WO2022166570A1 - 同时诱导egfr和parp蛋白降解的化合物及制备方法和应用 - Google Patents

同时诱导egfr和parp蛋白降解的化合物及制备方法和应用 Download PDF

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WO2022166570A1
WO2022166570A1 PCT/CN2022/072346 CN2022072346W WO2022166570A1 WO 2022166570 A1 WO2022166570 A1 WO 2022166570A1 CN 2022072346 W CN2022072346 W CN 2022072346W WO 2022166570 A1 WO2022166570 A1 WO 2022166570A1
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compound
acid
egfr
degradation
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李华
陈丽霞
周宜荣
霍峻锋
刘洋
顾小霞
张文波
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沈阳药科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

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  • the invention belongs to the field of medicinal chemistry, and in particular relates to a bifunctional compound that simultaneously induces the degradation of EGFR and PARP proteins based on PROTAC technology, and a preparation method and application thereof.
  • Chemotherapy resistance in advanced cancer is mediated by multiple factors including epidermal growth factor receptor (EGFR) overexpression and DNA repair enzymes (PARP).
  • EGFR epidermal growth factor receptor
  • PARP DNA repair enzymes
  • Epidermal growth factor receptor tyrosine kinase overexpression is associated with increased DNA repair activity and activation of anti-apoptotic signaling.
  • EGFR is a transmembrane protein tyrosine kinase that acts as a receptor for a member of the EGF family to trigger the epidermal growth factor receptor signaling pathway in human epithelial cells, thereby regulating cell proliferation, invasion, metastasis, and apoptosis.
  • EGFR is overexpressed in many tumors, including lung, brain, colon, and prostate cancer, and is often associated with poor prognosis.
  • EGFR inhibitors have been developed and approved by the FDA for the treatment of non-small cell lung cancer, but their efficacy may be compromised by acquired resistance caused by mutant EGFR variants. Therefore, the development of new small-molecule inhibitors or therapeutics to overcome multiple point mutations in EGFR remains an unmet medical need.
  • PARP plays a key role in DNA damage signaling and repair.
  • PARP is the cleavage substrate of caspase, a core member of apoptosis. Therefore, it plays an important role in DNA damage repair and apoptosis.
  • tumor cells In the past few decades, the research and development of cancer-targeted drugs has mainly focused on the design of single-function molecules.
  • tumor cells In cancer, tumor cells often upregulate different growth-promoting factors, which can act independently or interfere with each other intracellularly through signaling networks. Tumor cells can easily acquire drug resistance by upregulating alternative factors or switching signaling pathways that promote proliferation. Therefore, treatments targeting only a single target have limitations.
  • single-targeted drugs can also lead to reduced efficacy and reduced quality of life of patients due to side effects and tissue toxicity.
  • Combination therapy can achieve additive or synergistic effects and reduce the generation of drug resistance.
  • combination therapy improves the efficacy, often only a smaller dose of each single drug is required, thereby reducing side effects.
  • Another strategy to improve efficacy is to design a single hybrid molecule that fuses two or more pharmacophore to target two or more antitumor epitopes or targets simultaneously. These hybrid molecules generally have better efficacy and fewer side effects due to their ability to modulate multiple targets or pathways simultaneously.
  • Single-molecule dual-target or multi-target drugs are superior to single-target drugs or combination therapy in the following aspects; compared with single-target drugs, it is not easy to produce drug resistance, and compared with combined administration, it is more likely Predicted pharmacokinetics, reduced risk of drug interactions, simpler dosing regimens, improved patient compliance, fewer potential intellectual property conflicts, simpler regulatory approval process, and more. Over the past few decades, these hybrid molecules have attracted considerable interest and considerable success due to their advantages in the treatment of complex diseases, gradually becoming an alternative to combination therapy or the use of mixtures, including dual Specific antibodies and other dual-target or multi-target small molecule drugs, etc.
  • a proteolytic targeting chimera is a bifunctional small molecule in which a target protein ligand and an E3 ubiquitin ligase ligand are linked together by a linker to form a triplet compound.
  • proteolytic targeting chimeras can target specific proteins for degradation.
  • a proteolytic targeting chimera is a specific biological functional molecule, usually composed of a compound molecule that binds to a protein target, a small molecule ligand that recruits E3 ligase, and a linker. Induced by PROTACs, resulting in selective polyubiquitination of target proteins and subsequent degradation at the proteasome.
  • PROTAC Compared with traditional small molecule inhibitors, PROTAC has many advantages, including its ability to function without binding to the active site of the target protein, its ability to degrade difficult-to-drug targets, its event-driven effect and its catalytic properties, and its ability to act at lower It acts at different doses, so it has great potential especially in the development of anticancer drugs.
  • the present invention provides a bifunctional compound based on PROTAC technology that can simultaneously induce the degradation of EGFR and PARP proteins, or a stereoisomer, hydrate and pharmaceutically acceptable salt or prodrug of the compound. Also provided are the preparation method of the compound and the application of the compound in the preparation of medicaments for treating and/or preventing tumors.
  • the present invention is supported by PROTAC technology, using existing EGFR inhibitor (Gefitinib) and PARP inhibitor (Olaparib) as raw materials to synthesize EGFR recruited by different Linker lengths and different E3 ligases (CRBN- and VHL-). and PARP protein dual targeting degradation chimeric molecules (Dual Protacs).
  • amino acids are used as star-shaped linker units, through which different inhibitors and E3 ligand small molecules are connected. Amino acids are multifunctional molecules of non-toxic and harmless natural origin with very high biocompatibility.
  • various amino acid molecules also have a third reaction site. According to the different reactivity of the three reaction sites, three different small molecules can be artificially and controllably introduced on demand.
  • the primary object of the present invention is to provide a compound that simultaneously induces the degradation of EGFR and PARP proteins, which is a compound represented by formula (I) or (II) or a stereoisomer of a compound represented by formula (I) or (II) Forms, hydrates, and pharmaceutically acceptable salts or prodrugs.
  • Another object of the present invention is to provide a preparation method of the compound represented by formula (I) or (II).
  • Another object of the present invention is to provide the use of the compound represented by formula (I) or (II) in the preparation of a medicament for treating or preventing tumors.
  • A is the PARP selective inhibitor Olaparib; its structure is:
  • Gefitinib the EGFR selective inhibitor Gefitinib (gefitinib); its structure is:
  • E3 is a CRBN or VHL small molecule ligand in the E3 ubiquitin ligase complex, specifically thalidomide and its derivatives, lenalidomide and its derivatives, or pomalidomide and its derivatives; the E3 The structure is
  • X is O or S
  • Z is -alkyl, -cycloalkyl, -Cl, -F or -H;
  • G and G' are each independently -H, alkyl, -OH or -CH2 -heterocycle;
  • R 1 is -H, -D, -F, -Cl, -Br, -I, -NO 2 , -CN, -NH 2 , -OH, -CH 3 , -CH 2 F, -CHF 2 , -CF 3 , -CH2D , -CHD2 , -CD3 or -CH2CH3 ;
  • L is a connecting arm, which is an aliphatic chain, an aromatic chain, an ether chain or an amide chain; it is connected to A, B and E3 through covalent bonds, respectively, to form a compound represented by formula (I) or (II) or formula (I) Or the stereoisomer, hydrate and pharmaceutically acceptable salt or prodrug of the compound shown in (II); its structure is
  • a compound that simultaneously induces the degradation of EGFR and PARP proteins which is any one of the compounds represented by formulas (a) to (d) or a stereoisomer, hydrate and pharmaceutically acceptable salt of the compound or prodrug;
  • R 1 is -H, -D, -F, -Cl, -Br, -I, -NO 2 , -CN, -NH 2 , -OH, -CH 3 , -CH 2 F, -CHF 2 , -CF 3 , -CH2D , -CHD2 , -CD3 or -CH2CH3 ;
  • L is any one of the aforementioned structures; and 1 ⁇ n ⁇ 10.
  • a compound that simultaneously induces the degradation of EGFR and PARP proteins which is any one of the compounds represented by formulae (a') to (d') or a stereoisomer, hydrate and pharmaceutically acceptable compound of the compound salts or prodrugs;
  • a compound that simultaneously induces the degradation of EGFR and PARP proteins which is any one of the following compounds or a stereoisomer, hydrate and pharmaceutically acceptable salt or prodrug of the compound;
  • the compound that induces the degradation of EGFR and PARP proteins simultaneously contains asymmetric or chiral centers and exists in different stereoisomeric forms.
  • the present invention includes all stereoisomeric forms including, but not limited to, diastereomers, enantiomers, and atropisomers, as well as mixtures thereof, such as racemates.
  • the pharmaceutically acceptable salt of the compound that induces the degradation of EGFR and PARP proteins at the same time is the compound with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and benzenesulfonic acid.
  • the prodrug of a compound that induces the degradation of EGFR and PARP proteins at the same time refers to derivatives of compounds represented by formula (a), formula (b), formula (c) or formula (d), which themselves may have The weaker ones are not even active, but are converted to the corresponding biologically active form under physiological conditions (eg by metabolism, solvolysis or otherwise) after administration.
  • a pharmaceutical composition comprising one or more of the compound of the present invention, a stereoisomer of the compound, a hydrate of the compound and a pharmaceutically acceptable salt or prodrug of the compound; also Include pharmaceutically acceptable carriers, diluents, adjuvants, vehicles or combinations thereof.
  • the dosage form of the pharmaceutical composition is injection, tablet or capsule.
  • a preparation method of a compound for simultaneously inducing the degradation of EGFR and PARP proteins comprising the following steps:
  • Step 1 dissolving N-Boc amino acid methyl ester and propyne bromide in a solvent, adding potassium carbonate or sodium hydride as a base, and performing hydroxyl etherification protection to obtain intermediate A;
  • Step 2 The intermediate A and olaparib are subjected to amide condensation under the conditions of adding EDCI, HOBt and DIPEA, followed by deprotection, and the obtained product undergoes the same amide condensation as gefitinib to obtain intermediate B ;
  • Step 3 The intermediate B and the azide-linked E3 ligand are dissolved in a solvent, and connected by a Click reaction to obtain a Dual Protacs compound.
  • the solvent is one or a combination of DMF, dichloromethane, THF and water;
  • the amino acid is tyrosine, serine, threonine, cysteine, asparagine, Glutamine, histidine, arginine, lysine, tryptophan, aspartic acid, glutamic acid or hydroxyproline.
  • the tumor is multiple myeloma, gastric cancer, lung cancer, breast cancer, esophageal cancer, colon cancer, medulloblastoma, acute myeloid leukemia, chronic leukemia, melanoma, prostate cancer, hepatoma, renal cell tumor, Cervical, skin, ovarian, colon, glioma, thyroid or pancreatic cancer.
  • the present invention proposes the following four compounds for comparing the degradation effects.
