WO2023168830A1 - 一种hemtac小分子降解剂及其应用 - Google Patents

一种hemtac小分子降解剂及其应用 Download PDF

Info

Publication number
WO2023168830A1
WO2023168830A1 PCT/CN2022/093374 CN2022093374W WO2023168830A1 WO 2023168830 A1 WO2023168830 A1 WO 2023168830A1 CN 2022093374 W CN2022093374 W CN 2022093374W WO 2023168830 A1 WO2023168830 A1 WO 2023168830A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
protein
hsp90
small molecule
equiv
Prior art date
Application number
PCT/CN2022/093374
Other languages
English (en)
French (fr)
Inventor
李敏勇
杜吕佩
李臻臻
Original Assignee
山东大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东大学 filed Critical 山东大学
Priority to EP22930459.7A priority Critical patent/EP4306528A1/en
Publication of WO2023168830A1 publication Critical patent/WO2023168830A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the invention belongs to the field of biomedicine technology, and specifically relates to a HEMTAC small molecule degradation agent and its application.
  • HSP90 heat shock protein 90
  • HSP90 The heat shock protein 90 family is a type of ATP-dependent molecular chaperone with a molecular weight of approximately 90kDa.
  • HSP90 exists in cells in the form of dimers. The dimerization of HSP90 is necessary for its intracellular functions. Under non-stress conditions, the expression of HSP90 accounts for approximately 1% to 2% of the total intracellular protein. , which is thousands of times the average protein content; under stress conditions, the content of HSP90 can increase to 4% to 6% of the total cellular protein.
  • HSP90 proteins are divided into HSP90 ⁇ (inducible) and HSP90 ⁇ (constitutive). The expression of HSP90 ⁇ increases after being induced by heat, which is related to maintaining cell homeostasis under stress conditions; while HSP90 ⁇ is continuously expressed and must exist in mammals, and is related to the life activities of mammals.
  • HSP90 can activate different substrate proteins and then participate in the regulation of various life activities.
  • molecular chaperones can activate newly synthesized proteins, assemble and depolymerize molecular complexes, help refold abnormally folded proteins, coordinate with the ubiquitin-proteasome system to regulate and degrade misfolded proteins, etc.
  • heat shock protein family is closely related to tumors. The expression of HSP90 is abnormally increased in tumor cells induced by some oncogenes and their products, and is involved in tumor growth, invasion, metastasis, etc.
  • the purpose of the present invention is to provide a HEMTAC small molecule degradation agent based on HSP90 that induces target protein degradation and its application.
  • HSP90 which has stable client proteins, can utilize the ubiquitin-proteasome system to degrade erroneous proteins, and is highly expressed in tumor cells, the present invention designs and develops a new protein degradation technology, and names it heat shock protein 90 mediator.
  • a first aspect of the present invention provides a compound having the structure described in the following formula (I):
  • R1 is the ligand/binding compound of the target protein
  • R2 is the ligand of HSP90
  • L is a connecting chain, and the R1 and R2 are connected through L.
  • HSP90 heat shock protein 90
  • HEMTACs targeting chimeras
  • Small molecule HEMTACs contain three parts: a ligand that binds to the target protein, a linker chain, and a ligand that binds to HSP90.
  • the linking chain connects two ligands to form small molecule HEMTACs.
  • small molecule HEMTACs can induce the formation of HSP90-HEMTAC-target protein ternary complexes, position the target protein in a spatially favorable position that is mistakenly recognized as an "abnormal" protein by HSP90, and then introduce it In the ubiquitin-proteasome system and leading to the degradation of target proteins, HEMTACs are released and participate in the next degradation cycle. Therefore, small molecule HEMTACs have a catalytic role in inducing ubiquitin and degradation of target proteins.
  • the target proteins include, but are not limited to, kinases, G protein-coupled receptors, transcription factors, phosphatases and RAS superfamily members;
  • said R1 is selected from: compounds targeting kinases, G protein-coupled receptors, transcription factors, phosphatases and members of the RAS superfamily.
  • the R1 is Palbociclib.
  • the R2 is the ligand BIIB021 of HSP90 or a derivative thereof.
  • the compounds also include pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds thereof.
  • a second aspect of the present invention provides the use of the above compounds in the preparation of drugs for tumors, inflammation, and immune-related diseases.
  • the disease may also be a disease related to cell cycle-dependent protein kinase 4 and 6 (CDK4/6).
  • a third aspect of the present invention provides a pharmaceutical composition comprising the above compound.
  • a fourth aspect of the present invention provides a pharmaceutical preparation comprising the above compound and at least one pharmaceutically acceptable pharmaceutical inactive ingredient.
  • the above pharmaceutical composition or pharmaceutical preparation can be used to treat tumors, inflammation, and immune-related diseases, especially diseases related to cell cycle-dependent protein kinases 4 and 6 (CDK4/6).
  • a fifth aspect of the present invention provides a method for treating tumors, inflammation, and immune-related diseases, which method includes administering a therapeutically effective amount of the above-mentioned compound, pharmaceutical composition, or pharmaceutical preparation to a subject.
  • the above technical solution reports for the first time a HSP90-based HEMTAC small molecule degrader that induces target protein degradation and its application.
  • the present invention found through research that the HEMTAC small molecule degrader can induce the formation of HSP90-HEMTAC-target protein ternary complex, and position the target protein in a position that is conducive to being mistakenly recognized as an "abnormal" protein by HSP90. , which are then introduced into the ubiquitin-proteasome system and lead to the degradation of the target protein.
  • HEMTACs are released and participate in the next degradation cycle. Therefore, they have a catalytic role in inducing ubiquitin and degradation of the target protein, which can be used for therapy. It is related to diseases such as tumors, inflammation, and immunity, so it has good prospects for practical application.
  • Figure 1 is a functional schematic diagram of HSP90 chaperones and a schematic diagram of the protein degradation mechanism of small molecule HEMTACs.
  • Figure 2 shows the surface plasmon resonance (SPR) method to determine the binding affinity of compounds to HSP90 ⁇ .
  • Figure 3 shows the effect of HEMTAC 26 on CDK4 and CDK6 protein levels in B16F10 cells at different concentrations and times.
  • Figure 4 shows the anti-tumor activity of small molecule HEMTAC 26 in the mouse B16F10 melanoma model.
  • the experimental results show that small molecule HEMTAC 26 has significant anti-melanoma effects in vivo.
  • R1 is the ligand/binding compound of the target protein
  • R2 is the ligand of HSP90
  • L is a connecting chain, and the R1 and R2 are connected through L.
  • HSP90 heat shock protein 90
  • HEMTACs heat shock protein 90-mediated targeting chimeras
  • Small molecule HEMTACs contain three parts: a ligand that binds to the target protein, a linker chain, and a ligand that binds to HSP90.
  • the linking chain connects two ligands to form small molecule HEMTACs.
  • small molecule HEMTACs can induce the formation of HSP90-HEMTAC-target protein ternary complexes, position the target protein in a spatially favorable position that is mistakenly recognized as an "abnormal" protein by HSP90, and then introduce it In the ubiquitin-proteasome system and leading to the degradation of target proteins, HEMTACs are released and participate in the next degradation cycle. Therefore, small molecule HEMTACs have a catalytic role in inducing ubiquitin and degradation of target proteins.
  • the target proteins include, but are not limited to, kinases, G protein-coupled receptors, transcription factors, phosphatases and RAS superfamily members;
  • said R1 is selected from: compounds targeting kinases, G protein-coupled receptors, transcription factors, phosphatases and members of the RAS superfamily.
  • the R1 is Palbociclib.
  • the R2 is the ligand BIIB021 of HSP90 or a derivative thereof,
  • HSP90 ligand BIIB021 The general formula of the HSP90 ligand BIIB021 or its derivative is as follows:
  • the compound includes any one or more of the following:
  • the compound further includes pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds thereof.
  • the disease may also be a disease related to cell cycle-dependent protein kinases 4 and 6 (CDK4/6).
  • a pharmaceutical composition which contains the above compound.
  • the pharmaceutical composition can be used alone or in combination with other types of drugs to treat tumors, inflammation, immunity and other diseases.
  • a pharmaceutical preparation which contains the above-mentioned compound and at least one pharmaceutically acceptable pharmaceutical inactive ingredient.
  • the pharmaceutical inactive ingredients may be carriers, excipients, diluents, etc. commonly used in pharmaceuticals. Moreover, it can be prepared into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral use, external preparations, suppositories, and sterile injection solutions according to usual methods. .
  • non-pharmaceutical active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.
  • the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc Powder, magnesium stearate and mineral oil, etc.
  • the medicament of the present invention can be administered into the body in a known manner. Delivery to the tissue of interest may occur, for example, via intravenous systemic delivery or local injection. Administration may optionally be via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary to a large extent depending on a variety of factors, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the administration Drug routes, administration methods, etc.
  • the drug administration subjects can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc.
  • the above pharmaceutical composition or pharmaceutical preparation can be used to treat tumors, inflammation, and immune-related diseases, especially diseases related to cell cycle-dependent protein kinases 4 and 6 (CDK4/6).
  • a fifth aspect of the present invention provides a method for treating tumors, inflammation, and immune-related diseases, which method includes administering a therapeutically effective amount of the above-mentioned compound, pharmaceutical composition, or pharmaceutical preparation to a subject.
  • the subject refers to an animal that has been the subject of treatment, observation or experiment, preferably a mammal, and most preferably a human.
  • the “therapeutically effective amount” refers to the amount of active compounds or agents, including the compounds of the present invention, that can cause the biological or biological effects of tissue systems, animals or humans pursued by researchers, veterinarians, doctors or other medical personnel. Medical response, which includes alleviation or partial alleviation of symptoms of the disease, syndrome, condition or disorder being treated.
  • Dissolve compound 1 (2g, 11.86mmol, 1.0equiv.) in 20mL pyridine, then add trimethylacetyl chloride (4.45mL, 35.59mmol, 3equiv.), and stir at room temperature overnight to obtain N(2) monoacylation and Mixture of N(2) and N(7) bis-acylation.
  • the solvent was removed, and the residue was dissolved in a mixed solvent of ammonia (25% NH 3 , 5 mL) and methanol (20 mL), and stirred at room temperature for 30 minutes to selectively cleave the pivaloyl group at the N (7) position.
  • the reaction solution was heated to 75°C and stirred for 3 hours.
  • the reaction solution was filtered through celite, washed with ethyl acetate, and then the filtrate was diluted with 100 mL of ethyl acetate.
  • the organic phase was washed with water (40 mL ⁇ 3) and brine (40 mL ⁇ 3), and dried over anhydrous Na 2 SO 4 .
  • compound 13 (300 mg, 593.38 ⁇ mol, 1.0 equiv.) and 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamic acid tert-butyl ester (176.82 mg, 712.06 ⁇ mol, 1.2 equiv.) was used as raw material to obtain 310 mg of compound 17 as a yellow solid, with a yield of 71%.
  • Example 7 Surface plasmon resonance (SPR) method to determine the binding affinity of compounds to HSP90 ⁇
  • start detection change the buffer to PBS (pH 7.4) containing 1% DMSO, set the analyte binding time to 120s, the flow rate to 30 ⁇ L/min; the dissociation time to 300s, the flow rate to 30 ⁇ L/min; regeneration The time is 30s and the flow rate is 30 ⁇ L/min.
  • PBS pH 7.4 containing 1% DMSO
  • the intracellular CDK4 and CDK6 protein levels were determined by Western blot experiment.
  • extract cellular proteins and perform protein quantification lyse cells with RIPA cell lysis buffer (plus protease inhibitors), collect proteins, and quantify them with a BCA protein quantitative assay kit to the same concentration.
  • Protein denaturation Place the protein in a 100°C water bath for 8 minutes to denature the protein.
  • Load the sample and run the gel The sample volume per well is generally 20 ⁇ L.
  • the electrophoresis voltage of the upper stacking gel is 75V
  • the electrophoresis voltage of the lower separation gel is 120V.
  • Film transfer Use a wet film transfer instrument for film transfer.
  • the PVDF membrane must be soaked in methanol in advance, with a constant current of 250mA and a film transfer time of 90 minutes.
  • Blocking Place the PVDF membrane in BSA blocking solution for 1 hour (slow shaking on a shaker).
  • Incubate the primary antibody Dilute the primary antibody according to the instructions, add the primary antibody, and incubate at 4°C overnight.
  • Membrane washing Wash 3 times with TBST membrane washing solution, 10 minutes each time.
  • Incubate the secondary antibody After adding the secondary antibody, incubate on a shaking table at room temperature for 1 hour.
  • Membrane washing Wash 3 times with TBST membrane washing solution, 10 minutes each time.
  • Exposure Mix equal volumes of ECL developer A and B and perform exposure.
  • B16F10 cells (10 6 cells/mouse) were subcutaneously injected into the forelimb axilla of C57BL/6 mice to establish a mouse B16F10 melanoma xenograft model.
  • Compounds were injected intraperitoneally every day for two consecutive weeks, and mouse body weight and tumor volume were measured every two days.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明属于生物医药技术领域，具体涉及一种HEMTAC小分子降解剂及其应用。本发明通过研究发现，所述HEMTAC小分子降解剂可以诱导HSP90-HEMTAC-靶蛋白三元络合物的形成，将靶蛋白定位在空间上有利于被HSP90错误地识别为"异常"蛋白质的位置，随后将其导入泛素-蛋白酶体系统中，并导致靶蛋白的降解，HEMTACs被释放并参与下一个降解循环，因此，其在诱导靶蛋白泛素和降解方面具有催化作用，从而可用于治疗与肿瘤、炎症、免疫等相关疾病，因此具有良好的实际应用之前景。

