hz,1H),7.03–6.95(m,1H),4.21(t,J＝6.9Hz,2H),3.70(t,J＝6.9Hz,2H)。ESI-MS:m/z:[M+H]+269.97。
2，3-二氢-[1，4]噁嗪并[2，3，4-hi]吲哚-5，6-二酮(4)
2,3-dihydro-[1,4]oxazino[2,3,4-hi]indole-5,6-dione(4)
将中间体3(1.00g，3.70mmol)加入至圆底烧瓶中，加入30mL乙腈溶液，随后加入碳酸铯(2.41g，7.41mmol)，60℃加热反应1h，TLC监测反应结束。撤去加热，待反应液冷却至室温，抽滤，乙酸乙酯洗，浓缩，残留物经过柱层析(洗脱体系为石油醚：乙酸乙酯＝4：1，v/v)纯化得中间体4，为红色固体(0.57g，产率：81.8％)。1H NMR(300MHz,DMSO-d6)δ7.24–7.11(m,2H),6.99(t,J＝7.8Hz,1H),4.32(t,J＝4.7Hz,2H),3.79(t,J＝4.9Hz,2H)。ESI-MS:m/z:[M+H]+190.04。
2，3-二氢-[1，4]噁嗪并[2，3，4-hi]吲哚-5，6-二酮(5)
2,3-dihydro-[1,4]oxazino[2,3,4-hi]indole-5,6-dione(5)
将中间体4(2.40g，12.69mmol)加入至圆底烧瓶中，冰浴搅拌15min，再缓慢加入20mL浓硫酸溶液，冰浴搅拌0.5h，随后缓慢加入硝酸钾(1.28g，12.69mmol)，室温反应6h，TLC监测反应结束。将反应溶液缓慢倒入搅拌的冰水中，加入乙酸乙酯，萃取三遍，合并有机相，减压浓缩，残留物经过柱层析(洗脱体系为石油醚：乙酸乙酯＝4：1，v/v)纯化得中间体5，为橙红色固体(1.80g，产率：60.6％)。1H NMR(300MHz,DMSO-d6)δ8.06(d,J＝2.0Hz,1H),7.98(d,J＝2.0Hz,1H),4.41(t,J＝4.7Hz,2H),3.86(t,J＝4.6Hz,2H)。ESI-MS:m/z:[M+H]+235.03。
6-羟基-9-硝基-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-7-羧酸乙酯(6)
Ethyl-6-hydroxy-9-nitro-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinoline-7-carboxylate(6)
将中间体5(1.10g,4.70mmol)加入至圆底烧瓶中，加入EtOH:DMF＝20mL:4mL的混合溶剂，氩气置换，迅速加入DBU(107mg,0.70mmol)，氩气置换，溶液迅速变稠，待溶液由橙红色变为棕黄色(约15分钟)，再迅速加入重氮乙酸乙酯(1.07g,9.40mmol)，氩气置换，室温搅拌16h。TLC监测反应结束后，减压浓缩除去大部分乙醇，用滴液漏斗缓慢滴加稀盐酸(16mL,1.2M)，再加入20mL水，室温搅拌2小时。加入乙酸乙酯，萃取两遍，合并有机相，减压浓缩得粗品中间体6，为橙黄色油状物(1.40g,产率：93.0％)。ESI-MS:m/z:[M+H]+321.06。
6-羟基-9-硝基-2，3-二氢-5H-[1，4]噁嗪并[2，3，4-ij]喹啉-5-酮(7)
6-hydroxy-9-nitro-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-5-one(7)
将中间体6(1.40g，4.40mmol)加入至圆底烧瓶中，加入氢氧化钠固体(0.35g，8.80mmol)，再加入44mL水，115℃搅拌24h，TLC监测反应结束。缓慢滴加1M的稀盐酸溶液调节pH为2～3，抽滤，水洗，得滤饼，干燥得粗品中间体7，为黄色固体(0.78g，产率：71.9％)。ESI-MS:m/z:[M+H]+249.05。
N-甲基-2-(9-硝基-5-氧代-2，3-二氢-5H-[1，4]噁嗪并[2，3，4-ij]喹啉-6-基)氧基)乙酰胺(8)
N-methyl-2-((9-nitro-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)acetamide(8)
将中间体7(1.00g，4.03mmol)加入至圆底烧瓶中，再加入7a(0.74g，4.83mmol)，以及碳酸铯(2.63g，8.06mmol)，再加入DMF(15mL)，室温搅拌4h，TLC监测反应结束。先加入50mL的水，抽滤，水洗，滤饼烘干，向滤液中加入等体积的乙酸乙酯和水分液萃取3次，合并有机层并低压浓缩除去溶剂，残留物与滤饼合并，经过柱层析(洗脱体系为二氯甲烷：甲醇＝60：1，v/v)纯化得中间体8，为白色固体(0.48g，产率：37.3％)。1H NMR(300MHz,DMSO-d6)δ8.27(d,J＝2.5Hz,1H),7.96(s,1H),7.75(d,J＝2.5Hz,1H),7.54(s,1H),4.62(s,2H),4.48(t,J＝4.8Hz,2H),4.25(t,J＝4.8Hz,2H),2.69(d,J＝4.6Hz,3H)。ESI-MS:m/z:[M+H]+320.08。
2-(9-氨基-5-氧代-2，3-二氢-5H-[1，4]噁嗪并[2，3，4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(9)
2-((9-amino-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(9)
将8(0.40g，1.25mmol)加入至含有无水乙醇：水＝20mL:4mL的圆底烧瓶中，加入氯化铵(0.35g，6.26mmol)，在搅拌的状态下加入还原铁粉(0.34g，6.26mmol)，80℃加热反应3h，TLC监测反应结束。撤去加热，待反应液冷却至室温，加入2mL氨甲醇溶液调节溶液pH值大于7，硅藻土抽滤，二氯甲烷：甲醇＝20：1(1000mL)洗，收集滤液，低压浓缩，得到淡黄色固体9(0.24g，产率：67.0％)。ESI-MS:m/z:[M+H]+290.11。
2-((9-((2,5-二氯嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(11)
2-((9-((2,5-dichloropyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(11)
将中间体9(0.40g，1.38mmol)、原料10(0.51g，2.77mmol)、DIPEA(0.54g，4.15mmol)加入到圆底烧瓶中，加入8mL的DMSO溶液，100℃搅拌3h后TLC监测反应完全结束。加入35mL的水搅拌半小时，抽滤，水洗，干燥得淡黄色固体中间体11(0.43g，产率：71.6％)。ESI-MS:m/z:[M+H]+436.04。
合成路线2：
实施例1～3按照合成路线2合成。
合成路线2:试剂和条件:(a)NaBH(OAc)3,DCE,rt,3h；(b)H2,Pd/C,EtOH,rt,16h；(c)DIPEA,DMSO,100℃,3h；(d)TFA,DCM,rt,1h；(e)DIPEA,NMP,170℃，微波，3h.
4-((1-((苄氧基)羰基)哌啶-4-基)甲基)哌嗪-1-羧酸叔丁酯(21)
tert-butyl 4-((1-((benzyloxy)carbonyl)piperidin-4-yl)methyl)piperazine-1-carboxylate(21)
合成通法1：将中间体20a(151mg，0.81mmol)与20b(100mg，0.40mmol)加入至含有4mL无水1，2-二氯乙烷溶液的圆底烧瓶中，室温搅拌15分钟，再加入NaBH(OAc)3(171mg，0.81mmol)，室温搅拌过夜，TLC监测反应完全结束。将反应液低压浓缩，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体21，为白色固体(201mg，产率：59.4％)。1H NMR(300MHz,DMSO-d6)δ7.36(qd,J＝6.7,2.6Hz,5H),5.08(s,2H),3.32(dd,J＝12.5,7.6Hz,6H),2.28(t,J＝5.0Hz,4H),2.13(d,J＝6.8Hz,2H),2.01(s,1H),1.70(d,J＝12.1Hz,3H),1.41(s,9H),1.28–1.16(m,1H).ESI-MS:m/z:[M+H]+418.26。
4-(哌啶-4-基甲基)哌嗪-1-羧酸叔丁酯(22)
tert-butyl 4-(piperidin-4-ylmethyl)piperazine-1-carboxylate(22)
合成通法2：将中间体21(201mg，0.48mmol)溶于含有5mL无水乙醇溶液的圆底烧瓶中，加入钯碳(30mg，15％m/m)，氢气置换，室温搅拌16小时，TLC监测反应完全结束。反应液经硅藻土抽滤，二氯甲烷洗涤，滤液低压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体22，为白色油状物(108mg，产率：79.2％)。ESI-MS:m/z:[M+H]+283.23。
4-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-4-基)哌啶-4-基)甲基)哌嗪-1-羧酸叔丁酯(24a)
tert-butyl-4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperidin-4-yl)methyl)piperazine-1-carboxylate(24a)
合成通法3：将中间体23a(400mg，1.45mmol)与22(534mg，1.88mmol)加入至含有6mLDMSO溶液的圆底烧瓶中，再加入DIPEA(374mg，2.90mmol)，100℃搅拌3小时，TLC监测反应完全结束。待反应冷却至室温后，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体24a，为黄色固体(690mg，产率：88.3％)。1H NMR(300MHz,DMSO-d6)δ11.13(s,1H),7.69(dd,J＝8.5,7.1Hz,1H),7.34(dd,J＝8.0,4.1Hz,2H),5.11(dd,J＝13.1,5.7Hz,1H),3.70(d,J＝11.7Hz,1H),2.88(s,2H),2.56(s,6H),2.32(s,3H),2.21(d,J＝6.8Hz,1H),2.03(d,J＝8.9Hz,1H),1.83(d,J＝13.2Hz,2H),1.41(s,9H),1.37–1.23(m,3H).ESI-MS:m/z:[M+H]+540.27。
4-((1-(2-(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)甲基)哌嗪-1-羧酸叔丁酯(24b)
tert-butyl-4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperazine-1-carboxylate(24b)
按合成通法3，以23b(400mg，1.45mmol)为反应原料，得到目标产物黄色固体24b(690mg，产率：88.3％)。1H NMR(300MHz,DMSO-d6)δ7.68(dd,J＝8.5,7.1Hz,1H),7.33(dd,J＝7.8,5.4Hz,2H),5.09(dd,J＝12.7,5.3Hz,1H),2.88(t,J＝12.4Hz,3H),2.62(s,1H),2.56(s,1H),2.43(s,3H),2.23(d,J＝6.8Hz,2H),2.02(d,J＝11.4Hz,1H),1.81(t,J＝16.9Hz,3H),1.42–1.30(m,2H),1.25(s,9H),1.24(d,J＝4.4Hz,1H).ESI-MS:m/z:[M+H]+540.27。
4-((1-(2-(2-(2,6-二氧代哌啶-3-基)-6-氟-1,3-二氧代异吲哚啉-5-基)哌啶-4-基)甲基)哌嗪-1-羧酸叔丁酯(24c)
tert-butyl-4-((1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperazine-1-carboxylate(24c)
按合成通法3，以23c(400mg，1.36mmol)为反应原料，得到目标产物黄色固体24c(670mg，产率：91.3％)。1H NMR(300MHz,DMSO-d6)δ11.15(s,1H),7.73(d,J＝11.4Hz,1H),7.45(d,J＝7.4Hz,1H),5.12(dd,J＝12.9,5.3Hz,1H),3.61(d,J＝12.0Hz,2H),2.88(dd,J＝15.4,7.6Hz,3H),2.65–2.53(m,2H),2.30(d,J＝6.2Hz,4H),2.19(d,J＝6.8Hz,2H),2.07–2.00(m,1H),1.83(d,J＝13.3Hz,2H),1.41(d,J＝4.4Hz,10H),1.26(d,J＝9.7Hz,2H).ESI-MS:m/z:[M+H]+558.26。
2-(2,6-二氧代哌啶-3-基)-4-(4-(哌嗪-1-基甲基)哌啶-1-基)异吲哚啉-1,3-二酮(25a)
2-(2,6-dioxopiperidin-3-yl)-4-(4-(piperazin-1-ylmethyl)piperidin-1-yl)isoindoline-1,3-dione(25a)
合成通法4：将中间体24a(300mg，0.56mmol)加入至含有5mL二氯甲烷溶液的圆底烧瓶中，再加入三氟乙酸(1.5mL)，室温搅拌1小时，TLC监测反应完全结束。减压浓缩，得中间体25a，为黄色油状物(230mg，产率：94.1％)。ESI-MS:m/z:[M+H]+440.22。
2-(2,6-二氧代哌啶-3-基)-5-(4-(哌嗪-1-基甲基)哌啶-1-基)异吲哚啉-1,3-二酮(25b)
2-(2,6-dioxopiperidin-3-yl)-5-(4-(piperazin-1-ylmethyl)piperidin-1-yl)isoindoline-1,3-dione(25b)
按合成通法4，以24b(300mg，0.56mmol)为反应原料，得到目标产物黄色油状物25b(230mg，产率：94.1％)。ESI-MS:m/z:[M+H]+440.22。
2-(2,6-二氧代哌啶-3-基)-5-氟-6-(4-(哌嗪-1-基甲基)哌啶-1-基)异吲哚啉-1,3-二酮(25c)
2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(4-(piperazin-1-ylmethyl)piperidin-1-yl)isoindoline-1,3-dione(25c)
按合成通法4，以24c(300mg，0.54mmol)为反应原料，得到目标产物黄色油状物25c(210mg，产率：85.3％)。ESI-MS:m/z:[M+H]+458.21。
实施例1
2-((9-((5-氯-2-(4-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-4-基)哌啶-4-基)甲基)哌嗪-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例1)
2-((9-((5-chloro-2-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例1)
合成通法5：将中间体25a(121mg，0.28mmol)与11(60mg，0.14mmol)加入至含有5mL NMP溶液的圆底烧瓶中，再加入DIPEA(71mg，0.55mmol)，170℃微波反应3小时，TLC监测反应完全结束。待反应冷却至室温后，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过制备层析(洗脱体系为二氯甲烷：甲醇＝20：1，v/v)纯化得目标产物实施例1，为黄色固体(18mg，产率：15.6％)。1H NMR(300MHz,DMSO-d6)δ11.12(s,1H),8.79(s,1H),8.03(d,J＝13.0Hz,2H),7.75–7.54(m,2H),7.32(d,J＝10.8Hz,3H),7.10(s,1H),5.09(dd,J＝13.6,5.2Hz,1H),4.60(s,2H),4.38(s,2H),4.16(s,2H),3.66(s,6H),2.84(d,J＝12.9Hz,3H),2.66(d,J＝4.4Hz,6H),2.40(s,3H),2.22(s,2H),2.01(d,J＝9.9Hz,1H),1.83(d,J＝13.1Hz,3H),1.27(d,J＝31.9Hz,2H).13C NMR(75MHz,DMSO-d6)δ173.38,170.60,167.93,167.61,159.58,155.68,155.15,147.59,142.95,136.25,134.43,134.15,124.42,120.38,118.91,116.78,114.88,113.51,112.48,109.28,102.71,68.34,64.20,53.42,51.49,49.23,44.34,32.53,31.47,30.99,25.93,22.57.HRMS(ESI):calcd for C41H43ClN10O8[M+H]+839.2954,found 839.3023.纯度：96.63％by HPLC(MeOH/H2O＝80:20,tR＝4.042min)。
实施例2
2-((9-((5-氯-2-(4-((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)甲基)哌嗪-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例2)
2-((9-((5-chloro-2-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例2)
按合成通法5，以25b(121mg，0.28mmol)为反应原料，得到目标产物黄色固体实施例2(18mg，产率：15.6％)。1H NMR(300MHz,DMSO-d6)δ11.10(s,1H),8.78(s,1H),8.07(s,1H),7.98(d,J＝4.8Hz,1H),7.71–7.58(m,2H),7.41–7.30(m,2H),7.24(d,J＝8.6Hz,1H),7.12(s,1H),5.08(dd,J＝12.7,5.4Hz,1H),4.61(s,2H),4.40(t,J＝4.7Hz,2H),4.19(d,J＝4.6Hz,2H),4.06(d,J＝12.9Hz,2H),3.66(s,4H),2.94(dt,J＝24.8,12.4Hz,3H),2.70–2.55(m,4H),2.41(s,3H),2.19(s,2H),2.10–1.99(m,1H),1.84(d,J＝13.2Hz,2H),1.31–1.12(m,3H).13C NMR(75MHz,DMSO-d6)δ173.31,170.62,168.14,167.94,167.46,159.61,155.71,155.50,155.15,155.07,147.63,142.96,134.54,134.45,125.50,120.38,118.96,118.07,117.84,113.69,112.47,109.30,108.23,102.73,68.45,64.22,53.40,49.24,47.76,44.37,40.74,32.89,31.50,30.13,25.92,22.70.HRMS(ESI):calcd for C41H43ClN10O8[M+H]+839.2954,found 839.3026.纯度：98.43％by HPLC(MeOH/H2O＝80:20,tR＝4.012min)。
实施例3
2-((9-((5-氯-2-(4-((1-(2-(2,6-二氧代哌啶-3-基)-6-氟-1,3-二氧代异吲哚啉-5-基)哌啶-4-基)甲基)哌嗪-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例3)
2-((9-((5-chloro-2-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例3)
按合成通法5，以25c(126mg，0.28mmol)为反应原料，得到目标产物黄色固体实施例3(21mg，产率：17.8％)。1H NMR(300MHz,DMSO-d6)δ11.13(s,1H),8.80(s,1H),8.07(s,1H),7.99(d,J＝4.8Hz,1H),7.72(d,J＝11.4Hz,1H),7.61(d,J＝2.2Hz,1H),7.45(d,J＝7.4Hz,1H),7.36(d,J＝2.1Hz,1H),7.13(s,1H),5.11(dd,J＝13.0,5.3Hz,1H),4.61(s,2H),4.40(s,2H),4.18(s,2H),3.63(d,J＝14.9Hz,6H),2.91(t,J＝12.3Hz,3H),2.67(d,J＝4.6Hz,3H),2.61(d,J＝14.8Hz,2H),2.42(s,3H),2.23(s,2H),2.03(d,J＝7.8Hz,1H),1.86(d,J＝12.6Hz,2H),1.25(s,4H).13C NMR(75MHz,DMSO-d6)δ173.27,170.43,167.95,167.21,166.72,155.73,155.16,155.07,147.63,142.96,134.43,129.30,120.39,118.98,113.68,112.51,109.32,102.79,68.44,64.22,53.34,50.40,49.54,44.24,31.46,30.71,25.93,22.58.HRMS(ESI):calcd for C41H42ClFN10O8[M+H]+857.2860,found 857.2930。纯度：95.79％by HPLC(MeOH/H2O＝80:20,tR＝4.172min)。
合成路线3：
实施例4按照合成路线3合成。
合成路线3:试剂和条件:(a)Pd(OAc)2,Xantphos,Cs2CO3,二氧六环,110℃,10h；(b)LiOH,MeOH,H2O,40℃,10h；(c)EDCI,HOBt,DIPEA,DMF,25℃,10h；(d)TFA,DCM,rt,1h；(e)DIPEA,NMP,170℃,微波,3h.
