A targeting hsp90-a ha1 inhibitor, its preparation method and application in antitumor drugs

Abstract

The present application provides a compound shown in formula (I) and a prodrug thereof, the compound has good AHA1 inhibiting effect, can be used for treating HSP90-AHA1 mediated diseases and / or disorders, such as multiple myeloma and / or colorectal cancer, and preparing a medicine for the diseases or disorders. In particular, the compound containing a phenolic ester structure has higher bioavailability, as a prodrug, can release an AHA1 inhibitor with high activity in an animal body, and has good drug property.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a class of quinolinone AHA1 inhibitor compounds, prodrugs, pharmaceutical compositions, their preparation methods, and their antitumor applications. Background Technology

[0002] Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone protein that plays a central role in maintaining cellular protein homeostasis. It regulates the folding, stability, and functional activity of client proteins through dynamic conformational changes, forming a highly efficient "molecular chaperone machine." Under stress conditions (such as heat shock and oxidative damage), HSP90 expression levels are significantly upregulated, protecting cells from stress damage by stabilizing key signaling proteins (such as kinases and transcription factors) (Chiosis G, Digwal CS, Trepel JB, et al. Structural and functional complexity of HSP90 in cellular homeostasis and disease. Nat Rev Mol CellBiol. 2023, (24): 797-815.).

[0003] To cope with the harsh tumor microenvironment, cancer cells often overexpress HSP90 (accounting for approximately 4-6% of total intracellular protein). Its function extends beyond stress protection; it also maintains a malignant phenotype and drug resistance by maintaining the stability of mutated oncogenic proteins, activating compensatory pathways, promoting DNA repair, and regulating tumor immunity and metabolism (Garg G, Khandelwal A, Blagg BS. Anticancer Inhibitors of Hsp90 Function: Beyond the Usual Suspects. AdvCancer Res. 2016, (129): 51-88.) (Jarosz DF, Lindquist S. Hsp90 and environmental stress transform the adaptive value of natural genetic variation. Science. 2010, (330): 1820-1824.). Abnormal expression of HSP90 is closely related to various pathological processes, including tumors and neurodegenerative diseases, and has become an important target for anti-tumor drug development. Since the advent of the first geldmycin derivative 17-AAG to enter clinical trials in 1999, more than 20 HSP90 ATP competitive inhibitors with different chemical skeletons have entered clinical research, mainly including resorcinol, purine, and benzamide compounds (Wang L, Xu X, Jiang Z, et al. Modulation of protein fate decision by small molecules:targeting molecular chaperone machinery. Acta Pharm Sin B. 2020, (10): 1904-1925.). However, the strong blocking of HSP90 ATPase activity by ATP competitive inhibitors leads to irreversible heat shock response (HSR) and subsequent severe toxicity, limiting their application.

[0004] Recent studies have found that targeting protein-protein interactions (PPIs) between co-chaperone proteins and HSP90, and interfering with the regulation of molecular chaperone cycles by co-chaperone proteins, has developed into an innovative drug design strategy (Zhang X, Li S, Li Z, et al. The Therapeutic Potential of Targeting Hsp90-Cdc37 Interactions in Several Diseases. Curr Drug Targets. 2022, (23): 1023-1038.). This strategy not only avoids the toxic side effects related to client proteins caused by strongly inhibiting the ATP pocket of HSP90, but also benefits from the specificity of each co-chaperone protein to different substrate proteins, enabling precision treatment of diseases that highly express specific client proteins (Dutta Gupta S, Bommaka MK, Banerjee A. Inhibiting protein-protein interactions of Hsp90 as a novel approach for targeting cancer. Eur J Med Chem. 2019, (178): 48-63.). HSP90 ATPase activator homolog 1 (AHA1) is a key regulator of HSP90 ATPase activity. By binding to the intermediate and N-terminal domains of HSP90, it promotes ATP hydrolysis and drives conformational changes in HSP90. AHA1 is overexpressed in various cancers and is closely associated with tumor progression and poor prognosis. Developing inhibitors of the HSP90-AHA1 molecular chaperone system holds promise for providing more precise and personalized treatment strategies for various diseases. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a small molecule inhibitor that targets the HSP90-AHA1 molecular chaperone system, that is, to provide an AHA1 inhibitor with anti-multiple tumor effects.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the AHA1 inhibitor that is easy to operate.

[0007] Another technical problem to be solved by the present invention is to provide the use of the AHA1 inhibitor, namely, its use in the preparation of antitumor drugs.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: An AHA1 inhibitor with antitumor activity, said inhibitor being a compound of formula (I), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, or prodrug compound thereof: , R1 is selected from -H, hydroxyl, sulfonamide, amide, ester, alkoxy, and carboxyl groups; R2 is selected from -H, hydroxyl, sulfonamide, halogen, alkyl, alkoxy, amino, substituted amino, and five- or six-membered aromatic rings containing 0 to 2 nitrogen, sulfur or oxygen atoms; R3 is selected from -H, alkoxy, hydroxy, sulfonamide, halogen, alkyl, amino, substituted amino, and carboxyl groups; X is CH or N.

[0009] Preferably, the compound is selected from:

[0010] Another aspect of the present invention relates to a method for preparing an AHA1 inhibitor with antitumor activity, specifically comprising the following steps: a. Using substituted benzaldehyde (2-1) as the starting material, a nitration reaction is carried out at low temperature in fuming nitric acid to generate a nitro-substituted benzoyl derivative (2-2), which is then set aside. b. Take the nitro-substituted benzoyl derivative (2-2) obtained in step a and reduce it with iron powder in ethanol solution to obtain the amino-substituted benzoyl derivative (2-3), for later use; c. Using the amino-substituted benzoyl derivative (2-3) obtained in step b as a raw material, react it with ethyl nitroacetic acid in water and glacial acetic acid to form a cyclization intermediate (2-4), which is then set aside. d. Take the intermediate (2-4) obtained in step c and reduce it with iron powder in an ethanol solution to obtain intermediate (2-5), which is then set aside. e. React iodoaromatic acids with substituted aromatic boric acids under the catalysis of palladium to obtain intermediate (2-6), for later use; f. Take the intermediate (2-6) obtained in step e and prepare an acyl chloride using sulfoxide as a solvent, and then react it with the intermediate (2-5) to obtain the target compound (I). g. For compounds where R1 is an ester group, the compound (I) obtained in step f is hydrolyzed in a solution of potassium hydroxide in water and methanol to obtain the target compound (IA).

[0011] Furthermore, the concentrated nitric acid used in step a is fuming nitric acid, the molar volume ratio of substituted benzaldehyde to fuming nitric acid is 1 mol / 500 mL to 1 mol / 900 mL, the reaction temperature is -15 ℃ to -5 ℃, and the reaction time is 1 to 2 h.

