Heat shock protein 90-based bivalent inhibitor and its preparation method and application

Abstract

The present invention relates to an inhibitor of heat shock protein 90, and discloses a bivalent inhibitor based on heat shock protein 90, a preparation method, and an application thereof. The compound has 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 linking group of PEG and alkane, or a rigid linking group having an aryl, heteroalkyl, or heteroaryl group. The bivalent inhibitor can effectively inhibit the activity of the molecular chaperone protein HSP90, hinder the folding and modification of substrate proteins, degrade substrate proteins through the ubiquitin-proteasome pathway, induce non-native dimerization of HSP90, and interfere with protein-protein interactions associated with HSP90. At the same time, it also reduces the heat shock effect caused by HSP90 inhibition, and exhibits potent activity in degrading substrate proteins.

Description

Technical Field

[0001] The present invention relates to an inhibitor of heat shock protein 90, and in particular to a bivalent inhibitor based on heat shock protein 90, and a preparation method and application thereof. Background Art

[0002] Heat shock protein 90 (HSP90) is a major class of molecular chaperones and a key regulator of protease inhibition in eukaryotic cells under physiological and stress conditions. HSP90 has hundreds of protein substrates and, beyond protein folding, is involved in numerous cellular processes, 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. In the absence of ATP, HSP90 primarily adopts a v-shaped open conformation. Upon ATP binding, it undergoes a major, ATP-regulated conformational rearrangement, resulting in a closed state. Following ATP hydrolysis, the NTDs dissociate, releasing ADP and inorganic phosphate (Pi), allowing HSP90 to return to the open conformation. In summary, HSP90 maintains different dimerization states when interacting with its co-chaperone proteins at different times under the regulation of ATP, playing different roles in different parts of the chaperone cycle. Therefore, the dimerization regulation of HSP90 is crucial.

[0003] Currently, 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. ATP inhibitor small molecules designed for the ATP binding pocket of HSP90 have shown significant effects in inhibiting HSP90 activity, inducing substrate protein degradation, and blocking cell growth, proliferation, and signal transduction. However, the significant toxic side effects of these inhibitors limit their use in scientific research and clinical treatment. Therefore, in order to overcome these shortcomings, new HSP90 inhibitors are urgently needed to be developed. Summary of the Invention

[0004] In order to overcome the defects of the above-mentioned existing technologies, bivalent molecules were designed based on ATP inhibitors, in order to solve problems such as large toxic side effects, so as to be able to exert therapeutic effects in clinical treatment.

[0005] The present invention aims to provide a bivalent inhibitor based on heat shock protein 90 (HSP90), wherein the bivalent inhibitor is a compound having a structural formula as shown in Formula I:

[0006] ALB

[0007] Formula I

[0008] or a pharmaceutically acceptable salt, solvate, or optical isomer thereof,

[0009] Wherein: A and B are ATP inhibitors of heat shock protein 90 (HSP90), and L represents a connecting chain.

[0010] Furthermore, A and B can be selected from the following compounds separately or together:

[0011]

[0012] The Chinese name of GDA is geldanamycin; the Chinese name of 17-AAG is tanspiramycin, the Chinese name of AT13387 is onaspib, the Chinese name of NVP-AUY922 is lumispir, the Chinese name of PU-H71 is zeravispibu, the chemical name of BIIB021 is 1-hydroxytriacontol, the chemical name of SNX-2112 is 4-[6,6-dimethyl-4-oxo-3-(trifluoromethyl)-5,7-dihydroindole-1-yl]-2-[(4-hydroxycyclohexyl)amino]benzamide, and the chemical name of 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.

[0013] Furthermore, the motif structure of L is a flexible linking group of PEG and alkane or a rigid linking group having an aryl, heteroalkyl or heteroaryl group,

[0014] The specific structural formula is selected from one of the following:

[0015]

[0016] Where n represents any independent natural number between 1 and 12.

[0017] Furthermore, the compounds represented by Formula I are DDO-4104 to DDO-4114, and their structural formulas are as follows:

[0018]

[0019]

[0020] Another object of the present invention is to provide a method for preparing the bivalent inhibitor based on heat shock protein 90 (HSP90), the method comprising the following steps:

[0021] Dissolve (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone in 50 mL of anhydrous DCM, add a dicarboxylic acid compound and HATU under ice bath conditions, then slowly add Et3N and stir at room temperature. After the reaction is completed, water is added to the reaction solution for extraction. The separated organic layer is dried over anhydrous sodium sulfate and then evaporated to dryness. The crude product is separated and purified by column chromatography, wherein the stoichiometric ratio of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone, dicarboxylic acid compound, HATU, and Et3N is 2:1:2:4;

[0022] The crude product after separation and purification 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 50 minutes. After the reaction was completed, it was cooled to room temperature, and the catalyst was removed by filtration through a diatomaceous earth pad. The filtrate was concentrated to 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 separated and purified by column chromatography to obtain a divalent inhibitor based on heat shock protein 90, wherein the stoichiometric ratio of the crude product after separation and purification to ammonium formate was 1:80.

[0023] The dicarboxylic acid compounds include pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tetradecanedioic acid, 3,3'-oxydipropionic acid, triethylene glycol diacetic acid, 2,4-pyridinedicarboxylic acid, and 4-carboxyphenylacetic acid.

[0024] Another object of the present invention is to provide a use of a bivalent inhibitor based on heat shock protein 90 (HSP90) in the preparation of gastric cancer or colon cancer drugs.

