CN116803984A

CN116803984A - Targeting heat shock protein 110kDa (HSP 110) inhibitor and application thereof

Info

Publication number: CN116803984A
Application number: CN202310591948.8A
Authority: CN
Inventors: 叶向阳; 许丽; 谢恬; 张航; 白仁仁; 何兴瑞; 徐月莹; 谢玉娟; 杜晓利; 王媛
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2022-05-26
Filing date: 2023-05-24
Publication date: 2023-09-26

Abstract

The invention discloses a targeted heat shock protein 110kDa (HSP 110) inhibitor and application thereof. The invention provides a heat shock protein 110kDa inhibitor with a novel structure shown in a formula (I), a pharmaceutical composition and hydrate containing the compound shown in the formula (I), and isotope derivatives, chiral isomers, allosteric isomers, different salts, prodrugs, preparations and the like of the compound. The invention also provides a preparation method and application of the heat shock protein 110kDa inhibitor with novel structure, and activity of the compounds in inhibiting proliferation of colon cancer tumor cell strains. The heat shock protein 110kDa inhibitor with novel structure is expected to become an antitumor drug candidate.

Description

Targeting heat shock protein 110kDa (HSP 110) inhibitor and application thereof

Technical Field

The invention belongs to the field of preparation and application of a targeted heat shock protein 110kDa inhibitor, and particularly relates to a targeted heat shock protein 110kDa (HSP 110) inhibitor and application thereof.

Background

Heat shock proteins (heat shock proteins, HSPs) are a class of highly conserved proteins that are rapidly synthesized in large quantities when cells are stimulated by certain adverse factors (e.g., hyperthermia, infection, ischemia, hypoxia, chemicals, etc.), bind to intracellular denatured proteins, aid in renaturation of the denatured proteins or transport of the denatured proteins to lysosomal degradation, and protect the cells. The intracellular presence of HSP's under stress therefore marks the initiation of the cell's own protective mechanisms. HSP plays an important role in regulating the physiological function and the stable state of protein and changing the expression level of the protein, and is an important direction for researching medicines with new mechanisms.

HSP110: also known as HSP105 or HSPH1, is a molecular chaperone with anti-aggregation properties, which, through interaction with HSP70, participates in the correct folding of newly synthesized or misfolded proteins. In cancer cells, HSP110 may maintain protein conformation by preventing misfolded proteins from aggregating to achieve proper binding of ligands, thereby promoting protein stability and function. In 2021, the review articles published by Shonhai and Chakafana on "Cells" summaries the biological research effort of HSP110 in detail, indicating that modulation of HSP110 may affect cancer cell growth, metastasis and escape from three aspects: (1) angiogenesis; (2) transformation of epithelial cells into the stroma; (3) immune escape. In 2019, the professor team of Carmen Garrido, national institute of health and medicine, france, discovered that in colon cancer cells, HSP110 was expressed in large amounts, and that the Tyr705 site of the protein that activates signaling and transcriptional activator (signal transducer and activator oftranscription, STAT 3) was phosphorylated, promoting cell proliferation, disrupting phosphorylation-mediated degradation of β -catenin, maintaining activity of the Wnt pathway, resulting in proliferation of colon cancer cells. Direct inhibition of STAT3 activity, which is involved in a variety of physiological activities in humans, causes serious adverse effects, and can inhibit proliferation of colorectal cancer cells by inhibiting HSP110 expression, affecting HSP110/STAT3 interactions.

HSP110 belongs to a large molecular weight heat shock protein, and since HSP110 can bind to antigen presenting cells, previous studies have focused on developing antitumor vaccines. In 2020, the professor Carmen Garrido reported for the first time that small molecule inhibitors have an inhibitory effect on HSP 110. Compound a was resolved by the Carmen Garrido professor team by study of crystal structure, computational chemistry, and in vitro screening (compound a was found in literature (Gozzi GJ, et al, selection the first chemical molecule inhibitor of HSP110 for colorectal cancer therapy [ J]Cell Death Differ.2020,27 (1): 117-129.) under the number Compound 33, the synthesis of which is described in WO2015/132727A1 and in the literature (Gloaguen C, et al first evidence that oligopyridines,<-helix foldamers,inhibit Mcl-1and sensitize ovarian carcinoma cells to Bcl-xL-targeting strategies[J]j Med Chem,2015,58 (4): 1644-1668), chemical structure of formula II below) and HSP110, and the in vitro bioactivity and in vivo antitumor efficacy of compound A were studied. The compound has better drug effect for inhibiting tumor growth on a colon cancer grafted mouse model. The disadvantage is that compound A generally blocks the ability of HSP110 to block anti-aggregation activity, IC ₅₀ =58.3±1.7 μm. Ability to block HSP110/STAT3 interactions IC ₅₀ Because of the fact that =35.9±1.1 μm, more active compounds were required for the study. Thus, this study was conducted. On one hand, the development of the work is expected to develop new-generation new anti-colon cancer drugs with better anti-colon tumor effect than that of the compound A; on the other hand, the development of better targeted inhibitors blocking the anti-aggregation activity of HSP110 is expected.

Disclosure of Invention

The invention provides a targeting heat shock protein 110 inhibitor with a novel structure aiming at the existing targeting heat shock protein 110kDa inhibitor.

A targeted heat shock protein 110kDa inhibitor, or an optical isomer, racemate, single enantiomer, possible diastereoisomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate and solvate thereof, having the chemical structural formula shown in formula (I):

in formula (I):

R ¹ selected from-X, C _1-6 Alkyl, C _3-6 Cycloalkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, -COR ⁰ One of 2-phenylvinyl;

each R is ² And each R ³ Each independently selected from-X, C _1-6 Alkyl, C _3-6 Cycloalkyl, C _3-6 Heterocyclyl, C _1-6 Alkoxy, C _2-6 Alkenyl, C _2-6 Alkynyl, -COR ⁰ One of 6-to 10-membered aryl, 5-to 10-membered heteroaryl; wherein the 5-to 10-membered heteroaryl group contains 1-3 heteroatoms selected from N, O, S, C _3-6 Heterocyclyl contains 1-3 heteroatoms selected from N, O, S;

x is H, F, cl, br, I, -OCH ₃ 、-OH、-CN、-CF ₃ One of the following;

R ⁰ is H, -OH, -NH ₂ 、C _1-6 Alkyl, C _3-6 Cycloalkyl, C _2-6 Alkenyl, C _2-6 One of the alkynyl groups;

m is an integer of 0 to 3; n is an integer of 0 to 3;

selected from 5-to 10-membered heterocyclyl, said 5-to 10-membered heterocyclyl containing 1-3 heteroatoms selected from N, O, S;

selected from->C _2-6 Alkenyl, C _2-6 Alkynyl, -CO, C _2-6 alkenyl-CO, C _2-6 Alkynyl group-CO、-SO ₂ Q is an integer of 0 to 2.

-L-is selected fromC _2-6 Alkenyl, C _2-6 One of alkynyl groups, q is an integer of 0 to 2.

The other groups are as defined above.

Further, in the compound (I):

R ¹ selected from-CH ₃ 、-CN、-CHO、-CONH ₂ One of 2-phenylvinyl;

-L-is selected fromQ=0;

selected from->One of the following;

selected from-CO, & lt + & gt>Q=0, 1,2,3.

Wherein'"means>And->The site of ligation.

Further, the targeted heat shock protein 110kDa inhibitor is any one of compounds 1 to 60 shown in the following structures:

it is a second object of the invention to provide a method for preparing a heat shock protein 110kDa inhibitor targeting. For a novel class of heat shock protein 110kDa inhibitors of formula (I) structure, the following synthetic route may be employed:

When R is ¹ The preparation of the compound of formula (I) as 2-phenylvinyl by route one, comprises the following steps:

(1) Compound a in Pd (PPh ₃ ) ₄ Carrying out Suzuki coupling reaction with trans-styrene boric acid under the catalysis to obtain a compound b;

(2)carrying out Buchwald reaction or Suzuki coupling reaction on the compound d;

(3) Removing tert-butyloxycarbonyl from the compound d to obtain a compound e;

(4) Performing nucleophilic substitution reaction on the compound b and the compound e to obtain a compound f;

(5) Compound f in Pd (PPh ₃ ) ₄ And (3) carrying out Suzuki coupling reaction with the compound g under the catalysis to obtain the compound shown in the formula (I).

When R is ¹ For CHO-orThe preparation of the compound of formula (I) by route two, comprises the following steps:

(1) the synthesis of compounds a to c is the same route as steps (2) - (3);

(2) Carrying out nucleophilic substitution reaction on the compound c and 4-bromine, 2-chlorine and 3-cyanopyridine to obtain a compound d;

(3) Compound d in Pd (PPh ₃ ) ₄ Carrying out Suzuki coupling reaction with a compound g under the catalysis to prepare a compound containing cyano groups and having a formula (I);

(4) Further reduction of cyano groups to aldehyde groups with diisobutylaluminum hydride (DIBAL-H); wherein the feeding mole ratio of the compound of the formula (I) containing cyano to diisobutyl aluminum hydride is 1.1-2: 1, a step of;

(5) With 30% hydrogen peroxide (H) ₂ O ₂ ) And potassium carbonate further oxidizes cyano groups to amides.

When R is ¹ When other groups are adopted, the compound of the formula (I) is prepared by adopting a route III, and the method specifically comprises the following steps:

(1) Compound a and compound b undergo nucleophilic substitution reaction to prepare compound c;

(2) Compound c and compound d are subjected to Suzuki coupling reaction to prepare compound e;

(3) Removing tert-butyloxycarbonyl from the compound e to obtain a compound f;

(4) The compound f and the compound g are subjected to amide condensation, nucleophilic substitution or reductive amination reaction to prepare the compound shown in the formula (I).

The compound represented by the formula (I) of the present invention can be produced by the above-mentioned method, however, the conditions of the method, such as reactants, solvents, amounts of the compounds used, reaction temperature, time required for the reaction, etc., are not limited to the above-mentioned explanation. The compounds of the present invention may also optionally be conveniently prepared by combining the various synthetic methods described in this specification or known in the art, such combinations being readily apparent to those skilled in the art to which the present invention pertains.

The third object of the invention is to provide the application of the targeted heat shock protein 110kDa inhibitor with novel structure, or optical isomer, racemate, single enantiomer, possible diastereoisomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate and solvate thereof in preparing antitumor drugs.

The fourth object of the present invention is to provide an antitumor drug, which contains a safe and effective amount of the targeted heat shock protein 110kDa inhibitor with novel structure, or optical isomer, racemate, single enantiomer, possible diastereoisomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate and solvate thereof.

