A compound for targeted degradation of hmgb1 and preparation method and application thereof

Abstract

The application discloses a compound for targeted degradation of HMGB1 and a preparation method and application thereof, and the compound for targeted degradation of HMGB1 is a compound shown in formula (I) or a pharmaceutically acceptable salt thereof, wherein R is -(CH2) m -, -C(=O)-(CH2) n - or -(CH2CH2O) k -, m is any integer from 6 to 10, n is any integer from 5 to 9, and k is 2 or 3. The compound is a PROTAC compound spliced together by a linker from glycyrrhizic acid and an E3 ubiquitin ligase CRBN protein ligand, can specifically degrade a target protein HMGB1 through a ubiquitin-protease pathway, has the characteristics of low dosage, low toxicity and high drug efficacy, is used for preparing a drug for treating diseases related to abnormal activity or expression of HMGB1, can effectively degrade the expression level of HMGB1 protein in tumor cells, and reduces clinical drug resistance of an EGFR inhibitor.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a compound that targets and degrades HMGB1, its preparation method, and its application. Background Technology

[0002] Resistance to third-generation epidermal growth factor receptor (EGFR) inhibitors is a significant cause of treatment failure in non-small cell lung cancer (NSCLC) and a critical clinical challenge. High-Mobility Group Box 1 (HMGB1) is a highly conserved nuclear protein that participates in the regulation of cancer development through multiple cellular signaling pathways. Studies have shown that upregulation of HMGB1 can lead to resistance to EGFR inhibitors (such as osimertinib). Therefore, degrading the expression level of HMGB1 protein in tumor cells can be considered a strategy to reduce resistance to EGFR-targeting inhibitors. Research has found that glycyrrhizic acid, a clinically used natural anti-inflammatory and antiviral triterpenoid, can form a hydrophobic interaction with the shallow concave surface of the HMG box of HMGB1 through its terpene ring structure. Furthermore, it can form electrostatic interactions and hydrogen bonds with positively charged residues via carboxyl and carbonyl groups. This allows glycyrrhizic acid to bind to HMGB1 and form a stable complex. This binding can inhibit the chemotactic and proliferative activities of cancer cells to some extent, but this inhibitory effect needs improvement and lacks selectivity. Therefore, it is necessary to develop a highly efficient and selective degrader for the HMGB1 protein to reduce clinical resistance to third-generation EGFR inhibitors. Summary of the Invention

[0003] In view of this, the present application provides a compound that targets the degradation of HMGB1, its preparation method and application. The compound is a protein degradation targeting chimeric (PROTAC) compound spliced ​​together with glycyrrhizic acid and E3 ubiquitin ligase Cereblon (CRBN) protein ligand. It can target the degradation of HMGB1 with high specificity and good efficacy. It can be used to prepare anti-tumor drugs and can effectively reduce the protein level of HMGB1 in tumor cells, thereby inhibiting the proliferation of tumor cells.

[0004] In a first aspect, embodiments of this application provide a compound that targets and degrades HMGB1, which is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0005]

[0006] Where R is -(CH2) m -、-C(=O)(CH2) n -or-(CH2CH2O) k-, m is any integer from 6 to 10, n is any integer from 5 to 9, and k is 2 or 3.

[0007] Secondly, embodiments of this application provide a method for preparing a compound that targets and degrades HMGB1, comprising:

[0008] The compound shown in formula (VII), glycyrrhizic acid, and a condensing agent are mixed to form a condensation reaction solution. The condensation reaction yields the compound provided in the first aspect of this application for targeted degradation of HMGB1.

[0009]

[0010] Where R is -(CH2) m -、-C(=O)(CH2) n -or-(CH2CH2O) k -, m is any integer from 6 to 10, n is any integer from 5 to 9, and k is 2 or 3.

[0011] In the embodiments of this application, the method for preparing the compound represented by formula (VII) includes:

[0012] The compound represented by formula (II), the compound represented by formula (III), and the first catalyst are mixed to form a first substitution reaction solution. The first substitution reaction yields the compound represented by formula (IV), wherein X is selected from F, Cl, or Br.

[0013] (II) (III) (IV)

[0014] The compound shown in formula (Ⅳ), the compound shown in formula (Ⅴ), and the second catalyst are mixed to form a second substitution reaction solution. The compound shown in formula (Ⅵ) is obtained by the second substitution reaction, wherein R is -(CH2). m -、-C(=O)(CH2) n -or-(CH2CH2O) k -, m is any integer from 6 to 10, n is any integer from 5 to 9, and k is 2 or 3;

[0015] (V) (VI)

[0016] Finally, the compound shown in formula (VI) was subjected to a deBoc reaction to obtain the compound shown in formula (VII).

[0017] In some embodiments of this application, the molar ratio of the compound represented by formula (II) to the compound represented by formula (III) in the first substitution reaction solution is (1-2):1; the first catalyst comprises potassium acetate; the temperature of the first substitution reaction is 100℃-130℃, and the time of the first substitution reaction is 15h-20h; and / or,

[0018] In the second substitution reaction solution, the molar ratio of the compound represented by formula (Ⅳ) to the compound represented by formula (Ⅴ) is (1-2):1; the second catalyst includes triethylamine; the temperature of the second substitution reaction is 100℃-130℃, and the reaction time is 2h-4h; and / or,

[0019] The temperature for the deBoc reaction is 20℃-30℃, and the time is 1h-3h.

[0020] In some embodiments of this application, the condensation reaction is carried out under nitrogen protection; the molar ratio of the compound represented by formula (VII) to glycyrrhizic acid in the condensation reaction solution is (1-2):1; the condensing agent includes one or more of benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, propylphosphonic anhydride, and N,N-carbazide; the temperature of the condensation reaction is 20°C-30°C, and the time of the condensation reaction is 1h-5h.

[0021] Thirdly, embodiments of this application also provide a medicament for treating diseases related to abnormal HMGB1 activity or expression, including compounds that target and degrade HMGB1 as provided in the first aspect of this application or compounds that target and degrade HMGB1 prepared by the preparation method provided in the second aspect of this application.

[0022] In this application, compounds that target and degrade HMGB1 are used as a single active ingredient or together with other pharmaceutically acceptable active ingredients to constitute a drug for treating diseases related to abnormal HMGB1 activity or expression.

[0023] In this application, the medicament for treating diseases associated with abnormal HMGB1 activity or expression also includes one or more pharmaceutically acceptable carriers and excipients.

[0024] Fourthly, the embodiments of this application also provide the use of the compounds that target HMGB1 degradation provided in the first aspect or the compounds that target HMGB1 degradation prepared by the preparation method provided in the second aspect in the preparation of medicaments for treating diseases related to abnormal HMGB1 activity or expression.

[0025] In the embodiments of this application, diseases associated with abnormal HMGB1 activity or expression include one or more of the following: lung cancer, liver cancer, pancreatic cancer, breast cancer, cervical cancer, colon cancer, nasopharyngeal carcinoma, glioma, thyroid cancer, head and neck cancer, gastric cancer, kidney cancer, prostate cancer, testicular cancer, endometrial cancer, ovarian cancer, skin cancer, and lymphoma. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments of this application will be described below.

[0027] Figure 1 The LC-MS spectrum of CPD-4-intermediate 1 shown in formula (Ⅳ-1) in Example 1 of this application;

[0028] Figure 2 The 1H NMR spectrum of CPD-4-intermediate 1 as shown in formula (Ⅳ-1) in Example 1 of this application;

[0029] Figure 3 The LC-MS spectrum of CPD-4-intermediate 2 shown in formula (VI-1) in Example 1 of this application;

[0030] Figure 4 The 1H NMR spectrum of CPD-4-intermediate 2 as shown in formula (VI-1) in Example 1 of this application;

[0031] Figure 5 The LC-MS spectrum of CPD-4-intermediate 3 shown in formula (Ⅶ-1) in Example 1 of this application;

[0032] Figure 6 The LC-MS spectrum of CPD-4 shown in formula (Ⅰ-1) in Example 1 of this application;

[0033] Figure 7 The HPLC spectrum of CPD-4 shown in formula (Ⅰ-1) in Example 1 of this application;

[0034] Figure 8 The 1H NMR spectrum of CPD-4 shown in formula (Ⅰ-1) in Example 1 of this application;

[0035] Figure 9 This is a three-dimensional structural diagram of the CPD-4 molecule shown in formula (Ⅰ-1) in Example 1 of this application;

[0036] Figure 10 This is a graph showing the degradation activity of CPD-4 on HMGB1 protein as shown in formula (Ⅰ-1) in Example 1 of this application;

[0037] Figure 11The above is a SPPIER experiment result diagram of CPD-4 shown in formula (Ⅰ-1) in Example 1 of this application, wherein (a) is the fluorescence diagram of each group of cells, (b) is the fluorescence peak diagram of the area pointed to by the arrow in (a) control group, and (c) is the fluorescence peak diagram of the area pointed to by the arrow in (a) CPD-4 group;

[0038] Figure 12 This is a graph showing the cytotoxicity detection results of CPD-4 as shown in formula (Ⅰ-1) in Example 1 of this application;

[0039] Figure 13 This is a graph showing the antitumor cell activity test results of CPD-4 as shown in formula (Ⅰ-1) in Example 1 of this application;

[0040] Figure 14 This is a diagram showing the effect of CPD-4 inhibiting tumor cell proliferation, as shown in formula (Ⅰ-1) in Example 1 of this application;

[0041] Figure 15 The image shows the results of CPD-4 inhibiting tumor cell colony formation as shown in formula (Ⅰ-1) in Example 1 of this application. (a) shows the colony formation of tumor cells on a plate after treatment with CPD-4, and (b) shows the statistical results of the number of tumor cell colonies after treatment with CPD-4.

[0042] Figure 16 This is a diagram showing the effect of CPD-4 on tumor cell apoptosis, as shown in formula (Ⅰ-1) in Example 1 of this application. Figure 16 (a) shows a cell cycle analysis of tumor cells after CPD-4 treatment. Figure 16 (b) shows the statistical results of the number of tumor cells in different cell cycles after CPD-4 treatment;

[0043] Figure 17 The figure shows the results of CPD-4 inhibiting tumor growth in a mouse xenograft model experiment as shown in formula (Ⅰ-1) in Example 1 of this application, where (a) is the tumor removed from each group of mice and (b) is the statistical result of the tumor weight of each group.

[0044] Figure 18 The LC-MS spectrum of CPD-1-intermediate 2 shown in formula (VI-2) in Comparative Example 1 of this application is shown.

[0045] Figure 19 The LC-MS spectrum of CPD-1-intermediate 3 shown in formula (Ⅶ-2) in Comparative Example 1 of this application is shown.

