A dual-target inhibitor of histone deacetylase and heat shock protein 110 kda and application thereof

Abstract

The application discloses a histone deacetylase and heat shock protein 110kDa double-target inhibitor and application. The application provides a kind of structural novel histone deacetylase and heat shock protein 110kDa double-target inhibitor shown in structure of formula (I), pharmaceutical composition and hydrate containing compound of formula (I), and isotopic derivatives, chiral isomer, allosteric, different salt, prodrug and preparation of the compounds and the like. The application also provides a kind of structural novel histone deacetylase and heat shock protein 110kDa double-target inhibitor of preparation method, purposes, and the activity of these compounds to colon cancer tumor cell proliferation inhibition. A kind of structural novel histone deacetylase and heat shock protein 110kDa double-target inhibitor in the application is expected to become an antitumor candidate drug.

Description

Technical Field

[0001] This invention belongs to the field of histone deacetylase inhibitor synthesis technology, specifically, it relates to a dual-target inhibitor of histone deacetylase and heat shock protein 110kDa and its application. Background Technology

[0002] Cancer is a disease caused by multiple factors, requiring multifaceted treatment interventions. Chemotherapy remains the primary treatment option for cancer. However, most chemotherapy drugs act on only one target, which can lead to drug resistance after a period of treatment and ultimately treatment failure. Given the increasing success of treatments that inhibit two specific targets in clinical trials, the idea of ​​designing a drug to regulate two or more related targets to achieve a synergistic therapeutic effect in tumor treatment is feasible.

[0003] In cancer cells, overexpression of histone deacetylases (HDACs) leads to enhanced acetylation, causing histones to become positively charged. This increases the attraction between DNA and histones, making loose nucleosomes more compact and affecting the expression of specific genes, such as tumor suppressor genes. Applying HDAC inhibitors can regulate apoptosis and differentiation by increasing histone acetylation in specific chromatin regions. Based on structural characteristics, reported HDAC inhibitors can be broadly classified into four categories: hydroxamic acids, benzamides, cyclic peptides, and short-chain fatty acids. Hydroxamic acids include trichostatin and vorinostat (SAHA) and its derivatives. This class of HDAC inhibitors has attracted considerable attention, as it has been shown to induce tumor differentiation at low doses and concentrations, selectively inhibit tumor growth, and have no toxic side effects on normal cells, making it more suitable for clinical use. Approved HDAC inhibitors are primarily indicated for hematologic malignancies, such as lymphoma and myeloma. The reasons for the failure of HDAC inhibitors in treating solid tumors are currently not agreed upon. Recent studies have shown that inhibition of HDACs leads to compensatory activation of STAT3, a well-known cancer-related drug target in breast cancer, which may limit the antiproliferative effects of HDAC inhibitors in solid tumors. Zeng et al. demonstrated that HDAC inhibitors indirectly activate leukemia suppressor receptor (LIFR) by promoting BRD4 activation. Subsequently, LIFR activates the downstream JAK-STAT3 pathway, ultimately inhibiting the antitumor effects of HDAC inhibitors in breast cancer cells. Inhibition of BRD4 or JAK using inhibitors or siRNA can enhance the sensitivity of breast cancer, especially triple-negative breast cancer, to HDAC inhibitors. Yao et al. designed a JAK-HDAC dual-target inhibitor that showed broad-spectrum antiproliferative activity in cells, including hematology cell lines and breast cancer. Pan et al. synthesized several BRD4-HDAC inhibitors that inhibit colorectal cancer in vitro and in vivo. These studies indicate the existence of a signaling cascade, namely the HDAC-BRD4-LIFR-JAK-STAT3 signaling cascade, which begins with HDAC inhibition and ends with STAT3 activation. This cascade reaction may explain why HDAC inhibitors are ineffective against solid tumors. Inhibiting HDAC activation of STAT3 weakens the inherent anti-cancer ability of HDAC inhibitors. Inhibition of this cascade factor can sensitize breast and colorectal cancers to HDAC inhibitors.

[0004] The heat shock protein of 110 kDa (HSP110) is a member of the heat shock protein family and exhibits strong molecular chaperone activity, preventing protein aggregation (“holdase” activity). HSP110 can influence the growth, metastasis, and escape of cancer cells through three aspects: angiogenesis, epithelial-to-mesenchymal transition, and immune escape. Therefore, HSP110 is considered an important target for anti-tumor drug development. Studies have found that HSP110 is abnormally abundant in colorectal cancer, while it is not expressed or is weakly expressed in normal tissues and pre-cancerous cells. HSP110 is a molecular chaperone of the signal transducer and activator of transcription (STAT3), and it can promote the proliferation of colorectal cancer cells by mediating STAT3 phosphorylation, nuclear translocation, and increasing the activity of STAT3 transcription factors. Therefore, by designing inhibitors targeting HSP110, the phosphorylation and other activities of STAT3 can be inhibited, thereby suppressing the proliferation of colorectal cancer cells.

[0005] In 2020, Professor Garrido first reported the inhibitory effect of small molecule inhibitors on HSP110. Through studies in crystal structure, computational chemistry, and in vitro screening, Professor Garrido resolved the co-crystal structure of compound A (compound A is designated as compound 33 in the literature (Gozzi GJ, et al. Selecting the first chemical molecule inhibitor of HSP110 for colorectal cancer therapy[J]. Cell Death Differ. 2020, 27(1):117-129.), whose synthesis method is shown in WO2015 / 132727 A1 and the literature (Gloaguen C, et al. First evidence that oligopyridines, <-helix foldamers, inhibit Mcl-1 and sensitize ovarian carcinoma cells to Bcl-xL-targeting strategies[J]. J Med Chem, 2015, 58(4):1644-1668.), with the chemical structure shown in Formula II.) with HSP110, and studied the in vitro bioactivity and in vivo antitumor efficacy of compound A. This compound exhibited good inhibitory efficacy against tumor growth in a mouse model grafted with colon cancer. Compound A blocked the anti-aggregation activity of HSP110 (IC50). 50=58.3±1.7μM, IC50 value for disrupting the HSP110 / STAT3 complex 50 =35.9±1.1μM.

[0006] The hydroxamic acid fragment is a well-defined pharmacophore for HDACs inhibitors. This invention aims to indirectly inhibit STAT3 phosphorylation and other activities by designing an inhibitor targeting HDACs-HSP110, thereby disrupting the HSP110 / STAT3 interaction. This would increase the application of HDACs inhibitors in the treatment of solid tumors. Based on fragment drug design and bioisosteric principles, this invention involves a series of designs and modifications to the structure of compound A, introducing the hydroxamic acid pharmacophore, with the potential to obtain a novel class of anti-solid tumor compounds. Results: Through HDAC enzyme inhibition rate tests and activity tests on three types of colon cancer cells (COLO205, SW480, and HCT116), compounds with inhibitory effects on HDAC1 and HDAC6 enzymes and in vitro anti-colon cancer activity comparable to SAHA, but significantly superior to compound A, were screened.

[0007] Summary of the Invention

[0008] The first objective of this invention is to provide a novel dual-target inhibitor of histone deacetylase and heat shock protein 110kDa.

[0009] A dual-target inhibitor of histone deacetylase and heat shock protein 110kDa, or its optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate, having the structure shown in Formula (I):

[0010]

[0011] In formula (I) Choose the following structural fragment independently:

[0012]

[0013] in" "express With L 1 The site of connection;

[0014] Each is independently selected from one of a 4- to 10-membered heterocyclic group, a 6- to 10-membered aryl group, or a 5- to 10-membered heteroaryl group; wherein the 4- to 10-membered heterocyclic group selectively contains 1 to 3 heteroatoms selected from N, O, and S; and the 5- to 10-membered heteroaryl group selectively contains 1 to 3 heteroatoms selected from N, O, and S.

[0015] R 0 Selected from -X, C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, -COR z One of 2-phenylvinyl;

[0016] Each R 1 and each R 2 Each is independently selected from H and C 1-6 Alkyl, C 3-6 cycloalkyl, C 3-6 Heterocyclic group, C 1-6 One of alkoxy, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl; wherein C 3-6 The heterocyclic group contains 1-3 heteroatoms selected from N, O, and S; the 5- to 10-membered heteroaryl group contains 1-3 heteroatoms selected from N, O, and S; the C 3-6 Heterocyclic groups, 6- to 10-membered aryl groups, and 5- to 10-membered heteroaryl groups are selectively bound by m independent R groups. 3 Group substitution, m is an integer from 0 to 3, R 3 Selected from -X, C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, -COR z One of the following, X = H, F, Cl, Br, I, -OCH3, -OH, -CN, -CF3;

[0017] R z For H, -OH, -NH2, C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 One of the alkynyl groups;

[0018] p is an integer between 0 and 3; q is an integer between 0 and 3;

[0019] L 1 Selected from One of them;

[0020] in" " indicates L 1 and The site of connection; " indicates L 1 With L 2 The site of connection;

[0021] L 2 Selected from One of them; n is an integer between 0 and 6;

[0022] in" " indicates L 2 With L 3 The site of connection; " indicates L 2 With L 1 The site of connection;

[0023] L 3 Selected from One of them, where R 4 It is one of -CN, -OCH3, -H, -F, and -Cl.

[0024] The prerequisite is that L 1 L 2 L 3 and Together they can form a stable chemical structure.

[0025] Furthermore, R 0 Selected from -CN, -CHO, or -CONH2;

[0026] Selected from One of them;

[0027] Selected from One of them;

[0028] Furthermore, the aforementioned dual-target inhibitor of histone deacetylase and heat shock protein 110kDa is any one of compounds 1 to 42 with the following structures:

[0029]

[0030]

[0031]

[0032] A second objective of this invention is to provide a method for preparing the aforementioned dual-target inhibitor of histone deacetylase and heat shock protein 110kDa.