4-((1-(3-氟-4-(甲氧羰基)苯基)哌啶-4-基)甲基)哌嗪-1-羧酸叔丁酯(26)
tert-butyl-4-((1-(3-fluoro-4-(methoxycarbonyl)phenyl)piperidin-4-yl)methyl)piperazine-1-carboxylate(26)
合成通法6：将中间体22a(360mg，1.54mmol)与22(482mg，1.70mmol)加入至含有10mL无水二氧六环溶液的封管中，再依次加入碳酸铯(1.01g，3.09mmol)、醋酸钯(17mg，0.08mmol)、XantPhos(89mg，0.15mmol)，氩气置换，110℃反应16小时，TLC监测反应完全结束。待反应冷却至室温后，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过制备层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体26，为白色固体(390mg，产率：58.0％)。ESI-MS:m/z:[M+H]+436.25。
4-(4-((4-(叔丁氧羰基)哌嗪-1-基)甲基)哌啶-1-基)-2-氟苯甲酸(27)
4-(4-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-fluorobenzoic acid(27)
合成通法7：将中间体26(200mg，0.46mmol)加入至含有甲醇：水＝8mL：2mL混合溶液的圆底烧瓶中，再加入氢氧化锂(55mg，2.30mmol)，40℃反应10小时，TLC监测反应完全结束。待反应冷却至室温后，加入稀盐酸调节溶液pH值为5～6，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体27，为白色固体(180mg，产率：93.0％)。ESI-MS:m/z:[M+H]+421.24。
4-((1-(4-((2,6-二氧代哌啶-3-基)氨基甲酰基)-3-氟苯基)哌啶-4-基)甲基)哌嗪-1-羧酸叔丁酯(28)
tert-butyl-4-((1-(4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3-fluorophenyl)piperidin-4-yl)methyl)piperazine-1-carboxylate(28)
合成通法8：将中间体27(180mg，0.43mmol)加入至含有5mLDMF溶液的圆底烧瓶中，再依次加入DIPEA(276mg，2.14mmol)、EDCI(123mg，0.64mmol)、HOBt(87mg，0.64mmol)，室温搅拌半小时，再加入27a(83mg，0.51mmol)，室温反应过夜，TLC监测反应完全结束。使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝50：1，v/v)纯化得中间体28，为白色固体(160mg，产率：70.5％)。ESI-MS:m/z:[M+H]+532.28。
N-(2,6-二氧代哌啶-3-基)-2-氟-4-(4-(哌嗪-1-基甲基)哌啶-1-基)苯甲酰胺(29)
N-(2,6-dioxopiperidin-3-yl)-2-fluoro-4-(4-(piperazin-1-ylmethyl)piperidin-1-yl)benzamide(29)
按合成通法4，以28(200mg，0.38mmol)为反应原料，得到目标产物白色油状物29(130mg，产率：80.1％)。ESI-MS:m/z:[M+H]+432.23。
实施例4
4-(4-((4-(5-氯-4-((6-(2-(甲基氨基)-2-氧代乙氧基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-9-基)氨基)嘧啶-2-基)哌嗪-1-基)甲基)哌啶-1-基)-N-(2,6-二氧代哌啶-3-基)-2-氟苯甲酰胺(实施例4)
4-(4-((4-(5-chloro-4-((6-(2-(methylamino)-2-oxoethoxy)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-9-yl)amino)pyrimidin-2-yl)piperazin-1-yl)methyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2-fluorobenzamide(实施例4)
按合成通法5，以29(119mg，0.28mmol)为反应原料，得到目标产物黄色固体实施例4(25mg，产率：21.9％)。1H NMR(300MHz,DMSO-d6)δ10.87(s,1H),8.79(s,1H),8.07(s,1H),8.00(q,J＝4.1,3.7Hz,1H),7.92(t,J＝7.8Hz,1H),7.66(t,J＝8.9Hz,1H),7.59(d,J＝2.3Hz,1H),7.40(d,J＝2.2Hz,1H),7.12(s,1H),6.43(dd,J＝9.1,2.2Hz,1H),6.35(d,J＝15.1Hz,1H),4.74(dt,J＝12.4,6.4Hz,1H),4.60(s,2H),4.51(d,J＝12.0Hz,2H),4.41(d,J＝4.4Hz,2H),4.21–4.16(m,2H),4.03(d,J＝12.9Hz,1H),3.17(s,2H),2.97(t,J＝11.9Hz,2H),2.67(d,J＝4.6Hz,3H),2.15(d,J＝11.7Hz,2H),1.86(m,1H),1.76(m,2H),1.32(m,1H).HRMS(ESI):calcd for C40H44ClFN10O7[M+H]+831.3067,found 831.3149.纯度：96.19％by HPLC(MeOH/H2O＝80:20,tR＝4.022min)。
合成路线4：
实施例5～6按照合成路线4合成。
合成路线4:试剂和条件:(a)NaBH(OAc)3,DCE,rt,12h；(b)TFA,DCM,rt,3h；(c)DIPEA,DMSO,100℃,3h；(d)H2,Pd/C,EtOH,rt,16h；(e)DIPEA,NMP,微波,170℃,3h.
4-((4-(2-((叔丁氧羰基)氨基)乙基)哌啶-1-基)甲基)哌啶-1-羧酸苄酯(31a)
benzyl-4-((4-(2-((tert-butoxycarbonyl)amino)ethyl)piperidin-1-yl)methyl)piperidine-1-carboxylate(31a)
按合成通法1，以中间体30a(400mg，1.75mmol)为反应原料，得到目标产物白色固体31a(610mg，产率：75.8％)。ESI-MS:m/z:[M+H]+460.31。
4-((4-((叔丁氧羰基)氨基)哌啶-1-基)甲基)哌啶-1-羧酸苄酯(31b)
benzyl-4-((4-((tert-butoxycarbonyl)amino)piperidin-1-yl)methyl)piperidine-1-carboxylate(31b)
按合成通法1，以中间体30b(400mg，2.00mmol)为反应原料，得到目标产物白色固体31b(610mg，产率：75.8％)。ESI-MS:m/z:[M+H]+432.28。
4-((4-(2-氨基乙基)哌啶-1-基)甲基)哌啶-1-羧酸苄酯(32a)
benzyl 4-((4-(2-aminoethyl)piperidin-1-yl)methyl)piperidine-1-carboxylate(32a)
按合成通法4，以中间体31a(400mg，0.87mmol)为反应原料，得到目标产物白色油状物32a(290mg，产率：92.7％)。ESI-MS:m/z:[M+H]+360.26。
4-((4-氨基哌啶-1-基)甲基)哌啶-1-羧酸苄酯(32b)
benzyl 4-((4-aminopiperidin-1-yl)methyl)piperidine-1-carboxylate(32b)
按合成通法4，以中间体31b(400mg，0.87mmol)为反应原料，得到目标产物白色油状物32b(270mg，产率：93.6％)。ESI-MS:m/z:[M+H]+332.23。
4-((4-(2-((2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-4-基)氨基)乙基)哌啶-1-基)甲基)哌啶-1-羧酸苄酯(33a)
Benzyl-4-((4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)piperidin-1-yl)methyl)piperidine-1-carboxylate(33a)
按合成通法3，以23a(400mg，1.45mmol)为反应原料，得到目标产物黄色固体33a(840mg，产率：94.2％)。ESI-MS:m/z:[M+H]+616.31。
4-((4-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-4-基)氨基)哌啶-1-基)甲基)哌啶-1-羧酸苄酯(33b)
Benzyl-4-((4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)piperidin-1-yl)methyl)piperidine-1-carboxylate(33b)
按合成通法3，以23a(400mg，1.45mmol)为反应原料，得到目标产物黄色固体33b(810mg，产率：95.2％)。ESI-MS:m/z:[M+H]+588.27。
2-(2,6-二氧代哌啶-3-基)-4-((2-(1-(哌啶-4-基甲基)哌啶-4-基)乙基)氨基)异吲哚啉-1,3-二酮(34a)
2-(2,6-dioxopiperidin-3-yl)-4-((2-(1-(piperidin-4-ylmethyl)piperidin-4-yl)ethyl)amino)isoindoline-1,3-dione(34a)
按合成通法2，以33a(300mg，0.49mmol)为反应原料，得到目标产物黄色油状物34a(190mg，产率：81.0％)。ESI-MS:m/z:[M+H]+482.27。
2-(2,6-二氧代哌啶-3-基)-4-((1-(哌啶-4-基甲基)哌啶-4-基)氨基)异吲哚啉-1,3-二酮(34b)
2-(2,6-dioxopiperidin-3-yl)-4-((1-(piperidin-4-ylmethyl)piperidin-4-yl)amino)isoindoline-1,3-dione(34b)
按合成通法2，以33b(300mg，0.49mmol)为反应原料，得到目标产物黄色油状物34b(180mg，产率：76.7％)。ESI-MS:m/z:[M+H]+454.24。
实施例5
2-((9-((5-氯-2-(4-((4-(2-(2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-4-基)氨基)乙基)哌啶-1-基)甲基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例5)
2-((9-((5-chloro-2-(4-((4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)piperidin-1-yl)methyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例5)
按合成通法5，以34a(132mg，0.28mmol)为反应原料，得到目标产物黄色固体实施例5(18mg，产率：9.1％)。1H NMR(300MHz,DMSO-d6)δ11.17(s,1H),8.77(s,1H),8.03(d,J＝11.6Hz,2H),7.64–7.55(m,2H),7.41(d,J＝2.2Hz,1H),7.13–7.09(m,2H),7.05(d,J＝7.1Hz,1H),6.54(s,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.61(s,2H),4.50(d,J＝12.5Hz,2H),4.40(d,J＝5.4Hz,2H),4.20(d,J＝4.7Hz,2H),2.89(d,J＝14.4Hz,5H),2.68(d,J＝4.6Hz,5H),2.57(s,4H),2.09–1.97(m,3H),1.73(s,2H),1.54(s,2H),1.26(d,J＝4.4Hz,8H).HRMS(ESI):calcd for C44H49ClN10O8[M+H]+881.3423,found 881.3478.纯度：97.53％by HPLC(MeOH/H2O＝80:20,tR＝3.998min)。
实施例6
2-((9-((5-氯-2-(4-((4-((2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-4-基)氨基)哌啶-1-基)甲基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例6)
2-((9-((5-chloro-2-(4-((4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)piperidin-1-yl)methyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例6)
按合成通法5，以34b(125mg，0.28mmol)为反应原料，得到目标产物黄色固体实施例6(13mg，产率：11.1％)。1H NMR(300MHz,DMSO-d6)δ11.15(s,1H),9.00(s,1H),8.28(d,J＝10.7Hz,2H),7.89–7.81(m,2H),7.67(d,J＝2.2Hz,1H),7.44(d,J＝8.6Hz,1H),7.36(s,1H),7.31(d,J＝7.0Hz,1H),6.52(d,J＝8.0Hz,1H),5.33(dd,J＝12.9,5.4Hz,1H),4.86(s,2H),4.76(d,J＝12.7Hz,2H),4.65(d,J＝5.3Hz,2H),4.45(d,J＝4.8Hz,2H),3.12(t,J＝11.5Hz,3H),2.94(d,J＝4.6Hz,3H),2.88(s,1H),2.81(s,1H),2.41(s,3H),2.23(dd,J＝17.8,9.7Hz,3H),2.01(d,J＝12.6Hz,2H),1.76(s,2H),1.59–1.47(m,5H),1.29(d,J＝12.3Hz,2H).HRMS(ESI):calcd for C42H45ClN10O8[M+H]+853.3110,found 853.3174.纯度：96.13％by HPLC(MeOH/H2O＝80:20,tR＝4.018min)。
合成路线5：
实施例7～9按照合成路线5合成。
合成路线5：试剂和条件:(a)NaBH(OAc)3,DCE,rt,3h；(b)H2,Pd/C,EtOH,rt,16h；(c)DIPEA,DMSO,100℃,3h；(d)TFA,DCM,rt,1h；(e)DIPEA,DMSO,100℃,10h；(f)DIPEA,EDCI,HOBt,DMF,rt,12h.