Description

一种HEMTAC小分子降解剂及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种HEMTAC小分子降解剂及其应用。
背景技术
本发明背景技术中公开的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
在对药物化学的不断探索中，新的靶向疾病驱动蛋白的策略正在不断的被开发，其中由小分子诱导的靶蛋白降解策略是近年来最值得关注的，该策略可以靶向无活性位点或者不可成药的蛋白靶标，使得降解任意一种蛋白成为了可能。目前开发的降解技术主要有蛋白水解靶向嵌合体(proteolysis-targeting chimeras,PROTACs)，溶酶体靶向嵌合体(lysosome-targeting chimeras,LYTACs)和基于抗体的PROTACs(antibody-based PROTACs,AbTACs)。PROTAC技术相关的候选药物ARV-110和ARV-471已进入临床II期，PROTAC技术主要针对细胞内蛋白，而LYTAC和AbTAC技术可以降解细胞外分泌蛋白和膜蛋白。蛋白降解技术在靶向不可成药靶点和疾病治疗方面展现出了非常大的潜力。
虽然这些新的降解技术的出现可以克服小分子抑制剂的局限性，同时，新兴的降解技术也存在一些局限性，其可能导致在许多组织和器官中不参与疾病过程的靶蛋白的降解，从而在治疗过程中可能导致严重的副作用。另外一方面，小分子PROTAC通过“劫持”E3连接酶发挥其降解作用，然而已有研究表明基于VHL和CRBN的小分子PROTAC在细胞中会引起脱靶效应和耐药机制的产生。因此，迫切需要扩大靶向降解技术的范围，开发新的蛋白降解技术。
热休克蛋白90(heat shock protein 90,HSP90)家族是一类ATP依赖的分子伴侣，分子量约90kDa。HSP90以二聚体形式存在于细胞中，HSP90的二聚化是其行使细胞内功能所必须的，在非应激状态下，HSP90的表达量约占细胞内蛋白总量的1％～2％，是蛋白平均含量的数千倍；在应激条件下，HSP90的含量可升高到细胞蛋白总量的4％～6％。在人细胞中，HSP90蛋白分为HSP90α(诱导型)和HSP90β(组成型)。HSP90α在受到热诱导后表达增加，与压力条件下维持细胞稳态相关；而HSP90β可持续表达且在哺乳动物体内必须存在，与哺乳动物的生命活动相关。
作为一种重要的分子伴侣，HSP90可以激活不同的底物蛋白，进而参与多种生命活动的调节。为了使蛋白质行使其正常的生物学功能，分子伴侣可以激活新合成的蛋白质、组装和解聚分子复合物、帮助异常折叠的蛋白质重新折叠、与泛素-蛋白酶体系统协同调节并降解错误折叠蛋白等。此外，研究发现热休克蛋白家族与肿瘤关系密切，HSP90在一些癌基因及其产物诱导的肿瘤细胞中表达异常增高，并参与肿瘤的生长、侵袭、转移等。
发明内容
本发明的目的在于提供一种基于HSP90的诱导靶蛋白降解的HEMTAC小分子降解剂及其应用。本发明根据HSP90具有稳定客户蛋白，能够利用泛素-蛋白酶体系统降解错误蛋白，并在肿瘤细胞中高表达等特点，设计开发一种新型的蛋白降解技术，并将其命名为热休克蛋白90介导的靶向嵌合体(heat shock protein 90(HSP90)-mediated targeting chimeras,HEMTACs)。基于上述研究成果，从而完成本发明。
本发明的技术方案如下：
本发明的第一个方面，提供一种化合物，其具有如下式(I)所述结构：
R 1—L—R 2
式(I)
其中：
(a)R1是靶蛋白的配体/结合物；
(b)R2是HSP90的配体；
(c)L是连接链，所述R1和R2通过L连接起来。
上述化合物实际为一种热休克蛋白90介导的靶向嵌合体(heat shock protein 90(HSP90)-mediated targeting chimeras,HEMTACs)小分子降解剂。
小分子HEMTACs包含三个部分：与靶蛋白结合的配体、连接链和与HSP90结合的配体。连接链连接两个配体构成小分子HEMTACs。和PROTAC技术类似，小分子HEMTACs可以诱导HSP90-HEMTAC-靶蛋白三元络合物的形成，将靶蛋白定位在空间上有利于被HSP90错误地识别为“异常”蛋白质的位置，随后将其导入泛素-蛋白酶体系统中，并导致靶蛋白的降解，HEMTACs被释放并参与下一个降解循环，因此，小分子HEMTACs在诱导靶蛋白泛素和降解方面具有催化作用。
所述靶蛋白包括但不限于激酶、G蛋白偶联受体、转录因子、磷酸酶及RAS超家族成员；
因此，所述R1选自：靶向以下的化合物：激酶、G蛋白偶联受体、转录因子、磷酸酶及RAS超家族成员。
具体的，所述R1是帕博西尼(Palbociclib)。
所述R2是HSP90的配体BIIB021或其衍生物。
所述化合物还包括其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物。
本发明的第二个方面，提供上述化合物在制备肿瘤、炎症、免疫相关疾病药物中的应用。
进一步的，所述疾病还可以是与细胞周期依赖性蛋白激酶4和6(CDK4/6)相关的疾病。
本发明的第三个方面，提供一种药物组合物，其包含上述化合物。
本发明的第四个方面，提供一种药物制剂，其包含上述化合物和至少一种药学上可接受的药物非活性成分。
显而易见的，上述药物组合物或药物制剂可用于治疗肿瘤、炎症、免疫相关疾病，特别是与细胞周期依赖性蛋白激酶4和6(CDK4/6)相关的疾病。
本发明的第五个方面，提供一种治疗肿瘤、炎症、免疫相关疾病的方法，所述方法包括向受试者施用治疗有效量的上述化合物、药物组合物或药物制剂。
上述一个或多个技术方案的有益技术效果：
上述技术方案首次报道了一种基于HSP90的诱导靶蛋白降解的HEMTAC小分子降解剂及其应用。本发明通过研究发现，所述HEMTAC小分子降解剂可以诱导HSP90-HEMTAC-靶蛋白三元络合物的形成，将靶蛋白定位在空间上有利于被HSP90错误地识别为“异常”蛋白质的位置，随后将其导入泛素-蛋白酶体系统中，并导致靶蛋白的降解，HEMTACs被释放并参与下一个降解循环，因此，其在诱导靶蛋白泛素和降解方面具有催化作用，从而可用于治疗与肿瘤、炎症、免疫等相关疾病，因此具有良好的实际应用之前景。
附图说明
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。
图1为HSP90伴侣分子的功能示意图及小分子HEMTACs降解蛋白机制示意图。
图2为表面等离子共振法(SPR)测定化合物与HSP90α的结合亲和力。
图3为HEMTAC 26在不同的浓度和时间对B16F10细胞中CDK4和CDK6蛋白水平的影响。
图4为小分子HEMTAC 26在小鼠B16F10黑色素瘤模型中的抗肿瘤活性，实验结果表明小分子HEMTAC 26具有显著的体内抗黑色素瘤的作用。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
现结合具体实例对本发明作进一步的说明，以下实例仅是为了解释本发明，并不对其内容进行限定。如果实施例中未注明的实验具体条件，通常按照常规条件，或按照试剂公司所推荐的条件；下述实施例中所用的试剂、耗材等，如无特殊说明，均可从商业途径得到。
本发明的一个典型具体实施方式中，提供一种化合物，其具有如下式(I)所述结构：
R 1—L—R 2
式(I)
其中：
(a)R1是靶蛋白的配体/结合物；
(b)R2是HSP90的配体；
(c)L是连接链，所述R1和R2通过L连接起来。
上述化合物实际为一种热休克蛋白90介导的靶向嵌合体(heat shock protein 90(HSP90)-mediated targeting chimeras,HEMTACs)小分子降解剂。小分子HEMTACs包含三个部分：与靶蛋白结合的配体、连接链和与HSP90结合的配体。连接链连接两个配体构成小分子HEMTACs。和PROTAC技术类似，小分子HEMTACs可以诱导HSP90-HEMTAC-靶蛋白三元络合物的形成，将靶蛋白定位在空间上有利于被HSP90错误地识别为“异常”蛋白质的位置，随后将其导入泛素-蛋白酶体系统中，并导致靶蛋白的降解，HEMTACs被释放并参与下一个降解循环，因此，小分子HEMTACs在诱导靶蛋白泛素和降解方面具有催化作用。
所述靶蛋白包括但不限于激酶、G蛋白偶联受体、转录因子、磷酸酶及RAS超家族成员；
因此，所述R1选自：靶向以下的化合物：激酶、G蛋白偶联受体、转录因子、磷酸酶及RAS超家族成员。
具体的，所述R1是帕博西尼(Palbociclib)。
所述R2是HSP90的配体BIIB021或其衍生物，
所述HSP90的配体BIIB021或其衍生物的通式如下所示：
Figure PCTCN2022093374-appb-000001
本发明的又一具体实施方式中，所述化合物包括如下任意一种或多种：
Figure PCTCN2022093374-appb-000002
Figure PCTCN2022093374-appb-000003
本发明的又一具体实施方式中，所述化合物还包括其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物。
本发明的又一具体实施方式中，提供上述化合物在制备肿瘤、炎症、免疫相关疾病药物中的应用。
本发明的又一具体实施方式中，所述疾病还可以是与细胞周期依赖性蛋白激酶4和6(CDK4/6)相关的疾病。
本发明的又一具体实施方式中，提供一种药物组合物，其包含上述化合物。所述药物组合物可单独使用，或与其他种类的药物联合使用，用于治疗肿瘤、炎症、免疫等疾病。
本发明的又一具体实施方式中，提供一种药物制剂，其包含上述化合物和至少一种药学上可接受的药 物非活性成分。
所述药物非活性成分可以是药学上通常使用的载体、赋形剂及稀释剂等。而且，根据通常的方法，可以制作成粉剂、颗粒剂、片剂、胶囊剂、混悬剂、乳剂、糖浆剂、喷雾剂等的口服剂、外用剂、栓剂及无菌注射溶液形式的剂型使用。
所述可以包含的载体、赋形剂及稀释剂等非药物活性成分在领域内是熟知的，本领域普通技术人员能够确定其符合临床标准。