Preferably, the antitumor drug may further comprise a pharmaceutically acceptable carrier.

Pharmaceutical compositions and methods of administration

Because the compounds of the present invention have activity in inhibiting proliferation of various tumor cell lines, the compounds of the present invention and various crystalline forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compounds of the present invention as a main active ingredient are useful for treating, preventing and alleviating various diseases, including various cancers.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of the compound of the invention per dose, more preferably 5-1000mg of the compound of the invention per dose. Preferably, the "one dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifying agents (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or solubilisers, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, such as glycerin; (d) Disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents such as paraffin; (f) absorption accelerators, such as quaternary amine compounds; (g) humectants, such as cetyl alcohol and glycerol monostearate; (h) adsorbents such as kaolin; (i) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain location within the gut. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

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 nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredients are mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 1 to 5000mg, preferably 5 to 2000mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Compared with the prior art, the invention has the main advantages that: the invention provides a novel targeted heat shock protein 110kDa inhibitor with a structure shown in a formula (I), a pharmaceutical composition and hydrate containing the compound shown in the formula (I), and isotope derivatives, chiral isomers, allosteric isomers, different salts, prodrugs, preparations and the like of the compound. The invention also provides a preparation method and application of the targeted heat shock protein 110kDa inhibitor with novel structure, and activity of the compounds in inhibiting proliferation of various tumor cell strains. The targeted heat shock protein 110kDa inhibitor with novel structure is expected to become an antitumor candidate medicament for treating colon cancer.

Drawings

FIGS. 1 (A) - (B) are schematic molecular docking diagrams of compound 7 interacting with HSP110, respectively, and partial enlarged views thereof.

FIG. 2 IC for detecting the effect of Compound 7 (also known as HSP-007) and Compound A (also known as Compound 33) on HCT cells and SW480 cells by MTT assay ₅₀ A value; (A-B) Compound 7 at different concentrations after 48 hours of action on HCT116 cells (A) and SW480 cells (B), the MTT method detects cell viability and calculates IC ₅₀ The method comprises the steps of carrying out a first treatment on the surface of the (C-D) Compound A at different concentrations after 48h of action on HCT116 cells (C) and SW480 cells (D), the MTT method detects cell viability and calculates IC ₅₀ 。

FIG. 3 Compound 7 (also known as HSP-007) inhibits STAT3 phosphorylation levels in HCT116 cells and SW480 cells, and inhibits STAT3 phosphorylation in colorectal cancer cells more significantly than Compound A (also known as Compound 33); (A, B) Western Blot method to detect protein levels of HSP110, STAT3 and p-STAT3 in HCT116 cells (A) and SW480 cells (B) under the action of different concentrations of compound 7 (0,1.25,2.5,5, 10. Mu.M), with B2M as an internal reference. The protein levels of HSP110, STAT3 and p-STAT3 in HCT116 cells (C) and SW480 cells (D) were detected by a Western Blot method under the action of compound 7 and compound A at different concentrations (0, 5, 10. Mu.M), with B2M as an internal reference.

FIG. 4 affinity of Compound A for HSP110 protein (K _D Characterization).

Detailed Description

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The methods of operation, under which specific conditions are not noted in the examples below, are generally in accordance with conventional conditions, or in accordance with the conditions recommended by the manufacturer.

Example 1: preparation of Compound 1

Intermediate 1c:

compound 1a (2 g,6.29 mmol), 1b (1.02 g,6.92 mmol) and sodium carbonate (Na ₂ CO ₃ 1.67g,2.50 mmol) was placed in a 75mL tube sealer and 1,4-dioxane (1, 4-dioxane,30 mL) and water (H ₂ O,10 mL). The air in the reaction mixture was purged (about 15 minutes) with a nitrogen stream. Next, pd (PPh) was added ₃ ) ₄ (560 mg,0.51 mmol). And then nitrogen is introduced for about 10 min. The reaction solution was heated to 100℃in a closed system for 24 hours. The reaction was cooled to room temperature, filtered and the filter cake was washed with dichloromethane (DCM, 5 mL). The filtrate was concentrated under reduced pressure. Adding H to the concentrate ₂ O (10 mL) and extracted with ethyl acetate (EtOAc, 5 mL. Times.3). Combining the organic phases with H in sequence ₂ O (10 mL. Times.2) and saturated saline (aq. NaCl,10 mL) were washed and placed in anhydrous sodium sulfate (Na ₂ SO ₄ ) And (5) drying. The drying agent is removed by filtration, the filtrate is concentrated under reduced pressure, and the crude product obtained is purified by silica gel column chromatography (EtOAc/Petroleum ether) system elution ]White solid 1c (945 mg, yield 51%) was obtained.

Intermediate 1g:

compound 1d (2 g,9.22 mmol), compound 1e (2.15 g,11.53 mmol)Compound 1f (574 mg,0.92 mmol) and sodium tert-butoxide (2.21 g,23.05 mmol) were placed in a 75mL tube seal and toluene (tolue, 20 mL) was added. The reaction mixture was purged of air (about 15 min) with a stream of nitrogen and then charged with tris (dibenzylideneacetone) dipalladium [ Pd ] ₂ (dba) ₃ ，422mg，0.46mmol]. And then nitrogen is introduced for about 10 min. The reaction mixture was heated to 100℃in a closed system and reacted overnight. The reaction was cooled to room temperature, filtered and the filter cake washed with DCM (5 mL). The filtrate was concentrated under reduced pressure. Adding H to the concentrate ₂ O (10 mL) and extracted with EtOAc (5 mL. Times.3). The combined organic phases are successively treated with H ₂ O (10 mL. Times.2) and aq.NaCl (10 mL. Times.1), and placed in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (EtOAc/Petroleum ether system elution) to give 1g (2.41 g, yield 81%) of a tan solid.

Intermediate 1h:

transfer 1g (2.41 g,7.49 mmol) of compound into a 100mL round bottom flask, dissolve with methanol (MeOH, 8 mL), add dropwise hydrogen chloride in dioxane (HCl in dioxane,4M,8 mL) with ice bath stirring, remove ice bath and stir overnight at room temperature. The reaction solution was concentrated under reduced pressure to give a crude hydrochloride salt as an off-white solid (2.58 g) for 1h, which was directly used in the next reaction.

Intermediate 1i:

compound 1c (250 mg,0.85 mmol), 1h (209 mg,0.94 mmol) and cesium carbonate (Cs ₂ CO ₃ 210mg,0.64 mmol) was placed in a 100mL round bottom flask, anhydrous N, N-dimethylformamide (DMF, 10 mL) was added and the reaction was heated to 110℃in a closed system for 48h. The reaction solution was cooled to room temperature, and H was added ₂ After O (5 mL) quenching, extracted with EtOAc (5 mL. Times.3), the combined organic phases were washed with water (5 mL. Times.2) and aq.NaCl (5 mL) and placed in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (EtOAc/Petroleum ether system elution) to give 1i as an off-white solid (31 mg, yield 27%). Compound 1:

reference intermediate1c, the solvent is changed into 1, 2-dimethoxy ethane, and the reaction temperature is set to be 85 ℃. Compound 1 (33 mg, yield 70%) was obtained as an off-white solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.43(d,J＝2.3Hz,1H),7.93(d,J＝2.4Hz,1H),7.56(d,J＝7.4Hz,2H),7.40(t,J＝7.6Hz,2H),7.33–7.27(m,1H),7.26(s,1H),7.15(d,J＝16.4Hz,1H),6.83(d,J＝8.7Hz,1H),6.77(s,2H),6.65(d,J＝2.6Hz,1H),6.54(dd,J＝8.7,2.6Hz,1H),3.94(s,6H),3.90(d,J＝7.5Hz,6H),3.85(s,3H),3.61–3.43(m,4H),3.37–3.21(m,4H).LCMS m/z[M+H] ⁺ :568.3.

Example 2: preparation of Compound 4

Intermediate 4d:

referring to the procedure for the synthesis of intermediate 1g of example 1, compound 4d (2.41 g, 81% yield) was obtained as a tan solid. Intermediate 4e:

referring to the procedure for the synthesis of intermediate 1h of example 1, crude compound 4e (592 mg) was obtained as a pale yellow solid.

Intermediate 4f:

Referring to the procedure for the synthesis of intermediate 1i of example 1, the solvent was changed to acetonitrile and the reaction temperature was set at 90 ℃. Compound 4f (74.4 mg, yield 46%) was obtained as a pale yellow solid.

Compound 4:

referring to the synthetic procedure of compound 1 of example 1, compound 4 (85.7 mg, yield 90%) was obtained as an off-white solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.50(d,J＝2.5Hz,1H),7.89(s,1H),6.75(d,J＝8.7Hz,1H),6.59(s,2H),6.56(d,J＝2.6Hz,1H),6.45(s,1H),3.89–3.86(m,4H),3.85(s,6H),3.82(s,6H),3.78(s,3H),3.29–3.09(m,4H).LCMS m/z[M+H] ⁺ :491.2.

Example 3: preparation of Compound 5

Compound 5:

compound 4 (520 mg,1.06 mmol) was dissolved in dry DCM (7 mL) and placed in a 25mL round bottom flask, with a nitrogen balloon attached, the whole device was placed in an ice bath (iced ethanol + ice) and kept at about-20℃under nitrogen protection, diisobutylaluminum hydride (DIBAL-H, 1M in hexane,2.5mL,2.5mmol) was slowly added dropwise, and the temperature was gradually raised to room temperature, and the reaction was continued for two hours. Then dilute hydrochloric acid (1M, 4 mL) was added to continue the reaction for about 1h until the reaction mixture was significantly delaminated (the upper layer of bubbles was cloudy, and the lower layer was clear). The reaction was extracted with DCM (5 mL. Times.3) and the combined organic phases were taken up in the order H ₂ O (10 mL. Times.2) and aq.NaCl (10 mL), placed in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (EtOAc/Petroleum ether system elution) to give compound 5 (240 mg, yield 54%) as an off-white solid. ¹ H NMR(500MHz,CDCl ₃ )δ10.17(s,1H),8.63(d,J＝2.5Hz,1H),8.18(d,J＝2.5Hz,1H),6.83(d,J＝8.7Hz,1H),6.74(s,2H),6.64(d,J＝2.5Hz,1H),6.53(dd,J＝8.6,2.6Hz,1H),3.93(s,6H),3.90(s,6H),3.86(s,3H),3.74–3.59(m,4H),3.38–3.19(m,4H).LCMS m/z[M+H] ⁺ :494.2.