[0046] Figure 20 The LC-MS spectrum of CPD-1 shown in formula (1) in Comparative Example 1 of this application is shown.

[0047] Figure 21The hydrogen NMR spectrum of CPD-1 shown in formula (1) of Comparative Example 1 of this application;

[0048] Figure 22 This is a three-dimensional structural diagram of the CPD-1 molecule in Comparative Example 1 of this application;

[0049] Figure 23 The LC-MS spectrum of CPD-2-intermediate 2 shown in formula (VI-3) of Comparative Example 2 of this application is shown.

[0050] Figure 24 The LC-MS spectrum of CPD-2-intermediate 3 shown in formula (Ⅶ-3) of Comparative Example 2 of this application is shown.

[0051] Figure 25 The LC-MS spectrum of CPD-2 shown in formula (2) of Comparative Example 2 of this application;

[0052] Figure 26 The 1H NMR spectrum of CPD-2 shown in formula (2) of Comparative Example 2 of this application;

[0053] Figure 27 This is a three-dimensional structural diagram of the CPD-2 molecule in Comparative Example 2 of this application;

[0054] Figure 28 The LC-MS spectrum of CPD-3-intermediate 2 shown in formula (VI-4) of Comparative Example 3 of this application is shown.

[0055] Figure 29 The LC-MS spectrum of CPD-3-intermediate 3 shown in formula (Ⅶ-4) of Comparative Example 3 of this application is shown.

[0056] Figure 30 The LC-MS spectrum of CPD-3 shown in formula (3) of Comparative Example 3 of this application is shown.

[0057] Figure 31 The 1H NMR spectrum of CPD-3 shown in formula (3) of Comparative Example 3 of this application;

[0058] Figure 32 This is a three-dimensional structural diagram of the CPD-3 molecule in Comparative Example 3 of this application;

[0059] Figure 33 This is a comparison chart showing the degradation activity of CPD-1, CPD-2, CPD-4 and CPD-4 on HMGB1 protein in the embodiments of this application. Detailed Implementation

[0060] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0061] Unless otherwise specified, the raw materials used in the embodiments of this application are commercially available products.

[0062] Epidermal growth factor receptor (EGFR) is a common gene mutation in lung cancer, and it is highly or abnormally expressed in various malignant tumors.

[0063] High-mobility group box 1 (HMGB1): A highly conserved nuclear protein widely distributed in mammalian cells, it can participate in the regulation of cancer development through multiple cell signaling pathways.

[0064] PROTAC (Proteolysis Targeting Chimera) compounds are hybrid bifunctional small molecule compounds composed of a target protein ligand and an E3 ligase ligand spliced ​​together via a linker. After entering the cell, the target ligand in the PROTAC compound structure specifically binds to the corresponding target protein, while the other end recruits the E3 ligase. The E3 ligase mediates ubiquitination of the target protein by a ubiquitin-conjugating enzyme. The ubiquitinated target protein is then recognized and degraded by the proteasome. PROTAC compounds specifically degrade target proteins via the ubiquitin-protease pathway, requiring no prolonged occupation of the binding site; the ubiquitination of the target protein can be completed instantaneously with only a brief formation of the ternary complex.

[0065] Half-maximal inhibitory concentration (IC50) refers to the concentration at which a drug inhibits a specific biological response by 50%. This indicator is often used to measure the toxicity of a drug to cells or the tolerance of cells to a drug. The smaller the IC50 value, the stronger the inhibitory effect of the drug on cells.

[0066] Third-generation EGFR inhibitors (such as AZD9291, also known as osimertinib) are primarily indicated for non-small cell lung cancer (NSCLC) patients with confirmed EGFR mutations and resistance to other EGFR inhibitors. They work by irreversibly binding to EGFR and preventing its phosphorylation, thereby blocking downstream signaling pathways and inhibiting tumor cell proliferation and survival. However, long-term use can lead to clinical resistance, a major cause of treatment failure in NSCLC. One key resistance mechanism is that cancer cells bypass the inhibitory effect of AZD9291 by activating other signaling pathways. Studies have shown a correlation between HMGB1 and chemotherapy resistance to third-generation EGFR inhibitors. Based on this finding, targeting HMGB1 degradation could be a core strategy for improving the sensitivity of EGFR inhibitor chemotherapy and reducing clinical resistance. Therefore, blocking the HMGB1-mediated resistance signaling pathway holds promise for providing an innovative intervention pathway to address the resistance dilemma in EGFR-targeted therapy.

[0067] Therefore, this application provides a compound that targets and degrades HMGB1, its preparation method, and its application. This compound is a protein degradation-targeting chimeric compound (PROTAC) composed of glycyrrhizic acid and an E3 ubiquitin ligase Cereblon (CRBN) protein ligand. It can target and degrade HMGB1, possessing the advantages of low toxicity, high specificity, and good efficacy. It can exert its therapeutic effect at low dosages. When used in the preparation of antitumor drugs, it can effectively reduce the protein level of HMGB1 in tumor cells, thereby inhibiting the proliferation of tumor cells.

[0068] This application provides a compound that targets and degrades HMGB1, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0069]

[0070] Where R is -(CH2) m -、-C(=O)(CH2) n -or-(CH2CH2O) k -, m is any integer from 6 to 10, n is any integer from 5 to 9, and k is 2 or 3.

[0071] The HMGB1-targeting degradation compound provided in this application is a PROTAC compound composed of glycyrrhizic acid, an E3 ubiquitin ligase CRBN protein ligand, and a linker, wherein the linker is defined as R in formula (I) and the amino groups at its ends. This PROTAC compound specifically recognizes HMGB1 and tags it with ubiquitination. The ubiquitinated HMGB1 binds to glycyrrhizic acid and undergoes degradation. The linker molecule used in this compound is a classic alkyl chain linker with a suitable chain length, which allows for the binding of the CRBN protein ligand to glycyrrhizic acid without affecting the structure of either, and also improves the flexibility and freedom of the compound molecule, thus enhancing its selectivity and degradation efficiency for HMGB1. Furthermore, this HMGB1-targeting degradation compound can scavenge both active and inactive HMGB1, and its efficacy is not easily affected by increases or mutations in HMGB1, thus overcoming EGFR inhibitor resistance to some extent.

[0072] In some embodiments of this application, R is -(CH2). m -, m can be 6, 7, 8, 9, or 10, meaning R is C6-C 10 Linear alkylene compounds. In some specific embodiments of this application, R is -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, or -(CH2) 10 - One of them.

[0073] In some embodiments of this application, R is -C(=O)(CH2). n -, n can be 5, 6, 7, 8, or 9, meaning R is C6-C 10 Linear alkylene acyl group. In some specific embodiments of this application, R is -C(=O)(CH2)5-, -C(=O)(CH2)6-, -C(=O)(CH2)7-, or -C(=O)(CH2). 8- One of -C(=O)(CH2)9-.

[0074] In other embodiments of this application, R is -(CH2CH2O)2- or -(CH2CH2O)3-.

[0075] The R in the following text is the same as here, and will not be repeated.

[0076] It should be noted that the structural formulas described in this application include all stereoisomers (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, and (Z) and (E) conformational isomers. Therefore, any single stereochemical isomer of the compound of this application or its enantiomers, diastereomers, or mixtures of geometric isomers (or conformational isomers) are within the scope of this application.

[0077] In embodiments of this application, pharmaceutically acceptable salts include acid addition salts formed by the compound of formula (I) and one or more of the following acids: hydrochloric acid, hydrobromic acid, and sulfuric acid. In some embodiments of this application, compounds that target the degradation of HMGB1 include one or more of the compound of formula (I), the hydrochloride salt of the compound of formula (I), the hydrobromide salt of the compound of formula (I), and the sulfate salt of the compound of formula (I).

[0078] In the embodiments of this application, the half-maximal inhibitory concentration (IC50) of the compound represented by formula (I) or its pharmaceutically acceptable salt for inhibiting tumor cell growth can be 13.68 μmol / L. In some embodiments of this application, the IC50 of the compound represented by formula (I) for inhibiting the growth of human lung cancer cells PC-9R is 13.68 μmol / L.

[0079] The PROTAC compounds provided in this application use glycyrrhizic acid as a target protein ligand, which specifically recognizes HMGB1 and tags it with ubiquitination. The ubiquitinated HMGB1 is precisely recognized and bound by intracellular proteases. Once binding occurs, it induces the interaction between glycyrrhizic acid and the two shallow depressions formed by the two arms of two HMGB1 molecules, leading to degradation. Each PROTAC molecule can degrade multiple target protein molecules (HMGB1), thus achieving significant efficacy even at low doses. Furthermore, the specificity of PROTAC compounds gives them high safety in vivo. Unlike traditional small molecule inhibitors, PROTAC compounds do not exert their effects by inhibiting protein function but directly mediate the degradation of disease proteins. Therefore, high concentrations of inhibitors are not required to occupy the target site. This mechanism reduces the occurrence of side effects, thereby avoiding interference and toxicity to normal cells.

[0080] The choice of linker in the PROTAC compounds provided in this application is crucial. Firstly, the linker must be able to bind to both glycyrrhizic acid and the E3 ubiquitin ligase CRBN protein ligand without affecting their structures. Secondly, the linker length of the PROTAC compound significantly affects its activity, selectivity, and degradation efficiency. The linker length influences the docking angle between the PROTAC compound and the HMGB1 and E3 ligase CRBN proteins. Short linkers have strong rigidity, limiting molecular freedom and making it difficult for the PROTAC compound to flexibly adjust itself to adapt to the protein binding site. Long linkers, on the other hand, have a longer distance and more degrees of freedom. The linker length of a PROTAC compound makes it easier to bypass spatial barriers and reach specific target proteins, thus improving the selectivity between the ligands at both ends and the target protein, and consequently increasing the efficiency of targeted degradation. Thirdly, the linker length of a PROTAC compound affects drug metabolism. In the cellular environment, long linkers increase the hydrophobicity of the PROTAC compound, affecting cell permeability. Long linkers also typically increase the molecular weight of the PROTAC compound, affecting its metabolic and distribution characteristics. Compared to PROTAC compounds with short linkers, those with long linkers exhibit a longer half-life in vivo, requiring a longer time to complete metabolism or clearance. Therefore, the linker backbone of the PROTAC compound for targeted degradation of HMGB1 provided in this application has a moderate length, ensuring both high selectivity and degradation efficiency for HMGB1 while also accommodating a shorter drug metabolism time in vivo.