  • Dual Protacs (compound DP-1-DP-8) synthesized by the method of the present invention and 4 single PROTACs corresponding to VHL- and CRBN-ligands (compound MP-1-MP-4) were evaluated by the protein degradation activity evaluation system
  • the results of the comparative experiments showed that the compounds DP-1-DP-8 had obvious effects on the targeted degradation of EGFR and PARP proteins, and the effect was better than that of single PROTAC.
  • the present invention designs a series of novel dual targeting degradation molecules Dual Protacs (compound DP-1-DP) with two independent inhibitor units (inhibitors of EGFR and PARP) and one E3 ligase ligand (CRBN or VHL). -8), multiple functions (eg, inhibition of EGFR phosphorylation, induction of DNA damage, and prevention of its repair) can be assembled into one molecule. Compared with the negative control, these compounds can effectively induce the E3 ligase-dependent degradation of EGFR and PARP simultaneously in pancreatic cancer cell lines and 1299 cells, and effectively inhibit the growth of cancer cells.
  • the dual-targeted degradation molecule (Dual Protacs) of the present invention is superior to single PROTAC in that, as a PROTAC molecule with dual EGFR-PARP targeting, it can simultaneously degrade EGFR and PARP proteins in cancer cells, and this method may inspire a new generation of PROTAC molecules are designed to address tumor heterogeneity and reverse chemoresistance target diversity in advanced cancers.
  • the dual targeting degradation molecules (Dual Protacs) of the present invention have advantages over combined administration in that they can effectively target and degrade EGFR and PARP proteins; similar to catalytic reactions, the effective dose of the drug is low; only binding activity is provided, which is an event Drive, different from traditional occupation drive, does not need to directly inhibit the functional activity of the target protein; the drug does not need to bind to the target protein for a long time and with high intensity.
  • the method of the present invention provides a new treatment modality for the treatment of tumors and/or other diseases mediated by EGFR and PARP.
  • Figure 1 Degradation characteristics of EGFR and PARP proteins mediated by different concentrations of DP-1 after administration in SW1990 cells for 24 hours;
  • Figure 7 Degradation effect of different concentrations of DP-8 on EGFR and PARP proteins in H1299 cells at 36h;
  • FIG. 10 In A431 cells, 36h after administration, the degradation effects of EGFR and PARP proteins mediated by DP-5, DP-6, DP-7, and DP-8 were compared.
  • a preparation method of compound DP-1 that induces the degradation of EGFR and PARP proteins at the same time comprising the following steps:
  • Step 1 Preparation of intermediate A: N-Boc tyrosine methyl ester (19) (5.90 g, 20 mmol) and 2 equivalents of propargyl bromide (20) were dissolved in 20 mL of anhydrous DMF, and 2 equivalents were added with stirring Potassium carbonate, stirred at room temperature for 6 hours, and protected by hydroxyl etherification. After the reaction was completed, 200 mL of ethyl acetate and 200 mL of saturated aqueous sodium chloride solution were added, and the layers were extracted.
  • Step 2 Intermediate A undergoes amide condensation with olaparib and gefitinib in turn to prepare intermediate B;
  • gefitinib (1) (0.64 g, 2 mmol) was dissolved in 10 mL of acetone, and under stirring conditions, successively 1.1 equivalents of bromoester (2a) and 2 equivalents of potassium carbonate were added, heated to reflux overnight, cooled to room temperature after the reaction was completed, the solid was removed by suction filtration, the filtrate was spin-dried and concentrated to obtain a crude product, which was purified by silica gel column chromatography, Compound (3a) was obtained as a white solid in 42% yield.
  • Step 3 The intermediate B is connected with the azide-linked E3 ligand to obtain compound DP-1;
  • a preparation method of compound DP-2 which induces EGFR and PARP protein degradation at the same time comprising the following steps:
  • Step 1 Preparation of intermediate A: N-Boc serine methyl ester (23) (4.10 g, 20 mmol) and 2 equivalents of propargyl bromide (20) were dissolved in 20 mL of anhydrous DMF, and the reaction was added at 0 °C with stirring 2 equivalents of sodium hydride, the reaction was naturally raised to room temperature and stirred overnight for hydroxyl etherification protection. After the reaction was completed, saturated aqueous sodium chloride solution was added dropwise to quench the reaction, 200 mL of ether was added, the layers were extracted, and the organic layer was dried over anhydrous sodium sulfate. After concentration, it was purified by silica gel column chromatography to obtain 3.61 g of yellow oily intermediate A (24) with a yield of 74%.
  • Step 2 Intermediate A undergoes amide condensation with olaparib and gefitinib in turn to prepare intermediate B;
  • Step 3 The intermediate B is connected with the azide-linked E3 ligand to obtain compound DP-2;
  • a preparation method of compounds DP-3 and DP-4 which can induce the degradation of EGFR and PARP proteins at the same time comprising the following steps:
  • Step 1 Preparation of Intermediate A: The method of Example 2 was used to prepare Intermediate A (24).
  • Step 2 Intermediate A undergoes amide condensation with olaparib and gefitinib in turn to prepare intermediate B;
  • Step 3 The intermediate B is connected with the azide-linked E3 ligand to obtain compounds DP-3 and DP-4;
  • a preparation method of compound DP-5 which induces EGFR and PARP protein degradation at the same time comprising the following steps:
  • Step 1 Preparation of Intermediate A: Intermediate A (22) was prepared by the method of Example 1;
  • Step 2 Preparation of Intermediate B: Intermediate B (27) was prepared by the method of Example 1;
  • Step 3 The intermediate B is connected with the azide-linked E3 ligand to obtain compound DP-5;
  • the prepared compound DP-5 was detected by hydrogen NMR and mass spectrometry, as follows:
  • a preparation method of compound DP-6 that induces the degradation of EGFR and PARP proteins at the same time comprising the following steps:
  • Step 1 Preparation of Intermediate A: Intermediate A (24) was prepared by the method of Example 2;
  • Step 2 Preparation of Intermediate B: Intermediate B (30) was prepared by the method of Example 2;
  • Step 3 The intermediate B is connected with the azide-linked E3 ligand to obtain compound DP-6;
  • the prepared compound DP-6 was detected by hydrogen NMR and mass spectrometry, as follows:
  • a preparation method for compounds DP-7 and DP-8 that induces EGFR and PARP protein degradation simultaneously comprising the following steps:
  • Step 1 Preparation of Intermediate A: Intermediate A (24) was prepared by the method of Example 3;
  • Step 2 Preparation of Intermediate B: Intermediate B (33b) and Intermediate B (33c) were prepared by the method of Example 3;
  • Step 3 The intermediate B is connected with the azide-linked E3 ligand to obtain compound DP-6;
  • Intermediates B (33b) and (33c) were prepared using the reaction route in Example 3, and the yield of intermediate B (33b) was 45%. Dissolve 0.02 mmol of intermediates B (33b) and (33c) with 1 equivalent of azide-linked E3 ligand (18) in 2 mL of tetrahydrofuran, and add 0.5 mL of 1 equivalent of copper sulfate and 2 equivalents of aqueous sodium ascorbate solution dropwise with stirring at room temperature. , the reaction was stirred at room temperature for 0.5 h, after the reaction was completed, 10 mL of ethyl acetate and 10 mL of saturated aqueous sodium chloride were added, respectively, and the layers were extracted and separated.
  • the prepared compound MP-1 was detected by hydrogen NMR and mass spectrometry, as follows:
  • the prepared compound MP-2 was detected by hydrogen NMR and mass spectrometry, as follows:
  • the prepared compound MP-3 was detected by hydrogen NMR and mass spectrometry, as follows:
  • the prepared compound MP-4 was detected by hydrogen NMR and mass spectrometry, as follows:
  • SW1990, H1299 or A431 cells in log phase were prepared into a cell suspension with a concentration of 2 ⁇ 10 5 /mL in fresh medium, and then added to a 6-well plate after mixing;
  • H1299 cells grown in log phase were taken, digested with trypsin, plated in a 96-well plate with a density of 5000 cells/well, and treated with different concentration gradients (0-200 ⁇ M) of DP-8, Gefitinib and Olaparib after 24 hours .
  • the number of viable cells was detected with CCK-8 at a wavelength of 450 nm 24 hours after administration.
  • IC50 values were calculated with GraphPad Prism.
  • the degradation of EGFR and PARP proteins by compounds DP-5-DP-8 was detected by Western Blot method.
  • Figure 6 after 36 hours of administration, it can be observed that the degradation activity of DP-8 is significantly stronger than that of the other three compounds, especially the degradation ability of EGFR protein.
  • the degradation ability of DP-8 on PARP was similar to that of the other three compounds, but they all had a certain degradation effect.
  • Figure 7 the degradation effects of different concentrations of DP-8 on EGFR and PARP proteins at 36h.
  • the compound DP-8 had the best degradation effect on the two proteins EGFR and PARP at 6 hours and 12 hours.
  • the H1299 cell line was used to detect the killing effect of DP-8 on cancer cells.
  • the molecular weight of DP-8 is much larger than that of olaparib and gefitinib, and it still maintains a good tumor cell killing effect, it can be seen that it is a good lead compound.
  • the dual targeted degradation compounds DP-1-DP-8 designed in the present invention can simultaneously degrade EGFR and PARP through the proteasome pathway, which is superior to MP-3 and MP-4 that can only degrade a single target protein. , can complete the role of a single drug molecule to degrade two targets, providing a new idea for the research and development of anti-tumor drugs.

