Heat shock protein 90-based bivalent inhibitor, and preparation method therefor and use thereof

Abstract

The present invention relates to an inhibitor of heat shock protein 90. Disclosed are a heat shock protein 90-based bivalent inhibitor, and a preparation method therefor and the use thereof. The bivalent inhibitor is a compound having a structural formula as shown in formula I: A-L-B Formula I, or a pharmaceutically acceptable salt, solvate, or optical isomer thereof, wherein A and B are ATP inhibitors of heat shock protein 90, and the motif structure of L is a flexible linker group of PEG or alkanes, or a rigid linker group comprising aryl, heteroalkyl, or heteroaryl. The bivalent inhibitor can effectively inhibit the activity of molecular chaperone HSP90, hinder the folding and modification of a substrate protein, degrade the substrate protein via a ubiquitin-proteasome degradation pathway, induce non-native dimerization of HSP90, and interfere with protein-protein interactions associated with HSP90. In addition, the bivalent inhibitor reduces the heat shock response induced by HSP90 inhibition, and exhibits potent activity in degrading the substrate protein.

Description

Bivalent inhibitors based on heat shock protein 90, their preparation methods and applications Technical Field

[0001] This invention relates to inhibitors of heat shock protein 90, specifically to bivalent inhibitors based on heat shock protein 90, their preparation methods, and applications. Background Technology

[0002] Heat shock protein 90 (HSP90) is one of the most important molecular chaperone proteins and a key regulator of protease inhibition in eukaryotic cells under physiological and stress conditions. HSP90 has hundreds of protein substrates, thus participating in many cellular processes beyond protein folding, playing a crucial role in DNA repair, development, immune responses, and neurodegenerative diseases. HSP90 functions in the chaperone cycle by interacting with numerous co-chaperone proteins, regulating the ATPase conformation of the HSP90 dimer during client protein processing. Without ATP binding, HSP90 primarily adopts a V-shaped open conformation. Upon ATP binding, it undergoes a significant ATP-regulated conformational rearrangement, forming a closed state. After ATP hydrolysis, NTDs dissociate, ADP and inorganic phosphate (Pi) are released, allowing HSP90 to return to its open conformation. In summary, under the regulation of ATP, HSP90 interacts with its co-chaperone proteins at different times to maintain different dimer states, thus playing different roles in different parts of the chaperone cycle. Therefore, the dimerization regulation of HSP90 is crucial.

[0003] Heat shock protein 90 (HSP90) is closely related to the occurrence and development of diseases such as tumors, and is an attractive target for cancer treatment. Small ATP inhibitors designed targeting the ATP binding pocket of HSP90 have shown significant effects in inhibiting HSP90 activity, inducing substrate protein degradation, blocking cell growth, proliferation and signal transduction. However, the high toxicity of these inhibitors limits their use in scientific research and clinical treatment. Therefore, in order to overcome these shortcomings, novel HSP90 inhibitors urgently need to be developed. Summary of the Invention

[0004] To overcome the shortcomings of the existing technologies, a divalent molecule based on ATP inhibitors was designed in order to solve problems such as high toxicity and side effects, thereby enabling it to exert therapeutic effects in clinical treatment.

[0005] The purpose of this invention is to provide a bivalent inhibitor based on heat shock protein 90 (HSP90), wherein the bivalent inhibitor is a compound with the structural formula shown in Formula I:

[0006] ALB

[0007] Formula I

[0008] Or its pharmaceutically acceptable salts, solvates, or optical isomers.

[0009] Where: A and B are ATP inhibitors of heat shock protein 90 (HSP90), and L represents the linker chain.

[0010] [Correction 16.05.2025 based on Rule 91] Further, A and B may be selected individually or jointly from the following compounds:

[0011] The Chinese name for GDA is Gerdemycin; the Chinese name for 17-AAG is Tanspiramycin; the Chinese name for AT13387 is Onaspic; the Chinese name for NVP-AUY922 is Lumispiramycin; the Chinese name for PU-H71 is Zelavispicb; the chemical name for BIIB021 is 1-hydroxytrianetriane beeswaxol; the chemical name for SNX-2112 is 4-[6,6-dimethyl-4-oxo-3-(trifluoromethyl)-5,7-dihydroindoleazol-1-yl]-2-[(4-hydroxycyclohexyl)amino]benzamide; and the chemical name for XL-888 is 2-(butyl-2-amino)-4-N-[(1R,5S)-8-[5-(cyclopropanecarbonyl)pyridin-2-yl-8azabicyclo[3.2.1]octane-3-yl]-5-methylbenzene-1,4-dicarboxamide.

[0012] Furthermore, the motif structure of L consists of flexible linking groups of PEG-type and alkane-type or rigid linking groups with aryl, heteroalkyl, or heteroaryl groups.

[0013] [Corrected according to Rule 91, May 16, 2025] The specific structural formula is selected from one of the following;

[0014] Where n represents any independent natural number between 1 and 12.

[0015] [Correction 16.05.2025 based on Rule 91] Further, the compound shown in Formula I is DDO-4104 to DDO-4114, and its structural formula is as follows:

[0016] Another object of the present invention is to provide a method for preparing the aforementioned divalent inhibitor based on heat shock protein 90 (HSP90), the method comprising the following steps:

[0017] (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindoline-2-yl) methyl ketone was dissolved in 50 mL of anhydrous DCM. A dicarboxylic acid compound and HATU were added under ice bath conditions, followed by slow addition of Et3N. The mixture was stirred at room temperature. After the reaction was complete, water was added to the reaction solution for extraction. The resulting organic layer was dried over anhydrous sodium sulfate and then evaporated to dryness. The crude product was purified by column chromatography. The stoichiometric ratio of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindoline-2-yl) methyl ketone, dicarboxylic acid compound, HATU, and Et3N was 2:1:2:4.

[0018] The purified crude product was dissolved in a mixed solution of 1,2-dichloroethane and methanol, and ammonium formate was added. The mixture was stirred at 40°C for 10 minutes, and then 10% Pd / C was added. The mixture was stirred at 40°C for another 50 minutes. After the reaction was completed, the mixture was cooled to room temperature, and the catalyst was removed by filtration through a diatomaceous earth pad. The filtrate was concentrated into a solid, and the solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried with anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography to obtain a divalent inhibitor based on heat shock protein 90. The stoichiometric ratio of the purified crude product to ammonium formate was 1:80.

[0019] The dicarboxylic acid compounds include heptaic acid, octanoic acid, azelaic acid, sebacic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, 3,3'-oxydipropionic acid, triethylene glycol diacetic acid, 2,4-pyridine dicarboxylic acid, and 4-carboxyphenylacetic acid.

[0020] Another object of the present invention is to provide the use of a bivalent inhibitor of heat shock protein 90 (HSP90) in the preparation of drugs for gastric or colon cancer.

[0021] The present invention has the following beneficial effects: The divalent inhibitor molecule of divalent heat shock protein 90 (HSP90) provided by the present invention can effectively inhibit the activity of the molecular chaperone protein HSP90, hinder the folding modification of substrate proteins, and degrade substrate proteins through the ubiquitin-proteasome pathway. This divalent inhibitor molecule possesses a mechanism of action different from previous HSP90 ATP inhibitors, capable of inducing non-natural dimerization of HSP90 and interfering with HSP90-related protein-protein interactions. Simultaneously, it reduces the heat shock effect induced by HSP90 inhibition, exhibiting potent substrate protein degradation activity, and can be used to prepare therapeutic drugs for gastric cancer, colon cancer, and other related diseases. The present invention also discloses a method for synthesizing this series of HSP90 divalent ATP inhibitor compounds.

[0022] Specifically, there is no existing bivalent strategy for ATP inhibitors. This invention proposes a novel bivalent strategy. Based on the classic ATP inhibitor AT13387, this invention designs a bivalent molecule that can induce the N-terminus of HSP90 to approach, forming a non-natural dimer. This results in a mechanism of action different from classic ATP inhibitors, interfering with the interaction of HSP90-related co-chair proteins and, to some extent, weakening the heat shock effect of ATP inhibitors. It exhibits strong anti-proliferative activity and can exert a tumor growth inhibitory effect, showing great promise for clinical application.