本发明的又一具体实施方式中，所述载体、赋形剂及稀释剂包括但不限于有乳糖、葡萄糖、蔗糖、山梨糖醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石粉、硬脂酸镁和矿物油等。
本发明的又一具体实施方式中，本发明的药物可通过已知的方式施用至体内。例如通过静脉全身递送或者局部注射递送到感兴趣组织中。可选地经由静脉内、经皮、鼻内、粘膜或其他递送方法进行施用。这样的施用可以经由单剂量或多剂量来进行。本领域技术人员理解的是，本发明中有待施用的实际剂量可以在很大程度上取决于多种因素而变化，如靶细胞、生物类型或其组织、待治疗受试者的一般状况、给药途径、给药方式等等。
本发明的又一具体实施方式中，所述药物施用对象可以是人和非人哺乳动物，如小鼠、大鼠、豚鼠、兔、狗、猴、猩猩等。
显而易见的，上述药物组合物或药物制剂可用于治疗肿瘤、炎症、免疫相关疾病，特别是与细胞周期依赖性蛋白激酶4和6(CDK4/6)相关的疾病。
本发明的第五个方面，提供一种治疗肿瘤、炎症、免疫相关疾病的方法，所述方法包括向受试者施用治疗有效量的上述化合物、药物组合物或药物制剂。
所述受试者是指已经是治疗、观察或实验的对象的动物，优选指哺乳动物，最优选指人。
所述“治疗有效量”是指包括本发明化合物在内的活性化合物或药剂的量，该量可引起研究者、兽医、医生或其他医疗人员所追求的组织系统、动物或人的生物学或医学响应，这包括减轻或部分减轻受治疗的疾病、综合征、病症或障碍的症状。
本领域的研究者、兽医、医生或其他医疗人员可根据临床试验或者本领域其他公知的手段获知可使用的治疗有效量的范围。
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。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。下列实施例中为注明具体条件的试验方法，通常按照常规条件进行。
实施例1：化合物24的制备
化合物2的合成
Figure PCTCN2022093374-appb-000004
将化合物1(2g,11.86mmol,1.0equiv.)溶于20mL吡啶中,然后加入三甲基乙酰氯(4.45mL,35.59mmol,3equiv.)，室温搅拌过夜,得到N(2)单酰化和N(2),N(7)双酰化的混合物。旋除溶剂，残渣溶于氨水(25％NH 3,5mL)和甲醇(20mL)的混合溶剂中，室温搅拌30分钟，选择性的断开N(7)位的新戊酰基。过滤固体，用水洗涤滤饼(10mL×3)，真空干燥，得到2.55g化合物2，黄色固体，产率85％。 1H NMR(400MHz,DMSO-d 6)δ12.32(s,1H),10.01(s,1H),7.54(d,J＝3.6Hz,1H),6.53(d,J＝3.5Hz,1H),1.24(s,9H).ESI-MS:m/z[M+H] +calculated for C 11H 14ClN 4O +253.09,found 253.06.
化合物3的合成
Figure PCTCN2022093374-appb-000005
将化合物2(200mg,791.45μmol,1.0equiv.)溶于干燥的四氢呋喃(10mL)中，然后在N 2保护条件下加入N-碘代琥珀酰亚胺(213.68mg,949.74μmol,1.2equiv.)，室温反应1小时。旋除溶剂，残渣溶于二氯甲烷(60mL)中，用饱和Na 2S 2O 3溶液(40mL×3)和盐水(30mL×3)洗涤，有机层用无水Na 2SO 4干燥。减压除去有机溶剂，粗产物用硅胶柱层析进行纯化(二氯甲烷/甲醇＝60:1-30:1)，得到260mg化合物3，棕色固体，产率87％。 1H NMR(400MHz,DMSO-d 6)δ12.69(s,1H),10.09(s,1H),7.77(d,J＝1.2Hz,1H),1.23(s,9H).ESI-MS:m/z[M-H] -calculated for C 11H 11ClIN 4O -376.97,found 377.08.化合物5的合成
Figure PCTCN2022093374-appb-000006
将化合物3(255mg,673.54μmol,1.0equiv.)，化合物4(157.08mg,707.21μmol,1.1equiv.)和K 2CO 3(279.26mg,2.02mmol,3.0equiv.)溶于干燥的DMF(5mL)中，室温搅拌24小时。将反应液逐滴加入水(15mL)和异丙醇(15mL)的混合溶液中。过滤固体，用水(15mL)洗涤滤饼，经真空干燥得到 323mg化合物5，白色固体，产率91％。 1H NMR(400MHz,DMSO-d 6)δ10.14(s,1H),8.05(s,1H),7.73(s,1H),5.46(s,2H),3.74(s,3H),2.33(s,3H),2.16(s,3H),1.21(s,9H).ESI-MS:m/z[M+H] +calculated for C 20H 24ClIN 5O 2 +528.07,found 528.31.
化合物6的合成
Figure PCTCN2022093374-appb-000007
将化合物5(2.0g,3.79mmol,1.0equiv.)溶于乙醇/水(20:1,42mL)的混合溶剂中，然后将ZnCl 2(2.58g,18.95mmol,5.0equiv.)加入到反应液中，加热至80℃搅拌20小时。冷却至室温，然后将反应液倒入水(50mL)中，过滤固体，用水(10mL×3)洗涤滤饼，经真空干燥得到1.44g化合物6，白色固体，产率86％。 1H NMR(400MHz,DMSO-d 6)δ8.07(s,1H),7.26(s,1H),6.71(s,2H),5.29(s,2H),3.73(s,3H),2.26(s,3H),2.17(s,3H).ESI-MS:m/z[M+H] +calculated for C 15H 16ClIN 5O +444.01,found 444.11.
化合物7的合成
Figure PCTCN2022093374-appb-000008
将化合物6(1.0g,2.25mmol,1.0equiv.),CuI(42.93mg,225.39μmol,0.1equiv.),PPh 3(59.12mg,225.39μmol,0.1equiv.),10％Pd/C(66.73mg,56.35μmol,0.025equiv.)和K 2CO 3(342.65mg,2.48mmol,1.1equiv.)溶于DMF(12mL)和水(4mL)的混合溶剂中，N 2保护条件下，将溶于DMF(4mL)的对甲苯磺酸3-丁炔酯滴加至反应液中。将反应液加热至75℃搅拌3小时。反应液用硅藻土过滤，用乙酸乙酯洗涤硅藻土，然后用100mL乙酸乙酯稀释滤液。有机相用水(40mL×3)和盐水洗涤(40mL×3)，无水Na 2SO 4干燥。减压除去有机溶剂，粗产物用硅胶柱层析进行纯化(石油醚/乙酸乙酯＝1:1-1:3)，得到700mg化合物7，白色固体，产率74％。 1H NMR(400MHz,DMSO-d 6)δ8.10(s,1H),7.80(d,J＝7.7Hz,2H),7.41(d,J＝7.6Hz,2H),7.28(s,1H),6.75(s,2H),5.31(s,2H),4.14(t,J＝ 5.1Hz,2H),3.73(s,3H),2.77(t,J＝5.1Hz,2H),2.33(s,3H),2.27(s,3H),2.17(s,3H).ESI-MS:m/z[M+H] +calculated for C 26H 27ClN 5O 4S +540.15,found 540.13.
化合物8的合成
Figure PCTCN2022093374-appb-000009
将化合物7(406mg,751.80μmol,1.0equiv.)溶于DMF(8mL)中，然后将NaN 3(146.63mg,2.26mmol,3.0equiv.)加入至反应液中，将反应液加热至60℃搅拌4小时。冷却至室温后，将反应液倒入水中，乙酸乙酯(40mL×3)萃取三次，合并有机相，水洗盐洗，有机相用无水Na 2SO 4干燥。减压除去有机溶剂，粗产物用硅胶柱层析进行纯化(石油醚/乙酸乙酯＝1:1-1:3)，得到290mg化合物8，白色固体，产率94％。 1H NMR(400MHz,DMSO-d 6)δ8.06(s,1H),7.28(s,1H),6.71(s,2H),5.29(s,2H),3.73(s,3H),3.51(t,J＝6.5Hz,2H),2.72(t,J＝6.5Hz,2H),2.25(s,3H),2.17(s,3H).ESI-MS:m/z[M+H] +calculated for C 19H 20ClN 8O +411.14,found 411.03.
化合物9的合成
Figure PCTCN2022093374-appb-000010
将化合物8(810mg,1.97mmol,1.0equiv.)溶于四氢呋喃(30mL)和水(2mL)的混合溶剂中，然后将PPh 3(1.55g,5.91mmol,3.0equiv.)加入至反应液中，室温搅拌过夜，旋除溶剂，粗产物用硅胶柱层析进行纯化(二氯甲烷/甲醇＝30:1-5:1)，得到480mg化合物9，白色固体，产率63％。 1H NMR(400MHz,DMSO-d 6)δ8.06(s,1H),7.30(s,1H),6.73(s,2H),5.28(s,2H),3.73(s,3H),2.92(t,J＝6.9Hz,2H),2.65(t,J＝7.0Hz,2H),2.25(s,3H),2.16(s,3H),1.23(s,2H).ESI-MS:m/z[M+H] +calculated for C 19H 22ClN 6O +385.15,found 385.10.
化合物11的合成
Figure PCTCN2022093374-appb-000011
