Bivalent inhibitor based on heat shock protein 90 as well as preparation method and application of bivalent inhibitor

Abstract

The invention relates to an inhibitor of heat shock protein 90, and discloses a divalent inhibitor based on heat shock protein 90 and a preparation method and application thereof, the divalent inhibitor is a compound with a structural formula shown as a formula I: A-L-B formula I, or pharmaceutically acceptable salts, solvates and optical isomers thereof, A and B are ATP inhibitors of the heat shock protein 90, and L is an integer of 1-3. The motif structure of L is a flexible linking group of PEG and alkane or a rigid linking group with aryl, hetero-alkyl or hetero-aryl; the bivalent inhibitor can effectively inhibit the activity of molecular chaperone protein HSP90, hinder folding modification of substrate protein, induce non-natural dimerization of the HSP90 through ubiquitin-proteasome degradation pathway substrate protein, interfere protein-protein interaction related to the HSP90, reduce the heat shock effect caused by inhibition of the HSP90, and improve the activity of the HSP90. The activity of strongly degrading the substrate protein is shown.

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 the most important type of protein in molecular chaperones and is a key regulator of protease inhibition in eukaryotic cells under physiological and stress conditions. HSP90 has hundreds of protein substrates, so in addition to protein folding, it is also involved in many cellular processes and plays an important role in DNA repair, development, immune response and neurodegenerative diseases. HSP90 can function in the chaperone cycle by interacting with a large number of co-chaperone proteins and HSP90 to regulate the ATPase conformation of HSP90 dimers during client protein processing. In the absence of ATP binding, HSP90 mainly adopts a v-shaped open conformation. After ATP binding, it undergoes a large, ATP-regulated conformational rearrangement to form a closed state. After ATP hydrolysis, NTDs dissociate, ADP and inorganic phosphate (Pi) are released, and HSP90 can return to the open conformation. In summary, HSP90 interacts with its co-chaperone proteins at different times under the regulation of ATP and maintains different dimerization states, playing different roles in different parts of the chaperone cycle. Therefore, the dimerization regulation of HSP90 is crucial.

[0003] At present, 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 the activity of HSP90, inducing substrate protein degradation, blocking cell growth, proliferation and signal transduction, etc. However, the large toxic side effects of these inhibitors limit their use in scientific research and clinical treatment. Therefore, in order to overcome these defects, new HSP90 inhibitors are urgently needed to be developed. Summary of the invention

[0004] In order to overcome the defects of the above-mentioned prior art, a bivalent molecule was designed based on ATP inhibitors, in order to solve the problems such as large toxic side effects, so as to exert therapeutic effects in clinical treatment.

[0005] The object of the present invention is 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 zeravispib, the chemical name of BIIB021 is 1-hydroxytriacontanol, the chemical name of 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 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] Wherein, n represents any independent natural number between 1 and 12.

[0017] Furthermore, the compound represented by formula I is DDO-4104 to DDO-4114, and its structural formula is 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 dicarboxylic acid compounds and HATU under ice bath conditions, then slowly add Et3N, stir at room temperature, add water to the reaction solution after the reaction is completed for extraction, separate the obtained organic layer, dry it with anhydrous sodium sulfate, and evaporate it to dryness, and separate and purify the obtained crude product by column chromatography, wherein the stoichiometric ratio of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone, dicarboxylic acid compounds, 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, then 10% Pd / C was added, and stirring was continued 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 an application 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 molecule 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 modification of the substrate protein, and degrade the substrate protein through the ubiquitin-proteasome pathway. The bivalent inhibitor molecule has a different mechanism of action from the previous HSP90 ATP inhibitor, can induce non-natural dimerization of HSP90, and interfere with the protein-protein interaction related to HSP90, while also reducing the heat shock effect caused by HSP90 inhibition, showing a strong activity of degrading substrate proteins, and can be used to prepare therapeutic drugs for gastric cancer, colon cancer and other related diseases. 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 the classic ATP inhibitors, interfering with the interaction of co-chaperone proteins related to HSP90, and to a certain extent weakening the heat shock effect brought about by the ATP inhibitor, showing strong cell anti-proliferation activity, being able to exert a tumor growth inhibitory effect, and having great clinical application prospects.

[0027] Definition of terms:

[0028] The term "alkyl" used in the present invention includes saturated straight 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" used in the present invention 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 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, thienyl, furanyl, pyrimidinyl, imidazolyl, quinolinyl and morpholine rings.