[0025] The present invention has the following beneficial effects: The bivalent inhibitor molecules of the bivalent heat shock protein 90 (HSP90) provided by the present invention can effectively inhibit the activity of the molecular chaperone protein HSP90, hinder the folding and modification of substrate proteins, and degrade substrate proteins through the ubiquitin-proteasome pathway. This bivalent inhibitor molecule has a different mechanism of action from previous HSP90 ATP inhibitors, can induce non-native dimerization of HSP90, and interfere with protein-protein interactions related to HSP90. At the same time, it also reduces the heat shock effect caused by HSP90 inhibition, exhibits strong activity in degrading substrate proteins, and can be used to prepare therapeutic drugs for related diseases such as gastric cancer and colon cancer. The present invention also discloses a preparation method for synthesizing this series of HSP90 bivalent ATP inhibitor compounds.

[0026] Specifically, there is no bivalent strategy for ATP inhibitors in the prior art. The present invention proposes a new bivalent strategy. Based on the classic ATP inhibitor AT13387, the present invention designs a bivalent molecule that can induce the HSP90 N-terminus to approach and form a non-natural dimer, resulting in a mechanism of action different from that of classic ATP inhibitors, interfering with the interaction with co-chaperone proteins related to HSP90, and to a certain extent weakening the heat shock effect brought about by ATP inhibitors, showing strong cell anti-proliferative activity, and can exert tumor growth inhibitory effects, with great clinical application prospects.

[0027] Definition of terms:

[0028] The term "alkyl" used in the present invention includes saturated linear or branched monovalent hydrocarbon groups of 1 to 12 carbon atoms, some of which are alkyl groups of 5 to 12 carbon atoms.

[0029] As used herein, the term "PEG" refers to a linker chain containing an ethylene glycol linking motif.

[0030] The term "heteroalkyl" as used herein includes 3-6 membered saturated cycloalkanes containing a heteroatom selected from nitrogen, oxygen and sulfur. Some examples thereof are cyclobutylamine and cyclopentylamine.

[0031] The term "aryl" as used in the present invention is 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 following groups: (1) C1-C6 alkyl; (2) halogen; (3) C1-C6 alkoxy; (4) amino; (5) nitro; (6) cyano; (7) trifluoromethyl.

[0032] The term "heteroaryl" as used herein includes 5- or 6-membered rings containing one 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 such heteroaryl groups are pyridyl, thienyl, furanyl, pyrimidinyl, imidazolyl, quinolinyl and morpholine rings.

[0033] The term "halogen" as used herein is a group selected from fluoro (-F), chloro (-Cl) and bromo (-Br).

[0034] The term "amino" as used herein refers to a -NH2 group.

[0035] The term "nitro" as used herein refers to a -NO2 group.

[0036] The term "cyano" as used herein refers to a -CN group.

[0037] The term "aldehyde" as used herein refers to a -CHO group.

[0038] As used herein, the term "hydroxyl" refers to an -OH group.

[0039] Unless otherwise indicated, the structural formulas described herein include all isomeric forms (e.g., enantiomers, diastereomers, and geometric isomers (or conformers)); for example, R and S configurations containing asymmetric centers, (Z) and (E) isomers of double bonds, and (Z) and (E) conformers. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformers) are all within the scope of the present invention.

[0040] The term "pharmaceutically acceptable" as used herein includes any substance that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host to which it is administered.

[0041] Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are included within the scope of the present invention. In addition, unless otherwise indicated, the structural formulas of the compounds described herein may include enriched isotopes of one or more different atoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : A graph showing the activity of bivalent inhibitor molecules DDO-4105, DDO-4110, and DDO-4113 in inducing HSP90 NTD dimerization with ATP inhibitors;

[0043] Figure 2 : The crystal structure of a complex in which the bivalent inhibitor molecule DDO-4105 induces HSP90 dimerization to form a non-native dimer;

[0044] Figure 3 :The bivalent inhibitor molecule DDO-4113 interferes with protein interactions related to HSP90;

[0045] Figure 4 :The results of the antiproliferative activity of bivalent inhibitor molecules on HCT116 cells;

[0046] Figure 5 : The bivalent inhibitor molecule showed the same effect as AT13387 in inducing substrate protein degradation in the HCT116 cell line;

[0047] Figure 6 : The bivalent inhibitor molecules DDO-4113 and AT13387 exhibited heat shock effects in a concentration-dependent manner on HCT116 cells;

[0048] Figure 7 : The bivalent inhibitor molecules DDO-4113 and AT13387 exhibit heat shock effects in a time-dependent manner on HCT116 cells;

[0049] Figure 8 : Survival curves of the bivalent inhibitor molecules DDO-4113 and AT13387 measured at high and low doses;

[0050] Figure 9 : Body weight changes after high and low doses of the bivalent inhibitor molecule DDO-4113;

[0051] Figure 10 :Changes in tumors after high and low doses of the bivalent inhibitor molecule DDO-4113. DETAILED DESCRIPTION

[0052] Unless otherwise specified, the starting materials can be obtained from commercial sources or prepared by methods known in the art or according to the methods described herein. The structures of the compounds were determined by nuclear magnetic resonance ( 1 Confirmation was by H-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 used 200-300 mesh silica gel from Qingdao Ocean Chemical Plant.