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Abstract

一系列带有两个独立抑制剂单元和一个E3连接酶配基的新型双重靶向降解化合物或其在药学上可接受的盐、水合物、立体异构体或前药，及其制备方法和在制备治疗或预防肿瘤的药物中的应用。该化合物能同时有效诱导EGFR 和PARP 在胰腺癌细胞株和1299 细胞中E3 连接酶依赖性的降解，并有效抑制癌细胞的生长；能够解决肿瘤异质性和逆转化疗耐药性的晚期癌症的靶标多样性；为EGFR和PARP 介导的肿瘤和/或其他疾病的治疗，提供新的治疗方式。

Description

同时诱导EGFR和PARP蛋白降解的化合物及制备方法和应用 技术领域
本发明属于药物化学领域，具体涉及一种基于PROTAC技术的同时诱导EGFR和PARP蛋白降解的双功能化合物及其制备方法和用途。
背景技术
晚期癌症化疗耐药是由细胞表皮生长因子受体(EGFR)过表达和DNA修复酶(PARP)等多种因素介导的。细胞表皮生长因子受体酪氨酸激酶的过表达与DNA修复活性的增加和抗凋亡信号的激活有关。
EGFR是一个跨膜蛋白酪氨酸激酶，作为EGF家族成员的受体触发人类上皮细胞表皮生长因子受体信号通路，从而调节细胞增殖、侵袭、转移、凋亡。表皮生长因子受体活动增加造成过度，突变，或放大的表皮生长因子受体基因导致激活突变，如在19外显子的框内缺失和L858R突变，被归类为非小细胞肺癌的致癌因素。EGFR在许多肿瘤，包括肺、脑、结肠癌、前列腺癌中过表达，并且经常与不良预后相关。EGFR抑制剂已经被FDA开发并批准用于治疗非小细胞肺癌，但它们的疗效可能会因EGFR突变变种带来的获得性耐药性而受到影响。因此，开发新的小分子抑制剂或治疗方法来克服EGFR的多点突变仍然是一个未满足的医学需求。
PARP作为一种依赖于DNA的核酶，在DNA损伤的信号传导和修复中发挥着关键的作用。PARP又是细胞凋亡核心成员胱天蛋白酶(caspase)的切割底物。因此，它在DNA损伤修复与细胞凋亡中发挥着重要作用。
过去几十年中，癌症靶向药物的研发主要是针对单功能分子的设计。在癌症中，肿瘤细胞常常上调不同的促生长因子，这些因子可以独立发挥作用，也可以通过信号网络在细胞内相互干扰。肿瘤细胞很容易通过上调替代因子或者转换促进增值的信号通路而获得抗药性。因此，仅针对单一靶点的治疗具有局限性。
除了耐药性之外，单靶点治疗药物还会因为副作用和组织毒性等，导致疗效降低、病人生活质量下降。为了克服单靶点药物的不足，针对两个不同的与疾病发展有关途径进行联合用药，已成为一种公认的有效方法。联合用药可以得到加合或协同效应，并降低耐药性的产生，另外因为联合用药提高了疗效，常常只需各个单药的更小剂量，从而减少了副作用。
另一种提高疗效的策略是设计融合了两个或多个药效团的单一杂合分子，以同时靶向两种或两种以上的抗肿瘤表位或靶点。这些杂合分子因为能够同时调节多个靶点或通路，通常 有更好疗效并只有较少的副作用。单一分子的双靶点或多靶点药物优于单一靶点药物或联合治疗，还表现在以下几个方面；和单一靶点药物相比不容易产生抗药性，和联合给药相比有着可预测的药代动力学、减少的药物相互作用风险、更简单的给药方案、提高的患者依从性、更少的潜在知识产权矛盾、更简单的监管批准程序等。在过去数十年里，这些杂合分子因为其治疗复杂疾病方面的优势引起了人们的极大兴趣并获得了相当大的成功，逐渐成为联合治疗或使用混合物的一种替代方法，这包括双特异性抗体和其他双靶点或多靶点小分子药物等。
蛋白水解靶向嵌合体(PROTAC)是一种双功能小分子，其中目标蛋白配体和E3泛素连接酶配体通过连接臂连接在一起形成三重态化合物。作为一种潜在的治疗方法，蛋白水解靶向嵌合体(PROTACs)能够针对特定的蛋白进行降解。蛋白水解靶向嵌合体是一种特异生物功能分子，通常一个与蛋白靶点结合的化合物分子，一个招募E3连接酶的小分子配体以及linker组成。由PROTACs诱导，导致目标蛋白的选择性多聚泛素化以及随后在蛋白酶体上降解。与传统小分子抑制剂相比，PROTAC有着多种优势，包括无需与靶蛋白活性位点结合就能发挥作用、可以降解难于成药靶点、其作用是事件驱动而具有催化性质，可以在更低剂量下起作用，因此特别在抗癌药物的开发中具有巨大的潜力。
但药物联合使用存在一些问题或缺点，如，联合用药时由于制剂不合理，发生直接的物理或化学反应，导致药物作用的改变；用药品种偏多，使药物相互作用的发生率增加，影响药物疗效或毒性增加，不仅会减弱疗效，甚至可能引起严重不良反应；药理效应或毒性反应相同的药物联合应用，如各药不减量使用，就有产生药物中毒的可能；联合用药很难确定药物的最佳剂量；联合治疗还存在联合时序的问题，恰当的联合药物和合理的序贯用药才能提高疗效。
发明内容
针对上述存在的问题，本发明提供一种基于PROTAC技术的能同时诱导EGFR和PARP蛋白降解的双功能化合物或该化合物的立体异构体、水合物以及药学上可接受的盐或前药，另外还提供该化合物的制备方法和该化合物在制备治疗和/或预防肿瘤的药物中的应用。
具体的，本发明以PROTAC技术为支撑，以现有的EGFR抑制剂(Gefitinib)和PARP抑制剂(Olaparib)为原料，合成不同Linker长度和不同E3连接酶(CRBN-和VHL-)招募的EGFR和PARP蛋白的双重靶向降解嵌合分子(Dual Protacs)。本发明以氨基酸为星型链接单元(linker)，通过它连接不同的抑制剂和E3配体小分子。氨基酸是一种无毒无害的天然来源的多官能化分子，具有非常高的生物相容性。多种氨基酸分子除了常见的氨基和羧基，还存在第三个反应位点，可以根据三个反应位点反应活性不同，人为可控按需引入三个不同小分子。
本发明的首要目的在于提供一种同时诱导EGFR和PARP蛋白降解的化合物，其为如式(I)或(II)所示的化合物或式(I)或(II)所示化合物的立体异构体、水合物以及药学上可接受的盐或前药。
本发明的另一目的在于提供一种式(I)或(II)所示化合物的制备方法。
本发明的再一目的在于提供式(I)或(II)所示化合物在制备治疗或预防肿瘤的药物中的应用。
本发明的目的通过以下技术方案实现：
本发明所述的式(I)和(II)分别为：
Figure PCTCN2022072346-appb-000001
其中：
A是PARP选择性抑制剂Olaparib(奥拉帕尼)；其结构为：
Figure PCTCN2022072346-appb-000002
B是EGFR选择性抑制剂Gefitinib(吉非替尼)；其结构为：
Figure PCTCN2022072346-appb-000003
E3是E3泛素连接酶复合体中CRBN或VHL小分子配体，具体为沙利度胺及其衍生物、来那度胺及其衍生物或泊马度胺及其衍生物；所述E3的结构为
Figure PCTCN2022072346-appb-000004
其中：
W为CH 2、C＝O、SO 2、NH或N-烷基；
X为O或S；
Z为-烷基、-环烷基、-Cl、-F或-H；
G和G′各自独立地为-H、烷基、-OH或-CH 2-杂环；
R 1为-H、-D、-F、-Cl、-Br、-I、-NO 2、-CN、-NH 2、-OH、-CH 3、-CH 2F、-CHF 2、-CF 3、-CH 2D、-CHD 2、-CD 3或-CH 2CH 3；
L是连接臂，为脂肪链、芳香链、醚链或酰胺链；通过共价键分别与A、B和E3相连，共同构成式(I)或(II)所示的化合物或式(I)或(II)所示化合物的立体异构体、水合物以及药学上可接受的盐或前药；其结构为
Figure PCTCN2022072346-appb-000005
且1≤n≤10。
一种同时诱导EGFR和PARP蛋白降解的化合物，其为如式(a)～(d)所示的化合物中任一 种或该化合物的立体异构体、水合物以及药学上可接受的盐或前药；
Figure PCTCN2022072346-appb-000006
其中：
R 1为-H、-D、-F、-Cl、-Br、-I、-NO 2、-CN、-NH 2、-OH、-CH 3、-CH 2F、-CHF 2、-CF 3、-CH 2D、-CHD 2、-CD 3或-CH 2CH 3；
L为前述结构中的任一种；且1≤n≤10。
一种同时诱导EGFR和PARP蛋白降解的化合物，其为如式(a')～(d')所示的化合物中任一种或该化合物的立体异构体、水合物以及药学上可接受的盐或前药；
Figure PCTCN2022072346-appb-000007
其中，L为
Figure PCTCN2022072346-appb-000008
且1≤n≤5。
一种同时诱导EGFR和PARP蛋白降解的化合物，其为下列化合物中任一种或该化合物的立体异构体、水合物以及药学上可接受的盐或前药；
Figure PCTCN2022072346-appb-000009
Figure PCTCN2022072346-appb-000010
Figure PCTCN2022072346-appb-000011
Figure PCTCN2022072346-appb-000012
所述的一种同时诱导EGFR和PARP蛋白降解的化合物含有不对称或手性中心，以不同立体异构形式存在。本发明包括所有的立体异构形式，包括但不限于非对映异构体、对映异构体和阻转异构体以及他们的混合物，如外消旋物。
所述的一种同时诱导EGFR和PARP蛋白降解的化合物在药学上可接受的盐为该化合物与盐酸、氢溴酸、硫酸、磷酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘二磺酸、乙酸、 丙酸、乳酸、三氟乙酸、马来酸、柠檬酸、富马酸、草酸、酒石酸、苯甲酸、丙酮酸或琥珀酸形成的加成盐。
所述的一种同时诱导EGFR和PARP蛋白降解的化合物的前药是指，式(a)、式(b)、式(c)或式(d)所示化合物的衍生物，它们自身可能具有较弱的活性甚至没有活性，但是在给药后，在生理条件下(如通过代谢、溶剂分解或另外的方式)被转化成相应的生物活性形式。
一种药物组合物，其包括本发明所述的化合物、该化合物的立体异构体、该化合物的水合物及该化合物在药学上可接受的盐或前药中的一种或多种；还包括药学上可接受的载体、稀释剂、辅剂、媒介物或它们的组合。
其中，所述药物组合物的剂型为注射剂、片剂或胶囊剂。
一种同时诱导EGFR和PARP蛋白降解的化合物的制备方法，包括以下步骤：
步骤1：将N-Boc氨基酸甲酯与丙炔溴溶于溶剂中，加入碳酸钾或者氢化钠做碱，进行羟基醚化保护，得到中间体A；
步骤2：将中间体A和奥拉帕尼，在加入EDCI、HOBt以及DIPEA的条件下进行酰胺缩合，随后脱除保护，得到的产物与吉非替尼发生同样的酰胺缩合，得到中间体B；
步骤3：中间体B和叠氮连接的E3配体溶于溶剂中，通过Click反应进行连接，得到Dual Protacs化合物。
上述制备方法中，所述溶剂为DMF、二氯甲烷、THF和水中的一种或两种组合；所述的氨基酸为酪氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺、谷胺酰胺、组氨酸、精氨酸、赖氨酸、色氨酸、天冬氨酸、谷氨酸或羟基脯氨酸。
以N-Boc酪氨酸或丝氨酸甲酯为起始原料的反应路径如下：
Figure PCTCN2022072346-appb-000013
一种同时诱导EGFR和PARP蛋白降解的化合物或其立体异构体、其水合物、其在药学上可接受的盐或前药或前述的药物组合物在制备治疗和/或预防肿瘤的药物中的应用。