[0033] The term "halogen" as used herein is a group selected from fluorine (-F), chlorine (-Cl) and bromine (-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] As used herein, the term "aldehyde group" 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 in the present invention include all isomeric forms (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)); for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, and (Z), (E) conformational isomers. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformational isomers) are all within the scope of the present invention.

[0040] The term "pharmaceutically acceptable" as used herein refers to 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 in the present invention include enriched isotopes of one or more different atoms. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0043] Figure 2 :The crystal structure of the 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 associated with 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 exhibited 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] Fig. 9 :The changes in body weight after high and low doses of the bivalent inhibitor molecule DDO-4113;

[0051] Fig.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 prepared according to the methods described herein. The structures of the compounds were determined by nuclear magnetic resonance ( 1 H-NMR) and / or mass spectrometry (MS) to confirm. NMR determination was performed using a Varian INOVA (300 MHz) or Bruker Advance (400 MHz) nuclear magnetic resonance instrument, the determination solvent was deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (Chloroform-d), and TMS was used as an internal standard. MS determination was 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 (5.0g, 21.45mmol, 1.0eq) was dissolved in CH3CN solvent (about 40mL), benzyl bromide (7.65mL, 64.35mmol, 3eq), potassium carbonate (K2CO3) (20.75g, 150.15mmol, 6eq), and the heating temperature was kept at 80°C, and heated and stirred for 8 hours. During the reaction, TLC was used to monitor whether the reaction was complete in real time by UV spot plate (developing agent PE: EA (V / V) = 10:1). After the reaction was completed, the solvent was removed by rotary evaporator to obtain a white solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with Na2SO4, filtered, and concentrated. The concentrate was separated and purified by column chromatography (eluent: PE / EA (V / V) = 300 / 1-20 / 1, gradient elution) to obtain 9.99g of 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).

[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) (about 150 mL), and potassium isopropenyl trifluoroborate (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), cesium carbonate (CsCO3) (19.42 g, 59.58 mmol, 3.0 eq) were added, and the heating temperature was maintained at 80°C, N2 was protected, and heated for 12 hours. During the reaction, TLC was used to monitor whether the reaction was complete in real time by UV spot plate (developing agent PE:EA = 2:1). After the reaction was completed, it was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to an oil. The solid was extracted three times with CH2Cl2 and water, the organic phases were combined, dried with anhydrous Na2SO4, filtered and concentrated to obtain 8.98 g of white solid powder with a yield of 97.33%. 1H NMR (300MHz, DMSO-d6) δ7.72 (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) (about 50 mL), lithium hydroxide (1.65 g, 68.88 mmol, 4.0 eq) was added, and the mixture was stirred at 40°C for 12 h. During the reaction, TLC was used to monitor whether the reaction was complete in real time using a UV spot plate (developing agent PE:EA=3:1). After the reaction was completed, 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 about 60 mL of ethyl acetate was added for extraction. The organic layer was taken, dried over anhydrous sodium sulfate, and concentrated to obtain 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).

[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 (about 50 mL), and palladium-carbon catalyst (1.48 g) was added. The reaction mixture was reacted at room temperature for 12 h under H2 reducing environment. During the reaction, TLC was used to monitor whether the reaction was complete in real time by UV spot plate (developing agent PE: EA = 1: 1). After the reaction was completed, the catalyst was removed by suction through a diatomaceous earth pad, ethyl acetate was added for elution, and the eluate was concentrated to obtain 3.0 g of a white solid substance with a yield of 97.04%. 1H NMR (300 MHz, DMSO-d6) δ10.36 (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-isopropylbenzoic acid benzyl ester (AT6)

[0065] Compound AT5 (3.0 g, 15.29 mmol, 1.0 eq) was dissolved in CH3CN solvent (about 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 heating temperature was kept at 80 ° C and heated and stirred for 8 hours. During the reaction, TLC was used to monitor whether the reaction was complete in real time by UV spot plate (developing agent PE: EA = 4:1). After the reaction was completed, the solvent was removed by rotary evaporator to obtain a white solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with Na2SO4, filtered, and concentrated. The concentrate was separated and purified by column chromatography (eluent: PE / EA (V / V) = 200 / 1-1 / 1, gradient elution) to obtain 6.5 g of 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).