[0081] The linker sites between the linker and the ligands at both ends of the PROTAC compound provided in this application also determine the degradation effect of the compound on HMGB1 protein. Studies have found that small changes in the linker sites of isomer compounds can significantly affect the selectivity and binding efficiency of the target protein ligands to the target protein. The specific linker sites between the linker and the ligands at both ends of the compound provided in this application can simultaneously take into account the selectivity for HMGB1 and the metabolic clearance rate of the compound, thus exhibiting superior pharmacological performance.

[0082] In summary, the PROTAC compound targeting HMGB1 degradation provided in this application exhibits superior flexibility, specificity, efficiency, and safety compared to traditional HMGB1 inhibitors. It also features low dosage, low toxicity, and high efficacy. When used to prepare antitumor drugs, it can effectively reduce the protein level of HMGB1 in tumor cells, overcoming the multifunctionality of HMGB1 in the cell nucleus and preventing it from helping tumor cells evade treatment by regulating DNA repair or autophagy. This reduces clinical resistance to EGFR inhibitors (AZD9291) and thus inhibits tumor cell proliferation.

[0083] This application provides a method for preparing a compound that targets and degrades HMGB1, comprising:

[0084] S101: The compound shown in formula (II), the compound shown in formula (III), and the first catalyst are mixed to form a first substitution reaction solution. The compound shown in formula (IV) is obtained through a first substitution reaction, wherein X is selected from F, Cl, or Br.

[0085] (II) (III) (IV)

[0086] S102: The compound shown in formula (Ⅳ), the compound shown in formula (Ⅴ), and the second catalyst are mixed to form a second substitution reaction solution, and the compound shown in formula (Ⅵ) is obtained through a second substitution reaction.

[0087] (V) (VI)

[0088] S103: The compound shown in formula (VI) is subjected to a deBoc reaction to obtain the compound shown in formula (VII);

[0089]

[0090] S104: Finally, the compound shown in formula (VII), glycyrrhizic acid, and a condensing agent are mixed to form a condensation reaction solution. After condensation reaction, the compound shown in formula (I) that targets and degrades HMGB1 is obtained.

[0091]

[0092] The preparation method of the compound that targets and degrades HMGB1 provided in this application is simple in steps, easy to operate, has low pollution, high yield of target substance, low requirements for reaction time and temperature, and low preparation cost. It is suitable for producing drugs to treat diseases related to abnormal HMGB1 activity or expression.

[0093] In step S101, the compound represented by formula (II), the compound represented by formula (III), and the first catalyst are soluble in a first organic solvent to carry out a first substitution reaction. The first organic solvent may be, for example, but is not limited to, acetic acid. A suitable solvent is beneficial to accelerate the reaction rate and enhance the product selectivity.

[0094] In this application, the first catalyst may be, but is not limited to, potassium acetate. The cations in the first catalyst can provide the active sites required for the first substitution reaction, reducing the activation energy of the reaction and thus accelerating the reaction rate. In some embodiments of this application, the molar number of the first catalyst in the first substitution reaction solution is 0.8 to 1.3 times the sum of the molar numbers of the reactants, for example, but not limited to, 0.8, 0.9, 0.95, 1, 1.1, 1.2, and 1.3 times. An appropriate amount of catalyst helps reduce the formation of by-products, increase the yield of the target product per unit time, save the time and heat energy required to complete the reaction, and avoid waste of raw materials.

[0095] In this application, the temperature of the first substitution reaction is 100℃-120℃, for example, but not limited to 100℃, 105℃, 110℃, 115℃, and 120℃; the time of the first substitution reaction is 13h-18h, for example, but not limited to 13h, 14h, 15h, 16h, 17h, and 18h. In some embodiments of this application, the temperature of the first substitution reaction is 110℃-120℃, and the time is 15h-17h. Suitable reaction temperature and reaction time can provide the necessary activation energy for the first substitution reaction, which is beneficial to the complete reaction of the reactants, while avoiding the occurrence of side reactions.

[0096] In this embodiment of the application, step S101 further includes, after the first substitution reaction is completed, filtering, evaporating, washing and vacuum drying the reaction solution in sequence to obtain the solid compound shown in formula (Ⅳ).

[0097] In this application, the synthetic route of the compound represented by formula (Ⅳ) is as follows:

[0098]

[0099] In step S102, the compound represented by formula (IV), the compound represented by formula (V), and the second catalyst are soluble in a second organic solvent to carry out the second substitution reaction. The second organic solvent helps to dissolve and disperse the reactants, thereby accelerating the substitution reaction. In some embodiments of this application, the second organic solvent may be, for example, but not limited to, one or more of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and acetonitrile (ACN).

[0100] In this embodiment, the second catalyst includes a base catalyst, such as, but not limited to, triethylamine. The base catalyst is nucleophilic and basic, and can combine with electrophilic centers in the reactants to form nucleophilic substitution reaction intermediates, thereby lowering the activation energy of the reaction and promoting the second substitution reaction. In some embodiments of this application, the molar number of the second catalyst in the second substitution reaction solution is 0.8 to 1.3 times the sum of the molar numbers of the reactants, for example, but not limited to, 0.8, 0.9, 0.99, 1, 1.1, 1.2, and 1.3 times.

[0101] In the embodiments of this application, the temperature of the second substitution reaction is 110℃-130℃, for example, but not limited to 110℃, 115℃, 120℃, 125℃, and 130℃; the time of the second substitution reaction is 1h-3h, for example, but not limited to 1h, 1.5h, 2h, 2.5h, and 3h. In some embodiments of this application, the temperature of the second substitution reaction is 115℃-125℃, and the time is 1.5h-2.5h. Suitable reaction temperature and reaction time can provide the necessary activation energy for the second substitution reaction, which is beneficial to the complete reaction of the reactants, while avoiding the occurrence of side reactions.

[0102] In this embodiment of the application, step S102 further includes concentrating and purifying the reaction solution after the second substitution reaction is completed to obtain a solid compound of formula (VI).

[0103] In this application, the synthetic route of the compound represented by formula (VI) is as follows:

[0104]

[0105] In step S103, the compound shown in formula (VI) is dissolved in a third organic solvent, and then an acid catalyst is slowly added dropwise to the solution to carry out the de-tert-butyloxycarbonyl (Boc) reaction. The third organic solvent may be, for example, but not limited to, one or more of dichloromethane, dioxane, and methanol; the acid catalyst may be, for example, but not limited to, one or more of trifluoroacetic acid (TFA), hydrochloric acid, formic acid, and p-toluenesulfonic acid, which can break and hydrolyze the Boc group into tert-butanol and amine salt, thereby removing the Boc group from the compound shown in formula (VI). The slow dropwise addition helps to control the rate of the de-Boc reaction and avoids excessively high local concentrations that could lead to side reactions or the generation of large amounts of gas.

[0106] In the embodiments of this application, the concentration of the compound represented by formula (VI) in the third organic solvent is 25 mg / mL to 35 mg / mL, for example, but not limited to, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 31.25 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, and 35 mg / mL. In some specific embodiments of this application, the concentration of the compound represented by formula (VI) in the third organic solvent is 30 mg / mL to 33 mg / mL.

[0107] In the embodiments of this application, the volume ratio of the acid catalyst to the third organic solvent is 1:(3-5), for example, but not limited to 1:3, 1:3.5, 1:4, 1:4.5, 1:5.

[0108] In this embodiment, the Boc removal reaction is carried out at room temperature, such as, but not limited to, 20°C, 22°C, 24°C, 26°C, 28°C, or 30°C; the Boc removal reaction time is 1-2 hours.

[0109] In this embodiment of the application, step S103 further includes drying the reaction solution by rotary evaporation after the Boc removal reaction is completed to obtain a solid compound of formula (VII).

[0110] In this application, the synthetic route of the compound represented by formula (VII) is as follows:

[0111]

[0112] In step S104, the compound represented by formula (VII), the glycyrrhizic acid represented by formula (VIII), and the condensing agent are soluble in a fourth organic solvent to carry out the condensation reaction. The fourth organic solvent can increase the interaction between reactant molecules, thereby accelerating the reaction rate. In some embodiments of this application, the fourth organic solvent may be, for example, but not limited to, one or more of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and N-methylpyrrolidone (NMP).

[0113] In this embodiment, the condensation reaction solution also includes an alkaline catalyst, which may be, but is not limited to, one or more of triethylamine, N,N-diisopropylethylamine (DIEA), pyridine, and sodium carbonate. The alkaline catalyst can react with carboxyl groups to generate amide intermediates, which can reduce the activation energy of the reaction while maintaining the stability of the system, neutralizing the acidic byproducts generated during the reaction, promoting the forward condensation reaction, and helping to improve the selectivity and yield of the condensation reaction.

[0114] In the embodiments of this application, the condensing agent may be, for example, but not limited to, one or more of benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), propylphosphonic anhydride, and N,N-carbazide. The condensing agent can activate the reactants, stabilize the transition state and avoid side reactions by forming intermediates with the reactants, making the condensation reaction between the reactants easier. In addition, the condensing agent can lower the activation energy of the reaction and also has a certain catalytic effect, allowing the condensation reaction to proceed at a lower temperature, which helps to improve the yield and selectivity of the condensation product.

[0115] In the embodiments of this application, the molar concentration ratio of the compound represented by formula (VII) to glycyrrhizic acid in the condensation reaction solution is 1:(1-2); the molar concentration ratio of the base catalyst to the total molar concentration of the reactants is 1:(1-2).

[0116] In the embodiments of this application, the condensation reaction is carried out at room temperature, such as, but not limited to, 20°C, 22°C, 24°C, 26°C, 28°C, or 30°C; the time for the deBoc reaction is 1-3 hours, such as, but not limited to, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours.

[0117] In this embodiment of the application, step S104 further includes concentrating and purifying the reaction solution after the condensation reaction to obtain a solid compound of formula (I).

[0118] In this application, the synthetic route of the compound represented by formula (Ⅰ) is as follows:

[0119]

[0120] The molar ratios of the compounds represented by formula (II) and (III) in the first substitution reaction solution, the molar ratios of the compounds represented by formula (IV) and (V) in the second substitution reaction solution, and the molar ratios of the compound represented by formula (VII) and glycyrrhizic acid in the condensation reaction solution are all independently 1:(1-5), for example, but not limited to 1:1, 1:1.1, 1:2, 1:3, 1:4, and 1:5. Appropriate molar ratios of reactants ensure rapid reaction without wasting reactants, thus helping to control drug production costs. In some embodiments of this application, the molar ratios of the compounds represented by formula (II) and (III) in the first substitution reaction solution, the molar ratios of the compounds represented by formula (IV) and (V) in the second substitution reaction solution, and the molar ratios of the compound represented by formula (VII) and glycyrrhizic acid in the condensation reaction solution are all independently 1:(1-2).