[0012] Furthermore, in step b, the molar ratio of (2-2) to iron powder is 1:4 to 6, the molar ratio of (2-2) to glacial acetic acid is 1:8 to 12, the volume ratio of (2-2) to ethanol is 1 mmol / 10 mL to 1 mmol / 20 mL, the reaction temperature is reflux, and the reaction time is 2 to 8 h.

[0013] Furthermore, in step c, the molar ratio of (2-3) to ethyl nitroacetate is 1:2 to 3, the volume ratio of glacial acetic acid to water is 1:1, the molar ratio of amino-substituted benzaldehyde to solvent is 1 mmol / 3 mL to 1 mmol / 10 mL, the reaction temperature is 50 ℃ to 80 ℃, and the reaction time is 10 to 20 h.

[0014] Furthermore, in step d, the molar ratio of (2-4) to iron powder is 1:4 to 6, the molar ratio of intermediate (2-4) to glacial acetic acid is 1:8 to 12, the molar volume ratio of intermediate (2-4) to ethanol is 1 mmol / 10 mL to 1 mmol / 20 mL, the reaction temperature is reflux, and the reaction time is 2 to 8 h.

[0015] Furthermore, in step e, the molar ratio of iodoaromatic acid to substituted aromatic boric acid is 1:1.5–3; the catalyst is Pd(dppf)Cl2, the molar ratio of iodoaromatic acid to Pd(dppf)Cl2 is 1:0.03–0.08, the base used in the reaction is potassium phosphate or potassium carbonate solution, nitrogen protection is applied, the reaction temperature is 40 ℃–60 ℃, and the reaction time is 12–24 h.

[0016] Furthermore, in step f, the molar ratio of intermediate (2-5) to intermediate (2-6) is 1:1.5-3; the molar volume ratio of intermediate (2-6) to SOCl2 is 1 mmol / 10 mL to 1 mmol / 50 mL; the reaction temperature is reflux; and the reaction time is 2-8 h. The solvent used for the reaction of intermediate (2-6) after preparation as acyl chloride with intermediate (2-5) is DCM; the molar volume ratio of intermediate (2-5) and (2-6) to DCM is 1 mmol / 10 mL to 1 mmol / 50 mL; the reaction temperature is room temperature; and the reaction time is 2-8 h.

[0017] Furthermore, the compound (I) used in step g is hydrolyzed in a solution of potassium hydroxide in water and methanol to obtain the target compound (IA). The molar ratio of reactant to potassium hydroxide is 1:10~20, the volume ratio of methanol to water in the reaction solvent is 1:10~50, the molar volume ratio of reactant to total solvent is 1 mmol / 20 mL~1 mmol / 50 mL, the reaction temperature is 30 ℃~50 ℃, and the reaction time is 8~16 h.

[0018] Another aspect of the present invention relates to the prevention and treatment of tumor diseases caused by the HSP90-AHA1 axis. According to embodiments of the present invention, the tumor diseases include at least one of the following: leukemia, chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, hairy cell leukemia, lymphoblastic T-cell leukemia, lymphoma, B-cell lymphoma, Burkitt lymphoma, Hodgkin's lymphoma, chondrosarcoma, fibrosarcoma, Ewing sarcoma, rhabdomyosarcoma, small cell lung cancer, non-small cell lung cancer, myeloma, glioma, prostate cancer, breast cancer, bone cancer, neuroblastoma, gastric cancer, ovarian cancer, intestinal cancer, kidney cancer, medulloblastoma, pancreatic cancer, thyroid cancer, mesothelioma, cervical cancer, bladder cancer, liver cancer, skin cancer, and tumors of the central or peripheral nervous system.

[0019] In one embodiment, the AHA1 inhibitor (I) has a hydroxyl group in its molecular structure, thus exhibiting a certain acidity, and can react with alkali metal, alkaline earth metal hydroxide, alkali metal carbonate or alkaline earth metal carbonate, alkali metal bicarbonate or alkaline earth metal bicarbonate to form a salt.

[0020] In one embodiment, the AHA1 inhibitor (I) contains a nitrogen atom in its structure, thus exhibiting a certain degree of weak basicity and being able to react with hydrochloric acid, sulfuric acid, and phosphoric acid to form salts.

[0021] Furthermore, the drug is formulated into tablets, capsules, granules, sprays, injections, microcapsules, ointments, or transdermal controlled-release patches by combining an AHA1 inhibitor (I) salt with a pharmaceutically acceptable carrier.

[0022] In one embodiment, when the AHA1 inhibitor (I) salt provided by the present invention is made into tablets, the AHA1 inhibitor (I) salt and carrier lactose or corn starch, and the lubricant magnesium stearate are added as needed, mixed evenly, and then compressed into tablets.

[0023] In one embodiment, when the AHA1 inhibitor (I) salt provided by the present invention is made into capsules, the AHA1 inhibitor (I) salt and the carrier lactose or corn starch are mixed evenly, granulated, and then encapsulated to make capsules.

[0024] In one embodiment, when the AHA1 inhibitor (I) salt provided by the present invention is made into granules, the composition and diluent lactose or corn starch are mixed evenly, granulated, dried, and made into granules.

[0025] In one embodiment, when preparing the AHA1 inhibitor (I) salt provided by the present invention into an injection solution, the AHA1 inhibitor (I) salt is added to a solubilizer, stirred evenly, heated at 80°C for 30 min, filtered, pH value adjusted, filtered through a sintered glass funnel or other filter until clear, filled, and sterilized at 100 to 115°C for 30 min to prepare the injection solution.

[0026] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0027] 1. The series of small molecule inhibitors (I) targeting the HSP90-AHA1 molecular chaperone system provided by this invention can not only avoid the toxic side effects related to client proteins caused by strong inhibition of the ATP pocket of HSP90, but also benefit from the specificity of each co-chaperone protein to different substrate proteins, so as to achieve precise treatment of diseases with high expression of specific client proteins.

[0028] 2. The preparation method of the series of AHA1 inhibitors (I) provided by the present invention has a reasonable process design. It uses substituted benzaldehyde as raw material and undergoes nitration, reduction, cyclization and coupling reactions in sequence to obtain the target compound. The reaction conditions at each step are mild, highly operable, have high production efficiency, low cost and high yield and high purity of the finished product.

[0029] The beneficial technical effects of this invention are detailed in the experimental examples below.

[0030] definition

[0031] The term "halogen" or the prefix "halogen" refers to F, Cl, Br, or I.

[0032] "Alkoxy" refers to the group -OR, where R is a substituted or unsubstituted alkyl group.