[0033] when Independently selected from 5- to 10-membered heterocyclic groups that selectively contain 1-3 N atoms (preferably) When ), the derivative shown in formula (I) is prepared via routes one, two, and three. When When the radical is other than the radical, the derivative shown in formula (I) is prepared via route four.

[0034] Route 1 specifically includes the following steps:

[0035] (1) Compound a-1 and compound a-2 were reacted by the Buchwald reaction to prepare compound a-3;

[0036] (2) Compound a-3 was prepared by removing the tert-butyloxycarbonyl group from dioxane hydrochloride (HCl-dioxane) or trifluoroacetic acid (TFA) to obtain compound a-4;

[0037] (3) Compound a-4 and compound a-5 were reacted via a nucleophilic substitution reaction to prepare compound a-6;

[0038] (4) Compound a-6 and compound a-7 were coupled via a suzuki reaction to prepare compound a-8;

[0039] (5) Place the DCM solution of compound a-8 in a liquid nitrogen bath (-78℃), and under nitrogen protection, slowly drip diisobutylaluminum hydride (DIBAL-H, 6equiv) into the solution with a syringe and react until room temperature.

[0040] Post-treatment: Add 1M HCl dropwise under ice bath to neutralize excess DIBAL-H, then add 2M NaOH dropwise, separate the layers, extract three times with dichloromethane (DCM) and water, combine the organic phases, wash once with saturated brine, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and dry under vacuum to obtain compound a-9, which is directly added to the next step without purification.

[0041] (6) Dissolve compounds a-10 and a-11 in N,N-dimethylformamide (DMF), and add N,N-diisopropylethylamine (DIPEA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) in sequence under ice bath, and stir overnight.

[0042] Post-processing: The reaction solution was extracted three times with DCM and water, the organic phases were combined, and then washed once with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound a-12.

[0043] (7) Compound a-12 was dissolved in methanol solution, 2M NaOH solution was added, and the mixture was stirred at room temperature. Post-treatment: the reaction was checked by TLC to confirm that the reaction was complete. The reaction solution was dried by rotary evaporation, and the pH was adjusted to about 6 by adding 1M HCl. The mixture was extracted three times with ethyl acetate / water, the organic phases were combined, and the mixture was washed once with saturated brine. The organic phase was dried with anhydrous sodium sulfate and concentrated under reduced pressure to prepare compound a-13.

[0044] (8) Compounds a-9 and a-13 were dissolved in DMF, and DIPEA, EDCI and HOBt were added sequentially under ice bath and stirred overnight. Post-treatment: The reaction solution was extracted three times with DCM and water, the organic phases were combined, and then washed once with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound a-14.

[0045] (9) Dissolve compound a-14 in MeOH solution, add p-toluenesulfonic acid monohydrate (pTsOHH2O) with stirring, react at room temperature for about 1 hour, and monitor by TLC.

[0046] Post-processing: Add saturated sodium bicarbonate solution to the reaction solution to adjust the pH to around 7. Then, remove DCM and MeOH under reduced pressure. Extract three times with organic solvent and water, combine the organic phases, wash once with saturated brine, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, purify by preparative plate, scrape off the target product, rinse with MeOH:DCM = 1:9, concentrate the filtrate under reduced pressure, and dry to obtain compound (I).

[0047] or,

[0048] (1) Preparation of compound a-9: Steps (1)-(5) above;

[0049] (2) Compounds a-10 and a-11 were amide condensed to give compound a-12; compound a-12 was hydrolyzed to give compound a-13;

[0050] (3) Compound a-9 and compound a-13 were amide condensed to obtain compound a-15;

[0051] (4) Dissolve compound a-15 in MeOH and DCM (MeOH:DCM = 1:1), add MeOH solution of NaOH (10 equiv) under stirring in an ice bath, and then add aqueous solution of hydroxylamine (30 equiv). Maintain the reaction at 0°C and stir for 1 h. Monitor the reaction by spotting on a TLC plate.

[0052] Post-treatment: Add 1M HCl to the reaction solution to adjust the pH to about 7, then remove DCM and MeOH under reduced pressure, extract three times with organic solvent and water, combine the organic phases, wash once with saturated brine, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, purify by preparative plate, scrape off the target product, rinse with MeOH:DCM = 1:9, concentrate the filtrate under reduced pressure, and dry to obtain compound (I).

[0053]

[0054] Route 2 specifically includes the following steps:

[0055] (1) The preparation of compounds c-1 to c-6 is the same as that of compounds a-1 to a-6 in the same route.

[0056] (2) Compounds c-6 and c-7 are coupled by Suzuki reaction or Buchwald reaction to obtain compound c-8. Compound c-8 is de-tert-butyloxycarbonyl by trifluoroacetic acid (TFA) or dioxane hydrochloride (HCl dioxane) to obtain compound c-9.

[0057] (3) Compound c-9 and compound c-10 or c-11 are amide condensed to obtain compound c-12 or c-13;

[0058] (4) The method for preparing target compound I from compound c-12 or c-13 is the same as the method for preparing target compound I from compounds a-14 and a-15 in route one.

[0059]

[0060] Route 3 specifically includes the following steps:

[0061] (1) Compound c-9 and compound c-14 were subjected to a nucleophilic substitution reaction to prepare compound c-15; compound c-15 was subjected to urethane exchange to prepare compound Ic;

[0062] Alternatively, (2) compound c-9 and compound c-16 are subjected to nucleophilic substitution reaction to obtain compound c-17; compound c-17 is subjected to ester hydrolysis to obtain compound c-18; compound c-18 and compound c-19 are subjected to amide condensation to obtain compound c-20; compound c-20 is subjected to urethane exchange to obtain compound I.

[0063]

[0064] Route 4 specifically includes the following steps:

[0065] (1) Compound b-1 and compound b-2 were reacted via a nucleophilic substitution reaction to prepare compound b-3;

[0066] (2) Compound b-3 and compound b-4 were combined via a Suzuki coupling reaction to prepare compound b-5;

[0067] (3) Compound b-5 was de-tert-butyloxycarbonyl group to obtain compound b-6;

[0068] (4) Compound b-6 and compound b-7 are synthesized by nucleophilic substitution or amide condensation to obtain compound b-8 or compound I;

[0069] (5) Compound I was prepared by urethane exchange or by removing the hydroxyl protecting group (THP) from compound b-8.

[0070]

[0071] The compounds of formula (I) of this invention can be prepared by the method described above; however, the conditions of this method, such as reactants, solvents, amounts of compounds used, reaction temperature, and reaction time, are not limited to those explained above. The compounds of this 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.

[0072] A third objective of this invention is to provide the use of the aforementioned novel histone deacetylase and dual-target inhibitors targeting 110 kDa heat shock protein, or their optical isomers, racemates, single enantiomers, possible diastereomers, or pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, or solvates in the preparation of antitumor drugs.

[0073] Preferably, the tumor includes colon cancer.

[0074] A fourth objective of this invention is to provide an antitumor drug containing a safe and effective amount of the novel histone deacetylase and a dual-target inhibitor targeting a 110 kDa heat shock protein, or an optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof.

[0075] Preferably, the antitumor drug may also include a pharmaceutically acceptable carrier.

[0076] Pharmaceutical Compositions and Administration

[0077] Because the compounds of the present invention have the activity of inhibiting the proliferation of various tumor cell lines, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate various diseases, including various cancers.

[0078] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 5-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0079] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0080] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and local administration.

[0081] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0082] Solid dosage forms, such as tablets, sugar pills, capsules, pellets, and granules, can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such compositions may be delayed at a specific site within the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0083] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0084] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0085] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances.

[0086] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0087] Dosage forms of the compounds of this invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.

[0088] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0089] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–5000 mg, preferably 5–2000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician. Attached Figure Description

[0090] Figure 1 Compound A affinity (K) for HSP110 protein D (characterization);

[0091] Figure 2 Compound 12 has a high affinity (K) for HSP110 protein. D (Characteristics). Detailed Implementation

[0092] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0093] Example 1: Preparation of Compound 1

[0094]

[0095] Intermediate 1c:

[0096] Compounds 1a (2 g, 9.22 mmol), 1b (2.15 g, 11.53 mmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) (574 mg, 0.92 mmol), and sodium tert-butoxide (2.21 g, 23.05 mmol) were placed in a 75 mL sealed tube, and xylene (20 mL) was added. Air was purged from the reaction mixture with a nitrogen stream (approximately 15 min), followed by the addition of tris(dibenzylacetone)dipalladium (Pd2(dba)3, 422 mg, 0.46 mmol). Nitrogen was then purged again for 10 min. The reaction mixture was heated to 100 °C overnight in a sealed system. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with dichloromethane (DCM, 5 mL). The filtrate was concentrated under reduced pressure, and water (H2O, 10 mL) was added to the concentrate, followed by extraction with ethyl acetate (EtOAc, 5 mL × 3). The combined organic phases were washed successively with H2O (10 mL × 2) and saturated brine (aq. NaCl, 10 mL × 1), and dried in anhydrous sodium sulfate (Na2SO4). The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography [eluting with EtOAc / petroleum ether system] to give a brownish-yellow solid compound 1c (2.41 g, yield 81%).

[0097] Intermediate 1d:

[0098] Compound 1c (2.41 g, 7.49 mmol) was transferred to a 100 mL round-bottom flask, dissolved in DCM (8 mL), and TFA (8 mL) was added dropwise with stirring in an ice bath. After the addition was complete, the ice bath was removed and the mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to obtain a crude dark brown solid compound 1d (2.58 g), which was used directly in the next reaction.