所述肿瘤为多发性骨髓瘤、胃癌、肺癌、乳腺癌、食管癌、结肠癌、髓母细胞瘤、急性髓细胞性白血病、慢性白血病、黑色素瘤、前列腺癌、肝细胞瘤、肾细胞瘤、宫颈癌、皮肤癌、卵巢癌、结肠癌、神经胶质瘤、甲状腺癌或胰腺癌。
为了验证本发明所述化合物对癌细胞内的EGFR和PARP蛋白的同时降解效果，本发明提出以下4个化合物，用于对比降解效果。
Figure PCTCN2022072346-appb-000014
通过蛋白降解活性评价体系评价本发明方法合成的8个Dual Protacs(化合物DP-1-DP-8)以及4个VHL-和CRBN-配体对应的单PROTAC(化合物MP-1-MP-4)对EGFR和PARP蛋白的靶向降解活性，从对比实验结果可知化合物DP-1-DP-8对EGFR和PARP蛋白靶向降解有明显效果，且其效果优于单PROTAC。
本发明的优点：
本发明设计了一系列带有两个独立抑制剂单元(EGFR和PARP的抑制剂)和一个E3连接酶配基(CRBN或VHL)的新型双重靶向降解分子Dual Protacs(化合物DP-1-DP-8)，可以将多种功能(例如抑制EGFR磷酸化、诱导DNA损伤和阻止其修复)组装到一个分子中。与阴性对照相比，这些化合物能同时有效诱导EGFR和PARP在胰腺癌细胞株和1299细胞中E3连接酶依赖性的降解，并有效抑制癌细胞的生长。
本发明的双重靶向降解分子(Dual Protacs)优于单PROTAC的特点在于，作为EGFR-PARP双重靶向的PROTAC分子，能够同时降解癌细胞内的EGFR和PARP蛋白，这种方法可能激发新一代PROTAC分子的设计，能够解决肿瘤异质性和逆转化疗耐药性的晚期癌症的靶标多样性。
本发明的双重靶向降解分子(Dual Protacs)优于联合给药的特点在于，有效地靶向并降解EGFR和PARP蛋白；类似于催化反应，药物起效剂量低；只提供结合活性，是事件驱动，区别于传统的占有驱动，不需直接抑制目标蛋白的功能活性；药物不需要与目标蛋白长时间和高强度的结合。
本发明方法为EGFR和PARP介导的肿瘤和/或其他疾病的治疗，提供新的治疗方式。
附图说明
图1在SW1990细胞中给药24h后，不同浓度DP-1介导的EGFR和PARP蛋白的降解特性；
图2在SW1990细胞中，比较给药24h后，DP-1,DP-2,DP-3，DP-4介导的EGFR和PARP蛋白的降解效果；
图3在SW1990细胞中，不同浓度(1μM,3μM,5μM)的DP-1作用6-48小时后，EGFR和PARP蛋白的降解效果；
图4 700nM的MG132对不同浓度DP-1介导的EGFR和PARP蛋白的降解的逆转作用；
图5在H1299细胞中，给药36h后，MP-3和MP-4对EGFR和PARP蛋白的降解效果；
图6在H1299细胞中，给药36h后，比较DP-5,DP-6,DP-7,DP-8介导的EGFR和PARP蛋白的降解效果；
图7在H1299细胞中，不同浓度的DP-8在36h对EGFR和PARP蛋白的降解效果；
图8在H1299细胞中，4μM的DP-8作用6-48h后，对EGFR和PARP蛋白的降解效果；
图9在H1299细胞中，1μM的MG132对不同浓度DP-8介导的EGFR和PARP蛋白降解的逆转效果；
图10在A431细胞中，给药36h后，比较DP-5,DP-6,DP-7,DP-8介导的EGFR和PARP 蛋白的降解效果。
图11在H1299细胞中，gefitinib，olaparib，DP-8介导的抗肿瘤细胞增殖效果评价。
具体实施方式
下面结合附图及实施例对本发明的上述内容做进一步的详细说明。
本发明的同时诱导EGFR和PARP蛋白降解的化合物DP-1-DP-8及单PROTAC化合物MP-1-MP-4的制备过程如下所示。
以下所有实施例中所涉及的原料及试剂均市售可得。
实施例1
一种同时诱导EGFR和PARP蛋白降解的化合物DP-1的制备方法，包括以下步骤：
步骤1：中间体A的制备：将N-Boc酪氨酸甲酯(19)(5.90g，20mmol)与2当量的丙炔溴(20)溶于20mL无水DMF中，搅拌下加入2当量碳酸钾，室温搅拌6h，进行羟基醚化保护，反应完全后加入200mL乙酸乙酯和200mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到6.50g黄色油状产物(21)，产率98％。取2mmol化合物(21)溶于30mL体积比为1:1的甲醇和四氢呋喃的混合溶剂中，搅拌条件下，加入5当量的氢氧化钠饱和水溶液，室温搅拌过夜，反应完全后酸化，浓缩后得黄色油状中间体A(22)，产率91％。
步骤2：中间体A依次与奥拉帕尼、吉非替尼发生酰胺缩合，制备中间体B；
(1)与奥拉帕尼结合：将奥拉帕尼(7)(0.73g，2mmol)和1当量的中间体A(22)溶于50mL无水二氯甲烷，在0℃搅拌下依次加入1.1当量EDCI、HOBt及2当量DIPEA，反应室温搅拌过夜，反应完全后加入50mL二氯甲烷和50mL饱和碳酸氢钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到0.60g白色固体化合物(25)，产率45％；
(2)将0.2mmol化合物(25)溶于5mL无水二氯甲烷中，搅拌条件下，加入2mL氯化氢饱和乙酸乙酯溶液，室温搅拌过夜脱除保护，反应完全后直接旋干除去溶剂，得到白色固体化合物(26)，产率92％；
(3)与吉非替尼结合：为了进行酰胺缩合，先进行羟基羧基化反应，具体为，将吉非替尼(1)(0.64g，2mmol)溶解于10mL丙酮中，搅拌条件下，依次加入1.1当量的溴代酸酯(2a)和2当量的碳酸钾，加热回流过夜，反应完全后自然冷却到室温，抽滤除去固体，滤液旋干浓缩后得到粗品，经硅胶柱层析纯化，得到白色固体化合物(3a)，产率42％。然后将1mmol化合物(3a)溶于20mL体积比1:1的甲醇和四氢呋喃混合溶剂中，搅拌条件下，加入5当量的氢氧化钠饱和水溶液，室温搅拌过夜，反应完全后酸化，浓缩后得含羧基的白色 固体产物(4a)，产率90％。将化合物(4a)(75mg，0.2mmol)和1当量化合物(26)溶于15mL无水二氯甲烷，在0℃搅拌下依次加入1.1当量EDCI、HOBt以及2当量DIPEA，室温搅拌过夜，酰胺缩合反应完全后加入20mL二氯甲烷和20mL饱和碳酸氢钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到81mg白色固体中间体B(27)，产率44％。
步骤3：中间体B和叠氮连接的E3配体连接，得到化合物DP-1；
(1)叠氮连接的E3配体的制备：将化合物(13)(0.27g，1mmol)和1当量化合物(14a)溶于5mL无水DMF中，搅拌下加入2当量DIPEA，反应85℃搅拌过夜，反应完全后加入50mL乙酸乙酯和50mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到0.11g黄色固体产物即叠氮连接的E3配体(15a)，产率29％；
(2)中间体B和叠氮连接的E3配体的连接：将中间体B(27)(18mg，0.02mmol)和1当量叠氮连接的E3配体(15a)溶于2mL四氢呋喃，室温搅拌下滴加0.5mL1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到11mg黄色固体产物DP-1，产率42％。
Figure PCTCN2022072346-appb-000015
Figure PCTCN2022072346-appb-000016
上述制备过程中得到的化合物进行核磁共振氢谱及质谱检测，具体如下：
3a.  1H NMR(400MHz,DMSO-d 6)δ9.66(brs,1H),8.48(d,J＝4.4Hz,1H),8.10(d,J＝6.8Hz,1H),7.87(s,1H),7.74(d,J＝5.0Hz,1H),7.15(td,J＝13.2,2.8Hz,1H),7.22(d,J＝3.2Hz,1H),4.94(s,2H),4.21(q,J＝7.2Hz,2H),3.96(s,3H),1.24(t,J＝7.2Hz,3H).
4a.  1H NMR(400MHz,DMSO-d 6)δ13.15(brs,1H),12.06(s,1H),8.85(s,1H),8.78(s,1H),8.12(dd,J＝6.8,2.4Hz,1H),7.86(ddd,J＝9.0,4.4,2.6Hz,1H),7.60(s,1H),7.53(t,J＝13.2Hz,1H),5.09(s,2H),4.00(s,3H).
15a.  1H NMR(400MHz,CDCl 3)δ8.31(s,1H),7.50(t,J＝8.0Hz,1H),7.11(d,J＝7.2Hz,1H),6.94(d,J＝8.4Hz,1H),6.50(t,J＝5.6Hz,1H),4.92(dd,J＝12.0,5.2Hz,1H),3.73(t,J＝5.2Hz,2H),3.68(t,J＝5.2Hz,2H),3.50(q,J＝5.6Hz,2H),3.41(t,J＝5.2Hz,2H),2.87-2.72(m,3H),2.14-2.10(m,1H).
21.  1H NMR(400MHz,CDCl 3)δ7.06(d,J＝8.4Hz,2H),6.91(d,J＝8.8Hz,2H),4.98(d,J＝8.0Hz,1H),4.67(d,J＝2.4Hz,2H),4.55(dd,J＝14.0,6.0Hz,1H),3.72(s,3H),3.03(qd,J＝14.0,6.0Hz,2H),2.53(t,J＝2.4Hz,1H),1.42(s,9H).
25.  1H NMR(400MHz,CDCl 3)δ10.76(brs,1H),8.49-8.46(m,1H),7.80-7.69(m,3H), 7.30-7.28(m,2H),7.13(t,J＝8.8Hz,2H),7.02(dd,J＝15.2,8.8Hz,1H),6.99(dd,J＝15.6,8.4Hz,2H),5.44(t,J＝8.0Hz,1H),4.84-4.71(m,1H),4.67(dd,J＝10.8,6.0Hz,2H),4.28(s,2H),3.83(brs,1H),3.60-3.35(m,3H),3.27-3.21(m,1H),3.08-3.01(m,2H),2.97(dd,J＝13.6,5.6Hz,1H),2.89(dd,J＝12.8,5.6Hz,1H),2.53(t,J＝2.4Hz,1H),1.42(s,9H).
27.  1H NMR(400MHz,CDCl 3)δ12.58(s,1H),9.50(d,J＝2.4Hz,1H),8.53(s,1H),8.25(d,J＝6.4Hz,1H),8.22(t,J＝6.8Hz,1H),8.11(dd,J＝5.2,2.0Hz,1H),7.96-7.93(m,1H),7.90-7.86(m,1H),7.84-7.77(m,3H),7.47-7.42(m,2H),7.33(d,J＝4.0Hz,1H),7.22(t,J＝7.2Hz,1H),7.11(dd,J＝11.2,6.4Hz,2H),6.81(dd,J＝11.2,6.4Hz,2H),5.01(ddd,J＝44.8,13.8,6.6Hz,1H),4.71-4.69(m,4H),4.32(s,2H),3.94(s,3H),3.56-3.44(m,7H),3.11-3.08(m,2H),2.97-2.84(m,3H).