Example 4: preparation of Compound 6

Compound 6:

compound 4 (100 mg,0.20 mmol) and potassium carbonate (k) ₂ CO ₃ 57mg,0.41 mmol) was placed in a 25mL round bottom flask, dissolved in dimethyl sulfoxide (DMSO, 3 mL) and added dropwise with 30% hydrogen peroxide (H) under ice-bath stirring ₂ O ₂ 1.5 mL) to room temperature. Post-treatment: 2mL of water was added to quench the reaction, and the resulting crude product was purified by silica gel column chromatography (MeOH/DCM system elution) to give compound 6 (90 mg, 87% yield) as a pale pink solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ8.59(s,1H),8.00(d,J＝11.5Hz,2H),7.62(s,1H),6.92(s,2H),6.81(d,J＝8.8Hz,1H),6.66(d,J＝2.6Hz,1H),6.43(dd,J＝8.7,2.7Hz,1H),3.85(s,6H),3.74(s,3H),3.67(d,J＝5.1Hz,6H),3.51–3.42(m,4H),3.21–3.12(m,4H).LCMS m/z[M+H] ⁺ :509.2.

Example 5: preparation of Compound 7

Intermediate 7d:

referring to the procedure for the synthesis of intermediate 1g of example 1, a tan solid 7d (2.17 g, 68% yield) was obtained.

Intermediate 7e:

referring to the procedure for the synthesis of intermediate 1h of example 1, pale yellow solid 7e (592 mg) was obtained.

Intermediate 7g:

referring to the procedure for the synthesis of intermediate 4f of example 2, 7g (1.56 g, yield 45%) of a pale yellow solid was obtained.

Compound 7:

referring to the synthetic procedure of compound 1 of example 1, compound 7 (188.5 mg, yield 90%) was obtained as an off-white solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.50(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),6.82(d,J＝8.7Hz,1H),6.64(s,2H),6.21(d,J＝2.6Hz,1H),6.09(dd,J＝8.7,2.6Hz,1H),3.92(s,6H),3.88(d,J＝1.2Hz,6H),3.88–3.84(m,2H),3.82(s,3H),3.57(dd,J＝9.2,7.1Hz,2H),3.31(dd,J＝9.4,3.5Hz,2H),3.23–3.13(m,2H),1.61(s,2H).LCMS m/z[M+H] ⁺ :517.2.

Example 6: preparation of Compound 8

Compound 8:

referring to the synthetic procedure of compound 1 of example 1, compound 8 (106.4 mg, yield 47%) was obtained as an off-white solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.90(d,J＝2.5Hz,1H),6.82(d,J＝8.7Hz,1H),6.58(d,J＝2.2Hz,2H),6.45(t,J＝2.2Hz,1H),6.21(d,J＝2.6Hz,1H),6.09(dd,J＝8.7,2.7Hz,1H),4.15(dd,J＝11.3,7.3Hz,2H),3.87(d,J＝4.0Hz,4H),3.83(s,6H),3.81(s,3H),3.71(q,J＝7.0Hz,1H),3.56(dd,J＝9.3,7.1Hz,2H),3.30(dd,J＝9.4,3.6Hz,2H),3.15(s,2H).LCMS m/z[M+H] ⁺ :487.2.

Example 7: preparation of Compound 9

Compound 9:

referring to the synthetic procedure of compound 1 of example 1, compound 9 (107.5 mg, yield 47%) was obtained as an off-white solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.50(d,J＝2.5Hz,1H),8.00–7.80(m,1H),7.02(dd,J＝8.3,2.1Hz,1H),6.97–6.90(m,2H),6.81(t,J＝8.9Hz,1H),6.15(d,J＝62.8Hz,2H),4.14(dt,J＝14.7,7.3Hz,2H),3.94(s,3H),3.91(s,3H),3.89–3.86(m,3H),3.85–3.81(m,3H),3.71(q,J＝7.0Hz,2H),3.58(d,J＝7.0Hz,2H),3.41–3.24(m,2H),3.17(s,2H).LCMS m/z[M+H] ⁺ :487.2.

Example 8: preparation of Compound 10

Compound 10:

referring to the synthetic procedure of compound 1 of example 1, compound 10 (90 mg, yield 85%) was obtained as an off-white solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.39(d,J＝2.4Hz,1H),7.88(d,J＝2.4Hz,1H),7.47(d,J＝7.3Hz,2H),7.35(t,J＝7.6Hz,2H),7.24(s,1H),6.92(d,J＝4.2Hz,2H),6.82(d,J＝8.7Hz,1H),6.20(d,J＝2.6Hz,1H),6.09(dd,J＝8.7,2.7Hz,1H),4.14(dd,J＝11.4,7.3Hz,2H),3.88(s,3H),3.84(dd,J＝11.5,4.0Hz,2H),3.81(s,3H),3.56(dd,J＝9.3,7.1Hz,2H),3.29(dd,J＝9.4,3.6Hz,2H),3.15(dq,J＝7.6,4.4Hz,2H).LCMS m/z[M+H] ⁺ :453.2.

Example 9: preparation of Compound 11

Intermediate 11c:

referring to the synthetic procedure for intermediate 1c of example 1, the reaction solvent was replaced with acetonitrile. Yellow solid 11c (1.41 g, 58% yield) was obtained.

Intermediate 11d:

referring to the synthetic procedure for intermediate 1h of example 1, 11d (1083 mg) was obtained as a pale yellow solid.

Intermediate 11f:

referring to the procedure for the synthesis of intermediate 4f of example 2, 11f (728 mg, yield 52%) was obtained as a pale yellow solid.

Compound 11:

referring to the synthetic procedure of compound 1 of example 1, compound 11 (230.4 mg, yield 82%) was obtained as an off-white solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.56(d,J＝2.5Hz,1H),7.95(d,J＝2.5Hz,1H),7.04–6.94(m,2H),6.86(d,J＝8.1Hz,1H),6.67(s,2H),6.10(s,1H),4.43(s,2H),4.01–3.94(m,2H),3.92(s,9H),3.89(d,J＝2.3Hz,6H),2.76(s,2H).LCMS m/z[M+H] ⁺ :488.2.

Example 10: preparation of Compound 12

Compound 12:

referring to the synthetic procedure of compound 1 of example 1, compound 12 (256.22 mg, yield 90%) was obtained as an off-white solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.57(d,J＝2.5Hz,1H),7.96(d,J＝2.5Hz,1H),7.03–6.94(m,2H),6.86(d,J＝8.2Hz,1H),6.61(d,J＝2.2Hz,2H),6.47(t,J＝2.2Hz,1H),6.09(s,1H),4.42(d,J＝3.0Hz,2H),4.06(t,J＝5.5Hz,2H),3.92(s,3H),3.90(s,3H),3.85(s,6H),2.77(s,2H).LCMS m/z[M+H] ⁺ :458.2.

Example 11: preparation of Compound 13

Compound 13:

referring to the synthetic procedure for compound 1 of example 1, a yellow color was obtained Solid compound 13 (69.5 mg, 66% yield). ¹ H NMR(500MHz,CDCl ₃ )δ8.46(d,J＝2.4Hz,1H),7.96(d,J＝2.4Hz,1H),7.49(d,J＝7.4Hz,2H),7.37(t,J＝7.6Hz,2H),7.29(t,J＝7.3Hz,1H),7.05–6.91(m,2H),6.82(d,J＝8.7Hz,1H),6.63(d,J＝2.2Hz,1H),6.51(dd,J＝8.6,2.6Hz,1H),4.02–3.91(m,4H),3.87(d,J＝21.6Hz,6H),3.40–3.13(m,4H).LCMS m/z[M+H] ⁺ :427.2.

Example 12: preparation of Compound 14

Compound 14:

referring to the synthetic procedure for compound 1, example 1, the base is exchanged for K ₂ CO ₃ The solvent is changed into DMF, and the mixture is heated to 150 ℃ by a microwave synthesizer and reacted for 1h. Compound 14 (50 mg, yield 45%) was obtained as a pale yellow solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ8.86(d,J＝2.5Hz,1H),8.52(d,J＝2.5Hz,1H),8.04(s,1H),7.96(d,J＝8.4Hz,2H),7.83(d,J＝8.4Hz,2H),7.41(s,1H),6.83(d,J＝8.8Hz,1H),6.69(d,J＝2.7Hz,1H),6.47(dd,J＝8.7,2.7Hz,1H),3.88–3.83(m,4H),3.76(s,3H),3.69(s,3H),3.25–3.18(m,4H).LCMS m/z[M+H] ⁺ :444.2.

Example 13: preparation of Compound 15

Compound 15:

referring to the synthetic procedure of compound 1 of example 1, compound 15 (85 mg, yield 80%) was obtained as a white solid. ¹ HNMR(500MHz,CDCl ₃ )δ8.61(d,J＝2.5Hz,1H),8.00(d,J＝2.5Hz,1H),7.55–7.45(m,4H),6.88–6.68(m,2H),6.63(s,1H),6.52(d,J＝10.9Hz,1H),5.81(d,J＝17.6Hz,1H),5.31(d,J＝10.9Hz,1H),4.04–3.92(m,4H),3.90(s,3H),3.85(s,3H),3.35–3.17(m,4H).LCMS m/z[M+H] ⁺ :427.2.

Example 14: preparation of Compound 16

Intermediate 16c:

compounds 16a (2 g,9.22 mmol), 16b (3.95 g,9.22 mmol) and Cs were combined ₂ CO ₃ (3.31 g,10.14 mmol) was placed in a 125mL round bottom flask, acetonitrile (MeCN, 15 mL) was added and refluxed overnight in an oil bath at 90 ℃. Post-treatment: the reaction was cooled to room temperature, filtered and the filter cake washed with DCM (5 mL). The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (EtOAc/Petroleum ether system elution) to give 16c (2.9 g, 86% yield) as a white solid.

Intermediate 16e:

referring to the synthetic procedure of compound 1 of example 1, pale yellow solid 16e (1.22 g, yield 98%) was obtained.

Intermediate 16f:

intermediate 16e (1.20 g,2.64 mmol) was placed in a 100mL round bottom flask, trifluoroacetic acid (TFA, 10 mL) was added dropwise and reacted at room temperature for 1h. TLC detection reaction was complete. The reaction solution was concentrated under reduced pressure to give 16f (1.1 g) as a yellow solid.