[0099] Intermediate 1f:

[0100] Compounds 1d (600 mg, 1.88 mmol), 1e (409 mg, 1.88 mmol), and cesium carbonate (Cs₂CO₃, 1.53 g, 4.70 mmol) were placed in a 100 mL round-bottom flask, and N,N-dimethylformamide (DMF, 10 mL) was added. The reaction mixture was heated to 90 °C overnight in a sealed system. The reaction mixture was cooled to room temperature, quenched with H₂O (5 mL), and extracted with EtOAc (5 mL × 3). The combined organic phases were washed successively with H₂O (5 mL × 2) and aq. NaCl (5 mL), and then dried with anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with EtOAc / Petroleumether system) to give a yellowish-white solid compound 1f (322 mg, 80% yield).

[0101] Intermediate 1h:

[0102] Compound 1f (300 mg, 0.74 mmol), compound 1g (197 mg, 0.93 mmol), and sodium carbonate (Na₂CO₃, 392 mg, 1.85 mmol) were placed in a 15 mL sealed tube, and 1,2-dimethoxyethane [(CH₂OMe)₂, 5 mL) and H₂O (0.3 mL) were added. Air was purged from the reaction mixture with a nitrogen stream (approximately 15 min), followed by the addition of Pd(PPh₃)₄ (44 mg, 0.038 mmol). Nitrogen was then purged again for approximately 10 min. The reaction mixture was heated to 85 °C overnight in a sealed system. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with DCM. The filtrate was concentrated under reduced pressure. H₂O (10 mL) was added to the concentrate, and the mixture was extracted with EtOAc (5 mL × 3). The combined organic phases were washed with aq. NaCl (10 mL) and then dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with EtOAc / Petroleumether system) to give a yellowish-white solid compound 1h (417 mg, yield 85%).

[0103] Intermediate 1i:

[0104] A solution of compound 1h (200 mg, 0.48 mmol) in anhydrous DCM was placed in a liquid nitrogen bath at -78 °C under nitrogen protection. Diisobutylaluminum hydride (DIBAL-H, 1 M in hexane, 2.5 mL) was added dropwise, and the reaction was allowed to proceed to room temperature. The reaction was confirmed to be complete by TLC. Post-treatment: 1 M HCl was slowly added dropwise at low temperature, and the mixture was then brought to room temperature. 2 M sodium hydroxide was added to adjust the pH to approximately 7. The mixture was extracted with DCM (15 mL x 3). The combined organic phases were washed successively with H₂O (6 mL x 2) and aq. NaCl (6 mL), and then dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give a yellowish-brown solid compound 1i (190 mg). The crude product was used directly in the next reaction without further purification.

[0105] Intermediate 1l:

[0106] Compound 1k (736 mg, 6.91 mmol) was added to an anhydrous DCM solution of compound 1j (1 g, 5.31 mmol) under ice bath conditions. After stirring and clarifying, N,N-diisopropylethylamine (DIPEA, 2.07 g, 16 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 2.68 g, 14 mmol), and 1-hydroxybenzotriazole (HOBt, 934 mg, 6.91 mmol) were added sequentially, and the mixture was stirred overnight. The reaction was quenched with ice-cold H2O (40 mL) and extracted with DCM (15 mL × 3). The combined organic phases were washed sequentially with H2O (10 mL × 2) and aq. NaCl (10 mL × 2), and then dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (elution with EtOAc / Petroleum ether / triethylamine (Et3N, 0.1%) system) to give 1l (1.06g, yield 70%) of colorless and transparent liquid compound.

[0107] Intermediate 1m:

[0108] Under ice bath conditions, 2M sodium hydroxide solution was added to a methanol (MeOH, 10mL) solution of compound 1l (2g, 6.97mmol). After stirring for about 10min, the ice bath was removed, and the mixture was stirred at room temperature for about 4 hours. The reaction solution was concentrated under reduced pressure, and a white solid precipitated. This solid was first dissolved with 1M HCl, and then the pH was adjusted to about 7 with 6M HCl, resulting in a white solid precipitating on the flask wall. The solid was extracted with EtOAc (5mL × 3), and the combined organic phases were washed successively with H2O (6mL × 2) and aq. NaCl (6mL × 2), and then dried with anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give a bright yellow liquid compound 1m (1.34g, yield 70%).

[0109] Intermediate 1n:

[0110] Compound 1m (74 mg, 0.27 mmol) was added to an anhydrous DMF solution of compound 1i (110 mg, 0.22 mmol) under ice bath conditions. After stirring and clarifying, DIPEA (43 mg, 0.33 mmol), EDCI (105 mg, 0.55 mmol), and HOBt (39 mg, 0.29 mmol) were added sequentially, and the mixture was stirred overnight. The post-treatment procedure was the same as for compound 1l, yielding a pale yellow solid 1n (138 mg, 83% yield).

[0111] Compound 1:

[0112] Under ice bath stirring conditions, p-toluenesulfonic acid monohydrate (p-TsOH·H2O, 140 mg, 0.75 mmol) was added to a MeOH solution of compound 1n (138 mg, 0.18 mmol), and the mixture was stirred for approximately 2 hours. The pH was adjusted to approximately 6 by adding saturated sodium bicarbonate solution (aq. NaHCO3, 2 mL), and the solution was concentrated under reduced pressure. Extraction was performed with DCM (6 mL × 3) and H2O (5 mL × 2), followed by washing with aq. NaCl (5 mL). The combined organic phases were dried over anhydrous Na2SO4, and the desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was purified by thin-layer chromatography (developing solvent: DCM:MeOH = 9:1). The target band was washed with 10% MeOH solution, and then concentrated under reduced pressure to obtain a pale yellow solid, compound 1 (50 mg, yield 41%). 1H NMR (500MHz, DMSO-d6) δ10.32(s,1H),8.66(s,1H),8.54(d,J=2.4Hz,1H),8.36(t,J=5.7Hz,1H),7.8 1(d,J=2.4Hz,1H),6.86(d,J=16.2Hz,3H),6.69(d,J=2.7Hz,1H),6.48(dd,J=8.7,2.7Hz,1H),4.38(d ,J=5.6Hz,2H),3.86(s,6H),3.77(s,3H),3.70(d,J=3.8Hz,6H),3.24(q,J=5.7Hz,8H),2.18(t,J=7.4 Hz,2H),1.91(q,J=8.7,7.3Hz,2H),1.57–1.52(m,2H),1.45–1.40(m,2H),1.24(q,J=7.9Hz,4H).LCMS m / z[M+H] + 666.3.

[0113] Example 2: Preparation of Compound 2

[0114]

[0115] Intermediate 2c:

[0116] Following the synthesis steps of intermediate 1n in Example 1, a pale yellow solid 2c (200 mg, yield 75%) was obtained.

[0117] Compound 2:

[0118] Intermediate 2c (163 mg, 0.25 mmol) was placed in a 25 mL round-bottom flask, and MeOH (1 mL) / DCM (1 mL) was added. The mixture was stirred in an ice bath for 5 min. Then, MeOH (1 mL) of sodium hydroxide and 50% hydroxylamine aqueous solution (aq. NH2OH (50%), 162 mg, 5.40 mmol) were added sequentially, and the mixture was stirred in an ice bath for 1 h. The reaction was checked by TLC to confirm completeness. The pH was adjusted to approximately 6 by adding 1 M HCl (0.6 mL), and the mixture was concentrated under reduced pressure. The mixture was extracted with DCM (6 mL × 3) and aq. NaHCO3 (5 mL × 2), washed with aq. NaCl (5 mL), and the combined organic phases were dried in anhydrous Na2SO4. Anhydrous Na2SO4 was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by thin-layer chromatography (developing solvent: DCM:MeOH = 11:1). The target band was washed with a solution of eluent: DCM:MeOH = 10:1, and then concentrated under reduced pressure to obtain a pale yellow solid 2 (56 mg, yield 35%). 1H NMR (500MHz, DMSO-d6) δ10.34(s,1H),8.66(s,1H),8.55(d,J=2.2Hz,1H),8.35(t,J=5.3Hz, 1H),7.93–7.71(m,1H),6.94–6.77(m,3H),6.69(d,J=2.4Hz,1H),6.48(dd,J=8.7,2.5Hz,1H ),4.39(d,J=5.4Hz,2H),3.87(s,6H),3.77(s,3H),3.70(d,J=5.1Hz,6H),3.31–3.13(m,8H) ,2.19(t,J=7.4Hz,2H),1.91(t,J=7.4Hz,2H),1.58–1.44(m,4H),1.27(d,J=7.9Hz,2H).LCMS m / z[M+H] + 652.3.

[0119] Example 3: Preparation of Compound 3

[0120]

[0121] Intermediate 3c:

[0122] Following the synthesis steps of intermediate 1c in Example 1, a pale yellow solid intermediate 3c (1g, yield 80%) was obtained.

[0123] 3D intermediate:

[0124] Following the synthesis steps of intermediate 1d in Example 1, crude yellow solid intermediate 3d (1g) was obtained.

[0125] Intermediate 3f:

[0126] Following the synthesis steps of intermediate 1f in Example 1, a light yellow solid intermediate 3f (500 mg, yield 82%) was obtained.

[0127] Intermediate 3h:

[0128] Following the synthesis steps of intermediate 1h in Example 1, a light yellow solid intermediate 3h (300 mg, yield 84%) was obtained.

[0129] Intermediate 3i:

[0130] Following the synthesis steps of intermediate 1i in Example 1, crude yellow solid intermediate 3i (200 mg) was obtained.