DP-1.  1H NMR(400MHz,DMSO-d 6)δ12.58(s,1H),11.08(s,1H),9.49(s,1H),8.53(s,1H),8.25(d,J＝5.2Hz,1H),8.21(t,J＝6.8Hz,1H),8.11-8.10(m,2H),7.94(t,J＝7.2Hz,1H),7.87(t,J＝7.2Hz,1H),7.84-7.82(m,1H),7.80-7.76(m,1H),7.54(t,J＝6.0Hz,1H),7.44-7.42(m,2H),7.35(brs,1H),7.22(t,J＝6.8Hz,1H),7.13-7.07(m,3H),7.01(d,J＝6.0Hz,1H),6.84(t,J＝7.6Hz,2H),6.57(t,J＝4.8Hz,1H),5.04-4.99(m,4H),4.70(brs,2H),4.54(brs,2H),4.32(brs,2H),4.06-4.02(m,1H),3.96(s,3H),3.84(t,J＝4.0Hz,2H),3.60(t,J＝4.0Hz,2H),3.51(brs,2H),3.43(q,J＝4.0Hz,4H),3.10(brs,2H),2.94-2.81(m,4H),1.99-1.97(m,2H),1.76-1.71(m,1H).
HRMS(pos.ESI):m/z[M+H] +for C 66H 60ClF 2N 14O 12calcd:1313.4166,found:1313.4152.
实施例2
一种同时诱导EGFR和PARP蛋白降解的化合物DP-2的制备方法，包括以下步骤：
步骤1：中间体A的制备：将N-Boc丝氨酸甲酯(23)(4.10g，20mmol)与2当量的丙炔溴(20)溶于20mL无水DMF中，反应在0℃搅拌下加入2当量氢化钠，反应自然升至室温搅拌过夜，进行羟基醚化保护，反应完全后滴加饱和氯化钠水溶液猝灭反应，加入200mL乙醚，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到3.61g黄色油状中间体A(24)，产率74％。
步骤2：中间体A依次与奥拉帕尼、吉非替尼发生酰胺缩合，制备中间体B；
(1)与奥拉帕尼结合：将奥拉帕尼(7)(0.73g，2mmol)和1当量的中间体A(24)溶于50mL无水二氯甲烷，在0℃搅拌下依次加入1.1当量EDCI、HOBt及2当量DIPEA，反应室温搅拌过夜，反应完全后加入50mL二氯甲烷和50mL饱和碳酸氢钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到0.59g白色固体化合物(28)，产率50％；
(2)将0.2mmol化合物(28)溶于5mL无水二氯甲烷中，搅拌条件下，加入2mL氯化氢饱和乙酸乙酯溶液，室温搅拌过夜脱除保护，反应完全后直接旋干除去溶剂，得白色固体化合物(29)，产率90％；
(3)与吉非替尼结合：将吉非替尼(1)(0.64g，2mmol)溶解于10mL丙酮中，搅拌条件下，依次加入1.1当量的溴代酸酯(2b)和2当量的碳酸钾，加热回流过夜，反应完全后自然冷却到室温，抽滤除去固体，滤液旋干浓缩后得到粗品，经硅胶柱层析纯化，得到含有酯基的白色固体化合物(3b)，产率51％。然后将1mmol化合物(3b)溶于20mL体积比1:1的甲醇和四氢呋喃混合溶剂中，搅拌条件下，加入5当量的氢氧化钠饱和水溶液，室温搅拌过夜，反应完全后酸化，浓缩后得到含有羧基的白色固体产物(4b)，产率92％(具体反应路径见实施例1)。制备得到的化合物(4b)(81mg，0.2mmol)和1当量化合物(29)溶于15mL无水二氯甲烷，在0℃搅拌下依次加入1.1当量EDCI、HOBt以及2当量DIPEA，室温搅拌过夜，酰胺缩合反应完全后加入20mL二氯甲烷和20mL饱和碳酸氢钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到81mg白色固体中间体B(30)，产率64％。
步骤3：中间体B和叠氮连接的E3配体连接，得到化合物DP-2；
(1)叠氮连接的E3配体的制备：按照实施例1中的制备方法制备得到叠氮连接的E3配体(15a)；
(2)中间体B和叠氮连接的E3配体的连接：将中间体B(30)(18mg，0.02mmol)和1当量叠氮连接的E3配体(15a)溶于2mL四氢呋喃，室温搅拌下滴加0.5mL1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到12mg黄色固体产物DP-2，产率48％。
Figure PCTCN2022072346-appb-000017
Figure PCTCN2022072346-appb-000018
上述制备过程中得到的化合物进行核磁共振氢谱及质谱检测，具体如下：
24.  1H NMR(400MHz,CDCl 3)δ9.51(brs,1H),5.42(d,J＝8.4Hz,1H),4.50(dd,J＝5.2,3.2Hz,1H),4.19-4.18(m,2H),4.00(dd,J＝9.2,3.2Hz,1H),3.81(dd,J＝9.2,3.2Hz,1H),2.48(t,J＝2.4Hz,1H),1.46(s,9H).
28.  1H NMR(400MHz,CDCl 3)δ10.86(brs,1H),8.49-8.47(m,1H),7.79-7.71(m,3H),7.35-7.32(m,2H),7.05(t,J＝8.8Hz,2H),5.51(d,J＝8.0Hz,1H),4.83(d,J＝28.8Hz,1H),4.30(s,2H),4.15-4.11(m,2H),3.98(brs,1H),3.81-3.36(m,9H),2.44(d,J＝11.2Hz,1H),1.43(s,9H).
4b.  1H NMR(400MHz,DMSO-d 6)δ12.03(s,2H),8.84(s,1H),8.69(s,1H),8.10(dd,J＝6.8,2.4Hz,1H),7.87-7.84(m,1H),7.56(s,1H),7.53(t,J＝9.2Hz,1H),4.32(t,J＝6.4Hz,2H), 3.99(s,3H),2.46(t,J＝7.2Hz,2H),2.03(p,J＝6.8Hz,2H).
30.  1H NMR(400MHz,CDCl 3)δ12.60(s,1H),9.57(brs,1H),8.51(s,1H),8.37(d,J＝8.0Hz,1H),8.28-8.25(m,2H),8.13(dd,J＝6.8,2.4Hz,1H),7.98-7.91(m,3H),7.87-7.78(m,3H),7.40-7.36(m,1H),7.24(t,J＝9.2Hz,1H),5.00-4.90(m,1H),4.32(s,2H),4.16(s,4H),3.95(s,3H),3.63-3.53(m,8H),3.47(s,2H),3.16-3.13(m,2H),2.39(q,J＝6.8Hz,2H),2.05(q,J＝6.0Hz,2H).
DP-2.  1H NMR(400MHz,DMSO-d 6)δ12.59(s,1H),11.09(s,1H),9.52(s,1H),8.50(s,1H),8.32(d,J＝8.0Hz,1H),8.26-8.24(m,1H),8.11(dd,J＝6.8,2.4Hz,1H),8.01-7.99(m,1H),7.94-7.92(m,1H),7.88(td,J＝7.2,1.2Hz,1H),7.82-7.76(m,2H),7.53(t,J＝8.0Hz,1H),7.45-7.41(m,2H),7.35(brs,1H),7.20(brs,2H),7.08-7.02(m,2H),7.01(d,J＝7.2Hz,1H),6.56(brs,1H),5.05(dd,J＝12.8,5.2Hz,1H),4.97-4.92(m,1H),4.50(brs,4H),4.32(brs,2H),4.14-4.13(m,2H),3.94(s,3H),3.83(brs,2H),3.58-3.42(m,12H),3.12(brs,2H),2.93-2.85(m,1H),2.51-2.50(m,2H),2.49-2.36(m,2H),2.04(brs,2H),1.83-1.81(m,1H).
HRMS(pos.ESI):m/z[M+H] +for C 62H 60ClF 2N 14O 12calcd:1265.4166,found:1265.4172.
实施例3
一种同时诱导EGFR和PARP蛋白降解的化合物DP-3和DP-4的制备方法，包括以下步骤：
步骤1：中间体A的制备：采用实施例2的方法制备中间体A(24)。
步骤2：中间体A依次与奥拉帕尼、吉非替尼发生酰胺缩合，制备中间体B；
(1)与奥拉帕尼结合：将奥拉帕尼(7)(0.73g，2mmol)分别与1当量的化合物(8b)和(8c)溶于50mL无水二氯甲烷，在0℃搅拌下分别依次加入1.1当量EDCI、HOBt及2当量DIPEA，反应室温搅拌过夜，反应完全后分别加入50mL二氯甲烷和50mL饱和碳酸氢钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，分别得到白色固体化合物(9b)和(9c)，产率分别为(9b)，76％；(9c)，85％。然后将0.2mmol化合物(9b)和(9c)分别溶于5mL无水二氯甲烷中，搅拌条件下，分别加入2mL氯化氢饱和乙酸乙酯溶液，室温搅拌过夜，反应完全后直接旋干除去溶剂，分别得白色固体产物(10b)和(10c)，产率分别为(10b)，91％；(10c)，92％。取2mmol化合物(10b)和(10c)分别和1当量中间体A(24)溶于30mL无水二氯甲烷，反应在0℃搅拌下分别依次加入1.1当量EDCI、HOBt以及2当量DIPEA，反应室温搅拌过夜，反应完全后分别加入30mL二氯甲烷和30mL饱和碳酸氢钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，分别得白色固体产物(31b)和(31c)，产率分别为(31b)，61％；(31c)，76％；
(2)将0.2mmol化合物(31b)和(31c)分别溶于5mL无水二氯甲烷中，搅拌条件下，分别加入2mL氯化氢饱和乙酸乙酯溶液，室温搅拌过夜脱除保护，反应完全后直接旋干除去溶剂，分别得到白色固体化合物(32b)和(32c)，产率分别为(32b)，91％；(32c)，94％；
(3)与吉非替尼结合：采用实施例2的方法制备化合物(4b)和(4c)，得到的化合物(4c)的产率为96％。将化合物(4b)(75mg，0.2mmol)和1当量化合物(32b)、化合物(4c)(75mg，0.2mmol)和1当量化合物(32c)分别溶于15mL无水二氯甲烷，在0℃搅拌下分别依次加入1.1当量EDCI、HOBt以及2当量DIPEA，室温搅拌过夜，酰胺缩合反应完全后加入20mL二氯甲烷和20mL饱和碳酸氢钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，分别得到白色固体中间体B(33a)和(33c)，产率分别为(33a)，38％；(33c)，52％。
步骤3：中间体B和叠氮连接的E3配体连接，得到化合物DP-3和DP-4；
(1)叠氮连接的E3配体的制备：按照实施例1中的制备方法制备得到叠氮连接的E3配体(15a)和(15b)，其中化合物(15b)的产率33％；
(2)中间体B和叠氮连接的E3配体的连接：将0.02mmol中间体B(33a)和(33c)分别与1当量叠氮连接的E3配体(15a)和(15b)溶于2mL四氢呋喃，室温搅拌下分别滴加0.5mL1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后分别加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，分别得到黄色固体产物DP-3和DP-4，产率分别为DP-3，45％；DP-4，41％。
Figure PCTCN2022072346-appb-000019
上述制备过程中得到的化合物进行核磁共振氢谱及质谱检测，具体如下：
4c.  1H NMR(400MHz,DMSO-d 6)δ12.13(s,2H),8.84(s,1H),8.73(s,1H),8.12(dd,J＝6.8,2.4Hz,1H),7.89-7.86(m,1H),7.56(s,1H),7.52(t,J＝8.8Hz,1H),4.29(t,J＝6.4Hz,2H),3.98(s,3H),2.26(t,J＝7.2Hz,2H),1.81(p,J＝7.2Hz,2H),1.60(p,J＝7.2Hz,2H),1.48(p,J＝7.2Hz,2H).