[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) (about 40 mL), and lithium hydroxide (1.34 g, 55.72 mmol, 4.0 eq) was added, and stirred at room temperature for 12 h. During the reaction, TLC was used to monitor whether the reaction was complete in real time using a UV spot plate (developing agent PE: EA = 5: 1). After the reaction was completed, the mixture was concentrated, 1 M HCl was added to the residue to adjust the pH to 5, and then about 60 mL of CH2Cl2 was added for extraction 3 times. The organic layer was taken, 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-bromoisoindol-2-yl)methanone (AT8)

[0069] Compound AT7 (4.9 g, 13.02 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (about 60 mL), and 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 bath conditions, and stirred at room temperature for 8 hours. During the reaction, TLC was used to monitor whether the reaction was complete in real time by ultraviolet spot plate (developing agent PE: EA = 6:1). After the reaction was completed, the solvent was removed by rotary evaporator to obtain a yellow solid. The solid was extracted three times with CH2Cl2 and water, and the organic phases were combined, dried with Na2SO4, filtered and concentrated. The concentrate was separated and purified by column chromatography (eluent: PE / EA (V / V) = 100 / 1-5 / 1, gradient elution) to obtain 6.5 g of yellow solid powder with a yield of 89.73%. 1H NMR (300MHz, DMSO-d6) δ7.62–7.46 (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) (about 40 mL), and potassium (4-tert-butyloxycarbonylpiperazine-1-yl)methyl trifluoroborate (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.), tri-tert-butylphosphine tetrafluoroborate ((t-Bu)3P-HBF4) (339 mg, 1.168 mmol, 0.1 eq.) were added, and N2 was charged for protection, and heated and stirred at 100°C for 14 hours. During the reaction, TLC was carried out in real time to monitor whether the reaction was complete by ultraviolet spot plate (developing agent PE:EA = 1:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by filtration through a diatomaceous earth pad, and the filtrate was concentrated to an oil. The solid was extracted three times with CH2Cl2 and water, the organic phases were combined, dried with anhydrous Na2SO4, filtered, and concentrated to obtain 7.4 g of a light yellow solid powder with a yield of 93.74%. 1H NMR (300MHz, DMSO-d6) δ7.52–7.37 (m, 5H), 7.35–7.30 (m, 3H), 7.28–7.17 (m, 5H), 7.10 (d, J=1.4Hz, 1H), 6.96 (d, J=1.7Hz, 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 (about 10 mL), and 2M HCl / EA (60 mL) was added. The mixture was stirred at room temperature for 4 hours. During the reaction, TLC was used to monitor whether the reaction was complete in real time by UV spot plate (developing agent DCM: MeOH (V / V) = 10:1). After the reaction was completed, the solvent was discarded, leaving an orange solid. The pH of the solid was adjusted to alkaline using a saturated NaHCO3 solution, and ethyl acetate was added to extract the aqueous phase 3 times. The organic phases were combined, dried with anhydrous Na2SO4, filtered, and concentrated. The concentrate was separated and purified by column chromatography (eluent: DCM / MeOH (V / V) = 250 / 1-15 / 1 + 1% triethylamine, gradient elution) to obtain 6.0 g of 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).

[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, and 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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 920 mg of 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).

[0080] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazine-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 (about 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, and then 10% Pd / C (920 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate to see if the reaction was complete (developing agent DCM: MeOH (V / V) = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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 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)isoindolyl-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)isoindolyl-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 was completed, water (about 40 mL) was added to the reaction solution for extraction. The separated organic layer was dried with anhydrous sodium sulfate and evaporated to dryness. The crude product was separated by column chromatography (DCM / MeOH, 100 / 1 to 20 / 1) 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).

[0087] (2) Preparation of 1,3-bis(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolyl-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) (about 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, and then 10% Pd / C (900 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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)isoindolyl-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)isoindolyl-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, and 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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 910 mg of orange solid with a yield of 80.23%. 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)isoindolyl-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) (about 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, and then 10% Pd / C (910 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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 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)isoindolyl-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)isoindolyl-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, and 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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 920 mg of orange solid with a yield of 80.25%. 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)isoindolyl-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) (about 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, and then 10% Pd / C (920 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate to see if the reaction was complete (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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 16.46%. 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)isoindolyl-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)isoindolyl-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, undecane dioic 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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 922 mg of orange solid 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).

[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) (about 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, and then 10% Pd / C (922 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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)isoindolyl-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)isoindolyl-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, and 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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 930 mg of orange solid with 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)piperazine-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) (about 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, and then 10% Pd / C (930 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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)isoindolyl-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)isoindolyl-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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 950 mg of orange solid with 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)isoindolyl-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) (about 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, and then 10% Pd / C (950 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate to see if the reaction was complete (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 887 mg of orange solid 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).

