A class of tetrahydrobenzofuran[2,3-c]pyridine kinase inhibitors and preparation method and use thereof

Abstract

The present application relates to a kind of tetrahydrobenzofuran [2,3-c] pyridine kinase inhibitors and preparation method and purposes thereof.The specific compound of the present application has the structure shown in formula I, wherein the definition of each group and substituent group is described in the specification.The preparation method of the compound and its use in antitumor are also disclosed.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to a class of tetrahydrobenzofuran[2,3-c]pyridine kinase inhibitors, their preparation methods, and uses. Background Technology

[0002] p21-activated kinases (PAKs) are serine / threonine-specific protein kinases that play crucial roles in cytoskeleton organization, cell morphogenesis, and cell survival. They are involved in regulating a variety of diseases, including cancer, infectious diseases, and neurological disorders. PAKs are located at the intersection of signaling pathways essential for the development of many tumors. When PAKs are overexpressed, mutated, or abnormally activated by upstream signals such as the cell division control protein Cdc42 or RAC, they affect pathways such as ERK, AKT, and WNT, generating oncogenic signals within the cell, including signals that promote autonomous cell growth, apoptosis evasion, and the promotion of invasion and metastasis. Furthermore, PAK proteins are upregulated in various human cancers and are positively correlated with the degree of tumor progression and reduced patient survival. Therefore, the development of PAK inhibitors is an important strategy for cancer treatment.

[0003] The PAK family consists of six members, divided into two types based on sequence and structural homology: type I (PAK1-3) and type II (PAK4-6). However, type I PAKs have proven to be highly challenging targets for small molecule drugs. Furthermore, a recent study indicated that PAK2 inhibition is associated with acute cardiovascular toxicity, which is enhanced by PAK1 inhibition. This study raises concerns about the development of pan-type I PAK inhibitors. Meanwhile, type II PAK4 promotes BRAF or KRAS-driven tumor invasion, metastasis, and proliferation, attracting significant interest. Recent studies also show that PAK4 is involved in tumor immunity. PAK4 is enriched in tumor biopsies with low T cell and dendritic cell infiltration and is negatively correlated with immune marker responses and T cell infiltration. In addition, PAK4 gene knockout induces the re-expression of endothelial cell adhesion proteins, reduces vascular abnormalities, and improves T cell infiltration. In a B16 melanoma mouse model, PAK4 knockout increased susceptibility to PD-1 antibody therapy and improved T cell infiltration.

[0004] Currently, only one PAK4 inhibitor, ATG-019 (KPT-9274), developed by Karyopharm, is in Phase I clinical trials for the treatment of advanced solid tumors or non-Hodgkin's lymphoma. KPT-9274 is a PAK4 allosteric inhibitor that does not directly inhibit PAK4 kinase activity but downregulates the phosphorylation levels of PAK4 protein and downstream signaling in tumor cells; furthermore, KPT-9274 has no significant inhibitory effect on type I PAKs. It also exhibits good inhibitory activity against various tumor cell lines and demonstrates good antitumor activity in renal cell carcinoma and pancreatic cancer models. In addition, in mouse models of B16 melanoma, MC38 colon cancer, and glioblastoma multiforme, the combination of KPT-9274 with PD-1 monoclonal antibodies or CAR-T therapy improved the antitumor efficacy compared to monotherapy.

[0005] In summary, PAK4 plays a crucial role in tumor development and progression, as well as in tumor immune escape. Therefore, developing small molecule drugs targeting PAK4 has a promising market prospect. Currently, there are no marketed drugs in this field, and only one drug is in clinical development. There is an urgent need to develop more PAK4 inhibitors with novel structures to meet clinical needs. Therefore, the inventors hope to develop a highly selective PAK4 inhibitor with high efficacy, low toxicity, and good metabolic properties, providing a new option for tumor treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a compound of Formula I, its preparation method, and its use in antitumor activity.

