WO2022268148A1 - Compound for degrading btk protein, and preparation method therefor and use thereof - Google Patents
Compound for degrading btk protein, and preparation method therefor and use thereof Download PDFInfo
- Publication number
- WO2022268148A1 WO2022268148A1 PCT/CN2022/100642 CN2022100642W WO2022268148A1 WO 2022268148 A1 WO2022268148 A1 WO 2022268148A1 CN 2022100642 W CN2022100642 W CN 2022100642W WO 2022268148 A1 WO2022268148 A1 WO 2022268148A1
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- WIPO (PCT)
- Prior art keywords
- phenoxyphenyl
- pyrazolo
- pyrimidin
- amino
- dioxoisoindol
- Prior art date
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
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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
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the invention relates to the field of organic compound synthesis and medical application, in particular to a compound for degrading BTK protein and its preparation method and application.
- B-cell lymphoma is a kind of non-Hodgkin lymphoma (Non-Hodgkin lymphoma, NHL) with poor clinical curability and prognosis, and some malignant B-cell lymphomas are incurable (see Armitage, J.O.; Gascoyne, R.D.; Lunning, M.A.; Cavalli, F., Non-Hodgkin lymphoma. Lancet 2017, 390(10091), 298-310.).
- the B cell receptor (BCR) signaling pathway is a specific pathway for the growth and survival of B cells, and its abnormal expression and overactivation are key factors in the occurrence of B cell lymphoma.
- BTK Bacton's tyrosine kinase
- IBN Ibrutinib
- IBN is the first irreversible covalent BTK inhibitor launched in 2013. Although it has achieved good clinical efficacy, it is prone to drug resistance and side effects (see Liang, C.; Tian, D.
- Ubiquitin-proteasome system (ubiquitin-proteasome system, UPS) is the main way to degrade ubiquitinated proteins in eukaryotic organisms. Protein degradation targeting chimera technology is an emerging technology that has developed rapidly in recent years.
- target protein ubiquitin Proteolysis-targeting chimera (PROTACs) for targeted protein degradation and cancer therapy. J Hematol Oncol 2020,13(1),50.), can provide a new strategy for the treatment of B-cell lymphoma.
- the object of the present invention is to provide a compound for degrading BTK protein and its preparation method and application.
- the compound for degrading BTK protein can effectively degrade the BTK protein of lymphoma cells and inhibit the proliferation of tumor cells.
- the technical solution of the present invention is as follows:
- X is selected from methylene, pyrrolidinyl, benzyl, piperidinyl, pyridyl, pyrimidinyl, imidazolyl or oxadiazolyl; m is selected from 1-4; n is selected from 1-5.
- piperidinyl when X is piperidinyl, piperidinyl is The carbon connection end of piperidinyl is connected with the nitrogen connection end of pyrazole; when X is pyrrolidinyl, pyrrolidinyl is The carbon connection end of pyrrolidinyl is connected with the nitrogen connection end of pyrazole; m is selected from 1, 2, 3, 4; n is selected from 1, 3, 4, 5.
- a compound that degrades BTK protein according to the present invention has the following structure:
- the method specifically includes the following steps: using 4-aminopyrazolo[3,4-d]pyrimidine, namely compound 1, as a starting material, in DMF with N-bromosuccinimide (NBS) through bromination Free radical reaction gives intermediate 2; intermediate 2 reacts with N-Boc-4-hydroxyl X or 1-Boc-3-hydroxyl X by Mitsunobu to give intermediate 3; intermediate 3 is in tetrahydrofuran/concentrated hydrochloric acid Remove the tert-butoxycarbonyl protecting group to obtain intermediate 4; intermediate 2 or 4 reacts with different bromine-substituted carboxylate methyl and ethyl esters to obtain intermediate 5 through nucleophilic substitution; intermediate 5 and 4-phenoxy Phenylboronic acid was catalyzed by tetrakis (triphenylphosphine) palladium, and intermediate 6 was obtained through Suzuki-Miyaura reaction; intermediate 6 was hydrolyzed under basic conditions to obtain intermediate 7; final intermediate 7 and intermediate
- the third aspect of the present invention is a pharmaceutical composition, which comprises the BTK protein-degrading compound described in the first aspect above.
- the fourth aspect of the present invention is a pharmaceutical preparation, which includes an active ingredient and a pharmaceutically acceptable adjuvant and/or carrier, and the active ingredient includes the above-mentioned BTK protein-degrading compound, or includes the above-mentioned pharmaceutical composition.
- the application of the BTK protein-degrading compound described in the above-mentioned first aspect or the pharmaceutical composition described in the above-mentioned third aspect in the preparation of a BTK protein-degrading compound drug in the fifth aspect of the present invention, the application of the BTK protein-degrading compound described in the above-mentioned first aspect or the pharmaceutical composition described in the above-mentioned third aspect in the preparation of a BTK protein-degrading compound drug.
- the application of a BTK protein-degrading compound described in the above-mentioned first aspect or the pharmaceutical composition described in the above-mentioned third aspect in the preparation of a drug for treating B-cell lymphoma in the seventh aspect of the present invention, the application of a BTK protein-degrading compound described in the above-mentioned first aspect or the pharmaceutical composition described in the above-mentioned third aspect in the preparation of a drug for treating B-cell lymphoma.
- the compounds of the present invention have the effect of significantly degrading BTK protein on B cell lymphoma cells, wherein compounds I-6, I-7, I-8, I-10, I-11, I-14, I-15, I-16, I-17, I-18, I-21, I-23 and I-24 can effectively degrade BTK protein, especially compounds I-7, I-21 and I-23 on B-cell lymphoma cell beads Jeko-1 showed concentration-dependent and time-dependent significant degradation of BTK protein.
- reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in accordance with conventional methods in the art or according to product instructions. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention.
- Embodiment 1 the preparation of intermediate 2
- Embodiment 2 the preparation of intermediate 3
- Embodiment 3 the preparation of intermediate 4
- intermediate 3a-3b in a 250ml eggplant-shaped bottle, directly add 20mL of anhydrous THF and stir to dissolve, slowly add 5mL of concentrated hydrochloric acid into the eggplant-shaped bottle, and react at room temperature for 4 hours, a white solid precipitates, TLC detects that the reaction is complete, and directly suction filter , the filter cake was washed with ethyl acetate and dried to give intermediate 4a-4b.
- Embodiment 4 the preparation of intermediate 5
- Embodiment 6 the preparation of intermediate 7
- Embodiment 7 the preparation of intermediate 10
- Embodiment 8 the preparation of intermediate 11
- Embodiment 9 the preparation of intermediate 12
- Embodiment 10 the preparation of intermediate 13
- Embodiment 11 the preparation of intermediate 14
- reaction was detected by TLC, and the reaction solution was poured into a 150mL ice-water bath to precipitate a white solid, cooled to room temperature, filtered with suction, the filter cake was washed with cold water, and dried to obtain a white solid.
- dichloromethane:methanol 100:1 ⁇ 60:1 ⁇ 20:1, to obtain the target final products I-1 to I-24.
- Lymphoma cell lines Jeko-1, K562, HEL Cell Bank of Chinese Academy of Sciences
- 1640 medium was purchased from Israel Biological Industries
- PBS phosphate buffer (powder) was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.
- penicillin-streptomycin mixed solution (100 ⁇ double antibody) was purchased from American Genview Company, poloxamer (F-127) was purchased from Amresco, cell proliferation and toxicity detection kit (Cell Counting Kit-8, CCK8 ) was purchased from Dalian Meilun Biotechnology Co., Ltd., fetal bovine serum (FBS) was purchased from Gibco Company of the United States, the refrigerated centrifuge was purchased from Eppendorf Company of Germany, the Infinite F50 absorbing optical microplate reader was purchased from Tecan Company of Austria, and the cell culture box was purchased from Japan Panasonic Corporation (Model: MCO-170AICUVL-PC).
- FBS fetal bovine serum
- Experimental method take the cells in the logarithmic growth phase, centrifuge at 1000rpm for 5min, discard the upper medium, and dilute the centrifuged cells with 1640 medium containing 20% FBS to form a uniform cell suspension, according to the number of cells 4 ⁇ 10 4 /well Calculate the required number of cells, inoculate the cell suspension in a 96-well culture plate at 100 ⁇ L/well, and set up a 100% cell control at the same time Add 100 ⁇ L of cell suspension and 100 ⁇ L of blank medium, blank control Add 200 ⁇ L blank medium. Then the medium of different concentrations of compounds ( F127 ⁇ 1 %) was added to the corresponding 96-well plate, and 4 replicate wells were set for each concentration. Incubate for 48h.
- the half-inhibitory concentration (IC 50 value) of tumor cells the results showed that the IC 50 values of compound I-7, I-21, I-23 and I-24 inhibited Jeko-1 cells were 4.6 ⁇ M, 4.1 ⁇ M, 3.6 ⁇ M, respectively ⁇ M and 4.2 ⁇ M, the IC 50 value of the positive control drug IBN was 4.7 ⁇ M; the half-inhibitory concentration (IC 50 value) of compound I-21 and I-23 on K562 cells was 8 ⁇ M and 7 ⁇ M, respectively, and the IC 50 value of the positive control drug IBN The 50 value was 10 ⁇ M; the growth inhibitory effect of compounds I-21 and I-23 on HEL cells was twice that of the positive control drug IBN (IC 50 values were 16 ⁇ M, 15 ⁇ M and 31 ⁇ M, respectively).
- Experimental example 14 Western blot experiment of the target compound on the B-cell lymphoma cell line Jeko-1
- SDS-PAGE electrophoresis take gradient separation gel, take 20 ⁇ g protein samples for each group to load, and take protein markers to load at the same time.
- Membrane transfer Soak the qualitative filter paper and PVDF membrane together in the electrophoresis solution; take out the electrophoresed glass plate separately, and then transfer the SDS-PAGE gel to the electrotransfer solution; sandwich the gel and PVDF between the filter paper and soak in the electrophoresis solution. In the electroporation fluid of the transfer device.
- Blocking the membrane containing the target protein is placed in 5% skimmed milk powder solution to block non-specific binding.
Abstract
Provided in the present invention are a compound for degrading a BTK protein, and a preparation method therefor and the use thereof. The compound for degrading a BTK protein has a structure represented by general formula I. The present invention further relates to a pharmaceutical composition containing the compound having the structure represented by formula I. The activity screening experiments show that the compound of the present invention can effectively degrade a BTK protein, and has a growth inhibition effect on B-cell lymphoma cells. Therefore, further provided in the present invention is the use of the compound in the preparation of an anti-tumor drug.
Description
本发明涉及有机化合物合成及医药应用领域，尤其涉及降解BTK蛋白的化合物及其制备方法和应用。The invention relates to the field of organic compound synthesis and medical application, in particular to a compound for degrading BTK protein and its preparation method and application.
B细胞淋巴瘤是一种临床治愈性和预后性均较差的非霍奇金淋巴瘤(Non-Hodgkin lymphoma，NHL)，部分恶性B细胞淋巴瘤存在不可治愈性(参见Armitage,J.O.；Gascoyne,R.D.；Lunning,M.A.；Cavalli,F.,Non-Hodgkin lymphoma.Lancet 2017,390(10091),298-310.)。B细胞受体(B cell receptor，BCR)信号通路是B细胞生长存活的特异性通路，其异常表达和过度活化是B细胞淋巴瘤发生的关键因素。BTK(Bruton’s tyrosine kinase)在BCR信号通路中起重要作用，能够激活下游信号级联中的PI3K、NF-κB等多个通路，促进B细胞存活、增殖和分化。依鲁替尼(Ibrutinib，IBN)是2013年上市的第一个不可逆共价BTK抑制剂，虽然临床取得不错的疗效，但易产生耐药性和副作用(参见Liang,C.；Tian,D.；Ren,X.；Ding,S.；Jia,M.；Xin,M.；Thareja,S.,The development of Bruton's tyrosine kinase(BTK)inhibitors from 2012 to 2017:A mini-review.Eur.J.Med.Chem.2018,151,315-326.)。泛素-蛋白酶体系统(ubiquitin-proteasome system，UPS)是真核生物体内降解泛素化蛋白的主要途径，蛋白降解靶向嵌合体技术是近年来迅速发展的新兴技术，通过UPS将目标蛋白泛素化，进而被蛋白酶体识别降解，具有克服耐药性、提高选择性及催化降解靶蛋白等优势(参见Li,X.；Song,Y.,Proteolysis-targeting chimera(PROTACs)for targeted protein degradation and cancer therapy.J Hematol Oncol 2020,13(1),50.)，可以为B细胞淋巴瘤的治疗提供新的策略。B-cell lymphoma is a kind of non-Hodgkin lymphoma (Non-Hodgkin lymphoma, NHL) with poor clinical curability and prognosis, and some malignant B-cell lymphomas are incurable (see Armitage, J.O.; Gascoyne, R.D.; Lunning, M.A.; Cavalli, F., Non-Hodgkin lymphoma. Lancet 2017, 390(10091), 298-310.). The B cell receptor (BCR) signaling pathway is a specific pathway for the growth and survival of B cells, and its abnormal expression and overactivation are key factors in the occurrence of B cell lymphoma. BTK (Bruton's tyrosine kinase) plays an important role in the BCR signaling pathway and can activate multiple pathways such as PI3K and NF-κB in the downstream signaling cascade to promote the survival, proliferation and differentiation of B cells. Ibrutinib (IBN) is the first irreversible covalent BTK inhibitor launched in 2013. Although it has achieved good clinical efficacy, it is prone to drug resistance and side effects (see Liang, C.; Tian, D. ; Ren, X.; Ding, S.; Jia, M.; Xin, M.; Thareja, S., The development of Bruton's tyrosine kinase(BTK) inhibitors from 2012 to 2017: A mini-review. Eur.J. Med. Chem. 2018, 151, 315-326.). Ubiquitin-proteasome system (ubiquitin-proteasome system, UPS) is the main way to degrade ubiquitinated proteins in eukaryotic organisms. Protein degradation targeting chimera technology is an emerging technology that has developed rapidly in recent years. Through UPS, target protein ubiquitin Proteolysis-targeting chimera (PROTACs) for targeted protein degradation and cancer therapy.J Hematol Oncol 2020,13(1),50.), can provide a new strategy for the treatment of B-cell lymphoma.
发明内容Contents of the invention
本发明的目的是提供一种降解BTK蛋白的化合物及其制备方法和应用，所述降解BTK蛋白的化合物能有效降解淋巴瘤细胞的BTK蛋白，抑制肿瘤细胞的增殖。具体地，本发明的技术方案如下所述：The object of the present invention is to provide a compound for degrading BTK protein and its preparation method and application. The compound for degrading BTK protein can effectively degrade the BTK protein of lymphoma cells and inhibit the proliferation of tumor cells. Specifically, the technical solution of the present invention is as follows:
在本发明的第一方面，提供了一种降解BTK蛋白的化合物，结构如通式Ⅰ 所示：In the first aspect of the present invention, a compound for degrading BTK protein is provided, the structure of which is shown in general formula I:
其中，X选自亚甲基、吡咯烷基、苄基、哌啶基、吡啶基、嘧啶基、咪唑基或噁二唑基；m选自1～4；n选自1～5。Wherein, X is selected from methylene, pyrrolidinyl, benzyl, piperidinyl, pyridyl, pyrimidinyl, imidazolyl or oxadiazolyl; m is selected from 1-4; n is selected from 1-5.
根据本发明优选的，X为哌啶基时，哌啶基为
哌啶基的碳连接端与吡唑的氮连接端相连；X为吡咯烷基时，吡咯烷基为
吡咯烷基的碳连接端与吡唑的氮连接端相连；m选自1、2、3、4；n选自1、3、4、5。
Preferably according to the present invention, when X is piperidinyl, piperidinyl is The carbon connection end of piperidinyl is connected with the nitrogen connection end of pyrazole; when X is pyrrolidinyl, pyrrolidinyl is The carbon connection end of pyrrolidinyl is connected with the nitrogen connection end of pyrazole; m is selected from 1, 2, 3, 4; n is selected from 1, 3, 4, 5.
根据本发明进一步优选的，本发明所述一种降解BTK蛋白的化合物具有以下结构：According to further preference of the present invention, a compound that degrades BTK protein according to the present invention has the following structure:
2-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-1)；2-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl)piperazine-1 -yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide (I-1);
4-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-2)；4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)butanamide (I-2);
5-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-3)；5-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide (I-3);
6-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺(Ⅰ-4)；6-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide (I-4);
2-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-5)；2-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide (I-5);
4-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-6)；4-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyramide (I-6);
5-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-7)；5-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide (Ⅰ-7);
6-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪 -1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺(Ⅰ-8)；6-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide (I-8);
2-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-9)；2-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide (I-9);
4-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-10)；4-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyramide (I-10);
5-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-11)；5-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide (I-11);
6-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺(Ⅰ-12)；6-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide (I-12);
2-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-13)；2-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide (I-13);
4-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-14)；4-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyramide (I-14);
5-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-15)；5-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide (I-15);
6-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺(Ⅰ-16)；6-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide (I-16);
5-(4-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-17)；5-(4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1 -yl)butanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentyl Amide (Ⅰ-17);
2-(4-(5-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-18)；2-(4-(5-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1 -yl)pentanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)ethyl Amide (Ⅰ-18);
2-(4-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-19)；2-(4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1 -yl)butyryl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)ethyl Amide (Ⅰ-19);
2-(4-(5-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-20)；2-(4-(5-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)pentanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)ethyl Amide (Ⅰ-20);
4-(4-(5-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-21)；4-(4-(5-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)pentanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyl Amide (Ⅰ-21);
2-(4-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧代异吲哚-4-基)乙酰胺(Ⅰ-22)；2-(4-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)butanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl) Acetamide (Ⅰ-22);
5-(4-(3-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-23)；5-(4-(3-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)propionyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentyl Amide (Ⅰ-23);
4-(4-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-24)。4-(4-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)butyryl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyl Amide (I-24).
本发明的第二方面，提供了一种制备上述第一方面所述降解BTK蛋白的化合物及其制备方法，其包括：以4-氨基吡唑并[3,4-d]嘧啶即化合物1作为起始原料，经溴代自由基反应得到中间体2；中间体2与N-Boc-4-羟基X或1-Boc-3-羟基X经光延反应，得到中间体3；中间体3脱去叔丁氧羰基保护基，得到中间体4；中间体2或4与溴取代的羧酸甲酯、乙酯经取代反应得中间体5；中间体5与4-苯氧基苯基硼酸经Suzuki-Miyaura反应得到中间体6；中间体6经过酯的水解得中间体7；中间体7与中间体14经酰胺缩合反应得通式(Ⅰ)化合物，所述合成路线如下所示：The second aspect of the present invention provides a compound for preparing the BTK protein degradation described in the first aspect and its preparation method, which includes: using 4-aminopyrazolo[3,4-d]pyrimidine, that is, compound 1 as Starting material, intermediate 2 is obtained by bromo radical reaction; intermediate 2 is reacted with N-Boc-4-hydroxyl X or 1-Boc-3-hydroxyl X by Mitsunobu to obtain intermediate 3; intermediate 3 is removed tert-butoxycarbonyl protecting group to obtain intermediate 4; intermediate 2 or 4 and bromine-substituted methyl carboxylate, ethyl ester were substituted to obtain intermediate 5; intermediate 5 and 4-phenoxyphenylboronic acid were obtained by Suzuki -Miyaura reaction obtains intermediate 6; intermediate 6 obtains intermediate 7 through hydrolysis of ester; intermediate 7 and intermediate 14 undergo amide condensation reaction to obtain the compound of general formula (I), and the synthetic route is as follows:
其中，X、m和n如上述第一方面中所定义。Wherein, X, m and n are as defined in the first aspect above.
所述方法具体地包括如下：以4-氨基吡唑并[3,4-d]嘧啶即化合物1为起始原料，在DMF中与N-溴代丁二酰亚胺(NBS)经溴代自由基反应得到中间体2；中间体2与N-Boc-4-羟基X或1-Boc-3-羟基X经光延(Mitsunobu)反应，得到中间体3；中间体3在四氢呋喃/浓盐酸下脱去叔丁氧羰基保护基，得到中间体4；中间体2或4与不同溴取代的羧酸甲酯、乙酯经亲核取代反应得中间体5；中间体5与4-苯氧基苯基硼酸在四(三苯基膦)钯催化下，经Suzuki-Miyaura反应得到中间体6；中间体6在碱性条件下经过酯的水解得中间体7；最终中间体7与中间体14经酰胺缩合反应得到式Ⅰ化合物。The method specifically includes the following steps: using 4-aminopyrazolo[3,4-d]pyrimidine, namely compound 1, as a starting material, in DMF with N-bromosuccinimide (NBS) through bromination Free radical reaction gives intermediate 2; intermediate 2 reacts with N-Boc-4-hydroxyl X or 1-Boc-3-hydroxyl X by Mitsunobu to give intermediate 3; intermediate 3 is in tetrahydrofuran/concentrated hydrochloric acid Remove the tert-butoxycarbonyl protecting group to obtain intermediate 4; intermediate 2 or 4 reacts with different bromine-substituted carboxylate methyl and ethyl esters to obtain intermediate 5 through nucleophilic substitution; intermediate 5 and 4-phenoxy Phenylboronic acid was catalyzed by tetrakis (triphenylphosphine) palladium, and intermediate 6 was obtained through Suzuki-Miyaura reaction; intermediate 6 was hydrolyzed under basic conditions to obtain intermediate 7; final intermediate 7 and intermediate 14 The compound of formula I can be obtained through amide condensation reaction.