[0131] Intermediate 3k:

[0132] Following the synthesis steps of intermediate 1n in Example 1, brown oily intermediate 3k (180 mg, yield 75%) was obtained.

[0133] Compound 3:

[0134] Following the synthetic steps of compound 2 in Example 2, compound 3 (50 mg, yield 45%), a pale yellow solid, was obtained. 1 HNMR (500MHz, DMSO-d6) δ10.31(s,1H),8.65(s,1H),8.41(d,J=2.3Hz,1H),8.20(t,J=5.2Hz,1H),7.73(d ,J=2.1Hz,1H),6.84(s,2H),6.79(s,1H),6.27(d,J=2.5Hz,1H),6.08(dd,J=8.7,2.6Hz,1H),4.34(d,J=5. 1Hz,2H),3.84(s,6H),3.74(s,3H),3.72–3.68(m,2H),3.64(s,3H),3.48–3.40(m,4H),3.14(s,2H),3.04( s,2H),2.11(t,J=7.4Hz,2H),1.90(t,J=7.4Hz,2H),1.45(dt,J=31.1,6.7Hz,4H),1.22–1.15(m,4H).LCMS m / z[M+H] + 692.4.

[0135] Example 4: Preparation of Compound 5

[0136]

[0137] Intermediate 5c:

[0138] Following the synthetic steps of intermediate 1c in Example 1, a yellow-green solid compound 5c (3g, yield 80%) was obtained.

[0139] Intermediate 5d:

[0140] Following the synthetic steps of intermediate 1d in Example 1, a yellow-green solid compound 5d (1.74 g, 90% yield) was obtained.

[0141] Intermediate 5f:

[0142] Following the synthesis steps of intermediate 1n in Example 1, a pale yellow solid 5f (100 mg, yield 79%) was obtained.

[0143] Compound 5:

[0144] Following the synthetic steps of Compound 1 in Example 1, a yellow-green solid compound 5 (15 mg, yield 18%) was obtained. 1HNMR(500MHz,DMSO-d6)δ10.34(s,1H),8.66(s,1H),8.27(d,J=3.0Hz,1H),7.80(d,J=3.0H z,1H),6.82(d,J=8.8Hz,1H),6.67(d,J=2.6Hz,1H),6.46(dd,J=8.7,2.6Hz,1H),3.76(s,3 H),3.68(s,3H),3.58(s,4H),3.53–3.47(m,4H),3.23–3.17(m,4H),3.14(s,2H),3.08(s,2 H),2.33(t,J=7.4Hz,2H),1.94(t,J=7.3Hz,2H),1.51–1.45(m,4H),1.30–1.24(m,4H).LCMS m / z[M+H] + 580.3.

[0145] Example 5: Preparation of Compound 6

[0146]

[0147] Intermediate 6c:

[0148] Following the synthetic steps of intermediate 1n in Example 1, a yellow-green solid compound 6c (180 mg, yield 86%) was obtained.

[0149] Compound 6:

[0150] Following the synthetic steps of compound 2 in Example 2, a yellow-green solid compound 6 (107 mg, yield 44%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.34(s,1H),8.67(s,1H),8.27(d,J=2.9Hz,1H),7.80(d,J=2.9Hz ,1H),6.82(d,J=8.8Hz,1H),6.67(d,J=2.5Hz,1H),6.46(dd,J=8.7,2.6Hz,1H),3.76(s,3H), 3.68(s,3H),3.58(s,4H),3.54–3.48(m,4H),3.21–3.16(m,4H),3.11(d,J=28.5Hz,4H),2.3 3(t,J=7.4Hz,2H),1.94(t,J=7.3Hz,2H),1.54–1.46(m,4H),1.27(q,J=7.1,6.1Hz,2H).LCMS m / z[M+H] + 566.3.

[0151] Example 6: Preparation of Compound 7

[0152]

[0153] Intermediate 7c:

[0154] Following the synthetic steps of intermediate 1n in Example 1, a yellow-green solid compound 7c (170 mg, 80% yield) was obtained.

[0155] Compound 6:

[0156] Following the synthetic steps of compound 2 in Example 2, a yellow-green solid compound 7 (32 mg, yield 23%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.35(s,1H),8.67(s,1H),8.27(d,J=3.0Hz,1H),7.80( d,J=3.0Hz,1H),6.82(d,J=8.8Hz,1H),6.67(d,J=2.6Hz,1H),6.46(dd,J=8.7,2. 6Hz,1H),3.75(s,3H),3.68(s,3H),3.61–3.56(m,4H),3.53–3.48(m,4H),3.21–3 .08(m,8H),2.35(t,J=6.9Hz,2H),1.96(t,J=6.8Hz,2H),1.54–1.46(m,4H).LCMS m / z[M+H] + :552.3.

[0157] Example 7: Preparation of Compound 8

[0158]

[0159] Intermediate 8c:

[0160] Compounds 8a (600 mg, 1.49 mmol), 8b (491 mg, 2.24 mmol), and Na₂CO₃ (395 mg, 3.73 mmol) were placed in a 75 mL sealed tube, and DMF (5 mL) and H₂O (1 mL) were added. A nitrogen stream was used to purge air from the reaction mixture (approximately 15 min), followed by the addition of Pd(PPh₃)₄ (87 mg, 0.08 mmol). Nitrogen gas was then introduced for approximately 10 min. The reaction mixture was heated to 120 °C in a microwave reactor in a closed system for 1 h. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with DCM (5 mL). The filtrate was concentrated under reduced pressure. H₂O (10 mL) was added to the residue, and the mixture was extracted with EtOAc (5 mL × 3). The combined organic phases were washed successively with H₂O (10 mL × 2) and aq. NaCl (10 mL), and then dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluting with EtOAc / Petroleum ether system) to give a pale yellow solid compound 8c (500 mg, yield 81%).

[0161] Intermediate 8e:

[0162] Following the synthesis steps of intermediate 1n in Example 1, a pale yellow solid 8e (128 mg, yield 50%) was obtained.

[0163] Compound 8:

[0164] Following the synthetic steps of Compound 1 in Example 1, a yellowish-white solid compound 8 (24 mg, yield 27%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.34(s,1H),9.98(s,1H),8.76(d,J=2.5Hz,1H),8.66(s,1H),8.3 9(d,J=2.5Hz,1H),7.68(q,J=8.9Hz,4H),6.83(d,J=8.8Hz,1H),6.69(d,J=2.6Hz,1H),6.47( dd,J=8.7,2.6Hz,1H),3.85–3.77(m,4H),3.76(s,3H),3.69(s,3H),3.27–3.16(m,4H),2.32( t,J=7.4Hz,2H),1.95(t,J=7.3Hz,2H),1.55(dt,J=46.0,6.9Hz,4H),1.35–1.26(m,4H).LCMS m / z[M+H] + 587.3.

[0165] Example 8: Preparation of Compound 9

[0166]

[0167] Intermediate 9c:

[0168] Following the synthesis steps of intermediate 1n in Example 1, a pale yellow solid 9c (220 mg, yield 80%) was obtained.

[0169] Compound 9:

[0170] Following the synthetic steps of compound 2 in Example 2, compound 9 (43 mg, yield 17%), a yellow solid, was obtained. 1 H NMR(500MHz,DMSO-d6)δ10.34(s,1H),9.99(s,1H),8.76(d,J=2.5Hz,1H),8.67(s,1H),8.39 (d,J=2.5Hz,1H),7.70–7.65(m,4H),6.83(d,J=8.8Hz,1H),6.69(d,J=2.6Hz,1H),6.47(dd,J =8.7,2.6Hz,1H),3.81–3.78(m,4H),3.76(s,3H),3.68(s,3H),3.24–3.19(m,4H),2.32(t,J =7.3Hz,2H),1.96(t,J=7.3Hz,2H),1.56(dt,J=34.8,7.4Hz,4H),1.29(q,J=7.8Hz,2H).LCMS m / z[M+H] + 573.3.

[0171] Example 9: Preparation of Compound 10

[0172]

[0173] Intermediate 10c:

[0174] Following the synthetic steps of intermediate 1n in Example 1, a yellow solid compound 10c (205 mg, yield 82%) was obtained.

[0175] Compound 10:

[0176] Following the synthetic steps of compound 2 in Example 2, a yellow solid compound 10 (31 mg, yield 23%) was obtained. 1H NMR (400MHz, DMSO-d6) δ10.39(s,1H),10.02(s,1H),8.77(d,J=2.5Hz,1H),8.71(s,1 H),8.40(d,J=2.6Hz,1H),7.68(s,4H),6.83(d,J=8.8Hz,1H),6.69(d,J=2.7Hz,1H),6 .47(dd,J=8.8,2.7Hz,1H),3.79(t,J=4.9Hz,4H),3.76(s,3H),3.68(s,3H),3.21(t,J =5.0Hz,4H),2.33(t,J=6.8Hz,2H),1.99(t,J=6.7Hz,2H),1.56(d,J=6.3Hz,4H).LCMS m / z[M+H] + 559.3.

[0177] Example 10: Preparation of Compound 11

[0178]

[0179] Intermediate 11c:

[0180] Following the synthesis steps of intermediate 8c in Example 7, a pale yellow solid 11c (531 mg, yield 86%) was obtained.

[0181] Intermediate 11e:

[0182] Following the synthesis steps of intermediate 1n in Example 1, a yellow solid 11e (150 mg, 74% yield) was obtained.