[0053] 1) Preparation of intermediates

[0054] Its synthetic route:

[0055]

[0056] (1) Preparation of 2,4-bis(benzyloxy)-5-bromobenzoic acid benzyl ester (AT2)

[0057] The raw material, 1-bromo-2,4-dihydroxybenzoic acid (5.0 g, 21.45 mmol, 1.0 eq), was dissolved in CH3CN (approximately 40 mL). Benzyl bromide (7.65 mL, 64.35 mmol, 3 eq) and potassium carbonate (K2CO3) (20.75 g, 150.15 mmol, 6 eq) were added. The reaction was maintained at 80°C and stirred for 8 hours. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: PE:EA (v / v) = 10:1). After completion of the reaction, the solvent was removed using a rotary evaporator to obtain 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 (eluent: PE / EA (v / v) = 300 / 1 to 20 / 1, gradient elution) to obtain 9.99 g of a white solid powder in 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).

[0058] (2) Preparation of 2,4-bis(benzyloxy)-5-isopropenyl-benzoic acid benzyl ester (AT3)

[0059] Compound AT2 (10.0 g, 19.86 mmol, 1.0 eq) was dissolved in THF:H₂O ((V / V) = 9:1) (approximately 150 mL). Potassium isopropenyltrifluoroborate (4.41 g, 29.79 mmol, 1.5 eq), palladium acetate (Pd(OAc)₂) (222.94 mg, 0.993 mmol, 0.05 eq), XPhos (947.04 mg, 1.99 mmol, 0.1 eq), and cesium carbonate (CsCO₃) (19.42 g, 59.58 mmol, 3.0 eq) were added. The mixture was heated at 80°C under nitrogen for 12 hours. During the reaction, the reaction was monitored for completion by UV-spot chromatography (developing solvent: PE:EA = 2:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst and other components were removed by filtration through a pad of celite, and the filtrate was concentrated to an oil. The solid was extracted three times with CH2Cl2 and water. The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated to yield 8.98 g of a white solid powder (97.33% yield). 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).

[0060] (3) Preparation of 2,4-bis(benzyloxy)-5-isopropenylbenzoic acid (AT4)

[0061] Compound AT3 (8.0 g, 17.22 mmol, 1.0 eq) was dissolved in THF:MeOH:H2O ((V / V / V) = 2:1:1) (approximately 50 mL), and lithium hydroxide (1.65 g, 68.88 mmol, 4.0 eq) was added. The mixture was stirred at 40°C for 12 h. During the reaction, TLC was performed to monitor the reaction completion in real time using a UV-spot plate (developing solvent: PE:EA = 3:1). After completion of the reaction, the mixture was cooled to room temperature and concentrated. 1 M HCl was added to the residue to adjust the pH to 5, and then approximately 60 mL of ethyl acetate was added for extraction. The organic layer was dried over anhydrous sodium sulfate, and concentrated to yield 5.9 g of a white solid in 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).

[0062] (4) Preparation of 2,4-dihydroxy-5-isopropylbenzoic acid (AT5)

[0063] Compound AT4 (5.9 g, 15.76 mmol, 1.0 eq) was dissolved in methanol (approximately 50 mL) and palladium-carbon catalyst (1.48 g) was added. The reaction mixture was reacted at room temperature under a reducing atmosphere of H₂ for 12 h. During the reaction, TLC was performed to monitor the reaction completion using a UV-spot plate (developing solvent: PE:EA = 1:1). After completion, the catalyst and other components were removed by filtration through a pad of Celite. Ethyl acetate was added for elution, and the eluate was concentrated to yield 3.0 g of a white solid in a yield of 97.04%. 1H NMR (300 MHz, DMSO-d₂) 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).

[0064] (5) Preparation of 2,4-bis(benzyloxy)-5-isopropylbenzoate (AT6)

[0065] Compound AT5 (3.0 g, 15.29 mmol, 1.0 eq) was dissolved in CH3CN (approximately 40 mL) and benzyl bromide (5.44 mL, 45.87 mmol, 3.0 eq) and potassium carbonate (K2CO3) (14.79 g, 107.03 mmol, 7.0 eq) were added. The mixture was heated at 80°C and stirred for 8 hours. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: PE:EA = 4:1). After completion of the reaction, the solvent was removed using a rotary evaporator to obtain 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 (eluent: PE / EA (v / v) = 200 / 1 to 1 / 1, gradient elution) to obtain 6.5 g of a white solid powder in 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).

[0066] (6) Preparation of 2,4-bis(benzyloxy)-5-isopropylbenzoic acid (AT7)

[0067] The raw material AT6 (6.5 g, 13.93 mmol, 1.0 eq) was dissolved in THF:MeOH:H2O (2:1:1) (approximately 40 mL), and lithium hydroxide (1.34 g, 55.72 mmol, 4.0 eq) was added. The mixture was stirred at room temperature for 12 h. During the reaction, TLC was performed in real time to monitor the reaction completion using a UV-dot plate (developing solvent PE:EA=5:1). After completion of the reaction, the mixture was concentrated, and 1 M HCl was added to the residue to adjust the pH to 5. The residue was then extracted three times with approximately 60 mL of CH2Cl2. The organic layer was 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).