9b.  1H NMR(400MHz,CDCl 3)δ10.94(brs,1H),8.49-8.46(m,1H),7.78-7.73(m,3H), 7.34-7.27(m,2H),7.05(t,J＝8.8Hz,1H),4.81(brs,1H),4.30(s,2H),4.38(brs,3H),3.57-3.56(m,2H),3.43(brs,1H),3.30(brs,2H),3.20-3.17(m,2H),2.42(t,J＝6.8Hz,1H),2.35(t,J＝6.8Hz,1H),1.88-1.81(m,2H),1.44(s,9H).
9c.  1H NMR(400MHz,CDCl 3)δ10.80(brs,1H),8.49-8.47(m,1H),7.78-7.72(m,3H),7.33(brs,2H),7.05(t,J＝8.8Hz,1H),4.60(brs,1H),4.30(s,2H),3.78(brs,3H),3.57(brs,2H),3.43(brs,1H),3.29(brs,2H),3.11(brs,2H),2.37-2.30(m,2H),1.66-1.65(m,2H),1.51-1.50(m,2H),1.44(s,9H),1.37-1.36(m,2H).
31b.  1H NMR(400MHz,DMSO-d 6)δ12.60(s,1H),8.26(d,J＝7.6Hz,1H),7.97(d,J＝7.6Hz,1H),7.90(t,J＝7.6Hz,2H),7.84(t,J＝7.6Hz,1H),7.44(brs,1H),7.37(t,J＝6.4Hz,1H),7.24(t,J＝9.2Hz,1H),6.81(t,J＝6.4Hz,1H),4.33(s,2H),4.12(d,J＝12.4Hz,2H),4.06-4.02(m,1H),3.64(brs,1H),3.58-3.46(m,5H),3.41-3.38(m,1H),3.19(brs,1H),3.14(brs,1H),3.08(brs,2H),2.33(t,J＝6.8Hz,1H),2.26(t,J＝6.8Hz,1H),1.63(q,J＝5.6Hz,1H),1.35(d,J＝24.8Hz,9H),1.24(s,2H).
31c.  1H NMR(400MHz,CDCl 3)δ11.06(d,J＝46.8Hz,1H),8.49-8.46(m,1H),7.79-7.70(m,3H),7.34-7.32(m,2H),7.05(t,J＝9.2Hz,1H),6.57(brs,1H),5.51-5.46(m,1H),4.30(s,2H),4.28-4.23(m,1H),4.19-4.11(m,2H),3.93-3.89(m,1H),3.70-3.66(m,4H),3.55(brs,2H),3.43(brs,1H),3.28(brs,4H),2.48(brs,1H),2.37(t,J＝7.2Hz,1H),2.29(t,J＝7.2Hz,1H),1.66(p,J＝7.2Hz,2H),1.55(p,J＝7.2Hz,2H),1.45(s,9H),1.38(p,J＝7.2Hz,2H).
33a.  1H NMR(400MHz,DMSO-d 6)δ12.60(s,1H),9.56(s,1H),8.51(s,1H),8.26(dd,J＝7.6,1.2Hz,1H),8.15(t,J＝7.6Hz,1H),8.13(dd,J＝7.2,2.4Hz,1H),8.04-7.99(m,1H),7.95(t,J＝8.4Hz,1H),7.88(t,J＝7.6Hz,1H),7.85-7.77(m,3H),7.46-7.42(m,2H),7.36(brs,1H),7.25-7.21(m,2H),4.43(p,J＝6.8Hz,1H),4.32(s,2H),4.15-4.11(m,4H),3.94(s,3H),3.60-3.57(m,4H),3.50-3.99(m,4H),3.30(brs,1H),3.19(brs,1H),3.13(brs,1H),3.09-3.03(m,2H),2.43-2.41(m,2H),2.31(t,J＝7.2Hz,1H),2.24(t,J＝7.2Hz,1H),2.05(p,J＝7.2Hz,2H),1.61(brs,2H).
33c.  1H NMR(400MHz,CDCl 3)δ11.91(d,J＝53.2Hz,1H),9.15(s,1H),8.58(s,1H),8.40(d,J＝7.2,1H),7.88-7.86(m,1H),7.74-7.67(m,4H),7.64-7.62(m,2H),7.30-7.28(m,2H),7.23-7.19(m,2H),7.09-6.99(m,2H),4.70-4.64(m,1H),4.25(s,2H),4.15-4.00(m,2H),3.99(brs,2H),3.94(s,3H),3.82-3.65(m,6H),3.51(brs,3H),3.38-3.21(m,6H),2.45(s,1H),2.35-2.23(m,4H),1.81(t,J＝6.8Hz,2H),1.66(t,J＝6.8Hz,2H),1.57-1.43(m,5H).
DP-3.  1H NMR(400MHz,DMSO-d 6)δ12.59(s,1H),11.10(s,1H),9.56(s,1H),8.50(s,1H), 8.25(d,J＝7.6Hz,1H),8.12(dd,J＝6.8,2.4Hz,2H),8.00-7.76(m,6H),7.53(t,J＝8.0Hz,1H),7.43(t,J＝8.8Hz,2H),7.35(brs,1H),7.24-7.19(m,2H),7.07(d,J＝8.8Hz,1H),7.01(d,J＝7.2Hz,1H),6.56(t,J＝5.6Hz,1H),5.05(dd,J＝12.8,5.2Hz,1H),4.50-4.42(m,4H),4.31(d,J＝7.6Hz,2H),4.12(t,J＝5.2Hz,2H),3.94(s,3H),3.83(brs,2H),3.58-3.57(m,5H),3.49-3.42(m,5H),3.29(brs,1H),3.17(brs,1H),3.12(brs,1H),3.05-3.04(m,2H),2.93-2.85(m,1H),2.51-2.50(m,2H),2.42(t,J＝8.0Hz,2H),2.28(t,J＝7.2Hz,1H),2.21(t,J＝7.2Hz,1H),2.03(brs,3H),1.59(brs,2H),1.23(brs,2H).
HRMS(pos.ESI):m/z[M+H] +for C 66H 67ClF 2N 15O 13calcd:1350.4694,found:1350.4681.
DP-4.  1H NMR(400MHz,CDCl 3)δ11.06(d,J＝16.08Hz,1H),10.01(s,1H),8.99(s,1H),8.59(s,1H),8.41(d,J＝8.4Hz,1H),7.85(d,J＝6.4Hz,1H),7.74-7.70(m,4H),7.59-7.57(m,2H),7.46(t,J＝8.0Hz,1H),7.33-7.28(m,3H),7.08-7.04(m,4H),6.86(dd,J＝8.8,4.4Hz,1H),6.50(d,J＝4.4Hz,1H),4.94-4.89(m,1H),4.54-4.49(m,5H),4.25(s,2H),4.02(t,J＝7.2Hz,2H),3.98(s,3H),3.84(brs,3H),3.68-3.64(m,5H),3.60(s,4H),3.52(brs,2H),3.36(brs,2H),3.28(brs,2H),3.21(brs,3H),2.80-2.70(m,2H),2.32-2.29(m,3H),2.22-2.21(m,1H),1.84(brs,2H),1.69(brs,2H),1.57-1.43(m,7H).
HRMS(pos.ESI):m/z[M+H] +for C 72H 79ClF 2N 15O 14calcd:1450.5582,found:1450.5536.
实施例4
一种同时诱导EGFR和PARP蛋白降解的化合物DP-5的制备方法，包括以下步骤：
步骤1：中间体A的制备：采用实施例1的方法制备得到中间体A(22)；
步骤2：中间体B的制备：采用实施例1的方法制备得到中间体B(27)；
步骤3：中间体B和叠氮连接的E3配体连接，得到化合物DP-5；
将中间体B(27)(18mg，0.02mmol)和1当量叠氮连接的E3配体(18)溶于2mL四氢呋喃，室温搅拌下滴加0.5mL1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到14mg黄色固体产物DP-5，产率48％。
Figure PCTCN2022072346-appb-000020
制备得到的化合物DP-5进行核磁共振氢谱及质谱检测，具体如下：
DP-5.  1H NMR(400MHz,DMSO-d 6)δ12.60(s,1H),9.54(s,1H),8.98(s,1H),8.57(t,J＝6.0Hz,1H),8.54(s,1H),8.25(d,J＝7.6Hz,2H),8.18(d,J＝7.6Hz,1H),8.11(dd,J＝6.8,2.4Hz,1H),8.02(d,J＝9.2Hz,1H),7.94(t,J＝7.2Hz,1H),7.89-7.78(m,4H),7.47-7.37(m,6H),7.26(brs,1H),7.23(t,J＝8.8Hz,1H),7.11(t,J＝8.8Hz,2H),6.86(t,J＝8.4Hz,2H),5.14(d,J＝3.2Hz,1H),5.06-4.98(m,3H),4.71(brs,2H),4.54(d,J＝9.2Hz,1H),4.46-4.41(m,2H),4.35-4.33(m,5H),4.21(dd,J＝16.0,5.2Hz,1H),3.96(s,3H),3.66-3.43(m,7H),3.11(brs,2H),2.93-2.85(m,3H),2.44(s,3H),2.30-2.15(m,2H),2.04-2.02(m,3H),0.94(s,9H).
HRMS(pos.ESI):m/z[M+H] +for C 75H 77ClF 2N 15O 11S calcd:1468.5299,found:1468.5287.
实施例5
一种同时诱导EGFR和PARP蛋白降解的化合物DP-6的制备方法，包括以下步骤：
步骤1：中间体A的制备：采用实施例2的方法制备得到中间体A(24)；
步骤2：中间体B的制备：采用实施例2的方法制备得到中间体B(30)；
步骤3：中间体B和叠氮连接的E3配体连接，得到化合物DP-6；
将中间体B(30)(17mg，0.02mmol)和1当量叠氮连接的E3配体(18)溶于2mL四氢呋喃，室温搅拌下滴加0.5mL 1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到10mg黄色固体产物DP-6，产率36％。
Figure PCTCN2022072346-appb-000021
制备得到的化合物DP-6进行核磁共振氢谱及质谱检测，具体如下：
DP-6.  1H NMR(400MHz,DMSO-d 6)δ12.60(s,1H),9.57(s,1H),8.98(s,1H),8.57(t,J＝6.0Hz,1H),8.51(s,1H),8.34(d,J＝8.0Hz,1H),8.25(d,J＝7.6Hz,1H),8.11(dd,J＝6.8,2.4Hz,1H),8.05(d,J＝13.6Hz,1H),7.99(d,J＝9.2Hz,1H),7.97-7.93(m,1H),7.88(td,J＝8.0,1.2Hz,1H),7.84-7.76(m,3H),7.46-7.35(m,7H),7.21(s,2H),5.13(d,J＝3.6Hz,1H),4.97-4.92(m,1H),4.55-4.50(m,3H),4.46-4.41(m,2H),4.32(brs,5H),4.22(dd,J＝16.0,5.6Hz,1H),4.14(d,J＝5.6Hz,2H),3.94(s,3H),3.65-3.41(m,10H),3.13(brs,2H),2.44(s,3H),2.38(d,J＝6.4Hz,2H),2.28-2.15(m,2H),2.03-2.01(m,5H),0.92(s,9H).