[0033] As described in this invention, the ring system formed by a key connecting the substituent to the central ring (as shown in Formula I) means that R3 and R4 can be substituted at any substituted position on the ring.

[0034] In this invention, "pharmaceutically acceptable" means any substance that does not interfere with the bioactivity of the active ingredient and is non-toxic to the host to which it is given.

[0035] As used in this invention, the term "prodrug" refers to the conversion of a compound into a compound represented by Formula I in vivo. Such conversion is influenced by the hydrolysis of the prodrug in the blood or its enzymatic conversion into the parent structure in the blood or tissues. The prodrug compounds of this invention can be esters; in existing inventions, esters that can serve as prodrugs include phenyl esters, aliphatic (C1-24) esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, a compound in this invention contains a hydroxyl group, meaning it can be acylated to yield the prodrug form. Other prodrug forms include phosphate esters, such as those obtained by phosphorylation of a hydroxyl group on the parent compound.

[0036] Unless otherwise stated, all tautomer forms of the compounds of this invention are included within the scope of this invention. Additionally, unless otherwise stated, the structural formulas of the compounds described in this invention comprise enriched isotopes of one or more different atoms. The terms "tautomer" or "tautomer form" refer to isomers of structures with different energies that can interconvert through a low energy barrier. Examples of this include, but are not limited to, proton tautomers (i.e., proton-transfer tautomers) including interconversions via proton transfer, such as isomerizations of keto-enol and imine-enamine forms. Valence (combination valence) tautomers include some interconversions involving the recombination of bonding electrons.

[0037] The compounds of this invention may contain asymmetric or chiral centers, and therefore exist as different stereoisomers. All stereoisomers of the compounds of this invention, including but not limited to diastereomers, enantiomers, stoichiotropic isomers, and mixtures thereof, such as racemic mixtures, constitute a part of this invention. Many organic compounds exist in optically active forms, meaning they are capable of rotating the plane of polarized light. In describing optically active compounds, the prefixes D, L or R, S are used to indicate the absolute configuration of the chiral center of the molecule. The prefixes d, l or (+), (-) are used to name the symbols for the plane polarization rotation of the compound; the prefix (-) or l indicates that the compound is levorotatory, and the prefix (+) or d indicates that the compound is dextrorotatory. These stereoisomers have the same chemical structure, but their stereostructures are different. Specific stereoisomers can be enantiomers, and mixtures of isomers are generally called enantiomer mixtures. A 50:50 enantiomer mixture is called a racemic mixture or racemate, which may result in a lack of stereoselectivity or stereodirection during chemical reactions. The terms “racemic mixture” and “racemate” refer to a mixture of two equimolar enantiomers that lack optical activity.

[0038] The pharmaceutically acceptable carrier used in this invention refers to a non-toxic carrier, adjuvant, or mediator that does not impair the pharmacological activity of the co-formulated chemical contents. Attached Figure Description

[0039] Figure 1 This is a structural formula diagram of the compounds of the present invention.

[0040] Figure 2 This is a reaction flow diagram of the synthesis method of the compounds of the present invention.

[0041] Figure 3 The figure shows the experimental results of the affinity test between compound I-5 of the present invention and AHA1 protein.

[0042] Figure 4 The figure shows the experimental results of the affinity test between compound I-6 of the present invention and AHA1 protein.

[0043] Figure 5 The effect of I-6 on HSP90 enzyme activity.

[0044] Figure 6 The effect of I-6 on the interaction between HSP90 and AHA1.

[0045] Figure 7 For I-1 1 H-NMR spectrum.

[0046] Figure 8 For I-2 1 H-NMR spectrum.

[0047] Figure 9 For I-3 1 H-NMR spectrum.

[0048] Figure 10 For I-4 1 H-NMR spectrum.

[0049] Figure 11 For I-5 1 H-NMR spectrum.

[0050] Figure 12 For I-6 1 H-NMR spectrum.

[0051] Figure 13 The blood concentration curves for I-5 and I-6 are shown.

[0052] Figure 14 The ester-water distribution coefficients are I-1, I-5, and I-6 in 202210224472.X, and in this patent. Detailed Implementation

[0053] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0054] In this invention, the structure of the compound is determined by mass spectrometry (MS) and / or nuclear magnetic resonance (NMR). 1 It was determined by HNMR equipment.

[0055] The compounds of the present invention can be prepared according to conventional methods in the art, using suitable reagents, raw materials, and purification methods known to those skilled in the art. The preparation methods of the compounds of the present invention are described in more detail below, but these specific methods do not constitute any limitation on the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, such combinations being readily performed by those skilled in the art.

[0056] Example 1 Preparation of 4-formyl-2-methoxy-3-nitrophenylacetic acid ester (2-2)

[0057] Fuming nitric acid (450 mL) was added in portions to commercially available acetyl vanillin (98 g, 0.5 mol) at -10 ºC, and the mixture was stirred for 45 min. The acidic solution was slowly poured into ice water (4 L), and the precipitate was collected by filtration. The precipitate was washed several times with ice water (1 L) and dried. The crude product was purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixture (4:1 v / v) as the eluent to give 4-formyl-2-methoxy-3-nitrophenylacetic acid ester (2-2) (55 g, 46%), which was a yellow solid.

[0058] Example 2 Preparation of 3-amino-4-formyl-2-methoxyphenyl acetate (2-3)

[0059] The 4-formyl-2-methoxy-3-nitrophenylacetic acid ester (2-2) (1.4 g, 5.86 mmol) prepared above was dissolved in ethanol (88 mL), and reduced iron powder (1.634 g, 29.56 mmol) and glacial acetic acid (3.38 mL, 59 mmol) were added. The mixture was refluxed for 3.5 h. After the reaction was complete, the reduced iron powder was filtered off, the solvent was removed by vacuum evaporation, and the crude product was purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixture (6:1 v / v) as the eluent to obtain 3-amino-4-formyl-2-methoxyphenylacetic acid ester (2-3) (800 mg, 65.3%), which was a white solid.

[0060] Example 3 Preparation of 8-methoxy-3-nitro-2-oxy-1,2-dihydroquinoline-7-acetate (2-4)

[0061] 3-Amino-4-formyl-2-methoxyphenyl acetate (2-3) (540 mg, 2.58 mmol) and commercially available ethyl nitrate (0.859 mL, 7.75 mmol) were added to a mixture of glacial acetic acid (5 mL) and water (5 mL). The mixture was refluxed and stirred at 70 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the precipitate was collected and washed with water to obtain the product 8-methoxy-3-nitro-2-oxy-1,2-dihydroquinoline-7-acetic acid ester (2-4) (425 mg, 59%), which was a yellow solid.