[0068] (7) Preparation of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-bromoisoindolin-2-yl)methanone (AT8)

[0069] Compound AT7 (4.9 g, 13.02 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (approximately 60 mL). 5-Bromoisoindoline (3.87 g, 19.53 mmol, 1.5 eq.), N,N-diisopropylethylamine (DIPEA) (4.53 mL, 26.04 mmol, 2.0 eq.), N-hydroxy-7-azabenzotriazole (HOAt) (2.48 g, 18.23 mmol, 1.4 eq.), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (7.49 g, 39.06 mmol, 3.0 eq.) were added under ice-cooling conditions. The mixture was stirred at room temperature for 8 hours. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: PE:EA = 6:1). After completion of the reaction, the solvent was removed by rotary evaporation to yield a yellow solid. The solid was extracted three times with CH2Cl2 and water. The combined organic phases were dried over Na2SO4, filtered, and concentrated. The concentrate was purified by column chromatography (eluent: PE / EA (v / v) = 100 / 1-5 / 1, gradient elution) to afford 6.5 g of a yellow solid powder in an 89.73% yield. 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).

[0070] (8) Preparation of tert-butyl 4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazine-1-carboxylate (AT9)

[0071] Compound AT8 (6.5 g, 11.68 mmol, 1.0 eq) was dissolved in toluene:water ((V / V) = 10:3) (approximately 40 mL). Potassium (4-tert-butyloxycarbonylpiperazin-1-yl)methyltrifluoroborate (4.65 g, 15.18 mmol, 1.3 eq.), cesium carbonate (CsCO3) (11.42 g, 35.04 mmol, 3.0 eq.), palladium acetate (Pd(OAc)2) (97 mg, 0.584 mmol, 0.05 eq.), and tri-tert-butylphosphine tetrafluoroborate ((t-Bu)3P-HBF4) (339 mg, 1.168 mmol, 0.1 eq.) were added. The mixture was stirred under N2 protection and heated at 100°C for 14 hours. During the reaction, the reaction was monitored by TLC (developing solvent PE:EA = 1:1) to determine if the reaction was complete. After the reaction, the mixture was cooled to room temperature and the catalyst and other substances were removed by filtration through a celite pad. The filtrate was concentrated to an oil. 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 in 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).

[0072] (9) Preparation of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone (AT10)

[0073] Compound AT9 (7.4 g, 10.95 mmol, 1.0 eq.) was dissolved in a small amount of EA (approximately 10 mL). 2M HCl / EA (60 mL) was added and stirred at room temperature for 4 hours. During the reaction, TLC was performed to monitor the reaction completion in real time (developing solvent: DCM:MeOH (v / v) = 10:1). After completion of the reaction, the solvent was discarded, leaving an orange solid. The pH of the solid was adjusted to alkaline with saturated NaHCO₃ solution. 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 (eluent: DCM / MeOH (v / v) = 250 / 1 to 15 / 1 + 1% triethylamine, gradient elution) to obtain 6.0 g of a light yellow solid powder in 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).

[0074] 2) Preparation of each embodiment

[0075] Example 1

[0076] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)heptane-1,3-dione (DDO-4104)

[0077]

[0078] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)heptane-1,3-dione (DDO-4104-a).

[0079] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. Pimelic acid (139.35 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1 to 20 / 1, gradient elution) to obtain 920 mg of an orange solid in an 82.90% yield. 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).

[0080] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)heptane-1,3-dione (DDO-4104)

[0081] Compound DDO-5104-a (920 mg, 0.7212 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (v / v) = 1:1 (approximately 40 mL), and ammonium formate (3.64 g, 57.70 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes, after which 10% Pd / C (920 mg) was added. Stirring was continued at 40°C for 50 minutes. During the reaction, completion of the reaction was monitored by UV-spot TLC (developing solvent: DCM:MeOH (v / v) = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a Celite pad, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 60 / 1-20 / 1, gradient elution) to give 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).

[0082] Example 2

[0083] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)octane-1,3-dione (DDO-4105)

[0084]

[0085] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)octane-1,3-dione (DDO-4105-a)

[0086] In a 100 mL round-bottom flask, AT10 (1.0 g, 1.74 mmol, 2.0 eq.), suberic acid (151.55 mg, 0.87 mmol, 1.0 eq.), and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were dissolved in 50 mL of anhydrous DCM. The mixture was cooled to 0°C in an ice bath, and Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added. The mixture was then stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated by column chromatography (DCM / MeOH, 100 / 1 to 20 / 1) to obtain DDO-4105-a as an orange solid in 80% yield. 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).

[0087] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)octane-1,3-dione (DDO-4105)

[0088] Compound DDO-4105-a (900 mg, 0.6978 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL), and ammonium formate (3.52 g, 55.82 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes, after which 10% Pd / C (900 mg) was added. Stirring was continued at 40°C for 50 minutes. During the reaction, completion of the reaction was monitored by UV-spot chromatography (TLC) (developing solvent: DCM:MeOH = 15:1). After the reaction was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 60 / 1-20 / 1, gradient elution) to obtain 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).

[0089] Example 3

[0090] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)nonane-1,3-dione (DDO-4106)

[0091]

[0092] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)nonane-1,3-dione (DDO-4106-a)

[0093] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. Azelaic acid (163.75 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1 to 20 / 1, gradient elution) to obtain 910 mg of an orange solid in an 80.23% yield. 1 H 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).

[0094] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)nonane-1,3-dione (DDO-4106)

[0095] Compound DDO-4106-a (910 mg, 0.6980 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL) and ammonium formate (3.52 g, 55.84 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes before the addition of 10% Pd / C (910 mg). Stirring was continued at 40°C for 50 minutes. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: DCM:MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent DCM / MeOH (v / v) = 60 / 1-20 / 1, gradient elution) to give 100 mg of a white solid with a yield of 15.19%. 1H 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).