1-(5-氯-4-((6-(2-(甲基氨基)-2-氧代乙氧基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-9-基)氨基)嘧啶-2-基)哌啶-4-羧酸(35)
1-(5-chloro-4-((6-(2-(methylamino)-2-oxoethoxy)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-9-yl)amino)pyrimidin-2-yl)piperidine-4-carboxylic acid(35)
将中间体11(300mg，0.69mmol)，原料12i(178mg，1.39mmol)，DIPEA(151mg，1.17mmol)加入到圆底烧瓶中，加入8mL的DMSO溶液，100℃搅拌10h后TLC监测反应完全结束。缓慢滴加稀盐酸调节溶液pH至2～5，再加入50mL的水搅拌十分钟，抽滤，水洗，干燥得黄色固体35(310mg，产率：85.2％)。ESI-MS:m/z:[M+H]+259.15。
4-(1-((苄氧基)羰基)哌啶-4-基)哌嗪-1-羧酸叔丁酯(21c)
tert-butyl 4-(1-((benzyloxy)carbonyl)piperidin-4-yl)piperazine-1-carboxylate(21c)
按合成通法1，以中间体20c(400mg，1.71mmol)为反应原料，得到目标产物白色油状物21c(410mg，产率：59.2％)。ESI-MS:m/z:[M+H]+404.25。
4-((1-((苄氧基)羰基)氮杂环丁烷-3-基)甲基)哌嗪-1-羧酸叔丁酯(21d)
tert-butyl 4-((1-((benzyloxy)carbonyl)azetidin-3-yl)methyl)piperazine-1-carboxylate(21d)
按合成通法1，以中间体20d(400mg，1.82mmol)为反应原料，得到目标产物白色油状物21d(440mg，产率：61.9％)。ESI-MS:m/z:[M+H]+389.23。
4-(哌啶-4-基)哌嗪-1-羧酸叔丁酯(22c)
tert-butyl 4-(piperidin-4-yl)piperazine-1-carboxylate(22c)
按合成通法2，以21c(300mg，0.74mmol)为反应原料，得到目标产物无色油状物22c(170mg，产率：84.9％)。ESI-MS:m/z:[M+H]+270.21。
4-(氮杂环丁烷-3-基甲基)哌嗪-1-羧酸叔丁酯(22d)
tert-butyl 4-(azetidin-3-ylmethyl)piperazine-1-carboxylate(22d)
按合成通法2，以21d(300mg，0.77mmol)为反应原料，得到目标产物无色油状物22d(180mg，产率：91.5％)。ESI-MS:m/z:[M+H]+256.19。
4-(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)哌嗪-1-羧酸叔丁酯(24c)
tert-butyl-4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)piperazine-1-carboxylate(24c)
按合成通法3，以23b(400mg，1.45mmol)为反应原料，得到目标产物黄色固体24c(590mg，产率：77.5％)。1H NMR(300MHz,DMSO-d6)δ7.67(d,J＝8.5Hz,1H),7.33(d,J＝2.2Hz,1H),7.25(dd,J＝8.7,2.3Hz,1H),5.08(dd,J＝12.6,5.3Hz,1H),4.08(d,J＝13.2Hz,2H),2.92(dt,J＝25.2,9.1Hz,3H),2.63(s,1H),2.56(d,J＝3.8Hz,2H),2.45(t,J＝4.8Hz,4H),2.03(d,J＝12.1Hz,1H),1.84(d,J＝12.4Hz,2H),1.40(s,11H).ESI-MS:m/z:[M+H]+526.26。
4-((1-(2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)氮杂环丁烷-3-基)甲基)哌嗪-1-羧酸叔丁酯(24d)
tert-butyl-4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidin-3-yl)methyl)piperazine-1-carboxylate(24d)
按合成通法3，以23b(400mg，1.45mmol)为反应原料，得到目标产物黄色固体24d(610mg，产率：82.3％)。ESI-MS:m/z:[M+H]+526.26。
2-(2,6-二氧代哌啶-3-基)-5-(4-(哌嗪-1-基)哌啶-1-基)异吲哚啉-1,3-二酮(25c)
2-(2,6-dioxopiperidin-3-yl)-5-(4-(piperazin-1-yl)piperidin-1-yl)isoindoline-1,3-dione(25c)
按合成通法4，以24c(300mg，0.57mmol)为反应原料，得到目标产物黄色油状物25c(205mg，产率：84.4％)。ESI-MS:m/z:[M+H]+426.21。
2-(2,6-二氧代哌啶-3-基)-5-(3-(哌嗪-1-基甲基)氮杂环丁烷-1-基)异吲哚啉-1,3-二酮(25d)
2-(2,6-dioxopiperidin-3-yl)-5-(3-(piperazin-1-ylmethyl)azetidin-1-yl)isoindoline-1,3-dione(25d)
按合成通法4，以24d(300mg，0.56mmol)为反应原料，得到目标产物黄色油状物25d(195mg，产率：85.8％)。ESI-MS:m/z:[M+H]+412.19。
实施例7
2-((9-((5-氯-2-(4-(4-((1-(2-(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)甲基)哌嗪-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例7)
2-((9-((5-chloro-2-(4-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperazine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例7)
合成通法9：将中间体35(60mg，0.11mmol)加入至含有5mLDMF溶液的圆底烧瓶中，再依次加入DIPEA(71mg，0.55mmol)、EDCI(32mg，0.16mmol)、HOBt(23mg，0.16mmol)，室温搅拌半小时，再加入25b(100mg，0.23mmol)，室温反应过夜，TLC监测反应完全结束。使用乙酸乙酯萃取三次，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过制备层析(洗脱体系为二氯甲烷：甲醇＝15：1，v/v)纯化得目标产物实施例7，为黄色固体(16mg，产率：14.8％)。1H NMR(300MHz,DMSO-d6)δ11.10(s,1H),8.78(s,1H),8.07(s,1H),7.99(d,J＝4.8Hz,1H),7.66(s,1H),7.41(s,1H),7.33(s,2H),7.25(d,J＝8.7Hz,1H),7.12(s,1H),5.09(d,J＝7.3Hz,1H),4.60(s,2H),4.54–4.46(m,2H),4.40(s,2H),4.19(s,2H),4.05(s,2H),3.57(s,2H),3.46(s,2H),3.01–2.91(m,7H),2.70–2.59(m,5H),2.18(s,2H),2.07–2.00(m,2H),1.82(d,J＝12.7Hz,4H),1.67(d,J＝12.8Hz,3H),1.50(d,J＝12.3Hz,2H).HRMS(ESI):calcd for C47H52ClN11O9[M+H]+950.3638,found 950.3706。纯度：94.88％by HPLC(MeOH/H2O＝80:20,tR＝3.903min)。
实施例8
2-((9-((5-氯-2-(4-(4-(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)哌嗪-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例8)
2-((9-((5-chloro-2-(4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)piperazine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例8)
按合成通法9，以35(60mg，0.11mmol)为反应原料，得到目标产物黄色固体实施例8(13mg，产率：12.2％)。1H NMR(300MHz,DMSO-d6)δ11.11(s,1H),8.78(s,1H),8.07(s,1H),7.98(d,J＝5.0Hz,1H),7.68(d,J＝8.5Hz,1H),7.59(d,J＝2.2Hz,1H),7.40(d,J＝2.2Hz,1H),7.35(d,J＝2.2Hz,1H),7.27(dd,J＝8.7,2.2Hz,1H),7.12(s,1H),5.09(dd,J＝12.8,5.3Hz,1H),4.60(s,2H),4.50(d,J＝12.7Hz,2H),4.40(t,J＝4.7Hz,2H),4.19(t,J＝4.7Hz,2H),4.10(d,J＝12.7Hz,2H),3.55(s,2H),3.44(s,2H),3.03–2.89(m,6H),2.67(d,J＝4.6Hz,3H),2.63(s,1H),2.57(s,2H),2.45(s,2H),2.03(d,J＝11.9Hz,1H),1.85(d,J＝12.1Hz,2H),1.66(d,J＝12.5Hz,2H),1.59–1.38(m,5H),1.26(s,1H).HRMS(ESI):calcd for C46H50ClN11O9[M+H]+936.3481,found 936.3531。纯度：96.43％by HPLC(MeOH/H2O＝80:20,tR＝3.993min)。
实施例9
2-((9-((5-氯-2-(4-(4-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)氮杂环丁烷-3-基)甲基)哌嗪-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例9)
2-((9-((5-chloro-2-(4-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidin-3-yl)methyl)piperazine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例9)
按合成通法9，以35(60mg，0.11mmol)为反应原料，得到目标产物黄色固体实施例9(10mg，产率：9.6％)。1H NMR(300MHz,DMSO-d6)δ11.06(s,1H),8.77(s,1H),8.05(s,1H),7.96(d,J＝4.8Hz,1H),7.56(dd,J＝5.3,3.1Hz,2H),7.38(d,J＝2.2Hz,1H),7.09(s,1H),7.03(s,1H),6.90(dd,J＝8.6,2.1Hz,1H),5.03(dd,J＝12.7,5.3Hz,1H),4.64(s,1H),4.58(s,2H),4.48(d,J＝12.7Hz,2H),4.37(d,J＝5.4Hz,2H),4.17(d,J＝5.4Hz,2H),3.59(s,2H),3.48(d,J＝13.8Hz,4H),3.22(s,2H),2.94(t,J＝12.8Hz,4H),2.64(d,J＝4.6Hz,3H),2.59(s,1H),2.33(d,J＝22.3Hz,5H),1.96(dt,J＝13.4,6.6Hz,2H),1.65(d,J＝12.4Hz,2H),1.47(d,J＝10.8Hz,2H),1.23(s,1H).HRMS(ESI):calcd for C45H48ClN11O9[M+H]+922.3325,found 922.3402。纯度：95.49％by HPLC(MeOH/H2O＝80:20,tR＝4.001min)。
合成路线6：
实施例10～11按照合成路线6合成。
合成路线6：试剂和条件:(a)(PPh3)3RhCl,EtOH,80℃,18h；(b)KI,吡啶,115℃,12h；(c)TFA,DCM,rt,3h；(d)AcOH,NaBH(OAc)3,DCE,rt,3h；(e)TFA,DCM,rt,3h；(f)EDCI,HOBt,DIPEA,DMF,rt,12h.
2-(叔丁基)5,6-二甲基异吲哚啉-2,5,6-三羧酸酯(37)
2-(tert-butyl)5,6-dimethyl isoindoline-2,5,6-tricarboxylate(37)
将中间体36a(3.40g，17.59mmol)与36b(10.00g，70.37mmol)加入至含有50mL无水乙醇溶液的圆底烧瓶中，再加入(PPh3)3RhCl(163mg，0.18mmol)，氩气置换，85℃回流18小时，TLC监测反应完全结束。使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为石油醚：乙酸乙酯＝20：1，v/v)纯化得目标产物37，为白色固体(1.00g，产率：17.0％)。1H NMR(300MHz,DMSO-d6)δ7.72(d,J＝9.7Hz,2H),4.67(d,J＝4.7Hz,4H),3.84(s,6H),1.49(s,9H).ESI-MS:m/z:[M+H]+336.14。
6-(2,6-二氧代哌啶-3-基)-5,7-二氧代-3,5,6,7-四氢吡咯并[3,4-f]异吲哚-2(1H)-羧酸叔丁酯(38)
tert-butyl-6-(2,6-dioxopiperidin-3-yl)-5,7-dioxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindole-2(1H)-carboxylate(38)
将中间体37(820mg，2.45mmol)与27a(523mg，3.18mmol)加入至含有5mL吡啶溶液的圆底烧瓶中，再加碘化钾(1.22g，7.34mmol)，115℃回流过夜，TLC监测反应完全结束。减压浓缩反应液，使用乙酸乙酯和1M的柠檬酸水溶液萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得目标产物38，为黄色固体(460mg，产率：47.1％)。1H NMR(300MHz,DMSO-d6)δ11.15(s,1H),7.91(d,J＝1.7Hz,2H),5.17(dd,J＝12.8,5.3Hz,1H),4.74(d,J＝7.1Hz,4H),2.92(ddd,J＝17.0,13.8,5.4Hz,1H),2.60(t,J＝16.2Hz,2H),2.15–2.03(m,1H),1.49(s,9H).ESI-MS:m/z:[M+H]+400.14。
2-(2,6-二氧代哌啶-3-基)-6,7-二氢吡咯并[3,4-f]异吲哚-1,3(2H，5H)-二酮(39a)
2-(2,6-dioxopiperidin-3-yl)-6,7-dihydropyrrolo[3,4-f]isoindole-1,3(2H,5H)-dione(39a)
按合成通法4，以38(400mg，1.00mmol)为反应原料，得到目标产物黄色油状物39a(275mg，产率：91.8％)。ESI-MS:m/z:[M+H]+300.09。
4-((6-(2,6-二氧代哌啶-3-基)-5,7-二氧代-3,5,6,7-四氢吡咯并[3,4-f]异吲哚-2(1H)-基)甲基)哌啶-1-羧酸叔丁酯(40)
tert-butyl-4-((6-(2,6-dioxopiperidin-3-yl)-5,7-dioxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(1H)-yl)methyl)piperidine-1-carboxylate(40)
按合成通法1，以中间体39a(450mg，1.50mmol)为反应原料，得到目标产物白色固体40(345mg，产率：46.2％)。1H NMR(300MHz,DMSO-d6)δ11.13(s,1H),7.81–7.77(m,2H),5.20–5.10(m,1H),3.96(d,J＝13.6Hz,6H),3.85(d,J＝13.2Hz,4H),2.59(d,J＝5.4Hz,2H),2.41(t,J＝4.0Hz,1H),2.14–2.01(m,1H),1.78(t,J＝6.5Hz,5H),1.41–1.40(m,9H).ESI-MS:m/z:[M+H]+497.23。
2-(2,6-二氧代哌啶-3-基)-6-(哌啶-4-基甲基)-6,7-二氢吡咯并[3,4-f]异吲哚-1,3(2H，5H)-二酮(41)
2-(2,6-dioxopiperidin-3-yl)-6-(piperidin-4-ylmethyl)-6,7-dihydropyrrolo[3,4-f]isoindole-1,3(2H,5H)-dione(41)
按合成通法4，以中间体40(190mg，0.38mmol)为反应原料，得到目标产物白色油状物41(115mg，产率：75.8％)。ESI-MS:m/z:[M+H]+397.18。
实施例10
2-((9-((5-氯-2-(4-(6-(2,6-二氧代哌啶-3-基)-5,7-二氧代-1,2,3,5,6,7-六氢吡咯并[3,4-f]异吲哚-2-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例10)
2-((9-((5-chloro-2-(4-(6-(2,6-dioxopiperidin-3-yl)-5,7-dioxo-1,2,3,5,6,7-hexahydropyrrolo[3,4-f]isoindole-2-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例10)
按合成通法9，以35(60mg，0.11mmol)为反应原料，得到目标产物黄色固体实施例10(9mg，产率：9.8％)。1H NMR(300MHz,DMSO-d6)δ11.16(s,1H),8.80(s,1H),8.08(s,1H),7.97(d,J＝5.2Hz,1H),7.94(s,1H),7.89(s,1H),7.61(d,J＝2.0Hz,1H),7.41(d,J＝2.2Hz,1H),7.10(s,1H),5.18(dd,J＝12.9,4.3Hz,3H),4.79(s,2H),4.57(d,J＝11.8Hz,4H),4.39(t,J＝4.8Hz,2H),4.17(t,J＝4.8Hz,2H),3.04–2.86(m,4H),2.63(d,J＝4.8Hz,5H),2.15–2.06(m,1H),1.84(d,J＝12.5Hz,2H),1.56(d,J＝12.3Hz,2H).13C NMR(75MHz,DMSO-d6)δ173.35,173.27,170.34,167.91,167.41,159.53,155.76,155.23,155.06,147.61,144.91,144.23,142.95,134.54,131.44,131.39,120.37,119.18,118.94,118.85,113.66,112.36,109.31,102.52,68.42,64.20,52.44,52.25,49.57,44.05,31.45,27.71,25.85,22.53.HRMS(ESI):calcd for C39H36ClN9O9[M+Na]+832.2325,found 832.2228。纯度：96.41％by HPLC(MeOH/H2O＝80:20,tR＝4.301min)。
实施例11
2-((9-((5-氯-2-(4-(4-((6-(2,6-二氧代哌啶-3-基)-5,7-二氧代-3,5,6,7-四氢吡咯并[3,4-f]异吲哚-2(1H)-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例11)
2-((9-((5-chloro-2-(4-(4-((6-(2,6-dioxopiperidin-3-yl)-5,7-dioxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(1H)-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例11)
按合成通法9，以35(60mg，0.11mmol)为反应原料，得到目标产物黄色固体实施例11(12mg，产率：11.7％)。1H NMR(300MHz,DMSO-d6)δ11.16(s,1H),8.79(s,1H),8.07(d,J＝1.1Hz,1H),8.00(d,J＝5.1Hz,1H),7.79(d,J＝17.3Hz,2H),7.59(d,J＝2.2Hz,1H),7.41(d,J＝2.1Hz,1H),7.12(s,1H),5.16(dd,J＝12.7,5.3Hz,1H),4.61(s,2H),4.51(d,J＝12.0Hz,2H),4.40(d,J＝4.8Hz,2H),4.22–4.17(m,2H),4.00(s,4H),2.95(q,J＝15.7,14.1Hz,6H),2.70–2.66(m,4H),2.59(s,5H),2.13–2.05(m,1H),1.91–1.73(m,3H),1.65(s,2H),1.51(s,3H),1.26(s,1H).HRMS(ESI):calcd for C47H52ClN11O9[M+H]+907.3216,found 907.3258。纯度：97.32％by HPLC(MeOH/H2O＝80:20,tR＝4.108min)。
合成路线7：
实施例12～16按照合成路线7合成。
合成路线7：试剂和条件:(a)DIPEA,DMSO,100℃,3h；(b)TFA,DCM,rt,1h；(c)NaBH(OAc)3,DCE,rt,5h；(d)TFA,DCM,rt,1h；(e)DIPEA,EDCI,HOBt,DMF,rt,12h.