Compound 16:

compound 16f (100 mg,0.26 mmol) and 16g (46 mg,0.24 mmol) were placed in a 25mL round bottom flask, dissolved in DCM (5 mL) and slowly added dropwise triethylamine (Et) with stirring at room temperature ₃ N,29mg,0.29 mmol) to make the system slightly alkaline, and the reaction was completed at room temperature. Post-treatment: the reaction solution was dried by spin-drying, and the obtained crude product was purified by silica gel column chromatography (EtOAc/Petroleum ether system elution) to give compound 16 (115 mg, yield 87%) as a white solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.54(d,J＝2.5Hz,1H),7.93(d,J＝2.5Hz,1H),7.67(d,J＝8.2Hz,2H),7.35(d,J＝8.1Hz,2H),6.64(s,2H),3.91(s,6H),3.88(s,3H),3.83–3.77(m,4H),3.23–3.17(m,4H),2.44(s,3H).LCMS m/z[M+H] ⁺ :509.2.

Example 15: preparation of Compound 19

Intermediate 19d:

referring to the step of synthesizing intermediate 1g of example 1, compound 19d (667 mg, yield 75%) was obtained as a tan solid. Intermediate 19e:

referring to the synthetic procedure for intermediate 1h of example 1, crude compound 19e (750 mg) was obtained as a pale yellow solid.

Intermediate 19g:

referring to the procedure for the synthesis of intermediate 4f of example 2, 19g (600 mg, yield 80%) of a pale yellow solid was obtained.

Compound 19:

referring to the synthetic procedure of compound 1 of example 1, compound 19 (80 mg, yield 75%) was obtained as a pale yellow solid. ¹ HNMR(500MHz,CDCl ₃ )δ8.50(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),6.64(s,2H),5.79(s,2H),4.17–4.13(m,2H),3.92(s,6H),3.87(d,J＝9.0Hz,11H),3.77(s,3H),3.67–3.59(m,2H),3.33(dd,J＝9.5,3.6Hz,2H),3.18(s,2H).LCMS m/z[M+H] ⁺ :547.2.

Example 16: preparation of Compound 20

Intermediate 20c:

referring to the procedure for the synthesis of intermediate 16c of example 14, white solid 20c (1680 mg, yield 81%) was obtained.

Intermediate 20e:

Referring to the synthetic procedure of compound 1 of example 1, compound 20e (1890 mg, yield 77%) was obtained as a pale yellow solid. Intermediate 20f:

referring to the synthetic procedure for intermediate 16f of example 14, compound 20f (750 mg, 94% yield) was obtained as a tan solid. Compound 20:

compound 20f (269 mg,0.65 mmol), 20g (100 mg,0.43 mmol) and Cs ₂ CO ₃ (560 mg,1.72 mmol) was placed in a 25mL round bottom flask, meCN (10 mL) was added and dissolved, and the reaction was allowed to warm to 60℃with stirring overnight. TLC detection of completion of reaction, cooling the reaction solution to room temperature, filtering, washing with DCM (5 mL)The filter cake, filtrate, was concentrated under reduced pressure. Adding H to the concentrate ₂ O (5 mL) and extracted with EtOAc (5 mL. Times.3). Combining the organic phases with H in sequence ₂ O (5 mL. Times.2) and aq.NaCl (10 mL), placed in anhydrous Na ₂ SO ₄ And (5) drying. Filtering to remove anhydrous Na ₂ SO ₄ The filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (MeOH/DCM system elution) to give compound 20 (190 mg, yield 83%) as a pale yellow solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),6.88(d,J＝1.5Hz,1H),6.85–6.75(m,2H),6.65(s,2H),4.08–4.00(m,2H),3.92(s,6H),3.88(s,3H),3.86(d,J＝3.0Hz,6H),3.80(dd,J＝11.2,3.3Hz,2H),3.56(s,2H),2.98(s,2H),2.80–2.63(m,2H),2.57(dd,J＝9.4,2.9Hz,2H).LCMS m/z[M+H] ⁺ :531.3.

Example 17: preparation of Compound 22

Compound 22:

referring to the synthetic procedure of example 16, compound 22 (189 mg, yield 87%) was obtained as a white solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.54(d,J＝2.5Hz,1H),7.92(d,J＝2.5Hz,1H),6.93(d,J＝1.6Hz,1H),6.89–6.80(m,2H),6.65(s,2H),3.92(s,6H),3.91(s,3H),3.88(d,J＝1.5Hz,6H),3.80(q,J＝5.3Hz,4H),3.52(s,2H),2.67–2.54(m,4H).LCMS m/z[M+H] ⁺ :505.2.

Example 18: preparation of Compound 24

Intermediate 24d:

referring to the step of synthesizing intermediate 1g of example 1, a pale yellow solid was obtained 24d (975 mg, yield 71%).

Intermediate 24e:

referring to the synthetic procedure for intermediate 1h of example 1, crude white solid 24e (1120 mg) was obtained.

Intermediate 24g:

referring to the procedure for the synthesis of intermediate 4f of example 2, 24g (600 mg, 83% yield) of a pale brown solid was obtained.

Compound 24:

referring to the synthetic procedure of compound 1 of example 1, compound 24 (450 mg, yield 75%) was obtained as a yellow solid. ¹ H NMR(500MHz,CDCl ₃ )δ8.48(d,J＝2.4Hz,1H),7.84(d,J＝2.4Hz,1H),6.80(d,J＝8.7Hz,1H),6.63(s,2H),6.61(d,J＝2.6Hz,1H),6.49(dd,J＝8.7,2.7Hz,1H),4.14(s,4H),3.92(s,6H),3.88(s,6H),3.84(s,3H),3.22–2.95(m,4H),2.04–2.00(m,4H).LCMS m/z[M+H] ⁺ :531.3.

Example 19: preparation of Compound 26

Compound 26:

compound 26a (100 mg,0.23 mmol) and 26b (113 mg,0.69 mmol) were placed in a 25mL round bottom flask, dissolved in DCM (5 mL), stirred for about 10min, and sodium triacetoxyborohydride (NaBH (OAc) ₃ 2910 mg,1.38 mmol) was stirred overnight at room temperature. Post-treatment: to the reaction was added saturated sodium bicarbonate solution (aq. NaHCO ₃ 5 mL) to neutralize excess NaBH (OAc) ₃ With DCM (5 mL. Times.3) and aq. NaHCO ₃ (5 mL. Times.3) solution extraction, aq. NaCl (10 mL. Times.1) washing, combining organic phases placed in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give compound 26 (110 mg, yield 87%) as a yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),6.85–6.74(m,3H),6.65(s,2H),4.24(s,4H),4.05(dd,J＝11.1,8.0Hz,2H),3.92(s,6H),3.88(s,3H),3.77(dd,J＝11.2,3.3Hz,2H),3.53(s,2H),2.98(s,2H),2.82–2.68(m,2H),2.56(dd,J＝9.4,2.9Hz,2H).LCMS m/z[M+H] ⁺ :529.2.

Example 20: preparation of Compound 27

Compound 27:

referring to the synthetic procedure for compound 26 of example 19, compound 27a (100 mg,0.23 mmol) and 27b (102 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 27 as a yellow solid (105 mg, 89% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),7.15(s,1H),7.01(d,J＝9.8Hz,1H),6.70(d,J＝8.1Hz,1H),6.65(s,2H),4.55(t,J＝8.7Hz,2H),4.04(dd,J＝11.1,8.0Hz,2H),3.92(s,6H),3.88(s,3H),3.77(dd,J＝11.2,3.3Hz,2H),3.54(s,2H),3.18(t,J＝8.7Hz,2H),3.05–2.91(m,2H),2.79–2.67(m,2H),2.53(dd,J＝9.4,3.0Hz,2H).LCMS m/z[M+H] ⁺ :513.2.

Example 21: preparation of Compound 28

Compound 28:

referring to the synthetic procedure for compound 26 of example 19, compound 28a (100 mg,0.23 mmol) and 28b (94 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 28 as a yellow solid (110 mg, 95% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),7.36–7.30(m,1H),7.23(td,J＝8.1,1.6Hz,1H),6.99–6.82(m,2H),6.65(s,2H),4.06(dd,J＝11.1,8.1Hz,2H),3.92(s,6H),3.88(s,3H),3.82(s,3H),3.79(d,J＝3.3Hz,2H),3.69(s,2H),2.99(s,2H),2.88–2.75(m,2H),2.61(dd,J＝9.4,3.0Hz,2H).LCMS m/z[M+H] ⁺ :501.2.

Example 22: preparation of Compound 29

Compound 29:

referring to the synthetic procedure for compound 26 of example 19, compound 29a (60 mg,0.14 mmol) and 29b (64 mg,0.42 mmol) were subjected to one-step reductive amination to afford compound 29 as a brown solid (48 mg, yield 66%). ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),6.89(s,1H),6.78(s,2H),6.65(s,2H),5.30(s,1H),4.05(dd,J＝11.1,8.1Hz,2H),3.92(s,6H),3.88(s,3H),3.87(s,3H),3.77(dd,J＝11.2,3.3Hz,2H),3.53(s,2H),2.97(s,2H),2.80–2.63(m,2H),2.55(dd,J＝9.4,2.8Hz,2H).LCMS m/z[M+H] ⁺ :517.2.

Example 23: preparation of Compound 30

Compound 30:

referring to the synthetic procedure for compound 26 of example 19, compound 30a (100 mg,0.23 mmol) and 30b (118 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 30 as a pale brown solid (115 mg, yield 93%). ¹ H NMR(400MHz,CDCl ₃ )δ8.52(d,J＝2.5Hz,1H),7.90(d,J＝2.5Hz,1H),7.32(d,J＝2.0Hz,1H),7.16(dd,J＝8.4,2.0Hz,1H),6.86(d,J＝8.4Hz,1H),6.65(s,2H),4.06(dd,J＝11.2,8.1Hz,2H),3.92(s,6H),3.88(s,6H),3.77(dd,J＝11.2,3.4Hz,2H),3.53(s,2H),2.97(s,2H),2.74–2.62(m,2H),2.55(dd,J＝9.3,2.8Hz,2H).LCMS m/z[M+H] ⁺ :535.2.

Example 24: preparation of Compound 31

Compound 31:

referring to the synthetic procedure for compound 26 of example 19, compound 31a (100 mg,0.23 mmol) and 31b (120 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 31 as a yellow solid (100 mg, 81% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.53(d,J＝2.5Hz,1H),7.91(d,J＝2.5Hz,1H),7.56(d,J＝8.1Hz,2H),7.44(d,J＝8.0Hz,2H),6.66(s,2H),4.08(dd,J＝11.1,8.0Hz,2H),3.92(s,6H),3.89(s,3H),3.78(dd,J＝11.2,3.4Hz,2H),3.68(s,2H),3.06–2.94(m,2H),2.77–2.65(m,2H),2.62(dd,J＝9.4,2.6Hz,2H).LCMS m/z[M+H] ⁺ :539.2.