[0121] The first substitution reaction in step S101, the second substitution reaction in step S102, and the condensation reaction in step S104 are all carried out under a protective atmosphere, which may be, but is not limited to, nitrogen or argon. Carrying the reaction under a protective atmosphere helps to isolate oxygen and prevent the reactants from being oxidized, thus affecting the purity and yield of the product.

[0122] Steps S101, S102, S103 and S104 include, but are not limited to, monitoring the progress of each reaction by using liquid chromatography-mass spectrometry (LC-MS) and / or nuclear magnetic resonance hydrogen spectroscopy.

[0123] In summary, the synthetic route for the compound representing the targeted degradation of HMGB1, or its pharmaceutically acceptable salt, as shown in Formula (Ⅰ) of this application is as follows:

[0124]

[0125] Where R is -(CH2) m -、-C(=O)(CH2) n -or-(CH2CH2O) k -, m is any integer from 6 to 10, n is any integer from 5 to 9, k is 2 or 3; X is selected from F, Cl or Br.

[0126] The preparation method of the compound that targets and degrades HMGB1 provided in this application is simple in steps, easy to operate, has low pollution, high yield of target substance, low requirements for reaction time and temperature, and low preparation cost. It is suitable for producing drugs to treat diseases related to abnormal HMGB1 activity or expression.

[0127] In some embodiments of this application, a pharmaceutically acceptable salt of the compound represented by formula (I) can be prepared by reacting the compound represented by formula (I) with an acid, wherein the acid can be one or more of hydrochloric acid, hydrobromic acid, and sulfuric acid. In some embodiments of this application, the hydrochloride salt of the compound represented by formula (I) can be prepared by reacting the compound represented by formula (I) with hydrochloric acid. In some embodiments of this application, the hydrobromide salt of the compound represented by formula (I) can be prepared by reacting the compound represented by formula (I) with hydrobromic acid. In some embodiments of this application, the sulfate salt of the compound represented by formula (I) can be prepared by reacting the compound represented by formula (I) with sulfuric acid.

[0128] This application also provides a drug for treating diseases related to abnormal HMGB1 activity or expression. The drug contains the HMGB1-targeting degradative compound provided above or the HMGB1-targeting degradative compound prepared by the preparation method provided above. It can effectively reduce the HMGB1 protein level in tumor cells, further inhibit the upregulation of HER3 protein expression, reactivate the suppressed tumor suppressor gene, thereby inducing tumor cell differentiation and promoting tumor apoptosis, and achieving the effect of treating tumors.

[0129] In the embodiments of this application, the medicament for treating diseases related to abnormal HMGB1 activity or expression uses the compound that targets and degrades HMGB1 provided in this application as a single active ingredient; or, the medicament for treating diseases related to abnormal HMGB1 activity or expression includes the compound that targets and degrades HMGB1 provided in this application and other pharmaceutically acceptable active ingredients.

[0130] In this application, the medicament for treating diseases related to abnormal HMGB1 activity or expression further includes one or more pharmaceutically acceptable carriers and excipients. In this case, the medicament simultaneously contains a compound that targets and degrades HMGB1, as well as a pharmaceutically acceptable carrier and / or excipients. In some embodiments of this application, the role of the "pharmaceutically acceptable carrier" is to transport the medicament in this application so that it can exert its intended effect. Generally, transport is from one organ or part to another organ or part; the carrier must be compatible with the drug component, not affect the biological activity of the drug, and be relatively non-toxic. For example, once the carrier enters the body, it will not cause serious reactions due to its toxic side effects or the drug it carries, and it will not have a negative impact on the patient.

[0131] In the embodiments of this application, pharmaceutically acceptable carriers may be, but are not limited to, one or more of solvents, polymers, liposomes, recombinant viral vectors, and eukaryotic recombinant expression vectors. In some embodiments of this application, the solvent includes, but is not limited to, one or more of water, physiological saline, and other non-aqueous solvents; in other embodiments of this application, the polymer may be, but is not limited to, polylysine, polyethyleneimine (branched and / or chain-like) and its modified forms, polyamide-amine dendritic polymer (PAMAM) and its derivatives, polypropyleneimine dendritic polymer (PPI) and its derivatives, chitosan, polylactic-co-glycolic acid (PLGA), polylactic acid, gelatin, cyclodextrin, sodium alginate, albumin, and hemoglobin. Polyethyleneimine and its modified forms, PAMAM and its derivatives, PPI and its derivatives, chitosan, etc., may be referred to as cationic polymers. In other embodiments of this application, liposomes may be self-assembled from cationic lipids, neutral accessory lipids, cholesterol, and phospholipids (such as soybean lecithin, egg yolk lecithin, and cephalin), or they may be formed by distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) intercalating within a phospholipid layer formed by phospholipid molecules. In other embodiments of this application, the recombinant viral vector may be, but is not limited to, one or more of lentiviral vectors, adenoviral vectors, and retroviral vectors.

[0132] In this application, compounds that target and degrade HMGB1 in drugs used to treat diseases related to abnormal HMGB1 activity or expression can be dispersed or adsorbed in the above-mentioned carrier to form a dispersion system, or they can be encapsulated / encapsulated by the above-mentioned liposomes, polymers, etc. to form spherical structures (e.g., nanocapsules or microcapsules).

[0133] In this application, the excipients include one or more diluents and excipients. The main function of the diluent is to fill the weight or volume of the tablet to facilitate tableting. In some embodiments of this application, the diluent includes one or more of starches, sugars, celluloses, and inorganic salts. Excipients refer to other additives in the drug besides the main active ingredient. In some embodiments of this application, excipients include, for example, binders, fillers, disintegrants, and lubricants in tablets; wine, vinegar, and medicinal juice in pills; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0134] In this application, the dosage form of the drug for treating diseases related to abnormal HMGB1 activity or expression includes tablets, capsules, powders, granules, pills, syrups, solutions, or suspensions, with the specific application form depending on the actual situation. In some embodiments of this application, the drug for treating diseases related to abnormal HMGB1 activity or expression can be administered orally or by injection. When administered by injection, the drug dosage form is preferably a solution, such as dissolved in water, physiological saline, or other solvents. In some specific embodiments of this application, the injection can be administered via intraperitoneal injection, subcutaneous injection, intramuscular injection, or intravenous injection. In this application, the drug for treating diseases related to abnormal HMGB1 activity or expression can be administered locally or systemically. The dosage of the drug depends on various factors, including but not limited to the required biological activity and the subject's tolerance to the drug. In some specific embodiments of this application, the dosage of the drug for treating diseases related to abnormal HMGB1 activity or expression can be 25 mg / kg / day, administered by injection for 14 consecutive days.

[0135] This application also provides the application of compounds that target HMGB1 degradation in any of the foregoing embodiments, or compounds that target HMGB1 degradation prepared by the preparation methods in any of the foregoing embodiments, in the preparation of drugs for treating diseases related to abnormal HMGB1 activity or expression, especially in the preparation of antitumor active drugs, which have high pharmaceutical value.

[0136] In this application, diseases associated with abnormal HMGB1 activity or expression include tumors. In some embodiments of this application, tumors include one or more of the following: lung cancer, liver cancer, pancreatic cancer, breast cancer, cervical cancer, colon cancer, nasopharyngeal carcinoma, glioma, thyroid cancer, head and neck cancer, gastric cancer, kidney cancer, prostate cancer, testicular cancer, endometrial cancer, ovarian cancer, skin cancer, and lymphoma. In some specific embodiments of this application, tumors include lung cancer.

[0137] The effects of the technical solution in this application will be further illustrated below with several specific examples.

[0138] Example 1

[0139] The preparation method of CPD-4 is shown in formula (Ⅰ-1):

[0140]

[0141] (1) Preparation of CPD-4 intermediate 1

[0142] The compound shown in formula (II-1) (5-fluoroisobenzofuran-1,3-dione, 25 g, 1.0 eq, 150.5 mmol), the compound shown in formula (III) (3-aminopiperidine-2,6-dione, 27.25 g, 1.1 eq, 165.6 mmol), and potassium acetate (KOAc, 29.55 g, 2.0 eq, 301 mmol) were dissolved in acetic acid (250.00 mL) and reacted at 115°C for 16 hours under nitrogen protection. After the reaction was monitored by LC-MS, the reaction solution was filtered and evaporated to dryness to obtain a crude solid. The crude solid was washed with water (2 × 500 mL) and dried under vacuum to obtain 72 g of a yellow solid compound 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione, i.e., CPD-4-intermediate 1 shown in formula (IV-1).

[0143] The liquid chromatography-mass spectrometry (LC-MS) chromatogram of CPD-4 intermediate 1 is as follows: Figure 1 As shown, MS: m / z = 112.9 (M+1, ESI+). Figure 1 The upper chromatogram in the image is a liquid chromatography (LC) chromatogram of CPD-4-intermediate 1, with time (min) on the x-axis and response value (AU or mAU) on the y-axis. The lower chromatogram is a mass spectrum (MS) chromatogram corresponding to the highest peak of the upper chromatogram on the x-axis (time, also known as retention time, 1.23 min), with mass-to-charge ratio (m / z) on the x-axis and relative signal intensity (%) or absolute intensity (count) on the y-axis to reflect ion intensity. The LC-MS chromatograms in the following text are defined in this way and will not be repeated.

[0144] The 1H NMR spectrum of CPD-4 intermediate 1, as shown Figure 2 As shown, the chemical shift (ppm) is used as the horizontal axis, and the peak height reflects the relative intensity of the peak signal. The 1H NMR spectrum in the following text will be defined in this way, and will not be repeated here.

[0145] The structural characterization data of CPD-4 intermediate 1 are as follows:

[0146] 1 H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 8.03-8.00 (m, 1H), 7.86-7.84 (m, 1H), 7.74-7.70 (m, 1H), 5.20-5.5 (m, 1H), 2.90-2.87 (m, 1H), 2.64-2.55 (m, 2H), 2.10-2.07 (m, 1H).

[0147] (2) Preparation of CPD-4-intermediate 2

[0148] CPD-4 intermediate 1 (5 g, 1.0 eq, 18.2 mmol), the compound shown in formula (V-1) (tert-butyl(4-aminobutyroxyl)carbamate, 4 g, 1.02 eq, 18.5 mmol), and triethylamine (NEt3, 3.68 g, 5 mL, 2.0 eq, 36.4 mmol) were dissolved in dimethyl sulfoxide (DMSO, 25.00 mL) and stirred at 120°C for two hours under argon protection. After the reaction was completed by LC-MS, the reaction solution was concentrated to obtain a crude product, which was then purified by C-18 reversed-phase column chromatography (10-95% acetonitrile / water system) to obtain a yellow solid compound (tert-butyl(6-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)hexyl)carbamate, 2.5 g), i.e., CPD-4-intermediate 2 as shown in formula (VI-1).