将化合物10(150mg,335.16μmol,1.0equiv.)和丙炔酸(30.95μL,502.74μmol,1.5equiv.)溶于二氯甲烷(15mL)中，然后将HATU(156.05mg,402.20μmol,1.2equiv.)和DIPEA(169.58μL,1.01mmol,3.0equiv.)加入至上述混悬液中，室温搅拌3小时。用100mL二氯甲烷稀释反应液，水洗并盐洗，有机相用无水Na 2SO 4干燥。减压除去有机溶剂，粗产物用硅胶柱层析进行纯化(二氯甲烷/甲醇＝70:1-40:1)，得到105mg化合物11，黄色固体，产率63％。 1H NMR(400MHz,DMSO-d 6)δ10.15(s,1H),8.96(s,1H),8.09(d,J＝2.7Hz,1H),7.90(d,J＝9.0Hz,1H),7.52(dd,J＝9.1,2.7Hz,1H),5.94–5.77(m,1H),4.62(s,1H),3.96–3.79(m,2H),3.75–3.60(m,2H),3.32–3.20(m,2H),3.21–3.06(m,2H),2.43(s,3H),2.31(s,3H),2.29–2.14(m,2H),2.00–1.84(m,2H),1.85–1.69(m,2H),1.68–1.50(m,2H).ESI-MS:m/z[M+H] +calculated for C 27H 30N 7O 3 +500.24,found 500.17.
化合物12的合成
Figure PCTCN2022093374-appb-000012
将化合物10(1.0g,2.23mmol,1.0equiv.)溶于DMF(15mL)中，然后将溴乙酸叔丁酯(398.86μL,2.68mmol,1.2equiv.)和DIPEA(1.13mL,6.70mmol,3.0equiv.)依次加入至反应液中，室温搅拌过夜。然后将反应液倒入水(50mL)中，过滤固体，滤饼用水(30mL)洗涤，经真空干燥得1.2g化合物12，黄色固体，产率95％。 1H NMR(400MHz,CDCl 3)δ8.82(s,1H),8.23(s,1H),8.15(d,J＝9.1Hz,1H),8.06(d,J＝2.8Hz,1H),7.32(dd,J＝9.1,2.9Hz,1H),5.88(p,J＝8.9Hz,1H),3.34–3.22(m,4H),3.20(s,2H),2.92–2.64(m,4H),2.55(s,3H),2.41–2.30(m,5H),2.11–2.02(m,2H),1.92–1.84(m,2H),1.74–1.67(m,2H),1.49(s,9H).ESI-MS:m/z[M+H] +calculated for C 30H 40N 7O 4 +562.31,found 562.25.
化合物13的合成
Figure PCTCN2022093374-appb-000013
将化合物12(1.0g,1.78mmol,1.0equiv.)溶于三氟乙酸(5mL)和二氯甲烷(5mL)的混合溶剂中，室温搅拌2小时。旋除反应液中的大部分溶剂，加入少量甲苯然后旋除，反复多次直至除去残留的三氟乙酸。该粗产物未经进一步纯化直接用于下一步反应。
化合物14的合成
Figure PCTCN2022093374-appb-000014
将化合物13(255mg,504.37μmol,1.0equiv.)溶于干燥的二氯甲烷(20mL)中，然后依次将炔丙基胺(49.44μL,756.56μmol,1.5equiv.)，HATU(287.67mg,757.5μmol,1.5equiv.)和DIPEA(448.24μL,2.52mmol,5.0equiv.)加入至反应液中，室温搅拌5小时。用100mL二氯甲烷稀释反应液，水洗并盐洗，有机相用无水Na 2SO 4干燥。减压除去有机溶剂，粗产物用硅胶柱层析进行纯化(二氯甲烷/甲醇＝60:1-30:1)，得到185mg化合物14，黄色固体，产率68％。 1H NMR(400MHz,DMSO-d 6)δ10.09(s,1H),8.95(s,1H),8.20(t,J＝5.7Hz,1H),8.06(d,J＝2.7Hz,1H),7.85(d,J＝9.0Hz,1H),7.48(dd,J＝9.1,2.8Hz,1H),5.89–5.76(m,1H),3.89(dd,J＝5.7,2.3Hz,2H),3.21(t,4H),3.07(t,J＝2.3Hz,1H),3.02(s,2H),2.60(t,4H),2.42(s,3H),2.31(s,3H),2.28–2.14(m,2H),1.97–1.82(m,2H),1.82–1.68(m,2H),1.65–1.50(m,2H).ESI-MS:m/z[M+H] +calculated for C 29H 35N 8O 3 +543.28,found 543.22.
化合物15的合成
Figure PCTCN2022093374-appb-000015
将化合物13(257mg,508.33μmol,1.0equiv.)溶于干燥的二氯甲烷(20mL)中，然后依次将N-叔 丁氧羰基-1,2-乙二胺(97.73mg,609.99μmol,1.2equiv.)，HATU(231.94mg,609.99μmol,1.2equiv.)和DIPEA(451.76μL,2.54mmol,5.0equiv.)加入至反应液中，室温搅拌过夜。用100mL二氯甲烷稀释反应液，水洗并盐洗，有机相用无水Na 2SO 4干燥。减压除去有机溶剂，粗产物用硅胶柱层析进行纯化(二氯甲烷/甲醇＝60:1-30:1)，得到243mg化合物15，黄色固体，产率74％。 1H NMR(400MHz,DMSO-d 6)δ10.10(s,1H),8.96(s,1H),8.06(d,J＝2.8Hz,1H),7.96–7.73(m,2H),7.47(dd,J＝9.1,2.8Hz,1H),6.86(t,J＝5.3Hz,1H),5.88–5.77(m,1H),3.29–3.11(m,6H),3.08–2.90(m,4H),2.61(s,4H),2.43(s,3H),2.32(s,3H),2.29–2.18(m,2H),1.95–1.84(m,2H),1.83–1.72(m,2H),1.64–1.55(m,2H),1.38(s,9H).ESI-MS:m/z[M+H] +calculated for C 33H 46N 9O 5 +648.36,found 648.18.
化合物16的合成
Figure PCTCN2022093374-appb-000016
按照化合物15的合成方法，由化合物13(166.52mg,329.36μmol,1.0equiv.)和[2-(2-氨基乙氧基)乙基]氨基甲酸叔丁酯(82.38mg,395.24μmol,1.2equiv.)为原料，得到150mg化合物16，黄色固体，产率66％。 1H NMR(400MHz,DMSO-d 6)δ10.10(s,1H),8.96(s,1H),8.07(d,J＝2.6Hz,1H),7.85(d,J＝9.1Hz,1H),7.80(s,1H),7.48(dd,J＝9.0,2.6Hz,1H),6.77(t,J＝4.9Hz,1H),5.87–5.75(m,1H),3.48–3.38(m,4H),3.31–3.18(m,6H),3.11–2.94(m,4H),2.63(s,4H),2.43(s,3H),2.31(s,3H),2.29–2.18(m,2H),1.95–1.83(m,2H),1.82–1.70(m,2H),1.65–1.53(m,2H),1.37(s,9H).ESI-MS:m/z[M+H] +calculated for C 35H 50N 9O 6 +692.39,found 692.21.
化合物17的合成
Figure PCTCN2022093374-appb-000017
按照化合物15的合成方法，由化合物13(300mg,593.38μmol,1.0equiv.)和2-(2-(2-氨基乙氧基)乙氧基)乙基氨基甲酸叔丁酯(176.82mg,712.06μmol,1.2equiv.)为原料，得到310mg化合物17，黄色固体，产率71％。 1H NMR(400MHz,DMSO-d 6)δ10.09(s,1H),8.95(s,1H),8.06(d,J＝ 2.6Hz,1H),7.85(d,J＝9.0Hz,1H),7.77(s,1H),7.48(dd,J＝9.1,2.7Hz,1H),6.74(t,J＝5.4Hz,1H),5.87–5.77(m,1H),3.50(s,4H),3.45(t,J＝5.8Hz,2H),3.39–3.36(m,2H),3.30–3.26(m,2H),3.21(s,3H),3.12–2.81(m,6H),2.62(s,3H),2.42(s,3H),2.31(s,3H),2.28–2.18(m,2H),1.93–1.83(m,2H),1.81–1.72(m,2H),1.64–1.54(m,2H),1.36(s,9H).ESI-MS:m/z[M+H] +calculated for C 37H 54N 9O 7 +736.41,found 736.10.
化合物21的合成
Figure PCTCN2022093374-appb-000018
化合物15(245mg,378.21μmol,1.0equiv.)溶于二氯甲烷(3mL)和三氟乙酸(3mL)的混合溶剂中，室温搅拌30分钟。然后旋除反应液中的大部分溶剂，加入少量甲苯然后旋除，反复多次直至除去残留的三氟乙酸，得到的化合物18的粗产物未经进一步纯化直接用于下一步反应。将残留物溶于二氯甲烷(15mL)中，然后加入DIPEA(312.54μL,1.89mmol,5.0equiv.)，室温搅拌10分钟直至完全溶解，然后向反应液中加入丙炔酸(38.80μL,567.31μmol,1.5equiv.)和HATU(172.57mg,453.85μmol,1.2equiv.)，室温搅拌6小时。用100mL二氯甲烷稀释反应液，水洗并盐洗，有机相用无水Na 2SO 4干燥。减压除去有机溶剂，粗产物用硅胶柱层析进行纯化(二氯甲烷/甲醇＝60:1-30:1)，得到135mg化合物21，黄色固体，产率60％。 1H NMR(400MHz,DMSO-d 6)δ10.09(s,1H),8.95(s,1H),8.73(s,1H),8.06(d,J＝2.9Hz,1H),7.99–7.76(m,2H),7.48(dd,J＝9.1,2.9Hz,1H),5.90–5.77(m,1H),4.14(s,1H),3.32–3.07(m,8H),2.98(s,2H),2.60(s,4H),2.43(s,3H),2.31(s,3H),2.29–2.16(m,2H),1.97–1.83(m,2H),1.83–1.70(m,2H),1.66–1.53(m,2H).ESI-MS:m/z[M+H] +calculated for C 31H 38N 9O 4 +600.30,found 600.26.