6-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,6-二氮杂螺[3.3]庚烷-2-羧酸叔丁酯(43a)
tert-butyl-6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate(43a)
按合成通法3，以23b(400mg，1.45mmol)为反应原料，得到目标产物黄色固体43a(614mg，产率：93.3％)。1H NMR(300MHz,DMSO-d6)δ10.96(s,1H),7.52(d,J＝8.3Hz,1H),6.66(d,J＝2.1Hz,1H),6.52(dd,J＝8.3,2.2Hz,1H),4.93(dd,J＝12.8,5.3Hz,1H),4.07–4.01(m,4H),3.98–3.88(m,4H),2.85–2.67(m,1H),2.48(s,1H),2.42(s,1H),1.88(d,J＝11.6Hz,1H),1.28–1.22(m,9H).ESI-MS:m/z:[M+H]+455.18。
2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸叔丁酯(43b)
tert-butyl-2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonane-7-carboxylate(43b)
按合成通法3，以23b(400mg，1.45mmol)为反应原料，得到目标产物黄色固体43b(643mg，产率：92.0％)。ESI-MS:m/z:[M+H]+483.22。
7-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,7-二氮杂螺[4.4]壬烷-2-羧酸叔丁酯(43c)
tert-butyl-7-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[4.4]nonane-2-carboxylate(43c)
按合成通法3，以23b(400mg，1.45mmol)为反应原料，得到目标产物黄色固体43c(610mg，产率：87.3％)。ESI-MS:m/z:[M+H]+483.22。
2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸叔丁酯(43d)
tert-butyl-2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate(43d)
按合成通法3，以23b(400mg，1.45mmol)为反应原料，得到目标产物黄色固体43d(594mg，产率：82.6％)。ESI-MS:m/z:[M+H]+497.23。
9-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(43e)
tert-butyl-9-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate(43e)
按合成通法3，以23b(400mg，1.45mmol)为反应原料，得到目标产物黄色固体43e(605mg，产率：81.8％)。ESI-MS:m/z:[M+H]+511.25。
2-(2,6-二氧代哌啶-3-基)-5-(2,6-二氮杂螺[3.3]庚烷-2-基)异吲哚啉-1,3-二酮(44a)
2-(2,6-dioxopiperidin-3-yl)-5-(2,6-diazaspiro[3.3]heptan-2-yl)isoindoline-1,3-dione(44a)
按合成通法4，以43a(300mg，0.59mmol)为反应原料，得到目标产物黄色油状物44a(187mg，产率：79.9％)。ESI-MS:m/z:[M+H]+355.13。
2-(2,6-二氧代哌啶-3-基)-5-(2,7-二氮杂螺[3.5]壬烷-2-基)异吲哚啉-1,3-二酮(44b)
2-(2,6-dioxopiperidin-3-yl)-5-(2,7-diazaspiro[3.5]nonan-2-yl)isoindoline-1,3-dione(44b)
按合成通法4，以43b(300mg，0.62mmol)为反应原料，得到目标产物黄色油状物44b(171mg，产率：71.9％)。ESI-MS:m/z:[M+H]+383.16。
2-(2,6-二氧代哌啶-3-基)-5-(2,7-二氮杂螺[4.4]壬烷-2-基)异吲哚啉-1,3-二酮(44c)
2-(2,6-dioxopiperidin-3-yl)-5-(2,7-diazaspiro[4.4]nonan-2-yl)isoindoline-1,3-dione(44c)
按合成通法4，以43c(300mg，0.62mmol)为反应原料，得到目标产物黄色油状物44c(183mg，产率：77.0％)。ESI-MS:m/z:[M+H]+383.16。
2-(2,6-二氧代哌啶-3-基)-5-(2,8-二氮杂螺[4.5]癸烷-2-基)异吲哚啉-1,3-二酮(44d)
2-(2,6-dioxopiperidin-3-yl)-5-(2,8-diazaspiro[4.5]decan-2-yl)isoindoline-1,3-dione(44d)
按合成通法4，以43d(300mg，0.60mmol)为反应原料，得到目标产物黄色油状物44d(212mg，产率：88.5％)。ESI-MS:m/z:[M+H]+397.18。
2-(2,6-二氧代哌啶-3-基)-5-(3,9-二氮杂螺[5.5]十一烷-3-基)异吲哚啉-1,3-二酮(44e)
2-(2,6-dioxopiperidin-3-yl)-5-(3,9-diazaspiro[5.5]undecan-3-yl)isoindoline-1,3-dione(44e)
按合成通法4，以43e(300mg，0.59mmol)为反应原料，得到目标产物黄色油状物44e(215mg，产率：89.2％)。ESI-MS:m/z:[M+H]+411.20。
4-((6-(2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,6-二氮杂螺[3.3]庚烷-2-基)甲基)哌啶-1-羧酸叔丁酯(45a)
tert-butyl-4-((6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,6-diazaspiro[3.3]heptan-2-yl)methyl)piperidine-1-carboxylate(45a)
按合成通法1，以中间体44a(300mg，0.85mmol)为反应原料，得到目标产物黄色固体45a(248mg，产率：53.1％)。ESI-MS:m/z:[M+H]+552.27。
4-((2-(2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,7-二氮杂螺[3.5]壬烷-7-基)甲基)哌啶-1-羧酸叔丁酯(45b)
tert-butyl-4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidine-1-carboxylate(45b)
按合成通法1，以中间体44b(300mg，0.78mmol)为反应原料，得到目标产物黄色固体45b(263mg，产率：57.8％)。ESI-MS:m/z:[M+H]+580.30。
4-((7-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,7-二氮杂螺[4.4]壬烷-2-基)甲基)哌啶-1-羧酸叔丁酯(45c)
tert-butyl-4-((7-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[4.4]nonan-2-yl)methyl)piperidine-1-carboxylate(45c)
按合成通法1，以中间体44c(300mg，0.78mmol)为反应原料，得到目标产物黄色固体45c(211mg，产率：46.4％)。ESI-MS:m/z:[M+H]+580.30。
4-((2-(2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,8-二氮杂螺[4.5]癸烷-8-基)甲基)哌啶-1-羧酸叔丁酯(45d)
tert-butyl-4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,8-diazaspiro[4.5]decan-8-yl)methyl)piperidine-1-carboxylate(45d)
按合成通法1，以中间体44d(300mg，0.76mmol)为反应原料，得到目标产物黄色固体45d(224mg，产率：49.9％)。1H NMR(300MHz,DMSO-d6)δ11.20(s,1H),7.75(d,J＝8.4Hz,1H),7.01(d,J＝2.1Hz,1H),6.91(dd,J＝8.6,2.2Hz,1H),5.17(dd,J＝12.5,5.3Hz,1H),3.97(s,2H),3.57(s,3H),3.31–3.25(m,1H),2.98(d,J＝12.4Hz,1H),2.71(s,2H),2.66(s,2H),2.35(s,2H),2.19(d,J＝6.7Hz,5H),1.95–1.87(m,1H),1.71(d,J＝16.3Hz,7H),1.50(d,J＝2.0Hz,3H),1.48(s,9H).ESI-MS:m/z:[M+H]+594.32。
4-((9-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)哌啶-1-羧酸叔丁酯(45e)
tert-butyl-4-((9-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidine-1-carboxylate(45e)
按合成通法1，以中间体44e(300mg，0.73mmol)为反应原料，得到目标产物黄色固体45e(232mg，产率：52.2％)。ESI-MS:m/z:[M+H]+608.34。
2-(2,6-二氧代哌啶-3-基)-5-(6-(哌啶-4-基甲基)-2,6-二氮杂螺[3.3]庚烷-2-基)异吲哚啉-1,3-二酮(46a)
2-(2,6-dioxopiperidin-3-yl)-5-(6-(piperidin-4-ylmethyl)-2,6-diazaspiro[3.3]heptan-2-yl)isoindoline-1,3-dione(46a)
按合成通法4，以45a(300mg，0.54mmol)为反应原料，得到目标产物黄色油状物46a(213mg，产率：86.7％)。ESI-MS:m/z:[M+H]+452.22。
2-(2,6-二氧代哌啶-3-基)-5-(7-(哌啶-4-基甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)异吲哚啉-1,3-二酮(46b)
2-(2,6-dioxopiperidin-3-yl)-5-(7-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-2-yl)isoindoline-1,3-dione(46b)
按合成通法4，以45b(300mg，0.52mmol)为反应原料，得到目标产物黄色油状物46b(233mg，产率：93.9％)。ESI-MS:m/z:[M+H]+480.25。
2-(2,6-二氧代哌啶-3-基)-5-(7-(哌啶-4-基甲基)-2,7-二氮杂螺[4.4]壬烷-2-基)异吲哚啉-1,3-二酮(46c)
2-(2,6-dioxopiperidin-3-yl)-5-(7-(piperidin-4-ylmethyl)-2,7-diazaspiro[4.4]nonan-2-yl)isoindoline-1,3-dione(46c)
按合成通法4，以45c(300mg，0.54mmol)为反应原料，得到目标产物黄色油状物46c(221mg，产率：89.0％)。ESI-MS:m/z:[M+H]+480.25。
2-(2,6-二氧代哌啶-3-基)-5-(8-(哌啶-4-基甲基)-2,8-二氮杂螺[4.5]癸烷-2-基)异吲哚啉-1,3-二酮(46d)
2-(2,6-dioxopiperidin-3-yl)-5-(8-(piperidin-4-ylmethyl)-2,8-diazaspiro[4.5]decan-2-yl)isoindoline-1,3-dione(46d)
按合成通法4，以45d(300mg，0.50mmol)为反应原料，得到目标产物黄色油状物46d(217mg，产率：87.0％)。ESI-MS:m/z:[M+H]+494.27。
2-(2,6-二氧代哌啶-3-基)-5-(9-(哌啶-4-基甲基)-3,9-二氮杂螺[5.5]十一烷-3-基)异吲哚啉-1,3-二酮(46e)
2-(2,6-dioxopiperidin-3-yl)-5-(9-(piperidin-4-ylmethyl)-3,9-diazaspiro[5.5]undecan-3-yl)isoindoline-1,3-dione(46e)
按合成通法4，以中间体45e(300mg，0.73mmol)为反应原料，得到目标产物黄色固体46e(232mg，产率：52.2％)。ESI-MS:m/z:[M+H]+508.28。
实施例12
2-((9-((5-氯-2-(4-(4-((6-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,6-二氮杂螺[3.3]庚烷-2-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例12)
2-((9-((5-chloro-2-(4-(4-((6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,6-diazaspiro[3.3]heptan-2-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例12)
按合成通法9，以46a(60mg，0.11mmol)为反应原料，得到目标产物黄色固体实施例12(13mg，产率：11.9％)。1H NMR(300MHz,DMSO-d6)δ11.10(s,1H),8.78(s,1H),8.07(s,1H),7.98(q,J＝4.6,3.8Hz,1H),7.66(d,J＝8.3Hz,1H),7.58(d,J＝2.2Hz,1H),7.40(d,J＝2.2Hz,1H),7.12(s,1H),6.80(d,J＝2.0Hz,1H),6.66(dd,J＝8.4,2.0Hz,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.60(s,2H),4.50(d,J＝12.4Hz,2H),4.39(d,J＝5.3Hz,2H),4.34(s,1H),4.22–4.16(m,2H),4.12(s,4H),4.01(d,J＝12.7Hz,1H),3.30(s,5H),3.08–2.88(m,5H),2.67(d,J＝4.6Hz,3H),2.62(s,1H),2.56(s,1H),2.28(s,2H),2.08–1.96(m,1H),1.80–1.59(m,4H),1.59–1.36(m,3H),1.26(s,2H).HRMS(ESI):calcd for C48H52ClN11O9[M+H]+962.3638,found 962.3704。纯度：95.22％by HPLC(MeOH/H2O＝80:20,tR＝3.901min)。
实施例13
2-((9-((5-氯-2-(4-(4-((2-(2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,7-二氮杂螺[3.5]壬烷-7-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例13)
2-((9-((5-chloro-2-(4-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例13)
按合成通法9，以46b(60mg，0.11mmol)为反应原料，得到目标产物黄色固体实施例13(8mg，产率：7.1％)。1H NMR(300MHz,DMSO-d6)δ11.08(s,1H),8.77(s,1H),8.05(s,1H),7.97(d,J＝4.8Hz,1H),7.63(d,J＝8.2Hz,1H),7.56(d,J＝2.2Hz,1H),7.38(d,J＝2.2Hz,1H),7.10(s,1H),6.77(d,J＝2.0Hz,1H),6.64(dd,J＝8.4,2.1Hz,1H),5.05(dd,J＝12.7,5.4Hz,1H),4.58(s,2H),4.48(d,J＝11.7Hz,2H),4.38(s,2H),4.17(d,J＝4.8Hz,2H),4.01(d,J＝12.8Hz,1H),3.74(s,4H),3.05–2.80(m,6H),2.65(d,J＝4.6Hz,3H),2.60(s,1H),2.54(s,3H),2.29(d,J＝11.7Hz,3H),2.11(s,2H),2.02–1.96(m,1H),1.75(s,6H),1.63(d,J＝14.0Hz,3H),1.47(s,2H),1.23(s,1H).HRMS(ESI):calcd for C50H56ClN11O9[M+H]+990.3951,found 990.4034。纯度：96.64％by HPLC(MeOH/H2O＝80:20,tR＝3.817min)。
实施例14
2-((9-((5-氯-2-(4-(4-((7-(2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,7-二氮杂螺[4.4]壬烷-2-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例14)
2-((9-((5-chloro-2-(4-(4-((7-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[4.4]nonan-2-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例14)