[0149] LC-MS spectrum of CPD-4 intermediate 2, as shown Figure 3 As shown, MS: m / z = 473.5 (M+1, ESI+).

[0150] The 1H NMR spectrum of CPD-4 intermediate 2, as shown Figure 4 As shown, the structural characterization data are as follows:

[0151] 1 H NMR(400MHz, DMSO-d6) δ 11.05(s,1H), 7.56(d, J=4.2 Hz,1H), 7.11-7.09(m,1H),6.94(s,1H), 6.85-6.83(m,1H), 6.78-6.75(m,1H), 5.05-5.00(m,1H),3.16-3.13(m,2H), 2.91-2.88(m,2H),2.88-2.83(m,1H), 2.59-2.55(m,2H), 1.98-1.90(m,1H), 1.37-1.36(m,1H), 1.36(s,9H), 1.32-1.27(m,4H).

[0152] (3) Preparation of CPD-4 intermediate 3

[0153] CPD-4 intermediate 2 (500 mg, 1.1 mmol) was dissolved in dichloromethane (16 mL), and trifluoroacetic acid (TFA, 4 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for one hour. After the reaction was completed as monitored by LC-MS, the reaction mixture was evaporated to dryness to give a yellow solid compound (5-((6-aminohexyl)amino)-2-(2,6-dioxadiazin-3-yl)isoindoline-1,3-dione, 400 mg, 1.072 mmol), namely CPD-4 intermediate 3 as shown in formula (VII-1).

[0154] LC-MS spectrum of CPD-4 intermediate 3, as shown Figure 5 As shown, MS: m / z = 373.4 (M+1, ESI+).

[0155] (4) Preparation of CPD-4

[0156] CPD-4 intermediate 3 (200 mg, 1.0 eq, 0.538 mmol), as shown in formula (VIII), is (2S,3S,4S,5R,6R)-6-(((2R,3R,4S,5S,6S)-6-carboxy-2-(((4aR,6aR,6bS,8aS,11R,12aR,14aR,14bS)-11-carboxy-4,4,6a,6b,8a,11,14b-heptamethyl -14-Oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-eicosylpyran-3-yl)oxy)-4,5-dihydroxytetrahydro-2H-pyran-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (glycyrrhizic acid, 442mg, 1.0 eq (0.538 mmol) and triethylamine (NEt3, 109 mg, 2.0 eq, 1.076 mmol) were dissolved in N,N-dimethylformamide (DMF, 5 mL). Benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP condensing agent, 227 mg, 1.0 eq, 0.538 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for two hours under nitrogen protection. After the reaction was completed as monitored by LC-MS, the reaction mixture was concentrated and then purified by C-18 reversed-phase column chromatography (10-95% acetonitrile / water system) to give a yellow solid compound ((2S,3S,4S,5R,6R)-6-(((2R,3R,4S)) 5S,6S)-6-carboxy-2-(((4aR,6aR,6bS,8aS,11R,12aR,14aR,14bS)-11-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)hexyl)carbamoyl)-4,4,6a,6b,8a,11,14b-heptamethyl-14-oxo-1,2,3,4,4a,5,6,6a,7,8,8a,9,10,11,12,12a,14,14a,14b-eicosylpyran-3-yl)oxy)-4,5-dihydroxytetrahydro-2H-pyran-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, 12mg), namely the target product CPD-4 as shown in formula (Ⅰ-1).

[0157] LC-MS spectrum of CPD-4, such as Figure 6 As shown, MS:m / z=1177.7(M+1, ESI+).

[0158] HPLC chromatogram of CPD-4, such as Figure 7 As shown, the purity is 97.5%.

[0159] The proton NMR spectrum of CPD-4, as shown Figure 8 As shown, the structural characterization data are as follows:

[0160] 1 H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.66 (t, J=5.8 Hz, 1H), 7.54 (d, J=8.2 Hz, 1H), 7.11 (t, J=5.2 Hz, 1H), 6.93 (d, J=2.0 Hz, 1H), 6.82(d, J=8.4 Hz, 1H), 5.92 (s, 1H), 5.67 (s, 1H), 5.35 (d, J=1.8 Hz, 1H), 5.03(dd, J=13.0, 5.4 Hz, 3H), 4.44 (d, J=7.6 Hz, 1H), 4.38 (d, J=7.6 Hz, 1H),3.58 (d, J=9.6 Hz, 1H), 3.49 (d, J=9.6 Hz, 2H), 3.22-3.15 (m, 2H), 3.12-3.04 (m, 6H), 2.95-2.83 (m, 1H), 2.62-2.51 (m, 2H), 2.29 (s, 1H), 2.06-1.89 (m,4H), 1.78 (d, J=11.8 Hz, 1H), 1.65 (d, J=11.8Hz, 2H), 1.63-1.53 ​​(m, 4H), 1.48–1.43 (m, 2H), 1.43-1.19 (m, 14H), 1.08 (s, 3H), 0.99 (s, 3H), 0.96-0.90(m, 6H), 0.84 (d, J=12.2 Hz, 2H), 0.69 (d, J=14.2 Hz, 7H).

[0161] The three-dimensional structure of the CPD-4 molecule is as follows: Figure 9 As shown, its synthetic route is as follows:

[0162]

[0163] The compound CPD-4 obtained in Example 1 was subjected to the following tests:

[0164] (1) Western blot assay to detect the degradation activity of CPD-4 on HMGB1 protein in tumor cells.

[0165] 1) Treatment of human lung cancer cells (PC-9R) with CPD-4: Log-phase PC-9R cells were collected, and the cell suspension concentration was adjusted. 100 μL of cell suspension was added to each well of a 6-well plate to make the cell density 50,000-100,000 cells / well. Complete culture medium was added to 2 mL, and the cells were cultured at 37°C for 24 h with 5% CO2. Then, different concentrations of CPD-4 were added for 48 h: 0 μmol / L (control group), 3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L and 25 μmol / L.

[0166] 2) Collect protein samples: Take cells in good growth condition and gently discard the culture medium in the culture flask; add 2 mL of pre-chilled phosphate-buffered saline (PBS), wash twice to remove dead cells; add trypsin to digest the cells; add complete culture medium to stop digestion, collect the cell suspension, and centrifuge to collect the cell pellet; (for suspended cells, centrifuge directly to collect the cell pellet without digestion) rinse with PBS, centrifuge, repeat twice to wash away residual culture medium, discard residual PBS, and keep only the cell pellet (which can be frozen at -80℃ for protein extraction); lyse the cell pellet collected in the previous step, add radioimmunoprecipitation buffer (RIPA, strong) lysis buffer (protease inhibitors need to be added to the lysis buffer beforehand); lyse on ice for 30 min; after lysis, collect the sample, centrifuge at 4℃, 12000 rpm / 20 min; collect the supernatant and transfer to 1.5 mL In EP tubes, the entire process must be carried out on ice; aliquot and store at -20℃ or -80℃ for later use (adherent cells can also be washed three times with PBS and then directly added with an appropriate amount of RIPA containing protease inhibitors on ice for lysis, and then scraped off the lysed cells with a cell scraper and the supernatant collected).

[0167] 3) Prepare a suitable concentration of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): the concentration of the separating gel is 10%.

[0168] 4) Sample preparation: Prepare protein samples according to experimental requirements, centrifuge, mix, and load onto SDS-PAGE gel wells. Adjust the loading volume as needed based on protein quantification results.

[0169] 5) Electrophoresis: Turn on the power. The voltage for the protein sample in the stacking gel is 80 volts. When the protein sample enters the separating gel, adjust the voltage to 120 volts and continue electrophoresis. Stop electrophoresis when the bromophenol blue has almost completely run off the PAGE gel.

[0170] 6) Transfer: Remove the gel and cut a polyvinylidene fluoride (PVDF) membrane to the same size as the SDS-PAGE gel. The filter paper should be the same size as the fiber pad. Soak the PVDF membrane in methanol for 1 minute, then soak it and the filter paper in the electrotransfer buffer. Arrange the membrane in the following order: black plate - fiber pad - filter paper - gel - PVDF membrane - filter paper - fiber pad - white plate. Clamp the plates together and place them in the wet electrotransfer tank, with the black plate facing the black negative electrode. Fill the transfer tank with electrotransfer buffer and begin the transfer. (Place the transfer tank in water and place ice packs filled with frozen blocks inside. Use as many ice packs as possible to ensure the electrotransfer is carried out at a low temperature. If the transfer takes a long time and the temperature rises during the process, replace the ice packs.)

[0171] 7) Blocking: After the transfer is complete, remove the PVDF membrane and immerse it in Tris-buffered saline Tween-20 (TBST) buffer containing 5% skim milk powder. Shake on a shaker at room temperature for 1 hour.

[0172] 8) Primary antibody incubation: After blocking, wash three times with TBST buffer, then add the appropriately diluted primary antibody and incubate overnight at 4°C. Wash the PVDF membrane three times with TBST buffer, shaking for 10 min each time.

[0173] 9) Secondary antibody incubation: Discard the TBST buffer, add the diluted secondary antibody, and shake on a shaker at room temperature for 1 hour. Discard the secondary antibody, and wash the PVDF membrane three times with TBST buffer, shaking for 10 minutes each time.

[0174] 10) Exposure: The ECL substrate is evenly spread on the PVDF film and exposed for imaging.

[0175] The results of the CPD-4 degradation activity test on HMGB1 protein are as follows: Figure 10 As shown, the amount of β-actin as an internal control was constant in cells of the control group and the CPD-4 treatment group, while the amount of HMGB1 protein in cells with CPD-4 concentrations of 12.5 μmol / L and 25 μmol / L was significantly lower than that in the control group, indicating that CPD-4 can promote the degradation of HMGB1 protein in the PC-9R cell line.