[0062] Example 4 Preparation of 3-amino-8-methoxy-2-oxy-1,2-dihydroquinoline-7-acetic acid ester (2-5)

[0063] The 8-methoxy-3-nitro-2-oxy-1,2-dihydroquinoline-7-acetic acid ester (2-4) (443 mg, 1.59 mmol) prepared above was dissolved in ethanol (24 mL), and reduced iron powder (443 mg, 7.9 mmol) and glacial acetic acid (0.91 mL, 15.9 mmol) were added. The mixture was refluxed for 3.5 h. After the reaction was complete, the reduced iron powder was filtered off, the solvent was removed by vacuum evaporation, and the crude product was purified by silica gel column chromatography using a 1:1 (v / v) petroleum ether-ethyl acetate mixture as the eluent to obtain 3-amino-8-methoxy-2-oxy-1,2-dihydroquinoline-7-acetic acid ester (2-5) (266 mg, 67.3%), which was a yellow solid. 1H NMR(500 MHz, DMSO-d6) δ 11.36 (s, 1H), 7.11 (d, J = 8.6 Hz, 1H), 6.83 (d, J =8.6 Hz, 1H), 6.74 (d, J = 0.9 Hz, 1H), 5.51 (s, 2H), 3.75 (s, 3H), 2.32 (s, 3H).

[0064] Example 5 Preparation of 3-(5-fluoropyridin-3-yl)benzoic acid (2-6)

[0065] At room temperature, commercially available m-iodobenzoic acid (0.92 g, 4 mmol), 5-fluoro-3-pyridineboronic acid (1.13 g, 8 mmol), potassium phosphate (2.12 g, 10 mmol), Pd(dppf)Cl2 (293 mg, 0.4 mol), and anhydrous dioxane (30 mL) were added sequentially to a dry round-bottom flask. The mixture was purged with nitrogen and refluxed for 12 h. After the reaction was complete, the mixture was cooled to room temperature, acidified with 2N HCl, concentrated under reduced pressure, and the crude product was subjected to silica gel column chromatography using a mixed solvent of dichloromethane, methanol, and acetic acid (10:1:0.1) as eluent. The resulting product was then slurried with diethyl ether to obtain 3-(5-fluoropyridine-3-yl)benzoic acid (2-6) (480 mg, 55.3%), which was a yellow solid. 1 H NMR (500 MHz, DMSO) δ 13.10 (s, 1H), 8.83 (d, J = 2.1 Hz, 1H), 8.62(t, J = 2.2 Hz, 1H), 8.26 (s, 1H), 8.14 (dd, J = 10.2, 2.3 Hz, 1H), 8.03 (t,J = 6.8 Hz, 2H), 7.66 (t,J = 7.8 Hz, 1H).

[0066] Example 6 Preparation of 8-methoxy-2-oxo-3-(4-(pyridin-3-yl)benzoylamino)-1,2-dihydroquinoline-7-yl acetate (I-1)

[0067] Commercially available 4-pyridin-3-ylbenzoic acid (0.32 g, 1.6 mmol) was added to a dry round-bottom flask, and thionyl chloride (10 mL) was added under ice bath conditions. The mixture was then refluxed for 2 h after purging with nitrogen. In a new dry round-bottom flask, 3-amino-8-methoxy-2-oxy-1,2-dihydroquinoline-7-acetic acid ester (2-5) (0.2 g, 0.8 mmol), triethylamine (0.3 mL), and dichloromethane (4 mL) were added. After purging with nitrogen, the mixture was placed under ice bath conditions. After the acyl chloride reaction was complete, thionyl chloride was removed under reduced pressure, and the ester was reconstituted with anhydrous dichloromethane (4 mL). This solution was then added to the prepared solution of (2-5) and reacted at room temperature for 4 h. After completion, the product was concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography using a mixed solvent of dichloromethane and methanol (40:1) as the eluent. The crude product was then slurried with diethyl ether to obtain 8-methoxy-2-oxo-3-(4-(pyridin-3-yl)benzoylamino)-1,2-dihydroquinoline-7-yl acetate (I-1) (160 mg, 46.6%), which was a white solid. 1 H NMR (500 MHz, CDCl3) δ 9.59 (s, 1H),9.33 (s, 1H), 8.99 (s, 1H), 8.95 (s, 1H), 8.70 (s, 1H), 8.11 (d, J = 8.0 Hz,2H), 7.97 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 7.9 Hz, 2H), 7.49 – 7.43 (m, 1H), 7.41 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 4.02 (s, 3H), 2.43 (s,3H).

[0068] Example 7 Preparation of 8-methoxy-2-oxo-3-(3-(pyridin-3-yl)benzoylamino)-1,2-dihydroquinoline-7-yl acetate (I-2) Commercially available 3-pyridin-3-ylbenzoic acid (0.32 g, 1.6 mmol) was added to a dry round-bottom flask, and thionyl chloride (10 mL) was added under ice bath conditions. The mixture was then refluxed for 2 h after purging with nitrogen. In a new, dry round-bottom flask, the prepared 3-amino-8-methoxy-2-oxy-1,2-dihydroquinoline-7-acetic acid ester (2-5) (0.2 g, 0.8 mmol), triethylamine (0.3 mL), and dichloromethane (4 mL) were added. Nitrogen was purged, and the mixture was placed under ice bath conditions. After the acyl chloride reaction was complete, thionyl chloride was removed under reduced pressure, and the ester was reconstituted with anhydrous dichloromethane (4 mL). This reconstituted ester was then added to the prepared solution of (2-5), and the mixture was reacted at room temperature for 4 h. After completion, the product was concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography using a mixed solvent of dichloromethane and methanol (40:1) as the eluent. The crude product was then slurried with diethyl ether to obtain 8-methoxy-2-oxo-3-(3-(pyridin-3-yl)benzoylamino)-1,2-dihydroquinoline-7-yl acetate (I-2) (180 mg, 52.4%), which was a white solid. 1 H NMR (500 MHz, CDCl3) δ 9.68 (s, 1H), 9.33 (s, 1H), 9.00 – 8.92 (m, 2H), 8.70 – 8.65 (m, 1H), 8.20 (t, J = 1.9 Hz, 1H), 8.02 –7.95 (m, 2H), 7.82 (d, J = 7.7 Hz, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.44 (m, J= 7.9, 4.8 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 4.00(s, 3H), 2.42 (s, 2H).