进一步地，在本发明的实施方式中，式Ⅰ目标化合物的制备方法具体包括：Further, in an embodiment of the present invention, the preparation method of the target compound of formula I specifically includes:
(1)将原料1溶于DMF中，加入NBS，80℃油浴反应7h，TLC检测基本反应完全，将反应液冷却到室温，倒入冰水中，搅拌析出大量黄色固体，抽滤，滤饼水洗，干燥，得中间体2。(1) Dissolve raw material 1 in DMF, add NBS, react in an oil bath at 80°C for 7 hours, TLC detects that the reaction is basically complete, cool the reaction solution to room temperature, pour it into ice water, stir and precipitate a large amount of yellow solid, filter with suction, filter cake Washed with water and dried to obtain Intermediate 2.
(2)将中间体2、N-Boc-4-羟基哌啶或1-Boc-3-羟基吡咯烷和三苯基膦溶于无水THF中，冰浴下缓慢滴加偶氮二甲酸二异丙酯(DIAD)，搅拌10min，溶液由浑浊变澄清。TLC检测反应完全，加入乙酸乙酯萃取，合并有机相，加入NaCl洗涤，无水硫酸钠干燥，过滤，减压蒸除溶剂，硅胶柱层析纯化(乙酸乙酯:石油醚＝120:1-60:1)得中间体3a-3b。(2) Dissolve intermediate 2, N-Boc-4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine and triphenylphosphine in anhydrous THF, slowly add azodicarboxylic acid di Isopropyl ester (DIAD), stirred for 10min, the solution changed from cloudy to clear. TLC detected that the reaction was complete, added ethyl acetate to extract, combined the organic phases, added NaCl to wash, dried over anhydrous sodium sulfate, filtered, evaporated the solvent under reduced pressure, purified by silica gel column chromatography (ethyl acetate:petroleum ether=120:1- 60:1) intermediates 3a-3b were obtained.
(3)将中间体3a-3b溶于无水THF中，加入适量的浓盐酸，室温反应4h，析出白色固体，TCL检测反应完全，抽滤，滤饼用乙酸乙酯洗涤，干燥，得中间体4a-4b。(3) Dissolve the intermediates 3a-3b in anhydrous THF, add an appropriate amount of concentrated hydrochloric acid, react at room temperature for 4 hours, and a white solid precipitates out. TCL detects that the reaction is complete, filters with suction, washes the filter cake with ethyl acetate, and dries to obtain the intermediate Body 4a-4b.
(4)将中间体2或中间体4a-4b溶于DMF中，加入不同溴末端取代的羧酸甲酯和K
2CO
3，室温搅拌8h，反应完毕，反应液加入EA和水进行萃取，合并有机相，加入NaCl洗涤，无水Na
2SO
4干燥，过滤，减压蒸除溶剂，硅胶柱层析(二氯甲烷:甲醇＝120:1-60:1)得中间体5a-5i。
(4) Dissolve intermediate 2 or intermediate 4a-4b in DMF, add carboxylate methyl esters and K 2 CO 3 with different bromine terminal substitutions, stir at room temperature for 8 hours, the reaction is completed, and the reaction solution is extracted by adding EA and water, The organic phases were combined, washed with NaCl, dried over anhydrous Na 2 SO 4 , filtered, and the solvent was evaporated under reduced pressure. Silica gel column chromatography (dichloromethane:methanol=120:1-60:1) gave intermediates 5a-5i.
(5)将中间体5a-5i、4-苯氧基苯硼酸、四(三苯基膦)钯和磷酸钾置于微波管中，加入1,4-二氧六环和水(4:1)溶解，超声脱去溶液中的氧气，120℃进行微波反应20min。TLC检测反应完全，反应液加入EA/水萃取，合并有机相，加入食盐水洗涤，无水Na
2SO
4干燥，过滤，减压蒸除溶剂，硅胶柱层析(二氯 甲烷:甲醇＝100:1-50:1)得中间体6a-6i。
(5) Put intermediates 5a-5i, 4-phenoxyphenylboronic acid, tetrakis(triphenylphosphine)palladium and potassium phosphate in a microwave tube, add 1,4-dioxane and water (4:1 ) was dissolved, the oxygen in the solution was removed by ultrasonication, and microwave reaction was carried out at 120°C for 20min. TLC detected that the reaction was complete, the reaction solution was extracted with EA/water, the organic phases were combined, washed with brine, dried over anhydrous Na 2 SO 4 , filtered, the solvent was evaporated under reduced pressure, silica gel column chromatography (dichloromethane:methanol=100 :1-50:1) intermediates 6a-6i were obtained.
(6)将中间体6a-6i溶于无水乙醇中，加入3M NaOH调节pH为10-11左右，室温反应6h，反应完毕，加入1M HCl调节溶液的pH为5-6，析出白色固体，抽滤，滤饼用水洗涤，干燥，得到中间体7a-7i。(6) Intermediate 6a-6i was dissolved in absolute ethanol, and 3M NaOH was added to adjust the pH to about 10-11, and the room temperature was reacted for 6h. After the reaction was completed, 1M HCl was added to adjust the pH of the solution to be 5-6, and a white solid was precipitated. After suction filtration, the filter cake was washed with water and dried to obtain intermediates 7a-7i.
(7)将化合物8和化合物9置于醋酸溶液搅拌，加入醋酸钠，118℃油浴，回流反应8h，TLC检测反应完全，将反应液冷却至室温，倒入冰水中，搅拌析出黑紫色亮晶固体，抽滤，滤饼用甲醇洗涤，干燥，得中间体10。(7) Stir compound 8 and compound 9 in acetic acid solution, add sodium acetate, reflux for 8 hours in an oil bath at 118°C, TLC detects that the reaction is complete, cool the reaction solution to room temperature, pour it into ice water, and stir to precipitate a dark purple bright The crystalline solid was suction filtered, and the filter cake was washed with methanol and dried to obtain intermediate 10.
(8)将中间体10溶于体积比为1:1的乙醇和二氯甲烷混合溶液中，加入钯碳(Pd/C)，H
2保护，室温反应6h，反应完毕后，硅藻土过滤，二氯甲烷冲洗，合并滤液，减压蒸除溶剂，得中间体11。
(8) Dissolve intermediate 10 in a mixed solution of ethanol and dichloromethane with a volume ratio of 1:1, add palladium carbon (Pd/C), protect with H 2 , react at room temperature for 6 hours, after the reaction is completed, filter with diatomaceous earth , rinsed with dichloromethane, combined the filtrates, and evaporated the solvent under reduced pressure to obtain intermediate 11.
(9)将中间体11溶于无水四氢呋喃溶液，加入不同取代的卤代酰氯，N
2保护下，60℃油浴回流反应6h，TLC检测反应完毕，减压蒸除THF，加入适量无水乙醚，超声搅拌，析出淡黄色固体，抽滤，滤饼用无水乙醚洗涤，干燥，得中间体12a-12d。
(9) Dissolve intermediate 11 in anhydrous tetrahydrofuran solution, add different substituted haloacyl chlorides, under the protection of N2 , reflux the reaction in an oil bath at 60°C for 6 hours, TLC detects that the reaction is complete, evaporate THF under reduced pressure, and add an appropriate amount of anhydrous Ethyl ether, ultrasonic stirring, precipitated light yellow solid, suction filtration, the filter cake was washed with anhydrous ether, dried to obtain intermediates 12a-12d.
(10)将中间体12a-12d溶于N-甲基吡咯烷酮(NMP)中，加入N-Boc-哌嗪和碘化钠，滴加N,N-二异丙基乙胺(DIPEA)，N
2保护下，85℃油浴反应5h，TLC检测反应完毕，将反应液用EA/水萃取，合并有机相，加入NaCl洗涤，无水Na
2SO
4干燥，过滤，减压蒸除溶剂，硅胶柱层析(二氯甲烷:甲醇＝100:1-60:1)得中间体13a-13d。
(10) Dissolve intermediates 12a-12d in N-methylpyrrolidone (NMP), add N-Boc-piperazine and sodium iodide, drop N,N-diisopropylethylamine (DIPEA), N Under the protection of 2 , react in an oil bath at 85°C for 5 hours, TLC detects that the reaction is complete, extract the reaction solution with EA/water, combine the organic phases, add NaCl to wash, dry with anhydrous Na 2 SO 4 , filter, evaporate the solvent under reduced pressure, and silica gel Column chromatography (dichloromethane:methanol=100:1-60:1) yielded intermediates 13a-13d.
(11)将中间体13a-13d加入到饱和HCl的乙酸乙酯溶液，室温反应4h，析出白色固体，TCL检测反应完毕，抽滤，滤饼用乙酸乙酯洗涤，干燥，得中间体14a-14d。(11) Add intermediates 13a-13d to saturated ethyl acetate solution of HCl, react at room temperature for 4 hours, a white solid precipitates, TCL detects that the reaction is complete, filter with suction, wash the filter cake with ethyl acetate, and dry to obtain intermediates 14a- 14d.
(12)将中间体7a-7i和缩合剂2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)溶于无水DMF，滴加DIPEA，置于冰水浴中活化30min，得活化酯，再加入中间体14a-14d，N
2保护下，室温反应8h，TLC检测反应完毕，将反应液倒入冰水中，析出白色固体，冷却至室温，抽滤，滤饼用水洗涤，干燥，得白色固体，之后经过硅胶柱层析(二氯甲烷:甲醇＝100:1-50:1-20:1)得到目标化合物Ⅰ-1～Ⅰ-24。
(12) Dissolve intermediates 7a-7i and condensing agent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) in DMF in water, DIPEA was added dropwise, and activated in an ice-water bath for 30 minutes to obtain an activated ester, then intermediates 14a-14d were added, under the protection of N 2 , reacted at room temperature for 8 hours, TLC detected that the reaction was complete, poured the reaction solution into ice water, and precipitated The white solid was cooled to room temperature, suction filtered, the filter cake was washed with water, and dried to obtain a white solid, which was then subjected to silica gel column chromatography (dichloromethane:methanol=100:1-50:1-20:1) to obtain the target compound Ⅰ -1～Ⅰ-24.
具体地，其合成路线如下所示：Specifically, its synthetic route is as follows:
上述合成路线的试剂及条件：(a)NBS,DMF,80℃,7h；(b)不同溴末端取代的羧酸甲酯,K
2CO
3,DMF,r.t.,8h；(c)N-Boc-4-羟基哌啶或1-Boc-3-羟基吡咯烷,三苯基膦,DIAD,无水THF,0℃,10min；(d)四氢呋喃:浓盐酸＝1:1,r.t.,4h；(e)不同溴末端取代的羧酸甲酯,K
2CO
3,DMF,r.t.,8h；(f)Pd(PPh
3)
4,三水磷酸钾,1,4-二氧六环:水＝4:1,MW,120℃,20min；(g)无水甲醇,3M NaOH,r.t.,6h；(h)CH3COONa,CH3COOH,118℃,8h；(i)CH
3CH
2OH:CH
2Cl
2＝1:1,Pd/C,H
2,r.t.,6h；(j)不同取代的卤代酰氯,无水THF,N
2,60℃,6h；(k)N-Boc-哌嗪,NaI,DIPEA,NMP,N
2,85℃,5h；(l)饱和HCl的乙酸乙酯溶液,r.t.,4h；(q)DIPEA,HATU,N
2,无水DMF,r.t.,8h。
Reagents and conditions for the above synthetic route: (a) NBS, DMF, 80°C, 7h; (b) methyl carboxylate with different bromine terminal substitutions, K 2 CO 3 , DMF, rt, 8h; (c) N-Boc -4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine, triphenylphosphine, DIAD, anhydrous THF, 0°C, 10min; (d) tetrahydrofuran: concentrated hydrochloric acid = 1:1, rt, 4h; ( e) Carboxylic acid methyl esters with different bromine terminal substitutions, K 2 CO 3 , DMF, rt, 8h; (f) Pd(PPh 3 ) 4 , potassium phosphate trihydrate, 1,4-dioxane:water=4 :1, MW, 120°C, 20min; (g) anhydrous methanol, 3M NaOH, rt, 6h; (h) CH3COONa, CH3COOH, 118°C, 8h; (i) CH 3 CH 2 OH:CH 2 Cl 2 = 1:1, Pd/C, H 2 , rt, 6h; (j) different substituted haloacyl chlorides, anhydrous THF, N 2 , 60℃, 6h; (k) N-Boc-piperazine, NaI, DIPEA , NMP, N 2 , 85°C, 5h; (l) saturated HCl in ethyl acetate, rt, 4h; (q) DIPEA, HATU, N 2 , anhydrous DMF, rt, 8h.
本发明的第三方面，一种药物组合物，其包含上述第一方面所述的一种降解BTK蛋白的化合物。The third aspect of the present invention is a pharmaceutical composition, which comprises the BTK protein-degrading compound described in the first aspect above.
本发明的第四方面，一种药物制剂，其包括有效成分和药学上可接受的辅料和/或载体，所述有效成分包含上述降解BTK蛋白的化合物，或包含上述药物组合物。The fourth aspect of the present invention is a pharmaceutical preparation, which includes an active ingredient and a pharmaceutically acceptable adjuvant and/or carrier, and the active ingredient includes the above-mentioned BTK protein-degrading compound, or includes the above-mentioned pharmaceutical composition.
本发明的第五方面，上述第一方面中所述的降解BTK蛋白的化合物或上述 第三方面中所述的药物组合物在制备降解BTK蛋白的化合物药物中的应用。In the fifth aspect of the present invention, the application of the BTK protein-degrading compound described in the above-mentioned first aspect or the pharmaceutical composition described in the above-mentioned third aspect in the preparation of a BTK protein-degrading compound drug.
本发明的第六方面，上述第一方面中所述的一种降解BTK蛋白的化合物或上述第三方面中所述的药物组合物在制备抗肿瘤药物中的应用。In the sixth aspect of the present invention, the application of a BTK protein-degrading compound described in the above-mentioned first aspect or the pharmaceutical composition described in the above-mentioned third aspect in the preparation of anti-tumor drugs.
本发明的第七方面，上述第一方面中所述的一种降解BTK蛋白的化合物或上述第三方面中所述的药物组合物在制备治疗B细胞淋巴瘤的药物中的应用。In the seventh aspect of the present invention, the application of a BTK protein-degrading compound described in the above-mentioned first aspect or the pharmaceutical composition described in the above-mentioned third aspect in the preparation of a drug for treating B-cell lymphoma.
本发明具有以下有益效果：本发明所述的一种降解BTK蛋白的化合物对肿瘤细胞具有优良的抗增殖活性，其中化合物Ⅰ-7、Ⅰ-8、Ⅰ-10、Ⅰ-16、Ⅰ-17、Ⅰ-18对B细胞淋巴瘤细胞株Jeko-1的抑制率大于75％，化合物Ⅰ-21、Ⅰ-23和Ⅰ-24对B细胞淋巴瘤细胞株Jeko-1的抑制率大于80％；显著优于对照药依鲁替尼(IBN)，在相同测试条件下，IBN对B细胞淋巴瘤细胞珠Jeko-1的抑制率小于64％；进一步测定部分化合物对肿瘤细胞的半数生长抑制浓度(IC
50值)，结果显示化合物Ⅰ-7、Ⅰ-21、Ⅰ-23和Ⅰ-24抑制Jeko-1细胞的IC
50值分别为4.6μM、4.1μM、3.6μM和4.2μM，阳性对照药IBN的IC
50值为4.7μM；化合物Ⅰ-21和Ⅰ-23对K562细胞的半数生长抑制浓度(IC
50值)分别为8μM和7μM，阳性对照药IBN的IC
50值为10μM；化合物Ⅰ-21和Ⅰ-23对HEL细胞的生长抑制作用是阳性对照药IBN的两倍(IC
50值分别为16μM、15μM和31μM)。本发明所述的化合物对B细胞淋巴瘤细胞具有显著降解BTK蛋白的效果，其中化合物Ⅰ-6、Ⅰ-7、Ⅰ-8、Ⅰ-10、Ⅰ-11、Ⅰ-14、Ⅰ-15、Ⅰ-16、Ⅰ-17、Ⅰ-18、Ⅰ-21、Ⅰ-23和Ⅰ-24可有效降解BTK蛋白，特别是化合物Ⅰ-7、Ⅰ-21和Ⅰ-23对B细胞淋巴瘤细胞珠Jeko-1显示浓度依赖性和时间依赖性地显著降解BTK蛋白。具体的是，在化合物浓度为5μM时处理Jeko-1细胞24h，Ⅰ-7对BTK蛋白最大降解量(D
max)为73％，Ⅰ-21对BTK蛋白最大降解量(D
max)为92％，Ⅰ-23对BTK蛋白最大降解量(D
max)为94％，化合物Ⅰ-7、化合物Ⅰ-21和化合物Ⅰ-23对BTK的半数降解浓度(DC
50)分别为0.45μM、0.25μM和0.10μM。在相同实验条件下，对照药依鲁替尼和泊马度胺均未显示出对BTK蛋白的降解作用。
The present invention has the following beneficial effects: a compound that degrades BTK protein according to the present invention has excellent anti-proliferation activity on tumor cells, wherein compounds I-7, I-8, I-10, I-16, and I-17 , The inhibitory rate of I-18 on the B-cell lymphoma cell line Jeko-1 is greater than 75%, and the inhibitory rate of compounds I-21, I-23 and I-24 on the B-cell lymphoma cell line Jeko-1 is greater than 80%; Significantly better than the reference drug ibrutinib (IBN), under the same test conditions, the inhibitory rate of IBN to B cell lymphoma cell Jeko-1 is less than 64%; further measure the half median growth inhibitory concentration ( IC 50 value), the results showed that the IC 50 values of compounds Ⅰ-7, Ⅰ-21, Ⅰ-23 and Ⅰ-24 inhibiting Jeko-1 cells were 4.6 μM, 4.1 μM, 3.6 μM and 4.2 μM, and the positive control drug IBN The IC 50 value of the compound Ⅰ-21 and Ⅰ-23 on K562 cells was 8 μM and 7 μM respectively, and the IC 50 value of the positive control drug IBN was 10 μM; the compound Ⅰ-21 The growth inhibitory effect of HEL and Ⅰ-23 on HEL cells was twice that of the positive control drug IBN (IC 50 values were 16μM, 15μM and 31μM, respectively). The compounds of the present invention have the effect of significantly degrading BTK protein on B cell lymphoma cells, wherein compounds I-6, I-7, I-8, I-10, I-11, I-14, I-15, I-16, I-17, I-18, I-21, I-23 and I-24 can effectively degrade BTK protein, especially compounds I-7, I-21 and I-23 on B-cell lymphoma cell beads Jeko-1 showed concentration-dependent and time-dependent significant degradation of BTK protein. Specifically, when Jeko-1 cells were treated for 24 hours at a compound concentration of 5 μM, the maximum degradation (D max ) of BTK protein by Ⅰ-7 was 73%, and the maximum degradation (D max ) of BTK protein by Ⅰ-21 was 92% , the maximum degradation amount (D max ) of Ⅰ-23 to BTK protein was 94%, and the half-degradation concentrations (DC 50 ) of compound Ⅰ-7, compound Ⅰ-21 and compound Ⅰ-23 to BTK were 0.45 μM, 0.25 μM and 0.10 μM. Under the same experimental conditions, the control drugs ibrutinib and pomalidomide did not show any degradation effect on BTK protein.
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations to the present invention.
图1为目标化合物的蛋白质印迹法检测结果，其中IBN表示依鲁替尼， Pomalidomide表示泊马度胺。Fig. 1 is the Western blot detection result of the target compound, wherein IBN means ibrutinib, and Pomalidomide means pomalidomide.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer.
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。本发明所使用的试剂或原料均可通过常规途径购买获得，如无特殊说明，本发明所使用的试剂或原料均按照本领域常规方式使用或者按照产品说明书使用。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in accordance with conventional methods in the art or according to product instructions. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention.
为了使得本领域技术人员能够更加清楚地了解本公开的技术方案，以下将结合具体的实施例与对比例详细说明本公开的技术方案。In order to enable those skilled in the art to understand the technical solutions of the present disclosure more clearly, the technical solutions of the present disclosure will be described in detail below in conjunction with specific examples and comparative examples.