Example 25: preparation of Compound 32

Compound 32:

referring to the synthetic procedure for compound 26 of example 19, compound 32a (100 mg,0.23 mmol) and 32b (92 mg,0.69 mmol) were subjected to one-step reductive amination to give compound 32 (105 mg, yield 91%) as a pale yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),7.05(dd,J＝15.1,7.0Hz,3H),6.65(s,2H),4.04(dd,J＝11.1,8.0Hz,2H),3.92(s,6H),3.88(s,3H),3.78(dd,J＝11.2,3.3Hz,2H),3.56(s,2H),2.97(s,2H),2.80–2.68(m,2H),2.54(dd,J＝9.4,3.0Hz,2H),2.24(d,J＝2.0Hz,6H).LCMS m/z[M+H] ⁺ :499.3.

Example 26: preparation of Compound 33

Compound 33:

referring to the synthetic procedure for compound 26 of example 19, compound 33a (100 mg,0.23 mmol) and 33b (90 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 33 as a yellow solid (46 mg, 40% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.52(d,J＝2.5Hz,1H),7.90(d,J＝2.5Hz,1H),7.43(d,J＝8.1Hz,2H),7.27(d,J＝8.0Hz,2H),6.65(s,2H),4.12–4.03(m,2H),3.92(s,6H),3.89(s,3H),3.76(dd,J＝11.2,3.3Hz,2H),3.61(s,2H),2.97(s,2H),2.74–2.64(m,2H),2.58(dd,J＝9.3,2.5Hz,2H).LCMS m/z[M+H] ⁺ :495.2.

Example 27: preparation of Compound 34

Compound 33:

referring to the synthetic procedure for compound 26 of example 19, compound 34a (100 mg,0.23 mmol) and 34b (90 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 34 as a pale yellow solid (96 mg, 84% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.53(d,J＝2.5Hz,1H),7.91(d,J＝2.5Hz,1H),7.60(d,J＝8.2Hz,2H),7.44(d,J＝8.2Hz,2H),6.65(s,2H),4.14–4.06(m,2H),3.93(s,6H),3.89(s,3H),3.76(dd,J＝11.2,3.4Hz,2H),3.68(s,2H),3.00(s,2H),2.72–2.59(m,4H).LCMS m/z[M+H] ⁺ :496.2.

Example 28: preparation of Compound 35

Compound 35:

referring to the synthetic procedure for compound 26 of example 19, compound 35a (60 mg,0.14 mmol) and 35b (59 mg,0.42 mmol) were subjected to one-step reductive amination to give compound 35 (60 mg, yield 85%) as a pale yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.52(d,J＝2.5Hz,1H),7.90(d,J＝2.5Hz,1H),7.29–7.24(m,4H),6.65(s,2H),4.07(dd,J＝11.2,8.1Hz,2H),3.92(s,6H),3.89(s,3H),3.76(dd,J＝11.3,3.5Hz,2H),3.58(s,2H),2.98(s,2H),2.74–2.63(m,2H),2.57(dd,J＝9.3,2.7Hz,2H).LCMS m/z[M+H] ⁺ :505.2.

Example 29: preparation of Compound 36

Compound 36:

referring to the synthetic procedure for compound 26 of example 19, compound 36a (100 mg,0.23 mmol) and 36b (103 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 36 as a white solid (100 mg, 85% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),7.16(d,J＝8.6Hz,2H),6.69(d,J＝8.7Hz,2H),6.65(s,2H),4.03(dd,J＝11.2,8.1Hz,2H),3.92(s,6H),3.88(s,3H),3.78(dd,J＝11.2,3.3Hz,2H),3.54(s,2H),2.96(s,2H),2.93(s,6H),2.81–2.69(m,2H),2.53(dd,J＝9.4,3.0Hz,2H).LCMS m/z[M+H] ⁺ :514.3.

Example 30: preparation of Compound 37

Compound 37:

referring to the synthetic procedure for compound 26 of example 19, compound 37a (50 mg,0.12 mmol) and 37b (38 mg,0.36 mmol) were subjected to one-step reductive amination to afford compound 37 as a yellow solid (50 mg, 88% yield). ¹ H NMR(500MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.88(d,J＝2.5Hz,1H),7.37–7.29(m,5H),6.64(s,2H),4.02(dd,J＝11.2,8.0Hz,2H),3.92(s,6H),3.88(s,3H),3.80(dd,J＝11.2,3.2Hz,2H),3.68(s,2H),3.10–2.90(m,2H),2.83(dd,J＝9.3,6.8Hz,2H),2.60(dd,J＝9.6,3.3Hz,2H).LCMS m/z[M+H] ⁺ :471.2.

Example 31: preparation of Compound 38

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Compound 38:

referring to the synthetic procedure for compound 26 of example 19, compound 38a (100 mg,0.23 mmol) and 38b (92 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 38 as a brown oil (105 mg, 91% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.50(d,J＝2.4Hz,1H),7.88(d,J＝2.4Hz,1H),7.29(d,J＝7.3Hz,1H),7.22–7.15(m,4H),6.63(s,2H),4.04(dd,J＝11.1,8.0Hz,2H),3.91(s,6H),3.88(s,3H),3.79(dd,J＝11.2,2.9Hz,2H),2.97(s,2H),2.74–2.69(m,2H),2.64(t,J＝7.6Hz,2H),2.56(dd,J＝9.3,2.7Hz,2H),2.50–2.42(m,2H).LCMS m/z[M+H] ⁺ :499.3.

Example 32: preparation of Compound 39

Compound 39:

referring to the synthetic procedure for compound 26 of example 19, compound 39a (100 mg,0.23 mmol) and 39b (132 mg,0.69 mmol) were subjected to one-step reductive amination to give compound 39 (101 mg, yield 79%) as a pale yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),7.21(d,J＝8.5Hz,2H),6.86(d,J＝8.6Hz,2H),6.65(s,2H),4.05(dd,J＝11.1,8.0Hz,2H),3.92(s,6H),3.88(s,3H),3.87–3.83(m,4H),3.77(dd,J＝11.2,3.2Hz,2H),3.55(s,2H),3.17–3.10(m,4H),2.97(s,2H),2.78–2.66(m,2H),2.54(dd,J＝9.4,2.7Hz,2H).LCMS m/z[M+H] ⁺ :556.3.

Example 33: preparation of Compound 40

Compound 40:

referring to the synthetic procedure for compound 26 of example 19, compound 40a (100 mg,0.23 mmol) and 40b (126 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 40 as a pale yellow solid (120 mg, yield 95%). ¹ H NMR(400MHz,CDCl ₃ )δ8.52(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),7.55(dd,J＝19.1,7.7Hz,4H),7.37(ddt,J＝22.1,14.7,7.3Hz,5H),6.65(s,2H),4.07(dd,J＝11.0,8.0Hz,2H),3.91(s,6H),3.88(s,3H),3.79(dd,J＝11.2,3.1Hz,2H),3.66(s,2H),2.98(s,2H),2.80–2.67(m,2H),2.61(d,J＝11.6Hz,2H).LCMS m/z[M+H] ⁺ :547.3.

Example 34: preparation of Compound 41

Compound 41:

compounds 41a (100 mg,0.23 mmol) and 41b (47 mg,0.28 mmol) were placed in a 25mL round bottom flask, dissolved in DCM (3 mL) and N, N-diisopropylethylamine (DIPEA, 119mg,0.92 mmol) was added sequentially with ice-bath stirring, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 115mg,0.60 mmol), 1-hydroxybenzotriazole (HOBt, 41mg,0.30 mmol) and stirred under nitrogen to room temperature overnight. To the reaction solution was added aq. NaHCO dropwise ₃ (3 mL) with DCM (5 mL. Times.3) and aq. NaHCO ₃ (5 mL. Times.3) solution extraction, aq. NaCl (10 mL) wash, combined organic phases placed in anhydrous Na ₂ SO ₄ And (5) drying. Filtering the drying agent, concentrating the filtrate under reduced pressure, and passing through columnChromatography gave compound 41 (100 mg, yield 82%) as a pale yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.4Hz,1H),7.91(d,J＝2.4Hz,1H),7.16–6.97(m,2H),6.81(d,J＝8.0Hz,1H),6.66(s,2H),6.00(s,2H),4.09(dd,J＝16.4,9.2Hz,3H),3.93(s,6H),3.88(s,3H),3.85(s,2H),3.74–3.46(m,3H),3.09(d,J＝16.3Hz,2H).LCMS m/z[M+H] ⁺ :529.2.

Example 35: preparation of Compound 42

Compound 42:

referring to the synthetic procedure for compound 26 of example 19, compound 42a (100 mg,0.23 mmol) and 41b (90 mg,0.69 mmol) were subjected to one-step reductive amination to afford compound 41 (100 mg, 88% yield) as a pale yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.50(d,J＝2.5Hz,1H),7.88(d,J＝2.5Hz,1H),7.42(dd,J＝6.5,3.1Hz,2H),7.32–7.26(m,3H),6.64(s,2H),4.11(dd,J＝11.2,8.0Hz,2H),3.91(s,6H),3.88(s,3H),3.82(dd,J＝11.3,3.2Hz,2H),3.66(s,2H),3.03(s,2H),2.98–2.90(m,2H),2.73(d,J＝11.4Hz,2H).LCMS m/z[M+H] ⁺ :495.2.

Example 36: preparation of Compound 43

Compound 43:

referring to the synthetic procedure of compound 41 of example 34, compound 43a (80 mg,0.19 mmol) and 43b (34 mg,0.23 mmol) were subjected to amide condensation to give compound 43 (77 mg, yield 80%) as a pale yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.52(d,J＝2.5Hz,1H),7.91(d,J＝2.5Hz,1H),7.59–7.52(m,2H),7.46–7.34(m,3H),6.65(s,2H),4.12(td,J＝19.2,17.6,7.6Hz,4H),3.92(s,6H),3.88(s,3H),3.82(dq,J＝12.9,7.2,6.6Hz,3H),3.56(d,J＝12.8Hz,1H),3.14(s,2H).LCMS m/z[M+H] ⁺ :509.2.