[0096] Example 4

[0097] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)decane-1,3-dione (DDO-4107)

[0098]

[0099] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)decane-1,3-dione (DDO-4107-a)

[0100] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. Sebacic acid (175.96 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1 to 20 / 1, gradient elution) to obtain 920 mg of an orange solid in an 80.25% yield. 1H 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).

[0101] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)decane-1,3-dione (DDO-4107)

[0102] Compound DDO-4107-a (920 mg, 0.6982 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL) and ammonium formate (3.52 g, 55.86 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes before the addition of 10% Pd / C (920 mg). Stirring was continued at 40°C for 50 minutes. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: DCM:MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent: DCE / MeOH (v / v) = 60 / 1-20 / 1, gradient elution) to give 110 mg of a white solid with a yield of 16.46%. 1 H 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).

[0103] Example 5

[0104] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)undecane-1,3-dione (DDO-4108)

[0105]

[0106] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)undecane-1,3-dione (DDO-4108-a)

[0107] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. Undecanedioic acid (188.16 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1 to 20 / 1, gradient elution) to yield 922 mg of an orange solid in 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).

[0108] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)undecane-1,3-dione (9C-2)

[0109] Compound DDO-4108-a (922 mg, 0.6923 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL) and ammonium formate (3.49 g, 55.38 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes before the addition of 10% Pd / C (922 mg). Stirring was continued at 40°C for 50 minutes. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: DCM:MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent: DCE / MeOH (v / v) = 60 / 1-20 / 1, gradient elution) to obtain 115 mg of a 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).

[0110] Example 6

[0111] Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)dodecane-1,3-dione (DDO-4109)

[0112]

[0113] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)dodecane-1,3-dione (DDO-4109-a)

[0114] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. Dodecanedioic acid (200.36 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1 to 20 / 1, gradient elution) to obtain 930 mg of an orange solid in a yield of 79.43%. 1 H 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).

[0115] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)dodecane-1,3-dione (DDO-4109)

[0116] Compound DDO-4109-a (930 mg, 0.6910 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL), and ammonium formate (3.48 g, 55.28 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes, after which 10% Pd / C (930 mg) was added. Stirring was continued at 40°C for 50 minutes. During the reaction, completion of the reaction was monitored by TLC (developing solvent: DCM:MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent: DCE / MeOH (v / v) = 60 / 1-20 / 1, gradient elution) to obtain 110 mg of a 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).

[0117] Example 7

[0118] Preparation of 1,3-bis(4-((2-(2,4-bishydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)tetradecane-1,3-dione (DDO-4110)

[0119]

[0120] (1) Preparation of 1,3-bis(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)tetradecane-1,3-dione (DDO-4110-a)

[0121] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. Tetradecanedioic acid (224.77 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1 to 20 / 1, gradient elution) to obtain 950 mg of an orange solid in a yield of 79.48%. 1H 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).

[0122] (2) Preparation of 1,3-bis(4-((2-(2,4-bishydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)tetradecane-1,3-dione (DDO-4110)

[0123] Compound DDO-4110-a (950 mg, 0.6915 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL) and ammonium formate (3.48 g, 55.32 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes before the addition of 10% Pd / C (950 mg). Stirring was continued at 40°C for 50 minutes. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: DCM:MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent: DCE / MeOH (v / v) = 60 / 1-20 / 1, gradient elution) to obtain 120 mg of a white solid with a yield of 17.36%. 1 H 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).

[0124] Example 8

[0125] Preparation of 3,3'-oxybis(1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)propan-1-one)(DDO-4111)

[0126]

[0127] (1) Preparation of 3,3'-oxybis(1-(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)propan-1-one) (DDO-4111-a)

[0128] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. 3,3'-Oxydipropionic acid (140.81 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1 to 20 / 1, gradient elution) to obtain 887 mg of an orange solid in an 80.02% yield. 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).

[0129] (2) Preparation of 3,3'-oxybis(1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)propan-1-one) (DDO-4111)

[0130] Compound DDO-4111-a (887 mg, 0.6947 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL) and ammonium formate (3.49 g, 55.32 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes before the addition of 10% Pd / C (950 mg). Stirring was continued at 40°C for 50 minutes. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: DCM:MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (eluent: DCE / MeOH (v / v) = 60 / 1 to 20 / 1, gradient elution) to afford 110 mg of a white solid in a 17.19% yield. H NMR (300 MHz, DMSO-d6) δ 10.08 (s, 2H), 9.63 (s, 2H), 7.25 (d, J = 19.6 Hz, 7H), 7.04 (s, 2H), 6.40 (s, 2H), 4.76 (s, 8H), 3.57 (t, J = 6.5 Hz, 8H), 3.34 (s, 8H), 3.12–3.06 (m, 2H), 2.30 (d, J = 13.5 Hz, 12H), 1.14 (s, 6H), 1.12 (s, 6H).

[0131] Example 9

[0132] Preparation of 2,2'-(ethane-1,2-diylbis(oxy))bis(1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethan-1-one)(DDO-4112)

[0133]

[0134] (1) Preparation of 2,2'-(ethane-1,2-diylbis(oxy))bis(1-(4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethan-1-one)(DDO-4112-a)

[0135] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. Triethylene glycol diacetic acid (154.70 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1 to 20 / 1, gradient elution) to obtain 921 mg of an orange solid in an 82.00% yield. 1 H 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.