[0069] Example 8 Preparation of N-(7-hydroxy-8-methoxy-2-oxo-1,2-dihydroquinolin-3-yl)-3-(pyridin-3-yl)benzamide (I-3)

[0070] In a round-bottom flask, the 8-methoxy-2-oxo-3-(3-(pyridin-3-yl)benzoamide)-1,2-dihydroquinoline-7-yl acetate (I-2) (0.1 g, 0.23 mmol) and potassium hydroxide (0.13 g, 2.3 mmol) prepared above were added. Methanol (5 mL) and water (0.5 mL) were added to dissolve the solid, and the reaction was carried out overnight at 50°C. After completion, the reaction solution was concentrated, acidified with 2N HCl, and the solid was filtered. The filter cake was washed with water and dried to obtain N-(7-hydroxy-8-methoxy-2-oxo-1,2-dihydroquinoline-3-yl)-3-(pyridin-3-yl)benzamide (I-3) (40 mg, 44.9%), a yellow solid. 1 H NMR (500 MHz, DMSO) δ 11.62 (s, 1H), 10.01 (s, 1H), 9.58 (s, 1H), 9.03 (s, 1H), 8.65 (s,1H), 8.61 (s, 1H), 8.27 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 7.7Hz, 2H), 7.71 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.30 (d, J = 8.6Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 3.80 (s, 3H).

[0071] Example 9 Preparation of 8-methoxy-2-oxo-3-(4-(pyridin-4-yl)benzoylamino)-1,2-dihydroquinoline-7-yl acetate (I-4)

[0072] In a dry round-bottom flask, commercially available 4-pyridin-4-ylbenzoic acid (0.32 g, 1.6 mmol) was added, followed by the addition of 10 mL of thionyl chloride under ice bath conditions. The mixture was then refluxed for 2 h after purging with nitrogen. In a new, dry round-bottom flask, the prepared 3-amino-8-methoxy-2-oxy-1,2-dihydroquinoline-7-acetic acid ester (2-5) (0.2 g, 0.8 mmol), triethylamine (0.3 mL), and dichloromethane (4 mL) were added. After purging with nitrogen, the mixture was placed in an ice bath. After the acyl chloride reaction was complete, thionyl chloride was removed under reduced pressure, and the ester was reconstituted with anhydrous dichloromethane (4 mL). This reconstituted ester was then added to the prepared solution of (2-5) and reacted at room temperature for 4 h. After completion, the product was concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography using a mixed solvent of dichloromethane and methanol (40:1) as the eluent. The crude product was then slurried with diethyl ether to obtain 8-methoxy-2-oxo-3-(4-(pyridin-4-yl)benzoamide)-1,2-dihydroquinoline-7-yl acetate (I-4) (170 mg, 49.5%), which was a white solid. 1 H NMR (500 MHz, CDCl3) δ 9.45 (s, 1H),9.32 (s, 1H), 8.99 (s, 1H), 8.75 (d, J = 5.1 Hz, 2H), 8.12 (d, J = 8.0 Hz,2H), 7.81 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 5.1 Hz, 2H), 7.41 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 4.02 (s, 3H), 2.43 (s, 3H).

[0073] Example 10 Preparation of 3-(3-(5-fluoropyridin-3-yl)benzoylamino)-8-methoxy-2-oxo-1,2-dihydroquinoline-7-yl acetate (I-5)

[0074] Add the prepared 3-(5-fluoropyridin-3-yl)benzoic acid (2-6) (0.35 g, 1.6 mmol) to a dry round-bottom flask, add 10 mL of thionyl chloride under ice bath conditions, purge with nitrogen, and reflux for 2 h. Add the prepared 3-amino-8-methoxy-2-oxy-1,2-dihydroquinoline-7-acetic acid ester (2-5) (0.2 g, 0.8 mmol), triethylamine (0.3 mL), and dichloromethane (4 mL) to a new dry round-bottom flask, purge with nitrogen, and place under ice bath conditions. After the acyl chloride reaction is complete, remove the thionyl chloride under reduced pressure, redissolve in anhydrous dichloromethane (4 mL), add to the prepared solution of (2-5), and react at room temperature for 4 h. After completion, the product was concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography using a mixed solvent of dichloromethane and methanol (40:1) as the eluent. The crude product was then slurried with diethyl ether to obtain 3-(3-(5-fluoropyridin-3-yl)benzoamide)-8-methoxy-2-oxo-1,2-dihydroquinoline-7-yl acetate (I-5) (180 mg, 50.3%), which was a white solid. 1 H NMR (500 MHz, CDCl3) δ9.45 (s, 1H), 9.31 (s, 1H), 8.98 (s, 1H), 8.79 (s, 1H), 8.57 (s, 1H), 8.20(s, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.69 (m, J = 8.0Hz, 2H), 7.41 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 4.02 (s, 3H), 2.43 (s, 3H).

[0075] Example 11 Preparation of 3-(5-fluoropyridin-3-yl)-N-(7-hydroxy-8-methoxy-2-oxo-1,2-dihydroquinoline-3-yl)benzamide (I-6)

[0076] In a round-bottom flask, the prepared 3-(3-(5-fluoropyridin-3-yl)benzoamide)-8-methoxy-2-oxo-1,2-dihydroquinoline-7-yl acetate (I-5) (0.1 g, 0.23 mmol) and potassium hydroxide (0.13 g, 2.3 mmol) were added. Methanol (5 mL) and water (0.5 mL) were added to dissolve the solid, and the reaction was carried out overnight at 50°C. After completion, the reaction solution was concentrated, acidified with 2N HCl, and the solid was filtered. The filter cake was washed with water and dried to obtain 3-(5-fluoropyridin-3-yl)-N-(7-hydroxy-8-methoxy-2-oxo-1,2-dihydroquinoline-3-yl)benzamide (I-6) (35 mg, 37.6%), a yellow solid. 1 H NMR (500 MHz, DMSO) δ 11.63 (s, 1H), 10.02 (s, 1H), 9.60 (s, 1H), 8.93 (s, 1H), 8.65 (d, J = 2.6 Hz, 1H), 8.60 (s, 1H), 8.34 (s, 1H), 8.24 (d, J = 10.2 Hz, 1H), 8.03 (m, J = 14.4, 7.8 Hz, 2H), 7.71 (t, J = 7.8 Hz, 1H), 7.30 (d, J =8.5 Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 3.80 (s, 3H).

[0077] Example 12 Experiment on the affinity of the AHA1 inhibitor (I) of the present invention for AHA1 protein

[0078] The affinity of the synthesized compounds I-1 to I-6 for AHA1 in this invention was tested using microthermophoresis (MST) with a Monolith™ NT.115 series instrument manufactured by NanoTemper, and the accompanying kit was the Monolith™ RED-NHS second-generation protein labeling kit. First, the proteins were labeled according to the kit instructions. Then, the samples were analyzed: the AHA1 sample concentration, the concentration gradient of compounds I-1 to I-6, and the type of loading capillary were set, and the program was started. After the program finished, the MST curves were fitted using analysis software, and the Ka affinity of AHA1 for compounds I-1 to I-6 was calculated. d Values. Specific experimental results are shown in Table 1 below.