[0176] (2) Phase-separation protein-protein interaction experiment (SPPIER)

[0177] Denovo-designed coiled helices were used as homologous oligomeric tags (HO-tags), and enhanced green fluorescent protein (EGFP) and red fluorescent protein variant (mCherry) fluorescent tags were used as reporter genes. To detect the interaction between CRBN and HMGB1 induced by small molecules, a short helical-based homologous oligomeric tag 3 (HOTag3, hexamer, 30 amino acids) was fused to the HMGB1 gene; another coiled helical-based homologous oligomeric tag 6 (HOTag6, tetramer, 33 amino acids) was fused to the CRBN gene. When the small molecule induced the interaction between HMGB1 and CRBN, each hexamer HMGB1-EGFP-HOTag3 recruited six CRBN-mCherry-HOTag6; each tetramer CRBN-mCherry-HOTag6 recruited four HMGB1-EGFP-HOTag3, and so on. Ultimately, the introduction of multivalence in HO-Tag and the interaction between HMGB1 and CRBN led to fluorescence phase separation, forming high-fluorescence-intensity yellow droplets. The specific transfection steps were as follows: 1.0 μg HMGB1-EGFP-HOTag3 and 1 μg CRBN-mCherry-HOTag6 were co-transfected into HEK293T cells for 24 h, in duplicate. Then, 0 μmol / L CPD-4 (control group) and 5 μmol / L CPD-4 were added for 6 h, respectively. Cells were fixed with 4% paraformaldehyde for 15 min, perforated with 0.2% Triton 100 for 30 min, stained with the DNA fluorescent dye Hoechst 33342 for 5 min, and mounted with an anti-fluorescence quencher. Immunofluorescence analysis was performed using laser confocal microscopy, and co-localization fluorescence intensity analysis was performed using ImageJ software.

[0178] The results are as follows Figure 11 As shown, (a) is an image of the control group and the CPD-4 group under a laser confocal microscope, reflecting the immunofluorescence distribution of CRBN, HMGB1, the fluorescent dye Hoechst 33342, and the combination of the three in the control group and the CPD-4 group; (b) is the fluorescence peak diagram of the area pointed to by the arrow in (a) of the control group; (c) is the fluorescence peak diagram of the area pointed to by the arrow in (a) of the CPD-4 group. (b) and (c) are plotted with the distance along the scan line as the x-axis and the relative fluorescence intensity as the y-axis. Figure 11 It can be seen that yellow droplets with high fluorescence intensity appeared in the CPD-4 group, while no such phenomenon occurred in the control group. This indicates that CPD-4 induces HMGB1 to bind with CRBN to form a complex, proving that HMGB1 and CRBN interact under the action of CPD-4.

[0179] (3) CPD-4 cytotoxicity detection

[0180] Human normal bronchial epithelial cells (BEAS-2B) were seeded at a density of 2000 cells / well in 96-well plates, divided into three groups: 0 μmol / L CPD-4 (control group), 1 μmol / L CPD-4, and 5 μmol / L CPD-4. Incubation was performed for 1, 2, 3, 4, and 5 days, with three replicates per group. Then, 10 µL of CCK-8 reagent was added to each well, and incubation was carried out at 37°C for 1 h. The absorbance (optical density) of each well was measured at 450 nm using a microplate reader.

[0181] The results are as follows Figure 12 As shown, compared with the control group, after incubation for 1-5 days, the cell viability of BESA-2B cells in 1 μmol / L (μM) CPD-4 and 5 μmol / L CPD-4 was almost the same as that in the control group, indicating that CPD-4 at concentrations of 1 μmol / L and 5 μmol / L was not toxic to BESA-2B cells.

[0182] (4) CCK-8 assay for detecting IC50 of tumor cells

[0183] Log-phase human lung cancer cells (PC-9R) were collected, and the cell suspension concentration was adjusted. 100 μL was added to each well of a 96-well plate to achieve a cell density of 1000-10000 cells / well. After culturing the cells in 5% CO2 at 37°C for 24 h, they were treated for 48 h with either no CPD-4 (control group) or different concentration gradients of CPD-4, with three replicates for each concentration. 10 μL of CCK-8 solution was added to each well. If the initial culture volume was 200 μL, 20 μL of CCK-8 solution was added, and so on. The cells were incubated for another 1 h in a cell culture incubator. The absorbance of each well was measured at 450 nm using a microplate reader.

[0184] like Figure 13 As shown, when cell viability is 50%, the corresponding abscissa (logarithm of CPD-4 drug concentration) is 1.46, and its IC50 value is calculated to be 13.68 μmol / L (μM), indicating that CPD-4 exhibits good anti-tumor activity and can induce apoptosis in PC-9R cells at low doses.

[0185] (5) CCK-8 assay for tumor cell proliferation

[0186] PC-9R cells were seeded at a density of 2000 cells / well in 96-well plates, divided into three groups: 0 μmol / L CPD-4 (control group), 5 μmol / L CPD-4, and 25 μmol / L glycyrrhizin. Cells were incubated for 1, 2, 3, 4, and 5 days, with three replicates per group. Then, 10 µL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 1 h. The absorbance (optical density) of each well was measured at 450 nm using a microplate reader.

[0187] The results are as follows Figure 14 As shown, cell activity (OD) in the CPD-4 group and the glycyrrhizic acid group 450 The levels of both CPD-4 and glycyrrhizic acid were lower than those of the control group, indicating that both CPD-4 and glycyrrhizic acid have an inhibitory effect on the proliferation of tumor cells, with CPD-4 showing a more significant inhibitory effect.

[0188] (6) Colony formation assay to observe the effect of CPD-4 on inhibiting cell proliferation.

[0189] PC-9R cells were used at a rate of 5 × 10 3 Cells were seeded at a density of 10 cells / well in six-well plates, divided into three groups: 0 μmol / L CPD-4 (control group), 5 μmol / L CPD-4, and 25 μmol / L glycyrrhizic acid. Each group consisted of three wells. Treatment lasted 48 h, followed by replacement with normal medium. Cells were incubated at 37°C for 15 days, washed twice with PBS, incubated with methanol for 15 min, and stained with 0.1% crystal violet for 60 min. Clusters were assessed using an optical microscope; each cluster containing ≥50 cells was counted as a single colony.

[0190] like Figure 15 As shown, (a) shows the colony formation of tumor cells on the plate after treatment with the control group, CPD-4 and glycyrrhizic acid, respectively, and (b) shows the statistical results of the number of colonies formed by tumor cells after each group of treatment. It can be seen that both CPD-4 and glycyrrhizic acid can inhibit the formation of monoclonal colonies, with CPD-4 showing a more significant inhibitory effect.

[0191] (7) Flow cytometry detection of the effect of CPD-4 on tumor cell apoptosis

[0192] PC-9R cells were divided into three groups: 0 μmol / L CPD-4 (control group), 15 μmol / L CPD-4, and 50 μmol / L glycyrrhizic acid. After 48 h of intervention, cells were digested with trypsin, centrifuged at 1000 g for 5 min, the supernatant was discarded, and the cells were collected and washed three times with 1 mL of 1×PBS. 100 µL of the cell suspension was placed in a 5 mL flow cytometry tube, 5 µL of Annexin V-FITC was added, and the mixture was incubated at room temperature in the dark for 5 min. 5 µL of propidium iodide solution (PI) was added, followed by 400 µL of PBS, and flow cytometry or fluorescence microscopy were performed immediately. The cells were loaded onto the flow cytometer at low speed, and a total of 10,000 cells were recorded. Data collection and analysis followed.

[0193] like Figure 16 As shown, Figure 16 In (a), the cell cycle detection diagram of tumor cells after treatment with control group, CPD-4 and glycyrrhizic acid is shown. The log fluorescence intensity of Annexin V-FITC is used as the x-axis and the log fluorescence intensity of propidium iodide solution (PI) is used as the y-axis. In (a), the Q2 quadrant reflects the ratio of late-apoptotic tumor cells and the Q3 quadrant reflects the ratio of early-apoptotic tumor cells. Figure 16 Table (b) shows the statistical results of tumor cell apoptosis rate (%) after each treatment group. Figure 16 It is known that both CPD-4 and glycyrrhizic acid can induce tumor cell apoptosis, with CPD-4 showing a more significant effect.

[0194] (8) Mouse xenograft model experiment

[0195] 1) Sample pretreatment: Prepare a sufficient amount of PC-9R cells for expansion culture in a culture dish before inoculation.

[0196] 2) Sample inoculation: Mice were fixed with a restraint device, disinfected with alcohol swabs, and 0.2 mL of highly viable tumor cell suspension (3 × 10⁻⁶ cells) was injected into the lower axillary region of the mice. 6 (units / mL).

[0197] 3) Grouping: When the tumor volume reaches 30mm 3 -50mm 3 Mice with good tumor homogeneity were randomly divided into a control group (containing 0.1% dimethyl sulfoxide) and a drug intervention group (CPD-4 group and glycyrrhizic acid group), with 6 mice in each group. The mice were administered the drug via tail vein injection daily for 14 consecutive days at a dose of 25 mg / kg / day.

[0198] 4) Experimental endpoints: i) 21 days after drug intervention; ii) tumor volume in mice >1500 mmHg. 3iii) Tumor ulceration and necrosis in mice; iv) Weight loss in mice exceeding 20% ​​of normal animal weight (the tumor's proportion should be taken into account). Finally, the tumors of the mice that have completed the experiment are removed and weighed.

[0199] The results are as follows Figure 17 As shown, (a) represents the tumors removed from mice in each group, and (b) represents the statistical results of tumor weight in each group. Figure 17 It is known that both CPD-4 and glycyrrhizic acid can inhibit the growth of tumors in mice, with CPD-4 showing a better inhibitory effect.

[0200] Comparative Example 1

[0201] The preparation method of CPD-1 is shown in formula (1):

[0202]

[0203] (1) Preparation of CPD-1 intermediate 1

[0204] The compound shown in formula (II-2) (4-fluoroisobenzofuran-1,3-dione, 25 g, 1.0 eq, 150.5 mmol), the compound shown in formula (III) (3-aminopiperidine-2,6-dione, 27.25 g, 1.1 eq, 165.6 mmol), and potassium acetate (KOAc, 29.55 g, 2.0 eq, 301 mmol) were dissolved in acetic acid (AcOH, 250.00 mL) and reacted at 115°C for 16 hours under nitrogen protection. After the reaction was monitored by LC-MS, the reaction solution was filtered and evaporated to obtain a crude solid. The crude solid was washed with water (2×500mL) and then dried under vacuum to obtain a yellow solid compound 2-(2,6-dioxadiazine-3-yl)-4-fluoroisoindoline-1,3-dione (72g, 260.8mmol), which is CPD-1 intermediate 1 shown in formula (Ⅳ-2).

[0205] (2) Preparation of CPD-1 intermediate 2

[0206] CPD-1 intermediate 1 (6 g, 1.0 eq, 21.72 mmol), the compound shown in formula (V-2) (tert-butyl(4-aminobutyl)carbamate, 4.98 g, 1.02 eq, 22.16 mmol), and triethylamine (NEt3, 6 mL, 2.0 eq, 43.44 mmol) were dissolved in dimethyl sulfoxide (DMSO, 30 mL), and the reaction solution was stirred at 120°C under argon protection for two hours. After the reaction was completed as monitored by LC-MS, the reaction solution was concentrated to obtain a crude product, which was then purified by C-18 reversed-phase column chromatography (10-95% acetonitrile / water system) to obtain a yellow solid compound (tert-butyl(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)butyl)carbamate, 6.32 g), i.e., CPD-1-intermediate 2 as shown in formula (VI-2).