按合成通法9，以46c(60mg，0.11mmol)为反应原料，得到目标产物黄色固体实施例14(14mg，产率：12.5％)。1H NMR(300MHz,DMSO-d6)δ10.87(s,1H),8.56(s,1H),7.83(s,1H),7.76(d,J＝4.9Hz,1H),7.43(d,J＝8.4Hz,1H),7.35(d,J＝2.2Hz,1H),7.17(d,J＝2.2Hz,1H),6.88(s,1H),6.68(s,1H),6.58(d,J＝8.7Hz,1H),4.84(dd,J＝12.5,5.4Hz,1H),4.36(s,2H),4.27(d,J＝12.2Hz,2H),4.19–4.15(m,2H),4.12(s,1H),3.98–3.92(m,2H),3.79(d,J＝12.4Hz,1H),3.23(d,J＝6.7Hz,3H),3.17(s,2H),2.85–2.60(m,6H),2.43(d,J＝4.6Hz,5H),2.33(s,5H),2.06(s,1H),1.86–1.74(m,3H),1.62–1.38(m,7H),1.31–1.20(m,2H),1.02(s,1H).HRMS(ESI):calcd for C50H56ClN11O9[M+H]+990.3951,found 990.4016。纯度：95.82％by HPLC(MeOH/H2O＝80:20,tR＝3.842min)。
实施例15
2-((9-((5-氯-2-(4-(4-((2-(2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,8-二氮杂螺[4.5]癸烷-8-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例15)
2-((9-((5-chloro-2-(4-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,8-diazaspiro[4.5]decan-8-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例15)
按合成通法9，以46d(60mg，0.11mmol)为反应原料，得到目标产物黄色固体实施例15(11mg，产率：9.6％)。1H NMR(300MHz,DMSO-d6)δ11.10(s,1H),8.79(s,1H),8.10–8.05(m,1H),8.00(d,J＝4.8Hz,1H),7.66(d,J＝8.5Hz,1H),7.59(d,J＝2.2Hz,1H),7.40(d,J＝2.2Hz,1H),7.12(s,1H),6.94(d,J＝2.1Hz,1H),6.83(dd,J＝8.7,2.1Hz,1H),5.08(dd,J＝12.6,5.4Hz,1H),4.60(s,2H),4.51(d,J＝12.5Hz,2H),4.41(d,J＝4.5Hz,2H),4.37(s,1H),4.19(t,J＝4.7Hz,2H),4.03(d,J＝12.7Hz,1H),3.49(s,3H),3.40(s,3H),3.31(s,1H),3.10–2.85(m,6H),2.67(d,J＝4.6Hz,3H),2.62(s,1H),2.57(s,3H),2.40–2.12(m,2H),2.06–1.98(m,1H),1.94–1.87(m,2H),1.79(d,J＝13.8Hz,3H),1.65(d,J＝12.0Hz,6H),1.51(d,J＝6.6Hz,2H),1.26(s,1H).HRMS(ESI):calcd for C51H58ClN11O9[M+H]+1004.4108,found 1004.4176。纯度：95.02％by HPLC(MeOH/H2O＝80:20,tR＝3.813min)。
实施例16
2-((9-((5-氯-2-(4-(4-((9-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例16)
2-((9-((5-chloro-2-(4-(4-((9-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例16)
按合成通法9，以46e(60mg，0.11mmol)为反应原料，得到目标产物黄色固体实施例16(17mg，产率：14.7％)。1H NMR(300MHz,DMSO-d6)δ11.12(s,1H),8.80(s,1H),8.07(s,1H),8.00(q,J＝4.2Hz,1H),7.67(d,J＝8.5Hz,1H),7.59(d,J＝2.2Hz,1H),7.40(d,J＝2.0Hz,1H),7.32(d,J＝2.1Hz,1H),7.24(d,J＝8.9Hz,1H),7.12(s,1H),5.09(dd,J＝12.8,5.4Hz,1H),4.60(s,2H),4.51(d,J＝12.4Hz,2H),4.40(t,J＝4.3Hz,2H),4.36(s,1H),4.18(t,J＝4.2Hz,2H),4.02(d,J＝12.3Hz,1H),3.47(s,6H),2.96(s,2H),2.67(d,J＝4.6Hz,3H),2.63(s,1H),2.57(s,2H),2.36(s,4H),2.17(s,2H),2.06–1.98(m,2H),1.77(d,J＝14.7Hz,4H),1.65(d,J＝13.8Hz,2H),1.52(s,11H).HRMS(ESI):calcd for C52H60ClN11O9[M+H]+1018.4264,found 1018.4340。纯度：95.29％by HPLC(MeOH/H2O＝80:20,tR＝3.702min)。
合成路线8：
实施例17～18按照合成路线8合成。
合成路线8：试剂和条件:(a)Cs2CO3,Xantphos,Pd2(dba)3,二氧六环,100℃ 4h；(b)Cs2CO3,PdCl2(dppf)CH2Cl2,二氧六环,100℃,4h；(c)H2,Pd/C,THF,12h；(d)TFA,DCM,3h；(e)NaBH(OAc)3,DCE,6h；(f)TFA,DCM,3h；(g)EDCI,HOBt,DIPEA,DMF,12h.
7-(4-溴-2-氟苯基)-2,7-二氮杂螺[4.4]壬烷-2-羧酸叔丁酯(48a)
tert-butyl 7-(4-bromo-2-fluorophenyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate(48a)
合成通法10：将中间体47(500mg，1.66mmol)与47a(376mg，1.66mmol)加入至含有10mL无水二氧六环溶液的封管中，再依次加入碳酸铯(1.08g，3.32mmol)、Pd2(dba)3(106mg，0.12mmol)、XantPhos(144mg，0.25mmol)，氩气置换，100℃反应4小时，TLC监测反应完全结束。待反应冷却至室温后，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体48a，为白色固体(560mg，产率：84.4％)。ESI-MS:m/z:[M+H]+399.10。
2-(4-溴-2-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸叔丁酯(48b)
tert-butyl 2-(4-bromo-2-fluorophenyl)-2,8-diazaspiro[4.5]decane-8-carboxylate(48b)
按合成通法10，以47b(500mg，1.66mmol)为反应原料，得到目标产物白色固体48b(490mg，产率：71.3％)。ESI-MS:m/z:[M+H]+413.12。
7-(4-(2,6-双(苄氧基)吡啶-3-基)-2-氟苯基)-2,7-二氮杂螺[4.4]壬烷-2-羧酸叔丁酯(50a)
tert-butyl-7-(4-(2,6-bis(benzyloxy)pyridin-3-yl)-2-fluorophenyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate(50a)
合成通法11：将中间体48a(560mg，1.40mmol)与49(761mg，1.82mmol)加入至含有二氧六环：水＝6mL：2mL混合溶液的封管中，再依次加入碳酸铯(1.08g，3.32mmol)和PdCl2(dppf)CH2Cl2(110mg，0.14mmol)，氩气置换，100℃反应4小时，TLC监测反应完全结束。待反应冷却至室温后，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为石油醚：乙酸乙酯＝5：1，v/v)纯化得中间体50a，为白色固体(750mg，产率：87.7％)。1H NMR(300MHz,DMSO-d6)δ7.77(d,J＝8.2Hz,1H),7.50(d,J＝2.0Hz,1H),7.47(q,J＝1.9Hz,2H),7.45(t,J＝1.9Hz,2H),7.42–7.40(m,2H),7.38(d,J＝1.7Hz,2H),7.37(d,J＝1.3Hz,1H),7.35–7.32(m,1H),7.28(dd,J＝8.4,2.1Hz,1H),6.76(dd,J＝9.9,8.5Hz,1H),6.57(d,J＝8.1Hz,1H),5.43(d,J＝12.6Hz,4H),3.49(td,J＝7.0,2.6Hz,2H),3.41(s,1H),3.35(d,J＝6.3Hz,3H),3.28(d,J＝4.4Hz,2H),1.98–1.85(m,4H),1.45(d,J＝2.7Hz,9H).ESI-MS:m/z:[M+H]+610.30。
2-(4-(2,6-双(苄氧基)吡啶-3-基)-2-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸叔丁酯(50b)
tert-butyl-2-(4-(2,6-bis(benzyloxy)pyridin-3-yl)-2-fluorophenyl)-2,8-diazaspiro[4.5]decane-8-carboxylate(50b)
按合成通法11，以48b(540mg，1.31mmol)为反应原料，得到目标产物白色固体50b(710mg，产率：87.1％)。ESI-MS:m/z:[M+H]+624.32。
7-(4-(2,6-二氧代哌啶-3-基)-2-氟苯基)-2,7-二氮杂螺[4.4]壬烷-2-羧酸叔丁酯(51a)
tert-butyl-7-(4-(2,6-dioxopiperidin-3-yl)-2-fluorophenyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate(51a)
合成通法12：将中间体50a(750mg，1.23mmol)加入至含有10mL四氢呋喃溶液的圆底烧瓶中，加入钯碳(150mg，20％m/m)，氢气置换，室温搅拌16小时，TLC监测反应完全结束。反应液经硅藻土抽滤，二氯甲烷洗涤，滤液低压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体51a，为白色油状物(451mg，产率：85.1％)。ESI-MS:m/z:[M+H]+432.22。
2-(4-(2,6-二氧代哌啶-3-基)-2-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸叔丁酯(51b)
tert-butyl-2-(4-(2,6-dioxopiperidin-3-yl)-2-fluorophenyl)-2,8-diazaspiro[4.5]decane-8-carboxylate(51b)
按合成通法12，以50b(680mg，1.09mmol)为反应原料，得到目标产物白色油状物51b(462mg，产率：95.1％)。ESI-MS:m/z:[M+H]+446.24。
3-(3-氟-4-(2,7-二氮杂螺[4.4]壬烷-2-基)苯基)哌啶-2,6-二酮(52a)
3-(3-fluoro-4-(2,7-diazaspiro[4.4]nonan-2-yl)phenyl)piperidine-2,6-dione(52a)
按合成通法4，以51a(440mg，1.02mmol)为反应原料，得到目标产物白色油状物52a(315mg，产率：93.2％)。ESI-MS:m/z:[M+H]+332.17。
3-(3-氟-4-(2,8-二氮杂螺[4.5]癸烷-2-基)苯基)哌啶-2,6-二酮(52b)
3-(3-fluoro-4-(2,8-diazaspiro[4.5]decan-2-yl)phenyl)piperidine-2,6-dione(52b)
按合成通法4，以51b(430mg，0.96mmol)为反应原料，得到目标产物白色油状物52b(322mg，产率：96.6％)。ESI-MS:m/z:[M+H]+346.18。
4-((7-(4-(2,6-二氧代哌啶-3-基)-2-氟苯基)-2,7-二氮杂螺[4.4]壬烷-2-基)甲基)哌啶-1-羧酸叔丁酯(53a)
tert-butyl-4-((7-(4-(2,6-dioxopiperidin-3-yl)-2-fluorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)methyl)piperidine-1-carboxylate(53a)
按合成通法1，以中间体52a(300mg，0.90mmol)为反应原料，得到目标产物白色固体53a(302mg，产率：63.1％)。ESI-MS:m/z:[M+H]+529.31。
4-((2-(4-(2,6-二氧代哌啶-3-基)-2-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-基)甲基)哌啶-1-羧酸叔丁酯(53b)
tert-butyl-4-((2-(4-(2,6-dioxopiperidin-3-yl)-2-fluorophenyl)-2,8-diazaspiro[4.5]decan-8-yl)methyl)piperidine-1-carboxylate(53b)
按合成通法1，以中间体52b(300mg，0.87mmol)为反应原料，得到目标产物白色固体53b(290mg，产率：61.5％)。ESI-MS:m/z:[M+H]+543.33。
3-(3-氟-4-(7-(哌啶-4-基甲基)-2,7-二氮杂螺[4.4]壬烷-2-基)苯基)哌啶-2,6-二酮(54a)
3-(3-fluoro-4-(7-(piperidin-4-ylmethyl)-2,7-diazaspiro[4.4]nonan-2-yl)phenyl)piperidine-2,6-dione(54a)
按合成通法4，以53a(290mg，0.55mmol)为反应原料，得到目标产物白色油状物54a(210mg，产率：89.3％)。ESI-MS:m/z:[M+H]+429.26。
3-(3-氟-4-(8-(哌啶-4-基甲基)-2,8-二氮杂螺[4.5]癸烷-2-基)苯基)哌啶-2,6-二酮(54b)
3-(3-fluoro-4-(8-(piperidin-4-ylmethyl)-2,8-diazaspiro[4.5]decan-2-yl)phenyl)piperidine-2,6-dione(54b)
按合成通法4，以53b(270mg，0.50mmol)为反应原料，得到目标产物白色油状物54b(192mg，产率：87.2％)。ESI-MS:m/z:[M+H]+443.27。
实施例17
2-((9-((5-氯-2-(4-(4-(4-((7-(4-(2,6-二氧代哌啶-3-基)-2-氟苯基)-2,7-二氮杂螺[4.4]壬烷-2-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例17)
2-((9-((5-chloro-2-(4-(4-((7-(4-(2,6-dioxopiperidin-3-yl)-2-fluorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例17)
按合成通法9，以54a(60mg，0.11mmol)为反应原料，得到目标产物白色固体17(10mg，产率：9.4％)。1H NMR(300MHz,DMSO-d6)δ10.80(s,1H),8.79(s,1H),8.06(s,1H),7.99(d,J＝5.1Hz,1H),7.58(d,J＝2.2Hz,1H),7.40(d,J＝2.2Hz,1H),7.12(s,1H),7.02–6.83(m,2H),6.65(t,J＝9.0Hz,1H),4.60(s,2H),4.50(d,J＝12.6Hz,2H),4.40(t,J＝4.6Hz,2H),4.35(s,1H),4.18(t,J＝4.5Hz,2H),4.03(d,J＝13.0Hz,1H),3.74(dd,J＝11.4,4.8Hz,1H),3.39(s,3H),3.33(s,4H),3.25(d,J＝8.3Hz,1H),3.00(dt,J＝23.8,13.0Hz,5H),2.67(d,J＝4.7Hz,4H),2.61(d,J＝5.5Hz,3H),2.34–2.11(m,2H),2.03–1.89(m,3H),1.85–1.73(m,4H),1.64(d,J＝9.6Hz,2H),1.56–1.43(m,2H),1.26(d,J＝3.6Hz,2H).HRMS(ESI):calcd for C48H56ClFN10O7[M+H]+939.4006,found 939.4068。纯度：95.55％by HPLC(MeOH/H2O＝80:20,tR＝3.782min)。
实施例18
2-((9-((5-氯-2-(4-(4-((2-(4-(2-(2-(2,6-二氧代哌啶-3-基)-2-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例18)
2-((9-((5-chloro-2-(4-(4-((2-(4-(2,6-dioxopiperidin-3-yl)-2-fluorophenyl)-2,8-diazaspiro[4.5]decan-8-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例18)
按合成通法9，以54b(60mg，0.11mmol)为反应原料，得到目标产物白色固体实施例18(7mg，产率：6.5％)。1H NMR(300MHz,DMSO-d6)δ10.81(s,1H),8.79(s,1H),8.06(s,1H),7.99(d,J＝5.3Hz,1H),7.58(d,J＝2.2Hz,1H),7.40(d,J＝2.2Hz,1H),7.12(s,1H),6.94(dd,J＝15.0,2.0Hz,1H),6.86(d,J＝8.5Hz,1H),6.66(t,J＝9.1Hz,1H),4.60(s,2H),4.50(d,J＝12.2Hz,2H),4.42–4.38(m,2H),4.36(s,1H),4.21–4.16(m,2H),4.02(d,J＝13.0Hz,1H),3.74(dd,J＝11.4,4.8Hz,1H),3.46(s,4H),3.16(s,2H),2.98(q,J＝11.7Hz,4H),2.67(d,J＝4.6Hz,4H),2.60(d,J＝4.7Hz,1H),2.35(d,J＝20.4Hz,3H),2.17(d,J＝12.2Hz,3H),2.04–1.97(m,1H),1.82–1.72(m,5H),1.65(d,J＝14.0Hz,3H),1.53(d,J＝19.7Hz,6H),1.25(s,1H).HRMS(ESI):calcd for C49H58ClFN10O7[M+H]+953.4163,found 953.4231.