[0023] Definition of terminology:

[0024] As used in this invention, the term "alkyl" includes a monovalent hydrocarbon group consisting of a saturated straight-chain or branched chain of 1-12 carbon atoms. Some examples are alkyl groups consisting of 5-12 carbon atoms.

[0025] As used in this invention, the term "PEG" refers to a linker chain containing an ethylene glycol linker motif.

[0026] As used in this invention, the term "heteroalkyl" includes 3- to 6-membered saturated cycloalkanes containing a heteroatom selected from nitrogen, oxygen, and sulfur. Some examples include cyclobutylamine and cyclopentylamine.

[0027] As used in this invention, the term "aryl" refers to an unsaturated aromatic group having 6 carbon atoms. The aryl group may be unsubstituted or substituted by one or two substituents independently selected from the group consisting of: (1) C1-C6 alkyl; (2) halogen; (3) C1-C6 alkoxy; (4) amino; (5) nitro; (6) cyano; and (7) trifluoromethyl.

[0028] As used in this invention, the term "heteroaryl" includes a 5- or 6-membered ring containing a heteroatom selected from nitrogen, oxygen, and sulfur; the 5-membered ring has two double bonds, and the 6-membered ring has three double bonds; some examples of said heteroaryl are pyridyl, thiophenyl, furanyl, pyrimidinyl, imidazolyl, quinolinyl, and morpholine rings.

[0029] As used in this article, “halogen” refers to a group selected from fluorine (-F), chlorine (-Cl), and bromine (-Br); “amino” means -NH2 group; “nitro” means -NO2 group; “cyano” means -CN group; “aldehyde” means -CHO group; and “hydroxyl” means -OH group.

[0030] Unless otherwise indicated, the structural formulas described in this invention include all isomers (e.g., 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 compounds of this invention, or its enantiomers, diastereomers, or mixtures of geometric isomers (or conformational isomers), is within the scope of this invention.

[0031] In this invention, "pharmaceutically acceptable" means any substance that does not interfere with the bioactivity of the active ingredient and is non-toxic to the host to which it is given.

[0032] Unless otherwise stated, all tautomeristic forms of the compounds of this invention are included within the scope of this invention. Furthermore, unless otherwise stated, the structural formulas of the compounds described in this invention comprise enriched isotopes of one or more different atoms. Attached Figure Description

[0033] Figure 1: Graph of the activity of divalent inhibitor molecules DDO-4105, DDO-4110 and DDO-4113 in inducing HSP90NTD dimerization by ATP inhibitors;

[0034] Figure 2: Crystal structure of the complex formed by the dimerization of HSP90 induced by the divalent inhibitor molecule DDO-4105 to form a non-natural dimer;

[0035] Figure 3: Interference between the bivalent inhibitor molecule DDO-4113 and HSP90-related protein interactions;

[0036] Figure 4: Results of the antiproliferative activity of the bivalent inhibitor molecule against HCT116 cells;

[0037] Figure 5: Results of bivalent inhibitor molecules inducing substrate protein degradation with the same effect as AT13387 in the HCT116 cell line;

[0038] Figure 6: The heat shock effect of the bivalent inhibitor molecules DDO-4113 and AT13387 in HCT116 cells in a concentration-dependent manner;

[0039] Figure 7: The time-dependent heat shock effects of the bivalent inhibitor molecules DDO-4113 and AT13387 on HCT116 cells;

[0040] Figure 8: Survival curves of the bivalent inhibitor molecules DDO-4113 and AT13387 at high and low doses;

[0041] Figure 9: Body weight changes after high and low dose administration of the bivalent inhibitor molecule DDO-4113;

[0042] Figure 10: Changes in tumors after administration of the bivalent inhibitor molecule DDO-4113 at high and low doses. Detailed Implementation

[0043] Unless otherwise specified, the raw materials may be obtained commercially, or prepared by methods known in the art, or prepared according to the methods described herein. The structure of the compounds was determined by nuclear magnetic resonance (NMR). 1 Confirmation was performed using 1H-NMR and / or mass spectrometry (MS). NMR measurements were performed using a Varian INOVA (300 MHz) or Bruker Advance (400 MHz) NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (Chloroform-d) as the solvent, and TMS as the internal standard. MS measurements were performed using an Alilent time-of-flight mass spectrometer. Silica gel column chromatography was performed using 200-300 mesh silica gel from Qingdao Marine Chemical Plant.

[0044] 1) Preparation of intermediates

[0045] [Corrected according to Rule 91, May 16, 2025] Its synthetic route:

[0046] (1) Preparation of 2,4-bis(benzyloxy)-5-bromobenzoate benzyl ester (AT2)

[0047] 5.0 g (21.45 mmol, 1.0 eq) of 1-bromo-2,4-dihydroxybenzoic acid was dissolved in approximately 40 mL of CH3CN solvent. Then, 7.65 mL (64.35 mmol, 3 eq) of benzyl bromo and 20.75 g (150.15 mmol, 6 eq) of potassium carbonate (K2CO3) were added. The mixture was heated and stirred at 80 °C for 8 hours. During the reaction, UV TLC was used to monitor the completeness of the reaction. The developing solvent was PE:EA (V / V) = 10:1. After the reaction, the solvent was removed using a rotary evaporator, yielding a white solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over Na2SO4, filtered, and concentrated. The concentrate was purified by column chromatography using a gradient elution of PE / EA (V / V) = 300 / 1-20 / 1, yielding 9.99 g of a white solid powder, with a yield of 92.57%. 1H NMR (300MHz, DMSO-d6) δ7.97(s,1H),7.54–7.45(m,5H),7.42–7.33(m,10H),7.13(s,1H),5.35(s,2H),5.28(s,4H).

[0048] (2) Preparation of 2,4-bis(benzyloxy)-5-isopropenyl-benzoate (AT3)

[0049] 10.0 g (19.86 mmol, 1.0 eq) of compound AT2 was dissolved in approximately 150 mL of THF:H2O (V / V = 9:1). Then, 4.41 g (29.79 mmol, 1.5 eq) of potassium isopropenyltrifluoroborate, 222.94 mg (0.993 mmol, 0.05 eq) of palladium acetate (Pd(OAc)2), 947.04 mg (1.99 mmol, 0.1 eq) of XPhos, and 19.42 g (59.58 mmol, 3.0 eq) of cesium carbonate (CsCO3) were added. The mixture was heated at 80 °C under N2 protection for 12 hours. During the reaction, UV TLC was used to monitor the completeness of the reaction in real time, with a PE:EA ratio of 2:1 as the developing solvent. After the reaction was complete, the mixture was cooled to room temperature, and the catalyst and other contaminants were removed by filtration through a diatomaceous earth mat. The filtrate was concentrated into an oily solution. The solid was extracted three times with CH₂Cl₂ and water. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain 8.98 g of a white solid powder, with a yield of 97.33%. ¹H NMR (300 MHz, DMSO-d₆) δ 7.72 (s, 1H), 7.55–7.40 (m, 15H), 7.09 (s, 1H), 5.37 (s, 2H), 5.34 (s, 4H), 5.16 (dt, J = 11.2, 1.9 Hz, 2H), 2.12 (d, J = 1.3 Hz, 3H).