[0079] Table 1. K-response of compounds to AHA1 d value

[0080] a " / " indicates that the value has not yet been determined.

[0081] Among them, compounds I-5 and I-6 exhibited strong binding affinity to the target protein AHA1, and I-5 showed a high K-axis binding affinity to AHA1. d The value is 0.33 μM; K of I-6 and AHA1 d The value is 0.28 μM.

[0082] Furthermore, this invention expressed and purified truncated N-terminal and C-terminal variants of the AHA1 protein, and the results showed that this series of compounds exerts antitumor activity by binding to the C-terminus of AHA1 (I-5 binds to the K+ at the C-terminus of AHA1). d The value is 0.45 μM; K at the C-terminus of I-6 and AHA1 d The value was 0.24 μM; I-5, I-6 and the N-terminus of AHA1 had no affinity.

[0083] Example 13 Experiment on the effect of the AHA1 inhibitor (I) of the present invention on HSP90 enzyme activity

[0084] The ATPase activity of HSP90 was determined using the ATPase activity assay kit (catalog number: BC0065) from Beijing Solarbio Science & Technology Co., Ltd., via a micro-method.

[0085] Operating steps: (1) Sample preparation: After treating cells with I-6 or Geldanamycin (GA, HSP90 ATP inhibitor), collect the cells into centrifuge tubes, centrifuge and discard the supernatant; add the corresponding reagents according to the instructions, and sonicate to break up the bacteria or cells (power 200 w, sonication for 3 s, interval 10 s, repeat 30 times); centrifuge at 8000 g, 4 ℃ for 10 min, take the supernatant and place it on ice for testing; (2) Measurement steps: The visible spectrophotometer or microplate reader should be preheated for at least 30 minutes, and the wavelength should be adjusted to 660 nm. The visible spectrophotometer needs to be zeroed with distilled water. Set up the reaction system according to the instructions to carry out the enzymatic reaction and phosphorus determination; (3) Enzyme activity calculation: Na + K + -ATPase activity (U / mg prot) = C standard × (A assay - A control) ÷ (A standard - A blank) × V total ÷ (Cpr × V sample) ÷ T × F = 7.5 × (A assay - A control) ÷ (A standard - A blank) ÷ Cpr × F.

[0086] Experimental Results: In wild-type (WT) and ARP1 AHA1-overexpressing multiple myeloma cells, compared with the GA group, compound I-6 could inhibit the enhanced HSP90 enzyme activity induced by AHA1 overexpression, but had no significant effect on HSP90 enzyme activity in wild-type ARP1 cells. GA, however, strongly inhibited HSP90 ATPase activity in both cell types, indicating that the AHA1 inhibitor (I) of this invention can target the HSP90-AHA1 chaperone system without causing the toxic side effects caused by strong HSP90 inhibition (see [link to relevant documentation]). Figure 5 ).

[0087] Example 14 Effect of the AHA1 inhibitor (I) of the present invention on the HSP90-AHA1 interaction

[0088] The effect of the AHA1 inhibitor (I) of this invention on the interaction between HSP90 and AHA1 was investigated using Western blotting (WB) and co-immunoprecipitation (Co-IP).

[0089] Preparation of reagents for protein immunoblotting:

[0090] (1) 1.5 M Tris-HCl (pH=8.8): Weigh 18.16 g Tris, add it to 80 mL of ultrapure water and dissolve it completely. Adjust the pH to 8.8, add ultrapure water to make up to 100 mL, and store at 4℃.

[0091] (2) 1.0 M Tris-HCl (pH=6.8): Weigh 12.12 g Tris, add it to 80 mL of ultrapure water and dissolve it completely. Adjust the pH to 6.8, add ultrapure water to make up to 100 mL, and store at 4℃.

[0092] (3) 1.0 M Tris-HCl (pH=7.5): Weigh 12.11 g Tris, add it to 80 mL of ultrapure water and dissolve it completely. Adjust the pH to 7.5, add ultrapure water to make up to 100 mL, and store at 4℃.

[0093] (4) 30% Acrylamide: Weigh 290 g of acrylamide and 10 g of N,N-methyleneacrylamide, add them to 800 mL of ultrapure water, use a magnetic stirrer to accelerate dissolution, make up to 1000 mL, filter with qualitative filter paper, and store in a brown bottle at 4°C in the dark.

[0094] (5) 10% Sodium dodecyl sulfate (SDS): Weigh 10 g of SDS powder, add it to 100 mL of ultrapure water, heat it in a water bath at 65°C to dissolve it completely, and store it at room temperature.

[0095] (6) 10% ammonium persulfate (AP): Weigh 0.1 g of ammonium persulfate, add it to 1 mL of ultrapure water and dissolve it completely. Store at 4°C.

[0096] (7) 5× Electrophoresis buffer: Weigh 15.1 g Tris, 94 g glycine powder and 5 g SDS powder respectively and dissolve them in 1 L of ultrapure water. When using, dilute it to 1× with ultrapure water according to the ratio.

[0097] (8) 10× transfer buffer: Weigh 30.38 g Tris and 151.18 g g glycine powder respectively, add them to 1 L of ultrapure water and dissolve them completely. When using, dilute it to 1× according to the ratio of 10× transfer buffer: methanol: ultrapure water = 1:1:8.

[0098] (9) 10×TBST: Take 100 mL of Tris-HCl (pH=7.5) solution, 88 g of NaCl and 20 mL of Tween 20 respectively, add 1 L of ultrapure water and store at room temperature.

[0099] (10) 5× loading buffer: Weigh 0.5 g SDS powder and 25 mg bromophenol blue, add 1.25 mL Tris-HCl (pH=6.8) and 2.5 mL glycerol, add an appropriate amount of ultrapure water and stir until fully dissolved, bring the volume to 5 mL and store at -20℃.

[0100] (11) Blocking solution (5% skim milk powder / bovine serum albumin): Weigh 1 g of skim milk powder / bovine serum albumin, add 20 mL of 1×TBST solution, vortex to dissolve it completely, and store at 4℃.

[0101] Operating steps:

[0102] (1) Install the prepared gel correctly, add 1× electrophoresis buffer, remove the comb, load the sample in sequence, turn on the power, first use 80 V constant voltage for 30 min to flatten the upper gel, then use 120 V constant voltage for 90 min to separate the lower gel until the target band is completely separated.