Example 37: preparation of Compound 44

Compound 44:

compound 44a (80 mg,0.19 mmol) was placed in a 25mL round bottom flask, dissolved in DCM (2 mL), stirred in an ice bath and Et added ₃ N (0.3 mL), under the protection of nitrogen, a solution of 44b (38 mg,0.23 mmol) in DCM (1 mL) was slowly added dropwise, and the reaction was gradually warmed to room temperature and stirred for about 2 h. Post-treatment: to the reaction solution was added aq. NaHCO dropwise ₃ (3 mL) with DCM (5 mL. Times.3) and aq. NaHCO ₃ (5 mL. Times.3) solution extraction, aq. NaCl (10 mL) wash, put in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (DCM/MeOH system elution) to give compound 44 (89 mg, yield 91%) as a pale yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.42(d,J＝2.4Hz,1H),7.81(d,J＝2.4Hz,1H),7.63(d,J＝15.5Hz,1H),7.48–7.43(m,2H),7.32–7.25(m,3H),6.63(d,J＝15.5Hz,1H),6.56(s,2H),4.08–3.99(m,2H),3.94–3.85(m,2H),3.83(s,6H),3.79(s,3H),3.72(td,J＝11.0,4.9Hz,2H),3.56(td,J＝14.3,13.0,4.9Hz,2H),3.04(dd,J＝44.1,6.8Hz,2H).LCMS m/z[M+H] ⁺ :511.2.

Example 38: preparation of Compound 45

Compound 45:

referring to the synthetic procedure for compound 41 of example 34, compound 45a (100 mg,0.23 mmol) and 45b (41 mg,0.28 mmol) were subjected to amide condensation to give compound 45 (100 mg, yield 85%) as a pale yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.91(d,J＝2.5Hz,1H),7.50(d,J＝8.3Hz,2H),7.43(d,J＝8.2Hz,2H),6.66(s,2H),5.81(d,J＝17.6Hz,1H),5.32(d,J＝11.1Hz,1H),4.17–4.08(m,2H),4.02(d,J＝15.6Hz,2H),3.92(s,6H),3.88(s,3H),3.78(s,1H),3.70(s,2H),3.50(d,J＝4.4Hz,1H),3.26–2.97(m,2H).LCMS m/z[M+H] ⁺ :511.2.

Example 39: preparation of Compound 46

Compound 46:

referring to the synthetic procedure for compound 41 of example 34, compound 46a (100 mg,0.23 mmol) and 46b (52 mg,0.28 mmol) were subjected to amide condensation to give compound 46 as a dark yellow solid (80 mg, yield 63%). ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.91(d,J＝2.5Hz,1H),7.59(d,J＝2.0Hz,1H),7.47(dd,J＝8.5,2.1Hz,1H),6.95(d,J＝8.5Hz,1H),6.65(s,2H),4.10(dd,J＝16.7,9.5Hz,4H),3.94(s,3H),3.93(s,6H),3.89(s,3H),3.69(s,4H),3.10(d,J＝19.2Hz,2H).LCMS m/z[M+H] ⁺ :549.2.

Example 40: preparation of Compound 47

Compound 47:

referring to the synthetic procedure for compound 41 of example 34, compound 47a (100 mg,0.23 mmol) and 47b (51 mg,0.28 mmol) were subjected to amide condensation to give compound 47 as a dark yellow solid (87 mg, 69% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.91(d,J＝2.5Hz,1H),7.21–7.05(m,2H),6.86(d,J＝8.2Hz,1H),6.65(s,2H),4.15–4.09(m,2H),4.06(s,2H),3.93(s,6H),3.92(s,6H),3.89(s,3H),3.71(d,J＝15.3Hz,4H),3.10(d,J＝18.8Hz,2H).LCMS m/z[M+H] ⁺ :545.2.

Example 41: preparation of Compound 48

Compound 48:

referring to the synthetic procedure for compound 41 of example 34, compound 48a (100 mg,0.23 mmol) and 48b (34 mg,0.28 mmol) were subjected to amide condensation to give compound 48 as a white solid (100 mg, yield 90%). ¹ H NMR(400MHz,CDCl ₃ )δ8.51(d,J＝2.4Hz,1H),7.91(d,J＝2.5Hz,1H),7.52(dd,J＝7.6,1.9Hz,2H),7.45–7.39(m,3H),6.65(s,2H),4.23–3.95(m,4H),3.92(s,6H),3.88(s,3H),3.79(dd,J＝11.2,6.9Hz,1H),3.73–3.61(m,2H),3.47(dd,J＝11.3,4.8Hz,1H),3.09(dt,J＝29.5,6.9Hz,2H).LCMS m/z[M+H] ⁺ :585.2.

Example 42: preparation of Compound 49

Compound 49:

referring to the synthetic procedure for compound 26 of example 19, compound 49a (100 mg,0.23 mmol) and 49b (121 mg,0.69 mmol) were subjected to one-step reductive amination to give compound 49 (99 mg, yield 77%) as a pale yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ8.52(d,J＝2.5Hz,1H),7.90(d,J＝2.5Hz,1H),7.42–7.34(m,2H),7.15(dd,J＝8.2,1.7Hz,1H),6.65(s,2H),4.07(dd,J＝11.1,8.0Hz,2H),3.92(s,6H),3.89(s,3H),3.77(dd,J＝11.2,3.4Hz,2H),3.56(s,2H),2.99(s,2H),2.73–2.62(m,2H),2.58(dd,J＝9.3,2.6Hz,2H).LCMS m/z[M+H] ⁺ :539.2.

Example 43: preparation of Compound 50

Intermediate 50c:

referring to the synthetic procedure for compound 1 of example 1, the base was exchanged for potassium carbonate. Compound 50c (295 mg, yield 85%) was obtained as a pale yellow solid.

Intermediate 50d:

referring to the step of synthesis of intermediate 1h of example 1, crude 50d (216 mg,0.56 mmol) was obtained as a pale yellow solid, which was directly added to the next step.

Compound 50:

referring to the synthetic procedure for compound 26 of example 19, compounds 50d (216 mg,0.56 mmol) and 50e (279 mg,1.68 mmol) were subjected to one-step reductive amination to afford compound 50 (1)80mg, 64% yield). ¹ H NMR(500MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),6.88(d,J＝1.3Hz,1H),6.84–6.77(m,2H),6.59(d,J＝2.2Hz,2H),6.44(t,J＝2.1Hz,1H),4.03(dd,J＝11.2,8.0Hz,2H),3.85(d,J＝2.2Hz,6H),3.83(s,6H),3.80(dd,J＝11.3,3.2Hz,2H),3.55(s,2H),2.96(s,2H),2.78–2.65(m,2H),2.56(d,J＝12.2Hz,2H).LCMS m/z[M+H] ⁺ :501.2.

Example 44: preparation of Compound 51

Intermediate 51c:

referring to the synthetic procedure for compound 1 of example 1, the base was exchanged for potassium carbonate. Yellow solid compound 51c (285 mg, yield 86%) was obtained.

Intermediate 51d:

referring to the synthesis step of intermediate 1h of example 1, crude 51d (196 mg,0.53 mmol) was obtained as a yellow solid. Compound 51:

Referring to the synthetic procedure for compound 26 of example 19, compound 51d (150 mg,0.40 mmol) and 51e (199mg, 1.20 mmol) were subjected to reductive amination to give compound 51 as a pale brown solid (120 mg, yield 61%). ¹ H NMR(500MHz,CDCl ₃ )δ8.45(d,J＝2.5Hz,1H),7.83(d,J＝2.5Hz,1H),6.94–6.90(m,2H),6.87(d,J＝7.9Hz,2H),6.81(q,J＝8.1Hz,2H),6.00(s,2H),4.02(dd,J＝11.2,8.1Hz,2H),3.86(s,3H),3.86(s,3H),3.79(dd,J＝11.2,3.3Hz,2H),3.56(s,2H),2.96(s,2H),2.79–2.66(m,2H),2.55(dd,J＝9.4,3.0Hz,2H).LCMS m/z[M+H] ⁺ :485.2.

Example 45: preparation of Compound 52

Intermediate 52c:

referring to the synthetic procedure for compound 1 of example 1, the base was exchanged for potassium carbonate. Compound 52c (300 mg, yield 82%) was obtained as a pale yellow solid.

Intermediate 52d:

referring to the synthetic procedure for intermediate 1h of example 1, crude 52d (210 mg,0.51 mmol) was obtained as a yellow solid. Compound 52:

referring to the synthetic procedure for compound 26 of example 19, compound 52d (150 mg,0.36 mmol) and 52e (178 mg,1.08 mmol) were subjected to reductive amination to give compound 52 as a yellowish green solid (121 mg, yield 63%). ¹ H NMR(500MHz,CDCl ₃ )δ8.51(d,J＝2.5Hz,1H),7.87(d,J＝2.5Hz,1H),7.39(d,J＝8.7Hz,2H),6.97(d,J＝8.8Hz,2H),6.88(d,J＝1.3Hz,1H),6.85–6.77(m,2H),4.01(dd,J＝11.1,8.0Hz,2H),3.90–3.87(m,4H),3.86(s,3H),3.85(s,3H),3.79(dd,J＝11.1,3.2Hz,2H),3.56(s,2H),3.25–3.17(m,4H),2.96(s,2H),2.79–2.67(m,2H),2.55(d,J＝9.4Hz,2H).LCMS m/z[M+H] ⁺ :526.3.

Example 46: preparation of Compound 53

Intermediate 53c:

referring to the synthetic procedure for compound 1 of example 1, the base was exchanged for potassium carbonate. Yellow solid compound 53c (239 mg, yield 65%) was obtained.

Intermediate 53d:

referring to the synthesis step of intermediate 1h of example 1, crude 53d (164 mg,0.40 mmol) was obtained as a pale yellow solid.

Compound 53:

referring to the synthetic procedure for compound 26 of example 19, compound 53d (164 mg,0.40 mmol) and 53e (166 mg,1.20 mmol) were subjected to reductive amination to afford compound 53 as a yellow solid (95 mg, 45% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.47(d,J＝2.5Hz,1H),8.33(d,J＝2.5Hz,1H),7.83(d,J＝2.5Hz,1H),7.61(dd,J＝8.8,2.6Hz,1H),6.90(d,J＝1.7Hz,1H),6.85–6.76(m,2H),6.71(d,J＝8.8Hz,1H),4.00(dd,J＝11.1,7.7Hz,2H),3.86(d,J＝2.5Hz,6H),3.85–3.77(m,6H),3.59(s,2H),3.57–3.52(m,4H),3.03–2.92(m,2H),2.85–2.69(m,2H),2.56(dd,J＝9.6,3.2Hz,2H).LCMS m/z[M+H] ⁺ 527.3 example 47: preparation of Compound 54

Intermediate 54c:

referring to the synthetic procedure for compound 1 of example 1, the base was exchanged for potassium carbonate. Yellow solid compound 54c (203 mg, yield 84%) was obtained.