[0136] (2) Preparation of 2,2'-(ethane-1,2-diylbis(oxy))bis(1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethan-1-one) (DDO-4112)

[0137] Compound DDO-4112-a (921 mg, 0.6915 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL) and ammonium formate (3.50 g, 55.87 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes before the addition of 10% Pd / C (950 mg). Stirring was continued at 40°C for 50 minutes. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: DCM:MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent: DCE / MeOH (v / v) = 60 / 1-20 / 1, gradient elution) to obtain 128 mg of a white solid with a yield of 20.02%.

[0138] Example 10

[0139] Preparation of (((pyridine-2,4-dicarbonyl)bis(piperazine-4,1-diyl))bis(methylene))bis(isoindoline-5,2-diyl))bis((2,4-dihydroxy-5-isopropylphenyl)methanone) (DDO-4113)

[0140] Synthesis route:

[0141]

[0142] (1) Preparation of (((pyridine-2,4-dicarbonyl)bis(piperazine-4,1-diyl))bis(methylene))bis(isoindoline-5,2-diyl))bis((2,4-bis(benzyloxy)-5-isopropylphenyl)methanone) (DDO-4113-a)

[0143] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. 2,4-pyridinedicarboxylic acid (145.39 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: PE / EA (v / v) = 100 / 1 to 20 / 1, gradient elution) to obtain 900 mg of an orange solid in an 80.66% yield. 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).

[0144] (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)methanone) (DDO-4113)

[0145] Compound DDO-4113-a (900 mg, 0.7017 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL), and ammonium formate (3.54 g, 56.136 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes, followed by the addition of 10% Pd / C (900 mg). Stirring was continued at 40°C for 50 minutes. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: DCM:MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent: PE / EA (v / v) = 60 / 1-20 / 1, gradient elution) to give 100 mg of a 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).

[0146] Example 11

[0147] 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)piperazine-1-carbonyl)phenyl)ethan-1-one (DDO-4114)

[0148]

[0149] (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)piperazine-1-carbonyl)phenyl)ethan-1-one (DDO-4114-a)

[0150] AT10 (1.0 g, 1.74 mmol, 2.0 eq.) was dissolved in 50 mL of anhydrous DCM. 4-Carboxyphenylacetic acid (168.64 mg, 0.87 mmol, 1.0 eq.) and HATU (661.62 mg, 1.74 mmol, 2.0 eq.) were added under ice-cooling conditions. Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was then slowly added and stirred at room temperature for 8 h. After the reaction, water (approximately 40 mL) was added to the reaction mixture for extraction. The separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1 to 20 / 1, gradient elution) to yield 956 mg of an orange solid in 85.12% yield. 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).

[0151] (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)piperazine-1-carbonyl)phenyl)ethan-1-one (DDO-4114)

[0152] Compound DDO-4114-a (956 mg, 0.7381 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane:methanol (1:1) (approximately 40 mL) and ammonium formate (3.52 g, 55.99 mmol, 80.0 eq.) was added. The mixture was stirred at 40°C for 10 minutes before the addition of 10% Pd / C (950 mg). Stirring was continued at 40°C for 50 minutes. During the reaction, the reaction was monitored by UV-spot chromatography (developing solvent: DCM:MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated to a solid. The solid was extracted three times with CH2Cl2 and water. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (eluent: DCE / MeOH (v / v) = 60 / 1 to 20 / 1, gradient elution) to afford 172 mg of a white solid (24.90% yield). H NMR (300 MHz, DMSO-d6) δ 10.05 (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 = 13.6 Hz, 12H), 3.11–3.06 (m, 2H), 2.32 (d, J = 29.7 Hz, 8H), 1.14 (s, 6H), 1.12 (s, 6H).

[0153] Experimental Example 1

[0154] Fluorescence polarization experiment (FP method)

[0155] Table 1 Competitive activity of compounds with FITC-GA molecules in binding to HSP90 NTD and inducing HSP90 NTD dimerization

[0156]

[0157]

[0158] *a:The concentration of HSP90 NTD=10μM, the concentration of compound:protein=1:2

[0159] Note: The structures of the compounds are shown in the specific examples.

[0160] As shown in Table 1, the example compounds all exhibited nM-level binding activity against HSP90 NTD and exhibited a structure-activity relationship. Short-chain molecules exhibited relatively good binding activity, essentially maintaining the same level as the positive control compound AT13387. This indicates that the example compounds of this patent retain strong HSP90 binding activity.

[0161] The FP method for testing the competitive binding activity of compounds with HSP90 NTD is as follows:

[0162] First, prepare 50 mL of a buffer solution containing 20 mM HEPES, 50 mM KCl, 5 mM MgCl2, and 20 mM NaMoO4, adjust the pH to 7.3-7.4, then add 0.01% NP40, 2 mM DTT, and 0.1 mg / ml BGG. Use this buffer solution to dissolve the test compound (DMSO content less than 10%), HSP90 NTD protein, and probe molecule. Using a 384-well plate, duplicate wells were added to each well. 20 μL of HSP90 NTD protein solution (final concentration 60 nM) and 20 μL of probe molecule solution (final concentration 40 nM) were added, followed by 20 μL of compound solution (maximum final concentration 3 μM, 3-fold dilution, 10 concentration gradients). In the [Probe + Protein] control wells, 20 μL of HSP90 NTD protein solution, 20 μL of probe molecule solution, and 20 μL of the system buffer solution were added. In the [Probe] control wells, 20 μL of probe molecule solution and 40 μL of buffer solution were added. The 384-well plate was incubated at room temperature (25°C) for 30 min. Data were collected using a microplate reader at a wavelength of 405 nm and analyzed using GraphPad software.