合成路线9：
实施例19按照合成路线9合成。
合成路线9：试剂和条件:(a)K2CO3,DMF,60℃,4h；(b)H2,Pd/C,EtOH,rt,6h；(c)NaHCO3,DMF,80℃,16h；(d)TFA,DCM,rt,3h；(e)NaBH(OAc)3,DCE,rt,5h；(f)TFA,DCM,rt,2h；(g)DIPEA,EDCI,HOBt,,rt,12h.
2-(2-氟-4-硝基苯基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸叔丁酯(56)
tert-butyl 2-(2-fluoro-4-nitrophenyl)-2,8-diazaspiro[4.5]decane-8-carboxylate(56)
合成通法13：将55(400mg,2.51mmol)，47b(604mg,2.51mmol)，碳酸钾(695mg,5.03mmol)加入到圆底烧瓶中，加入10mL的DMF溶液，60℃搅拌4h后TLC监测反应完全结束。待反应冷却至室温后，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为石油醚：乙酸乙酯＝4：1，v/v)纯化得中间体56，为白色固体(874mg，产率：91.6％)。ESI-MS:m/z:[M+H]+380.19。
2-(4-氨基-2-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸叔丁酯(57)
tert-butyl 2-(4-amino-2-fluorophenyl)-2,8-diazaspiro[4.5]decane-8-carboxylate(57)
按合成通法2，以中间体56(800mg，2.11mmol)为反应原料，得到目标产物白色油状物57(719mg，产率：97.6％)。ESI-MS:m/z:[M+H]+350.22。
2-(4-((2,6-二氧代哌啶-3-基)氨基)-2-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸叔丁酯(58)
tert-butyl-2-(4-((2,6-dioxopiperidin-3-yl)amino)-2-fluorophenyl)-2,8-diazaspiro[4.5]decane-8-carboxylate(58)
合成通法14：将57(800mg,2.29mmol)，57a(1.10g,5.72mmol)，碳酸氢钠(1.92g,22.90mmol)加入到圆底烧瓶中，加入15mL的DMF溶液，85℃搅拌16h后TLC监测反应完全结束。待反应冷却至室温后，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝50：1，v/v)纯化得中间体58，为白色固体(632mg，产率：59.9％)。ESI-MS:m/z:[M+H]+461.25。
3-((3-氟-4-(2,8-二氮杂螺[4.5]癸烷-2-基)苯基)氨基)哌啶-2,6-二酮(59)
3-((3-fluoro-4-(2,8-diazaspiro[4.5]decan-2-yl)phenyl)amino)piperidine-2,6-dione(59)
按合成通法4，以58(600mg，1.30mmol)为反应原料，得到目标产物白色油状物59(431mg，产率：91.8％)。ESI-MS:m/z:[M+H]+361.20。
4-((2-(4-((2,6-二氧代哌啶-3-基)氨基)-2-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-基)甲基)哌啶-1-羧酸叔丁酯(60)
tert-butyl-4-((2-(4-((2,6-dioxopiperidin-3-yl)amino)-2-fluorophenyl)-2,8-diazaspiro[4.5]decan-8-yl)methyl)piperidine-1-carboxylate(60)
按合成通法1，以中间体59(400mg，1.11mmol)为反应原料，得到目标产物白色固体60(301mg，产率：48.6％)。ESI-MS:m/z:[M+H]+558.34。
3-((3-氟-4-(8-(哌啶-4-基甲基)-2,8-二氮杂螺[4.5]癸烷-2-基)苯基)氨基)哌啶-2,6-二酮(61)
3-((3-fluoro-4-(8-(piperidin-4-ylmethyl)-2,8-diazaspiro[4.5]decan-2-yl)phenyl)amino)piperidine-2,6-dione(61)
按合成通法4，以60(300mg，0.54mmol)为反应原料，得到目标产物白色油状物61(208mg，产率：84.5％)。ESI-MS:m/z:[M+H]+458.28。
实施例19
2-((9-((5-氯-2-(4-(4-((2-(4-(4-((2,6-二氧代哌啶-3-基)氨基)-2-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例19)
2-((9-((5-chloro-2-(4-(4-((2-(4-((2,6-dioxopiperidin-3-yl)amino)-2-fluorophenyl)-2,8-diazaspiro[4.5]decan-8-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例19)
按合成通法9，以61(60mg，0.11mmol)为反应原料，得到目标产物褐色固体19(9mg，产率：8.2％)。1H NMR(300MHz,DMSO-d6)δ10.79(s,1H),8.79(s,1H),8.07(s,1H),7.99(d,J＝4.4Hz,1H),7.58(d,J＝2.3Hz,1H),7.40(d,J＝2.3Hz,1H),7.12(s,1H),6.66–6.48(m,2H),6.41(d,J＝8.7Hz,1H),5.58(d,J＝7.6Hz,1H),4.60(s,2H),4.50(d,J＝12.2Hz,2H),4.39(d,J＝4.9Hz,2H),4.36(s,1H),4.28–4.21(m,1H),4.19(d,J＝4.8Hz,2H),4.03(d,J＝12.3Hz,1H),3.19(t,J＝6.0Hz,2H),2.98(t,J＝16.9Hz,8H),2.76(d,J＝12.2Hz,1H),2.67(d,J＝4.6Hz,4H),2.60(d,J＝4.6Hz,1H),2.34(s,3H),2.11(dd,J＝8.8,4.7Hz,3H),1.73(s,4H),1.57(s,4H),1.35(s,2H),1.31(s,1H),1.27(s,4H).HRMS(ESI):calcd for C49H59ClFN11O7[M+H]+968.4271,found 968.4332。纯度：95.87％by HPLC(MeOH/H2O＝80:20,tR＝3.697min)。
合成路线10：
实施例20按照合成路线10合成。
合成路线10：试剂和条件:(a)K2CO3,DMF,60℃,4h；(b)H2,Pd/C,EtOH,rt,6h；(c)NaHCO3,DMF,80℃,16h；(d)TFA,DCM,rt,3h；(e)DIPEA,EDCI,HOBt,rt,12h.
4-((1-(2-氟-4-硝基苯基)哌啶-4-基)甲基)哌嗪-1-羧酸叔丁酯(56b)
tert-butyl 4-((1-(2-fluoro-4-nitrophenyl)piperidin-4-yl)methyl)piperazine-1-carboxylate(56b)
按合成通法13，以55(500mg，3.14mmol)为反应原料，得到目标产物白色固体56b(1.13g，产率：85.1％)。ESI-MS:m/z:[M+H]+423.23。
4-((1-(4-氨基-2-氟苯基)哌啶-4-基)甲基)哌嗪-1-羧酸叔丁酯(57b)
tert-butyl 4-((1-(4-amino-2-fluorophenyl)piperidin-4-yl)methyl)piperazine-1-carboxylate(57b)
按合成通法2，以56b(700mg，1.66mmol)为反应原料，得到目标产物白色油状物57b(626mg，产率：96.3％)。ESI-MS:m/z:[M+H]+393.26。
4-((1-(4-((2,6-二氧代哌啶-3-基)氨基)-2-氟苯基)哌啶-4-基)甲基)哌嗪-1-羧酸叔丁酯(58b)
tert-butyl-4-((1-(4-((2,6-dioxopiperidin-3-yl)amino)-2-fluorophenyl)piperidin-4-yl)methyl)piperazine-1-carboxylate(58b)
按合成通法14，以57b(700mg，1.27mmol)为反应原料，得到目标产物暗紫色固体58b(301mg，产率：46.9％)。ESI-MS:m/z:[M+H]+504.29。
3-((3-氟-4-(4-(哌嗪-1-基甲基)哌啶-1-基)苯基)氨基)哌啶-2,6-二酮(59b)
3-((3-fluoro-4-(4-(piperazin-1-ylmethyl)piperidin-1-yl)phenyl)amino)piperidine-2,6-dione(59b)
按合成通法4，以58b(283mg，0.56mmol)为反应原料，得到目标产物暗紫色油状物59b(199mg，产率：87.8％)。ESI-MS:m/z:[M+H]+404.24。
实施例20
2-((9-((5-氯-2-(4-(4-((1-(4-((2,6-二氧代哌啶-3-基)氨基)-2-氟苯基)哌啶-4-基)甲基)哌嗪-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例20)
2-((9-((5-chloro-2-(4-(4-((1-(4-((2,6-dioxopiperidin-3-yl)amino)-2-fluorophenyl)piperidin-4-yl)methyl)piperazine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例20)
按合成通法9，以35(60mg，0.11mmol)为反应原料，得到目标产物褐色固体实施例20(11mg，产率：10.2％)。1H NMR(300MHz,DMSO-d6)δ10.80(s,1H),8.79(s,1H),8.06(s,1H),7.99(q,J＝3.7Hz,1H),7.58(d,J＝2.2Hz,1H),7.40(d,J＝2.2Hz,1H),7.12(s,1H),6.84(t,J＝9.3Hz,1H),6.51(dd,J＝15.0,2.4Hz,1H),6.42(d,J＝8.9Hz,1H),5.81(d,J＝7.6Hz,1H),4.60(s,2H),4.50(d,J＝12.6Hz,2H),4.40(t,J＝4.7Hz,2H),4.28(dd,J＝10.4,6.2Hz,1H),4.19(d,J＝4.8Hz,2H),3.56(s,2H),3.46(s,2H),3.12(d,J＝10.8Hz,2H),2.96(t,J＝12.1Hz,3H),2.67(d,J＝4.6Hz,3H),2.60(d,J＝4.7Hz,1H),2.55(s,4H),2.38(s,2H),2.30(s,2H),2.20(d,J＝6.8Hz,2H),2.14–2.05(m,1H),1.78(d,J＝12.1Hz,2H),1.66(d,J＝12.6Hz,2H),1.51(t,J＝12.5Hz,2H),1.36–1.20(m,3H).HRMS(ESI):calcd for C45H53ClFN11O7[M+H]+914.3802,found 914.3814.
合成路线11：
实施例21按照合成路线11合成。
合成路线11：试剂和条件:(a)Cs2CO3,Xantphos,Pd(OAc)2,二氧六环,110℃,10h；(b)LiOH,MeOH,H2O,rt,12h；(c)EDCI,HOBt,TEA,DMF,12h；(d)TFA,DCM,3h；(e)NaBH(OAc)3,DCE,6h；(f)TFA,DCM,3h；(g)EDCI,HOBt,DIPEA,DMF,12h.