[0050] (3) Preparation of 2,4-bis(benzyloxy)-5-isopropenylbenzoic acid (AT4)

[0051] 8.0 g (17.22 mmol, 1.0 eq) of compound AT3 was dissolved in approximately 50 mL of THF:MeOH:H2O (V / V / V = 2:1:1). 1.65 g (68.88 mmol, 4.0 eq) of lithium hydroxide was added, and the mixture was stirred at 40 °C for 12 h. During the reaction, UV-Vis TLC was used to monitor the completeness of the reaction, with the developing solvent being PE:EA = 3:1. After the reaction was complete, the mixture was cooled to room temperature and concentrated. The residue was adjusted to pH 5 with 1 M HCl, and then extracted with approximately 60 mL of ethyl acetate. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated to give 5.9 g of a white solid, with a yield of 91.50%. 1H NMR (300MHz, DMSO-d6) δ12.31(s,1H),7.61(s,1H),7.54(d,J=6.9Hz,2H),7.47–7. 34(m,8H),6.95(s,1H),5.26(s,2H),5.23(s,2H),5.12–5.05(m,2H),2.05(s,3H).

[0052] (4) Preparation of 2,4-dihydroxy-5-isopropylbenzoic acid (AT5)

[0053] 5.9 g (15.76 mmol, 1.0 eq) of compound AT4 was dissolved in approximately 50 mL of methanol, and 1.48 g of palladium-carbon catalyst was added. The reaction mixture was reacted at room temperature for 12 h under H2 reduction. During the reaction, UV TLC was used to monitor the completeness of the reaction, with PE:EA as the developing solvent at a ratio of 1:1. After the reaction, the catalyst and other components were removed by filtration through a diatomaceous earth mat. Elute with ethyl acetate, and concentrate the eluent to give 3.0 g of a white solid, with a yield of 97.04%. ¹H NMR (300 MHz, DMSO-d6) δ 10.36 (s, 1H), 7.49 (s, 1H), 6.32 (s, 1H), 3.11–3.03 (m, 1H), 1.13 (d, J = 6.9 Hz, 6H).

[0054] (5) Preparation of 2,4-bis(benzyloxy)-5-isopropylbenzoate (AT6)

[0055] 3.0 g (15.29 mmol, 1.0 eq) of compound AT5 was dissolved in approximately 40 mL of CH3CN solvent. Then, 5.44 mL (45.87 mmol, 3.0 eq) of benzyl bromide and 14.79 g (107.03 mmol, 7.0 eq) of potassium carbonate (K2CO3) were added. The mixture was heated and stirred at 80 °C for 8 hours. During the reaction, UV TLC was used to monitor the completeness of the reaction. The developing solvent was PE:EA = 4:1. After the reaction, the solvent was removed using a rotary evaporator, yielding a white solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over Na2SO4, filtered, and concentrated. The concentrate was purified by column chromatography with a gradient elution of PE / EA (V / V) = 200 / 1-1 / 1, yielding 6.5 g of a white solid powder, with a yield of 91.11%. 1H NMR(300MHz,DMSO-d6)δ7.64(s,1H),7.50–7.42(m,6H),7.39–7.31(m,9H),6.95(s,1H), 5.28(s,2H),5.24(s,2H),5.21(s,2H),3.19(p,J=6.9Hz,1H),1.16(s,3H),1.14(s,3H).

[0056] (6) Preparation of 2,4-bis(benzyloxy)-5-isopropylbenzoic acid (AT7)

[0057] 6.5 g (13.93 mmol, 1.0 eq) of AT6 feedstock was dissolved in approximately 40 mL of THF:MeOH:H2O at a volume ratio of 2:1:1. 1.34 g (55.72 mmol, 4.0 eq) of lithium hydroxide was added, and the mixture was stirred at room temperature for 12 h. During the reaction, UV-Vis TLC was used to monitor the completeness of the reaction, with the developing solvent being PE:EA = 5:1. After the reaction was complete, the mixture was concentrated. The residue was adjusted to pH 5 with 1 M HCl, and then extracted three times with approximately 60 mL of CH2Cl2. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated to obtain 4.9 g of a white solid, with a yield of 93.43%. 1H NMR(300MHz,DMSO-d6)δ7.61(s,1H),7.57–7.46(m,5H),7.44–7.36(m,5H), 6.92(s,1H),5.23(s,4H),3.20(p,J=6.9Hz,1H),1.18(s,3H),1.15(s,3H).

[0058] (7) Preparation of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-bromoisoindolin-2-yl)methyl ketone (AT8)

[0059] 4.9 g (13.02 mmol, 1.0 eq.) of compound AT7 was dissolved in approximately 60 mL of anhydrous dichlorodichlorophenate. Under ice bath conditions, 3.87 g (19.53 mmol, 1.5 eq.) of 5-bromoisoindoline, 4.53 mL (26.04 mmol, 2.0 eq.) of N,N-diisopropylethylamine (DIPEA), 2.48 g (18.23 mmol, 1.4 eq.) of N-hydroxy-7-azabenzotriazole (HOAt), and 7.49 g (39.06 mmol, 3.0 eq.) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were added. The mixture was stirred at room temperature for 8 hours. During the reaction, UV TLC was used to monitor the completeness of the reaction. The developing solvent was PE:EA = 6:1. After the reaction was complete, the solvent was removed using a rotary evaporator, yielding a yellow solid. The solid was extracted three times with CH₂Cl₂ and water. The organic phases were combined, dried over Na₂SO₄, filtered, and concentrated. The concentrate was purified by column chromatography with a gradient elution of PE / EA (V / V) = 100 / 1–5 / 1, yielding 6.5 g of a yellow solid powder, with a yield of 89.73%. ¹H NMR (300 MHz, DMSO-d₆) δ 7.62–7.46 (m, 8H), 7.44–7.35 (m, 5H), 7.22 (s, 1H), 7.08 (s, 1H), 5.30 (s, 4H), 4.88 (d, J = 13.8 Hz, 2H), 4.62 (d, J = 16.4 Hz, 2H), 3.32 (q, J = 7.0 Hz, 1H), 1.28 (s, 3H), 1.26 (s, 3H).

[0060] (8) Preparation of 4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazine-1-carboxylic acid tert-butyl ester (AT9)

[0061] 6.5 g (11.68 mmol, 1.0 eq) of compound AT8 was dissolved in approximately 40 mL of toluene:water, v / v = 10:3. Then, 4.65 g (15.18 mmol, 1.3 eq. of potassium (4-tert-butyloxycarbonylpiperazin-1-yl)methyltrifluoroborate), 11.42 g (35.04 mmol, 3.0 eq. of cesium carbonate (CsCO3), 97 mg (0.584 mmol, 0.05 eq. of palladium acetate (Pd(OAc)2), 339 mg (1.168 mmol, 0.1 eq. of tritert-butylphosphine tetrafluoroborate ((t-Bu)3P-HBF4)) were added. The mixture was heated and stirred at 100 °C for 14 hours under N2 protection. During the reaction, UV TLC was used to monitor the completeness of the reaction. The developing solvent was PE:EA = 1:1. After the reaction was completed and cooled to room temperature, the catalyst and other components were removed by filtration through a diatomaceous earth pad. The filtrate was concentrated into an oily solution. The solid was extracted three times with CH₂Cl₂ and water. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain 7.4 g of a light yellow solid powder, with a yield of 93.74%. ¹H NMR (300 MHz, DMSO-d₆) δ 7.52–7.37 (m, 5H), 7.35–7.30 (m, 3H), 7.28–7.17 (m, 5H), 7.10 (d, J = 1.4 Hz, 1H), 6.96 (d, J = 1.7 Hz, 1H), 5.19 (s, 4H), 4.77 (s, 2H), 4.51 (s, 2H) ,3.46(d,J=11.4Hz,2H),3.29(t,J=5.4Hz,4H),3.25–3.17(m,1H),2.28(dt,J=10. 2,4.8Hz,4H),1.38(d,J=4.1Hz,9H),1.17(d,J=2.7Hz,3H),1.15(d,J=2.5Hz,3H).