化合物22的合成
Figure PCTCN2022093374-appb-000019
按照化合物21的合成方法，由化合物16(342mg,494.34μmol,1.0equiv.)为原料，得到148mg化合物22，黄色固体，产率47％。 1H NMR(400MHz,DMSO-d 6)δ10.09(s,1H),8.95(s,1H),8.74(t,1H),8.06(d,J＝2.8Hz,1H),7.85(d,J＝9.0Hz,1H),7.76(t,J＝5.8Hz,1H),7.47(dd,J＝9.1,2.9Hz,1H),5.88–5.77(m,1H),4.11(s,1H),3.47–3.40(m,4H),3.31–3.10(m,8H),2.99(s,2H),2.61(s,4H),2.42(s,3H),2.31(s,3H),2.29–2.17(m,2H),1.95–1.84(m,2H),1.82–1.71(m,2H),1.64–1.51(m,2H).ESI-MS:m/z[M+H] +calculated for C 33H 42N 9O 5 + 644.33,found 644.29.
化合物23的合成
Figure PCTCN2022093374-appb-000020
按照化合物21的合成方法，由化合物17(310mg,421.26μmol,1.0equiv.)为原料，得到120mg化合物23，黄色固体，产率41.5％。 1H NMR(400MHz,DMSO-d 6)δ10.10(s,1H),8.95(s,1H),8.86–8.62(m,1H),8.06(d,J＝2.7Hz,1H),7.85(d,J＝9.0Hz,1H),7.77(t,J＝5.6Hz,1H),7.48(dd,J＝9.0,2.7Hz,1H),5.90–5.75(m,1H),4.11(s,1H),3.59–3.40(m,10H),3.30–3.19(m,6H),2.99(s,2H),2.61(s,4H),2.42(s,3H),2.31(s,3H),2.28–2.14(m,2H),1.94–1.82(m,2H),1.82–1.67(m,2H),1.65–1.50(m,2H).ESI-MS:m/z[M+H] +calculated for C 35H 46N 9O 6 +688.36,found 688.20.
化合物24的合成
Figure PCTCN2022093374-appb-000021
化合物9(100mg,259.83μmol,1.0equiv.)和化合物13(197.05mg,389.74μmol,1.5equiv.)溶于干燥的二氯甲烷(15mL)中，然后将HATU(148.19mg,389.74μmol,1.5equiv.)和DIPEA(226.29μL,1.30mmol,5.0equiv.)加入到反应液中，室温搅拌过夜。用100mL二氯甲烷稀释反应液，水洗并盐洗，有机相用无水Na 2SO 4干燥。减压除去有机溶剂，粗产物用硅胶柱层析进行纯化(二氯甲烷/甲醇＝50:1-20:1)，得到80mg化合物24，黄色固体，产率35％。 1H NMR(400MHz,DMSO-d 6)δ10.07(s,1H),8.95(s,1H),8.01(s,1H),7.97(d,J＝2.5Hz,1H),7.93(t,J＝5.6Hz,1H),7.82(d,J＝9.0Hz,1H),7.33(dd,J＝9.1,2.4Hz,1H),7.25(s,1H),6.68(s,2H),5.82(p,J＝8.7Hz,1H),5.24(s,2H),3.70(s,3H),3.41–3.34(m,2H),3.13(s,4H),3.00(s,2H),2.68–2.54(m,6H),2.43(s,3H),2.31(s,3H),2.29–2.18(m,5H),2.13(s,3H),1.93–1.82(m,2H),1.81–1.71(m,2H),1.64–1.51(m,2H). 13C NMR(100MHz,DMSO)δ202.92,169.58,163.78,161.24,160.00,159.03,158.72,155.23,153.87,153.80,152.07,149.21,144.76,143.78,142.55,135.73,131.49,129.68,125.50,124.95,123.85,115.61,107.96,107.05,95.52,89.77,74.82,61.66,60.26,53.34,53.06,48.66,46.86,37.93,31.78,28.04,25.58,20.48,14.10,13.32,10.69. ESI-HRMS:m/z[M+H] +calculated for C 45H 51ClN 13O 4 +872.3870,found 872.3872.
实施例2：化合物25的制备
Figure PCTCN2022093374-appb-000022
将化合物8(123.36mg,300.26μmol,1.0equiv.)，化合物11(150mg,300.26μmol,1.0equiv.)和抗坏血酸钠(237.93mg,1.20mmol,4.0equiv.)溶解在t-BuOH/DCM(10mL/5mL)中，然后将CuSO 4(71.88mg,450.39μmol,1.5equiv.)溶解在5mL水中，滴加至上述反应液中，在N 2保护条件下将反应液加热至50℃并搅拌3小时。反应液冷却至室温后，旋除溶剂，残留物溶解在2M氨水(20mL)中，用二氯甲烷进行萃取，合并有机层，水洗并盐洗，有机相用无水Na 2SO 4干燥。减压除去有机溶剂，粗产物用硅胶柱层析进行纯化(二氯甲烷/甲醇＝50:1-20:1)，得到110mg化合物25，黄色固体，产率42％。 1H NMR(400MHz,DMSO-d 6)δ10.15(s,1H),8.96(s,1H),8.67(s,1H),8.10(s,1H),8.05(s,1H),7.90(s,1H),7.52(d,J＝7.7Hz,1H),7.26(s,1H),6.70(s,2H),5.90–5.76(m,1H),5.27(s,2H),4.65(t,J＝6.2Hz,2H),4.25(s,2H),3.81(s,2H),3.71(s,3H),3.23(s,4H),3.12(t,J＝6.1Hz,2H),2.43(s,3H),2.34–2.22(m,8H),2.14(s,3H),1.95–1.87(m,2H),1.82–1.74(m,2H),1.63–1.56(m,2H). 13C NMR(100MHz,DMSO)δ202.94,163.79,161.21,160.01,159.88,153.84,153.79,152.11,149.26,143.30,131.93,129.42,125.51,123.84,107.81,94.97,87.75,75.89,60.28,53.42,48.96,46.88,31.78,28.04,25.61,21.13,14.10,13.33,10.71.ESI-HRMS:m/z[M+H] +calculated for C 46H 49ClN 15O 4 +910.3775,found 910.3773.
实施例3：化合物26的制备
Figure PCTCN2022093374-appb-000023
按照化合物25的合成方法，以化合物8(113.57mg,276.42μmol,1.0equiv.)和化合物14(150mg,276.42μmol,1.0equiv.)为原料，合成得到115mg化合物25，黄色固体，产率43.6％。 1H NMR(400MHz,DMSO-d 6)δ10.09(s,1H),8.95(s,1H),8.26(t,J＝5.8Hz,1H),8.04(s,2H),7.99(s,1H),7.84(d,J＝9.0Hz,1H),7.44(d,J＝8.9Hz,1H),7.26(s,1H),6.69(s,2H),5.88–5.77(m,1H),5.26(s,2H),4.55(t,J＝6.6Hz,2H),4.36(d,J＝5.7Hz,2H),3.71(s,3H), 3.17(s,4H),3.10–2.89(m,4H),2.57(s,4H),2.42(s,3H),2.38–2.18(m,8H),2.14(s,3H),1.93–1.83(m,2H),1.81–1.71(m,2H),1.62–1.53(m,2H). 13C NMR(100MHz,DMSO)δ202.93,169.61,163.79,161.23,160.01,159.03,158.73,155.23,153.86,153.79,152.14,149.24,145.37,144.75,143.84,142.56,135.79,131.90,129.68,125.51,125.13,123.82,123.29,115.64,107.85,107.03,95.08,88.04,75.73,61.50,60.28,53.36,53.04,48.64,46.88,34.57,31.78,28.03,26.82,25.59,21.36,14.10,13.33,10.70.ESI-HRMS:m/z[M+H] +calculated for C 48H 54ClN 16O 4 +953.4197,found 953.4205.
实施例4：化合物27的制备
Figure PCTCN2022093374-appb-000024