[0183] Compound 11:

[0184] Following the synthetic steps of compound 2 in Example 2, a light brown solid compound 11 (20 mg, yield 10%) was obtained. 1HNMR(500MHz,DMSO-d6)δ10.33(s,1H),9.95(s,1H),8.66(d,J=2.5Hz,2H),8.23(d,J=2.5Hz,1H),7.69–7.57(m ,4H),6.80(d,J=8.7Hz,1H),6.24(d,J=2.6Hz,1H),6.05(dd,J=8.7,2.6Hz,1H),4.04(dd,J=10.9,7.0Hz,2H),3. 74(s,3H),3.70(dd,J=11.2,3.6Hz,2H),3.64(s,3H),3.45(dd,J=9.4,6.9Hz,2H),3.23(dd,J=9.7,3.3Hz,2H), 3.14(s,2H),2.31(t,J=7.4Hz,2H),1.94(t,J=7.4Hz,2H),1.54(dt,J=45.0,6.9Hz,4H),1.32–1.25(m,4H).LCMS m / z[M+H] + 613.3.

[0185] Example 11: Preparation of Compound 12

[0186]

[0187] Intermediate 12c:

[0188] Following the synthetic steps of intermediate 1c in Example 1, a yellow-green solid compound 12c (844 mg, yield 64%) was obtained.

[0189] Intermediate 12d:

[0190] Following the synthetic steps of intermediate 1d in Example 1, a brownish-yellow solid compound 12d (600 mg, yield 93%) was obtained.

[0191] Intermediate 12f:

[0192] Following the synthesis steps of intermediate 1n in Example 1, a yellow solid 12f (276 mg, yield 65%) was obtained.

[0193] Compound 12:

[0194] Following the synthetic steps of compound 2 in Example 2, a light brown solid compound 12 (80 mg, yield 70%) was obtained. 1HNMR(500MHz,DMSO-d6)δ10.32(s,1H),8.65(s,1H),7.92(d,J=3.1Hz,1H),7.36(d,J=3.1Hz,1H),6.82(d,J=8.8Hz,1H), 6.67(d,J=2.7Hz,1H),6.46(dd,J=8.7,2.8Hz,1H),3.76(s,3H),3.72(dd,J=10.6,7.6Hz,1H),3.68(s,3H),3.58(dd,J=12 .2,7.6Hz,1H),3.45(ddd,J=9.9,7.4,4.5Hz,2H),3.39–3.36(m,4H),3.25(dd,J=12.3,4.7Hz,1H),3.19(dd,J=9.7,4.6H z,7H),3.08–2.95(m,2H),2.20(t,J=7.4Hz,2H),1.92(t,J=7.3Hz,2H),1.47(q,J=7.1Hz,4H),1.25(t,J=8.4Hz,4H).LCMS m / z[M+H] + 606.3.

[0195] Example 12: Preparation of Compound 13

[0196]

[0197] Intermediate 13c:

[0198] Compounds 13a (200 mg, 0.49 mmol), 13b (109 mg, 0.54 mmol), DIPEA (159 mg, 1.23 mmol), and TBAI (18 mg, 0.05 mmol) were placed in a 50 mL round-bottom flask, and 4 mL of MeCN solvent was added. The mixture was reacted overnight at 60 °C. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with EtOAc / Petroleumether / Et3N (0.1%) system) to give a pale yellow solid compound 13c (200 mg, yield 74%).

[0199] Compound 13:

[0200] Following the synthetic steps of compound 2 in Example 2, a brown solid compound 13 (65 mg, yield 46%) was obtained. 1HNMR(500MHz,DMSO-d6)δ10.33(s,1H),8.67(s,1H),8.25(d,J=3.0Hz,1H),7.75(d,J=3.0 Hz,1H),6.83(d,J=8.8Hz,1H),6.67(d,J=2.6Hz,1H),6.46(dd,J=8.7,2.7Hz,1H),3.76(s, 3H),3.69(s,3H),3.53–3.45(m,4H),3.22–3.17(m,4H),3.12(s,4H),2.49(s,4H),2.30(s, 2H),1.95(t,J=7.4Hz,2H),1.47(dt,J=27.1,6.5Hz,4H),1.32–1.23(m,4H).LCMSm / z[M+H] + :552.3.

[0201] Example 13: Preparation of Compound 14

[0202]

[0203] Intermediate 14c:

[0204] Following the synthetic steps of intermediate 13c in Example 12, a pale yellow solid compound 14c (200 mg, yield 76%) was obtained.

[0205] Compound 14:

[0206] Following the synthetic steps of compound 2 in Example 2, a brown solid compound 14 (62 mg, yield 44%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.36(s,1H),8.71(s,1H),8.23(d,J=3.0Hz,1H),7.73(d,J=3.0Hz,1H), 6.82(d,J=8.7Hz,1H),6.67(d,J=2.6Hz,1H),6.45(dd,J=8.6,2.7Hz,1H),3.76(s,3H),3.68(s,3H ),3.47(t,J=4.6Hz,4H),3.19(d,J=4.5Hz,4H),3.11(t,J=4.9Hz,4H),2.47(t,J=4.8Hz,4H),2.28 (t,J=7.2Hz,2H),1.96(t,J=7.3Hz,2H),1.48(dt,J=39.8,7.5Hz,4H),1.26(q,J=7.5Hz,2H).LCMS m / z[M+H] + 538.3.

[0207] Example 14: Preparation of Compound 15

[0208]

[0209] Intermediate 15c:

[0210] Following the synthetic steps of intermediate 13c in Example 12, a pale yellow solid compound 15c (265 mg, yield 67%) was obtained.

[0211] Compound 15:

[0212] Following the synthetic steps of compound 2 in Example 2, a yellow-green solid compound 15 (103 mg, yield 65%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.41(s,1H),8.73(s,1H),8.23(d,J=2.9Hz,1H),7.73(d,J= 2.9Hz,1H),6.81(d,J=8.8Hz,1H),6.67(d,J=2.5Hz,1H),6.44(dd,J=8.7,2.7Hz,1H),3 .76(s,3H),3.68(s,3H),3.50–3.45(m,4H),3.16(d,J=8.0Hz,4H),3.11(s,4H),2.47( s,4H),2.29(t,J=7.0Hz,2H),1.99(t,J=7.2Hz,2H),1.47(dt,J=48.5,7.3Hz,4H).LCMS m / z[M+H] + :524.3.

[0213] Example 15: Preparation of Compound 16

[0214]

[0215] Intermediate 16c:

[0216] Compound 16a (2 g, 9.22 mmol), compound 16b (3.95 g, 9.22 mmol), and Cs₂CO₃ (3.31 g, 10.14 mmol) were placed in a 125 mL round-bottom flask, and MeCN (15 mL) was added. The mixture was then refluxed overnight in a 90 °C oil bath. Post-treatment: The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with DCM (5 mL). The filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluting with EtOAc / Petroleum ether system) to obtain a white solid, 16c (2.9 g, 86% yield).

[0217] Intermediate 16d:

[0218] Following the synthesis steps of intermediate 1h in Example 1, a pale yellow solid 16e (1.22 g, 98% yield) was obtained.

[0219] Intermediate 16f:

[0220] Following the synthesis steps of intermediate 1d in Example 1, a brownish-yellow solid 16f (700 mg, 90% yield) was obtained.

[0221] Intermediate 16h:

[0222] Following the synthesis steps of intermediate 1n in Example 1, a pale yellow solid 16h (220mg, yield 77%) was obtained.

[0223] Compound 16:

[0224] Following the synthetic steps of Compound 1 in Example 1, a pale yellow solid compound 16 (36 mg, yield 42%) was obtained. 1 HNMR (400MHz, DMSO-d6) δ10.35(s,1H),8.81(d,J=2.5Hz,1H),8.68(s,1H),8.50(d,J=2.5Hz,1H),6.99(s,2H),3.86(s,6H),3 .65(d,J=21.8Hz,11H),2.34(t,J=7.4Hz,2H),1.94(t,J=7.4Hz,2H),1.48(p,J=6.8Hz,4H),1.26(dq,J=10.0,6.0Hz,4H).LCMS m / z[M+H] + 526.3.

[0225] Example 16: Preparation of Compound 17

[0226]

[0227] Intermediate 17c:

[0228] Following the synthetic steps of intermediate 1n in Example 1, a yellow solid compound 17c (200 mg, 77% yield) was obtained.

[0229] Compound 17:

[0230] Following the synthetic steps of compound 2 in Example 2, a pale yellow solid compound 17 (117 mg, yield 39%) was obtained. 1HNMR(500MHz,DMSO-d6)δ10.35(s,1H),8.82(d,J=2.2Hz,1H),8.68(s,1H),8.51(d,J=2.2Hz,1H),7.00(s,2H),3.87(s,6H), 3.66(d,J=26.6Hz,11H),2.35(t,J=7.3Hz,2H),1.96(t,J=7.2Hz,2H),1.51(q,J=7.4Hz,4H),1.28(q,J=7.5,7.1Hz,2H).LCMS m / z[M+H] + 512.2.

[0231] Example 17: Preparation of Compound 18

[0232]

[0233] Intermediate 18c:

[0234] Following the synthetic steps of intermediate 1n in Example 1, a yellow solid compound 18c (468 mg, yield 89%) was obtained.

[0235] Compound 18:

[0236] Following the synthetic steps of compound 2 in Example 2, a pale yellow solid compound 18 (48 mg, yield 32%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.36(s,1H),8.83(d,J=2.5Hz,1H),8.68(s,1H),8.51(d,J=2.5Hz,1H),7.00(s,2H), 3.86(s,6H),3.66(d,J=25.2Hz,11H),2.36(t,J=6.8Hz,2H),1.97(t,J=6.7Hz,2H),1.53(d,J=10.2Hz,4H).LCMS m / z[M+H] + 498.2.