[0007] In a first aspect, the present invention provides a compound, said compound being a compound of formula I, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0008]

[0009] in,

[0010] A is selected from the following group: Among them, R 0 Selected from the following group: hydrogen, C1-C6 alkyl;

[0011] V, W, X, and Y are each independently selected from the following groups: N, CR 4 , where R 4 Selected from the following group: hydrogen, halogen, hydroxyl, C1-C6 alkoxy, amino, cyano, C1-C6 alkyl;

[0012] Z is selected from the following group:

[0013] R 1Selected from the group consisting of: hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted 4-8 membered heterocyclic alkyl containing 1, 2 or 3 heteroatoms selected from N, O, S, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O, S; wherein, the substitution independently refers to substitution by one or more substituents selected from the group consisting of: halogen, hydroxyl, cyano, C1-C6 alkoxy, amino, C1-C6 alkyl;

[0014] R 2 Selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 ynyl, substituted or unsubstituted 4-8 membered heterocyclic alkyl containing 1, 2 or 3 heteroatoms selected from N, O, and S, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O, and S. Wherein, n is selected from the group consisting of: 0, 1, 2, 3, and the substitution independently refers to being substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, amino, cyano, mercapto, difluoromethyl, oxo (=O), C1-C6 alkoxy, C1-C6 alkyl, and the heterocyclic alkyl is selected from the group consisting of: monocyclic, fused, spirocyclic, and bridged ring.

[0015] R 3 Selected from the following groups: hydrogen, amino.

[0016] In another preferred example, A is selected from the following group: Among them, R 0 Selected from the following group: H, C1-C6 alkyl;

[0017] V, W, X, and Y are each independently selected from the following groups: N, CR 4 , where R 4 Selected from the following group: hydrogen, halogen, C1-C6 alkyl;

[0018] Z is selected from the following group:

[0019] R 1 Selected from the group consisting of: hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C10 aryl; wherein the substitution independently refers to being substituted by one or more substituents selected from the group consisting of: halogen, C1-C6 alkyl;

[0020] R 2 Selected from the following group: substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted 4-8 membered heterocyclic alkyl groups containing 1, 2, or 3 heteroatoms selected from N, O, and S, substituted or unsubstituted C3-C8 cycloalkyl groups. Wherein, n is selected from the group consisting of: 0, 1, 2, and the substitution independently refers to being substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, cyano, mercapto, difluoromethyl, oxo (=O), C1-C6 alkoxy, C1-C6 alkyl, and the heterocyclic alkyl is selected from the group consisting of: monocyclic, fused, spirocyclic, and bridged ring.

[0021] R 3 Selected from the following groups: hydrogen, amino.

[0022] In another preferred example, A is selected from the following group: Among them, R 0 For H.

[0023] In another preferred embodiment, Selected from the following group:

[0024]

[0025] R 4 Selected from the following group: hydrogen, halogens.

[0026] In another preferred embodiment, R 2 Selected from the following group: substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted 4-8 membered heterocyclic alkyl groups containing 1, 2, or 3 heteroatoms selected from N, O, and S, substituted or unsubstituted C3-C8 cycloalkyl groups. Wherein, n is selected from the group consisting of: 0, 1, 2, 3, and the substitution independently refers to being substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, cyano, mercapto, difluoromethyl, oxo (=O), C1-C6 alkoxy, C1-C6 alkyl, and the heterocyclic alkyl is selected from the group consisting of: monocyclic, fused, spirocyclic, and bridged ring.

[0027] In another preferred embodiment, R 2 Selected from the following group of groups, whether substituted or unsubstituted: C1-C6 alkyl,

[0028] In another preferred embodiment, R 1 Selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C6-C10 aryl groups; wherein the substitution independently refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C6 alkyl.

[0029] In another preferred embodiment, the compound is selected from the group consisting of:

[0030]

[0031]

[0032]

[0033]

[0034] A second aspect of the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compounds described in the first aspect of the invention.

[0035] A third aspect of the invention provides the use of the compound described in the first aspect of the invention for preparing a medicament for preventing and / or treating diseases selected from the group consisting of: cancer, infectious diseases, and neurological diseases.

[0036] In a fourth aspect, the invention provides the use of the compound described in the first aspect for preparing a medicament for inhibiting PAK4 kinase activity.

[0037] A fifth aspect of the invention provides the use of the compound described in the first aspect of the invention for preparing a medicament for preventing and / or treating diseases related to PAK4 kinase activity.

[0038] In another preferred embodiment, the diseases associated with PAK4 kinase activity are selected from the group consisting of: cancer, infectious diseases, and neurological diseases.

[0039] In another preferred embodiment, the cancer is selected from the group consisting of: non-Hodgkin's lymphoma, kidney cancer, pancreatic cancer, melanoma, colon cancer, and glioblastoma.