HRMS(pos.ESI):m/z[M+H] +for C 71H 77ClF 2N 15O 11S calcd:1420.5299,found:1420.5281.
实施例6
一种同时诱导EGFR和PARP蛋白降解的化合物DP-7和DP-8的制备方法，包括以下步骤：
步骤1：中间体A的制备：采用实施例3的方法制备得到中间体A(24)；
步骤2：中间体B的制备：采用实施例3的方法制备得到中间体B(33b)和中间体B(33c)；
步骤3：中间体B和叠氮连接的E3配体连接，得到化合物DP-6；
中间体B(33b)和(33c)采用实施例3中的反应路径制备得到，中间体B(33b)的产率45％。将0.02mmol中间体B(33b)和(33c)分别与1当量叠氮连接的E3配体(18)溶于2mL四氢呋喃，室温搅拌下分别滴加0.5mL 1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后分别加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，分别得到黄色固体产物DP-7和DP-8，产率分别为DP-7，41％；DP-4，35％。
Figure PCTCN2022072346-appb-000022
上述制备过程中得到的化合物进行核磁共振氢谱及质谱检测，具体如下：
33b.  1H NMR(400MHz,DMSO-d 6)δ12.60(s,1H),9.77(s,1H),8.54(s,1H),8.26(d,J＝7.6Hz,1H),8.14-8.10(m,1H),8.02(t,J＝4.8Hz,1H),7.97-7.95(m,1H),7.90-7.78(m,3H),7.45(t,J＝8.8Hz,2H),7.36(brs,1H),7.24(brs,1H),7.21(s,1H),4.56(brs,1H),4.33(s,2H),4.16-4.13(m,3H),3.99(brs,1H),3.95(s,3H),3.63-3.44(m,6H),3.19(brs,1H),3.13(brs,1H),3.05-2.99(m,1H),2.75(d,J＝4.4Hz,1H),2.22(t,J＝6.0Hz,2H),1.86-1.82(m,2H),1.61(t,J＝6.4Hz,2H),1.49(p,J＝7.6Hz,2H).
DP-7.  1H NMR(400MHz,DMSO-d 6)δ12.60(s,1H),10.05(brs,1H),8.98(s,1H),8.59(brs,2H),8.25(d,J＝7.6Hz,1H),8.13(d,J＝7.6Hz,1H),8.11-8.08(m,2H),8.03-7.99(m,2H),7.97-7.93(m,2H),7.89(brs,1H),7.83(d,J＝7.6Hz,1H),7.79-7.76(m,1H),7.45-7.36(m,6H),7.22(s,2H),5.15(brs,1H),4.55-4.52(m,4H),4.45-4.50(m,2H),4.34-4.30(m,5H),4.21(dd,J＝16.0,5.6Hz,1H),4.14(t,J＝6.4Hz,2H),3.99(brs,2H),3.95(s,3H),3.64-3.57(m,6H),3.51-3.46(m,2H),3.19(brs,1H),3.13(brs,1H),2.43(s,3H),2.28-2.15(m,5H),2.06-2.01(m,3H),1.83-1.81(m,3H),1.60(brs,2H),1.46(brs,2H),0.92(s,9H).
HRMS(pos.ESI):m/z[M+H] +for C 75H 84ClF 2N 16O 12S calcd:1505.5826,found:1505.5829.
DP-8.  1H NMR(400MHz,DMSO-d 6)δ12.60(s,1H),10.29(brs,2H),8.98(s,1H),8.65(s,1H),8.59(t,J＝6.0Hz,1H),8.25(d,J＝7.6Hz,1H),8.08(dd,J＝6.8,2.0Hz,1H),8.05(brs,1H),8.02-7.94(m,4H),7.88(t,J＝7.6Hz,1H),7.82(t,J＝7.6Hz,1H),7.78-7.76(m,1H),7.49(t,J＝8,8Hz,1H),7.42-7.36(m,5H),7.21-7.21(m,2H),5.15(brs,1H),4.55-4.40(m,5H),4.32(brs,5H),4.21(dd,J＝15.6,5.6Hz,1H),4.14(t,J＝5.2Hz,2H),3.96(s,3H),3.65-3.62(m,3H),3.55-3.49(m,5H),3.15(d,J＝16.4Hz,3H),3.02(brs,3H),2.43(s,3H),2.28-2.19(m,7H),2.06-2.00(m,3H),1.89-1.83(m,1H),1.82(brs,2H),1.58(brs,2H),1.44-1.34(m,7H),0.92(s,9H).
HRMS(pos.ESI):m/z[M+H] +for C 79H 92ClF 2N 16O 12S calcd:1561.6452,found:1561.6462.
对比例1
单PROTAC化合物MP-1的制备过程：
将化合物(6)(22mg，0.05mmol)和1当量叠氮连接的E3配体(15a)溶于2mL四氢呋喃，室温搅拌下滴加0.5mL1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到13mg黄色固体产物MP-1，产率32％。
Figure PCTCN2022072346-appb-000023
制备得到的化合物MP-1进行核磁共振氢谱及质谱检测，具体如下：
MP-1.  1H NMR(400MHz,DMSO-d 6)δ11.10(s,1H),9.59(s,1H),8.51(s,1H),8.42(t,J＝5.6Hz,1H),8.13(dd,J＝6.8,2.4Hz,1H),7.89(s,1H),7.82(s,1H),7.80-7.77(m,1H),7.55(t,J＝8.0Hz,1H),7.44(t,J＝9.2Hz,1H),7.20(s,1H),7.07(d,J＝8.8Hz,1H),7.02(d,J＝7.2Hz,1H),6.56(t,J＝6.0Hz,1H),5.06(dd,J＝12.8,5.6Hz,1H),4.49(t,J＝4.8Hz,2H),4.31(d,J＝5.6Hz,2H),4.15(t,J＝6.0Hz,2H),3.94(s,3H),3.80(t,J＝5.2Hz,2H),3.58(t,J＝5.2Hz,2H),3.41(dd,J＝10.8,5.2Hz,2H),2.94-2.84(m,1H),2.61-2.54(m,2H),2.36(t,J＝7.2Hz,2H),2.10-2.02(m,3H).
HRMS(pos.ESI):m/z[M+H] +for C 39H 38ClFN 10O 8calcd:829.2619,found:829.2631.
对比例2
单PROTAC化合物MP-2的制备过程：
Figure PCTCN2022072346-appb-000024
将化合物(12)(22mg，0.05mmol)和1当量叠氮连接的E3配体(15a)溶于2mL四氢呋喃，室温搅拌下滴加0.5mL1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到16mg黄色固体产物MP-2，产率39％。
制备得到的化合物MP-2进行核磁共振氢谱及质谱检测，具体如下：
MP-2.  1H NMR(400MHz,DMSO-d 6)δ12.61(s,1H),11.11(s,1H),8.26(d,J＝8.0Hz,1H),7.96(d,J＝8.0Hz,1H),7.89(td,J＝8.0,1.6Hz,1H),7.85-7.80(m,2H),7.56(dd,J＝12.8,7.6Hz,1H),7.46-7.43(m,1H),7.37(d,J＝6.4Hz,1H),7.24(t,J＝8.8Hz,1H),7.11(dd,J＝8.4,5.6Hz,1H),7.03(dd,J＝6.8,4.4Hz,1H),6.58(d,J＝4.4Hz,1H),5.07(dd,J＝12.8,5.6Hz,1H), 4.49(t,J＝4.4Hz,2H),4.34(s,2H),3.82(d,J＝3.2Hz,2H),3.62-3.61(m,4H),3.52(brs,2H),3.45(brs,2H),3.37(brs,1H),3.30(brs,1H),3.16(brs,2H),2.90-2.82(m,3H),2.72-2.54(m,4H),2.06-2.03(m,1H).
HRMS(pos.ESI):m/z[M+H] +for C 42H 42FN 10O 8calcd:833.3166,found:833.3161.
对比例3
单PROTAC化合物MP-3的制备过程：
Figure PCTCN2022072346-appb-000025
将化合物(6)(22mg，0.05mmol)和1当量叠氮连接的E3配体(18)溶于2mL四氢呋喃，室温搅拌下滴加0.5mL1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到17mg黄色固体产物MP-3，产率35％。
制备得到的化合物MP-3进行核磁共振氢谱及质谱检测，具体如下：
MP-3.  1H NMR(400MHz,CDCl 3)δ8.95(brs,1H),8.67(s,1H),8.61(s,1H),8.04(d,J＝4.4Hz,1H),7.96(brs,1H),7.88(s,1H),7.46(s,1H),7.36-7.29(m,5H),7.20(s,1H),7.14(t,J＝8.8Hz,1H),7.07(d,J＝5.6Hz,1H),6.53(d,J＝8.0Hz,1H),4.74(t,J＝4.8Hz,1H),4.59-4.47(m,4H),4.44(d,J＝8.8Hz,1H),4.38-4.34(m,2H),4.25-4.22(m,1H),4.19-4.15(m,2H),4.07(d,J＝12.0Hz,1H),3.97(s,3H),3.62(dd,J＝11.2,3.2Hz,1H),2.50(s,3H),2.45(t,J＝4.8Hz,3H),2.22-2.01(m,7H),0.93(s,9H).
HRMS(pos.ESI):m/z[M+H] +for C 48H 56ClFN 11O 7S calcd:984.3752,found:984.3759.
对比例4
单PROTAC化合物MP-4的制备过程：
Figure PCTCN2022072346-appb-000026
将化合物(12)(22mg，0.05mmol)和1当量叠氮连接的E3配体(18)溶于2mL四氢呋喃，室温搅拌下滴加0.5mL 1当量硫酸铜和2当量抗坏血酸钠水溶液，反应室温搅拌0.5h，反应完全后加入10mL乙酸乙酯和10mL饱和氯化钠水溶液，萃取分层，有机层用无水硫酸钠干燥，浓缩后经硅胶柱层析纯化，得到14mg黄色固体产物MP-4，产率36％。
制备得到的化合物MP-4进行核磁共振氢谱及质谱检测，具体如下：
MP-4.  1H NMR(400MHz,CDCl 3)δ11.33(brs,1H),8.69(s,1H),8.43(s,1H),7.78-7.71(m,4H),7.44-7.29(m,6H),7.24-7.19(m,2H),7.07-7.00(m,1H),4.78-4.72(m,1H),4.64-4.55(m,2H),4.34-4.22(m,5H),4.15(t,J＝11.2Hz,1H),3.70-3.67(m,2H),3.48(brs,2H),3.31(brs,1H),3.17(brs,1H),3.02(brs,2H),2.76(brs,2H),2.48(s,3H),2.43-2.07(m,10H),0.99(s,9H).