[0062] (9) Preparation of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindololin-2-yl)methyl ketone (AT10)

[0063] 7.4 g (10.95 mmol, 1.0 eq.) of compound AT9 was dissolved in approximately 10 mL of EA, and 60 mL of 2M HCl / EA was added. The mixture was stirred at room temperature for 4 hours. During the reaction, UV TLC was used to monitor the completeness of the reaction. The volume ratio of the developing solvent DCM:MeOH was 10:1. After the reaction, the solvent was discarded, leaving an orange solid. The pH of the solid was adjusted to alkaline using saturated NaHCO3 solution, and the aqueous phase was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The concentrate was purified by column chromatography using a gradient elution with a DCM / MeOH volume ratio of 250 / 1 - 15 / 1 + 1% triethylamine to obtain 6.0 g of a light yellow solid powder, with a yield of 95.18%. 1H NMR(300MHz,DMSO-d6)δ7.51–7.38(m,5H),7.34–7.29(m,3H),7.24(dd,J=4.7 ,2.6Hz,4H),7.17(d,J=10.1Hz,2H),6.96(d,J=1.7Hz,1H),5.19(s,4H),4.77( s,2H),4.51(s,2H),3.46(s,1H),3.42(s,1H),3.26–3.20(m,1H),2.80(dt,J=1 0.1, 4.6Hz, 4H), 2.35 (s, 4H), 1.23 (t, J = 4.4Hz, 1H), 1.17 (s, 3H), 1.14 (s, 3H).

[0064] 2) Preparation of each embodiment

[0065] Example 1

[0066] [Corrected according to Rule 91, 16.05.2025] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)heptane-1,3-dione (DDO-4104)

[0067] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)heptane-1,3-dione (DDO-4104-a).

[0068] 1.0 g, 1.74 mmol, and 2.0 eq. of AT10 were dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, 139.35 mg, 0.87 mmol, and 1.0 eq. of pimecrolic acid, and 661.62 mg, 1.74 mmol, and 2.0 eq. of HATU were added. Then, 483.71 μL, 3.48 mmol, and 4.0 eq. of Et3N were slowly added, and the mixture was stirred at room temperature for 8 h. After the reaction was complete, approximately 40 mL of water was added to the reaction solution for extraction. The separated organic layer was dried over anhydrous sodium sulfate and then evaporated to dryness. The crude product was purified by column chromatography using a gradient elution with a DCM / MeOH volume ratio of 100 / 1 to 20 / 1, yielding 920 mg of an orange solid, with a yield of 82.90%. 1H NMR (300MHz, DMSO-d6) δ7.52–7.42(m,8H),7.34(d,J=7.7Hz,7H),7.28–7.16(m,11H),7.12(s,2H),6.98(s,2H),5.21(s,8H),4.79 (s,4H),4.53(s,4H),3.51–3.39(m,12H),3.23(q,J=6.8Hz,2H),2.29(s,12H),1.47(s,4H),1.27(s,2H),1.18(s,6H),1.16(s,6H).

[0069] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)heptane-1,3-dione (DDO-4104)

[0070] 920 mg (0.7212 mmol, 1.0 eq.) of compound DDO-5104-a was dissolved in approximately 40 mL of 1,2-dichloroethane:methanol, V / V = 1:1, and 3.64 g (57.70 mmol, 80.0 eq.) of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 920 mg Pd / C was added. Stirring continued at 40 °C for 50 minutes. During the reaction, UV TLC was used to monitor the completeness of the reaction. The developing solvent was DCM:MeOH at a volume ratio of 15:1. After the reaction was complete, the mixture was cooled to room temperature, and the catalyst was removed by filtration through a diatomaceous earth mat. The filtrate was concentrated into a solid. The solid was extracted three times with CH2Cl2 and water. The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography with a gradient elution of DCM / MeOH at a volume ratio of 60 / 1 to 20 / 1, yielding 110 mg of a white solid, with a yield of 15.46%. 1H NMR (300MHz, DMSO-d6) δ10.09(s,2H),9.65(s,2H),7.28(d,J=19.5Hz,6H),7.06(s,2H),6.42(s,2H),4.78(s ,8H),3.44(s,12H),3.15–3.07(m,2H),2.38–2.24(m,12H),1.49(s,4H),1.27(s,2H),1.16(d,J=6.9Hz,12H).

[0071] Example 2

[0072] [Corrected according to Rule 91, 16.05.2025] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)octane-1,3-dione (DDO-4105)

[0073] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)octane-1,3-dione (DDO-4105-a)

[0074] The rest is the same as step (1) in Example 1. The difference from step (1) in Example 1 is that 151.55 mg, 0.87 mmol, and 1.0 eq. of octanoic acid were added to obtain orange solid DDO-4105-a with a yield of 80%. 1 H NMR (300MHz, DMSO-d6) δ7.52–7.42(m,8H),7.34(d,J=7.7Hz,7H),7.28–7.16(m,11H),7.12(s,2H),6.98(s,2H),5.21(s,8H),4.79 (s,4H),4.53(s,4H),3.51–3.39(m,12H),3.23(q,J=6.8Hz,2H),2.29(s,12H),1.47(s,4H),1.27(s,4H),1.18(s,6H),1.16(s,6H).

[0075] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)octane-1,3-dione (DDO-4105)

[0076] The procedure was the same as in step (2) of Example 1, except that 900 mg, 0.6978 mmol, and 1.0 eq. of compound DDO-4105-a were dissolved in approximately 40 mL of 1,2-dichloroethane:methanol at a volume ratio of 1:1, and 3.52 g, 55.82 mmol, and 80.0 eq. of ammonium formate were added. The mixture was stirred at 40°C for 10 minutes, and then 10% of 900 mg Pd / C was added, yielding 115 mg of a white solid, with a yield of 17.74%. 1 H NMR (300MHz, DMSO-d6) δ10.09(s,2H),9.64(s,2H),7.27(d,J=15.3Hz,6H),7.06(s,2H),6.42(s,2H),4.78(s,8H),3.48(d ,J=24.2Hz,12H),3.14–3.09(m,2H),2.32(dd,J=23.1,10.7Hz,12H),1.47(s,4H),1.27(s,4H),1.16(s,6H),1.14(s,6H).

[0077] Example 3

[0078] [Corrected according to Rule 91, 16.05.2025] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)nonane-1,3-dione (DDO-4106)

[0079] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)nonane-1,3-dione (DDO-4106-a)

[0080] The rest is the same as step (1) in Example 1. The difference from step (1) in Example 1 is that 163.75 mg, 0.87 mmol, and 1.0 eq. of azelaic acid were added to obtain 910 mg of orange solid, with a yield of 80.23%. 1H NMR(300MHz,DMSO-d6)δ7.50(d,J=1.9Hz,2H),7.36–7.29(m,9H),7.26–7.13(m,14H),7.11–7.04(m,3H),6.93(d,J=13.2Hz,2H),5.18 (s,8H),4.77(s,4H),4.50(s,4H),3.42(s,12H),3.25–3.18(m,2H),2.26(s,12H),1.45(s,4H),1.23(s,6H),1.16(s,6H),1.14(s,6H).

[0081] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)nonane-1,3-dione (DDO-4106)

[0082] The procedure was the same as in Example 1, except that 910 mg, 0.6980 mmol, 1.0 eq. of compound DDO-4106-a was dissolved in about 40 mL of 1,2-dichloroethane:methanol, and 3.52 g, 55.84 mmol, 80.0 eq. of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 910 mg of Pd / C was added to give 100 mg of white solid, with a yield of 15.19%. 1 H NMR (300MHz, DMSO-d6) δ10.06(s,2H),9.66(s,2H),7.26(d,J=22.4Hz,6H),7.03(s,2H),6.41(s,2H),4.76(s,8H),3.44(d ,J=16.4Hz,12H),3.08(q,J=6.9Hz,2H),2.27(q,J=10.8,7.4Hz,12H),1.45(s,4H),1.23(s,6H),1.14(s,6H),1.12(s,6H).