[0207] LC-MS spectrum of CPD-1-intermediate 2, as shown Figure 18 As shown, MS: m / z = 467.4 (M+1, ESI+).

[0208] (3) Preparation of CPD-1 intermediate 3

[0209] CPD-1 intermediate 2 (270 mg, 1.0 eq, 0.608 mmol) was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (TFA, 1.5 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for one hour. After the reaction was completed as monitored by LC-MS, the reaction mixture was evaporated to dryness to obtain a yellow solid compound (4-((4-aminobutyl)amino)-2-(2,6-dioxadiazin-3-yl)isoindoline-1,3-dione, 210 mg), namely CPD-1 intermediate 3 as shown in formula (Ⅶ-2).

[0210] LC-MS spectrum of CPD-1-intermediate 3, as shown Figure 19 As shown, MS: m / z = 345.2 (M+1, ESI+).

[0211] (4) Preparation of CPD-1

[0212] CPD-1 intermediate 3 (210 mg, 1.0 eq, 0.61 mmol), glycyrrhizic acid (502 mg, 1.0 eq, 0.61 mmol) as shown in formula (VIII), and triethylamine (NEt3, 0.17 mL, 2.0 eq, 1.22 mmol) were dissolved in N,N-dimethylformamide (DMF, 10 mL). BOP condensing agent (270 mg, 1.0 eq, 0.610 mmol) was added at room temperature, and the reaction mixture was stirred at room temperature for two hours under nitrogen protection. LC-MS After the reaction was completed, the reaction solution was concentrated and then purified by C-18 reversed-phase column chromatography (10-95% acetonitrile / water system) to obtain a yellow solid compound ((2S,3S,4S,5R,6R)-6-(((2R,3R,4S)5S,6S)-6-carboxy-2-(((4aR,6aR,6bS,8aS,11R,12aR,14aR,14bS)-11-((4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-) (12mg) , i.e., CPD-1 as shown in formula (1).

[0213] LC-MS spectrum of CPD-1, such as Figure 20 As shown, MS: m / z = 1147.8 (M+1, ESI+).

[0214] The proton NMR spectrum of CPD-1, as shown Figure 21 As shown, the structural characterization data are as follows:

[0215] 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.88 (s, 1H), 7.57 (t, J=8.0Hz, 1H), 7.07 (d, J=8.8Hz, 1H), 7.01 (d, J=6.8Hz, 1H), 6.51 (t, J=5.6Hz,1H), 5.38 (s, 2H), 5.04 (dd, J=12.8Hz, 2H), 4.42 (dd, J=45.6Hz, 2H), 3.17-2.84 (m, 15H), 2.61-2.54 (m, 3H), 2.28 (s, 1H), 2.07-2.02 (m, 4H), 1.81-1.49 (m, 14H), 1.31 (s, 7H), 1.24-1.19 (m, 2H), 1.15-1.13 (m, 1H), 1.09 (s, 3H), 1.01 (d, J=2.8Hz, 7H), 0.91 (s, 4H), 0.75 (s, 3H), 0.72 (s, 3H).

[0216] The three-dimensional structure of the CPD-1 molecule is as follows: Figure 22 As shown, its synthetic route is as follows:

[0217]

[0218] Comparative Example 2

[0219] The preparation method of CPD-2 is shown in formula (2):

[0220]

[0221] (1) Preparation of CPD-2 intermediate 1

[0222] Similar to Comparative Example 1, CPD-2-Intermediate 1 is identical to CPD-1-Intermediate 1.

[0223] (2) Preparation of CPD-2 intermediate 2

[0224] CPD-2 intermediate 1 (6.68 g, 1.0 eq, 24.20 mmol), the compound shown in formula (V-1) (tert-butyl(6-aminohexyl)carbamate, 5.34 g, 1.02 eq, 24.69 mmol), and triethylamine (NEt3, 6.72 mL, 2.0 eq, 48.40 mmol) were dissolved in dimethyl sulfoxide (DMSO, 30 mL), and the reaction mixture was stirred at 120 °C for two hours under nitrogen protection. After the reaction was completed as monitored by LC-MS, the reaction solution was concentrated to obtain a crude product, which was then purified by C-18 reversed-phase column chromatography (10-95% acetonitrile / water system) to obtain a yellow solid compound (tert-butyl(6-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)hexyl)carbamate, 6.54 g), i.e., CPD-2-intermediate 2 as shown in formula (VI-3).

[0225] LC-MS spectrum of CPD-2-intermediate 2, as shown Figure 23 As shown, MS: m / z = 495.4 (M+1, ESI+).

[0226] (3) Preparation of CPD-2-intermediate 3

[0227] CPD-2 intermediate 2 (287 mg, 1.0 eq, 0.608 mmol) was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (TFA, 1.5 mL) was slowly added dropwise at room temperature. The reaction mixture was stirred at room temperature for one hour. After the reaction was completed as monitored by LC-MS, the reaction mixture was evaporated to dryness to obtain a yellow solid compound (4-((6-aminohexyl)amino)-2-(2,6-dioxadiazin-3-yl)isoindoline-1,3-dione, 250 mg), namely CPD-2 intermediate 3 as shown in formula (VII-3).

[0228] LC-MS spectrum of CPD-2-intermediate 3, as shown Figure 24 As shown, MS: m / z = 373.3 (M+1, ESI+).

[0229] (4) Preparation of CPD-2

[0230] CPD-2 intermediate 3 (250 mg, 1.0 eq, 0.672 mmol), glycyrrhizic acid (553 mg, 1.0 eq, 0.672 mmol) as shown in formula (VIII), and triethylamine (NEt3, 0.19 mL, 2.0 eq, 1.344 mmol) were dissolved in N,N-dimethylformamide (DMF, 10 mL). BOP condensing agent (297 mg, 1.0 eq, 0.672 mmol) was added at room temperature, and the reaction solution was stirred at room temperature for two hours under nitrogen protection. After the reaction was completed as monitored by LC-MS, the reaction solution was concentrated and then purified by C-18 reversed-phase column chromatography (10-95% acetonitrile / water system) to obtain 25 mg of a yellow solid compound ((2S,3S,4S,5R,6R)-6-(((2R,3R,4S,)5S,6S)-6-carboxy-2-(((4aR,6aR,6))-6-carboxyl ...aR))-6-carboxyl-2-(((4aR,6aR,6aR))-6-carboxyl-2-(((4aR,6aR,6aR))-6-carboxyl-2-(((4aR,6aR,6aR))-6-carboxyl-2-(((4aR,6aR))-6-carboxyl-2-(((4aR,6aR))-6-carboxyl-2-(((4aR,6aR))-6-carboxyl-2-(((4aR,6aR))-6-carboxyl-2-(((4aR,6aR)) bS,8As,11R,12aR,14aR,14bS)-11-((6-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)hexyl)carbamoyl)-4,4,6a,6b,8a,11,14b-heptamethyl-14-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-eicosylpyran-3-yl)oxy)-4,5-dihydroxytetrahydro-2H-pyran-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid), i.e., CPD-2 as shown in formula (2).

[0231] LC-MS spectrum of CPD-2, such as Figure 25 As shown, MS: m / z = 1175.65 (M+1, ESI+).

[0232] The proton NMR spectrum of CPD-2, as shown Figure 26 As shown, the compound structure characterization data are as follows:

[0233] 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.58-7.54 (m, 2H), 7.06 (d, J=8.8 Hz, 1H), 7.00 (d, J=7.2 Hz, 1H), 6.52 (s, 1H), 5.48 (s, 1H), 5.07-5.02 (m, 2H), 4.50-4.39 (m, 3H), 3.64 (d, J=9.2 Hz, 1H), 3.56 (d, J=9.6 Hz,1H), 3.42-2.85 (m, 16H), 2.30 (s, 1H), 2.04-1.41 (m, 14H) , 1.33 (s, 12H), 1.01-0.94 (m, 16H), 0.70 (d, J=4.0 Hz, 7H).

[0234] The three-dimensional structure of the CPD-2 molecule is as follows: Figure 27 As shown, its synthetic route is as follows:

[0235]

[0236] Comparative Example 3

[0237] The preparation method of CPD-3 is shown in formula (3):

[0238]

[0239] (1) Preparation of CPD-3 intermediate 1

[0240] Similar to Example 1, CPD-3-intermediate 1 is the same as CPD-1-intermediate 1.

[0241] (2) Preparation of CPD-3 intermediate 2

[0242] CPD-3 intermediate 1 (5 g, 1.0 eq, 18.2 mmol), the compound shown in formula (V-2) (tert-butyl(4-aminobutyl)carbamate, 4 g, 1.02 eq, 18.5 mmol), and triethylamine (NEt3, 3.68 g, 5 mL, 2.0 eq, 36.4 mmol) were dissolved in dimethyl sulfoxide (DMSO, 25.00 mL), and the reaction solution was stirred at 120°C for two hours under nitrogen protection. After the reaction was monitored by LC-MS, the reaction solution was evaporated to obtain crude product, which was purified by C-18 reversed-phase column chromatography (10-95% acetonitrile / water system) to obtain a yellow solid compound (tert-butyl(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)butyl)amino)carbamate, 2.5 g), i.e., CPD-3-intermediate 2 as shown in formula (VI-4).

[0243] LC-MS spectrum of CPD-3-intermediate 2, as shown Figure 28 As shown, MS: m / z = 443.3 (M+1, ESI+).

[0244] The structural characterization data of CPD-3 intermediate 2 by 1H NMR spectroscopy are as follows:

[0245] 1 H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H),7.56 (d, J = 4.2 Hz, 1H),7.11-7.09 (m, 1H), 6.94 (s, 1H),6.85-6.83 (m, 2H), 5.05-5.00 (m, 1H),3.16-3.13(m, 2H),2.97-2.92 (m, 2H), 2.88-2.84 (m, 1H), 2.59-2.55 (m, 2H), 2.01-1.98 (m, 1H), 1.54-1.36 (m, 4H),1.22(s, 9H).