按照化合物25的合成方法，以化合物8(68.5mg,166.75μmol,1.0equiv.)和化合物21(100mg,166.75μmol,1.0equiv.)为原料，合成得到77mg化合物27，黄色固体，产率45.7％。 1H NMR(400MHz,DMSO-d 6)δ10.09(s,1H),8.92(s,1H),8.64(s,1H),8.58(t,J＝5.3Hz,1H),8.04(s,1H),8.00(s,1H),7.95–7.78(m,2H),7.42(dd,J＝8.8,1.7Hz,1H),7.20(s,1H),6.69(s,2H),5.88–5.77(m,1H),5.24(s,2H),4.60(t,J＝6.5Hz,2H),3.71(s,3H),3.48–3.35(m,4H),3.16–3.03(m,6H),2.96(s,2H),2.55(s,4H),2.43(s,3H),2.30–2.20(m,8H),2.15(s,3H),1.94–1.85(m,2H),1.83–1.73(m,2H),1.63–1.54(m,2H). 13C NMR(100MHz,DMSO)δ202.95,169.84,163.79,161.22,160.50,159.98,158.99,158.65,155.20,153.84,153.74,152.11,149.24,144.81,143.78,143.18,142.55,135.78,131.82,129.67,127.00,125.51,125.13,123.84,115.50,107.79,107.01,94.96,87.61,75.80,65.49,60.28,53.40,52.98,48.99,48.61,46.86,38.75,38.62,31.78,28.04,25.61,21.13,14.06,13.33,10.70.ESI-HRMS:m/z[M+H] +calculated for C 50H 57ClN 17O 5 +1010.4412,found 1010.4421.
实施例5：化合物28的制备
Figure PCTCN2022093374-appb-000025
按照化合物25的合成方法，以化合物8(89.35mg,217.48μmol,1.0equiv.)和化合物22(140mg, 217.48μmol,1.0equiv.)为原料，合成得到90mg化合物28，黄色固体，产率39.2％。 1H NMR(400MHz,DMSO-d 6)δ10.09(s,1H),8.94(s,1H),8.62(s,1H),8.40(t,J＝5.0Hz,1H),8.05(s,2H),7.86(d,J＝8.3Hz,1H),7.79(s,1H),7.46(d,J＝7.7Hz,1H),7.23(s,1H),6.69(s,2H),5.88–5.75(m,1H),5.25(s,2H),4.61(t,J＝6.0Hz,2H),3.72(s,3H),3.54–3.41(m,6H),3.31–3.27(m,2H),3.19(s,4H),3.11–3.04(m,2H),3.00(s,2H),2.60(s,4H),2.43(s,3H),2.33–2.20(m,8H),2.15(s,3H),1.92–1.84(m,2H),1.81–1.73(m,2H),1.61–1.53(m,2H). 13C NMR(100MHz,DMSO)δ202.92,167.43,163.79,161.23,160.22,159.99,159.02,158.69,155.22,153.84,153.76,152.13,149.25,144.76,143.83,143.14,142.55,135.82,131.99,129.13,126.94,125.50,125.16,123.84,115.61,107.82,107.03,94.98,87.67,75.80,69.25,69.06,65.50,60.28,53.38,52.99,49.01,48.63,46.85,38.74,38.66,31.77,28.03,25.58,21.11,14.07,13.32,10.70.ESI-HRMS:m/z[M+H] +calculated for C 52H 61ClN 17O 6 +1054.4674,found 1054.4683.
实施例6：化合物29的制备
Figure PCTCN2022093374-appb-000026
按照化合物25的合成方法，以化合物8(73.47mg,178.83μmol,1.0equiv.)和化合物23(123mg,178.83μmol,1.0equiv.)为原料，合成得到88mg化合物29，黄色固体，产率44.8％。 1H NMR(400MHz,DMSO-d 6)δ10.06(s,1H),8.94(s,1H),8.62(s,1H),8.36(t,J＝4.9Hz,1H),8.05(s,2H),7.85(d,J＝8.8Hz,1H),7.76(s,1H),7.46(d,J＝8.1Hz,1H),7.23(s,1H),6.67(s,2H),5.88–5.74(m,1H),5.26(s,2H),4.61(t,J＝6.0Hz,2H),3.72(s,3H),3.53(s,6H),3.47–3.39(m,4H),3.29–3.25(m,2H),3.19(s,4H),3.09(t,J＝6.0Hz,2H),2.99(s,2H),2.60(s,4H),2.42(s,3H),2.34–2.19(m,8H),2.15(s,3H),1.96–1.84(m,2H),1.83–1.72(m,2H),1.64–1.53(m,2H). 13C NMR(100MHz,DMSO)δ202.89,169.55,163.81,161.24,160.19,160.00,159.03,158.69,155.23,153.84,153.78,152.14,149.27,144.78,143.84,143.15,142.55,135.84,131.84,129.69,126.93,125.50,125.16,123.86,115.62,107.84,107.05,95.00,87.69,75.81,70.02,69.97,69.48,69.28,61.57,60.28,53.40,53.00,49.02,48.70,46.87,38.69,38.61,31.76,28.03,25.57,21.13,14.07,13.32,10.71.ESI-HRMS:m/z[M+H] +calculated for C 54H 65ClN 17O 7 +1098.4936,found 1098.4947.
实施例7：表面等离子共振法(SPR)测定化合物与HSP90α的结合亲和力
打开Biacore T200控制软件按照标准流程安装CM5芯片，准备开始正式实验，缓冲液以较高的流速冲洗整个系统内部的流路系统。根据样本量选择合适的程序。开始捕获芯片，偶联缓冲液为PBS(pH7.4)。准备足够体积的样品，EDC/NHS，blocking buffer。开始偶联程序，最终配体偶联量约为12500RU。偶偶联完成后开始检测，缓冲液换为含1％DMSO PBS(pH7.4)，设定分析物结合时间为120s，流速为30μL/min；解离时间为300s，流速为30μL/min；再生时间30s，流速为30μL/min。按照要求准备需要检测的对应样品，开始自动运行程序进行检测。结果分析，根据运行结果，进行数据的拟合分析，得到最终的亲和力拟合KD值。
图2结果表明，与HSP90抑制剂BIIB021相比，设计合成的小分子HEMTACs对HSP90均有很好的亲和力，表明对BIIB021的N7位的改造并在该位点引入linker对小分子HEMTACs与HSP90结合的影响很小，证明了前期设计的合理性。
实施例8：Western blot实验
用化合物对B16F10细胞进行不同浓度和不同时间的处理后，通过Western blot实验测定细胞内CDK4和CDK6蛋白的水平。首先提取细胞蛋白并进行蛋白定量：用RIPA细胞裂解液(加蛋白酶抑制剂)裂解细胞，收集蛋白，并用BCA蛋白定量测定试剂盒进行定量，定量成同一浓度。蛋白变性：将蛋白置于100℃水浴8分钟，使蛋白变性。上样并跑胶：每孔上样量一般为20μL，上层浓缩胶的电泳电压为75V，下层分离胶的电泳电压为120V，待40KD的蛋白marker下至凝胶末端处1cm左右，停止电泳。转膜：使用湿转转膜仪进行转膜，PVDF膜要事先用甲醇浸泡，恒流250mA，转膜90分钟。封闭：将PVDF膜置于BSA封闭液中封闭1h(摇床慢摇)。孵育一抗：按照说明书稀释一抗，加入一抗后，4℃孵育过夜。洗膜：用TBST洗膜液洗涤3次，每次洗涤10分钟。孵育二抗：加入二抗后，室温下摇床孵育1h。洗膜：用TBST洗膜液洗涤3次，每次洗涤10分钟。曝光：将ECL显影液A液和B液等体积混合，进行曝光。
用小分子HEMTACs处理B16F10黑色素瘤细胞24h后，Western blot结果(图3)表明CDK4/6蛋白水平呈剂量依赖性降低，其中化合物26的降解活性最好，大大增强了BIIB021的降解活性。化合物26诱导B16F10细胞CDK4和CDK6降解的DC 50值(导致50％蛋白质降解的药物浓度)分别约为26nM和19nM。此外，用0.5μM化合物26处理细胞可在12小时内诱导CDK4/6的降解。以上结果说明了我们基于HSP90抑制剂BIIB021成功的建立了一种新的蛋白降解方法。
实施例9：小鼠体内抗肿瘤实验
在C57BL/6小鼠前肢腋下皮下注射B16F10细胞(10 6个细胞/小鼠)，建立小鼠B16F10黑色素瘤异种移植模型。当肿瘤体积达到100-200mm 3时，将小鼠随机分为四组(n＝6)。连续两周每天腹腔注射化合物，每两天测量小鼠体重和肿瘤体积。肿瘤体积是通过用游标卡尺沿两个正交轴来测量肿瘤的长度和宽度，并用公式V(mm 3)＝(长×宽 2)/2计算小鼠的肿瘤体积。
根据图4可知，化合物26(40mg/kg)能够显著抑制黑色素瘤的生长，并且化合物26(20mg/kg)的体内抑瘤作用优于HSP90抑制剂BIIB021(20mg/kg)。此外，化合物26导致小鼠的体重有轻微减轻，但在所有治疗组中都没有显示出其他的毒性迹象。该实验说明了我们设计合成的靶向CDK4/6的小分子HEMTAC 26具有体内抗黑色素瘤的作用。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。