[0103] (2) Prepare 1× transfer buffer in advance. After electrophoresis, cut the required molecular weight range of gel strips according to the visible markers. Cut the PVDF membrane to the appropriate size according to the size of the gel strips and activate it in methanol for 1 min. Place the gel strips and PVDF membranes in the order of "black gel, white membrane". Install the transfer clamp and place the transfer clamps into the transfer tank according to the principle of "black to black, red to red". Pour in 1× transfer buffer, place on ice, turn on the power, and transfer at a constant current of 300 mA for 90 min.

[0104] (3) After the transfer is complete, turn off the power, remove the PVDF membrane with tweezers, with the protein side down, add 5% blocking solution and place it on a shaker at room temperature for 1 h.

[0105] (4) After the blocking was completed, the PVDF membrane was washed three times with 1×TBST solution for 5 min each time. The target primary antibody was added and incubated overnight on a shaker at 4°C. The next day, the primary antibody was recovered, the PVDF membrane was washed three times with 1×TBST solution for 5 min each time, the secondary antibody was added, and the membrane was incubated at room temperature on a shaker for 2 h.

[0106] (5) After antibody incubation, the secondary antibody was recovered, and the PVDF membrane was washed three times with 1×TBST solution for 5 min each time. The membrane was then developed using ECL ultrasensitive chromogenic solution.

[0107] The co-immunoprecipitation (Co-IP) assay was performed using the Pierce Direct Magnetic IP / Co-IP kit (ThermoScientific) and was performed strictly in accordance with the manufacturer’s instructions.

[0108] Experimental results showed that treatment with the AHA1 inhibitor (I-6) of this invention reduced the phosphorylation levels of kinases such as AKT and ERK in tumor cells. Figure 6 A). Immunoprecipitation showed that (I-6) disrupted the interaction between HSP90 and AHA1 intracellularly. Figure 6 B).

[0109] Example 15 Effect of the AHA1 inhibitor (I) of the present invention on MM cell proliferation

[0110] Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT (5 mg / mL; Sigma-Aldrich, USA) to water-insoluble blue-purple formazan crystals, which are then deposited in the cells. Dead cells lack this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells. The absorbance value is measured at 570 nm using a microplate reader. Within a certain cell number range, the amount of MTT crystals formed is directly proportional to the cell number. The number of live cells is determined based on the measured absorbance value (OD); the higher the OD, the higher the cell viability. MM cell lines ARP1 and H929 were cultured in RPMI-1640.

[0111] Specific steps: 1) Wild-type (WT) ARP1 and H929, and overexpressing (OE) ARP1 and H929 AHA1, in the logarithmic growth phase, were seeded into sterile 96-well plates, with three replicates per group. Six concentration gradients of the drug were added, and the plates were incubated at 37°C and 5% CO2 for 48 h; 2) Freshly prepared MTT reagent was added to each well, and the plates were incubated at 37°C and 5% CO2 for 4 h; 3) The plates were centrifuged, and 150 μL of DMSO was added to each well; 4) The OD of each well was measured at 570 nm using a microplate reader. The growth inhibition rate of the drug on myeloma cells was calculated using the following formula: Inhibition rate = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%, and the IC50 was calculated. 50 .

[0112] Operating steps: (1) Take the constructed ARP1 and H929 wild-type and ARP1 and H929 AHA1 overexpressing cell lines in the logarithmic growth phase, adjust the cell suspension density, and seed them into sterile 96-well plates with 6000-8000 cells per well and 3 replicates per group. Add 6 concentration gradients of drugs, with a total volume of 180 μL per well. Then incubate in a 37 ℃, 5% CO2 incubator for 48 h. (2) Add 20 μL of freshly prepared MTT reagent to each well and incubate at 37 °C in a 5% CO2 incubator for 4 h; (3) Centrifuge at 4000 rpm for 12 min, aspirate the culture medium from the well plate, and add 150 μL of DMSO to each well; (4) After shaking for 5 s, the OD value of each well at a wavelength of 570 nm was measured using an ELISA reader. The growth inhibition rate of the drug on myeloma cells was calculated according to the following formula, and the half-maximal concentration (IC50) was calculated using Graphpad. 50 The specific results are shown in Table 2.

[0113] Table 2. IC50 of compounds on MM cells 50 value

[0114] a " / " indicates that the value was not measured.

[0115] Example 16 Pharmacokinetic Study of the AHA1 Inhibitor (I) of the Present Invention

[0116] Pharmacokinetic results of compounds I-5 and I-6 in mice: Compounds I-5 and I-6 were administered to mice by gavage at a dose of 10 mg / kg, and the plasma concentrations of the compounds were detected at 0.08, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 10 h.

[0117] Mice were intravenously injected with compounds I-5 and I-6, and the plasma concentrations of the compounds were measured at 0.03, 0.08, 0.25, 0.5, 0.75, 1, 2, 4, 6, 10, and 24 h.

[0118] Pharmacokinetic analysis revealed that the preferred target compound I-6 exhibited low bioavailability (1.9%) during oral administration, while plasma stability analysis showed that compound I-5 was rapidly converted to compound I-6 in vivo. Figure 11 The experiment found that administering compound I-5 as a prodrug via gavage significantly improved the bioavailability of I-6 (to 37.9%), thus compound I-5 has good prospects for anti-tumor development.

[0119] Table 3 Pharmacokinetic parameters of compounds I-5 and I-6

[0120] Example 17 Determination of the ester-water distribution coefficient LogP of the AHA1 inhibitor (I) of the present invention

[0121] The ester-water distribution coefficient (LogP) of I-1 in patent 202210224472.X and I-5 and I-6 in this patent was determined using the "shake flask method". 2 mg of standard was weighed and dissolved in n-octanol to prepare six standard solutions of different concentrations. HPLC was performed with an injection volume of 10 μL, and the peak areas A1-A6 were measured. A standard curve of peak area versus concentration was plotted. A sample of concentration C1 (dissolved in n-octanol) was mixed with an equal volume of water, suspended, and shaken overnight. The aqueous and n-octanol phases were separated. 10 μL of the n-octanol phase was injected into the HPLC system, and the peak area was substituted into the standard curve to determine the concentration Co of the n-octanol phase. The concentration of the aqueous phase was Cw. LogP = LogCo / Cw.

[0122] The results show that, after structural optimization, the water solubility of the compounds in this patent is enhanced. The LogP of I-1 in patent 202210224472.X is increased from 1.4 to 1.2 for I-5 and 0.7 for I-6 in this patent. The introduction of heterocycles such as pyridine enhances the absorption capacity of the compounds. Furthermore, consistent with the description in Example 16, administering compound I-5 as a prodrug via gavage can significantly improve the bioavailability of I-6.