[0040] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0041] Figure 1 This is a linear relationship graph of KPT-9274 (hydrochloride) solution concentration and peak area.

[0042] Figure 2 This is a linear relationship graph of the concentration and peak area of ​​compound S21 (hydrochloride) solution.

[0043] Figure 3 This is a linear relationship graph of the concentration and peak area of ​​compound S23 (hydrochloride) solution. Detailed Implementation

[0044] Through long-term and in-depth research and structural optimization, the inventors unexpectedly prepared a novel compound with high inhibitory activity and high water solubility. Based on this, the inventors completed this invention.

[0045] the term

[0046] In this invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0047] In this invention, the term "halogen" refers to F, Cl, Br, or I.

[0048] In this invention, "C1-C6 alkyl" refers to a straight-chain or branched alkyl group comprising 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, neopentyl, pterpentyl, or similar groups.

[0049] In this invention, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2-6 carbon atoms and containing a double bond, and includes, without limitation, vinyl, propenyl, butenyl, isobutenyl, pentenyl, and hexenyl groups.

[0050] In this invention, the term "C2-C6 ynyl" refers to a straight-chain or branched ynyl group having 2-6 carbon atoms and containing a triple bond, and includes, without limitation, ethynyl, propynyl, butynyl, isobutynyl, pentylyl, and hexynyl.

[0051] In this invention, the term "C3-C8 cycloalkyl" refers to a cyclic alkyl group having 3-8 carbon atoms on a ring, and non-limitingly includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The term "C3-C6 cycloalkyl" has a similar meaning.

[0052] In this invention, the term "C1-C6 alkoxy" refers to a straight-chain or branched alkoxy group having 1-6 carbon atoms, and includes, without limitation, methoxy, ethoxy, propoxy, isopropoxy, and butoxy. Preferably, it is a C1-C4 alkoxy group.

[0053] In this invention, the term "heterocyclic group" refers to a 4-8 membered heterocyclic group containing 1, 2, or 3 heteroatoms selected from N, O, and S, including (but not limited to) the following groups:

[0054] In this invention, the terms "aromatic ring" or "aryl" have the same meaning, and are preferably "C6-C10 aryl". The term "C6-C10 aryl" refers to an aromatic cyclic group with 6-10 carbon atoms that does not contain heteroatoms on the ring, such as phenyl, naphthyl, etc.

[0055] In this invention, the terms "aromatic heterocycle" or "heteroaryl" have the same meaning, referring to a heteroaromatic group containing one or more heteroatoms. For example, "C3-C10 heteroaryl" refers to an aromatic heterocycle containing 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen, and 3 to 10 carbon atoms. Non-limiting examples include: furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0056] In this invention, the term "halogenated" refers to being replaced by a halogen.

[0057] In this invention, the term "deuterium substitution" refers to being replaced by deuterium.

[0058] In this invention, the term "substitution" refers to the substitution of one or more hydrogen atoms on a specific group by a specific substituent. The specific substituent is the substituent described accordingly above, or the substituent appearing in the various embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substituted site of that group, and the substituents may be the same or different at each position. Those skilled in the art will understand that the combinations of substituents contemplated in this invention are stable or chemically feasible combinations. Such substituents include, but are not limited to: halogens, hydroxyl groups, carboxyl groups (-COOH), C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, C3-C8 cycloalkyl groups, 3- to 12-membered heterocyclic groups, aryl groups, heteroaryl groups, C1-C8 aldehyde groups, C2-C10 acyl groups, C2-C10 ester groups, amino groups, C1-C6 alkoxy groups, C1-C10 sulfonyl groups, etc.

[0059] In this invention, the terms 1-6 refer to 1, 2, 3, 4, 5, or 6. Other similar terms each have a similar meaning independently. The term "multiple" refers to 2-6, such as 2, 3, 4, 5, or 6.

[0060] It should be understood that when a group exists simultaneously at multiple different positions in a compound, its definition at each position is independent and can be the same or different. That is, the term "selected from the following group:" and the term "each independently selected from the following group:" have the same meaning.

[0061] compound

[0062] This invention relates to a class of compounds having p21-activated kinase 4 (PAK4) inhibitory activity and pharmaceutically acceptable salts or solvates thereof, methods for their preparation, pharmaceutical compositions comprising the compounds, and the use of these compounds in the preparation of medicaments for the prevention or treatment of tumors, infectious diseases, and neurological disorders.