[0083] Example 4

[0084] [Corrected according to Rule 91, 16.05.2025] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)decane-1,3-dione (DDO-4107)

[0085] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)decane-1,3-dione (DDO-4107-a)

[0086] The rest of the steps are the same as in Example 1, except that 1.0 g, 1.74 mmol, and 2.0 eq. AT10 are dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, 175.96 mg, 0.87 mmol, and 1.0 eq. sebacic acid, and 661.62 mg, 1.74 mmol, and 2.0 eq. HATU are added, followed by the slow addition of 483.71 μL, 3.48 mmol, and 4.0 eq. Et3N. The mixture is stirred at room temperature for 8 h. 920 mg of an orange solid is obtained, with a yield of 80.25%. 1 H NMR (300MHz, DMSO-d6) δ7.52–7.36(m,13H),7.28–7.14(m,13H),7.11(d,J=1.4Hz,2H),6.97(s,2H),5.20(s,8H),4.78(s,4H),4.5 2(s,4H),3.43(d,J=11.3Hz,12H),3.28–3.19(m,2H),2.27(t,J=6.8Hz,12H),1.46(s,4H),1.24(s,8H),1.17(s,6H),1.15(s,6H).

[0087] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)decane-1,3-dione (DDO-4107)

[0088] The procedure was the same as in step (2) of Example 1, except that 920 mg, 0.6982 mmol, 1.0 eq. of compound DDO-4107-a was dissolved in about 40 mL of 1,2-dichloroethane:methanol, and 3.52 g, 55.86 mmol, 80.0 eq. of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 920 mg of Pd / C was added. The mixture was stirred at 40 °C for another 50 minutes to obtain 110 mg of a white solid, with a yield of 16.46%. 1H NMR(300MHz,DMSO-d6)δ10.09(s,2H),9.63(s,2H),7.24(s,6H),7.06(s,2H),6.42(s,2H),4.78(s,8H),3.49(s,4 H),3.43(s,8H),3.14–3.07(m,2H),2.31(t,J=12.0Hz,12H),1.47(s,4H),1.26(s,8H),1.17(s,6H),1.14(s,6H).

[0089] Example 5

[0090] [Corrected according to Rule 91, 16.05.2025] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)undecane-1,3-dione (DDO-4108)

[0091] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)undecane-1,3-dione (DDO-4108-a)

[0092] The procedure was the same as in Example 1, except that 1.0 g, 1.74 mmol, and 2.0 eq. AT10 were dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, 188.16 mg, 0.87 mmol, and 1.0 eq. undecanoic acid, and 661.62 mg, 1.74 mmol, and 2.0 eq. HATU were added, followed by slow addition of 483.71 μL, 3.48 mmol, and 4.0 eq. Et3N. The mixture was stirred at room temperature for 8 hours. 922 mg of an orange solid was obtained, with a yield of 79.58%. 1 H NMR(300MHz,DMSO-d6)δ7.54–7.46(m,6H),7.45(s,2H),7.32(s,7H),7.25(dd ,J=4.6,2.4Hz,8H),7.20(s,2H),7.17(s,1H),7.11(d,J=1.3Hz,2H),6.97(s,2 H),5.20(s,8H),4.79(s,4H),4.52(s,4H),3.50–3.38(m,12H),3.28–3.19(m, 2H),2.35–2.22(m,12H),1.47(s,4H),1.25(s,10H),1.18(s,6H),1.16(s,6H).

[0093] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)undecane-1,3-dione (9C-2)

[0094] The procedure was the same as in step (2) of Example 1, except that 922 mg, 0.6923 mmol, 1.0 eq. of compound DDO-4108-a was dissolved in about 40 mL of 1,2-dichloroethane:methanol, and 3.49 g, 55.38 mmol, 80.0 eq. of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 922 mg of Pd / C was added to give 115 mg of white solid, with a yield of 17.10%. 1 H NMR (300MHz, DMSO-d6) δ10.16(s,2H),7.26(d,J=15.6Hz,6H),7.06(s,2H),6.43(s,2H),4.78(s,8H),3.46(d,J= 16.4Hz,12H),3.15–3.08(m,2H),2.30(t,J=11.8Hz,12H),1.47(s,4H),1.26(s,10H),1.17(s,6H),1.14(s,6H).

[0095] Example 6

[0096] [Corrected according to Rule 91, 16.05.2025] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)dodecane-1,3-dione (DDO-4109)

[0097] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)dodecane-1,3-dione (DDO-4109-a)

[0098] The procedure was the same as in Example 1, except that 1.0 g, 1.74 mmol, and 2.0 eq. AT10 were dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, 200.36 mg, 0.87 mmol, and 1.0 eq. dodecanoic acid, and 661.62 mg, 1.74 mmol, and 2.0 eq. HATU were added, followed by the slow addition of 483.71 μL, 3.48 mmol, and 4.0 eq. Et3N. The mixture was stirred at room temperature for 8 hours. 930 mg of an orange solid was obtained, with a yield of 79.43%. 1H NMR (300MHz, DMSO-d6) δ7.56–7.45(m,11H),7.39–7.33(m,7H),7.28(tt,J=4.6,3.3,2.5Hz,8H),7.15(d,J=1.4Hz,2H),7.01(s,2H),5.24(s,8H),4 .82(s,4H),4.56(s,4H),3.50(d,J=19.3Hz,12H),3.31–3.23(m,2H),2.3 2(d,J=6.5Hz,12H),1.50(s,4H),1.28(s,12H),1.21(s,6H),1.19(s,6H).

[0099] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)dodecane-1,3-dione (DDO-4109)

[0100] The procedure was the same as in step (2) of Example 1, except that 930 mg, 0.6910 mmol, 1.0 eq. of compound DDO-4109-a was dissolved in about 40 mL of 1,2-dichloroethane:methanol, and 3.48 g, 55.28 mmol, 80.0 eq. of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 930 mg of Pd / C was added to give 110 mg of white solid, with a yield of 16.16%. 1 H NMR(300MHz,DMSO-d6)δ10.09(s,2H),9.67(s,2H),7.24(s,4H),7.05(s,2H),6.43(s,2H),5.78(s,2H),4.78(s,8H),3.46(d ,J=17.1Hz,12H),3.11(p,J=6.9Hz,2H),2.29(q,J=11.8,7.5Hz,12H),1.47(s,4H),1.25(s,12H),1.16(s,6H),1.14(s,6H).

[0101] Example 7

[0102] [Corrected according to Rule 91, 16.05.2025] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)tetradecane-1,3-dione (DDO-4110)

[0103] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)tetradecane-1,3-dione (DDO-4110-a)

[0104] The procedure was the same as in Example 1, except that 1.0 g, 1.74 mmol, and 2.0 eq. AT10 were dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, 224.77 mg, 0.87 mmol, and 1.0 eq. tetradecanoic acid, and 661.62 mg, 1.74 mmol, and 2.0 eq. HATU were added, followed by the slow addition of 483.71 μL, 3.48 mmol, and 4.0 eq. Et3N. The mixture was stirred at room temperature for 8 hours. 950 mg of an orange solid was obtained, with a yield of 79.48%. 1 H NMR (300MHz, DMSO-d6) δ7.52–7.36(m,13H),7.28–7.14(m,13H),7.11(d,J=1.4Hz,2H),6.97(s,2H),5.20(s,8H),4.78(s,4H),4.5 2(s,4H),3.43(d,J=11.3Hz,12H),3.28–3.19(m,2H),2.27(t,J=6.8Hz,12H),1.46(s,4H),1.24(s,16H),1.17(s,6H),1.15(s,6H).

[0105] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)tetradecane-1,3-dione (DDO-4110)

[0106] The procedure was the same as in step (2) of Example 1, except that 950 mg, 0.6915 mmol, 1.0 eq. of compound DDO-4110-a was dissolved in about 40 mL of 1,2-dichloroethane:methanol, and 3.48 g, 55.32 mmol, 80.0 eq. of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 950 mg Pd / C was added to give 120 mg of white solid, with a yield of 17.36%. 1H NMR (300MHz, DMSO-d6) δ10.07(s,1H),9.64(s,1H),7.24(d,J=15.4Hz,6H),7.04(s,2H),6.39(s,2H),4.76(s,8H),3.46 (s,4H),3.40(s,8H),3.15–3.02(m,2H),2.28(t,J=11.8Hz,12H),1.45(s,4H),1.23(s,16H),1.14(s,6H),1.12(s,6H).