[0237] Example 18: Preparation of Compound 19

[0238]

[0239] Intermediate 19c:

[0240] Under ice bath conditions, Et3N (3 mL) was added to 10 mL of DCM containing 19b (1.36 g, 10.58 mmol). After dissolution, 6 mL of DCM containing 19a (2 g, 10.58 mmol) was added dropwise using a syringe under nitrogen protection, and the mixture was stirred overnight. The reaction was quenched with ice-cold H2O (40 mL) and extracted with DCM (15 mL × 3). The combined organic phases were washed successively with H2O (10 mL × 2) and aq. NaCl (10 mL × 2), and then dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with EtOAc / Petroleum ether / Et3N (0.1%) system) to give a white solid 19c (1.51 g, yield 53%).

[0241] Intermediate 19e:

[0242] 19c (285 mg, 1.06 mmol) and 19d (365 mg, 0.96 mmol) were placed in a 50 mL round-bottom flask, and MeCN (5 mL) was added. DIPEA (372 mg, 2.88 mmol) was added with stirring, and the mixture was heated to 50 °C and reacted for approximately 4 hours. The reaction solution was cooled and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with a MeOH / DCM / Et3N (0.1%) system) to obtain a yellow solid 19e (390 mg, yield 69%).

[0243] Compound 19:

[0244] Following the synthetic steps of Compound 1 in Example 1, a light brown solid compound 19 (107 mg, yield 32%) was obtained. 1 HNMR (500MHz, DMSO-d6) δ11.20(s,1H),9.02(s,1H),8.79(d,J=2.5Hz,1H),8.47(d,J=2.5Hz,1H),7.74(d,J=8.2H z,2H),7.42(d,J=8.1Hz,2H),6.99(s,2H),3.86(s,6H),3.68(s,3H),3.67(s,4H),3.59(s,2H),2.55(s,4H).LCMS m / z[M+H] + 504.2.

[0245] Example 19: Preparation of Compound 20

[0246]

[0247] Intermediate 20c:

[0248] Compounds 20a (500 mg, 1.3 mmol), 20b (357 mg, 1.6 mmol), and tetrabutylammonium iodide (TBAI, 48 mg, 0.13 mmol) were placed in a 50 mL round-bottom flask, dissolved in MeCN (6 mL), and then DIPEA (351 mg, 2.6 mmol) was added. The mixture was heated to 60 °C and reacted overnight. The reaction solution was cooled and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with a MeOH / DCM / Et3N (0.1%) system) to give a yellow solid 20c (442 mg, yield 68%).

[0249] Compound 20:

[0250] Following the synthetic steps of Compound 2 in Example 2, a light brown solid compound 20 (442 mg, yield 42%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.33(s,1H),8.79(d,J=2.5Hz,1H),8.66(s,1H),8.46(d,J=2.5Hz,1H),6.99(s,2H),3.86(s,6H),3.68(s,3H),3.65 LCMS m / z[M+H] + 498.3.

[0251] Example 20: Preparation of Compound 21

[0252]

[0253] Intermediate 21c:

[0254] Following the synthetic steps of intermediate 20c in Example 19, a yellow solid compound 21c (208 mg, 78% yield) was obtained.

[0255] Compound 21:

[0256] Following the synthetic steps of compound 2 in Example 2, a light brown solid compound 21 (28 mg, yield 47%) was obtained. 1HNMR (500MHz, DMSO-d6) δ10.34(s,1H),8.79(d,J=2.5Hz,1H),8.67(s,1H),8.47(d,J=2.5Hz,1H),6.99(s,2H),3.86(s,6H),3.68(s,3H),3.6 7–3.62(m,4H),2.51(d,J=4.5Hz,4H),2.35–2.27(m,2H),1.95(t,J=7.3Hz,2H),1.49(ddd,J=29.7,14.7,7.3Hz,4H),1.30–1.25(m,2H).LCMS m / z[M+H] + 484.2.

[0257] Example 21: Preparation of compound 22

[0258]

[0259] Intermediate 22c:

[0260] Following the synthetic steps of intermediate 20c in Example 19, a yellow solid compound 22c (380 mg, 77% yield) was obtained.

[0261] Compound 22:

[0262] Following the synthetic steps of compound 2 in Example 2, a light brown solid compound 22 (60 mg, yield 30%) was obtained. 1 HNMR (500MHz, DMSO-d6) δ10.38(s,1H),8.78(d,J=2.4Hz,1H),8.72(s,1H),8.45(d,J=2.4Hz,1H),6.98(s,2H),3.87(s,6H),3. 69(s,3H),3.66(s,4H),2.55–2.51(m,4H),2.33(t,J=6.9Hz,2H),2.00(t,J=7.1Hz,2H),1.49(dq,J=37.3,6.5,5.9Hz,4H).LCMS m / z[M+H] + 497.2.

[0263] Example 22: Preparation of compound 23

[0264]

[0265] Intermediate 23c:

[0266] Following the synthetic steps of intermediate 20c in Example 19, a yellow solid compound 23c (439 mg, 77% yield) was obtained.

[0267] Compound 23:

[0268] Following the synthetic steps of compound 2 in Example 2, a light brown solid compound 23 (60 mg, yield 40%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.75(s,1H),9.03(s,1H),8.79(d,J=2.6Hz,1H),8.47(d,J=2.5Hz,1H),7.54(d,J=7.8Hz,2H),7.46(d,J=15.8Hz,1H), 7.38(d,J=7.8Hz,2H),6.99(s,2H),6.45(d,J=15.9Hz,1H),3.85(s,6H), 3.68(s,3H),3.68–3.62(m,4H),3.57(s,2H),2.55(t,J=4.8Hz,4H).LCMS m / z[M+H] + :530.2.

[0269] Example 23: Preparation of Compound 24

[0270]

[0271] Intermediate 24c:

[0272] Following the synthesis steps of intermediate 16c in Example 15, white solid 24c (1.68 g, yield 81%) was obtained.

[0273] Intermediate 24e:

[0274] Following the synthesis steps of intermediate 16e in Example 15, a pale yellow solid 24e (1.89 g, 77% yield) was obtained.

[0275] Intermediate 24f:

[0276] Following the synthetic steps of intermediate 1d in Example 1, a brownish-yellow solid compound 24f (750 mg, 94% yield) was obtained.

[0277] Intermediate 24h:

[0278] Following the synthesis steps of intermediate 1n in Example 1, a yellow solid (81 mg, yield 66%) was obtained over 24 hours.

[0279] Compound 24:

[0280] Following the synthetic steps of compound 2 in Example 2, a light brown solid compound 24 (36 mg, yield 43%) was obtained. 1HNMR(500MHz,DMSO-d6)δ10.34(s,1H),8.73(d,J=2.5Hz,1H),8.67(s,1H),8.36(d,J=2.5Hz,1 H),6.95(s,2H),4.02–3.96(m,2H),3.86(s,6H),3.75–3.70(m,1H),3.68(s,3H),3.61(ddd,J=2 0.6,10.4,5.8Hz,3H),3.42(dd,J=10.7,4.9Hz,1H),3.29(dd,J=12.3,4.8Hz,1H),3.11–2.95( m,2H),2.23(q,J=7.4Hz,2H),1.93(t,J=7.4Hz,2H),1.51–1.44(m,4H),1.28–1.21(m,4H).LCMS m / z[M+H] + :552.3.

[0281] Example 24: Preparation of Compound 25

[0282]

[0283] Intermediate 25c:

[0284] Following the synthesis steps of intermediate 20c in Example 19, a brownish-yellow solid 25c (450 mg, yield 62%) was obtained.

[0285] Compound 25:

[0286] Following the synthetic steps of compound 2 in Example 2, a light brown solid compound 25 (201 mg, yield 51%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.75(s,1H),9.05(s,1H),8.75(s,1H),8.37(s,1H),7.47(dd,J=29.2,11.6Hz,3H),7.32(d,J=7.7Hz,2H),6.97(s,2H),6. 45(d,J=15.4Hz,1H),3.99–3.93(m,2H),3.87(s,6H),3.69(s,3H),3.66(d ,J=11.6Hz,2H),3.59(s,2H),2.93(s,2H),2.60(s,2H),2.54(s,2H).LCMS m / z[M+H] + 556.2.

[0287] Example 25: Preparation of Compound 26

[0288]

[0289] Intermediate 26c:

[0290] Following the synthetic steps of intermediate 19e in Example 18, a pale yellow solid compound 26c (200 mg, 74% yield) was obtained.

[0291] Compound 26:

[0292] Following the synthetic steps of Compound 1 in Example 1, a brown solid compound 26 (148 mg, 72% yield) was obtained. 1 HNMR (500MHz, DMSO-d6) δ11.16(s,1H),9.00(s,1H),8.75(d,J=2.4Hz,1H),8.37(d,J=2.4Hz,1H),7.70(d,J=7.8Hz,2H),7.36(d,J=7.8Hz,2H),6. 97(s,2H),3.96(t,J=9.0Hz,2H),3.87(s,6H),3.68(s,3H),3.66(s,2H), 3.62(s,2H),3.35(s,2H),2.93(s,2H),2.60(dd,J=9.4,5.6Hz,2H).LCMS m / z[M+H] + 530.2.

[0293] Example 26: Preparation of Compound 27

[0294]

[0295] Intermediate 27c:

[0296] Compounds 27a (500 mg, 1.41 mmol), 27b (237 mg, 1.55 mmol), and Cs₂CO₃ (1.38 g, 4.23 mmol) were placed in a 25 mL round-bottom flask, and 6 mL of MeCN solvent was added. The mixture was reacted overnight at 60 °C. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with EtOAc / Petroleum ether / Et₃N (0.1%) system) to give a yellow solid 27c (350 mg, yield 82%).