实施例1：中间体2的制备Embodiment 1: the preparation of intermediate 2
将原料4-氨基吡唑并[3,4-d]嘧啶(15.0g，111.0mmol)置于250mL茄形瓶中，加入55mL N,N-二甲基甲酰胺(DMF)溶液，搅拌至溶解。室温条件下加入N-溴代丁二酰亚胺(23.7g，133.2mmol)，反应瓶置于80℃油浴，加热反应6h，溶液由黄棕色浑浊液体变为红褐色透明液体。TLC检测基本反应完全，将反应液冷却至室温，倒入300mL冰水中淬灭，搅拌下析出大量黄棕色固体，抽滤，滤饼水洗，干燥得中间体2，黄棕色固体20.8g，产率86.9％。Mp：272-274℃；
1H NMR(400MHz,DMSO-d
6)δ13.76(s,1H),8.16(s,1H),7.94(s,1H),6.85(s,1H)。
Put the raw material 4-aminopyrazolo[3,4-d]pyrimidine (15.0g, 111.0mmol) in a 250mL eggplant-shaped flask, add 55mL of N,N-dimethylformamide (DMF) solution, stir until dissolved . N-bromosuccinimide (23.7 g, 133.2 mmol) was added at room temperature, and the reaction flask was placed in an oil bath at 80° C., and heated for 6 h. The solution changed from a yellowish-brown turbid liquid to a reddish-brown transparent liquid. TLC detected that the basic reaction was complete, cooled the reaction solution to room temperature, poured it into 300mL ice water to quench, and precipitated a large amount of yellow-brown solid under stirring, filtered it with suction, washed the filter cake with water, and dried to obtain intermediate 2, 20.8g of yellow-brown solid, the yield 86.9%. Mp: 272-274° C.; 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.76 (s, 1H), 8.16 (s, 1H), 7.94 (s, 1H), 6.85 (s, 1H).
实施例2：中间体3的制备Embodiment 2: the preparation of intermediate 3
取中间体2(3.0g，14.0mmol)、N-Boc-4-羟基哌啶或1-Boc-3-羟基吡咯烷(21.0mol)和三苯基膦(8.1g，30.8mol)于100mL茄形瓶中，加入30mL无水四氢呋喃，置于0℃冰浴中，搅拌溶解，缓慢滴加DIAD(8.5g，42.1mol)，冰浴下反应10min，溶液由红褐色浑浊逐渐变澄清。TCL检测反应完毕，加入EA/水萃取(30ml×3)，合并有机相，加入食盐水(20ml)洗涤，无水硫酸钠干燥，过滤，减压蒸除溶剂，硅胶柱层析纯化，石油醚:乙酸乙酯＝80:1～20:1～5:1，得中间体3a-3b。Take intermediate 2 (3.0g, 14.0mmol), N-Boc-4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine (21.0mol) and triphenylphosphine (8.1g, 30.8mol) in 100mL solanum Add 30 mL of anhydrous tetrahydrofuran to a shaped flask, place in an ice bath at 0°C, stir to dissolve, slowly add DIAD (8.5 g, 42.1 mol) dropwise, and react under ice bath for 10 min, the solution gradually turns from reddish-brown turbid to clear. TCL detects that the reaction is complete, add EA/water to extract (30ml×3), combine the organic phases, add brine (20ml) to wash, dry over anhydrous sodium sulfate, filter, evaporate the solvent under reduced pressure, purify by silica gel column chromatography, petroleum ether : Ethyl acetate=80:1～20:1～5:1 to obtain intermediate 3a-3b.
实施例3：中间体4的制备Embodiment 3: the preparation of intermediate 4
将中间体3a-3b置于250ml茄形瓶，直接加入20mL无水THF搅拌溶解，将5mL浓盐酸缓慢滴加茄形瓶中，室温反应4h，析出白色固体，TLC检测反应完毕，直接抽滤，滤饼用乙酸乙酯洗，干燥，得中间体4a-4b。Put the intermediate 3a-3b in a 250ml eggplant-shaped bottle, directly add 20mL of anhydrous THF and stir to dissolve, slowly add 5mL of concentrated hydrochloric acid into the eggplant-shaped bottle, and react at room temperature for 4 hours, a white solid precipitates, TLC detects that the reaction is complete, and directly suction filter , the filter cake was washed with ethyl acetate and dried to give intermediate 4a-4b.
实施例4：中间体5的制备Embodiment 4: the preparation of intermediate 5
取中间体2(11.98mmol)分别加入3-溴丙酸甲酯、4-溴丁酸甲酯、5-溴戊酸甲酯、6-溴己酸甲酯(14.38mmol)，或取中间体4a-4b，分别加入3-溴丙酸甲酯、4-溴丁酸甲酯、5-溴戊酸甲酯(14.38mmol)，再加入K
2CO
3粉末(29.98mmol)置于100mL茄形瓶中，加入30mL DMF溶液搅拌溶解，室温反应8h。TCL检测反应完毕，将反应液倒入60mL冷水淬灭，乙酸乙酯萃取(30mL×3)，有机相合并，饱和NaCl溶液(30mL)洗涤，无水Na
2SO
4干燥，过滤，减压蒸除溶剂，硅胶柱层析纯化，二氯甲烷:甲醇＝120:1～60:1～40:1，得中间体5a-5i。
Take intermediate 2 (11.98mmol) and add methyl 3-bromopropionate, methyl 4-bromobutyrate, methyl 5-bromovalerate, methyl 6-bromohexanoate (14.38mmol), or take intermediate 4a-4b, add 3-bromopropionate methyl ester, 4-bromobutyrate methyl ester, 5-bromovalerate methyl ester (14.38mmol), then add K 2 CO 3 powder (29.98mmol) into 100mL eggplant In the bottle, add 30mL DMF solution and stir to dissolve, and react at room temperature for 8h. The completion of the reaction was detected by TCL, the reaction solution was poured into 60 mL of cold water to quench, extracted with ethyl acetate (30 mL × 3), the organic phases were combined, washed with saturated NaCl solution (30 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated under reduced pressure. Remove the solvent and purify by silica gel column chromatography, dichloromethane:methanol=120:1~60:1~40:1 to obtain intermediate 5a-5i.
实施例5：中间体6的制备Embodiment 5: the preparation of intermediate 6
取中间体5a-5i(10mmol)，4-苯氧基苯基硼酸(16mmol)，四(三苯基膦)钯(Pd(PPh3)4，0.522mmol)和磷酸钾三水(K
3PO
4·3H
2O，21mmol)于35mL微波管中，加入1,4-二氧六环/水(4:1)溶剂搅拌溶解，超声脱去溶液中的氧气，微波反应设置反应温度时间分别为120℃和20min。反应完毕后，TLC检测反应完全，将反应液用乙酸乙酯/水萃取(25mL×3)，有机相合并，NaCl溶液(20mL)洗涤，无水硫酸钠干燥，过滤，减压蒸除溶剂，硅胶柱层析纯化，二氯甲烷:甲醇＝120:1～100:1～50:1，得中间体6a-6i。
Take intermediates 5a-5i (10mmol), 4-phenoxyphenylboronic acid (16mmol), tetrakis(triphenylphosphine) palladium (Pd(PPh3)4, 0.522mmol) and potassium phosphate trihydrate (K 3 PO 4 3H 2 O, 21mmol) into a 35mL microwave tube, add 1,4-dioxane/water (4:1) solvent and stir to dissolve, ultrasonically remove the oxygen in the solution, set the reaction temperature and time for the microwave reaction to 120 ℃ and 20min. After the reaction was complete, TLC detected that the reaction was complete, and the reaction solution was extracted with ethyl acetate/water (25mL×3), the organic phases were combined, washed with NaCl solution (20mL), dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Silica gel column chromatography purification, dichloromethane: methanol = 120:1 ~ 100:1 ~ 50:1, to obtain intermediates 6a-6i.
实施例6：中间体7的制备Embodiment 6: the preparation of intermediate 7
取中间体6a-6i(16mmol)置于100mL的茄形瓶中，加入25mL无水乙醇溶解，滴加1mL 3M NaOH溶液，搅拌使溶液呈碱性，pH约为10～11，室温反应6h。取样点板，TCL检测反应完毕，减压蒸除溶剂，加入15mL纯化水溶解，滴加1M HCl调节溶液pH约为5～6，搅拌，析出白色固体，抽滤，滤饼水洗，得中间体7a-7i。Take intermediate 6a-6i (16mmol) in a 100mL eggplant-shaped bottle, add 25mL of absolute ethanol to dissolve, add dropwise 1mL of 3M NaOH solution, stir to make the solution alkaline, pH is about 10-11, and react at room temperature for 6h. Sampling point plate, TCL detection reaction is complete, evaporate the solvent under reduced pressure, add 15mL purified water to dissolve, add dropwise 1M HCl to adjust the pH of the solution to about 5-6, stir, precipitate a white solid, filter with suction, wash the filter cake with water, and obtain the intermediate 7a-7i.
实施例7：中间体10的制备Embodiment 7: the preparation of intermediate 10
将化合物8(3g，18.4mmol)和化合物9(3.3g，20.2mmol)置于250mL茄形瓶中，用40mL醋酸溶液溶解，加入醋酸钠(1.8g，22.0mmol)，反应瓶置 于118℃回流，加热反应8h。TLC检测反应完全，将反应液倒入200mL冰水中，析出黑紫色亮晶固体，抽滤，滤饼用甲醇洗涤，干燥，得中间体10，黑紫色固体4.16g，产率88.32％。Mp：274-276℃；
1H NMR(400MHz,DMSO)δ11.19(s,1H),8.36(d,J＝8.0Hz,1H),8.25(d,J＝7.4Hz,1H),8.13(t,J＝7.7Hz,1H),5.22(dd,J＝12.7,5.1Hz,1H),3.02–2.80(m,1H),2.72–2.59(m,1H),2.58–2.49(m,1H),2.17–2.03(m,1H)。
Compound 8 (3g, 18.4mmol) and compound 9 (3.3g, 20.2mmol) were placed in a 250mL eggplant-shaped flask, dissolved with 40mL acetic acid solution, sodium acetate (1.8g, 22.0mmol) was added, and the reaction flask was placed at 118°C Reflux, heating reaction 8h. TLC detected that the reaction was complete, and the reaction solution was poured into 200 mL of ice water to precipitate a black-purple shiny solid, which was filtered with suction, and the filter cake was washed with methanol and dried to obtain Intermediate 10, 4.16 g of a black-purple solid, with a yield of 88.32%. Mp: 274-276°C; 1 H NMR (400MHz, DMSO) δ11.19(s, 1H), 8.36(d, J=8.0Hz, 1H), 8.25(d, J=7.4Hz, 1H), 8.13( t,J=7.7Hz,1H),5.22(dd,J=12.7,5.1Hz,1H),3.02–2.80(m,1H),2.72–2.59(m,1H),2.58–2.49(m,1H) ,2.17–2.03(m,1H).
实施例8：中间体11的制备Embodiment 8: the preparation of intermediate 11
取中间体10(4g，13.2mmol)和钯碳(Pd/C，400mg)置于250mL茄形瓶中，加入35mL乙醇和二氯甲烷(1:1)溶液，搅拌溶解，H
2保护，室温反应6h。TLC检测反应完毕，硅藻土过滤，合并滤液，减压蒸除有机溶剂，得中间体11，黄色固体3.3g，产率93％。Mp：278-280℃；
1H NMR(400MHz,DMSO-d
6)δ11.10(s,1H),7.47(t,J＝7.7Hz,1H),7.01(t,J＝6.9Hz,2H),6.53(s,2H),5.05(dd,J＝12.9,5.1Hz,1H),2.95–2.82(m,1H),2.70–2.52(m,2H),2.09–1.95(m,1H)。
Take intermediate 10 (4g, 13.2mmol) and palladium carbon (Pd/C, 400mg) in a 250mL eggplant-shaped flask, add 35mL of ethanol and dichloromethane (1:1) solution, stir to dissolve, H 2 protection, room temperature Reaction 6h. The completion of the reaction was detected by TLC, filtered with diatomaceous earth, the filtrates were combined, and the organic solvent was evaporated under reduced pressure to obtain intermediate 11, 3.3 g of a yellow solid, with a yield of 93%. Mp: 278-280°C; 1 H NMR (400MHz, DMSO-d 6 ) δ11.10(s, 1H), 7.47(t, J=7.7Hz, 1H), 7.01(t, J=6.9Hz, 2H) , 6.53(s, 2H), 5.05(dd, J=12.9, 5.1Hz, 1H), 2.95–2.82(m, 1H), 2.70–2.52(m, 2H), 2.09–1.95(m, 1H).
实施例9：中间体12的制备Embodiment 9: the preparation of intermediate 12
取中间体11(3.7mmol)分别加入氯乙酰氯、4-氯丁酰氯、5-氯戊酰氯、6-溴己酰氯(10.98mmol)于100mL茄形瓶中，加入25mL无水四氢呋喃溶液，搅拌溶解，N
2保护，60℃回流，加热反应6h。TLC检测反应完毕，减压蒸除溶剂，加入15mL无水乙醚，超声搅拌，析出淡黄色固体，抽滤，滤饼用无水乙醚洗涤，干燥，得中间体12a-12d。
Take intermediate 11 (3.7mmol) and add chloroacetyl chloride, 4-chlorobutyryl chloride, 5-chloropentanoyl chloride, 6-bromohexanoyl chloride (10.98mmol) respectively to a 100mL eggplant-shaped bottle, add 25mL of anhydrous tetrahydrofuran solution, and stir Dissolved, protected by N 2 , refluxed at 60°C, and heated for 6h. TLC detected that the reaction was complete, evaporated the solvent under reduced pressure, added 15 mL of anhydrous diethyl ether, stirred ultrasonically, and precipitated a pale yellow solid, filtered with suction, washed the filter cake with anhydrous diethyl ether, and dried to obtain intermediates 12a-12d.
实施例10：中间体13的制备Embodiment 10: the preparation of intermediate 13
取中间体12a-12d(16mmol)，N-Boc-哌嗪(79mmol)和碘化钠(3.176mmol)于250mL茄形瓶中，加入20mL N-甲基吡咯烷酮(NMP)溶液，搅拌溶解，滴加N,N-二异丙基乙胺(DIPEA，16mmol)，N
2保护，85℃油浴，加热反应8h。TLC检测反应完毕，反应液用乙酸乙酯/水萃取(30mL×3)，合并有机相，食盐水(30mL)洗涤，无水Na
2SO
4干燥，过滤，减压蒸除溶剂，硅胶柱层析纯化，二氯甲烷:甲醇＝120:1～100:1～50:1，得中间体13a-13d。
Take intermediate 12a-12d (16mmol), N-Boc-piperazine (79mmol) and sodium iodide (3.176mmol) in a 250mL eggplant-shaped flask, add 20mL N-methylpyrrolidone (NMP) solution, stir to dissolve, drop Add N,N-diisopropylethylamine (DIPEA, 16mmol), N 2 protection, 85 ° C oil bath, heating reaction for 8h. The completion of the reaction was detected by TLC, the reaction solution was extracted with ethyl acetate/water (30mL×3), the organic phases were combined, washed with brine (30mL), dried over anhydrous Na2SO4 , filtered, the solvent was evaporated under reduced pressure, and the silica gel column layer was Analysis and purification, dichloromethane: methanol = 120:1 ~ 100:1 ~ 50:1, to obtain intermediates 13a-13d.
实施例11：中间体14的制备Embodiment 11: the preparation of intermediate 14
称取中间体13a-13d(8.01mmol)置于100mL茄形瓶中，加入15mL饱和 HCl的乙酸乙酯溶液，搅拌溶解，室温下反应4h，逐渐析出白色固体。取样点板，TLC检测反应完全，抽滤，滤饼用乙酸乙酯洗涤，干燥，得中间体(14a-14d)。Intermediates 13a-13d (8.01 mmol) were weighed and placed in a 100 mL eggplant-shaped flask, 15 mL of saturated HCl in ethyl acetate was added, stirred and dissolved, reacted at room temperature for 4 h, and a white solid gradually precipitated. Sampling was done on the plate, TLC was used to detect the completion of the reaction, and the filter cake was washed with ethyl acetate and dried to obtain intermediates (14a-14d).
实施例12：目标化合物Ⅰ-1至Ⅰ-24的制备Example 12: Preparation of Target Compounds I-1 to I-24
取中间体7a-7i(5.05mmol)和缩合剂HATU(5.05mmol)于100mL茄形瓶中，加入15mL无水DMF，滴加N,N-二异丙基乙胺(14mmol)，搅拌溶解，置于冰水浴(0℃)中活化30min，后再加入中间体14a-14d(4.59mmol)，N
2保护，室温反应8h。TLC检测反应完毕，将反应液倒入150mL冰水浴中，析出白色固体，冷却至室温，抽滤，滤饼用冷水洗涤，干燥，得白色固体，固体用二氯甲烷溶解拌样，硅胶柱层析纯化，二氯甲烷:甲醇＝100:1～60:1～20:1，得到目标终产物Ⅰ-1至Ⅰ-24。
Take intermediate 7a-7i (5.05mmol) and condensing agent HATU (5.05mmol) in a 100mL eggplant-shaped flask, add 15mL of anhydrous DMF, dropwise add N,N-diisopropylethylamine (14mmol), stir to dissolve, Place in an ice-water bath (0°C) for activation for 30 min, then add intermediates 14a-14d (4.59 mmol), protect with N 2 , and react at room temperature for 8 h. The reaction was detected by TLC, and the reaction solution was poured into a 150mL ice-water bath to precipitate a white solid, cooled to room temperature, filtered with suction, the filter cake was washed with cold water, and dried to obtain a white solid. Analysis and purification, dichloromethane:methanol=100:1～60:1～20:1, to obtain the target final products I-1 to I-24.
Ⅰ-1：2-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺Ⅰ-1: 2-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide
白色固体，产率47.4％，Mp:166-168℃。
1H-NMR(400MHz,CDCl
3)δ11.14(s,1H),9.74(s,1H),8.87(d,J＝8.4Hz,1H),8.42(s,1H),7.72(t,J＝7.9Hz,1H),7.64(d,J＝8.0Hz,2H),7.57(d,J＝7.2Hz,1H),7.39(t,J＝7.6Hz,2H),7.23–7.10(m,3H),7.08(d,J＝7.7Hz,2H),5.04–4.93(m,1H),4.80(t,J＝7.1Hz,2H),3.78(s,2H),3.58(s,2H),3.21(s,2H),3.17–3.08(m,1H),3.07–2.98(m,1H),2.95–2.87(m,1H),2.86–2.73(m,2H),2.59(s,4H),2.25–2.11(m,1H)；
13C-NMR(100MHz,CDCl
3)δ171.99(s),169.73(s),168.88(s),168.69(s),168.41(s),166.92(s),158.56(s),157.95(s),156.29(s),155.74(s),154.36(s),144.11(s),136.85(s),136.29(s),131.42(s),129.99(s),129.89(s),127.59(s),125.10(s),124.09(s),119.56(s),119.10(s),118.68(s),116.13(s),98.41(s),61.70(s),53.34(s),53.10(s),49.35(s),45.28(s),43.54(s),41.51(s),32.80(s),31.53(s),22.68(s).HRMS(ESI):calcd for C
39H
36N
10O
7[M+H]
+757.2841,found 757.2838。
White solid, yield 47.4%, Mp: 166-168°C. 1 H-NMR (400MHz, CDCl 3 ) δ11.14(s, 1H), 9.74(s, 1H), 8.87(d, J=8.4Hz, 1H), 8.42(s, 1H), 7.72(t, J =7.9Hz,1H),7.64(d,J=8.0Hz,2H),7.57(d,J=7.2Hz,1H),7.39(t,J=7.6Hz,2H),7.23–7.10(m,3H ),7.08(d,J=7.7Hz,2H),5.04–4.93(m,1H),4.80(t,J=7.1Hz,2H),3.78(s,2H),3.58(s,2H),3.21 (s,2H),3.17–3.08(m,1H),3.07–2.98(m,1H),2.95–2.87(m,1H),2.86–2.73(m,2H),2.59(s,4H),2.25 –2.11(m,1H); 13 C-NMR(100MHz,CDCl 3 )δ171.99(s),169.73(s),168.88(s),168.69(s),168.41(s),166.92(s), 158.56(s), 157.95(s), 156.29(s), 155.74(s), 154.36(s), 144.11(s), 136.85(s), 136.29(s), 131.42(s), 129.99(s), 129.89(s), 127.59(s), 125.10(s), 124.09(s), 119.56(s), 119.10(s), 118.68(s), 116.13(s), 98.41(s), 61.70(s), 53.34(s), 53.10(s), 49.35(s), 45.28(s), 43.54(s), 41.51(s), 32.80(s), 31.53(s), 22.68(s).HRMS(ESI): calcd for C 39 H 36 N 10 O 7 [M+H] + 757.2841, found 757.2838.