[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) (about 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, and then 10% Pd / C (950 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate to see if the reaction was complete (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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 17.19%. 1H 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)isoindol-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)isoindol-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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 921 mg of orange solid with a yield of 82.00%. 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)isoindol-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) (about 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, and then 10% Pd / C (950 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate to see if the reaction was complete (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: PE / EA (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 900 mg of orange solid 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).

[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) (about 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, and then 10% Pd / C (900 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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 obtain 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] Embodiment 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 bath conditions, and then Et3N (483.71 μL, 3.48 mmol, 4.0 eq.) was slowly added, and stirred at room temperature for 8 h. After the reaction was completed, water (about 40 mL) was added to the reaction solution for extraction, and the separated organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was separated and purified by column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1-20 / 1, gradient elution) to obtain 956 mg of orange solid 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).

[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) (about 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, and then 10% Pd / C (950 mg) was added. The mixture was stirred at 40°C for 50 minutes. During the reaction, the reaction was monitored by TLC in real time by UV spot plate (developing agent DCM: MeOH = 15:1). After the reaction was completed, the mixture was cooled to room temperature, the catalyst was removed by suction through a diatomaceous earth pad, and the filtrate was concentrated to a solid. The solid was extracted 3 times with CH2Cl2 and water, and the organic phases were combined, dried with 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 172 mg of a white solid with a yield of 24.90%. 1H 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 showed nM-level binding activity to HSP90 NTD and showed a structure-activity relationship, among which the molecules with short chain lengths showed relatively good binding activity, which was basically maintained at the same level as the positive control compound AT13387. This shows that the example compounds of this patent retain strong HSP90 binding activity.

[0161] Among them, the operation method of the FP method to test the competitive binding activity of the compound with HSP90 NTD is as follows:

[0162] First, prepare 50 mL of 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, and use the buffer solution to dissolve the test compound (DMSO content is less than 10%), HSP90 NTD protein and probe molecule. Use a 384-well plate, set up two duplicate wells, add 20μL HSP90 NTD protein solution (final concentration 60nM), 20μL probe molecule solution (final concentration 40nM) to each well, and finally add 20μL compound solution (highest final concentration 3μM, 3-fold dilution, 10 concentration gradients); add 20μL HSP90 NTD protein solution, 20μL probe molecule solution, and 20μL buffer solution of the system to the [probe + protein] control well; add 20μL probe molecule solution and 40μL buffer solution to the [probe] control well. Incubate the 384-well plate at room temperature (25℃) for 30min, use an ELISA reader to scan and collect data at a wavelength of 405nm, and analyze the results using GraphPad software.

[0163] Experimental Example 2

[0164] Native-page experiment

[0165] Native-page was used to test the activity of divalent compounds inducing HSP90 NTD dimerization

[0166] like Figure 1 As shown, the bivalent inhibitor molecules DDO-4105, DDO-4110 and DDO-4113 induced 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 dimerization to different extents 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] The Native-page dimerization experiment was performed according to the standard experimental procedure. The specific experimental process included protein extraction, setting different ratios of protein and compound co-incubation, sampling at different time points, preparing non-denaturing polyacrylamide gel, Native-page analysis, and scanning the results. In the experiment, 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-native 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 method)

[0178] Co-IP testing of bivalent inhibitor molecules for interference with 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] Among them, the operation method of Co-IP test is as follows:

[0182] The Western-Blots experiment followed the standard experimental procedure. The specific experimental process included cell administration, cell lysis, total protein collection, overnight incubation with primary antibody to recognize the complex, incubation with Protein A / G magnetic beads, preparation of polyacrylamide gel, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation, and result scanning. In the experiment, the expression of co-chaperone proteins (CDC37, HOP, and P23, etc.) was tested.

[0183] Experimental Example 5

[0184] Cell antiproliferation assay (CCK8 method)

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

[0186] like Figure 4 As shown, the cell proliferation inhibition activity of the bivalent inhibitor molecule exhibits cell antiproliferative activity consistent with that of AT13387.