[0123] The experimental results show that the compounds (I-1) to (I-6) synthesized in this invention have good anti-proliferative activity against MM cells, and they interfere with the HSP90-AHA1 interaction by binding to the C-terminus of AHA1. Furthermore, compound I-5 can be used as a prodrug of compound I-6 for oral administration, exhibiting good oral bioavailability, making it suitable for further transformation and development.

[0124] The compounds synthesized in this invention can target AHA1 to inhibit the proliferation of MM cells, showing promising potential for the treatment of multiple myeloma and other diseases. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An AHA1 inhibitor with antitumor activity, characterized in that, The inhibitor is a compound of formula (I) or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof: ， R1 is selected from -H, hydroxyl, sulfonamide, amide, ester, alkoxy, and carboxyl groups; R2 is selected from -H, hydroxyl, sulfonamide, halogen, alkyl, alkoxy, amino, substituted amino, and five- or six-membered aromatic rings containing 0 to 2 nitrogen, sulfur or oxygen atoms; R3 is selected from -H, alkoxy, hydroxy, sulfonamide, halogen, alkyl, amino, substituted amino, and carboxyl groups; X is CH or N.

2. The AHA1 inhibitor with antitumor activity according to claim 1, characterized in that, The compounds mentioned are selected from:

3. The method for preparing the AHA1 inhibitor according to claim 1 or 2, characterized in that, Includes the following steps: ， a. Using substituted benzaldehyde (2-1) as the starting material, a nitration reaction is carried out at low temperature in fuming nitric acid to generate a nitro-substituted benzoyl derivative (2-2), which is then set aside. b. Take the nitro-substituted benzoyl derivative (2-2) obtained in step a and reduce it with iron powder in ethanol solution to obtain the amino-substituted benzoyl derivative (2-3), for later use; c. Using the amino-substituted benzoyl derivative (2-3) obtained in step b as a raw material, react it with ethyl nitroacetic acid in water and glacial acetic acid to form a cyclization intermediate (2-4), which is then set aside. d. Take the intermediate (2-4) obtained in step c and reduce it with iron powder in an ethanol solution to obtain intermediate (2-5), which is then set aside. e. React iodoaromatic acids with substituted aromatic boric acids under the catalysis of palladium to obtain intermediate (2-6), for later use; f. Take the intermediate (2-6) obtained in step e and prepare an acyl chloride using sulfoxide as a solvent, and then react it with the intermediate (2-5) to obtain the target compound (I). g. For compounds where R1 is an ester group, the compound (I) obtained in step f is hydrolyzed in a solution of potassium hydroxide in water and methanol to obtain the target compound (IA).

4. The method for preparing the AHA1 inhibitor according to claim 3, characterized in that, The concentrated nitric acid used in step a is fuming nitric acid. The molar volume ratio of substituted benzaldehyde to fuming nitric acid is 1 mol / 500 mL to 1 mol / 900 mL. The reaction temperature is -15 ℃ to -5 ℃, and the reaction time is 1 to 2 h.

5. The method for preparing the AHA1 inhibitor according to claim 3 or 4, characterized in that, In step b, the molar ratio of the nitro-substituted benzoyl derivative (2-2) to iron powder is 1:4 to 6; the molar ratio of the nitro-substituted benzoyl derivative (2-2) to glacial acetic acid is 1:8 to 12; the molar ratio of the nitro-substituted benzoyl derivative (2-2) to ethanol is 1 mmol / 10 mL to 1 mmol / 20 mL; the reaction temperature is reflux; and the reaction time is 2 to 8 h. In step c, the molar ratio of the amino-substituted benzoyl derivative (2-3) to ethyl nitroacetate is 1:2-3, the volume ratio of glacial acetic acid to water is 1:1, the molar ratio of amino-substituted benzaldehyde to solvent is 1 mmol / 3 mL to 1 mmol / 10 mL, the reaction temperature is 50 ℃ to 80 ℃, and the reaction time is 10-20 h.

6. The method for preparing the AHA1 inhibitor according to claim 5, characterized in that, In step d, the molar ratio of intermediate (2-4) to iron powder is 1:4 to 6, the molar ratio of intermediate (2-4) to glacial acetic acid is 1:8 to 12, the molar volume ratio of intermediate (2-4) to ethanol is 1 mmol / 10 mL to 1 mmol / 20 mL, the reaction temperature is reflux, and the reaction time is 2 to 8 h. In step e, the molar ratio of iodoaromatic acid to substituted aromatic boric acid is 1:1.5-3; the catalyst is Pd(dppf)Cl2, the molar ratio of iodoaromatic acid to Pd(dppf)Cl2 is 1:0.03-0.08, the base used in the reaction is potassium carbonate solution, nitrogen protection is applied, the reaction temperature is 40 ℃-100 ℃, and the reaction time is 12-24 h.

7. The method for preparing the AHA1 inhibitor according to claim 6, characterized in that, In step f, the molar ratio of intermediate (2-6) to thionyl chloride is 1:5~15, the molar ratio of intermediate (2-5) to intermediate (2-6) is 1:1.1~3, DCM is used as the solvent for the reaction, and the molar volume ratio of (2-5), (2-6) to DCM is 1 mmol / 10 mL~1 mmol / 50 mL. The reaction temperature is room temperature, and the reaction time is 2~8 h.

8. The method for preparing the AHA1 inhibitor according to claim 7, characterized in that, In step g, compound (I) was hydrolyzed in a solution of potassium hydroxide in water and methanol to obtain a hydroxy derivative (IA). The molar ratio of compound (I) to potassium hydroxide was 1:10~20. The reaction solvent was methanol:water in a volume ratio of 1:10~50. The molar volume ratio of reactant to total solvent was 1 mmol / 20 mL to 1 mmol / 50 mL. The reaction temperature was 30 ℃~50 ℃. The reaction time was 8~16 h.

9. The use of the AHA1 inhibitor according to claim 1 or 2 in the preparation of drugs for preventing and treating HSP90-AHA1-mediated tumors.

10. The application according to claim 9, characterized in that, The tumors include at least one of the following: multiple myeloma, leukemia, chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, hairy cell leukemia, lymphoblastic T-cell leukemia, lymphoma, B-cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma, chondrosarcoma, fibrosarcoma, Ewing sarcoma, rhabdomyosarcoma, small cell lung cancer, non-small cell lung cancer, glioma, prostate cancer, breast cancer, bone cancer, neuroblastoma, gastric cancer, ovarian cancer, intestinal cancer, kidney cancer, medulloblastoma, pancreatic cancer, thyroid cancer, mesothelioma, cervical cancer, bladder cancer, liver cancer, skin cancer, and tumors of the central or peripheral nervous system.

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