Ⅰ-2：4-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺Ⅰ-2: 4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)butanamide
白色固体，产率43.5％，Mp:162-164℃。
1H NMR(400MHz,CDCl
3)δ9.52(s,1H),8.82(d,J＝8.6Hz,1H),8.42(s,1H),7.71(t,J＝8.0Hz,1H),7.63(d,J＝7.9Hz,2H),7.55(d,J＝7.2Hz,1H),7.39(t,J＝7.3Hz,2H),7.19–7.12(m,3H),7.08 (d,J＝7.8Hz,2H),4.99–4.90(m,1H),4.77(t,J＝7.4Hz,2H),3.58(s,2H),3.36(s,2H),3.08–2.98(m,2H),2.95–2.87(m,1H),2.85–2.73(m,2H),2.51(dd,J＝10.4,6.2Hz,2H),2.44–2.29(m,6H),2.20–2.14(m,1H),1.97–1.86(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.31(s),171.68(s),169.23(s),168.67(s),168.57(s),166.75(s),158.52(s),157.93(s),156.34(s),155.86(s),154.30(s),144.01(s),137.77(s),136.46(s),131.22(s),129.98(s),129.93(s),127.67(s),125.24(s),124.05(s),119.53(s),119.11(s),118.50(s),115.28(s),98.45(s),56.64(s),53.17(s),52.36(s),49.41(s),45.28(s),43.55(s),41.46(s),35.59(s),32.73(s),31.52(s),22.69(s),22.10(s).HRMS(ESI):calcd for C
41H
40N
10O
7[M+H]
+785.3154,found 785.3146。
White solid, yield 43.5%, Mp: 162-164°C. 1 H NMR (400MHz, CDCl 3 ) δ9.52(s, 1H), 8.82(d, J=8.6Hz, 1H), 8.42(s, 1H), 7.71(t, J=8.0Hz, 1H), 7.63 (d,J=7.9Hz,2H),7.55(d,J=7.2Hz,1H),7.39(t,J=7.3Hz,2H),7.19–7.12(m,3H),7.08 (d,J= 7.8Hz, 2H), 4.99–4.90(m, 1H), 4.77(t, J=7.4Hz, 2H), 3.58(s, 2H), 3.36(s, 2H), 3.08–2.98(m, 2H), 2.95–2.87(m,1H),2.85–2.73(m,2H),2.51(dd,J＝10.4,6.2Hz,2H),2.44–2.29(m,6H),2.20–2.14(m,1H), 1.97–1.86(m,2H); 13 C NMR(100MHz, CDCl 3 )δ172.31(s), 171.68(s), 169.23(s), 168.67(s), 168.57(s), 166.75(s), 158.52(s), 157.93(s), 156.34(s), 155.86(s), 154.30(s), 144.01(s), 137.77(s), 136.46(s), 131.22(s), 129.98(s), 129.93(s), 127.67(s), 125.24(s), 124.05(s), 119.53(s), 119.11(s), 118.50(s), 115.28(s), 98.45(s), 56.64(s), 53.17(s), 52.36(s), 49.41(s), 45.28(s), 43.55(s), 41.46(s), 35.59(s), 32.73(s), 31.52(s), 22.69(s), 22.10(s). HRMS(ESI): calcd for C 41 H 40 N 10 O 7 [M+H] + 785.3154, found 785.3146.
Ⅰ-3：5-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺Ⅰ-3: 5-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide
白色固体，产率45％，Mp:160-162℃。
1H NMR(400MHz,CDCl
3)δ10.30(s,1H),9.44(s,1H),8.80(d,J＝8.4Hz,1H),8.42(s,1H),7.71(t,J＝7.9Hz,1H),7.63(d,J＝7.9Hz,2H),7.54(d,J＝7.2Hz,1H),7.38(t,J＝7.4Hz,2H),7.19–7.11(m,3H),7.07(d,J＝7.9Hz,2H),5.00–4.90(m,1H),4.78(t,J＝7.0Hz,2H),3.59(t,J＝16.6Hz,2H),3.38(s,2H),3.01(t,J＝7.0Hz,2H),2.95–2.86(m,1H),2.84–2.70(m,2H),2.48(t,J＝6.9Hz,2H),2.40–2.33(m,4H),2.31(s,2H),2.20–2.14(m,1H),1.82–1.74(m,2H),1.60–1.52(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.13(s),171.58(s),169.29(s),168.61(s),168.58(s),166.73(s),158.52(s),157.88(s),156.35(s),155.87(s),154.39(s),143.99(s),137.77(s),136.46(s),131.15(s),129.98(s),129.92(s),127.70(s),125.25(s),124.05(s),119.53(s),119.13(s),118.52(s),115.34(s),98.46(s),57.64(s),53.14(s),52.70(s),49.35(s),45.35(s),43.57(s),41.51(s),37.68(s),32.75(s),31.48(s),25.80(s),23.23(s),22.76(s).HRMS(ESI):calcd for C
42H
42N
10O
7[M+H]
+799.3311,found 799.3301。
White solid, 45% yield, Mp: 160-162°C. 1 H NMR (400MHz, CDCl 3 ) δ10.30(s, 1H), 9.44(s, 1H), 8.80(d, J=8.4Hz, 1H), 8.42(s, 1H), 7.71(t, J= 7.9Hz, 1H), 7.63(d, J=7.9Hz, 2H), 7.54(d, J=7.2Hz, 1H), 7.38(t, J=7.4Hz, 2H), 7.19–7.11(m, 3H) ,7.07(d,J=7.9Hz,2H),5.00–4.90(m,1H),4.78(t,J=7.0Hz,2H),3.59(t,J=16.6Hz,2H),3.38(s, 2H), 3.01(t, J＝7.0Hz, 2H), 2.95–2.86(m, 1H), 2.84–2.70(m, 2H), 2.48(t, J＝6.9Hz, 2H), 2.40–2.33(m ,4H),2.31(s,2H),2.20–2.14(m,1H),1.82–1.74(m,2H),1.60–1.52(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.13 (s), 171.58(s), 169.29(s), 168.61(s), 168.58(s), 166.73(s), 158.52(s), 157.88(s), 156.35(s), 155.87(s), 154.39 (s), 143.99(s), 137.77(s), 136.46(s), 131.15(s), 129.98(s), 129.92(s), 127.70(s), 125.25(s), 124.05(s), 119.53 (s), 119.13(s), 118.52(s), 115.34(s), 98.46(s), 57.64(s), 53.14(s), 52.70(s), 49.35(s), 45.35(s), 43.57 (s), 41.51(s), 37.68(s), 32.75(s), 31.48(s), 25.80(s), 23.23(s), 22.76(s).HRMS(ESI): calcd for C 42 H 42 N 10 O 7 [M+H] + 799.3311, found 799.3301.
Ⅰ-4：6-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺Ⅰ-4: 6-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide
白色固体，产率57.49％，Mp:152-154℃。
1H NMR(400MHz,CDCl
3)δ9.86(s,1H),9.43(s,1H),8.82(d,J＝8.4Hz,1H),8.42(s,1H),7.71(t,J＝8.1Hz,1H),7.64(d,J＝8.1Hz,2H),7.55(d,J＝7.2Hz,1H),7.39(t,J＝7.5Hz,2H),7.20–7.12 (m,4H),7.08(d,J＝7.8Hz,2H),4.98–4.90(m,1H),4.83–4.75(m,2H),3.60(m,2H),3.39(m,2H),3.10–2.98(m,2H),2.94–2.86(m,1H),2.78(t,J＝11.3Hz,2H),2.46(t,J＝7.1Hz,2H),2.37–2.26(m,6H),2.20–2.13(m,1H),1.80–1.73(m,2H),1.55–1.48(m,2H),1.43–1.37(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.18(s),171.74(s),169.28(s),168.70(s),168.45(s),166.73(s),158.52(s),157.89(s),156.35(s),155.80(s),154.42(s),144.00(s),137.81(s),136.45(s),131.13(s),129.97(s),129.91(s),127.71(s),125.26(s),124.05(s),119.53(s),119.12(s),118.48(s),115.33(s),98.45(s),58.06(s),53.16(s),52.64(s),49.33(s),45.39(s),43.61(s),41.55(s),37.85(s),32.69(s),31.47(s),26.89(s),26.18(s),25.06(s),22.78(s).HRMS(ESI):calcd for C
43H
44N
10O
7[M+H]
+813.3467,found 813.3456。
White solid, yield 57.49%, Mp: 152-154°C. 1 H NMR (400MHz, CDCl 3 ) δ9.86(s, 1H), 9.43(s, 1H), 8.82(d, J=8.4Hz, 1H), 8.42(s, 1H), 7.71(t, J= 8.1Hz, 1H), 7.64(d, J=8.1Hz, 2H), 7.55(d, J=7.2Hz, 1H), 7.39(t, J=7.5Hz, 2H), 7.20–7.12 (m, 4H) ,7.08(d,J＝7.8Hz,2H),4.98–4.90(m,1H),4.83–4.75(m,2H),3.60(m,2H),3.39(m,2H),3.10–2.98(m ,2H),2.94–2.86(m,1H),2.78(t,J＝11.3Hz,2H),2.46(t,J＝7.1Hz,2H),2.37–2.26(m,6H),2.20–2.13( m,1H),1.80–1.73(m,2H),1.55–1.48(m,2H),1.43–1.37(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.18(s),171.74( s), 169.28(s), 168.70(s), 168.45(s), 166.73(s), 158.52(s), 157.89(s), 156.35(s), 155.80(s), 154.42(s), 144.00( s), 137.81(s), 136.45(s), 131.13(s), 129.97(s), 129.91(s), 127.71(s), 125.26(s), 124.05(s), 119.53(s), 119.12( s), 118.48(s), 115.33(s), 98.45(s), 58.06(s), 53.16(s), 52.64(s), 49.33(s), 45.39(s), 43.61(s), 41.55( s), 37.85(s), 32.69(s), 31.47(s), 26.89(s), 26.18(s), 25.06(s), 22.78(s).HRMS(ESI): calcd for C 43 H 44 N 10 O 7 [M+H] + 813.3467, found 813.3456.
Ⅰ-5：2-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺Ⅰ-5: 2-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide
白色固体，产率28.6％,Mp:164-166℃。
1H NMR(400MHz,CDCl
3)δ11.07(s,1H),8.84(d,J＝8.5Hz,1H),8.45(s,1H),7.73(t,J＝7.8Hz,1H),7.66(d,J＝7.9Hz,2H),7.58(d,J＝7.3Hz,1H),7.39(t,J＝7.6Hz,2H),7.21–7.13(m,3H),7.09(d,J＝8.0Hz,2H),5.04–4.91(m,1H),4.85–4.58(m,1H),4.54–4.36(m,1H),3.89(s,1H),3.50(s,1H),3.44–3.30(m,2H),3.18(dd,J＝72.3,16.8Hz,2H),2.99–2.82(m,1H),2.82–2.62(m,2H),2.62–2.44(m,4H),2.42–2.33(m,1H),2.33–2.28(m,2H),2.22–2.07(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.85(s),169.98(s),169.74(s),168.68(s),168.28(s),166.85(s),158.51(s),157.92(s),156.38(s),155.46(s),154.85(s),144.17(s),136.90(s),136.27(s),131.45(s),129.98(s),129.93(s),127.73(s),125.13(s),124.04(s),119.52(s),119.16(s),118.65(s),116.13(s),98.05(s),61.96(s),53.48(s),53.12(s),49.20(s),46.32(s),45.16(s),41.45(s),31.33(s),29.41(s),24.91(s),23.21(s).HRMS(ESI):calcd for C
40H
38N
10O
7[M+H]+771.2998,found 771.2994。
White solid, yield 28.6%, Mp: 164-166°C. 1 H NMR (400MHz, CDCl 3 ) δ11.07(s, 1H), 8.84(d, J=8.5Hz, 1H), 8.45(s, 1H), 7.73(t, J=7.8Hz, 1H), 7.66 (d, J=7.9Hz, 2H), 7.58(d, J=7.3Hz, 1H), 7.39(t, J=7.6Hz, 2H), 7.21–7.13(m, 3H), 7.09(d, J= 8.0Hz,2H),5.04–4.91(m,1H),4.85–4.58(m,1H),4.54–4.36(m,1H),3.89(s,1H),3.50(s,1H),3.44–3.30 (m,2H),3.18(dd,J＝72.3,16.8Hz,2H),2.99–2.82(m,1H),2.82–2.62(m,2H),2.62–2.44(m,4H),2.42–2.33 (m,1H),2.33–2.28(m,2H),2.22–2.07(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.85(s),169.98(s),169.74(s), 168.68(s), 168.28(s), 166.85(s), 158.51(s), 157.92(s), 156.38(s), 155.46(s), 154.85(s), 144.17(s), 136.90(s), 136.27(s), 131.45(s), 129.98(s), 129.93(s), 127.73(s), 125.13(s), 124.04(s), 119.52(s), 119.16(s), 118.65(s), 116.13(s), 98.05(s), 61.96(s), 53.48(s), 53.12(s), 49.20(s), 46.32(s), 45.16(s), 41.45(s), 31.33(s), 29.41(s), 24.91(s), 23.21(s). HRMS(ESI): calcd for C 40 H 38 N 10 O 7 [M+H]+771.2998, found 771.2994.
Ⅰ-6：4-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺Ⅰ-6: 4-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)butanamide
白色固体，产率22.8％，Mp:154-156℃。
1H NMR(400MHz,CDCl
3)δ10.94(s,1H),9.49(s,1H),8.81(d,J＝8.5Hz,1H),8.41(s,1H),7.71(t,J＝7.8Hz,1H), 7.64(d,J＝8.1Hz,2H),7.55(d,J＝7.2Hz,1H),7.39(t,J＝7.4Hz,2H),7.20–7.13(m,4H),7.08(d,J＝7.7Hz,2H),5.07–4.84(m,1H),4.57–4.50(m,2H),3.56(s,2H),3.28(s,2H),2.95–2.84(m,1H),2.84–2.67(m,2H),2.58–2.47(m,2H),2.42–2.35(m,4H),2.34–2.25(m,6H),2.14–2.06(m,1H),1.96–1.90(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.32(s),172.19(s),170.42(s),169.25(s),168.61(s),166.75(s),158.50(s),157.95(s),156.37(s),155.70(s),154.57(s),143.97(s),137.79(s),136.46(s),131.21(s),129.95(d,J＝5.1Hz),127.72(s),125.27(s),124.03(s),119.52(s),119.14(s),118.51(s),115.32(s),98.21(s),56.59(s),53.01(s),52.65(s),49.39(s),46.69(s),45.25(s),41.51(s),35.63(s),31.51(s),30.22(s),25.28(s),22.82(s),22.40(s).HRMS(ESI):calcd for C
42H
42N
10O
7[M+H]
+799.3311,found 799.3279。
White solid, yield 22.8%, Mp: 154-156°C. 1 H NMR (400MHz, CDCl 3 ) δ10.94(s, 1H), 9.49(s, 1H), 8.81(d, J=8.5Hz, 1H), 8.41(s, 1H), 7.71(t, J= 7.8Hz,1H), 7.64(d,J=8.1Hz,2H),7.55(d,J=7.2Hz,1H),7.39(t,J=7.4Hz,2H),7.20–7.13(m,4H) ,7.08(d,J＝7.7Hz,2H),5.07–4.84(m,1H),4.57–4.50(m,2H),3.56(s,2H),3.28(s,2H),2.95–2.84(m ,1H),2.84–2.67(m,2H),2.58–2.47(m,2H),2.42–2.35(m,4H),2.34–2.25(m,6H),2.14–2.06(m,1H),1.96 –1.90(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.32(s),172.19(s),170.42(s),169.25(s),168.61(s),166.75(s),158.50 (s), 157.95(s), 156.37(s), 155.70(s), 154.57(s), 143.97(s), 137.79(s), 136.46(s), 131.21(s), 129.95(d, J= 5.1Hz), 127.72(s), 125.27(s), 124.03(s), 119.52(s), 119.14(s), 118.51(s), 115.32(s), 98.21(s), 56.59(s), 53.01 (s), 52.65(s), 49.39(s), 46.69(s), 45.25(s), 41.51(s), 35.63(s), 31.51(s), 30.22(s), 25.28(s), 22.82 (s), 22.40 (s). HRMS (ESI): calcd for C 42 H 42 N 10 O 7 [M+H] + 799.3311, found 799.3279.
Ⅰ-7：5-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺Ⅰ-7: 5-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide
白色固体，产率29.3％，Mp:144-146℃。
1H NMR(400MHz,CDCl
3)δ11.06(s,1H),9.48(s,1H),8.82(d,J＝8.5Hz,1H),8.42(s,1H),7.72(t,J＝7.8Hz,1H),7.65(d,J＝7.9Hz,2H),7.56(d,J＝7.4Hz,1H),7.39(t,J＝7.5Hz,2H),7.16(t,J＝9.8Hz,3H),7.08(d,J＝7.9Hz,2H),5.02–4.90(m,1H),4.62–4.42(m,2H),3.72–3.49(m,2H),3.29(s,2H),2.95–2.85(m,1H),2.85–2.71(m,2H),2.47(dd,J＝13.2,7.0Hz,2H),2.36(s,6H),2.28(s,4H),2.21–2.14(m,1H),1.86–1.73(m,2H),1.59–1.52(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.41(s),172.15(s),170.33(s),169.32(s),168.71(s),166.76(s),158.48(s),157.94(s),156.38(s),155.61(s),154.62(s),143.98(s),137.79(s),136.44(s),131.14(s),129.97(s),129.92(s),127.77(s),125.28(s),124.02(s),119.50(s),119.15(s),118.52(s),115.40(s),98.14(s),57.63(s),53.12(s),52.89(s),49.36(s),46.60(s),45.28(s),41.54(s),37.86(s),31.52(s),29.81(s),25.96(s),25.14(s),23.30(s),22.83(s).HRMS(ESI):calcd for C
43H
44N
10O
7[M+H]
+813.3467,found 813.3442。
White solid, yield 29.3%, Mp: 144-146°C. 1 H NMR (400MHz, CDCl 3 ) δ11.06(s, 1H), 9.48(s, 1H), 8.82(d, J=8.5Hz, 1H), 8.42(s, 1H), 7.72(t, J= 7.8Hz, 1H), 7.65(d, J=7.9Hz, 2H), 7.56(d, J=7.4Hz, 1H), 7.39(t, J=7.5Hz, 2H), 7.16(t, J=9.8Hz ,3H),7.08(d,J＝7.9Hz,2H),5.02–4.90(m,1H),4.62–4.42(m,2H),3.72–3.49(m,2H),3.29(s,2H), 2.95–2.85(m,1H),2.85–2.71(m,2H),2.47(dd,J＝13.2,7.0Hz,2H),2.36(s,6H),2.28(s,4H),2.21–2.14( m,1H),1.86–1.73(m,2H),1.59–1.52(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.41(s),172.15(s),170.33(s),169.32 (s), 168.71(s), 166.76(s), 158.48(s), 157.94(s), 156.38(s), 155.61(s), 154.62(s), 143.98(s), 137.79(s), 136.44 (s), 131.14(s), 129.97(s), 129.92(s), 127.77(s), 125.28(s), 124.02(s), 119.50(s), 119.15(s), 118.52(s), 115.40 (s), 98.14(s), 57.63(s), 53.12(s), 52.89(s), 49.36(s), 46.60(s), 45.28(s), 41.54(s), 37.86(s), 31.52 (s),29.81(s),25.96(s),25.14(s),23.30(s),22.83(s).HRMS(ESI):calcd for C 43 H 44 N 10 O 7 [M+H] + 813.3467, found 813.3442.