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

[0188] Take cells in logarithmic growth phase and culture them in 96-well plates for 24 hours, with a volume of 100 μL per well (the total number of cells per well is 3000-8000 tumor cells). After 24 hours, the drug-treated group was added with compounds of different concentrations, and the compounds were diluted with the corresponding culture medium. Each group was set up with 8 concentrations and 3 replicates. The control group was added with the same volume of culture medium as the experimental group. Then it was placed in a cell culture incubator for culture. After 72 hours, the culture medium was discarded and 100 μL of cell culture medium solution containing 10% CCK8 was added to each well. 37°C, incubated in an incubator for 1-3 hours with mild shaking, and then the microplate reader was read (the optical density value (OD value) was measured under the conditions of reference wavelength 450nm and detection wavelength 570nm until the OD value of the blank control group>1.2). The tumor cells treated with culture medium were used as the control group, and the inhibition rate of the compound on 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 effect 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 a considerable activity in degrading substrate proteins.

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

[0195] Western-Blots experiments were performed according to the standard experimental procedures. The specific experimental process included cell administration, cell lysis, total protein collection, preparation of polyacrylamide gel, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation and result scanning. In the experiment, the expression level of the HSPs (HSP90, HSP70, HSP40 and HSP27) protein family was tested.

[0196] Experimental Example 7

[0197] Western blots to test the activity of bivalent inhibitor molecules on attenuation of heat shock effects

[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 showed that DDO-4113 reduced 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 showed that DDO-4113 reduced 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-Blots experiments were performed according to the standard experimental procedures. The specific experimental process included cell administration, cell lysis, total protein collection, preparation of polyacrylamide gel, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation and result scanning. In the experiment, the expression level of the HSPs (HSP90, HSP70, HSP40 and HSP27) protein family was tested.

[0203] Experimental Example 8

[0204] Pharmacokinetic study 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 in each group. The intraperitoneal injection dose was 5 mg / kg. 150 μL blood samples were taken 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] The in vivo pharmacodynamic study of compound DDO-4113 was conducted in balb / c-nu nude mice (5 weeks old). The mice were kept under standard conditions (12 / 12h light-dark cycle, 22±3℃, relative humidity 40%) for 5 days and fed with standard laboratory rodent chow and water. HCT116 cells (5*10 6 When the average tumor volume reaches about 100 mm 3The mice 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 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. When the 21st day was over or the tumor volume reached 1800mm 3 Afterwards, the nude mice were killed.

[0212] like Figure 8 and 9 As shown, high doses of AT13387 (50 mg / kg) were highly toxic, with deaths occurring on the third day after administration, and all mice dying on the ninth day. The mice also showed a significant decrease in body weight, indicating strong toxicity. High doses of DDO-4113 (50 mg / kg) also resulted in weight loss, but the result was weaker than that of AT13387, and the survival of mice was better than that of AT13387. Low doses of both compounds (25 mg / kg) had relatively weak toxic side effects.

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

Claims

1. A bivalent inhibitor based on heat shock protein 90, characterized in that: The bivalent inhibitor is a compound having a structural formula as shown in Formula I: ALB Formula I, or a pharmaceutically acceptable salt, solvate, or optical isomer thereof, Among them, A and B are ATP inhibitors of heat shock protein 90. 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.

2. The bivalent inhibitor based on heat shock protein 90 according to claim 1, characterized in that: A and B are selected from the following compounds respectively or together:

3. The bivalent inhibitor based on heat shock protein 90 according to claim 1, characterized in that: The motif structure of L is selected as one of the following: Wherein, n represents any independent natural number between 1 and 12.

4. The bivalent inhibitor based on heat shock protein 90 according to claim 1, characterized in that: The structural formula of the compound shown in Formula I is selected from the following:

5. The method for preparing a bivalent inhibitor based on heat shock protein 90 according to claim 4, 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 dicarboxylic acid compounds and HATU under ice bath conditions, then slowly add Et3N, stir at room temperature, add water to the reaction solution after the reaction is completed for extraction, separate the obtained organic layer, dry it with anhydrous sodium sulfate, and evaporate it to dryness, and separate and purify the obtained crude product by column chromatography, wherein the stoichiometric ratio of (2,4-bis(benzyloxy)-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone, dicarboxylic acid compounds, 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, then 10% Pd / C was added, and stirring was continued 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.

6. The method for preparing a bivalent inhibitor based on heat shock protein 90 according to claim 5, characterized in that: 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.

7. Use of the bivalent inhibitor based on heat shock protein 90 according to any one of claims 1 to 4 in the preparation of gastric cancer or colon cancer drugs.

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