[0107] Example 8

[0108] [Corrected according to Rule 91, 16.05.2025] Preparation of 3,3'-oxobis(1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)prop-1-one)(DDO-4111)

[0109] (1) Preparation of 3,3'-oxobis(1-(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)prop-1-one)(DDO-4111-a)

[0110] The procedure was the same as in Example 1, except that 1.0 g, 1.74 mmol, 2.0 eq. AT10 was dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, 140.81 mg, 0.87 mmol, 1.0 eq. 3,3'-oxydipropionic acid, and 661.62 mg, 1.74 mmol, 2.0 eq. HATU were added, followed by slow addition of 483.71 μL, 3.48 mmol, 4.0 eq. Et3N. The mixture was stirred at room temperature for 8 h. 887 mg of an orange solid was obtained, with a yield of 80.02%. 1H NMR (300MHz, DMSO-d6) δ7.50–7.43(m,10H),7.35–7.29(m,10H),7.24(d,J=3.2Hz,6H),7.10(d,J=1.5Hz,2H),6.96(s,2H),5.19( s,8H),4.77(s,4H),4.51(s,4H),3.61–3.47(m,8H),3.42(s,8H),3.21(d,J=6.9Hz,2H),2.29(s,12H),1.16(s,6H),1.14(s,6H).

[0111] (2) Preparation of 3,3'-Oxadiazine (1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)prop-1-one)(DDO-4111)

[0112] The procedure was the same as in Example 1, except that 887 mg, 0.6947 mmol, 1.0 eq. of compound DDO-4111-a was dissolved in about 40 mL of 1,2-dichloroethane:methanol, and 3.49 g, 55.32 mmol, 80.0 eq. of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 950 mg Pd / C was added to give 110 mg of white solid, with a yield of 17.19%. 1H NMR (300MHz, DMSO-d6) δ10.08(s,2H),9.63(s,2H),7.25(d,J=19.6Hz,7H),7.04(s,2H),6.40(s,2H),4.76(s, 8H),3.57(t,J=6.5Hz,8H),3.34(s,8H),3.12–3.06(m,2H),2.30(d,J=13.5Hz,12H),1.14(s,6H),1.12(s,6H).

[0113] Example 9

[0114] [Corrected according to Rule 91, 16.05.2025] Preparation of 2,2'-(ethane-1,2-diylbis(oxy))bis(1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)acet-1-one)(DDO-4112)

[0115] (1) Preparation of 2,2'-(ethane-1,2-diylbis(oxy))bis(1-(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)acet-1-one)(DDO-4112-a)

[0116] The procedure was the same as in Example 1, except that 1.0 g, 1.74 mmol, and 2.0 eq. AT10 were dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, 154.70 mg, 0.87 mmol, and 1.0 eq. triethylene glycol diacetic acid, and 661.62 mg, 1.74 mmol, and 2.0 eq. HATU were added, followed by the slow addition of 483.71 μL, 3.48 mmol, and 4.0 eq. Et3N. The mixture was stirred at room temperature for 8 h. 921 mg of an orange solid was obtained, with a yield of 82.00%. 1H NMR (300MHz, DMSO) δ7.50,7.49,7.47,7.45,7.43,7.40,7.38,7.35,7.33,7.31,7.30,7.24,7.23,7.22, 7.18,7.17,7.14,7.09,7.09,6.98,6.95,5.18,4.76,4.50,4.13,4.12,4.10,3.86,3.56,3.54,3.53,3. 47,3.43,3.33,3.24,3.22,3.19,3.18,3.16,3.11,3.08,3.06,3.03,2.51,2.51,2.50,2.49,2.49,2.31,2.01,1.99,1.77,1.33,1.30,1.26,1.25,1.23,1.20,1.17,1.16,1.15,1.13,1.01,0.99,0.85,-0.00.

[0117] (2) Preparation of 2,2′-(ethane-1,2-diylbis(oxy))bis(1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindololin-5-yl)methyl)piperazin-1-yl)acet-1-one)(DDO-4112)

[0118] The procedure was the same as in step (2) of Example 1, except that 921 mg, 0.6915 mmol, 1.0 eq. of compound DDO-4112-a was dissolved in about 40 mL of 1,2-dichloroethane:methanol, and 3.50 g, 55.87 mmol, 80.0 eq. of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 950 mg Pd / C was added to give 128 mg of white solid, with a yield of 20.02%.

[0119] Example 10

[0120] Preparation of (((pyridine-2,4-dicarbonyl)bis(piperazine-4,1-diyl))bis(methylene))bis(isoindoline-5,2-diyl))bis((2,4-dihydroxy-5-isopropylphenyl) methyl ketone) (DDO-4113)

[0121] [Corrected according to Rule 91, May 16, 2025] Crafting route:

[0122] Preparation of (1) (((pyridine-2,4-dicarbonyl)bis(piperazine-4,1-diyl))bis(methylene))bis(isoindoline-5,2-diyl))bis((2,4-bis(benzyloxy)-5-isopropylphenyl) ketone) (DDO-4113-a)

[0123] The procedure was the same as in Example 1, except that 1.0 g, 1.74 mmol, and 2.0 eq. AT10 were dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, 145.39 mg, 0.87 mmol, and 1.0 eq. 2,4-pyridinedicarboxylic acid, and 661.62 mg, 1.74 mmol, and 2.0 eq. HATU were added, followed by the slow addition of 483.71 μL, 3.48 mmol, and 4.0 eq. Et3N. The mixture was stirred at room temperature for 8 h. 900 mg of an orange solid was obtained, with a yield of 80.66%. 1H NMR (300MHz, DMSO-d6) δ8.66(q,J=4.3Hz,1H),7.50(t,J=6.6Hz,7H),7.35(q,J=5.5,5.0Hz,9H),7.28–7.17(m,12H),7.12(s,2H),6.98(s,2H),5 .21(s,8H),4.79(s,4H),4.53(s,4H),3.65(s,4H),3.51(d,J=11.7Hz,4 H),3.36–3.19(m,6H),2.40(d,J=27.3Hz,8H),1.18(s,6H),1.16(s,6H).

[0124] (2) Preparation of (((pyridine-2,4-dicarbonyl)bis(piperazine-4,1-diyl))bis(methylene))bis(isoindoline-5,2-diyl))bis((2,4-dihydroxy-5-isopropylphenyl) methyl ketone) (DDO-4113)

[0125] The procedure was the same as in step (2) of Example 1, except that 900 mg, 0.7017 mmol, 1.0 eq. of compound DDO-4113-a was dissolved in about 40 mL of 1,2-dichloroethane:methanol, and 3.54 g, 56.136 mmol, 80.0 eq. of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 900 mg Pd / C was added to give 100 mg of white solid, with a yield of 15.46%. 1 H NMR (300MHz, DMSO-d6) δ10.07(s,2H),9.63(s,2H),8.65(d,J=5.0Hz,1H),7.51(s,1H),7.45(d,J=5.1Hz,1H),7.23(s,4H),7.04(s,2H),6 .40(s,2H),4.76(s,8H),3.63(s,4H),3.51(s,4H),3.26(s,4H),3.09(t,J=6.9Hz,2H),2.40(d,J=24.9Hz,8H),1.14(s,6H),1.12(s,6H).