Ⅰ-8：6-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺Ⅰ-8: 6-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide
白色固体，产率60.5％，Mp:146-148℃。
1H NMR(400MHz,CDCl
3)δ11.52 (s,1H),9.45(s,1H),8.82(d,J＝8.4Hz,1H),8.42(s,1H),7.72(t,J＝7.8Hz,1H),7.64(d,J＝7.8Hz,2H),7.56(d,J＝7.1Hz,1H),7.39(t,J＝7.4Hz,2H),7.20–7.13(m,3H),7.08(d,J＝7.7Hz,2H),5.08–4.90(m,1H),4.60–4.44(m,2H),3.66–3.53(m,2H),3.30(s,2H),2.96–2.85(m,1H),2.85–2.68(m,2H),2.53–2.42(m,2H),2.37(s,2H),2.30(s,8H),2.18(s,1H),1.76(td,J＝13.8,7.0Hz,2H),1.56–1.48(m,2H),1.44–1.36(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.63(s),172.20(s),170.26(s),169.33(s),168.77(s),166.75(s),158.49(s),157.98(s),156.38(s),155.60(s),154.65(s),144.00(s),137.84(s),136.48(s),131.14(s),129.98(s),129.92(s),127.75(s),125.26(s),124.03(s),119.51(s),119.16(s),118.51(s),115.32(s),98.13(s),58.00(s),53.00(s),49.34(s),46.60(s),45.31(s),41.57(s),37.82(s),31.53(s),29.97(s),26.66(s),26.27(s),25.27(s),25.13(s),22.87(s).HRMS(ESI):calcd for C
44H
46N
10O
7[M+H]
+827.3624,found 827.3615。
White solid, yield 60.5%, Mp: 146-148°C. 1 H NMR (400MHz, CDCl 3 ) δ11.52 (s, 1H), 9.45 (s, 1H), 8.82 (d, J = 8.4Hz, 1H), 8.42 (s, 1H), 7.72 (t, J = 7.8Hz, 1H), 7.64(d, J=7.8Hz, 2H), 7.56(d, J=7.1Hz, 1H), 7.39(t, J=7.4Hz, 2H), 7.20–7.13(m, 3H) ,7.08(d,J＝7.7Hz,2H),5.08–4.90(m,1H),4.60–4.44(m,2H),3.66–3.53(m,2H),3.30(s,2H),2.96–2.85 (m,1H),2.85–2.68(m,2H),2.53–2.42(m,2H),2.37(s,2H),2.30(s,8H),2.18(s,1H),1.76(td,J =13.8,7.0Hz,2H),1.56–1.48(m,2H),1.44–1.36(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.63(s),172.20(s),170.26( s), 169.33(s), 168.77(s), 166.75(s), 158.49(s), 157.98(s), 156.38(s), 155.60(s), 154.65(s), 144.00(s), 137.84( s), 136.48(s), 131.14(s), 129.98(s), 129.92(s), 127.75(s), 125.26(s), 124.03(s), 119.51(s), 119.16(s), 118.51( s), 115.32(s), 98.13(s), 58.00(s), 53.00(s), 49.34(s), 46.60(s), 45.31(s), 41.57(s), 37.82(s), 31.53( s), 29.97(s), 26.66(s), 26.27(s), 25.27(s), 25.13(s), 22.87(s). HRMS(ESI): calcd for C 44 H 46 N 10 O 7 [M +H] + 827.3624, found 827.3615.
Ⅰ-9：2-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺Ⅰ-9: 2-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide
白色固体，产率41.2％，Mp:160-162℃。
1H NMR(400MHz,CDCl
3)δ11.21(s,1H),8.93(d,J＝8.5Hz,1H),8.46(s,1H),7.77–7.70(m,1H),7.66–7.58(m,3H),7.40(t,J＝7.9Hz,2H),7.20–7.13(m,3H),7.09(d,J＝7.8Hz,2H),5.05–4.93(m,1H),4.80–4.62(m,1H),4.41–4.32(m,1H),4.33–4.16(m,1H),3.88–3.64(m,1H),3.44–3.33(m,1H),3.32–3.09(m,3H),2.97–2.87(m,1H),2.88–2.56(m,6H),2.52–2.43(m,1H),2.40–2.29(m,2H),2.20–2.13(m,1H),2.12–2.02(m,1H),1.92–1.72(m,2H)；
13C NMR(100MHz,CDCl
3)δ173.08(s),171.20(s),169.89(s),168.72(s),168.36(s),166.99(s),158.54(s),158.06(s),156.37(s),155.25(s),154.66(s),144.49(s),136.90(s),136.24(s),131.44(s),129.98(s),129.91(s),127.63(s),125.08(s),124.03(s),119.53(s),119.17(s),118.67(s),116.13(s),98.09(s),61.80(s),53.62(s),53.38(s),49.34(s),44.97(s),44.93(s),41.51(s),31.56(s),31.16(s),28.20(s),23.13(s),20.93(s).HRMS(ESI):calcd for C
41H
40N
10O
7[M+H]
+785.3154,found 785.3146。
White solid, yield 41.2%, Mp: 160-162°C. 1 H NMR (400MHz, CDCl 3 ) δ11.21(s, 1H), 8.93(d, J=8.5Hz, 1H), 8.46(s, 1H), 7.77–7.70(m, 1H), 7.66–7.58( m,3H),7.40(t,J=7.9Hz,2H),7.20–7.13(m,3H),7.09(d,J=7.8Hz,2H),5.05–4.93(m,1H),4.80–4.62 (m,1H),4.41–4.32(m,1H),4.33–4.16(m,1H),3.88–3.64(m,1H),3.44–3.33(m,1H),3.32–3.09(m,3H) ,2.97–2.87(m,1H),2.88–2.56(m,6H),2.52–2.43(m,1H),2.40–2.29(m,2H),2.20–2.13(m,1H),2.12–2.02( m,1H),1.92–1.72(m,2H); 13 C NMR(100MHz,CDCl 3 )δ173.08(s),171.20(s),169.89(s),168.72(s),168.36(s), 166.99(s), 158.54(s), 158.06(s), 156.37(s), 155.25(s), 154.66(s), 144.49(s), 136.90(s), 136.24(s), 131.44(s), 129.98(s), 129.91(s), 127.63(s), 125.08(s), 124.03(s), 119.53(s), 119.17(s), 118.67(s), 116.13(s), 98.09(s), 61.80(s), 53.62(s), 53.38(s), 49.34(s), 44.97(s), 44.93(s), 41.51(s), 31.56(s), 31.16(s), 28.20(s), 23.13(s), 20.93(s). HRMS(ESI): calcd for C 41 H 40 N 10 O 7 [M+H] + 785.3154, found 785.3146.
Ⅰ-10：4-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺Ⅰ-10: 4-(4-(5-(4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)butanamide
白色固体，产率28.6％，Mp:138-140℃。
1H NMR(400MHz,CDCl
3)δ9.50(s,1H),8.81(d,J＝8.2Hz,1H),8.43(s,1H),7.72(t,J＝7.9Hz,1H),7.64(d,J＝8.1Hz,2H),7.56(d,J＝7.3Hz,1H),7.39(t,J＝7.6Hz,2H),7.19–7.13(m,3H),7.08(d,J＝7.9Hz,2H),5.06–4.86(m,1H),4.56–4.40(m,2H),3.71–3.54(m,1H),3.48–3.29(m,3H),3.00–2.83(m,1H),2.88–2.71(m,2H),2.58–2.46(m,2H),2.46–2.35(m,5H),2.34–2.23(m,3H),2.22–2.16(m,1H),2.14–2.07(m,1H),2.04–1.97(m,1H),1.97–1.88(m,2H),1.57–1.47(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.28(s),172.26(s),171.19(s),169.24(s),168.91(s),166.78(s),158.44(s),157.99(s),156.37(s),155.56(s),154.26(s),143.93(s),137.75(s),136.45(s),131.19(s),129.97(s),129.94(s),127.73(s),125.28(s),124.00(s),119.48(s),119.16(s),118.51(s),115.33(s),98.17(s),56.86(s),53.28(s),52.57(s),50.66(s),49.37(s),46.32(s),45.44(s),35.62(s),32.37(s),31.50(s),29.09(s),22.78(s),22.17(s),22.04(s).HRMS(ESI):calcd for C
43H
44N
10O
7[M+H]
+813.3467,found 813.3461。
White solid, yield 28.6%, Mp: 138-140°C. 1 H NMR (400MHz, CDCl 3 ) δ9.50(s, 1H), 8.81(d, J=8.2Hz, 1H), 8.43(s, 1H), 7.72(t, J=7.9Hz, 1H), 7.64 (d,J=8.1Hz,2H),7.56(d,J=7.3Hz,1H),7.39(t,J=7.6Hz,2H),7.19–7.13(m,3H),7.08(d,J= 7.9Hz,2H),5.06–4.86(m,1H),4.56–4.40(m,2H),3.71–3.54(m,1H),3.48–3.29(m,3H),3.00–2.83(m,1H) ,2.88–2.71(m,2H),2.58–2.46(m,2H),2.46–2.35(m,5H),2.34–2.23(m,3H),2.22–2.16(m,1H),2.14–2.07( m,1H),2.04–1.97(m,1H),1.97–1.88(m,2H),1.57–1.47(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.28(s),172.26( s), 171.19(s), 169.24(s), 168.91(s), 166.78(s), 158.44(s), 157.99(s), 156.37(s), 155.56(s), 154.26(s), 143.93( s), 137.75(s), 136.45(s), 131.19(s), 129.97(s), 129.94(s), 127.73(s), 125.28(s), 124.00(s), 119.48(s), 119.16( s), 118.51(s), 115.33(s), 98.17(s), 56.86(s), 53.28(s), 52.57(s), 50.66(s), 49.37(s), 46.32(s), 45.44( s), 35.62(s), 32.37(s), 31.50(s), 29.09(s), 22.78(s), 22.17(s), 22.04(s).HRMS(ESI): calcd for C 43 H 44 N 10 O 7 [M+H] + 813.3467, found 813.3461.
Ⅰ-11：5-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺Ⅰ-11: 5-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide
白色固体，产率45.2％，Mp:140-421℃。
1H NMR(400MHz,CDCl
3)δ9.48(s,1H),8.81(d,J＝8.3Hz,1H),8.40(s,1H),7.72(t,J＝7.7Hz,1H),7.64(d,J＝8.0Hz,2H),7.56(d,J＝7.1Hz,1H),7.39(t,J＝7.4Hz,2H),7.19–7.12(m,3H),7.08(d,J＝7.8Hz,2H),5.03–4.87(m,1H),4.56–4.40(m,2H),3.69–3.45(m,2H),3.47–3.26(m,2H),2.98–2.82(m,1H),2.86–2.63(m,2H),2.52–2.45(m,2H),2.45–2.24(m,8H),2.18(s,1H),2.10–2.00(m,2H),1.84–1.76(m,2H),1.64–1.51(m,4H)；
13C NMR(100MHz,CDCl
3)δ172.08(s),172.01(s),171.24(s),169.31(s),168.68(s),166.73(s),158.46(s),157.94(s),156.40(s),155.57(s),154.38(s),143.87(s),137.79(s),136.45(s),131.14(s),129.97(s),129.94(s),127.80(s),125.26(s),124.00(s),119.49(s),119.17(s),118.52(s),115.38(s),98.21(s),57.73(s),53.48(s),52.70(s),49.34(s),46.38(s),45.52(s),41.48(s),37.72(s),32.57(s),31.48(s),29.20(s),25.92(s),23.23(s),22.83(s),22.24(s).HRMS(ESI):calcd for C
44H
46N
10O
7[M+H]
+827.3624,found 827.3611。
White solid, yield 45.2%, Mp: 140-421°C. 1 H NMR (400MHz, CDCl 3 ) δ9.48(s, 1H), 8.81(d, J=8.3Hz, 1H), 8.40(s, 1H), 7.72(t, J=7.7Hz, 1H), 7.64 (d, J=8.0Hz, 2H), 7.56(d, J=7.1Hz, 1H), 7.39(t, J=7.4Hz, 2H), 7.19–7.12(m, 3H), 7.08(d, J= 7.8Hz,2H),5.03–4.87(m,1H),4.56–4.40(m,2H),3.69–3.45(m,2H),3.47–3.26(m,2H),2.98–2.82(m,1H) ,2.86–2.63(m,2H),2.52–2.45(m,2H),2.45–2.24(m,8H),2.18(s,1H),2.10–2.00(m,2H),1.84–1.76(m, 2H), 1.64–1.51(m, 4H); 13 C NMR (100MHz, CDCl 3 ) δ172.08(s), 172.01(s), 171.24(s), 169.31(s), 168.68(s), 166.73( s), 158.46(s), 157.94(s), 156.40(s), 155.57(s), 154.38(s), 143.87(s), 137.79(s), 136.45(s), 131.14(s), 129.97( s), 129.94(s), 127.80(s), 125.26(s), 124.00(s), 119.49(s), 119.17(s), 118.52(s), 115.38(s), 98.21(s), 57.73( s), 53.48(s), 52.70(s), 49.34(s), 46.38(s), 45.52(s), 41.48(s), 37.72(s), 32.57(s), 31.48(s), 29.20( s), 25.92(s), 23.23(s), 22.83(s), 22.24(s). HRMS(ESI): calcd for C 44 H 46 N 10 O 7 [M+H] + 827.3624, found 827.3611.
Ⅰ-12：6-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基) 哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺Ⅰ-12: 6-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide
白色固体，产率50.3％，Mp:134-136℃。
1H NMR(400MHz,CDCl
3)δ11.21–10.66(m,1H),9.47(s,1H),8.82(d,J＝8.4Hz,1H),8.41(s,1H),7.74–7.69(m,1H),7.64(d,J＝8.6Hz,2H),7.56(d,J＝7.3Hz,1H),7.38(t,J＝7.9Hz,2H),7.18–7.12(m,3H),7.08(d,J＝7.8Hz,2H),5.04–4.90(m,1H),4.48(dt,J＝12.2,6.8Hz,2H),3.63–3.49(m,2H),3.41–3.32(m,2H),3.05–2.86(m,1H),2.87–2.60(m,2H),2.49–2.43(m,2H),2.43–2.36(m,2H),2.36–2.27(m,6H),2.21–2.15(m,1H),2.12–2.02(m,2H),1.81–1.73(m,2H),1.59–1.48(m,4H),1.42(dd,J＝18.3,3.7Hz,2H)；
13C NMR(100MHz,CDCl
3)δ172.21(s),172.18(s),171.15(s),169.29(s),168.87(s),166.74(s),158.46(s),157.98(s),156.41(s),155.53(s),154.41(s),143.91(s),137.81(s),136.43(s),131.15(s),129.96(s),129.94(s),127.80(s),125.26(s),124.00(s),119.48(s),119.17(s),118.48(s),115.36(s),98.18(s),57.98(s),53.48(s),52.60(s),49.35(s),46.25(s),45.50(s),41.47(s),37.92(s),32.47(s),31.51(s),29.05(s),26.78(s),26.18(s),25.15(s),22.79(s),22.13(s).HRMS(ESI):calcd for C
45H
48N
10O
7[M+H]
+841.3780,found 841.3773。
White solid, yield 50.3%, Mp: 134-136°C. 1 H NMR (400MHz, CDCl 3 ) δ11.21–10.66 (m, 1H), 9.47 (s, 1H), 8.82 (d, J=8.4Hz, 1H), 8.41 (s, 1H), 7.74–7.69 ( m,1H),7.64(d,J=8.6Hz,2H),7.56(d,J=7.3Hz,1H),7.38(t,J=7.9Hz,2H),7.18–7.12(m,3H), 7.08(d,J=7.8Hz,2H),5.04–4.90(m,1H),4.48(dt,J=12.2,6.8Hz,2H),3.63–3.49(m,2H),3.41–3.32(m, 2H),3.05–2.86(m,1H),2.87–2.60(m,2H),2.49–2.43(m,2H),2.43–2.36(m,2H),2.36–2.27(m,6H),2.21– 2.15(m,1H),2.12–2.02(m,2H),1.81–1.73(m,2H),1.59–1.48(m,4H),1.42(dd,J=18.3,3.7Hz,2H); 13 C NMR(100MHz, CDCl 3 )δ172.21(s), 172.18(s), 171.15(s), 169.29(s), 168.87(s), 166.74(s), 158.46(s), 157.98(s), 156.41 (s), 155.53(s), 154.41(s), 143.91(s), 137.81(s), 136.43(s), 131.15(s), 129.96(s), 129.94(s), 127.80(s), 125.26 (s), 124.00(s), 119.48(s), 119.17(s), 118.48(s), 115.36(s), 98.18(s), 57.98(s), 53.48(s), 52.60(s), 49.35 (s), 46.25(s), 45.50(s), 41.47(s), 37.92(s), 32.47(s), 31.51(s), 29.05(s), 26.78(s), 26.18(s), 25.15 (s), 22.79 (s), 22.13 (s). HRMS (ESI): calcd for C 45 H 48 N 10 O 7 [M+H] + 841.3780, found 841.3773.
Ⅰ-13：2-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺Ⅰ-13: 2-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide
白色固体，产率50.3％，Mp:160-162℃。
1H NMR(400MHz,CDCl
3)δ11.16(s,1H),8.88(d,J＝8.4Hz,1H),8.42(s,1H),7.73(t,J＝7.7Hz,1H),7.65(d,J＝7.9Hz,2H),7.58(d,J＝7.2Hz,1H),7.39(t,J＝7.6Hz,2H),7.20–7.12(m,3H),7.08(d,J＝7.7Hz,2H),5.15–4.76(m,1H),4.55–4.36(m,2H),3.88(s,1H),3.80–3.44(m,3H),3.21(q,J＝17.0Hz,2H),2.98–2.85(m,1H),2.85–2.70(m,2H),2.68–2.46(m,4H),2.36–2.21(m,2H),2.16(s,1H),2.05–1.93(m,2H),1.66–1.59(m,2H),1.44–1.32(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.23(s),171.45(s),169.80(s),168.86(s),168.38(s),166.92(s),158.43(s),157.99(s),156.39(s),155.49(s),154.29(s),143.83(s),136.87(s),136.28(s),131.42(s),129.96(s),129.89(s),127.79(s),125.12(s),124.00(s),119.47(s),119.16(s),118.66(s),116.14(s),98.24(s),61.79(s),53.58(s),53.24(s),50.71(s),49.32(s),47.06(s),45.42(s),32.98(s),31.52(s),29.36(s),26.40(s),24.66(s),22.75(s).HRMS(ESI):calcd for C
42H
42N
10O
7[M+H]
+799.3311,found 799.3297。
White solid, yield 50.3%, Mp: 160-162°C. 1 H NMR (400MHz, CDCl 3 ) δ11.16(s, 1H), 8.88(d, J=8.4Hz, 1H), 8.42(s, 1H), 7.73(t, J=7.7Hz, 1H), 7.65 (d, J=7.9Hz, 2H), 7.58(d, J=7.2Hz, 1H), 7.39(t, J=7.6Hz, 2H), 7.20–7.12(m, 3H), 7.08(d, J= 7.7Hz, 2H), 5.15–4.76(m, 1H), 4.55–4.36(m, 2H), 3.88(s, 1H), 3.80–3.44(m, 3H), 3.21(q, J=17.0Hz, 2H ),2.98–2.85(m,1H),2.85–2.70(m,2H),2.68–2.46(m,4H),2.36–2.21(m,2H),2.16(s,1H),2.05–1.93(m ,2H), 1.66–1.59(m,2H), 1.44–1.32(m,2H); 13 C NMR (100MHz, CDCl 3 )δ172.23(s), 171.45(s), 169.80(s), 168.86( s), 168.38(s), 166.92(s), 158.43(s), 157.99(s), 156.39(s), 155.49(s), 154.29(s), 143.83(s), 136.87(s), 136.28( s), 131.42(s), 129.96(s), 129.89(s), 127.79(s), 125.12(s), 124.00(s), 119.47(s), 119.16(s), 118.66(s), 116.14( s), 98.24(s), 61.79(s), 53.58(s), 53.24(s), 50.71(s), 49.32(s), 47.06(s), 45.42(s), 32.98(s), 31.52( s), 29.36(s), 26.40(s), 24.66(s), 22.75(s). HRMS(ESI): calcd for C 42 H 42 N 10 O 7 [M+H] + 799.3311, found 799.3297.
Ⅰ-14：4-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺Ⅰ-14: 4-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)butanamide
白色固体，产率48.5％，Mp:126-128℃。
1H NMR(400MHz,CDCl
3)δ10.50(s,1H),9.47(s,1H),8.82(d,J＝8.4Hz,1H),8.40(s,1H),7.72(t,J＝7.8Hz,1H),7.65(d,J＝8.0Hz,2H),7.56(d,J＝7.2Hz,1H),7.39(t,J＝7.5Hz,2H),7.20–7.11(m,3H),7.08(d,J＝7.8Hz,2H),5.06–4.85(m,1H),4.52–4.33(m,2H),3.65–3.45(m,2H),3.38(s,2H),3.01–2.84(m,1H),2.88–2.66(m,2H),2.51(t,J＝6.5Hz,2H),2.49–2.30(m,6H),2.31–2.22(m,2H),2.21–2.13(m,1H),2.01–1.90(m,4H),1.71–1.62(m,2H),1.39–1.29(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.24(s),172.06(s),171.39(s),169.26(s),168.72(s),166.76(s),158.43(s),157.94(s),156.42(s),155.54(s),154.31(s),143.79(s),137.80(s),136.47(s),131.19(s),129.96(s),129.90(s),127.84(s),125.26(s),123.99(s),119.47(s),119.18(s),118.50(s),115.30(s),98.22(s),56.98(s),53.24(s),52.72(s),49.38(s),46.95(s),45.50(s),41.45(s),35.75(s),33.04(s),31.51(s),29.41(s),26.37(s),24.74(s),22.77(s),22.19(s).HRMS(ESI):calcd for C
44H
46N
10O
7[M+H]
+827.3624,found 827.3608。
White solid, yield 48.5%, Mp: 126-128°C. 1 H NMR (400MHz, CDCl 3 ) δ10.50(s, 1H), 9.47(s, 1H), 8.82(d, J=8.4Hz, 1H), 8.40(s, 1H), 7.72(t, J= 7.8Hz, 1H), 7.65(d, J=8.0Hz, 2H), 7.56(d, J=7.2Hz, 1H), 7.39(t, J=7.5Hz, 2H), 7.20–7.11(m, 3H) ,7.08(d,J＝7.8Hz,2H),5.06–4.85(m,1H),4.52–4.33(m,2H),3.65–3.45(m,2H),3.38(s,2H),3.01–2.84 (m,1H),2.88–2.66(m,2H),2.51(t,J＝6.5Hz,2H),2.49–2.30(m,6H),2.31–2.22(m,2H),2.21–2.13(m ,1H),2.01–1.90(m,4H),1.71–1.62(m,2H),1.39–1.29(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.24(s),172.06(s ),171.39(s),169.26(s),168.72(s),166.76(s),158.43(s),157.94(s),156.42(s),155.54(s),154.31(s),143.79(s ),137.80(s),136.47(s),131.19(s),129.96(s),129.90(s),127.84(s),125.26(s),123.99(s),119.47(s),119.18(s ),118.50(s),115.30(s),98.22(s),56.98(s),53.24(s),52.72(s),49.38(s),46.95(s),45.50(s),41.45(s ),35.75(s),33.04(s),31.51(s),29.41(s),26.37(s),24.74(s),22.77(s),22.19(s).HRMS(ESI):calcd for C 44 H 46 N 10 O 7 [M+H] + 827.3624, found 827.3608.