[0063] This invention provides a compound, which is a compound of Formula I, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0064]

[0065] The groups are as defined above.

[0066] In another preferred embodiment, in the compound, A, V, W, X, Y, Z, R 1 R 2 R 3 Each of the above can be independently a group corresponding to the specific compound described in this invention.

[0067] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

[0068] Another preferred class of salts are salts formed by the compounds of the present invention with a base, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (such as lower alkanol ammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0069] The term "solvent" refers to a complex formed by the coordination of the compound of the present invention with solvent molecules in a specific ratio. "Hydrate" refers to a complex formed by the coordination of the compound of the present invention with water.

[0070] The term "prodrug" refers to a compound that is biologically active or inactive on its own, and which, when taken in a suitable manner, is metabolized or chemically reacted in the human body to form a compound of Formula I, or a salt or solution of a compound of Formula I. Prodrugs include (but are not limited to) carboxylic acid esters, carbonates, phosphate esters, nitrate esters, sulfate esters, sulfone esters, sulfoxide esters, amino compounds, carbamates, azo compounds, phosphoramides, glucosides, ethers, acetals, etc.

[0071] Preparation method

[0072] The preparation method of the compound of Formula I of the present invention is described in more detail below, but these specific methods do not constitute any limitation on the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, such combinations can be easily performed by those skilled in the art.

[0073] Typically, the preparation process of the compounds of the present invention is as follows, wherein the raw materials and reagents used can be purchased commercially unless otherwise specified.

[0074] Step 1:

[0075]

[0076] Step Two:

[0077]

[0078] Or step three:

[0079]

[0080] Pharmaceutical Compositions and Administration

[0081] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compound.

[0082] Because the compounds of the present invention have excellent antitumor activity, 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 tumor-related diseases.

[0083] 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, 10-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0084] "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.), and emulsifiers. Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0085] The pharmaceutical composition is an injection, capsule, tablet, pill, powder, or granule.

[0086] 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.

[0087] 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 compatibilizers, 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; and (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.

[0088] 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 from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can 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.

[0089] 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, e.g., 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] Dosage forms of the compounds of the present 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 propellants that may be necessary.

[0094] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds (such as antitumor drugs).

[0095] The treatment method of the present invention can be used alone or in combination with other treatment methods or drugs.

[0096] 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 1–2000 mg, preferably 50–1000 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 skills of a skilled physician.

[0097] Compared with the prior art, the present invention has the following main advantages:

[0098] (1) The compound has excellent PAK4 inhibitory activity and excellent water solubility;

[0099] (2) The compound has excellent safety, pharmacokinetic properties, and PAK4 selective inhibition.

[0100] (3) The compound has excellent tumor cell proliferation inhibitory activity.

[0101] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0103] 1 ¹H-NMR was performed using a Varian Mercury AMX300 and a Varian MR-400 instrument. Reagents were purchased from J&K Chemical Co., Ltd., Shaoyuan Technology (Shanghai) Co., Ltd., Shanghai Bide Pharmaceutical Technology Co., Ltd., and Shanghai Haohong Biomedical Technology Co., Ltd., with other reagents purchased from Sinopharm Chemical Reagent Co., Ltd. All solvents were redistilled before use, and the anhydrous solvents used were dried according to standard methods. Unless otherwise specified, all reactions were carried out under nitrogen protection and monitored by TLC. Post-processing included washing with saturated sodium chloride aqueous solution and drying with anhydrous sodium sulfate. Unless otherwise specified, the products were purified using silica gel (200-300 mesh) column chromatography. The silica gel (200-300 mesh) was produced by Qingdao Ocean Chemical Plant, and the GF-254 thin-layer silica gel plates were produced by Yantai Jiangyou Silica Gel Development Co., Ltd.