[0297] Intermediate 27d:

[0298] Lithium hydroxide (67 mg, 2.80 mmol) was dissolved in MeOH (2 mL) and H2O (2 mL). After the solution was clear, compound 27c (300 mg, 0.70 mmol) was added and stirred at room temperature for about 3 h. The reaction was confirmed to be complete by TLC. The reaction solution was concentrated under reduced pressure, dissolved in H2O (5 mL), and extracted with a small amount of EtOAc (2 mL) to remove impurities. The EtOAc layer was discarded. The pH of the aqueous phase was adjusted to 6 with 1 M HCl, and then extracted two to three times with EtOAc (5 mL). The organic phases were combined and washed once with aq. NaCl (10 mL). The combined organic phases were dried in anhydrous Na2SO4 and filtered to remove anhydrous Na2SO4. The filtrate was concentrated under reduced pressure to give a yellow solid compound 27d (268 mg, yield 65%).

[0299] Intermediate 27f:

[0300] Under ice bath conditions, 27e (27 mg, 0.15 mmol) was added to 3 mL of 27d (250 mg, 0.12 mmol) DMF solution. After stirring and clarifying, DIPEA (77 mg, 0.60 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 49 mg, 0.13 mmol) were added sequentially, and the mixture was stirred overnight. The reaction was quenched with 5 mL of ice-cold H2O and extracted with DCM (10 mL × 3). The combined organic phases were washed sequentially with 2 mL of H2O and 2 mL of aq. NaCl, and then dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with EtOAc / Petroleum ether / Et3N (0.1%) system) to give a yellow solid compound 27f (378 mg, yield 70%).

[0301] Compound 27:

[0302] Following the synthetic steps of compound 2 in Example 2, a pale yellow solid compound 27 (67 mg, yield 45%) was obtained. 1HNMR(500MHz,DMSO-d6)δ10.36(s,1H),8.80(d,J=2.5Hz,1H),8.69(s,1H),8.47(d,J=2.5Hz,1H),7.81(t,J=5.7Hz,1H),6.99(s,2H),3.86 LCMS m / z[M+H] + 541.3.

[0303] Example 27: Preparation of Compound 28

[0304]

[0305] Intermediate 28c:

[0306] Following the synthetic steps of intermediate 27f in Example 26, a pale yellow solid compound 28c (400 mg, 74% yield) was obtained.

[0307] Compound 28:

[0308] Following the synthetic steps of compound 2 in Example 2, a pale yellow solid compound 28 (160 mg, 80% yield) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.33(s,1H),8.80(d,J=2.5Hz,1H),8.65(s,1H),8.48(d,J=2.5Hz,1H),6.99(s,2H),3.86(s,6H),3.74–3.69 LCMS m / z[M+H] + :555.3.

[0309] Example 28: Preparation of Compound 29

[0310]

[0311] Intermediate 29c:

[0312] Following the synthetic steps of intermediate 27c in Example 26, a pale yellow solid compound 29c (344 mg, 76% yield) was obtained.

[0313] Intermediate 29d:

[0314] Following the synthetic steps of intermediate 27d in Example 26, yellow solid compound 29d (290 mg, yield 66%) was obtained.

[0315] Intermediate 29f:

[0316] Following the synthetic steps of intermediate 27f in Example 26, a pale yellow solid compound 29f (200 mg, 70% yield) was obtained.

[0317] Compound 29:

[0318] Following the synthetic steps of compound 2 in Example 2, a pale yellow solid compound 29 (69 mg, yield 46%) was obtained. 1 HNMR(500MHz,DMSO-d6)δ10.32(s,1H),8.75(d,J=2.5Hz,1H),8.65(s,1H),8.37(d,J=2.5 Hz,1H),7.63(t,J=5.9Hz,1H),6.96(s,2H),3.99(dd,J=10.9,7.9Hz,2H),3.86(s,6H),3. 71–3.65(m,5H),3.04(d,J=11.3Hz,4H),2.94(s,2H),2.74–2.65(m,2H),2.60(d,J=9.1Hz ,2H),1.92(t,J=7.4Hz,2H),1.50–1.44(m,2H),1.41–1.33(m,2H),1.24–1.18(m,2H).LCMS m / z[M+H] + 567.3.

[0319] Example 29: Preparation of Compound 36

[0320]

[0321] Intermediate 36c:

[0322] Under ice bath conditions, Et3N (935 mg, 9.63 mmol) was added to a DCM solution (5 mL) of compound 36a (500 mg, 4.63 mmol). Under nitrogen protection, a DCM solution (5 mL) of compound 36b (875 mg, 4.63 mmol) was added dropwise using a syringe, and the mixture was stirred under ice bath conditions for approximately 3 hours. The reaction was confirmed to be complete by TLC. The reaction was quenched with ice-cold H2O (5 mL) and extracted with DCM (10 mL × 3). The combined organic phases were washed successively with H2O (5 mL × 2) and aq. NaCl (5 mL × 2), and dried in anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with an EtOAc / Petroleum ether / Et3N (0.1%) system) to give a yellow solid compound 36c (146 mg, yield 56%).

[0323] Compound 36:

[0324] Compounds 36c (130 mg, 0.50 mmol), 36d (285 mg, 0.75 mmol), and Cs₂CO₃ (489 mg, 1.50 mmol) were placed in a 25 mL round-bottom flask, and 4 mL of MeCN solvent was added. The mixture was reacted overnight at 60 °C. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with MeOH / DCM / Et₃N (0.1%) system) to give a brown solid compound 36 (242 mg, yield 40%). 1 H NMR(500MHz,DMSO-d6)δ9.63(s,1H),8.76(d,J=2.5Hz,1H),8.39(d,J=2.5Hz,1H ),7.94(d,J=8.0Hz,2H),7.43(d,J=8.1Hz,2H),7.17(d,J=7.6Hz,1H),6.98(s,3 H),6.79(d,J=9.0Hz,1H),6.60(t,J=8.0Hz,1H),4.89(s,2H),3.99(dd,J=11.0, 8.0Hz,2H),3.86(s,6H),3.76–3.61(m,7H),2.96(s,2H),2.65–2.54(m,4H).LCMS m / z[M+H] + 605.3.

[0325] Example 30: Results of the in vitro antiproliferative activity of some compounds against solid tumor cell lines

[0326] 1. Experimental Materials and Instruments

[0327] Experimental materials: DMEM (Zhejiang Senrui Biotechnology Co., Ltd.); RPMI 1640 (Zhejiang Senrui Biotechnology Co., Ltd.); Fatal Bovine Serum (BI); PBS (Zhejiang Senrui Biotechnology Co., Ltd.); Trypsin (Zhejiang Senrui Biotechnology Co., Ltd.); DMSO (Coolaber); CCK8 (Coolaber).

[0328] Experimental instruments: Biosafety cabinet (Shanghai Baiji Biotechnology Co., Ltd.); constant temperature carbon dioxide incubator (THERMO); enzyme-linked immunosorbent assay (ELISA) analyzer (Spark); inverted microscope (Nikon); a set of pipettes (Eppendorf); centrifuge (Beckman Coulter).

[0329] Human colon cancer cell lines: COLO205, SW480 and HCT116.

[0330] 2. Experimental Procedure

[0331] 1) Take test cells in the logarithmic growth phase, digest and count them with trypsin, and then dilute the tumor cell suspension to 5 × 10⁻⁶. 4 Inoculate the cells at a concentration of 100 cells / mL into 96-well plates. Except for the control group, which is given 100 μL of cell-free medium, add 100 μL of cell-containing medium to each well (1 × 10⁻⁶ cells / mL per well). 3 (cells);

[0332] 2) After incubating at 37°C for 8 hours in a humidified incubator containing 5% CO2, the original culture medium in the 96-well plate was aspirated. Except for the control and blank groups, which were added 100 μL of culture medium without the test compound, each well was added 100 μL of culture medium containing the test compound. Four replicates were set up for each concentration. Wells without cells and the test compound served as the blank group, wells with cells but no test compound served as the control group, and wells with cells and the test compound served as the experimental group. Compound A was selected as the positive control.

[0333] 3) Continue culturing in an incubator at 37℃ and 5% CO2 humidification for 72 hours;

[0334] 4) Under light-protected conditions, add 10 μL of CCK8 solution (5 mg / mL) to each well and continue culturing in an incubator at 37℃ and 5% CO2 for 1 h. Measure the absorbance (OD value) of each well at 450 nm using a microplate reader.

[0335] 5) Calculate the survival rate and inhibition rate using the following formulas.

[0336] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0337] Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0338] Single-concentration inhibition rate was calculated using Excel; GraphPadPrism 7.0 software was used to plot S-shaped dose-survival curves using a nonlinear regression model and to calculate IC50. 50 value.

[0339] As: Absorbance of the experimental wells (containing cell culture medium, CCK8, and the test drug)

[0340] Ac: Absorbance of control wells (containing cell culture medium, CCK8, and solvent (DMSO))

[0341] Ab: Absorbance of blank wells (cell-free culture medium, CCK8, solvent (DMSO))

[0342] 3. Experimental Results

[0343] The inhibitory effects of the prepared compound and the positive control compound (structure II, compound A) on the proliferation of three colon cancer cell lines were determined according to the above experimental method. The results are shown in Table 1.

[0344] Table 1 shows the single-concentration (75 μM) inhibitory rates of some target compounds against three types of colon cancer cells.

[0345]

[0346] Table 1 continues with the single-concentration (75 μM) inhibitory rates of some target compounds against three types of colon cancer cells.

[0347]

[0348] Note: a It indicates the results of two or more experiments. b ND indicates that it has not been tested.