Ⅰ-15：5-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺Ⅰ-15: 5-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide
白色固体，产率45.6％，Mp:130-132℃。
1H NMR(400MHz,CDCl
3)δ10.59(s,1H),9.46(s,1H),8.82(d,J＝8.5Hz,1H),8.40(s,1H),7.72(t,J＝7.8Hz,1H),7.65(d,J＝7.9Hz,2H),7.56(d,J＝7.0Hz,1H),7.39(t,J＝7.7Hz,2H),7.20–7.12(m,3H),7.08(d,J＝7.8Hz,2H),5.10–4.81(m,1H),4.53–4.37(m,2H),3.60(d,J＝17.5Hz,2H),3.42(s,2H),2.97–2.83(m,1H),2.87–2.66(m,2H),2.57–2.43(m,2H),2.45–2.31(m,6H),2.27(t,J＝7.4Hz,2H),2.21–2.13(m,1H),2.02–1.93(m,2H),1.88–1.74(m,2H),1.71–1.65(m,2H),1.59–1.55(m,3H),1.38–1.31(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.14(s),172.09(s),171.44(s),169.31(s),168.71(s),166.74(s),158.43(s),157.94(s),156.43(s),155.53(s),154.33(s),143.76(s),137.79(s),136.44(s),131.14(s),129.96(s),129.91(s),127.87(s), 125.26(s),123.99(s),119.47(s),119.18(s),118.51(s),115.38(s),98.22(s),57.70(s),53.35(s),52.81(s),49.36(s),46.93(s),45.56(s),41.48(s),37.73(s),33.04(s),31.50(s),29.40(s),26.37(s),25.90(s),24.81(s),23.17(s),22.80(s).HRMS(ESI):calcd for C
45H
48N
10O
7[M+H]
+841.3780,found 841.3770。
White solid, yield 45.6%, Mp: 130-132°C. 1 H NMR (400MHz, CDCl 3 ) δ10.59(s, 1H), 9.46(s, 1H), 8.82(d, J=8.5Hz, 1H), 8.40(s, 1H), 7.72(t, J= 7.8Hz, 1H), 7.65(d, J=7.9Hz, 2H), 7.56(d, J=7.0Hz, 1H), 7.39(t, J=7.7Hz, 2H), 7.20–7.12(m, 3H) ,7.08(d,J=7.8Hz,2H),5.10–4.81(m,1H),4.53–4.37(m,2H),3.60(d,J=17.5Hz,2H),3.42(s,2H), 2.97–2.83(m,1H),2.87–2.66(m,2H),2.57–2.43(m,2H),2.45–2.31(m,6H),2.27(t,J＝7.4Hz,2H),2.21– 2.13(m,1H),2.02–1.93(m,2H),1.88–1.74(m,2H),1.71–1.65(m,2H),1.59–1.55(m,3H),1.38–1.31(m,2H ); 13 C NMR (100MHz, CDCl 3 ) δ172.14(s), 172.09(s), 171.44(s), 169.31(s), 168.71(s), 166.74(s), 158.43(s), 157.94( s), 156.43(s), 155.53(s), 154.33(s), 143.76(s), 137.79(s), 136.44(s), 131.14(s), 129.96(s), 129.91(s), 127.87( s), 125.26(s), 123.99(s), 119.47(s), 119.18(s), 118.51(s), 115.38(s), 98.22(s), 57.70(s), 53.35(s), 52.81( s), 49.36(s), 46.93(s), 45.56(s), 41.48(s), 37.73(s), 33.04(s), 31.50(s), 29.40(s), 26.37(s), 25.90( s), 24.81(s), 23.17(s), 22.80(s). HRMS(ESI): calcd for C 45 H 48 N 10 O 7 [M+H] + 841.3780, found 841.3770.
Ⅰ-16：6-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺Ⅰ-16: 6-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl) Piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide
白色固体，产率30.6％，Mp:134-136℃。
1H NMR(400MHz,CDCl
3)δ9.43(s,1H),8.83(d,J＝8.2Hz,1H),8.42(s,1H),7.72(t,J＝7.7Hz,1H),7.65(d,J＝7.7Hz,2H),7.56(d,J＝7.2Hz,1H),7.39(t,J＝7.4Hz,2H),7.19–7.13(m,3H),7.08(d,J＝7.9Hz,2H),5.04–4.92(m,1H),4.53–4.37(m,2H),3.59(s,2H),3.42(s,2H),2.96–2.86(m,1H),2.86–2.69(m,2H),2.46(t,J＝7.2Hz,2H),2.42–2.30(m,6H),2.27(t,J＝7.6Hz,2H),2.22–2.14(m,1H),2.01–1.93(m,2H),1.84–1.73(m,2H),1.69–1.63(m,2H),1.54–1.49(m,2H),1.45–1.40(m,2H),1.38–1.32(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.16(s),172.06(s),171.32(s),169.29(s),168.77(s),166.73(s),158.44(s),157.94(s),156.44(s),155.55(s),154.34(s),143.75(s),137.84(s),136.44(s),131.15(s),129.95(s),129.86(s),127.87(s),125.25(s),123.99(s),119.47(s),119.18(s),118.47(s),115.32(s),98.25(s),58.04(s),53.25(s),52.89(s),49.36(s),47.04(s),45.56(s),41.48(s),37.78(s),33.07(s),31.52(s),29.44(s),26.81(s),26.46(s),26.12(s),25.02(s),24.85(s),22.81(s).HRMS(ESI):calcd for C
46H
50N
10O
7[M+H]
+855.3937,found 855.3910。
White solid, yield 30.6%, Mp: 134-136°C. 1 H NMR (400MHz, CDCl 3 ) δ9.43(s, 1H), 8.83(d, J=8.2Hz, 1H), 8.42(s, 1H), 7.72(t, J=7.7Hz, 1H), 7.65 (d, J=7.7Hz, 2H), 7.56(d, J=7.2Hz, 1H), 7.39(t, J=7.4Hz, 2H), 7.19–7.13(m, 3H), 7.08(d, J= 7.9Hz,2H),5.04–4.92(m,1H),4.53–4.37(m,2H),3.59(s,2H),3.42(s,2H),2.96–2.86(m,1H),2.86–2.69 (m,2H),2.46(t,J=7.2Hz,2H),2.42–2.30(m,6H),2.27(t,J=7.6Hz,2H),2.22–2.14(m,1H),2.01– 1.93(m,2H),1.84–1.73(m,2H),1.69–1.63(m,2H),1.54–1.49(m,2H),1.45–1.40(m,2H),1.38–1.32(m,2H ); 13 C NMR (100MHz, CDCl 3 ) δ172.16(s), 172.06(s), 171.32(s), 169.29(s), 168.77(s), 166.73(s), 158.44(s), 157.94( s), 156.44(s), 155.55(s), 154.34(s), 143.75(s), 137.84(s), 136.44(s), 131.15(s), 129.95(s), 129.86(s), 127.87( s), 125.25(s), 123.99(s), 119.47(s), 119.18(s), 118.47(s), 115.32(s), 98.25(s), 58.04(s), 53.25(s), 52.89( s), 49.36(s), 47.04(s), 45.56(s), 41.48(s), 37.78(s), 33.07(s), 31.52(s), 29.44(s), 26.81(s), 26.46( s), 26.12(s), 25.02(s), 24.85(s), 22.81(s). HRMS(ESI): calcd for C 46 H 50 N 10 O 7 [M+H] + 855.3937, found 855.3910.
Ⅰ-17：5-(4-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺Ⅰ-17: 5-(4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) Pyrrolidin-1-yl)butyryl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxisoindole-4 -yl)pentanamide
白色固体，产率33％，Mp:128-130℃。
1H NMR(400MHz,CDCl
3)δ9.46(s,1H),8.82(d,J＝8.4Hz,1H),8.40(s,1H),7.72(t,J＝7.8Hz,1H),7.65(d,J＝7.8Hz,2H),7.56(d,J＝7.2Hz,1H),7.39(t,J＝7.4Hz,2H),7.22–7.12(m,3H),7.08(d,J＝7.6Hz,2H),5.61–5.54(m,1H),5.06–4.85(m,1H),3.60(s,2H),3.47(s,2H),2.99(s,1H),2.93–2.87(m,1H),2.87–2.72(m,3H),2.54–2.44(m,5H),2.44–2.34(m,7H),2.20–2.13(m,1H),1.92–1.88(m,1H),1.86–1.75(m,3H),1.68– 1.63(m,2H),1.63–1.55(m,4H)；
13C NMR(100MHz,CDCl
3)δ172.10(s),171.79(s),171.05(s),169.29(s),168.61(s),166.74(s),158.59(s),157.92(s),156.31(s),155.21(s),154.24(s),144.16(s),137.77(s),136.43(s),131.16(s),129.98(s),129.90(s),127.68(s),125.26(s),124.07(s),119.56(s),119.08(s),118.49(s),115.38(s),98.58(s),58.05(s),57.60(s),55.15(s),55.03(s),53.24(s),53.20(s),52.78(s),49.38(s),45.41(s),41.49(s),37.68(s),31.51(s),30.73(s),25.71(s),23.43(s),23.36(s),23.19(s),22.74(s).HRMS(ESI):calcd for C
47H
51N
11O
7[M+H]
+882.4046,found 882.4037。
White solid, yield 33%, Mp: 128-130°C. 1 H NMR (400MHz, CDCl 3 ) δ9.46(s, 1H), 8.82(d, J=8.4Hz, 1H), 8.40(s, 1H), 7.72(t, J=7.8Hz, 1H), 7.65 (d, J=7.8Hz, 2H), 7.56(d, J=7.2Hz, 1H), 7.39(t, J=7.4Hz, 2H), 7.22–7.12(m, 3H), 7.08(d, J= 7.6Hz,2H),5.61–5.54(m,1H),5.06–4.85(m,1H),3.60(s,2H),3.47(s,2H),2.99(s,1H),2.93–2.87(m ,1H),2.87–2.72(m,3H),2.54–2.44(m,5H),2.44–2.34(m,7H),2.20–2.13(m,1H),1.92–1.88(m,1H),1.86 –1.75(m,3H),1.68–1.63(m,2H),1.63–1.55(m,4H); 13 C NMR(100MHz,CDCl 3 )δ172.10(s),171.79(s),171.05(s ),169.29(s),168.61(s),166.74(s),158.59(s),157.92(s),156.31(s),155.21(s),154.24(s),144.16(s),137.77(s ),136.43(s),131.16(s),129.98(s),129.90(s),127.68(s),125.26(s),124.07(s),119.56(s),119.08(s),118.49(s ),115.38(s),98.58(s),58.05(s),57.60(s),55.15(s),55.03(s),53.24(s),53.20(s),52.78(s),49.38(s ),45.41(s),41.49(s),37.68(s),31.51(s),30.73(s),25.71(s),23.43(s),23.36(s),23.19(s),22.74(s ). HRMS (ESI): calcd for C 47 H 51 N 11 O 7 [M+H] + 882.4046, found 882.4037.
Ⅰ-18：2-(4-(5-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺Ⅰ-18: 2-(4-(5-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) Pyrrolidin-1-yl)pentanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxinindole-4 -yl)acetamide
白色固体，产率18％，Mp:146-148℃。
1H NMR(400MHz,CDCl
3)δ11.40(d,J＝10.8Hz,1H),8.85(d,J＝8.4Hz,1H),8.36(s,1H),7.72(t,J＝7.9Hz,1H),7.65(d,J＝7.8Hz,2H),7.57(d,J＝5.6Hz,1H),7.39(t,J＝7.5Hz,2H),7.21–7.13(m,3H),7.08(d,J＝7.9Hz,2H),5.55–5.51(m,2H),4.98–4.86(m,1H),4.25–3.96(m,1H),3.90–3.53(m,3H),3.23(q,J＝17.1Hz,2H),3.00(s,2H),2.95–2.89(m,1H),2.89–2.76(m,2H),2.77–2.66(m,4H),2.64–2.49(m,4H),2.49–2.38(m,3H),2.37–2.30(m,1H),2.16–2.06(m,1H),1.76–1.66(m,4H)；
13C NMR(100MHz,CDCl
3)δ171.81(s),171.78(s),169.83(s),168.79(s),168.46(s),166.87(s),158.62(s),157.89(s),156.30(s),155.19(s),154.23(s),144.14(s),136.87(s),136.28(s),131.47(s),130.07(s),129.99(s),127.67(s),124.98(s),124.09(s),119.58(s),119.10(s),118.64(s),116.17(s),98.58(s),61.60(s),58.14(s),55.71(s),54.96(s),53.30(s),53.15(s),53.01(s),49.36(s),45.46(s),41.55(s),32.94(s),31.53(s),30.74(s),27.50(s),22.79(s),21.70(s).HRMS(ESI):calcd for C
45H
47N
11O
7[M+H]
+854.3733,found 854.3725。
White solid, yield 18%, Mp: 146-148°C. 1 H NMR (400MHz, CDCl 3 ) δ11.40(d, J=10.8Hz, 1H), 8.85(d, J=8.4Hz, 1H), 8.36(s, 1H), 7.72(t, J=7.9Hz ,1H),7.65(d,J=7.8Hz,2H),7.57(d,J=5.6Hz,1H),7.39(t,J=7.5Hz,2H),7.21–7.13(m,3H),7.08 (d,J=7.9Hz,2H),5.55–5.51(m,2H),4.98–4.86(m,1H),4.25–3.96(m,1H),3.90–3.53(m,3H),3.23(q ,J＝17.1Hz,2H),3.00(s,2H),2.95–2.89(m,1H),2.89–2.76(m,2H),2.77–2.66(m,4H),2.64–2.49(m,4H ),2.49–2.38(m,3H),2.37–2.30(m,1H),2.16–2.06(m,1H),1.76–1.66(m,4H); 13 C NMR(100MHz,CDCl 3 )δ171.81 (s), 171.78(s), 169.83(s), 168.79(s), 168.46(s), 166.87(s), 158.62(s), 157.89(s), 156.30(s), 155.19(s), 154.23 (s), 144.14(s), 136.87(s), 136.28(s), 131.47(s), 130.07(s), 129.99(s), 127.67(s), 124.98(s), 124.09(s), 119.58 (s), 119.10(s), 118.64(s), 116.17(s), 98.58(s), 61.60(s), 58.14(s), 55.71(s), 54.96(s), 53.30(s), 53.15 (s), 53.01(s), 49.36(s), 45.46(s), 41.55(s), 32.94(s), 31.53(s), 30.74(s), 27.50(s), 22.79(s), 21.70 (s). HRMS (ESI): calcd for C 45 H 47 N 11 O 7 [M+H] + 854.3733, found 854.3725.
Ⅰ-19：2-(4-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺Ⅰ-19: 2-(4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) Pyrrolidin-1-yl)butyryl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxisoindole-4 -yl)acetamide
白色固体，产率25.1％，Mp:164-166℃。
1H NMR(400MHz,CDCl
3)δ11.23 (s,1H),8.95–8.80(m,1H),8.38(d,J＝7.0Hz,1H),7.73(t,J＝8.4Hz,1H),7.64(d,J＝8.6Hz,2H),7.61–7.55(m,1H),7.39(t,J＝7.9Hz,2H),7.20–7.13(m,3H),7.08(d,J＝7.7Hz,2H),5.58–5.53(m,1H),5.01–4.83(m,1H),4.19–3.86(m,1H),3.75–3.50(m,3H),3.31–3.26(m,1H),3.21–3.13(m,1H),3.12–2.97(m,2H),2.96–2.83(m,2H),2.83–2.57(m,7H),2.58–2.31(m,6H),2.19–2.08(m,1H),1.93–1.83(m,2H)；
13C NMR(100MHz,CDCl
3)δ171.81(s),171.78(s),169.83(s),168.47(s),166.87(s),158.62(s),157.89(s),156.30(s),155.19(s),154.23(s),144.14(s),136.87(s),136.28(s),131.47(s),129.99(s),129.92(s),127.72(s),124.96(s),124.09(s),119.58(s),119.12(s),119.09(s),118.64(s),116.19(s),98.59(s),61.56(s),58.05(s),55.76(s),54.92(s),53.30(s),53.18(s),53.01(s),49.36(s),45.52(s),41.55(s),32.96(s),31.47(s),30.78(s),23.48(s),21.70(s).HRMS(ESI):calcd for C
44H
45N
11O
7[M+H]
+840.3576,found 840.3568。
White solid, yield 25.1%, Mp: 164-166°C. 1 H NMR (400MHz, CDCl 3 ) δ11.23 (s, 1H), 8.95–8.80 (m, 1H), 8.38 (d, J = 7.0Hz, 1H), 7.73 (t, J = 8.4Hz, 1H) ,7.64(d,J=8.6Hz,2H),7.61–7.55(m,1H),7.39(t,J=7.9Hz,2H),7.20–7.13(m,3H),7.08(d,J=7.7 Hz,2H),5.58–5.53(m,1H),5.01–4.83(m,1H),4.19–3.86(m,1H),3.75–3.50(m,3H),3.31–3.26(m,1H), 3.21–3.13(m,1H),3.12–2.97(m,2H),2.96–2.83(m,2H),2.83–2.57(m,7H),2.58–2.31(m,6H),2.19–2.08(m ,1H),1.93–1.83(m,2H); 13 C NMR(100MHz,CDCl 3 )δ171.81(s),171.78(s),169.83(s),168.47(s),166.87(s),158.62 (s), 157.89(s), 156.30(s), 155.19(s), 154.23(s), 144.14(s), 136.87(s), 136.28(s), 131.47(s), 129.99(s), 129.92 (s), 127.72(s), 124.96(s), 124.09(s), 119.58(s), 119.12(s), 119.09(s), 118.64(s), 116.19(s), 98.59(s), 61.56 (s), 58.05(s), 55.76(s), 54.92(s), 53.30(s), 53.18(s), 53.01(s), 49.36(s), 45.52(s), 41.55(s), 32.96 (s), 31.47(s), 30.78(s), 23.48(s), 21.70(s). HRMS(ESI): calcd for C 44 H 45 N 11 O 7 [M+H] + 840.3576, found 840.3568.