Claims (10)

  1. 一种化合物，其特征在于，其具有如下式(I)所述结构：
    R 1-L-R 2
    式(I)
    其中：
    (a)R1是靶蛋白的配体/结合物；
    (b)R2是HSP90的配体；
    (c)L是连接链，所述R1和R2通过L连接起来。
  2. 如权利要求1所述的化合物，其特征在于，所述靶蛋白包括激酶、G蛋白偶联受体、转录因子、磷酸酶及RAS超家族成员；
    所述R1选自：靶向以下的化合物：激酶、G蛋白偶联受体、转录因子、磷酸酶及RAS超家族成员；
    优选的，所述R1是帕博西尼。
  3. 如权利要求1所述的化合物，其特征在于，所述R2是HSP90的配体BIIB021或其衍生物；
    优选的，所述HSP90的配体BIIB021或其衍生物的通式如下所示：
    Figure PCTCN2022093374-appb-100001
  4. 如权利要求1所述的化合物，其特征在于，所述化合物包括如下任意一种或多种：
    Figure PCTCN2022093374-appb-100002
    Figure PCTCN2022093374-appb-100003
  5. 如权利要求1-4任一项所述化合物，其特征在于，所述化合物还包括其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物。
  6. 权利要求1-5任一项所述化合物在制备肿瘤、炎症、免疫相关疾病药物中的应用；
    优选的，所述疾病还包括与细胞周期依赖性蛋白激酶4和6相关的疾病。
  7. 一种药物组合物，其特征在于，其包含权利要求1-5任一项所述化合物。
  8. 一种药物制剂，其特征在于，其包含权利要求1-5任一项所述化合物和至少一种药学上可接受的药物非活性成分。
  9. 如权利要求8所述药物制剂，其特征在于，所述药物非活性成分是药学上通常使用的载体、赋形剂及稀释剂。
  10. 一种治疗肿瘤、炎症、免疫相关疾病的方法，其特征在于，所述方法包括向受试者施用治疗有效量的权利要求1-4任一项所述化合物、权利要求7所述药物组合物或权利要求8或9所述药物制剂。
PCT/CN2022/093374 2022-03-11 2022-05-17 一种hemtac小分子降解剂及其应用 WO2023168830A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP22930459.7A EP4306528A1 (en) 2022-03-11 2022-05-17 Hemtac small molecule degradation agent and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210241970.5 2022-03-11
CN202210241970.5A CN114907386B (zh) 2022-03-11 2022-03-11 一种hemtac小分子降解剂及其应用