Intermediate 54d:

referring to the synthetic procedure for intermediate 1h of example 1, crude 54d (140 mg,0.34 mmol) was obtained as a yellow solid. Compound 54:

referring to the synthetic procedure for compound 26 of example 19, compound 54d (140 mg,0.34 mmol) and 54e (169 mg,1.02 mmol) were reductively aminated to afford compound 54 as a gray solid (70 mg, 37% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.46(d,J＝2.5Hz,1H),8.30(d,J＝2.5Hz,1H),7.82(d,J＝2.5Hz,1H),7.54(dd,J＝8.9,2.6Hz,1H),6.93(s,1H),6.87–6.78(m,2H),6.71(d,J＝8.9Hz,1H),3.96(dd,J＝10.9,7.5Hz,2H),3.87(s,6H),3.81(dd,J＝11.1,2.6Hz,2H),3.62(s,2H),3.58(d,J＝5.3Hz,4H),3.00(s,2H),2.91–2.76(m,2H),2.57(dd,J＝9.4,2.8Hz,2H),1.66(s,6H).LCMS m/z[M+H] ⁺ :525.3.

Example 48: preparation of Compound 55

Intermediate 55c:

referring to the synthetic procedure for compound 1 of example 1, the base was exchanged for potassium carbonate. Compound 55c (326 mg, yield 77%) was obtained as a pale yellow solid.

Intermediate 55d:

referring to the synthesis step of intermediate 1h of example 1, crude 55d (250 mg,0.71 mmol) was obtained as a yellow solid. Compound 55:

referring to the synthetic procedure for compound 26 of example 19, reductive amination of compound 55d (200 mg,0.57 mmol) with 55e (284 mg,1.71 mmol) gives compound 55 as a pale yellow solid (150 mg, 57% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.37(d,J＝2.4Hz,1H),7.85(d,J＝2.4Hz,1H),7.49–7.44(m,2H),7.35(t,J＝7.6Hz,2H),7.26(d,J＝6.0Hz,1H),6.89(dd,J＝5.0,1.7Hz,3H),6.83–6.76(m,2H),4.00(dd,J＝11.2,8.0Hz,2H),3.85(s,6H),3.78(dd,J＝11.3,3.1Hz,2H),3.56(s,2H),3.00–2.89(m,2H),2.77–2.67(m,2H),2.55(dd,J＝9.4,2.9Hz,2H).LCMS m/z[M+H] ⁺ :467.2.

Example 49: preparation of Compound 56

Compound 56:

referring to the synthetic procedure for compound 41 of example 34, compound 56a (200 mg,0.48 mmol) and 56b (106 mg,0.58 mmol) were subjected to a one-step amide condensation reaction to give compound 56 (204 mg, yield 78%). ¹ H NMR(400MHz,CDCl ₃ )δ8.47(d,J＝2.5Hz,1H),8.33(d,J＝2.5Hz,1H),7.85(d,J＝2.5Hz,1H),7.62(dd,J＝8.8,2.6Hz,1H),7.18–7.08(m,2H),6.86(d,J＝8.2Hz,1H),6.72(d,J＝8.8Hz,1H),4.12(q,J＝7.1Hz,2H),4.09–3.97(m,2H),3.91(s,7H),3.87–3.83(m,4H),3.68(s,2H),3.57–3.54(m,4H),3.09(d,J＝22.8Hz,2H).LCMS m/z[M+H] ⁺ :541.2.

Example 50: preparation of Compound 57

Compound 57:

referring to the synthetic procedure for compound 41 of example 34, compound 57a (200 mg,0.49 mmol) and 57b (109 mg,0.59 mmol) were subjected to a one-step amide condensation reaction to give compound 57 (180 mg, yield 69%). ¹ H NMR(500MHz,CDCl ₃ )δ8.50(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),7.40(d,J＝8.7Hz,2H),7.18–7.05(m,2H),6.98(d,J＝8.8Hz,2H),6.85(d,J＝8.3Hz,1H),4.25–3.93(m,4H),3.91(s,6H),3.89–3.86(m,4H),3.61(d,J＝69.2Hz,4H),3.25–3.15(m,4H),3.08(d,J＝18.5Hz,2H).LCMS m/z[M+H] ⁺ :540.3.

Example 51: evaluation of in vitro anti-tumor Activity of the prepared Compounds

1. Experimental materials and instruments

Experimental materials: DMEM (biotech limited, biont, se, zhejiang); RPMI 1640 (Zhejiang Senrui Biotechnology Co., ltd.); fatal Bovine Serum (BI); PBS (zhejiang senrui biotechnology limited); trypsin (Zhejiang Senri Biotechnology Co., ltd.); DMSO (Coolaber); CCK-8 (Coolaber).

Experimental instrument: biosafety cabinet (Shanghai hundred Biotechnology Co., ltd.); a constant temperature carbon dioxide incubator (THERMO); an enzyme-linked immunoassay (Spark); inverted microscopes (Nikon); pipette set (Eppendorf); centrifuge (Beckman coulter).

Human colon cancer cell line: COLO205, SW480, HCT116.

2. Experimental procedure

1) Taking test cells in logarithmic growth phase, digesting and counting with pancreatin, diluting tumor cell suspension to 5×10 ⁴ The cells were inoculated into 96-well plates at a concentration of one/mL, and 100. Mu.L of the cell-containing medium was added to each well (1X 10 per well) except for the blank group, which was 100. Mu.L of the cell-free medium ³ Individual cells);

2) In wet condition containing 5% CO ₂ After incubation at 37 ℃ for 8 hours in the incubator, the original culture medium in the 96-well plate is sucked off, 100 mu L of culture medium without the compound to be tested is added to each well except the control and blank groups, 100 mu L of culture medium with the compound to be tested is added to each well (10% FBS/RPMI 1640 complete culture medium is used), 2 compound wells are arranged at each concentration, the wells without adding the compound to the cells are blank groups, the wells without adding the compound to the cells are control groups, and the wells with adding the compound to the cells are experimental groups. Compound a was selected as a positive control for the experiment;

3) At 37℃with 5% CO ₂ Culturing in a wet incubator for 72 hours;

4) Under dark conditions, 10. Mu.L of cck8 solution (5 mg/mL) was added per well at 37℃in 5% CO ₂ Culturing is continued for 1h in a wet incubator, and the absorbance value (OD value) of each well is measured at 450nm of the microplate reader;

5) The survival and inhibition were calculated using the following formula

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

Inhibition ratio = [ (Ac-As)/(Ac-Ab) ]. Times.100%

Calculating single concentration inhibition rate by using Excel; using GraphPadPrism 7.0 software, an S-type dose-survival curve was drawn using a nonlinear regression model and IC was calculated ₅₀ Values.

As: absorbance of experimental wells (cell-containing medium, cck8, test drug)

Ac: absorbance of control wells (cell-containing medium, cck8, vehicle (DMSO))

Ab: absorbance of blank wells (cell-free medium, cck8, vehicle (DMSO))

3. Experimental results

The proliferation inhibition effect of the prepared compound and the compound A with the positive control medicine structural formula of formula II on two colon cancer cell lines is measured according to the experimental method, and the results are shown in tables 1 and 2.

TABLE 1 Single concentration inhibition of target Compounds on three colon cancer cells

Table 1 shows the single concentration inhibition of three colon cancer cells by the target compounds

Note that: ^a results of two or more experiments are shown. ^b ND represents untested.

According to the single-concentration test result, most of the synthesized compounds have stronger antiproliferative capacity on three colorectal cancer cells than the positive control drug compound A. To further characterize antiproliferative capacity, we used compound a as a positive control, and determined half Inhibitory Concentrations (IC) of single-concentration high-inhibition compounds (5, 6, 15, 17, 20, 29) on three colorectal cancer cells ₅₀ ) The experimental results are shown in table 2.

TABLE 2 targetsHalf inhibitory concentration of compound on three colon cancer cells (IC ₅₀ )

4. Discussion of results

The proliferation test results of the colon cancer cell (HCT 116, SW480 and COLO 205) show that the anti-proliferation capacity of the compound 54 is improved by 9-10 times compared with that of the positive control medicine compound A, and the compound can be used as a lead compound for further optimization or for researching a preliminary verification test of an HSP110 protein target.

Example 52: molecular docking simulation determines that compound 7 (also known as HSP-007) has better binding effect with HSP110 protein

Compound 7 molecules were subjected to molecular docking and interaction pattern analysis with HSP110 protein using pymol2.3.0 software (as shown in fig. 1). The result shows that the binding energy of the compound 7 and the HSP110 protein is-8.6 kcal/mol, and the compound 7 and the HSP110 protein have better binding effect. As can be seen from the docking results, the compound 7 interacts with the HSP110 protein mainly through the formation of hydrogen bonds and hydrophobic forces, the compound 7 can form hydrogen bonds with Arg-73, ser-155 and Tyr-454 residues of the HSP110, and the lengths of the hydrogen bonds are respectively as followsThe method comprises the steps of carrying out a first treatment on the surface of the At the same time, the amino acid residues of Ala-151, tyr-604, phe-79, phe-230, pro-229, phe-147, gly-72, thr-149 and the like form a hydrophobic effect.

Example 53: protein quantification and Western Blot

1. Experimental materials and instruments

Experimental materials: antibodies (Table 3), BCA protein assay kit (Kagaku, applied biosciences), immobilon Western HRP substrate Millipore, immobilonWesternHRP substrate (Millipore), RIPA lysate (Soy Biotechnology Co., ltd.), PMSF (100 mM) (Soy Biotechnology Co., ltd.), phospho-enzyme inhibitorPreparation (Soy Biotechnology Co., ltd.), tween-20 (national medicine group chemical reagent Co., ltd.), 30% acr-Bis (Kagaku reagent Biotechnology Co., ltd.), fetal bovine serum (Biological Industries), 0.25% Trypsin-EDTA (Biological Industries), dimethyl sulfoxide DMSO (cell culture grade) (Thermo Fisher Scientific), 10X electrophoresis transfer buffer (transfer membrane liquid) (Soy Biotechnology Co., ltd.), 5X Tris-glycine electrophoresis buffer (Soy Biotechnology Co., ltd.), 1X TBST (Soy Biotechnology Co., ltd.), paraformaldehyde fixing solution (Soy Biotechnology Co., ltd.),Matrix(CORNING)、/>3000Reagent Transfections(TheroFisher SCIENTIFIC)、McCoy’s 5AMedium(Modified)(Biological Industries)、Leibovitz L-15Medium(Biological Industries)、PrimeScriptTM RT Master Mix(Perfect Real Time)(Takar)、TB Green ^TM Premix Ex Taq ^TM (Tli RNaseH Plus)(Takara)。

TABLE 3 antibodies related to the experiment

Experimental instrument: high speed centrifuges (Eppendorf), ultracentrifuges (Eppendorf), bench top high speed cryocentrifuges (Eppendorf), cell incubators (Thermo Fisher Scientific), chemiluminescent enzyme-labeled instruments (Sigma), ultra pure water instruments (Millipore), biosafety cabinet (Thermo Fisher Scientific), inverted fluorescence microscope (Nikon), upright fluorescence microscope (Nikon), ice maker (snowy ice maker), cell counter (Bio-rad), constant temperature water bath (shanghai Heng instruments inc.) and Western Blot electrophoresis instrument (Bio-rad).