Ⅰ-20：2-(4-(5-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺Ⅰ-20: 2-(4-(5-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) Piperidin-1-yl)pentanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxisoindole-4 -yl)acetamide
白色固体，产率25.1％，Mp:162-164℃。
1H NMR(400MHz,CDCl
3)δ11.34(s,1H),8.86(d,J＝8.5Hz,1H),8.37(s,1H),7.71(t,J＝7.9Hz,1H),7.64(d,J＝8.5Hz,2H),7.56(d,J＝7.2Hz,1H),7.39(t,J＝7.9Hz,2H),7.20–7.13(m,3H),7.09(d,J＝8.0Hz,2H),5.03–4.92(m,1H),4.90–4.66(m,1H),4.15–3.85(m,1H),3.79–3.61(m,3H),3.30–3.21(m,2H),3.21–3.13(m,2H),2.95–2.85(m,1H),2.83–2.74(m,2H),2.73–2.58(m,4H),2.51(s,2H),2.45–2.34(m,4H),2.26–2.16(m,2H),2.15(d,J＝10.0Hz,1H),2.01(d,J＝9.3Hz,2H),1.59–1.50(m,4H)；
13C NMR(100MHz,CDCl
3)δ172.08(s),171.83(s),169.81(s),168.77(s),168.42(s),166.92(s),158.44(s),157.88(s),156.38(s),155.46(s),153.86(s),143.53(s),136.86(s),136.25(s),131.49(s),129.96(s),129.91(s),127.92(s),124.96(s),124.01(s),119.54(s),119.09(s),118.61(s),116.19(s),98.60(s),61.63(s),57.51(s),54.05(s),53.38(s),53.15(s),52.58(s),52.27(s),49.37(s),45.51(s),41.58(s),32.73(s),31.51(s),30.62(s),30.58(s),26.14(s),23.02(s),22.70(s).HRMS(ESI):calcd for C
46H
49N
11O
7[M+H]
+868.3889,found 868.3878。
White solid, yield 25.1%, Mp: 162-164°C. 1 H NMR (400MHz, CDCl 3 ) δ11.34(s, 1H), 8.86(d, J=8.5Hz, 1H), 8.37(s, 1H), 7.71(t, J=7.9Hz, 1H), 7.64 (d, J=8.5Hz, 2H), 7.56(d, J=7.2Hz, 1H), 7.39(t, J=7.9Hz, 2H), 7.20–7.13(m, 3H), 7.09(d, J= 8.0Hz,2H),5.03–4.92(m,1H),4.90–4.66(m,1H),4.15–3.85(m,1H),3.79–3.61(m,3H),3.30–3.21(m,2H) ,3.21–3.13(m,2H),2.95–2.85(m,1H),2.83–2.74(m,2H),2.73–2.58(m,4H),2.51(s,2H),2.45–2.34(m, 4H), 2.26–2.16(m, 2H), 2.15(d, J＝10.0Hz, 1H), 2.01(d, J＝9.3Hz, 2H), 1.59–1.50(m, 4H); 13 C NMR (100MHz ,CDCl 3 )δ172.08(s), 171.83(s), 169.81(s), 168.77(s), 168.42(s), 166.92(s), 158.44(s), 157.88(s), 156.38(s) ,155.46(s),153.86(s),143.53(s),136.86(s),136.25(s),131.49(s),129.96(s),129.91(s),127.92(s),124.96(s) ,124.01(s),119.54(s),119.09(s),118.61(s),116.19(s),98.60(s),61.63(s),57.51(s),54.05(s),53.38(s) ,53.15(s),52.58(s),52.27(s),49.37(s),45.51(s),41.58(s),32.73(s),31.51(s),30.62(s),30.58(s) , 26.14(s), 23.02(s), 22.70(s). HRMS(ESI): calcd for C 46 H 49 N 11 O 7 [M+H] + 868.3889, found 868.3878.
Ⅰ-21：4-(4-(5-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺Ⅰ-21: 4-(4-(5-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) Piperidin-1-yl)pentanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxisoindole-4 -yl) butanamide
白色固体，产率26.3％，Mp:142-144℃。
1H NMR(400MHz,CDCl
3)δ9.51(s,1H),8.82(d,J＝8.5Hz,1H),8.38(s,1H),7.74–7.69(m,1H),7.65(d,J＝8.6Hz,2H),7.55(d,J＝7.3Hz,1H),7.41–7.36(m,2H),7.19–7.12(m,3H),7.08(d,J＝7.7Hz,2H),4.99–4.91(m,1H),4.82–4.72(m,1H),3.64–3.54(m,2H),3.49–3.40(m,2H),3.16–3.05(m,2H),2.95–2.85(m,1H),2.84–2.70(m,2H),2.54–2.49(m,2H),2.45–2.39(m,8H),2.38–2.33(m,3H),2.21–2.13(m,3H),2.06–1.97(m,3H),1.96–1.92(m,2H),1.61–1.55(m,4H)；
13C NMR(100MHz,CDCl
3)δ172.24(s),171.62(s),171.56(s),169.23(s),168.64(s),166.74(s),158.34(s),157.87(s),156.44(s),155.32(s),153.84(s),143.34(s),137.79(s),136.43(s),131.21(s),129.98(s),129.94(s),128.11(s),125.24(s),123.96(s),119.48(s),119.10(s),118.46(s),115.30(s),98.58(s),57.79(s),56.84(s),54.84(s),53.40(s),52.78(s),52.73(s),52.63(s),49.42(s),45.58(s),41.48(s),35.61(s),33.12(s),31.53(s),31.13(s),26.92(s),26.75(s),23.31(s),22.70(s),22.14(s).HRMS(ESI):calcd for C
48H
53N
11O
7[M+H]
+896.4202,found 896.4212。
White solid, yield 26.3%, Mp: 142-144°C. 1 H NMR (400MHz, CDCl 3 ) δ9.51(s, 1H), 8.82(d, J=8.5Hz, 1H), 8.38(s, 1H), 7.74–7.69(m, 1H), 7.65(d, J＝8.6Hz, 2H), 7.55(d, J＝7.3Hz, 1H), 7.41–7.36(m, 2H), 7.19–7.12(m, 3H), 7.08(d, J＝7.7Hz, 2H), 4.99–4.91(m,1H),4.82–4.72(m,1H),3.64–3.54(m,2H),3.49–3.40(m,2H),3.16–3.05(m,2H),2.95–2.85(m ,1H),2.84–2.70(m,2H),2.54–2.49(m,2H),2.45–2.39(m,8H),2.38–2.33(m,3H),2.21–2.13(m,3H),2.06 –1.97(m,3H),1.96–1.92(m,2H),1.61–1.55(m,4H); 13 C NMR(100MHz,CDCl 3 )δ172.24(s),171.62(s),171.56(s ),169.23(s),168.64(s),166.74(s),158.34(s),157.87(s),156.44(s),155.32(s),153.84(s),143.34(s),137.79(s ),136.43(s),131.21(s),129.98(s),129.94(s),128.11(s),125.24(s),123.96(s),119.48(s),119.10(s),118.46(s ),115.30(s),98.58(s),57.79(s),56.84(s),54.84(s),53.40(s),52.78(s),52.73(s),52.63(s),49.42(s ),45.58(s),41.48(s),35.61(s),33.12(s),31.53(s),31.13(s),26.92(s),26.75(s),23.31(s),22.70(s ), 22.14(s). HRMS(ESI): calcd for C 48 H 53 N 11 O 7 [M+H] + 896.4202, found 896.4212.
Ⅰ-22：2-(4-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧代异吲哚-4-基)乙酰胺Ⅰ-22: 2-(4-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) Piperidin-1-yl)butyryl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindole- 4-yl)acetamide
白色固体，产率28.6％，Mp:156-158℃。
1H NMR(400MHz,CDCl
3)δ11.23(s,1H),8.87(d,J＝8.4Hz,1H),8.32(s,1H),7.72(t,J＝7.9Hz,1H),7.62(d,J＝8.5Hz,2H),7.57(d,J＝7.2Hz,1H),7.39(t,J＝7.9Hz,2H),7.22–7.11(m,3H),7.08(d,J＝7.8Hz,2H),5.03–4.93(m,1H),4.89–4.78(m,1H),3.92–3.71(m,4H),3.31–3.19(m,4H),2.95–2.85(m,1H),2.85–2.74(m,2H),2.73–2.59(m,6H),2.52–2.40(m,6H),2.18–2.07(m,3H),1.98–1.89(m,2H)；
13C NMR(100MHz,CDCl
3)δ176.29(s),171.69(s),171.39(s),169.83(s),168.67(s),168.45(s),166.90(s),158.54(s),157.96(s),156.31(s),154.78(s),153.65(s),143.88(s),136.86(s),136.32(s),131.44(s),129.99(s),129.97(s),127.66(s),125.09(s),124.07(s),119.57 (s),119.08(s),118.69(s),116.15(s),98.47(s),61.69(s),57.09(s),53.40(s),53.15(s),51.98(s),50.70(s),49.33(s),45.32(s),41.57(s),31.51(s),30.89(s),30.23(s),22.71(s),21.88(s),21.70(s).HRMS(ESI):calcd for C
45H
47N
11O
7[M+H]
+854.3733,found 854.3743。
White solid, yield 28.6%, Mp: 156-158°C. 1 H NMR (400MHz, CDCl 3 ) δ11.23(s, 1H), 8.87(d, J=8.4Hz, 1H), 8.32(s, 1H), 7.72(t, J=7.9Hz, 1H), 7.62 (d, J=8.5Hz, 2H), 7.57(d, J=7.2Hz, 1H), 7.39(t, J=7.9Hz, 2H), 7.22–7.11(m, 3H), 7.08(d, J= 7.8Hz,2H),5.03–4.93(m,1H),4.89–4.78(m,1H),3.92–3.71(m,4H),3.31–3.19(m,4H),2.95–2.85(m,1H) ,2.85–2.74(m,2H),2.73–2.59(m,6H),2.52–2.40(m,6H),2.18–2.07(m,3H),1.98–1.89(m,2H); 13 C NMR( 100MHz, CDCl 3 )δ176.29(s), 171.69(s), 171.39(s), 169.83(s), 168.67(s), 168.45(s), 166.90(s), 158.54(s), 157.96(s ),156.31(s),154.78(s),153.65(s),143.88(s),136.86(s),136.32(s),131.44(s),129.99(s),129.97(s),127.66(s ),125.09(s),124.07(s),119.57(s),119.08(s),118.69(s),116.15(s),98.47(s),61.69(s),57.09(s),53.40(s ),53.15(s),51.98(s),50.70(s),49.33(s),45.32(s),41.57(s),31.51(s),30.89(s),30.23(s),22.71(s ), 21.88(s), 21.70(s). HRMS(ESI): calcd for C 45 H 47 N 11 O 7 [M+H] + 854.3733, found 854.3743.
Ⅰ-23：5-(4-(3-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺Ⅰ-23: 5-(4-(3-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) Piperidin-1-yl)propionyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxisoindole-4 -yl)pentanamide
白色固体，产率18.9％，Mp:134-136℃。
1H NMR(400MHz,CDCl
3)δ9.45(s,1H),8.84(d,J＝8.5Hz,1H),8.37(s,1H),7.73–7.69(m,1H),7.65(d,J＝8.6Hz,2H),7.55(d,J＝7.2Hz,1H),7.39(t,J＝7.9Hz,2H),7.18–7.13(m,3H),7.09(d,J＝7.8Hz,2H),5.03–4.91(m,1H),4.79(t,J＝10.8Hz,1H),3.78–3.61(m,2H),3.54–3.45(m,2H),3.17(s,2H),2.93–2.86(m,1H),2.80–2.74(m,2H),2.61–2.57(m,2H),2.55–2.50(m,2H),2.49–2.46(m,1H),2.45–2.40(m,4H),2.39–2.37(m,1H),2.37–2.26(m,4H),2.17–2.12(m,1H),2.05–1.98(m,2H),1.93–1.83(m,2H),1.81–1.76(m,2H),1.61–1.57(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.23(s),171.79(s),170.27(s),169.32(s),168.87(s),166.78(s),158.40(s),157.87(s),156.38(s),155.34(s),153.82(s),143.44(s),137.75(s),136.44(s),131.15(s),129.99(s),129.97(s),127.98(s),125.26(s),124.01(s),119.53(s),119.08(s),118.52(s),115.36(s),98.58(s),72.38(s),61.73(s),57.72(s),53.81(s),53.07(s),52.99(s),52.88(s),52.64(s),49.44(s),45.49(s),41.51(s),37.65(s),31.59(s),30.96(s),30.91(s),25.54(s),23.30(s),22.72(s).HRMS(ESI):calcd for C
47H
51N
11O
7[M+H]
+882.4046,found 882.4037。
White solid, yield 18.9%, Mp: 134-136°C. 1 H NMR (400MHz, CDCl 3 ) δ9.45(s, 1H), 8.84(d, J=8.5Hz, 1H), 8.37(s, 1H), 7.73–7.69(m, 1H), 7.65(d, J＝8.6Hz, 2H), 7.55(d, J＝7.2Hz, 1H), 7.39(t, J＝7.9Hz, 2H), 7.18–7.13(m, 3H), 7.09(d, J＝7.8Hz, 2H),5.03–4.91(m,1H),4.79(t,J＝10.8Hz,1H),3.78–3.61(m,2H),3.54–3.45(m,2H),3.17(s,2H),2.93 –2.86(m,1H),2.80–2.74(m,2H),2.61–2.57(m,2H),2.55–2.50(m,2H),2.49–2.46(m,1H),2.45–2.40(m, 4H),2.39–2.37(m,1H),2.37–2.26(m,4H),2.17–2.12(m,1H),2.05–1.98(m,2H),1.93–1.83(m,2H),1.81– 1.76(m,2H),1.61–1.57(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.23(s),171.79(s),170.27(s),169.32(s),168.87(s ),166.78(s),158.40(s),157.87(s),156.38(s),155.34(s),153.82(s),143.44(s),137.75(s),136.44(s),131.15(s ),129.99(s),129.97(s),127.98(s),125.26(s),124.01(s),119.53(s),119.08(s),118.52(s),115.36(s),98.58(s ),72.38(s),61.73(s),57.72(s),53.81(s),53.07(s),52.99(s),52.88(s),52.64(s),49.44(s),45.49(s ),41.51(s),37.65(s),31.59(s),30.96(s),30.91(s),25.54(s),23.30(s),22.72(s).HRMS(ESI):calcd for C 47 H 51 N 11 O 7 [M+H] + 882.4046, found 882.4037.
Ⅰ-24：4-(4-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺Ⅰ-24: 4-(4-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) Piperidin-1-yl)butyryl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxisoindole-4 -yl) butanamide
白色固体，产率23.9％，Mp:140-142℃。
1H NMR(400MHz,CDCl
3)δ9.53(s,1H),8.84(d,J＝8.6Hz,1H),8.37(s,1H),7.74–7.69(m,1H),7.65(d,J＝8.5Hz,2H),7.55(d,J＝7.1Hz,1H),7.39(t,J＝7.9Hz,2H),7.16(dd,J＝12.5,8.0Hz,3H),7.09(d,J＝7.9Hz,2H),5.07–4.91(m,1H),4.89–4.67(m,1H),3.68(s,1H),3.60 –3.28(m,3H),3.30–2.95(m,2H),2.95–2.86(m,1H),2.85–2.72(m,2H),2.58–2.47(m,4H),2.48–2.32(m,9H),2.33–2.19(m,3H),2.17–2.12(m,1H),2.07–2.00(m,2H),1.96–1.92(m,2H),1.87–1.84(m,2H)；
13C NMR(100MHz,CDCl
3)δ172.37(s),171.65(s),171.40(s),169.23(s),168.72(s),166.76(s),158.39(s),157.87(s),156.43(s),155.35(s),153.85(s),143.44(s),137.77(s),136.43(s),131.23(s),129.99(s),129.96(s),128.02(s),125.26(s),123.98(s),119.49(s),119.12(s),118.49(s),115.30(s),98.59(s),57.36(s),56.66(s),53.49(s),52.65(s),52.57(s),52.53(s),52.48(s),49.43(s),45.53(s),41.52(s),35.59(s),31.57(s),30.98(s),30.86(s),30.78(s),22.68(s),22.49(s),22.28(s).HRMS(ESI):calcd for C
47H
51N
11O
7[M+H]
+882.4046,found 882.4057。
White solid, yield 23.9%, Mp: 140-142°C. 1 H NMR (400MHz, CDCl 3 ) δ9.53(s, 1H), 8.84(d, J=8.6Hz, 1H), 8.37(s, 1H), 7.74–7.69(m, 1H), 7.65(d, J＝8.5Hz, 2H), 7.55(d, J＝7.1Hz, 1H), 7.39(t, J＝7.9Hz, 2H), 7.16(dd, J＝12.5, 8.0Hz, 3H), 7.09(d, J＝7.9Hz,2H),5.07–4.91(m,1H),4.89–4.67(m,1H),3.68(s,1H),3.60–3.28(m,3H),3.30–2.95(m,2H) ,2.95–2.86(m,1H),2.85–2.72(m,2H),2.58–2.47(m,4H),2.48–2.32(m,9H),2.33–2.19(m,3H),2.17–2.12( m,1H),2.07–2.00(m,2H),1.96–1.92(m,2H),1.87–1.84(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.37(s),171.65( s), 171.40(s), 169.23(s), 168.72(s), 166.76(s), 158.39(s), 157.87(s), 156.43(s), 155.35(s), 153.85(s), 143.44( s), 137.77(s), 136.43(s), 131.23(s), 129.99(s), 129.96(s), 128.02(s), 125.26(s), 123.98(s), 119.49(s), 119.12( s), 118.49(s), 115.30(s), 98.59(s), 57.36(s), 56.66(s), 53.49(s), 52.65(s), 52.57(s), 52.53(s), 52.48( s), 49.43(s), 45.53(s), 41.52(s), 35.59(s), 31.57(s), 30.98(s), 30.86(s), 30.78(s), 22.68(s), 22.49( s), 22.28(s). HRMS(ESI): calcd for C 47 H 51 N 11 O 7 [M+H] + 882.4046, found 882.4057.
实验例13：目标化合物对不同肿瘤细胞的生长抑制活性测定实验Experimental Example 13: Determination of the Growth Inhibitory Activity of the Target Compound on Different Tumor Cells
实验材料和仪器：淋巴瘤细胞株Jeko-1、K562、HEL(中国科学院细胞库)，1640培养基购买自以色列Biological Industries公司、PBS磷酸盐缓冲剂(粉剂)购买自北京鼎国昌盛生物技术有限责任公司，青霉素-链霉素混合液(100×双抗)购买自美国Genview公司，泊洛沙姆(F-127)购买自Amresco，细胞增殖及毒性检测试剂盒(Cell Counting Kit-8，CCK8)购买自大连美仑生物技术有限公司，胎牛血清(FBS)购买自美国Gibco公司，冷冻离心机购买自德国Eppendorf公司，Infinite F50吸收光酶标仪购买自奥地利Tecan公司，细胞培养箱购买自日本Panasonic公司(型号：MCO-170AICUVL-PC)。Experimental materials and instruments: Lymphoma cell lines Jeko-1, K562, HEL (Cell Bank of Chinese Academy of Sciences), 1640 medium was purchased from Israel Biological Industries, PBS phosphate buffer (powder) was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. Responsible company, penicillin-streptomycin mixed solution (100× double antibody) was purchased from American Genview Company, poloxamer (F-127) was purchased from Amresco, cell proliferation and toxicity detection kit (Cell Counting Kit-8, CCK8 ) was purchased from Dalian Meilun Biotechnology Co., Ltd., fetal bovine serum (FBS) was purchased from Gibco Company of the United States, the refrigerated centrifuge was purchased from Eppendorf Company of Germany, the Infinite F50 absorbing optical microplate reader was purchased from Tecan Company of Austria, and the cell culture box was purchased from Japan Panasonic Corporation (Model: MCO-170AICUVL-PC).
实验方法：取对数生长期细胞，1000rpm转速离心5min后，弃去上层培养基，用含20％FBS的1640培养基将离心后的细胞稀释吹打成均匀的细胞悬液，根据细胞数4×10
4/孔计算出所需的细胞数，按照100μL/孔将细胞悬液接种于96孔培养板中进行接种，同时设立100％细胞对照加入100μL细胞悬液和100μL空白培养基，空白对照加入200μL空白培养基。随后不同浓度化合物的培养基(F127≤1％)分别加入对应的96孔板中，每个浓度设置4个复孔，完毕后，将96孔板放置37℃，5％CO
2恒温培养箱中孵育48h。孵育48h之后，避光下向每孔加入20μL的CCK-8溶液，锡纸避光，置于培养箱内培养0.5～4h，2h后用Infinite F50吸收光酶标仪在波长为450nm条件下测定化合物的吸光度值。使用GraphPad Prism 6.01软件，以抑制率为纵坐标，化合物的浓度对数为横坐标， 拟合曲线，计算化合物的IC
50值。
Experimental method: take the cells in the logarithmic growth phase, centrifuge at 1000rpm for 5min, discard the upper medium, and dilute the centrifuged cells with 1640 medium containing 20% FBS to form a uniform cell suspension, according to the number of cells 4 ×10 4 /well Calculate the required number of cells, inoculate the cell suspension in a 96-well culture plate at 100 μL/well, and set up a 100% cell control at the same time Add 100 μL of cell suspension and 100 μL of blank medium, blank control Add 200 μL blank medium. Then the medium of different concentrations of compounds ( F127≤1 %) was added to the corresponding 96-well plate, and 4 replicate wells were set for each concentration. Incubate for 48h. After incubating for 48 hours, add 20 μL of CCK-8 solution to each well in the dark, protect from light with tin foil, and incubate in the incubator for 0.5 to 4 hours. After 2 hours, use the Infinite F50 absorption microplate reader to measure the compound at a wavelength of 450 nm. absorbance value. Using GraphPad Prism 6.01 software, with the inhibition rate as the ordinate and the logarithm of the concentration of the compound as the abscissa, curve fitting was used to calculate the IC 50 value of the compound.
实验结果：目标化合物的细胞生长抑制活性结果如表1所示。Experimental results: the results of the cytostatic activity of the target compounds are shown in Table 1.