[0126] Example 11

[0127] [Corrected according to Rule 91, 16.05.2025] Preparation of 1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)-2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-carbonyl)phenyl)ethyl-1-one (DDO-4114)

[0128] (1) Preparation of 1-(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)-2-(4-(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-carbonyl)phenyl)ethyl-1-one (DDO-4114-a)

[0129] The procedure was the same as in Example 1, except that 1.0 g, 1.74 mmol, and 2.0 eq. AT10 were dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, 168.64 mg, 0.87 mmol, and 1.0 eq. 4-carboxyphenylacetic acid, and 661.62 mg, 1.74 mmol, and 2.0 eq. HATU were added, followed by slow addition of 483.71 μL, 3.48 mmol, and 4.0 eq. Et3N. The mixture was stirred at room temperature for 8 h. 956 mg of an orange solid was obtained, with a yield of 85.12%. 1H NMR(300MHz, DMSO-d6)δ7.55–7.40(m,10H),7.34(dd,J=16.3,5.4Hz,10H),7.28–7.18(m,10H),7.16(s,1H),7.10(d,J=2.0Hz,2H),6.96(s, 1H),5.19(s,8H),4.78(s,4H),4.51(s,4H),4.05–3.97(m,2H),3.55(s ,12H),3.06(s,2H),2.31(d,J=21.5Hz,8H),1.17(s,6H),1.14(s,6H).

[0130] (2) Preparation of 1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)-2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-carbonyl)phenyl)ethyl-1-one (DDO-4114)

[0131] The procedure was the same as in step (2) of Example 1, except that 956 mg, 0.7381 mmol, 1.0 eq. of compound DDO-4114-a was dissolved in about 40 mL of 1,2-dichloroethane:methanol, and 3.52 g, 55.99 mmol, 80.0 eq. of ammonium formate was added. The mixture was stirred at 40 °C for 10 minutes, and then 10% of 950 mg Pd / C was added to give 172 mg of white solid, with a yield of 24.90%. 1H NMR(300MHz,DMSO-d6)δ10.05(s,2H),9.62(s,2H),7.30–7.22(m,10H),7.03(s,2H),6.39(s,2H),4.75(s,8H ),3.73(s,2H),3.47(d,J=13.6Hz,12H),3.11–3.06(m,2H),2.32(d,J=29.7Hz,8H),1.14(s,6H),1.12(s,6H).

[0132] Experimental Example 1

[0133] Fluorescence polarization experiment (FP method)

[0134] Table 1 shows the results of the compounds' competitive binding activity against FITC-GA molecules to HSP90 NTD and their activity in inducing HSP90 NTD dimerization.

[0135] *a:The concentration of HSP90 NTD=10μM, the concentration of compound:protein=1:2

[0136] Note: The structure of the compound is shown in the specific examples.

[0137] As shown in Table 1, the compounds in the examples all exhibited nM-level binding activity to HSP90 NTD and displayed a structure-activity relationship. Shorter chain length molecules showed relatively better binding activity, essentially maintaining a similar level to the positive control compound AT13387. This indicates that the compounds in the examples of this patent retain strong HSP90 binding activity.

[0138] The procedure for testing the competitive binding activity of the compound with HSP90 NTD using the FP method is as follows:

[0139] First, prepare a 50 mL buffer solution containing 20 mM HEPES, 50 mM KCl, 5 mM MgCl2, and 20 mM NaMoO4, and adjust the pH to 7.3-7.4. Then, add 0.01% NP40, 2 mM DTT, and 0.1 mg / mL BGG. Dissolve the test compound (DMSO content less than 10%), HSP90 NTD protein, and probe molecules in this buffer solution. Using a 384-well plate, set up two replicates. Add 20 μL of HSP90 NTD protein solution (final concentration 60 nM), 20 μL of probe molecule solution (final concentration 40 nM), and finally 20 μL of compound solution (maximum final concentration 3 μM, 3-fold dilution, 10 concentration gradients) to each well. For the [probe + protein] control wells, add 20 μL of HSP90 NTD protein solution, 20 μL of probe molecule solution, and 20 μL of the buffer solution for this system. For the [probe] control wells, add 20 μL of probe molecule solution and 40 μL of buffer solution. Incubate the 384-well plate at room temperature (25℃) for 30 min. Collect data using a microplate reader at a wavelength of 405 nm. Analyze the results using GraphPad software.

[0140] Experiment Example 2

[0141] Native-page experiment

[0142] The activity of divalent compound molecules in inducing HSP90 NTD dimerization was tested using native-page assays.

[0143] As shown in Figure 1, the divalent inhibitor molecules DDO-4105, DDO-4110 and DDO-4113 induce HSP90 dimerization, while the ATP inhibitor does not induce dimerization. The test results show that the compounds induce HSP90 NTD dimerization in a concentration-dependent and time-dependent manner, and exhibit a typical "Hook" effect at high concentrations.

[0144] As shown in Table 1, the compounds in the examples induced different degrees of dimerization when the compound to protein concentration ratio was 1:2, exhibiting a structure-activity relationship related to the linker chain length. Among them, compound DDO-4113 in the examples showed the strongest effect.

[0145] The operation method for Native-page testing is as follows:

[0146] The native-page dimerization experiment followed standard experimental procedures. The specific procedures included protein extraction, co-incubation with different ratios of protein and compound, sampling at different time points, preparation of non-denaturing polyacrylamide gels, native-page analysis, and result scanning. The dimerization effect of HSP90 NTD was tested in the experiment.

[0147] Experimental Example 3

[0148] X-ray crystal structure diffraction

[0149] Crystal structure diffraction confirmed that the divalent inhibitor molecule can act on the binding pocket of HSP90, induce the non-natural dimerization of HSP90NTD, and cause a significant change in the conformation of the HSP90 NTD dimer.

[0150] As shown in Figure 2, DDO-4105 induces non-natural dimerization of HSP90 NTD.

[0151] The operation method of crystal structure diffraction is as follows:

[0152] HSP90 NTD protein extraction, HSP90 NTD protein purification and monomer identification, HSP90 NTD monomer protein incubation and co-purification with small molecules, Native-page electrophoresis identification, crystal growth and culture, initial screening of co-crystallization conditions, multi-round optimization of crystallization conditions, crystal data collection, processing and model building, and crystal structure analysis.

[0153] Experiment Example 4

[0154] Immunoprecipitation assay (Co-IP method)

[0155] Co-IP assay to detect the interference of bivalent inhibitor molecules on HSP90-related protein-protein interactions.

[0156] The interactions of the compounds in the embodiments of the present invention with HSP90-related chaperone proteins (CDC37, HOP, and P23, etc.) are as follows:

[0157] As shown in Figure 3, the test results indicate that DDO-4113 at high concentrations inhibits the interaction between the co-chair protein and HSP90, suggesting that the divalent inhibitor molecule exerts a different mechanism of action than previous ATP inhibitors when it exerts strong anti-proliferative activity on tumor cells, and can block protein interactions related to HSP90.

[0158] The Co-IP testing procedure is as follows:

[0159] Western blotting experiments were conducted according to standard procedures. The specific procedures included cell drug administration, cell lysis, total protein collection, overnight incubation with primary antibody to identify the complex, incubation with Protein A / G magnetic beads, preparation of polyacrylamide gels, SDS-PAGE analysis, incubation with primary antibody, incubation with secondary antibody, and result scanning. The expression levels of co-chaperone proteins (CDC37, HOP, and P23, etc.) were also measured.

[0160] Experimental Example 5

[0161] Cell anti-proliferation assay (CCK8 assay)

[0162] The CCK8 assay was used to test the cell proliferation inhibitory activity of the bivalent inhibitor molecule in the HCT116 colon cancer cell line.

[0163] As shown in Figure 4, the cell proliferation inhibitory activity of the bivalent inhibitor molecule exhibited the same anti-proliferative activity as AT13387.

[0164] The operation method of the CCK8 method test is as follows:

[0165] Logarithmically growing cells were cultured in 96-well plates for 24 hours, with 100 μL per well (total number of tumor cells per well: 3000-8000). After 24 hours, different concentrations of the compound were added to the drug-treated groups, diluted with the corresponding culture medium, with 8 concentrations and 3 replicates per group. The control group received the same volume of culture medium as the experimental groups. The plates were then incubated in a cell culture incubator. After 72 hours, the culture medium was discarded, and 100 μL of cell culture medium containing 10% CCK8 was added to each well. The plates were incubated at 37°C for 1-3 hours with gentle shaking, and the optical density (OD) was measured using a microplate reader (reference wavelength 450 nm, detection wavelength 570 nm) until the OD value of the blank control group was >1.2. Tumor cells treated with the culture medium served as the control group. The inhibition rate of the compound on tumor cells was calculated using the following formula and analyzed using Graphpad Prism software.