HRMS(pos.ESI):m/z[M+Na] +for C 51H 58FN 11NaO 7S calcd:1010.4118,found:1010.4128.
对比例5
在肿瘤细胞(SW1990、H1299或A431)中，检测化合物MP-3、MP-4及DP-1－DP-8介导的EGFR和PARP蛋白的降解。
1.实验方法和步骤
(1)将处于对数期的SW1990、H1299或A431细胞用新鲜培养基制成浓度为2×10 5/mL的细胞悬液，混匀后加入6孔板中；
(2)①待细胞贴壁后加入不同浓度的化合物(DP-1－DP-8)，各处理24小时后，离心收集细胞；
②待细胞贴壁后加入不同浓度的DP-1或DP-8，各处理6,12,24,36,48小时后，离心收集细胞；
③待细胞贴壁后，不加或加入给定浓度MG 132(蛋白酶体抑制剂)预处理细胞12小时，再加入不同浓度的DP-1或DP-8，处理36小时后，离心收集细胞；
(3)将(2)中收集的细胞以western方法，用EGFR和PARP抗体检测蛋白的降解情况；
(4)取对数期生长的H1299细胞，用胰酶消化，铺96孔板，密度5000个/孔，24小时后，用不同浓度梯度(0-200μM)的DP-8以及Gefitinib、Olaparib处理。在给药24小时后用CCK-8在450nm波长下，检测活细胞数目。用GraphPad Prism计算IC 50值。
2.实验结果
以SW1990细胞，应用Western Blot方法检测给药24小时后化合物DP-1－DP-4对EGFR和PARP两种蛋白质的降解情况。如图1所示，DP-1作用SW1990细胞2小时后EGFR和PARP的表达随着给药浓度的提高而降低。如图2所示，可知DP-1、DP-2、DP-3、DP-4均能降解EGFR和PARP，其中DP-1和DP-3对EGFR的降解能力最强，而DP-1对PARP的降解能力是4个化合物中最好的。进一步的检测化合物DP-1在SW1990细胞系中的降解动力学。如图3所示，可知5μM的DP-1在6小时的时候开始降解EGFR和PARP，到24和36小时对EGFR和PARP的降解作用最为明显。为了进一步证明化合物DP-1是通过蛋白酶体系统进行蛋白质降解，引入了蛋白酶体抑制剂MG 132(700nM)和DP-1共同作用24小时后提取蛋白质并使 用Western Blot实验检测相应蛋白质的变化。如图4所示，可知各加药浓度蛋白质的表达量均和MG 132单用组保持一致，这说明化合物DP-1的降解作用被MG 132完全逆转。
以H1299细胞系，应用Western Blot方法检测化合物DP-5－DP-8对EGFR和PARP两种蛋白质的降解情况。如图6所示，给药36h后，可以观察到DP-8的降解活性明显强于其余三个化合物，尤其是对EGFR蛋白的降解能力。DP-8对PARP的降解能力和其余3个化合物效果相差不大，但都有一定的降解作用。如图7所示，不同浓度的DP-8在36h对EGFR和PARP蛋白的降解效果。如图8所示，化合物DP-8在6小时和12小时对两种蛋白质EGFR和PARP的降解效果最好。如图9所示，同样的加入MG 132(1μM)会完全逆转DP-8的降解作用。另外如图10所示，同时也在A431细胞中发现，DP-8具有显著的降解EGFR和PARP的作用。
另外，在H1299细胞中，给药36h后，单PROTAC(化合物MP-3、MP-4)对EGFR和PARP蛋白的降解效果如图5所示。从结果可知，化合物MP-3或MP-4的降解效果没有DP-1－DP-8明显，尤其比DP-1－DP-4、DP-8效果差很多。
以H1299细胞系，检测DP-8对于癌细胞的杀伤作用，如图11所示，DP-8的IC 50＝19.92±1.08μM，优于奥拉帕尼(IC50＝35.93±1.05μM)，略差于吉非替尼(IC50＝6.56±1.07μM)。鉴于DP-8的分子量大大大于奥拉帕尼和吉非替尼，且仍然保持良好的杀伤肿瘤细胞作用，可见其是一个很好的先导化合物。
综上所述，本发明设计的双重靶向降解化合物DP-1－DP-8可以同时通过蛋白酶体途径同时降解EGFR和PARP，优于只能降解单个靶点蛋白的MP-3和MP-4，能够完成一个药物单分子降解两个靶点的作用，为抗肿瘤药物的研发提供了新的思路。

Claims (10)

  1. 一种同时诱导EGFR和PARP蛋白降解的化合物，其特征在于，其为如式(I)或(II)所示的化合物或式(I)或(II)所示化合物的立体异构体、水合物以及药学上可接受的盐或前药；
    Figure PCTCN2022072346-appb-100001
    其中：
    A是PARP选择性抑制剂Olaparib(奥拉帕尼)；其结构为：
    Figure PCTCN2022072346-appb-100002
    B是EGFR选择性抑制剂Gefitinib(吉非替尼)；其结构为：
    Figure PCTCN2022072346-appb-100003
    E3是E3泛素连接酶复合体中CRBN或VHL小分子配体，具体为沙利度胺及其衍生物、来那度胺及其衍生物或泊马度胺及其衍生物；所述E3的结构为
    Figure PCTCN2022072346-appb-100004
    其中：
    W为CH 2、C＝O、SO 2、NH或N-烷基；
    X为O或S；
    Z为-烷基、-环烷基、-Cl、-F或-H；
    G和G′各自独立地为-H、烷基、-OH或-CH 2-杂环；
    R 1为-H、-D、-F、-Cl、-Br、-I、-NO 2、-CN、-NH 2、-OH、-CH 3、-CH 2F、-CHF 2、-CF 3、-CH 2D、-CHD 2、-CD 3或-CH 2CH 3；
    L是连接臂，为脂肪链、芳香链、醚链或酰胺链；通过共价键分别与A、B和E3相连，共同构成式(I)或(II)所示的化合物或式(I)或(II)所示化合物的立体异构体、水合物以及药学上可接受的盐或前药；其结构为
    Figure PCTCN2022072346-appb-100005
    Figure PCTCN2022072346-appb-100006
    且1≤n≤10。
  2. 根据权利要求1所述的一种同时诱导EGFR和PARP蛋白降解的化合物，其特征在于，其为如式(a)～(d)所示的化合物中任一种或该化合物的立体异构体、水合物以及药学上可接受的盐或前药；
    Figure PCTCN2022072346-appb-100007
    Figure PCTCN2022072346-appb-100008
    其中：
    R 1为-H、-D、-F、-Cl、-Br、-I、-NO 2、-CN、-NH 2、-OH、-CH 3、-CH 2F、-CHF 2、-CF 3、-CH 2D、-CHD 2、-CD 3或-CH 2CH 3；
    L为前述结构中的任一种；且1≤n≤10。
  3. 根据权利要求1所述的一种同时诱导EGFR和PARP蛋白降解的化合物，其特征在于，其为如式(a')～(d')所示的化合物中任一种或该化合物的立体异构体、水合物以及药学上可接受的盐或前药；
    Figure PCTCN2022072346-appb-100009
    Figure PCTCN2022072346-appb-100010
    其中，L为
    Figure PCTCN2022072346-appb-100011
    且1≤n≤5。
  4. 根据权利要求1所述的一种同时诱导EGFR和PARP蛋白降解的化合物，其特征在于，其为下列化合物中任一种或该化合物的立体异构体、水合物以及药学上可接受的盐或前药；
    Figure PCTCN2022072346-appb-100012
    Figure PCTCN2022072346-appb-100013
    Figure PCTCN2022072346-appb-100014
    Figure PCTCN2022072346-appb-100015
  5. 根据权利要求1-4任一项所述的一种同时诱导EGFR和PARP蛋白降解的化合物，其特征在于，所述化合物在药学上可接受的盐为该化合物与盐酸、氢溴酸、硫酸、磷酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘二磺酸、乙酸、丙酸、乳酸、三氟乙酸、马来酸、柠檬酸、 富马酸、草酸、酒石酸、苯甲酸、丙酮酸或琥珀酸形成的加成盐。
  6. 一种药物组合物，其特征在于，包括权利要求1-4任一项所述的一种同时诱导EGFR和PARP蛋白降解的化合物、该化合物的立体异构体、该化合物的水合物及该化合物在药学上可接受的盐或前药中的一种或多种；还包括药学上可接受的载体、稀释剂、辅剂、媒介物或它们的组合；其中，所述药物组合物的剂型为注射剂、片剂或胶囊剂。
  7. 一种权利要求1-4任一项所述的一种同时诱导EGFR和PARP蛋白降解的化合物的制备方法，其特征在于，包括以下步骤：
    步骤1：将N-Boc氨基酸甲酯与丙炔溴溶于溶剂中，加入碳酸钾或者氢化钠做碱，进行羟基醚化保护，得到中间体A；
    步骤2：将中间体A和奥拉帕尼，在加入EDCI、HOBt以及DIPEA的条件下进行酰胺缩合，随后脱除保护，得到的产物与吉非替尼发生同样的酰胺缩合，得到中间体B；
    步骤3：中间体B和叠氮连接的E3配体溶于溶剂中，通过Click反应进行连接，得到Dual Protacs化合物；
    以N-Boc酪氨酸或丝氨酸甲酯为起始原料的反应路径如下：
    Figure PCTCN2022072346-appb-100016
  8. 根据权利要求7所述的一种同时诱导EGFR和PARP蛋白降解的化合物的制备方法，其特征在于，所述溶剂为DMF、二氯甲烷、THF和水中的一种或两种组合；所述的氨基酸为酪氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺、谷胺酰胺、组氨酸、精氨酸、赖氨酸、色氨酸、天冬氨酸、谷氨酸或羟基脯氨酸。
  9. 一种权利要求1-4任一项所述的一种同时诱导EGFR和PARP蛋白降解的化合物或该化合物的立体异构体、水合物、药学上可接受的盐或前药或权利要求6所述的药物组合物在制备治疗和/或预防肿瘤的药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述肿瘤为多发性骨髓瘤、胃癌、肺癌、乳腺癌、食管癌、结肠癌、髓母细胞瘤、急性髓细胞性白血病、慢性白血病、黑色素瘤、前列腺癌、肝细胞瘤、肾细胞瘤、宫颈癌、皮肤癌、卵巢癌、结肠癌、神经胶质瘤、甲状腺癌或胰腺癌。
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