[0163] Experimental Example 2

[0164] Native-page experiment

[0165] Native-page assay to test the activity of bivalent compounds in inducing HSP90 NTD dimerization

[0166] like Figure 1 As shown, the bivalent inhibitor molecules DDO-4105, DDO-4110 and DDO-4113 induced the dimerization of HSP90, while ATP inhibitors did not induce dimerization. The test results showed that the compounds induced HSP90NTD dimerization in a concentration-dependent and time-dependent manner, and exhibited a typical "Hook" effect at high concentrations.

[0167] As shown in Table 1, the Example compounds induced different degrees of dimerization when the compound to protein concentration ratio was 1:2, showing a structure-activity relationship related to the length of the connecting chain, among which the Example compound DDO-4113 showed the strongest effect.

[0168] The operation method of Native-page test is as follows:

[0169] Native-PAGE dimerization experiments followed a standard protocol. The specific experimental process included protein extraction, incubation with different ratios of protein and compound, sampling at different time points, preparation of a non-denaturing polyacrylamide gel, Native-PAGE analysis, and scanning of the results. The dimerization effect of HSP90 NTD was tested.

[0170] Experimental Example 3

[0171] X-ray crystal structure diffraction

[0172] Crystal structure diffraction confirmed that the bivalent inhibitor molecule can act on the binding pocket of HSP90, induce non-natural dimerization of HSP90 NTD, and cause a significant change in the conformation of the HSP90 NTD dimer.

[0173] like Figure 2 As shown, DDO-4105 induces non-native dimerization of HSP90 NTD

[0174] Among them, the operation method of crystal structure diffraction is as follows:

[0175] HSP90 NTD protein extraction, HSP90 NTD protein purification and monomer confirmation, incubation and co-purification of HSP90 NTD monomer protein with small molecules, Native-page electrophoresis identification, crystal growth and cultivation, initial screening of co-crystal conditions, multiple rounds of optimization of crystallization conditions, crystal data collection, processing and model building, and crystal structure analysis.

[0176] Experimental Example 4

[0177] Co-immunoprecipitation assay (Co-IP)

[0178] Co-IP testing of bivalent inhibitors for the interference of HSP90-related protein-protein interactions

[0179] The interactions of the compounds of the present invention with co-chaperone proteins (CDC37, HOP, and P23, etc.) associated with HSP90 are as follows:

[0180] like Figure 3As shown, the test results showed that DDO-4113 blocked the interaction between co-chaperone proteins and HSP90 at high concentrations, indicating that the bivalent inhibitor molecule has a different mechanism of action from previous ATP inhibitors when exerting strong anti-proliferative activity on tumor cells, and can block protein interactions related to HSP90.

[0181] The operation method of the Co-IP test is as follows:

[0182] Western blot experiments followed standard protocols. The specific experimental process included cell administration, cell lysis, total protein collection, overnight incubation with the primary antibody to recognize the complex, incubation with Protein A / G magnetic beads, preparation of polyacrylamide gels, SDS-PAGE analysis, primary and secondary antibody incubations, and result scanning. During the experiment, the expression levels of co-chaperone proteins (CDC37, HOP, and P23, etc.) were measured.

[0183] Experimental Example 5

[0184] Cell anti-proliferation assay (CCK8 method)

[0185] CCK8 assay was used to test the cell proliferation inhibitory activity of bivalent inhibitor molecules in the colon cancer cell line HCT116 cells

[0186] like Figure 4 As shown, the cell proliferation inhibition activity of the bivalent inhibitor molecule showed consistent cell anti-proliferative activity with AT13387.

[0187] Among them, the operation method of CCK8 test is as follows:

[0188] Cells in logarithmic growth phase were cultured in 96-well plates for 24 hours, with a volume of 100 μL per well (3,000-8,000 tumor cells per well). After 24 hours, the treatment group was treated with compounds containing different concentrations, diluted with the corresponding culture medium. Eight concentrations were set for each group, with three replicates. The control group was treated with the same volume of culture medium as the experimental group. The cells were then cultured 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 cells were incubated at 37°C for 1-3 hours with gentle shaking, and the optical density (OD) was measured using a microplate reader (at a reference wavelength of 450 nm and a detection wavelength of 570 nm until the OD value of the blank control group was >1.2). Tumor cells treated with culture medium served as the control group. The inhibitory rate of the compounds against tumor cells was calculated using the following formula and analyzed using Graphpad Prism software.

[0189]

[0190] Experimental Example 6

[0191] Western blots to test the effects of bivalent inhibitor molecules on substrate proteins

[0192] The weakening effect of the compounds of the present invention on substrate proteins is as follows:

[0193] like Figure 5 As shown, the test results showed that DDO-4113 caused downregulation of substrate proteins in a concentration-dependent manner, and the effect was consistent with that of AT13387, indicating that the bivalent inhibitor molecule maintained considerable activity in degrading substrate proteins.

[0194] Among them, the operation method of Western blots test is as follows:

[0195] Western blot experiments were performed according to standard protocols. The specific experimental process included cell administration, cell lysis, total protein collection, polyacrylamide gel preparation, SDS-PAGE analysis, primary and secondary antibody incubations, and result scanning. The expression levels of the HSPs (HSP90, HSP70, HSP40, and HSP27) protein family were measured.