[0349] Based on the single-concentration test results, the synthesized target compounds showed inhibition rates of more than 90% against most of the three types of colon cancer cells. To further characterize their anti-proliferative capacity, SAHA was used as a positive control, and the half-maximal inhibitory concentrations (IC50) of compounds with high single-concentration inhibition rates (1, 2, 3, 12, 16, 17, 19, 21, 23 to 26, and 28) against the three types of colon cancer cells were determined. 50 The results are shown in Table 2.

[0350] Table 2 shows the half-maximal inhibitory concentrations (IC50) of some target compounds against colon cancer cells.

[0351]

[0352] Note: aIt indicates the results of two or more experiments. b ND indicates that it has not been tested.

[0353] 4. Results and Discussion

[0354] The test results show that all compounds exhibited significantly stronger anti-proliferative activity against the three colon cancer cell lines mentioned above than the positive control compound A. Furthermore, compound 25 showed significantly stronger anti-proliferative activity (IC50) against the three colon cancer cell lines mentioned above. 50 The concentrations of all compounds were less than 1 μM, comparable to the positive control drug SAHA. Therefore, compound 25 is a very promising compound worthy of further investigation.

[0355] Example 31: In vitro HDAC1 and HDAC6 enzyme inhibition rate test of some compounds

[0356] 1. Experimental objective: To determine the inhibitory activity of the synthesized final product on HDAC1 and HDAC6 enzymes and the selectivity of compound 25 for HDAC enzyme subtypes (including HDAC2, HDAC3, HDAC8 and HDAC11) using quantitative fluorescence analysis. SAHA was used as a positive control.

[0357] 2. Experimental materials and instruments

[0358] Experimental materials: HDAC1 (BPS, Cat. No. 50051); HDAC6 (BPS, Cat. No. 50006); Tris buffer; 384-well plate (Perkin Elmer, Cat. No. 6007279); DMSO (Coolaber).

[0359] Instrument: SpectraMax Paradigm multi-functional microplate detector.

[0360] 3. Experimental Methods

[0361] 1) Preparation of 1x concentration analytical buffer: This refers to diluting a stock solution prepared at 10x or higher concentration to the required concentration for the experiment. The buffer solution used here contains Tris, which makes it more stable;

[0362] 2) Preparation of the test compounds: Dissolve each test compound in 100% DMSO and transfer a certain volume to the test plate. Note that the DMSO content in each well should not exceed 1%.

[0363] 3) Preparation of HDAC enzyme solution: Prepare enzyme (HDAC1,2,3,6,8,11) solution in the prepared analytical buffer solution at 1x concentration;

[0364] 4) Preparation of substrate solutions: For HDAC1,2,3,6, trypsin and acetyl polypeptide substrate were added to an analytical buffer of 1x concentration to prepare substrate solutions; for HDAC8,11, acetyl polypeptide substrate was added to an analytical buffer of 1x concentration to prepare substrate solutions.

[0365] 5) Experiment: For the HDAC1,2,3,6 enzyme assay, the OD value was read on Paradigm with an excitation wavelength of 355 nm and an emission wavelength of 460 nm; for the HDAC8,11 enzyme assay, the enzyme was first incubated at room temperature for 240 min, then trypsin solution was added, and the enzyme was incubated at room temperature for another 120 min. Finally, the OD value was read on Paradigm with an excitation wavelength of 355 nm and an emission wavelength of 460 nm.

[0366] 6) Detection: Read the absorbance (OD value) of each well on the measurement plate using Paradigm at wavelengths of 355nm / 460nm. The blank control group does not contain any compound.

[0367] 7) Inhibition rate and IC 50 Calculation: Use the following formula in Excel to calculate the single-concentration inhibition rate;

[0368] Formula: Inh%=(Max-Signal) / (Max-Min)*100

[0369] Where Max: absorbance of the experimental well; Signal: absorbance of the control well; Min: absorbance of the blank well;

[0370] Using GraphPad Prism 7.0 software, a nonlinear regression model was used to plot an S-shaped dose-survival curve and calculate the IC50. 50 value.

[0371] 4. Experimental Results

[0372] The inhibitory effects of some target compounds on HDAC1 and HDAC6 enzymes were determined using the experimental methods described above. The results are shown in Tables 3 and 4.

[0373] Table 3. Single-concentration inhibition rates of the target compounds against HDAC1 and HDAC6 enzymes.

[0374]

[0375] Table 3 (continued) shows the single-concentration inhibition rates of the target compounds against HDAC1 and HDAC6 enzymes.

[0376]

[0377] Based on the single-concentration test results, compounds with single-concentration inhibition rates higher than 80% were selected to determine their half-maximal inhibitory concentrations (IC50) for HDAC1 and HDAC6. 50 The experimental results are shown in Table 4.

[0378] Table 4. Half-maximal inhibitory concentrations (IC50) of the target compounds against HDAC1 and HDAC6 enzymes.

[0379]

[0380] Note: a ND was not measured.

[0381] According to IC 50 The values ​​indicate that compounds 2, 9, 11, 16, 25 and the positive control drug SAHA have comparable inhibitory abilities against HDAC1 and HDAC6.

[0382] Compared to pan-HDAC inhibitors, subtype-selective HDAC inhibitors have attracted much attention due to their lower toxicity. Therefore, we further tested the enzymatic activities of compound 25 against HDAC2,3,8 (type I) and HDAC11 (type IV), respectively. SAHA or CUDC907 (type I PI3K and type I and II HDAC enzyme inhibitors) were used as positive controls (Table 5).

[0383] Table 5. HDAC enzyme subtype selectivity of compound 25

[0384]

[0385] Note: c ND indicates that it has not been tested.

[0386] As shown in Tables 4 and 5, compound 25 exhibits inhibitory effects on class I and class IIb HDACs comparable to SAHA. Its inhibitory effects on the four HDAC isotypes 1, 2, 3, and 6 are IC50. 50 The values ​​were 20.8, 46.8, 53.0, and 15.5 nM, respectively; while the 25-pair inhibitory activity against HDAC8 and HDAC11 was weak, IC 50 The value is greater than 1250 nM. In summary, 25, like SAHA, is a pan-HDACs inhibitor.

[0387] Example 32: Study on the affinity of compound 12 for HSP110

[0388] 1. Experimental Objective: To verify whether the designed and synthesized small molecule targeting HSP110-HDACs has an affinity for the target HSP110, we used compound 12, which has good in vitro anti-colorectal cancer proliferation activity, as the research object. We used the Biolayer interferometry technique (Octet Red96, Forté-Bio) to determine its affinity for HSP110 protein. The experimental results are expressed as the dissociation constant (K0). D The values ​​were used to characterize the compound, with compound A serving as the positive control.

[0389] 2. Experimental materials and instruments

[0390] 1) Experimental materials: HSP110, NBD-HSP110, NHS-PEG4-Biotin reagent (Thermo Scientific 21330), Tris (Zhejiang Senrui Biotechnology), DMSO (Coolaber) and 96-well plates.

[0391] 2) Experimental apparatus: Zeba™ spin desalination column (Thermo Scientific B2162579) and SuperStreptavidin biosensor (Forté-Bio).

[0392] 3. Experimental Methods

[0393] 1) Research subject: protein (HSP110). HSP110 was biotinylated using NHS-PEG4-Biotin reagent at a ratio of 1:3 to HSP110.

[0394] 2) Next, remove excess biotin (using a Zeba™ spin desalting column, 7 KMWCO, 0.5 mL).

[0395] 3) Before the kinetic experiment, the SuperStreptavidin biosensor (Forté-Bio) was hydrated for 15 min at room temperature in running buffer (Tris 50mM pH8, NaCl 300mM, 0.01% (w / v) Tween-20 and 0.1% (w / v) BSA).

[0396] 4) Experiments were performed in 96-well plates with a volume of 200 μL / well, under constant oscillation (1000 rpm) and at room temperature. Baseline measurements were taken in running buffer for 120 s, followed by a 600 s migration in 15 μg / mL HSP110.

[0397] 5) Wash the protein-linked sensor in run buffer for 60 s, and then measure the second baseline for 120 s in run buffer solution.

[0398] 6) Key steps (4 and 5) are performed in parallel with multiple compound concentrations; an appropriate amount of the compound is dissolved in DMSO and diluted in a flow buffer, with the DMSO content not exceeding 1%.

[0399] 7) Evaluate dissociation after running in buffer for 60 seconds. K D The value (1:1 binding model) was calculated using software provided by the manufacturer, with a loaded control protein (hemoglobin, 15 μg / mL) used as a reference for the sensor.

[0400] 4. Experimental Results

[0401] Experimental results show that compound 12 affects the K-axis of HSP110. D The value was 1.41 μM, compared to the K value of compound A for HSP110. D The K value was significantly increased to 89.2 μM (compounds A and 12 significantly improved the K of HSP110). D The value determination curves are attached in sequence. Figure 1 and 2 (As shown).

Claims

1. A compound that inhibits both histone deacetylase and heat shock protein 110 kDa dual pathways, characterized in that... The compound has a structural formula of any one of the following: 。 2. The application of the histone deacetylase and heat shock protein 110 kDa dual-pathway inhibitor compound according to claim 1 in the preparation of antitumor drugs.

3. The application according to claim 2, characterized in that, The tumor is colon cancer.

4. An antitumor drug, characterized in that... The compound containing a safe and effective amount of the histone deacetylase and heat shock protein 110 kDa dual pathway inhibitor compound of claim 1.

5. An antitumor drug according to claim 4, characterized in that, The antitumor drugs also include pharmaceutically acceptable carriers.

6. An antitumor drug according to claim 4 or 5, characterized in that, The tumor is colon cancer.

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