2-(3-氟-4-(甲氧羰基)苯基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸叔丁酯(63)
tert-butyl-2-(3-fluoro-4-(methoxycarbonyl)phenyl)-2,8-diazaspiro[4.5]decane-8-carboxylate(63)
按合成通法6，将中间体62(360mg，1.54mmol)与47b(482mg，1.70mmol)加入至含有10mL无水二氧六环溶液的封管中，再依次加入碳酸铯(1.01g，3.09mmol)、醋酸钯(17mg，0.08mmol)、XantPhos(89mg，0.15mmol)，氩气置换，110℃反应16小时，TLC监测反应完全结束。待反应冷却至室温后，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过制备层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体63，为白色固体(390mg，产率：58.0％)。ESI-MS:m/z:[M+H]+393.21。
4-(8-(叔丁氧羰基)-2,8-二氮杂螺[4.5]癸烷-2-基)-2-氟苯甲酸(64)
4-(8-(tert-butoxycarbonyl)-2,8-diazaspiro[4.5]decan-2-yl)-2-fluorobenzoic acid(64)
按合成通法7，将中间体63(200mg，0.46mmol)加入至含有甲醇：水＝8mL：2mL混合溶液的圆底烧瓶中，再加入氢氧化锂(55mg，2.30mmol)，40℃反应10小时，TLC监测反应完全结束。待反应冷却至室温后，加入稀盐酸调节溶液pH值为5～6，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体64，为白色固体(180mg，产率：93.0％)。ESI-MS:m/z:[M+H]+379.20。
2-(4-((2,6-二氧代哌啶-3-基)氨基甲酰基)-3-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸叔丁酯(65)
tert-butyl-2-(4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3-fluorophenyl)-2,8-diazaspiro[4.5]decane-8-carboxylate(65)
按合成通法8，将中间体64(180mg，0.43mmol)加入至含有5mLDMF溶液的圆底烧瓶中，再依次加入DIPEA(276mg，2.14mmol)、EDCI(123mg，0.64mmol)、HOBt(87mg，0.64mmol)，室温搅拌半小时，再加入27a(83mg，0.51mmol)，室温反应过夜，TLC监测反应完全结束。使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝50：1，v/v)纯化得中间体65，为白色固体(160mg，产率：70.5％)。ESI-MS:m/z:[M+H]+489.24
N-(2,6-二氧代哌啶-3-基)-2-氟-4-(2,8-二氮杂螺[4.5]癸烷-2-基)苯甲酰胺(66)
N-(2,6-dioxopiperidin-3-yl)-2-fluoro-4-(2,8-diazaspiro[4.5]decan-2-yl)benzamide(66)
按合成通法4，以65(320mg，0.65mmol)为反应原料，得到目标产物白色油状物66(237mg，产率：93.2％)。ESI-MS:m/z:[M+H]+389.19。
4-((2-(4-((2,6-二氧代哌啶-3-基)氨基甲酰基)-3-氟苯基)-2,8-二氮杂螺[4.5]癸烷-8-基)甲基)哌啶-1-羧酸叔丁酯(67)
tert-butyl-4-((2-(4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3-fluorophenyl)-2,8-diazaspiro[4.5]decan-8-yl)methyl)piperidine-1-carboxylate(67)
按合成通法1，以中间体66(230mg，0.59mmol)为反应原料，得到目标产物白色固体67(195mg，产率：56.2％)。ESI-MS:m/z:[M+H]+586.33。
N-(2,6-二氧代哌啶-3-基)-2-氟-4-(8-(哌啶-4-基甲基)-2,8-二氮杂螺[4.5]癸烷-2-基)苯甲酰胺(68)
N-(2,6-dioxopiperidin-3-yl)-2-fluoro-4-(8-(piperidin-4-ylmethyl)-2,8-diazaspiro[4.5]decan-2-yl)benzamide(68)
按合成通法4，以67(190mg，0.32mmol)为反应原料，得到目标产物白色油状物68(143mg，产率：90.8％)。ESI-MS:m/z:[M+H]+486.28。
实施例21
4-(8-((1-(1-(5-氯-4-((6-(2-(甲基氨基)-2-氧代乙氧基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-9-基)氨基)嘧啶-2-基)哌啶-4-羰基)哌啶-4-基)甲基)-2,8-二氮杂螺[4.5]癸烷-2-基)-N-(2,6-二氧代哌啶-3-基)-2-氟苯甲酰胺(实施例21)
4-(8-((1-(1-(5-chloro-4-((6-(2-(methylamino)-2-oxoethoxy)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-9-yl)amino)pyrimidin-2-yl)piperidine-4-carbonyl)piperidin-4-yl)methyl)-2,8-diazaspiro[4.5]decan-2-yl)-N-(2,6-dioxopiperidin-3-yl)-2-fluorobenzamide(实施例21)
按合成通法9，以35(60mg，0.11mmol)为反应原料，得到目标产物褐色固体实施例21(17mg，产率：15.0％)。1H NMR(300MHz,DMSO-d6)δ10.87(s,1H),8.79(s,1H),8.07(s,1H),8.00(q,J＝4.1,3.7Hz,1H),7.92(t,J＝7.8Hz,1H),7.66(t,J＝8.9Hz,1H),7.59(d,J＝2.3Hz,1H),7.40(d,J＝2.2Hz,1H),7.12(s,1H),6.43(dd,J＝9.1,2.2Hz,1H),6.35(d,J＝15.1Hz,1H),4.74(dt,J＝12.4,6.4Hz,1H),4.60(s,2H),4.51(d,J＝12.0Hz,2H),4.41(d,J＝4.4Hz,2H),4.33(s,1H),4.21–4.16(m,2H),4.03(d,J＝12.9Hz,1H),3.68–3.39(m,3H),3.17(s,2H),2.99(dd,J＝24.0,11.5Hz,4H),2.83–2.72(m,1H),2.67(d,J＝4.6Hz,3H),2.42–2.29(m,2H),2.15(d,J＝11.7Hz,2H),2.08–1.97(m,2H),1.86(s,3H),1.76(s,2H),1.68–1.49(m,8H),1.32(s,1H),1.26(d,J＝7.7Hz,4H).HRMS(ESI):calcd for C50H59ClFN11O8[M+H]+996.4221,found 996.4289.纯度：96.27％by HPLC(MeOH/H2O＝80:20,tR＝3.891min)。
合成路线12：
实施例22按照合成路线12合成。
合成路线12：试剂和条件:(a)DIPEA,DMSO,100℃,10h；(b)戴斯-马丁氧化剂,DMF,100℃,2h；(c)NaBH(OAc)3,DCE,6h.
2-((9-((5-氯-2-(4-(羟甲基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(12b)
2-((9-((5-chloro-2-(4-(hydroxymethyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(12b)
将中间体11(300mg，0.69mmol)，原料11c(158mg，1.38mmol)，DIPEA(151mg，1.17mmol)加入到圆底烧瓶中，加入6mL的DMSO溶液，100℃搅拌过夜后TLC监测反应完全结束。缓慢加入40mL的水搅拌十分钟，抽滤，水洗，干燥得黄色固体12b(212mg，产率：59.9％)。ESI-MS:m/z:[M+H]+515.17。
2-((9-((5-氯-2-(4-甲酰基哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(13b)
2-((9-((5-chloro-2-(4-formylpiperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(13b)
将中间体12b(200mg，0.39mmol)，戴斯-马丁氧化剂(496mg，1.17mmol)，加入到圆底烧瓶中，加入5mL的无水DMF溶液，室温搅拌2h后TLC监测反应完全结束。使用乙酸乙酯萃取两次，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩得黄色固体粗品13b(141mg，产率：70.8％)。ESI-MS:m/z:[M+H]+513.16。
实施例22
2-((9-((5-氯-2-(4-((4-((7-(4-(4-(2,6-二氧代哌啶-3-基)-2-氟苯基)-2,7-二氮杂螺[4.4]壬烷-2-基)甲基)哌啶-1-基)甲基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例22)
2-((9-((5-chloro-2-(4-((4-((7-(4-(2,6-dioxopiperidin-3-yl)-2-fluorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)methyl)piperidin-1-yl)methyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例22)
将中间体13b(80mg，0.16mmol)与54a(100mg，0.23mmol)加入至含有5mL无水DMF溶液的圆底烧瓶中，室温搅拌15分钟，再加入NaBH(OAc)3(99mg，0.47mmol)，室温搅拌过夜，TLC监测反应完全结束。使用乙酸乙酯萃取三次，合并有机相，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过制备层析(流动相体系为二氯甲烷：甲醇＝15：1，v/v)纯化得目标化合物实施例22，为棕色固体(10mg，产率：6.93％)。1H NMR(300MHz,DMSO-d6)δ9.21(s,1H),8.03(s,1H),7.79(s,1H),7.18(s,1H),7.07(d,J＝7.5Hz,1H),6.77(d,J＝8.8Hz,1H),6.61(d,J＝2.9Hz,2H),6.59(d,J＝7.5Hz,1H),4.77(s,2H),4.41(dt,J＝12.5,7.1Hz,2H),4.39(s,2H),4.05(s,2H),3.82(s,1H),3.71(dt,J＝9.5,7.1Hz,1H),3.66–3.51(m,3H),3.32(d,J＝9.5Hz,1H),3.17–3.10(m,2H),2.81(s,3H),2.63–2.54(m,2H),2.59–2.46(m,4H),2.33–2.19(m,2H),2.22–2.13(m,2H),2.00(d,J＝9.5Hz,1H),1.97–1.86(m,2H),1.91–1.77(m,2H),1.82–1.69(m,2H),1.72–1.55(m,2H),1.42–1.32(m,4H),1.39–1.30(m,2H),1.33–1.20(m,2H).HRMS(ESI):calcd for C48H58ClFN10O6[M+H]+925.4213,found 925.4217.纯度：96.02％by HPLC(MeOH/H2O＝80:20,tR＝3.797min)。
合成路线13：
实施例23按照合成路线13合成。
合成路线13：试剂和条件:(a)Na2CO3,Pd(PPh)3Cl2,二氧六环,110℃,4h；(b)Pd/C,EtOH,rt,16h；(c)LiOH,MeOH,H2O,rt,16h；(d)BuLi,THF,DMF,-78℃,6h；(e)K2CO3,DMF,rt,5h；(f)NaBH3CN,DIPEA,DCE；(g)TFA,DCM,3h；(h)二甲基氯硅烷,ACN,6h；(i)Pd/C,H2,rt,12h；Boc2O,EtOH,THF,rt,3h；(j)Tf2O,TEA,DCM,rt,3h；(k)K2CO3,DMF,70℃,4h；(l)TFA,DCM,3h；(m)DIPEA,NMP,170℃,3h.
4-(2-氟-4-(甲氧羰基)苯基)-3,6-二氢吡啶-1(2H)-羧酸叔丁酯(70)
tert-butyl-4-(2-fluoro-4-(methoxycarbonyl)phenyl)-3,6-dihydropyridine-1(2H)-carboxylate(70)
将中间体69a(5.00g，21.46mmol)与69b(8.62g，27.89mmol)加入至含有二氧六环：水＝60：20mL溶液的封管中，再依次加入碳酸钠(4.29g，42.92mmol)、Pd(PPh)3Cl2(1.51g，2.15mmol)，氩气置换，100℃反应4小时，TLC监测反应完全结束。待反应冷却至室温后，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为石油醚：乙酸乙酯＝5：1，v/v)纯化得中间体70，为白色固体(4.6g，产率：63.9％)。ESI-MS:m/z:[M+H]+336.15。
4-(2-氟-4-(甲氧羰基)苯基)哌啶-1-羧酸叔丁酯(71)
tert-butyl 4-(2-fluoro-4-(methoxycarbonyl)phenyl)piperidine-1-carboxylate(71)
按合成通法2，以70(4.50g，13.42mmol)为反应原料，得到目标产物无色油状物71(4.32g，产率：95.4％)。ESI-MS:m/z:[M+H]+338.17。
4-(1-(叔丁氧羰基)哌啶-4-基)-3-氟苯甲酸(72)
4-(1-(tert-butoxycarbonyl)piperidin-4-yl)-3-fluorobenzoic acid(72)
按合成通法7，以71(4.40g，13.04mmol)为反应原料，得到目标产物白色固体72(4.06g，产率：96.3％)。ESI-MS:m/z:[M+H]+324.15。
4-(4-氟-3-羟基-1-氧代-1,3-二氢异苯并呋喃-5-基)哌啶-1-羧酸叔丁酯(73)
tert-butyl-4-(4-fluoro-3-hydroxy-1-oxo-1,3-dihydroisobenzofuran-5-yl)piperidine-1-carboxylate(73)
将中间体72(5.00g，15.46mmol)加入至含有无水THF：无水DMF＝40mL:20mL混合溶液的双颈瓶中，氩气置换，-78℃下搅拌30分钟，再缓慢加入BuLi(23.19mL，23.19mmol，1M in THF)、室温反应6小时，TLC监测反应完全结束。待反应恢复至室温后，缓慢加入饱和氯化铵淬灭，再使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，未经进一步纯化得中间体73，为白色固体(2.40g，产率：44.2％)。ESI-MS:m/z:[M+H]+352.15。
4-(2-氟-3-甲酰基-4-(甲氧羰基)苯基)哌啶-1-羧酸叔丁酯(74)
tert-butyl 4-(2-fluoro-3-formyl-4-(methoxycarbonyl)phenyl)piperidine-1-carboxylate(74)
将中间体73(4.00g，11.38mmol)加入至含有40mL DMF溶液的圆底烧瓶中，加入碳酸钾(3.15g，22.77mmol)，室温反应4小时，TLC监测反应完全结束。使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为石油醚：乙酸乙酯＝5：1，v/v)纯化得中间体74，为白色固体(1.93g，产率：46.4％)。ESI-MS:m/z:[M+H]+366.16。
4-(2-氟-3-甲酰基-4-(甲氧羰基)苯基)哌啶-1-羧酸叔丁酯(75)
tert-butyl 4-(2-fluoro-3-formyl-4-(methoxycarbonyl)phenyl)piperidine-1-carboxylate(75)
将中间体74(5.00g，13.68mmol)加入至含有60mLDCE溶液的圆底烧瓶中，再依次加入DIPEA(3.54g，27.37mmol)、74a(2.10g，16.42mmol)，室温搅拌半小时，再加入NaBH3CN(1.72g，27.37mmol)，室温反应6小时，TLC监测反应完全结束。减压浓缩，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体75，为白色固体(2.17g，产率：35.6％)。ESI-MS:m/z:[M+H]+446.20。
3-(4-氟-1-氧代-5-(哌啶-4-基)异吲哚啉-2-基)哌啶-2,6-二酮(76)
3-(4-fluoro-1-oxo-5-(piperidin-4-yl)isoindolin-2-yl)piperidine-2,6-dione(76)
按合成通法4，以75(3.00g，6.73mmol)为反应原料，得到目标产物无色油状物76(2.19g，产率：94.16％)。ESI-MS:m/z:[M+H]+336.15。
4-((1s，3s)-3-(苄氧基)环丁氧基)哌啶-1-羧酸苄酯(78)
benzyl 4-((1s,3s)-3-(benzyloxy)cyclobutoxy)piperidine-1-carboxylate(78)
将中间体77(17.67g，75.74mmol)加入至含有100mL无水乙腈溶液的圆底烧瓶中，冰浴半小时，再依次加入二甲基氯硅烷(9.56g，100.99mmol)、77(9.00g，50.50mmol)，室温反应6小时，TLC监测反应完全结束。减压浓缩，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得中间体78，为白色固体(8.20g，产率：41.1％)。ESI-MS:m/z:[M+H]+396.21。
4-((1s，3s)-3-羟基环丁氧基)哌啶-1-羧酸叔丁酯(79)
tert-butyl 4-((1s,3s)-3-hydroxycyclobutoxy)piperidine-1-carboxylate(79)
将中间体78(8.00g，20.23mmol)加入至含有100mL甲醇溶液的圆底烧瓶中，加入钯碳(1.60g，20％m/m)，氢气置换，室温反应过夜，TLC监测反应完全结束。硅藻土抽滤，甲醇洗涤，减压浓缩，得无色油状物。再加入乙醇：四氢呋喃＝40mL:40mL混合溶液，加入Boc2O(6.62g，30.34mmol)，室温反应4h，TLC监测反应完全结束。使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝40：1，v/v)纯化得中间体79，为无色油状物(5.01g，产率：91.28％)。ESI-MS:m/z:[M+H]+272.18。
4-((1s，3s)-3-(((三氟甲基)磺酰基)氧基)环丁氧基)哌啶-1-羧酸叔丁酯(80)
tert-butyl-4-((1s,3s)-3-(((trifluoromethyl)sulfonyl)oxy)cyclobutoxy)piperidine-1-carboxylate(80)
将中间体79(5.00g，18.43mmol)加入至含有50mL无水DCM溶液的圆底烧瓶中，再依次加入TEA(3.72g，36.86mmol)，Tf2O(7.80g，27.64mmol)，室温反应3h，TLC监测反应完全结束。减压浓缩，使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝50：1，v/v)纯化得中间体80，为无色油状物(6.28g，产率：84.5％)。ESI-MS:m/z:[M+H]+404.13。
4-((1r，3r)-3-(4-(2-(2,6-二氧代哌啶-3-基)-4-氟-1-氧代异吲哚啉-5-基)哌啶-1-基)环丁氧基)哌啶-1-羧酸叔丁酯(81)
tert-butyl-4-((1r,3r)-3-(4-(2-(2,6-dioxopiperidin-3-yl)-4-fluoro-1-oxoisoindolin-5-yl)piperidin-1-yl)cyclobutoxy)piperidine-1-carboxylate(81)
将中间体76(1.00g，2.90mmol)加入至含有20mL无水DMF溶液的圆底烧瓶中，再依次加入碳酸钾(0.80g，5.79mmol)，80(1.28g，3.18mmol)，70℃反应4h，TLC监测反应完全结束。使用乙酸乙酯萃取，有机层经饱和食盐水洗涤后加入无水硫酸钠干燥，减压浓缩，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝50：1，v/v)纯化得中间体81，为无色油状物(1.29g，产率：74.4％)。ESI-MS:m/z:[M+H]+599.32。
3-(4-氟-1-氧代-5-(1-((1r，3r)-3-(哌啶-4-基氧基)环丁基)哌啶-4-基)异吲哚啉-2-基)哌啶-2,6-二酮(82)
3-(4-fluoro-1-oxo-5-(1-((1r,3r)-3-(piperidin-4-yloxy)cyclobutyl)piperidin-4-yl)isoindolin-2-yl)piperidine-2,6-dione(82)
按合成通法4，以81(1.10g，1.84mmol)为反应原料，得到目标产物无色油状物82(0.85g，产率：92.8％)。ESI-MS:m/z:[M+H]+499.26。
实施例23
2-((9-((5-氯-2-(4-((1r，3r)-3-(4-(2-(2,6-二氧代哌啶-3-基)-4-氟-1-氧代异吲哚啉-5-基)哌啶-1-基)环丁氧基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N-甲基乙酰胺(实施例23)
2-((9-((5-chloro-2-(4-((1r,3r)-3-(4-(2-(2,6-dioxopiperidin-3-yl)-4-fluoro-1-oxoisoindolin-5-yl)piperidin-1-yl)cyclobutoxy)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N-methylacetamide(实施例23)
按合成通法5，以82(171mg，0.34mmol)为反应原料，得到目标产物黄色固体实施例23(85mg，产率：41.3％)。1H NMR(300MHz,DMSO-d6)δ8.61(s,1H),8.12(s,1H),7.83(s,1H),7.58(d,J＝7.5Hz,1H),6.62–6.62(m,2H),5.11(dt,J＝8.6,7.0Hz,2H),4.91(s,2H),4.81(d,J＝19.4Hz,1H),4.50(m,J＝19.4Hz,1H),4.31(t,J＝4.6Hz,2H),4.22(m,J＝12.5,7.1,3.8Hz,2H),4.07(t,J＝4.7Hz,2H),3.62(d,J＝12.6,7.1,3.9Hz,2H),3.32–3.21(m,3H),2.97(m,J＝14.3,7.1Hz,1H),2.92–2.81(m,2H),2.80(s,3H),2.62–2.51(m,2H),2.51–2.36(m,2H),2.19–2.01(m,7H),1.91–1.72(m,4H),1.57(m,J＝13.7,7.1Hz,2H).HRMS(ESI):calcd for C45H49ClFN9O8[M+H]+898.3377,found 898.3366.纯度：96.33％by HPLC(MeOH/H2O＝80:20,tR＝4.147min)。
合成路线14：
实施例24按照合成路线14合成。
合成路线14：试剂和条件:(a)N,N-二甲基-溴乙酰胺,Cs2CO3,DMF,rt,3h；(b)Fe,NH4Cl,EtOH,H2O,80℃,3h；(c)DIPEA,DMSO,100℃,3h；(d)DIPEA,DMSO,100℃,10h；(e)DIPEA,EDCI,HOBt,rt,12h.