[0166] Experimental Example 6

[0167] Western blot analysis of the effect of divalent inhibitor molecules on substrate proteins

[0168] The attenuating effect of the compounds in the embodiments of the present invention on the substrate protein is as follows:

[0169] As shown in Figure 5, the test results indicate that DDO-4113 caused a concentration-dependent downregulation of the substrate protein, and the effect was consistent with that of AT13387, indicating that the bivalent inhibitor molecule maintained considerable activity in degrading the substrate protein.

[0170] The operation method for Western blot testing is as follows:

[0171] Western blotting experiments were conducted according to standard procedures. The specific procedures included cell drug administration, cell lysis, total protein collection, preparation of polyacrylamide gels, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation, and result scanning. The expression levels of HSPs (HSP90, HSP70, HSP40, and HSP27) protein families were measured in the experiments.

[0172] Experimental Example 7

[0173] Western blot analysis of the activity of divalent inhibitor molecules in reducing the heat shock effect

[0174] The effect of the compounds in the embodiments of the present invention on reducing the heat shock effect is as follows:

[0175] As shown in Figure 6, the test results show that DDO-4113 weakens the heat shock effect compared to AT13387 in a concentration-dependent manner, indicating that the divalent inhibitor molecule can overcome the heat shock response to a certain extent while having good inhibitory or activity.

[0176] As shown in Figure 7, the test results show that DDO-4113 weakens the heat shock effect compared to AT13387 in a concentration-dependent manner, indicating that the divalent inhibitor molecule can overcome the heat shock response to a certain extent while having good inhibitory or activity.

[0177] The operation method for Western blot testing is as follows:

[0178] Western blotting experiments were conducted according to standard procedures. The specific procedures included cell drug administration, cell lysis, total protein collection, preparation of polyacrylamide gels, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation, and result scanning. The expression levels of HSPs (HSP90, HSP70, HSP40, and HSP27) protein families were measured in the experiments.

[0179] Experimental Example 8

[0180] Pharmacokinetic studies of bivalent inhibitor molecules

[0181] Pharmacokinetic studies of compound DDO-4113 were conducted in SD rats. Rats were randomly assigned to groups of n=3. Intraperitoneal injection of 5 mg / kg was administered. 150 μL of blood samples were collected at predetermined time points (0, 0.017, 0.133, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, and 24 h) and immediately centrifuged at 4 °C and 3000 rpm for 10 min. Plasma samples were analyzed by LC-MS / MS (SHIMADZU LCMS-8050), and results were analyzed using Phoenix software.

[0182] Table 2 Pharmacokinetic data of compound DDO-4113

[0183] As shown in Table 2, after intraperitoneal administration of 5 mg / kg, the plasma Cmax was 16.2 ± 6 ng / mL, Tmax was 40 min, and AUC was [missing value]. (0-∞) It was 528±88 min*ng / mL, T 1 / 2 The duration was 118 ± 59 min, MRT (0-∞) The time to bioavailability was 190±69 min, Vz was 338449±172059 mL / kg, CLz was 1928±328 mL / min / kg, and the bioavailability F reached 25.98%. These experimental results indicate that the divalent molecule has good pharmacokinetic properties and can be further evaluated for pharmacodynamics in vivo.

[0184] Experimental Example 9

[0185] In vivo pharmacodynamic evaluation of compound DDO-4113

[0186] In vivo pharmacodynamic studies of compound DDO-4113 were conducted in balb / c-nu nude mice (5 weeks). Mice were fed standard laboratory rodent diet and water for 5 days under standard conditions (12 / 12h light / dark cycle, 22±3℃, 40% relative humidity). HCT116 cells (5*10⁻⁶ cells) were then subcutaneously injected into the right side of the mice. 6 (Number of tumors). When the average tumor volume reaches approximately 100 mm... 3 Mice were randomly divided into 5 groups (including a control group, high / low dose AT13387 groups, and high / low dose DDO-4113 groups, n=6 in each group). The drugs were administered intraperitoneally every two days (the control group received saline) for 21 consecutive days. Mice mortality was monitored every other day, and tumor volume and nude mouse weight were measured. Mice were considered dead at the end of the 21-day period or when the tumor volume reached 1800 mmHg. 3 Afterwards, the nude rats were euthanized.

[0187] As shown in Figures 8 and 9, high doses (50 mg / kg) of AT13387 exhibited strong toxicity, with mice beginning to die on the 3rd day after administration and all mice dying by the 9th day, showing significant weight loss and demonstrating strong toxicity. High doses (50 mg / kg) of DDO-4113 also resulted in weight loss, but the effect was weaker compared to AT13387, and the survival rate of mice was better than that of AT13387. Low doses (25 mg / kg) of both compounds showed relatively weak toxic side effects.

[0188] As shown in Figure 10, DDO-4113 and AT13387 have similar anti-tumor growth effects at both high and low doses, and can exert a good tumor-suppressing effect.

Claims

1. A bivalent inhibitor based on heat shock protein 90, characterized in that, The divalent inhibitor is a compound with the structural formula shown in Formula I: ALB Formula I. Or its pharmaceutically acceptable salts, solvates, or optical isomers. Among them, A and B are ATP inhibitors of heat shock protein 90. The motif of L is a flexible linking group of PEG and alkane or a rigid linking group with aryl, heteroalkyl or heteroaryl groups.

2. [Correction 07.01.2025 according to Rule 91] The bivalent inhibitor based on heat shock protein 90 according to claim 1, characterized in that, A and B are selected, or jointly selected, from the following compounds:

3. [Correction 07.01.2025 according to Rule 91] The bivalent inhibitor based on heat shock protein 90 according to claim 1, characterized in that, The motif structure of L can be chosen from one of the following: Where n represents any independent natural number between 1 and 12.

4. [Correction 07.01.2025 according to Rule 91] The bivalent inhibitor based on heat shock protein 90 according to claim 1, characterized in that, Choose one of the following structural formulas for the compound shown in Formula I:

5. The method for preparing the divalent inhibitor based on heat shock protein 90 according to claim 4, characterized in that, The method includes the following steps: (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindoline-2-yl) methyl ketone was dissolved in 50 mL of anhydrous DCM. Under ice bath conditions, a dicarboxylic acid compound and HATU were added, followed by the slow addition of Et3N. The mixture was stirred at room temperature. After the reaction was completed, water was added to the reaction solution for extraction. The separated organic layer was dried with anhydrous sodium sulfate and then evaporated to dryness. The crude product was purified by column chromatography. The stoichiometric ratio of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindoline-2-yl) methyl ketone, the dicarboxylic acid compound, HATU, and Et3N was 2:1:2:

4. The purified crude product was dissolved in a mixed solution of 1,2-dichloroethane and methanol, and ammonium formate was added. The mixture was stirred at 40°C for 10 minutes, and then 10% Pd / C was added. The mixture was stirred at 40°C for another 50 minutes. After the reaction was completed, the mixture was cooled to room temperature, and the catalyst was removed by filtration through a diatomaceous earth pad. The filtrate was concentrated into a solid, and the solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried with anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography to obtain a divalent inhibitor based on heat shock protein 90. The stoichiometric ratio of the purified crude product to ammonium formate was 1:

80.

6. The method for preparing the bivalent inhibitor based on heat shock protein 90 according to claim 5, characterized in that, The dicarboxylic acid compounds include heptaic acid, octanoic acid, azelaic acid, sebacic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, 3,3'-oxydipropionic acid, triethylene glycol diacetic acid, 2,4-pyridine dicarboxylic acid, and 4-carboxyphenylacetic acid.

7. The use of the divalent inhibitor based on heat shock protein 90 according to any one of claims 1-4 in the preparation of drugs for gastric or colon cancer.

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