[0196] Experimental Example 7

[0197] Western blots to test the activity of bivalent inhibitor molecules in attenuating the heat shock effect

[0198] The effects of the compounds of the present invention on reducing the heat shock effect are as follows:

[0199] like Figure 6 As shown, the test results show that DDO-4113 weakened the heat shock effect compared with AT13387 in a concentration-dependent manner, indicating that the bivalent inhibitor molecule can overcome the heat shock response to a certain extent while having good inhibition or activity.

[0200] like Figure 7 As shown, the test results show that DDO-4113 weakened the heat shock effect compared with AT13387 in a concentration-dependent manner, indicating that the bivalent inhibitor molecule can overcome the heat shock response to a certain extent while having good inhibition or activity.

[0201] Among them, the operation method of Western blots test is as follows:

[0202] Western blot experiments were performed according to standard protocols. The specific experimental process included cell administration, cell lysis, total protein collection, polyacrylamide gel preparation, SDS-PAGE analysis, primary and secondary antibody incubations, and result scanning. The expression levels of the HSPs (HSP90, HSP70, HSP40, and HSP27) protein family were measured.

[0203] Experimental Example 8

[0204] Pharmacokinetic studies of bivalent inhibitor molecules

[0205] The pharmacokinetic study of compound DDO-4113 was conducted in SD rats. Rats were randomly divided into groups with n=3 per group. A 5 mg / kg intraperitoneal injection was performed. 150 μL 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 the results were analyzed using Phoenix software.

[0206] Table 2 Pharmacokinetic data of compound DDO-4113

[0207]

[0208] As shown in Table 2, after intraperitoneal administration of 5 mg / kg, the Cmax in plasma was 16.2±6 ng / mL, Tmax was 40 min, and AUC (0-∞) 528±88min*ng / mL, T 1 / 2 118±59min, MRT (0-∞) The bioavailability F can reach 25.98%. The experimental results show that the bivalent molecule has good pharmacokinetic properties and can be further evaluated for in vivo pharmacodynamics.

[0209] Experimental Example 9

[0210] In vivo pharmacodynamic evaluation of compound DDO-4113

[0211] In vivo pharmacodynamic studies of compound DDO-4113 were conducted in 5-week-old balb / c nude mice. The mice were housed under standard conditions (12 / 12h light / dark cycle, 22±3°C, 40% relative humidity) for 5 days and fed a standard laboratory rodent chow and water. HCT116 cells (5 x 10 6 When the average tumor volume reaches about 100 mm 3Mice were randomly divided into 5 groups (including control group, AT13387 high / low dose group, DDO-4113 high and low dose group, n = 6 in each group). The drug was intraperitoneally injected every two days according to the dose (the control group was given normal saline) for 21 consecutive days. The death of mice was monitored every other day, and the tumor volume and weight of nude mice were measured. At the end of 21 days or when the tumor volume reached 1800mm, the mice were treated with 40% paracetamol. 3 Afterwards, the nude mice were killed.

[0212] like Figure 8 and 9 As shown, high-dose AT13387 (50 mg / kg) has strong toxicity. Deaths occurred one after another on the third day after administration, and all mice died on the ninth day. There was also a significant decrease in body weight, indicating strong toxicity. High-dose DDO-4113 (50 mg / kg) also caused weight loss, but the result was weaker than that of AT13387. The survival of mice was also better than that of AT13387. Low-dose (25 mg / kg) of both compounds had relatively weak toxic side effects.

[0213] like Figure 10 As shown, the anti-tumor growth effects of DDO-4113 and AT13387 at high and low doses are basically similar, and they can exert a good tumor inhibitory effect.

Claims

1. A bivalent inhibitor based on heat shock protein 90, characterized in that The structural formula of the bivalent inhibitor is selected from one of the following:

2. The method for preparing a bivalent inhibitor of heat shock protein 90 according to claim 1, characterized in that: The method comprises the following steps: Dissolve (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone in 50 mL of anhydrous DCM, add a dicarboxylic acid compound and HATU under ice bath conditions, then slowly add Et3N and stir at room temperature. After the reaction is completed, water is added to the reaction solution for extraction. The separated organic layer is dried over anhydrous sodium sulfate and then evaporated to dryness. The crude product is separated and purified by column chromatography, wherein the stoichiometric ratio of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone, the dicarboxylic acid compound, HATU, and Et3N is 2:1:2:4; The crude product after separation and purification 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 50 minutes. After the reaction was completed, it was cooled to room temperature, and the catalyst was removed by filtration through a diatomaceous earth pad. The filtrate was concentrated to 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 separated and purified by column chromatography to obtain a divalent inhibitor based on heat shock protein 90, wherein the stoichiometric ratio of the crude product after separation and purification to ammonium formate was 1:

80.

3. The method for preparing a bivalent inhibitor of heat shock protein 90 according to claim 2, characterized in that: The dicarboxylic acid compound is selected from one of pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tetradecanedioic acid, 3,3'-oxydipropionic acid, triethylene glycol diacetic acid, 2,4-pyridinedicarboxylic acid, and 4-carboxyphenylacetic acid.

4. Use of the bivalent inhibitor based on heat shock protein 90 according to claim 1 in the preparation of a drug for treating gastric cancer or colon cancer.

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