N，N-二甲基-2-((9-硝基-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)乙酰胺(8b)
N,N-dimethyl-2-((9-nitro-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)acetamide(8b)
将中间体7(1.00g，4.03mmol)加入至圆底烧瓶中，再加入7b(0.83g，4.83mmol)，以及碳酸铯(2.63g，8.06mmol)，再加入DMF(15mL)，室温搅拌4h，TLC监测反应结束。先加入50mL的水，抽滤，水洗，滤饼烘干，向滤液中加入等体积的乙酸乙酯和水分液萃取3次，合并有机层并低压浓缩除去溶剂，残留物与滤饼合并，经过柱层析(洗脱体系为二氯甲烷：甲醇＝60：1，v/v)纯化得中间体8b，为黄色固体(0.53g，产率：37.3％)。1H NMR(300MHz,DMSO-d6)δ8.23(d,J＝2.5Hz,1H),7.73(d,J＝2.5Hz,1H),7.45(s,1H),4.97(s,2H),4.49(t,J＝4.7Hz,2H),4.24(t,J＝4.8Hz,2H),3.05(s,3H),2.90(s,3H)。ESI-MS:m/z:[M+H]+334.10。
2-((9-氨基-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N，N-二甲基乙酰胺(9b)
2-((9-amino-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N,N-dimethylacetamide(9b)
将8b(0.40g，1.20mmol)加入至含有无水乙醇：水＝20mL:4mL的圆底烧瓶中，加入氯化铵(0.35g，6.26mmol)，在搅拌的状态下加入还原铁粉(0.34g，6.26mmol)，80℃加热反应3h，TLC监测反应结束。撤去加热，待反应液冷却至室温，加入2mL氨甲醇溶液调节溶液pH值大于7，硅藻土抽滤，二氯甲烷：甲醇＝20：1(1000mL)洗，收集滤液，低压浓缩，得到淡黄色固体9b(0.23g，产率：63.5％)。ESI-MS:m/z:[M+H]+304.12。
2-((9-((2,5-二氯嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N，N-二甲基乙酰胺(11b)
2-((9-((2,5-dichloropyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N,N-dimethylacetamide(11b)
将中间体9b(0.40g，1.38mmol)，10(0.51g，2.77mmol)，DIPEA(0.54g，4.02mmol)加入到圆底烧瓶中，加入8mL的DMSO溶液，100℃搅拌3h后TLC监测反应完全结束。加入35mL的水搅拌半小时，抽滤，水洗，干燥得淡黄色固体中间体11b(0.43g，产率：76.0％)。ESI-MS:m/z:[M+H]+450.06。
1-(5-氯-4-((6-(2-(二甲基氨基)-2-氧代乙氧基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-9-基)氨基)嘧啶-2-基)哌啶-4-羧酸(35b)
1-(5-chloro-4-((6-(2-(dimethylamino)-2-oxoethoxy)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-9-yl)amino)pyrimidin-2-yl)piperidine-4-carboxylic acid(35b)
将中间体11b(300mg，0.69mmol)，原料12i(178mg，1.39mmol)，DIPEA(151mg，1.17mmol)加入到圆底烧瓶中，加入8mL的DMSO溶液，100℃搅拌10h后TLC监测反应完全结束。缓慢滴加稀盐酸调节溶液pH至2～5，再加入50mL的水搅拌十分钟，抽滤，水洗，干燥得黄色固体35b(310mg，产率：85.2％)。ESI-MS:m/z:[M+H]+543.17。
实施例24
2-((9-((5-氯-2-(4-(4-((2-(2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚啉-5-基)-2,8-二氮杂螺[4.5]癸烷-8-基)甲基)哌啶-1-羰基)哌啶-1-基)嘧啶-4-基)氨基)-5-氧代-2,3-二氢-5H-[1,4]噁嗪并[2,3,4-ij]喹啉-6-基)氧基)-N，N-二甲基乙酰胺(实施例24)
2-((9-((5-chloro-2-(4-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,8-diazaspiro[4.5]decan-8-yl)methyl)piperidine-1-carbonyl)piperidin-1-yl)pyrimidin-4-yl)amino)-5-oxo-2,3-dihydro-5H-[1,4]oxazino[2,3,4-ij]quinolin-6-yl)oxy)-N,N-dimethylacetamide(实施例24)
按合成通法13，以35b(60mg，0.10mmol)为反应原料，得到目标产物黄色固体实施例24(13mg，产率：12.2％)。1H NMR(300MHz,DMSO-d6)δ11.10(s,1H),8.79(s,1H),8.10–8.05(m,1H),8.00(d,J＝4.8Hz,1H),7.66(d,J＝8.5Hz,1H),7.59(d,J＝2.2Hz,1H),7.40(d,J＝2.2Hz,1H),7.12(s,1H),6.94(d,J＝2.1Hz,1H),6.83(dd,J＝8.7,2.1Hz,1H),5.08(dd,J＝12.6,5.4Hz,1H),4.60(s,2H),4.51(d,J＝12.5Hz,2H),4.41(d,J＝4.5Hz,2H),4.37(s,1H),4.19(t,J＝4.7Hz,2H),4.03(d,J＝12.7Hz,1H),3.49(s,3H),3.40(s,3H),3.31(s,1H),3.10–2.85(m,6H),2.67(d,J＝4.6Hz,3H),2.62(s,1H),2.57(s,6H),2.40–2.12(m,2H),2.06–1.98(m,1H),1.94–1.87(m,2H),1.79(d,J＝13.8Hz,3H),1.65(d,J＝12.0Hz,6H),1.51(d,J＝6.6Hz,2H),1.26(s,1H).HRMS(ESI):calcd for C52H60ClN11O9[M+H]+1018.4264,found 1018.4270。纯度：95.49％by HPLC(MeOH/H2O＝80:20,tR＝4.203min)。
药理活性评价
BCL6蛋白降解测试：将OCI-LY1细胞(浙江美森细胞科技有限公司)接种于6孔板中，随后加入不同浓度的化合物。给药12小时后，离心收取细胞，加入中效RIPA裂解液(碧云天生物技术)混匀裂解，离心吸取上清液，BCA法测定蛋白浓度。将蛋白质样品与蛋白上样缓冲液(碧云天生物技术)混匀于100℃干热加热10min制样，随后将样品加入12％的聚丙烯酰胺凝胶SDS-PAGE中，以60V恒压电泳至marker离开浓缩胶，随后用120V恒压继续电泳，在含有10％甲醇的湿转缓冲液中转膜90min，随后将转膜后的PVDF膜剪出所需要的条带，用牛奶封闭2小时，在相应条带中分别加入用牛奶稀释后的anti-BCL6抗体(abcam)和β-Actin(Proteintech)抗体，于4℃孵育过夜。次日用TBST洗去一抗，加入二抗室温孵育45min。TBST继续洗去二抗溶液，Odyssey Infrared Imaging System(LI-COR,Lincoln,Nebraska,USA)下扫膜。DC50指BCL6降解剂达到降解50％的BCL6蛋白时所需的浓度，计算过程如下：将降解剂从1000nM开始，5倍梯度稀释9个浓度，以WB方法检测各浓度下对BCL6蛋白的降解。以Image J软件进行灰度分析，计算BCL6的剩余量。通过Graphpad 8.0软件拟合蛋白剩余量与浓度的对数(Log(C)),得到DC50值。
表1化合物对OCI-LY1细胞内BCL6的降解作用

细胞抗增殖活性测试：将处于对数生长期的细胞以每孔5000个的密度接种与96孔板(Coring,3799)中，含20％FBS的RPMI 1640培养基(Adamas)体积为100μL，于37℃，5％CO2的孵箱内过夜培养。次日，加入100μL培养基配制的不同浓度的药物溶液，每个浓度设三个复孔(记为RLUtest)，并设置对照孔和空白孔。对照孔为细胞、培养液与相同浓度的药物溶解介质(记为RLUcontrol)，空白孔为培养液(记为RLUblank)。9天后，悬浮细胞每孔吸出100μL至96孔黑板(上海卧宏生物科技有限公司,WHB-96-02)中，每孔再加入100μL的CellTiter-Lumi发光法细胞活力检测试剂盒(碧云天生物技术)检测试剂，放置在摇床上避光孵育10min后检测。贴壁细胞每孔吸出100μL培养基丢弃，在96孔板中各加入100μL的检测试剂，放置摇床上裂解2min后，将孔中溶液吸出至96孔黑板中，放置在摇床上避光孵育8min后检测。通过Thermo scientific varioskanflash上的化学发光模块检测化学发光值。通过下式计算细胞的存活百分比，计算公式为：细胞存活率(％)＝(RLUtest-RLUblank)/(RLUcontrol-RLUblank)×100％，采用Graphpad Prism 8.0软件计算IC50值。
表2化合物对OCI-LY1细胞的抗增殖活性

Claims (10)

  1. 一种化学结构如式Ⅰ所示的化合物或其药学上可接受的盐、溶剂化物：
    POI-L-E3L
    Ⅰ
    其中：
    L为连接POI和E3L的Linker；
    E3L独立地选自：
    其中：
    R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21独立地选自-H、卤素、-C1～C3烷基、-C1～C3卤代烷基、-C1～C3烷氧基；
    R22独立地选自-H、-C1～C3烷基；
    POI独立地选自：
    其中：
    R23独立地选自-H、卤素；
    R24、R25独立地选自-H、-C1～C3烷基；
    L独立地选自：
    其中：
    n＝1、2或3。
  2. 一种如下化学结构之一所示的化合物或其药学上可接受的盐、溶剂化物：


  3. 一种药物组合物，其特征在于：含有权利要求1或2所述的化合物或其药学上可接受的盐、溶剂化物。
  4. 权利要求1或2所述的化合物或其药学上可接受的盐、溶剂化物用于制备BCL6蛋白降解剂的用途。
  5. 权利要求1或2所述的化合物或其药学上可接受的盐、溶剂化物用于制备治疗疾病的药物的用途，所述疾病为通过降解BCL6蛋白而得到治疗或缓解的疾病。
  6. 根据权利要求5所述的用途，其特征在于：所述疾病为癌症。
  7. 根据权利要求6所述的用途，其特征在于：所述癌症为霍奇金淋巴瘤、B细胞源性非霍奇金淋巴瘤、T细胞源性非霍奇金淋巴瘤、NK/T细胞源性非霍奇金淋巴瘤或弥漫性大B细胞淋巴瘤。
  8. 权利要求3所述的药物组合物用于制备治疗疾病的药物的用途，所述疾病为通过降解BCL6蛋白而得到治疗或缓解的疾病。
  9. 根据权利要求8所述的用途，其特征在于：所述疾病为癌症。
  10. 根据权利要求9所述的用途，其特征在于：所述癌症为霍奇金淋巴瘤、B细胞源性非霍奇金淋巴瘤、T细胞源性非霍奇金淋巴瘤、NK/T细胞源性非霍奇金淋巴瘤或弥漫性大B细胞淋巴瘤。
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