[0246] (3) Preparation of CPD-3 intermediate 3

[0247] CPD-3 intermediate 2 (500 mg, 1.0 eq, 1.126 mmol) was dissolved in dichloromethane (6 mL), and trifluoroacetic acid (TFA, 1.5 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for one hour. After the reaction was completed as monitored by LC-MS, the reaction mixture was evaporated to dryness to give a yellow solid compound (5-((4-aminobutyl)amino)-2-(2,6-dioxopiridine-3-yl)isoindoline-1,3-dione, 400 mg), namely CPD-3 intermediate 3 as shown in formula (VII-4).

[0248] LC-MS spectrum of CPD-3-intermediate 3, as shown Figure 29 As shown, MS: m / z = 345.3 (M+1, ESI+).

[0249] (4) Preparation of CPD-3

[0250] CPD-3 intermediate 3 (194.0 mg, 1.0 eq, 0.564 mmol), glycyrrhizic acid (464 mg, 1.0 eq, 0.564 mmol), and triethylamine (NEt3, 114 mg, 2.0 eq, 1.128 mmol) were dissolved in N,N-dimethylformamide (DMF, 5.00 mL). BOP condensing agent (249 mg, 1.0 eq, 0.564 mmol) was added at room temperature, and the reaction mixture was stirred at room temperature for two hours under nitrogen protection. After the reaction was monitored by LC-MS, the reaction mixture was concentrated and then subjected to C-18 reversed-phase column chromatography (10-95% acetonitrile / water with 0.1% formic acid). Purification yielded a yellow solid compound ((2S,3S,4S,5R,6R)-6-(((2R,3R,4S)5S,6S)-2-(((4aR,6aR,6bS,8aS,11R,12aR,14aR,14bS)-11-carboxy-4,4,6a,6b,8a,11,14b-heptamethyl-14-oxo-1,2,3,4,4a,5,6,6a,6b,7 ,8,8a,9,10,11,12,12a,14,14a,14b-eicosylpyridin-3-yl)oxy)-6-((4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)butyl)carbamoyl)-4,5-dihydroxytetrahydro-2H-pyran-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, 54mg), i.e., CPD-3 as shown in formula (3).

[0251] LC-MS spectrum of CPD-3, such as Figure 30 As shown, MS: m / z = 1147.6 (M+1, ESI+).

[0252] The proton NMR spectrum of CPD-3, as shown Figure 31 As shown, the compound structure characterization data are as follows:

[0253] 1 H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.86 (t, J=5.8 Hz, 1H), 7.54 (dd, J=8.4, 2.6 Hz, 1H), 7.08(s,1H), 6.95 (s, 1H), 6.83 (dd, J=8.5, 2.2Hz, 1H), 5.39 (s, 1H), 5.03 (dd, J=12.8, 5.4 Hz, 1H), 4.48 (d, J=7.8 Hz, 1H), 4.35 (d, J=7.4 Hz, 1H), 3.61-3.52 (m, 2H), 3.43-3.37 (m, 2H), 3.35-3.25 (m,2H), 3.16 (q, J=7.8, 7.4 Hz, 6H), 3.04 (t, J=8.4 Hz, 2H), 2.94-2.81 (m, 1H), 2.57 (dd, J=18.8, 6.2 Hz, 4H), 2.29 (s, 1H), 2.08 (s, 1H), 2.06-1.95 (m, 2H),1.78 (t, J=14.2 Hz, 2H), 1.66 (d, J=11.5 Hz, 3H), 1.59-1.50 (m, 8H), 1.38-1.36(m,2H),1.35 (d, J=12.8 Hz, 6H), 1.26 (d, J=16.8 Hz, 2H), 1.09 (s, 3H), 1.05-0.89 (m, 9H), 0.75 (s, 3H), 0.69 (d, J=12.9 Hz, 6H).

[0254] The three-dimensional structure of the CPD-3 molecule is as follows: Figure 32 As shown, its synthetic route is as follows:

[0255]

[0256] The differences between CPD-1 and CPD-4 lie in the length of their linkers and the connection sites between their linkers and the E3 ubiquitin ligase ligand (thalidomide). Similarly, the differences between CPD-2 and CPD-4 lie in the connection sites between their linkers and the E3 ubiquitin ligase ligand (thalidomide). Furthermore, the differences between CPD-3 and CPD-4 lie in the length of their linkers and the connection sites between their linkers and the target protein ligand (glycyrrhizic acid). CPD-1 and CPD-3 are isomers, as are CPD-2 and CPD-4.

[0257] Similarly, CPD-1 prepared in Comparative Example 1, CPD-2 prepared in Comparative Example 2, and CPD-3 prepared in Comparative Example 3 were subjected to the aforementioned Western blotting experiment, with the specific operation method being the same as in Example 1, to compare the differences in the degradation activities of the four compounds against HMGB1 protein in tumor cells. Finally, the test results of the degradation activities of 25 μmol / L CPD-1, CPD-2, CPD-3, and CPD-4 against HMGB1 protein are as follows: Figure 33 As shown, using β-actin as an internal control, its protein level remained constant across all cell groups. Figure 33 It can be seen that in the PC-9R cell line, CPD-1, CPD-2 and CPD-4 all have a significant promoting effect on the degradation of HMGB1 protein. Among them, the reduction of HMGB1 protein in cells containing CPD-4 is significantly higher than that in cells containing CPD-1 or CPD-2. This indicates that the degradation activity of the four compounds on HMGB1 protein from strong to weak is: CPD-4 > CPD-1 > CPD-2 > CPD-3.

[0258] Figure 14 , Figure 15 , Figure 16 , Figure 17 "in "and" "Used to represent the significance level of the p-value in hypothesis testing, reflecting whether the result is statistically significant." "A p-value less than 0.01 indicates that the statistical result is highly significant." The corresponding p-value is less than 0.001, indicating that the statistical result is extremely significant.

[0259] In summary, the differences between CPD-1, CPD-2, CPD-3, and CPD-4 lie solely in the length of the linker or the connection sites between the linker and the ligands at both ends, resulting in varying degradation abilities. In PC-9R cells, CPD-4 exhibited the most significant degradation effect on HMGB1 protein, indicating that the PROTAC compound design for targeting HMGB1 degradation provided in this application is not a simple superposition of structures. This process requires not only synergistic optimization of the structures of each part to ensure the stable formation of the ternary complex, but also comprehensive consideration of the rationality of the connection sites.

[0260] In summary, the HMGB1-targeting compounds provided in this application are stable PROTAC compounds. The appropriate length of their linkers gives these PROTAC compounds greater freedom and flexibility, helping them to bypass spatial barriers and approach the HMGB1 target protein, thereby improving the adaptability between the ligands at both ends and the target protein. The length of the linker also greatly affects the connection sites between the linker and the ligands at both ends. The specific connection sites between the linker and the ligands at both ends in the HMGB1-targeting compounds provided in this application give the compounds superior HMGB1 protein degradation activity, enabling them to more effectively inhibit the growth and proliferation of tumor cells, and showing broad application prospects in the field of anti-tumor active drugs.

[0261] The above-disclosed embodiments are merely preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that implementing all or part of the processes of the above embodiments and making equivalent changes in accordance with the claims of this application still fall within the scope of this application.

Claims

1. A compound that targets and degrades HMGB1, characterized in that, The compound that targets and degrades HMGB1 is the compound shown in formula (I) or a pharmaceutically acceptable salt thereof. Where R is -(CH2) m - where m is any integer from 6 to 10.

2. A method for preparing a compound that targets and degrades HMGB1, characterized in that, include: The compound shown in formula (VII), glycyrrhizic acid, and a condensing agent are mixed to form a condensation reaction solution. The condensation reaction yields the compound as described in claim 1 that targets and degrades HMGB1. Where R is -(CH2) m - where m is any integer from 6 to 10.

3. The method for preparing the compound for targeted degradation of HMGB1 as described in claim 2, characterized in that, The method for preparing the compound represented by formula (VII) includes: The compound represented by formula (II), the compound represented by formula (III), and the first catalyst are mixed to form a first substitution reaction solution. The first substitution reaction yields the compound represented by formula (IV), wherein X is selected from F, Cl, or Br. （Ⅱ） （Ⅲ） （Ⅳ） The compound represented by formula (Ⅳ), the compound represented by formula (Ⅴ), and the second catalyst are mixed to form a second substitution reaction solution, and the compound represented by formula (Ⅵ) is obtained by the second substitution reaction. （Ⅴ） （Ⅵ） Where R is -(CH2) m -, m is any integer from 6 to 10; Finally, the compound shown in formula (VI) was subjected to a deBoc reaction to obtain the compound shown in formula (VII).

4. The method for preparing the compound for targeted degradation of HMGB1 as described in claim 3, characterized in that, The molar ratio of the compound represented by formula (II) to the compound represented by formula (III) in the first substitution reaction solution is (1-2):1; the first catalyst is potassium acetate; the temperature of the first substitution reaction is 100℃-130℃, and the time of the first substitution reaction is 15h-20h; The molar ratio of the compound represented by formula (Ⅳ) and the compound represented by formula (Ⅴ) in the second substitution reaction solution is (1-2):1; the second catalyst is triethylamine; the temperature of the second substitution reaction is 100℃-130℃, and the time of the second substitution reaction is 2h-4h; The temperature of the Boc removal reaction is 20℃-30℃, and the time of the Boc removal reaction is 1h-3h.

5. The method for preparing the compound for targeted degradation of HMGB1 as described in claim 2, characterized in that, The condensation reaction is carried out under nitrogen protection; the molar ratio of the compound represented by formula (VII) and the glycyrrhizic acid in the condensation reaction solution is (1-2):1; the condensing agent is one or more of benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, propylphosphonic anhydride, and N,N-carbazide; the temperature of the condensation reaction is 20℃-30℃, and the time of the condensation reaction is 1h-5h.

6. A medicament for treating diseases associated with abnormal HMGB1 activity or expression, characterized in that, This includes compounds that target HMGB1 degradation as described in claim 1 or compounds that target HMGB1 degradation prepared by any one of claims 2-5.

7. The medicament for treating diseases associated with abnormal HMGB1 activity or expression as described in claim 6, characterized in that, The compound that targets and degrades HMGB1 may be used as a single active ingredient or together with other pharmaceutically acceptable active ingredients to constitute the medicament for treating diseases associated with abnormal HMGB1 activity or expression.

8. The medicament for treating diseases associated with abnormal HMGB1 activity or expression as described in claim 6, characterized in that, It also includes one or more pharmaceutically acceptable carriers and excipients.

9. The use of the compound that targets and degrades HMGB1 as described in claim 1 or the compound that targets and degrades HMGB1 prepared by any one of claims 2-5 in the preparation of a medicament for treating diseases related to abnormal HMGB1 activity or expression, wherein the disease related to abnormal HMGB1 activity or expression is lung cancer.

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