2. Experimental procedure

(1) And (3) making a standard curve: 0.5mg/mL BSA was removed from the-20deg.C refrigerator and thawed at 4deg.C for use. They were diluted to 0, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL, respectively. The various reagents were added to the centrifuge tube in the proportions listed below.

Table 4 protein quantitative Standard Curve concentration preparation ratio

(2) Protein concentration determination: mu.L of double distilled water and 2. Mu.L of protein to be tested were added to each well of a 96-well plate, followed by 204. Mu.L of biuret reagent (solution A: solution B=50:1) per well.

(3) The sample is placed in a baking oven at 37 ℃ for 30min, and an absorbance value at 570nm is detected by an enzyme-labeled instrument.

(4) And drawing a standard curve and a standard equation according to the result, and solving the concentration of the protein according to the equation.

(5) Configuration: and drawing a standard curve according to the standard solution to measure the concentration of each protein, and preparing protein lysate with the same molar mass in different experimental groups. Heating at 100deg.C in a dry bath for 10min to denature protein.

(6) Glue making

(1) And cleaning the glass plate with double distilled water, airing, placing the glass plate on a glass plate frame, and adding the double distilled water for leak detection. And after water leakage is avoided, the double distilled water is sucked and abandoned.

(2) According to the detected molecular weight of the protein, preparing a separating gel solution with corresponding concentration, injecting the separating gel solution into a glass plate, covering an upper layer with isopropanol to remove bubbles, and standing for half an hour.

(3) After the gel had set, the upper isopropanol layer was sucked off with filter paper.

(4) The concentrated gel was poured onto the separation gel and slowly inserted into a suitable Teflon comb to prevent bubbles from being generated and allowed to stand for 10min.

TABLE 5 concentration of separation gel and corresponding protein molecular weight size

Table 6 formulation of separation gel and concentrated gel

(7) Loading sample

(1) After removing the comb and fixing the SDS-PAGE gel in the electrophoresis tank, 1 Xof electrophoresis buffer was poured.

(2) Protein samples are sequentially added according to the experimental sequence, and protein pre-dyeing markers are added at two ends of the protein samples to serve as references. Constant pressure 90V, about 30min. When the bromophenol blue front reached the boundary between the concentrate and the separator, the voltage was increased to 120V for about 60min until the bromophenol blue band reached the bottom of the separator.

(3) The glass plates were removed and the SDS-PAGE gel carefully removed.

(8) Transferring: the PVDF membrane was activated with methanol for about 10s. The transfer clips were placed in a 1 x transfer solution. The membrane transferring clamp is sequentially placed with sponge, filter paper, SDS-PAGE gel, PVDF membrane, filter paper and sponge, and the SDS-PAGE condensation and bubbles in the PVDF membrane are carefully removed. Constant pressure is 110V, and the rotation is carried out for 70-100min.

(9) Closing: and placing the PVDF film into a sealing liquid of 5% skimmed milk powder, placing the sealing liquid on a shaking table, and sealing the sealing liquid at room temperature for more than 1 h.

(10) Incubation resistance: the blocking solution was pipetted off, the corresponding primary antibody was added and incubated overnight in a shaker at 4 ℃.

(11) Secondary antibody incubation: the next day, primary antibodies were recovered and washed 3 times with 1 XTBE for 5min each. The corresponding secondary antibody was added and incubated for 2h at room temperature.

(12) Exposure: the secondary antibody was discarded and washed 3 times with 1 XTBE for 5min each. ECL chemiluminescent solution from BI company was used for the exposure. The working solution is measured and mixed according to the volume ratio of the solution A to the solution B of 1:1, the luminescent solution is placed in a dark place, the working solution is sucked by a gun head and uniformly dripped on the strips, the strips are sealed by a plastic packaging film, and the strips are exposed by a Bio-Rad chemiluminescent imager.

3. Experimental results

As shown in FIG. 2 (A, B), the protein levels of HSP110, STAT3 and p-STAT3 in HCT116 cells (A) and SW480 cells (B) under the action of different concentrations of compound 7 (0,1.25,2.5,5, 10. Mu.M) were detected by the WesternBlot method, taking B2M as an internal reference. The protein levels of HSP110, STAT3 and p-STAT3 in HCT116 cells (C) and SW480 cells (D) were detected by a Western Blot method under the action of compound 7 and compound A at different concentrations (0, 5, 10. Mu.M), with B2M as an internal reference.

4. Discussion of results

From this, it can be seen that compound 7 appears to inhibit STAT3 phosphorylation in colorectal cancer cells more significantly than compound a.

Example 54: research of affinity of Compound 7 for HSP110

1. The purpose of the experiment is as follows: in order to verify whether designed and synthesized targeted small HSP110 molecules have affinity for the target HSP110, we used compound 7 with better anti-colorectal cancer proliferation activity in vitro as a research object, and measured the affinity for HSP110 protein by using Biolayer interferometry (OctetRed 96, forte-Bio) method, and the experimental result was expressed in terms of dissociation constant (K _D Values), the experiment was characterized with compound a as positive control compound.

Experimental materials and instrumentation experimental materials: HSP110, NBD-HSP110, NHS-PEG 4-biotin reagent (Thermo Scientific 21330), tris (Zhejiang Seny Biotechnology), DMSO (Coolaber) and 96 well plates.

2) Experimental instrument: zebatM spin desalting columns (Thermo Scientific B2162579) and SuperStreptavidin biosensors (Forte-Bio).

2. Experimental method

1) Study object: protein (HSP 110), HSP110 was biotinylated at a ratio of HSP110 to biotin of 1:3 using NHS-PEG 4-biotin reagent.

2) The excess biotin was then removed (using a ZebatM spin-desalting column, 7KMWCO,0.5 mL).

3) Prior to kinetic experiments, superStrepitavidin biosensors (Fort-Bio) were hydrated for 15min in running buffer (Tris 50mM pH 8,NaCl 300mM,0.01% (w/v) Tween-20 and 0.1% (w/v) BSA) at room temperature.

4) Experiments were performed in 200 μl volume/well 96-well plates, with constant shaking (1000 rpm) and room temperature. Baseline 120s was measured in running buffer followed by 600s shift in 15 μg/mL HSP 110.

5) The protein-linked sensor was washed in running buffer for 60s, and then the second baseline 120s was measured in running buffer.

6) The key steps (4 and 5) are carried out in parallel with the concentration of a plurality of compounds; an appropriate amount of compound was dissolved in DMSO and diluted in running buffer, with no more than 1% DMSO.

7) Dissociation was assessed after 60s run in buffer. K (K) _D The values (1:1 binding model) were calculated by the software provided by the manufacturer using the loaded control protein (here using hemoglobin, 15 μg/mL) as a reference for the sensor.

3. Experimental results

Experimental results indicate that compound 7 has K to HSP110 _D The value is 7.642 multiplied by 10 ^-5 M, K against HSP110 compared with Compound A _D An increase in 89.2. Mu.M (K for HSP110 for Compounds A and 7, respectively _D The value measurement graphs are shown in fig. 3 and 4 in sequence).

Claims

1. An inhibitor compound targeting heat shock protein 110kDa, or an optical isomer, racemate, single enantiomer, possible diastereoisomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, characterized in that the structural formula of the compound is shown in formula (I):

in formula (I):

each R is ² And each R ³ Each independently selected from-X, C _1-6 Alkyl, C _3-6 Cycloalkyl, C _3-6 Heterocyclyl, C _1-6 Alkoxy, C _2-6 Alkenyl, C _2-6 Alkynyl, -COR ⁰ One of 6-to 10-membered aryl, 5-to 10-membered heteroaryl; wherein 5-to 10-membered heteroAryl contains 1-3 heteroatoms selected from N, O, S, C _3-6 Heterocyclyl contains 1-3 heteroatoms selected from N, O, S;

x is H, F, cl, br, I, -OCH ₃ 、-OH、-CN、-CF ₃ One of the following;

m is an integer of 0 to 3; n is an integer of 0 to 3;

selected from->C _2-6 Alkenyl, C _2-6 Alkynyl, -CO, C _2-6 alkenyl-CO, C _2-6 alkynyl-CO, -SO ₂ Q is an integer of 0 to 2;

2. An inhibitor compound targeting heat shock protein 110kDa as claimed in claim 1 wherein the compound has the structural formula (I):

R ¹ selected from-CH ₃ 、-CN、-CHO、-CONH ₂ One of 2-phenylvinyl.

3. An inhibitor compound targeting heat shock protein 110kDa according to claims 1-2, characterized in that the compound has the structural formula (I):

-L-is selected fromQ=0;

Selected from->One of the following;

selected from-CO, & lt + & gt>Q=0, 1,2,3.

Wherein the method comprises the steps ofRepresentation->And->The site of ligation.

4. A heat shock protein 110kDa targeting inhibitor compound according to claims 1-3, or an optical isomer, racemate, single enantiomer, possible diastereoisomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, characterized in that the compound has the structural formula as shown in any one of formulae 1-60:

5. use of an inhibitor compound targeting heat shock protein 110kDa according to any of claims 1-4, or an optical isomer, racemate, single enantiomer, possible diastereoisomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, for the preparation of an antitumor drug.

6. The use of claim 5, wherein the neoplasm comprises colon cancer.

7. An antitumor agent comprising a safe and effective amount of an inhibitor compound targeting 110kDa heat shock protein according to any one of claims 1-4, or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof.

8. The anti-tumor drug according to claim 7, wherein the anti-tumor drug further comprises a pharmaceutically acceptable carrier.

9. An anti-tumour agent according to claim 7 or 8 wherein the tumour comprises colon cancer.

10. An antitumor agent according to claim 7 or 8, characterized in that it is in a form suitable for oral, intratumoral, rectal, parenteral and topical administration.