Publications (1)

Publication Number Publication Date
WO2023168830A1 true WO2023168830A1 (zh) 2023-09-14

Family

ID=82762960

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/093374 WO2023168830A1 (zh) 2022-03-11 2022-05-17 一种hemtac小分子降解剂及其应用

Country Status (3)

Country Link
EP (1) EP4306528A1 (zh)
CN (1) CN114907386B (zh)
WO (1) WO2023168830A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114907386B (zh) * 2022-03-11 2023-03-31 山东大学 一种hemtac小分子降解剂及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106458993A (zh) * 2014-04-14 2017-02-22 阿尔维纳斯股份有限公司 基于酰亚胺的蛋白水解调节剂和相关使用方法
CN108137507A (zh) * 2015-07-10 2018-06-08 阿尔维纳斯股份有限公司 基于mdm2的蛋白水解调节剂和相关的使用方法
CN108601764A (zh) * 2015-03-18 2018-09-28 阿尔维纳斯股份有限公司 用于靶蛋白的增强降解的化合物和方法
CN109952299A (zh) * 2016-09-14 2019-06-28 邓迪大学 用于制备蛋白降解靶向嵌合体的氟代羟脯氨酸衍生物
WO2021143816A1 (zh) * 2020-01-16 2021-07-22 江苏恒瑞医药股份有限公司 稠合酰亚胺类衍生物、其制备方法及其在医药上的应用
CN113924123A (zh) * 2019-04-09 2022-01-11 珃诺生物医药科技(杭州)有限公司 用于靶向蛋白降解的方法和组合物
CN114907386A (zh) * 2022-03-11 2022-08-16 山东大学 一种hemtac小分子降解剂及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106458993A (zh) * 2014-04-14 2017-02-22 阿尔维纳斯股份有限公司 基于酰亚胺的蛋白水解调节剂和相关使用方法
CN108601764A (zh) * 2015-03-18 2018-09-28 阿尔维纳斯股份有限公司 用于靶蛋白的增强降解的化合物和方法
CN108137507A (zh) * 2015-07-10 2018-06-08 阿尔维纳斯股份有限公司 基于mdm2的蛋白水解调节剂和相关的使用方法
CN109952299A (zh) * 2016-09-14 2019-06-28 邓迪大学 用于制备蛋白降解靶向嵌合体的氟代羟脯氨酸衍生物
CN113924123A (zh) * 2019-04-09 2022-01-11 珃诺生物医药科技(杭州)有限公司 用于靶向蛋白降解的方法和组合物
WO2021143816A1 (zh) * 2020-01-16 2021-07-22 江苏恒瑞医药股份有限公司 稠合酰亚胺类衍生物、其制备方法及其在医药上的应用
CN114907386A (zh) * 2022-03-11 2022-08-16 山东大学 一种hemtac小分子降解剂及其应用

Also Published As

Publication number Publication date
CN114907386B (zh) 2023-03-31
CN114907386A (zh) 2022-08-16
EP4306528A1 (en) 2024-01-17

Similar Documents

Publication Publication Date Title
RU2285693C2 (ru) Производные пиридина и хинолина, способ их получения, фармацевтическая композиция, применение соединений для лечения заболеваний, ассоциированных с dpp-iv
KR20220151616A (ko) 에스트로겐 수용체-연관 질환의 치료 방법
AU2007238676A1 (en) Use of biarylcarboxamides in the treatment of hedgehog pathway-related disorders
WO2018219281A1 (zh) 一类4‐嘧啶二胺类小分子有机化合物及其衍生物及其应用
JP2019508475A (ja) 化合物（ｓ）−４−（（ｓ）−３−フルオロ−３−（２−（５，６，７，８−テトラヒドロ−１，８−ナフチリジン−２−イル）エチル）ピロリジン−１−イル）−３−（３−（２−メトキシエトキシ）フェニル）ブタン酸のクエン酸塩
CN113993845A (zh) 作为治疗cns障碍例如多发性硬化的gpr17调节剂的n-(苯基)-吲哚-3-磺酰胺衍生物和相关化合物
KR20220034129A (ko) 에스트로겐 수용체 길항제 요법
TWI293301B (en) A1 adenosine receptor antagonists
RU2727194C2 (ru) Гетероциклические соединения для лечения заболевания
WO2022174525A1 (zh) 一类化合物及其制备方法和用途
WO2022174526A1 (zh) 一类化合物及其用于结直肠癌的医药用途
WO2023168830A1 (zh) 一种hemtac小分子降解剂及其应用
WO2024031965A1 (zh) 一种具有Cyclophilin A降解活性的PROTAC化合物及其制备方法与应用
WO2023077739A1 (zh) 一种喹诺酮衍生物及其制备方法和用途
AU2005321915A1 (en) Xanthurenic acid derivative pharmaceutical compositoins and methods related thereto
JP6298172B2 (ja) Ｇｐｒ１４２アゴニスト化合物
CN115477634B (zh) 用于镇痛的化合物及其医药用途
WO2023098852A1 (en) Crystal forms of thienoimidazole compound and preparation method thereof
CN101253146A (zh) 用于治疗肥胖症及相关病症的新的氨基酸衍生物
CN117229291B (zh) 内体-溶酶体转运靶向嵌合体降解剂及其制备方法与应用
US20240252657A1 (en) Hemtac small-molecule degradation agent and application thereof
CN115433160B (zh) 镇痛活性化合物及其医药用途
KR102576102B1 (ko) Uch37 탐지용 프로브 및 이의 용도
WO2022184111A1 (zh) 与tau蛋白结合的小分子化合物
CN111559982B (zh) 2-(2-取代-4-羟基嘧啶-5-甲酰胺基)乙酸类化合物及其制备方法与用途

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 2022930459

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22930459

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 18557959

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2022930459

Country of ref document: EP

Effective date: 20231012