表1.目标化合物对不同肿瘤细胞的生长抑制活性结果Table 1. Results of growth inhibitory activity of target compounds on different tumor cells
IBN：依鲁替尼(Ibrutinib)IBN: Ibrutinib
由表1可知，大部分化合物对三株细胞系具有一定的生长抑制作用，有的化合物对肿瘤细胞的生长抑制作用优于阳性对照药IBN，其中化合物Ⅰ-7、Ⅰ-8、Ⅰ-10、Ⅰ-16、Ⅰ-17、Ⅰ-18、Ⅰ-21、Ⅰ-23和Ⅰ-24对Jeko-1细胞株的抑制率均在75％以上，IBN的抑制率为64％；进一步测定化合物对肿瘤细胞的半数生长抑制浓度(IC
50值)，结果显示化合物Ⅰ-7、Ⅰ-21、Ⅰ-23和Ⅰ-24抑制Jeko-1细胞的IC
50值分别为4.6μM、4.1μM、3.6μM和4.2μM，阳性对照药IBN的IC
50值为4.7μM；化合物Ⅰ-21和Ⅰ-23对K562细胞的半数生长抑制浓度(IC
50值)分别为8μM和7μM，阳性对照药IBN的IC
50值为10μM；化合物Ⅰ-21和Ⅰ-23对HEL细胞的生长抑制作用是阳性对照药IBN两倍(IC
50值分别为16μM、15μM和31μM)。
It can be seen from Table 1 that most of the compounds have certain growth inhibitory effects on the three cell lines, and some compounds have better growth inhibitory effects on tumor cells than the positive control drug IBN, among which compounds Ⅰ-7, Ⅰ-8, Ⅰ-10 , Ⅰ-16, Ⅰ-17, Ⅰ-18, Ⅰ-21, Ⅰ-23 and Ⅰ-24 had an inhibitory rate of over 75% on Jeko-1 cell line, and the inhibitory rate of IBN was 64%. The half-inhibitory concentration (IC 50 value) of tumor cells, the results showed that the IC 50 values of compound Ⅰ-7, Ⅰ-21, Ⅰ-23 and Ⅰ-24 inhibited Jeko-1 cells were 4.6 μM, 4.1 μM, 3.6 μM, respectively μM and 4.2μM, the IC 50 value of the positive control drug IBN was 4.7μM; the half-inhibitory concentration (IC 50 value) of compound Ⅰ-21 and Ⅰ-23 on K562 cells was 8 μM and 7 μM, respectively, and the IC 50 value of the positive control drug IBN The 50 value was 10 μM; the growth inhibitory effect of compounds Ⅰ-21 and Ⅰ-23 on HEL cells was twice that of the positive control drug IBN (IC 50 values were 16 μM, 15 μM and 31 μM, respectively).
实验例14：目标化合物对B细胞淋巴瘤细胞株Jeko-1的蛋白质印迹法实验Experimental example 14: Western blot experiment of the target compound on the B-cell lymphoma cell line Jeko-1
实验材料和仪器：蛋白裂解液-Pierce RIPA buffer、蛋白酶抑制剂-Protease&phosohatease inhibitor cocktail、BCA蛋白浓度测定试剂盒、Mini-protean TGXTM Gels、10×电泳缓冲液-10×Tris/Glycine/SDS Buffer、10×转膜缓冲液-10×Transfer buffer、牛血清白蛋白-Bovine serum albumin,BSA、20×TBST、吐温20-Tween-20、脱脂奶粉-Nonfat milk、硝酸纤维素杂交膜-nitrocellulose membrane；NC membrane、预染蛋白Marker-Rageruler prestained protein ladder、HRP标记山羊抗小鼠IgG、HRP标记山羊抗兔IgG、显影液、定影液、室温摇shaker、低温离心机、其他电泳和转膜相关装置。Experimental materials and instruments: protein lysate-Pierce RIPA buffer, protease inhibitor-Protease&phosohatease inhibitor cocktail, BCA protein concentration determination kit, Mini-protean TGXTM Gels, 10×electrophoresis buffer-10×Tris/Glycine/SDS Buffer, 10 ×Transfer buffer-10×Transfer buffer, bovine serum albumin-Bovine serum albumin,BSA, 20×TBST, Tween 20-Tween-20, skimmed milk powder-Nonfat milk, nitrocellulose hybrid membrane-nitrocellulose membrane; NC Membrane, pre-stained protein Marker-Rageruler prestained protein ladder, HRP-labeled goat anti-mouse IgG, HRP-labeled goat anti-rabbit IgG, developing solution, fixing solution, room temperature shaker, low-temperature centrifuge, other electrophoresis and membrane transfer related devices.
实验方法：(1)处理细胞：取对数生长期Jeko-1细胞，接种于T25培养瓶，细胞数为4×10
6/瓶，加入不同浓度的化合物(DMSO≤1‰)，将培养瓶置于37℃(5％CO
2)的恒温培养箱中培养一定时间。(2)提取蛋白：收集Jeko-1细胞，PBS洗涤，离心后加入蛋白裂解液，使细胞与裂解液均匀混合，反应40min。裂解反应完毕后，吸取上清液即所需蛋白样品，使用BSA蛋白定量法测定蛋白浓度，-80℃低温保存。(3)SDS-PAGE电泳：取梯度分离胶，每组取20μg蛋白样品进行上样，同时取蛋白marker上样。(4)转膜：将定性滤纸和PVDF膜一 起浸泡在电转液中；分开取出电泳好的玻璃板，后将SDS-PAGE胶移至电转液；将凝胶和PVDF夹在滤纸中间，浸泡在转移装置的电转液中。(5)封闭：将含目标蛋白的膜放置5％脱脂奶粉溶液中以封闭非特异结合。(6)抗体孵育：加入一抗4℃过夜；TBS-T洗涤一抗孵育后的PVDF膜；加入HRP标记的二抗以结合一抗；TBS-T洗涤二抗孵育后的PVDF膜。(7)蛋白质印迹法结果：使用化学发光法检测，经X胶片曝光显影，保存电脑图片，并用Image J软件将灰度值定量化。(8)结果分析：以内参β-actin灰度值作对照，以校正误差，计算目标蛋白的相对含量。
Experimental method: (1) Treatment of cells: Take Jeko-1 cells in the logarithmic growth phase, inoculate them in T25 culture flasks, the number of cells is 4×10 6 /bottle, add different concentrations of compounds (DMSO≤1‰), and inoculate the culture flasks Place them in a constant temperature incubator at 37°C (5% CO 2 ) for a certain period of time. (2) Protein extraction: Collect Jeko-1 cells, wash with PBS, centrifuge, add protein lysate, mix cells and lyse evenly, and react for 40 minutes. After the cleavage reaction is completed, draw the supernatant, that is, the desired protein sample, use the BSA protein quantification method to determine the protein concentration, and store it at -80°C. (3) SDS-PAGE electrophoresis: take gradient separation gel, take 20 μg protein samples for each group to load, and take protein markers to load at the same time. (4) Membrane transfer: Soak the qualitative filter paper and PVDF membrane together in the electrophoresis solution; take out the electrophoresed glass plate separately, and then transfer the SDS-PAGE gel to the electrotransfer solution; sandwich the gel and PVDF between the filter paper and soak in the electrophoresis solution. In the electroporation fluid of the transfer device. (5) Blocking: the membrane containing the target protein is placed in 5% skimmed milk powder solution to block non-specific binding. (6) Antibody incubation: add the primary antibody overnight at 4°C; TBS-T washes the PVDF membrane after the primary antibody incubation; adds HRP-labeled secondary antibody to bind the primary antibody; TBS-T washes the PVDF membrane after the secondary antibody incubation. (7) The results of Western blot method: detected by chemiluminescence, exposed and developed by X film, saved as computer pictures, and quantified the gray value with Image J software. (8) Analysis of the results: The gray value of the internal reference β-actin was used as a control to correct the error and calculate the relative content of the target protein.
实验结果：Ⅰ系列目标终产物中，部分化合物可有效降解Jeko-1细胞的BTK蛋白，化合物Ⅰ-6、Ⅰ-7、Ⅰ-8、Ⅰ-10、Ⅰ-11、Ⅰ-14、Ⅰ-15和Ⅰ-16在15μM浓度时BTK蛋白表达明显下降，且在化合物浓度为5μM条件下，化合物Ⅰ-7、Ⅰ-8和Ⅰ-10也能显著下调BTK蛋白；此外，化合物Ⅰ-17、Ⅰ-18、Ⅰ-21、Ⅰ-23和Ⅰ-24在浓度为5μM时明显下调BTK蛋白，在化合物浓度为0.5μM时，化合物Ⅰ-21和Ⅰ-23即能显著下调BTK蛋白(图1)。进一步测定化合物Ⅰ-7、Ⅰ-21和Ⅰ-23在不同浓度和不同时间下对多珠B细胞淋巴瘤细胞BTK蛋白的降解作用，结果表明：化合物Ⅰ-7、Ⅰ-21和Ⅰ-23对B细胞淋巴瘤细胞珠Jeko-1显示浓度依赖性和时间依赖性地显著降解BTK蛋白。在化合物浓度为5μM时处理Jeko-1细胞24h，化合物Ⅰ-7对BTK蛋白最大降解量(D
max)为72.84％，化合物Ⅰ-21对BTK蛋白最大降解量(D
max)为91.86％，化合物Ⅰ-23对BTK蛋白最大降解量(D
max)为94.44％；化合物Ⅰ-7、化合物Ⅰ-21和化合物Ⅰ-23对BTK的半数降解浓度(DC
50)分别为0.45μM、0.25μM和0.10μM。在相同实验条件下，对照药依鲁替尼和泊马度胺均未显示出对BTK蛋白的降解作用。
Experimental results: Among the target end products of series I, some compounds can effectively degrade the BTK protein of Jeko-1 cells, compounds I-6, I-7, I-8, I-10, I-11, I-14, I- 15 and Ⅰ-16 significantly decreased the expression of BTK protein at the concentration of 15 μM, and at the compound concentration of 5 μM, compounds Ⅰ-7, Ⅰ-8 and Ⅰ-10 could also significantly down-regulate BTK protein; in addition, compounds Ⅰ-17, Ⅰ-18, Ⅰ-21, Ⅰ-23 and Ⅰ-24 significantly down-regulated BTK protein when the concentration was 5 μM, and when the compound concentration was 0.5 μM, compounds Ⅰ-21 and Ⅰ-23 could significantly down-regulate BTK protein (Figure 1 ). The degradation effects of compounds Ⅰ-7, Ⅰ-21 and Ⅰ-23 on the BTK protein of multibead B-cell lymphoma cells were further determined at different concentrations and at different times. The results showed that: compounds Ⅰ-7, Ⅰ-21 and Ⅰ-23 B-cell lymphoma cell beads Jeko-1 showed concentration-dependent and time-dependent significant degradation of BTK protein. Jeko-1 cells were treated for 24 hours at a compound concentration of 5 μM, and the maximum degradation (D max ) of compound Ⅰ-7 to BTK protein was 72.84%, and the maximum degradation (D max ) of compound Ⅰ-21 to BTK protein was 91.86%. The maximum degradation capacity (D max ) of Ⅰ-23 to BTK protein was 94.44%; the half-degradation concentration (DC 50 ) of compound Ⅰ-7, compound Ⅰ-21 and compound Ⅰ-23 to BTK were 0.45 μM, 0.25 μM and 0.10 μM. Under the same experimental conditions, the control drugs ibrutinib and pomalidomide did not show any degradation effect on BTK protein.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改或替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still understand the foregoing embodiments The technical solutions described are modified or replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
Claims (9)
- 一种降解BTK蛋白的化合物，其特征在于，结构如通式Ⅰ所示：A compound for degrading BTK protein, characterized in that the structure is as shown in general formula I:
其中，X选自亚甲基、吡咯烷基、苄基、哌啶基、吡啶基、嘧啶基、咪唑基或噁二唑基；m选自1～4；n选自1～5。Wherein, X is selected from methylene, pyrrolidinyl, benzyl, piperidinyl, pyridyl, pyrimidinyl, imidazolyl or oxadiazolyl; m is selected from 1-4; n is selected from 1-5. - 根据权利要求1所述的一种降解BTK蛋白的化合物，其特征在于，X为哌啶基时，哌啶基为哌啶基的碳连接端与吡唑的氮连接端相连；X为吡咯烷基时，吡咯烷基为
吡咯烷基的碳连接端与吡唑的氮连接端相连；m选自1、2、3、4；n选自1、3、4、5。 A kind of compound that degrades BTK protein according to claim 1, is characterized in that, when X is piperidinyl, piperidinyl is
The carbon connection end of piperidinyl is connected with the nitrogen connection end of pyrazole; when X is pyrrolidinyl, pyrrolidinyl isThe carbon connection end of pyrrolidinyl is connected with the nitrogen connection end of pyrazole; m is selected from 1, 2, 3, 4; n is selected from 1, 3, 4, 5. - 根据权利要求1～2中任一项所述的一种降解BTK蛋白的化合物，其特征在于，选自下列化合物：A compound for degrading BTK protein according to any one of claims 1 to 2, characterized in that it is selected from the following compounds:2-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-1)；2-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl)piperazine-1 -yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide (I-1);4-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-2)；4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)butanamide (I-2);5-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-3)；5-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide (I-3);6-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺(Ⅰ-4)；6-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propionyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide (I-4);2-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-5)；2-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide (I-5);4-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-6)；4-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyramide (I-6);5-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪 -1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-7)；5-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide (Ⅰ-7);6-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺(Ⅰ-8)；6-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyryl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide (I-8);2-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-9)；2-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide (I-9);4-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-10)；4-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyramide (I-10);5-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-11)；5-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide (I-11);6-(4-(5-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺(Ⅰ-12)；6-(4-(5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide (I-12);2-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-13)；2-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide (I-13);4-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-14)；4-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyramide (I-14);5-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-15)；5-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentanamide (I-15);6-(4-(6-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)己酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺(Ⅰ-16)；6-(4-(6-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)hexanoyl)piperazine-1 -yl)-N-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-yl)hexanamide (I-16);5-(4-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-17)；5-(4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1 -yl)butanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentyl Amide (Ⅰ-17);2-(4-(5-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-18)；2-(4-(5-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1 -yl)pentanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)ethyl Amide (Ⅰ-18);2-(4-(4-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-19)；2-(4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1 -yl)butyryl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)ethyl Amide (Ⅰ-19);2-(4-(5-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基) 戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)乙酰胺(Ⅰ-20)；2-(4-(5-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)pentanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)ethyl Amide (Ⅰ-20);4-(4-(5-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)戊酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-21)；4-(4-(5-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)pentanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyl Amide (Ⅰ-21);2-(4-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧代异吲哚-4-基)乙酰胺(Ⅰ-22)；2-(4-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)butanoyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl) Acetamide (Ⅰ-22);5-(4-(3-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丙酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)戊酰胺(Ⅰ-23)；5-(4-(3-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)propionyl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pentyl Amide (Ⅰ-23);4-(4-(4-(4-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丁酰基)哌嗪-1-基)-N-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基)丁酰胺(Ⅰ-24)。4-(4-(4-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -yl)butyryl)piperazin-1-yl)-N-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)butyl Amide (I-24).
- 一种制备权利要求1～3中任一项所述降解BTK蛋白的化合物的方法，其制备方法包括：以4-氨基吡唑并[3,4-d]嘧啶即化合物1作为起始原料，经溴代自由基反应得到中间体2；中间体2与N-Boc-4-羟基X或1-Boc-3-羟基X经光延反应，得到中间体3；中间体3脱去叔丁氧羰基保护基，得到中间体4；中间体2或4与溴取代的羧酸甲酯、乙酯经取代反应得中间体5；中间体5与4-苯氧基苯基硼酸经Suzuki-Miyaura反应得到中间体6；中间体6经过酯的水解得中间体7；中间体7与中间体14经酰胺缩合反应得通式(Ⅰ)化合物，所述合成路线如下所示：A method for preparing a compound for degrading BTK protein according to any one of claims 1 to 3, the preparation method comprising: using 4-aminopyrazolo[3,4-d]pyrimidine, namely compound 1, as a starting material, Intermediate 2 is obtained by bromo radical reaction; intermediate 2 is reacted with N-Boc-4-hydroxyl X or 1-Boc-3-hydroxyl X by Mitsunobu to obtain intermediate 3; intermediate 3 removes tert-butoxycarbonyl Protecting group, intermediate 4 is obtained; intermediate 2 or 4 is substituted with bromo-substituted methyl carboxylate, ethyl ester to obtain intermediate 5; intermediate 5 is obtained by Suzuki-Miyaura reaction with 4-phenoxyphenylboronic acid Intermediate 6; Intermediate 6 obtains Intermediate 7 through hydrolysis of ester; Intermediate 7 and Intermediate 14 obtain the compound of general formula (I) through amide condensation reaction, and the synthetic route is as follows:
其中，X、m和n如上述权利要求中所定义。wherein X, m and n are as defined in the preceding claims. - 一种药物组合物，其特征包含权利要求1～3中任一项所述的降解BTK蛋白的化合物。A pharmaceutical composition characterized by comprising the compound for degrading BTK protein according to any one of claims 1-3.
- 一种药物组合物，其特征包含权利要求1～3中任一项所述的降解BTK蛋白的化合物和至少一种药学上可接受的辅料和/或载体。A pharmaceutical composition characterized by comprising the compound for degrading BTK protein according to any one of claims 1 to 3 and at least one pharmaceutically acceptable adjuvant and/or carrier.
- 一种权利要求1～3中任一项所述的降解BTK蛋白的化合物或权利要求6所述的药物组合物在制备降解BTK蛋白的化合物中的应用。A use of the compound for degrading BTK protein according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 6 in the preparation of a compound for degrading BTK protein.
- 一种权利要求1～3中任一项所述的降解BTK蛋白的化合物或权利要求6所述的药物制剂在制备抗肿瘤药物中的应用。A use of the BTK protein-degrading compound according to any one of claims 1 to 3 or the pharmaceutical preparation according to claim 6 in the preparation of antitumor drugs.
- 一种权利要求1～3任一所述的降解BTK蛋白的化合物或权利要求6所述的药物组合物在制备治疗B细胞淋巴瘤的药物中的应用。A use of the BTK protein-degrading compound as claimed in any one of claims 1 to 3 or the pharmaceutical composition as claimed in claim 6 in the preparation of a medicament for treating B-cell lymphoma.
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| CN111662294A (en) * | 2019-03-05 | 2020-09-15 | 上海美志医药科技有限公司 | Compound with activity of degrading Btk |
| CN112010858A (en) * | 2019-05-31 | 2020-12-01 | 四川海思科制药有限公司 | BTK inhibitor, preparation method and pharmaceutical application thereof |
| WO2020239103A1 (en) * | 2019-05-31 | 2020-12-03 | 四川海思科制药有限公司 | Btk inhibitor ring derivative, preparation method therefor and pharmaceutical application thereof |
| WO2020252397A1 (en) * | 2019-06-12 | 2020-12-17 | Baylor College Of Medicine | Small molecule proteolysis-targeting chimeras and methods of use thereof |
| WO2020263935A1 (en) * | 2019-06-24 | 2020-12-30 | Dana-Farber Cancer Institute, Inc. | Hck degraders and uses thereof |
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| CN109422752B (en) * | 2017-09-03 | 2023-04-07 | 上海美志医药科技有限公司 | Compound with functions of inhibiting and degrading Bruton tyrosine protein kinase Btk activity |
| US20200121684A1 (en) * | 2018-03-10 | 2020-04-23 | Yale University | Modulators of btk proteolysis and methods of use |
| CN110835345A (en) * | 2018-08-17 | 2020-02-25 | 中国科学院上海药物研究所 | Degradation agent of cell cycle dependent kinase, preparation method thereof, pharmaceutical composition and application thereof |
| CN109369654A (en) * | 2018-11-20 | 2019-02-22 | 山东大学 | 1,3- bis- substituted-4-amino Pyrazolopyrimidines and its preparation method and application |
| CN110845500B (en) * | 2019-10-09 | 2021-05-11 | 清华大学 | Application of target BTK degradation compound in treatment of autoimmune system diseases |
| CN111662296B (en) * | 2020-06-02 | 2021-12-31 | 山东大学 | Hydroxamic acid derivative containing pyrazolopyrimidine and preparation method and application thereof |
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| CN111662294A (en) * | 2019-03-05 | 2020-09-15 | 上海美志医药科技有限公司 | Compound with activity of degrading Btk |
| CN112010858A (en) * | 2019-05-31 | 2020-12-01 | 四川海思科制药有限公司 | BTK inhibitor, preparation method and pharmaceutical application thereof |
| WO2020239103A1 (en) * | 2019-05-31 | 2020-12-03 | 四川海思科制药有限公司 | Btk inhibitor ring derivative, preparation method therefor and pharmaceutical application thereof |
| WO2020252397A1 (en) * | 2019-06-12 | 2020-12-17 | Baylor College Of Medicine | Small molecule proteolysis-targeting chimeras and methods of use thereof |
| WO2020263935A1 (en) * | 2019-06-24 | 2020-12-30 | Dana-Farber Cancer Institute, Inc. | Hck degraders and uses thereof |
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