[0104] Preparation Example 1: Preparation of Compound S1

[0105]

[0106] Synthesis of compounds 1-2:

[0107] Compound 1-1 (4.62 g, 19.6 mmol) was dissolved in tetrahydrofuran (40 mL), cooled to 0 °C, and a 1 mol / L solution of the sodium borohydride tetrahydrofuran complex in tetrahydrofuran (39.2 mL, 39.2 mmol) was carefully added. The mixture was stirred for 20 minutes, heated to reflux, and stirred for 3 hours. The reaction was monitored by TLC until complete. After cooling to room temperature, the reaction was quenched with methanol and concentrated under reduced pressure. The solution was dissolved in ethyl acetate, extracted three times with 2 M hydrochloric acid, alkalized with excess ammonia, and extracted three times with dichloromethane. The organic layers were washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound 1-2 (2.2 g, 47%). 1 ¹H NMR (400MHz, deuterated chloroform) δ 7.68 (d, J = 1.7 Hz, 1H), 7.47 (s, 1H), 7.36 (dt, J = 17.6, 5.2 Hz, 2H), 3.03 (t, J = 6.8 Hz, 2H), 2.78 (t, J = 6.8 Hz, 2H).

[0108] Synthesis of compounds 1-3:

[0109] Compounds 1-2 (2 g, 8.33 mmol) were dissolved in formic acid (20 mL), and paraformaldehyde (750 mg, 8.33 mmol) was added. The mixture was heated to 50 °C and stirred for three hours (with continuous monitoring of the reaction). After the reaction was complete as determined by TLC, the mixture was cooled to room temperature, and the pH was adjusted to neutral by adding 1 mol / L sodium hydroxide aqueous solution. The mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give crude product 1-3, which was used directly in the next step without further processing.

[0110] Synthesis of compounds 1-4:

[0111] The crude products 1-3 were dissolved in dichloromethane (20 mL), cooled to 0 °C, and then di-tert-butyl dicarbonate (2.73 g, 12.5 mmol) and N,N-diisopropylethylamine (2.15 g, 16.66 mmol) were added. The mixture was stirred overnight. The reaction was monitored by TLC until complete. The mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography to give compounds 1-4 (1.46 g, 50%). 1 ¹H NMR (400 MHz, deuterated chloroform) δ 7.55 (s, 1H), 7.36–7.27 (m, 2H), 4.58 (s, 2H), 3.74 (s, 2H), 2.68 (s, 2H), 1.50 (s, 9H).

[0112] Synthesis of compounds 1-6:

[0113] Compounds 1-4 (85 mg, 0.24 mmol), 1-5 (94 mg, 0.266 mmol), and potassium carbonate (83 mg, 0.6 mmol) were dissolved in a mixed solution of 1,4-dioxane (2 mL) and water (0.5 mL). Tetra(triphenylphosphine)palladium (28 mg, 0.024 mmol) was added under nitrogen protection. The mixture was heated to reflux and stirred for 2 hours. The reaction was monitored by TLC until complete. After cooling to room temperature, the mixture was concentrated under reduced pressure. The mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography to give compounds 1-6 (105 mg, 88%). 1 ¹H NMR (400MHz, deuterated chloroform) δ 7.82 (q, J = 8.4 Hz, 4H), 7.67 (s, 1H), 7.61–7.47 (m, 2H), 4.65 (s, 2H), 3.88–3.75 (m, 6H), 3.13–2.99 (m, 4H), 2.79 (s, 2H), 1.52 (s, 9H).

[0114] Synthesis of compounds 1-7:

[0115] Compounds 1-6 (105 mg, 0.21 mmol) were dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete as detected by TLC, the solution was concentrated under reduced pressure to obtain crude compounds 1-7, which were used directly in the next step without further processing.

[0116] Synthesis of compound S1:

[0117] Compounds 1-7 (84 mg, 0.215 mmol), 1-8 (39 mg, 0.237 mmol), and 2-(7-azobenzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 122 mg, 0.32 mmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (139 mg, 1.08 mmol) was added. The mixture was stirred overnight at room temperature. After the reaction was complete as detected by TLC, the mixture was cooled to room temperature and concentrated under reduced pressure. The mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography to give compound S1 (43 mg, 37%) as a white solid. 1 H NMR(500MHz,DMSO-d6)δ8.20(s,1H),8.01(d,J=8.3Hz,2H),7.97(s,1H),7.92(d ,J=7.9Hz,1H),7.82(d,J=8.3Hz,2H),7.70(q,J=8.6Hz,2H),7.46(d,J=15.2Hz, 1H),7.16(d,J=15.0Hz,1H),6.49(d,J=8.7Hz,1H),6.44(s,2H),4.92(d,J=96.5 Hz, 2H), 4.00 (d, J = 45.3Hz, 2H), 3.70–3.60 (m, 4H), 2.98–2.75 (d, J = 38.4Hz, 6H).

[0118] Preparation Example 2: Preparation of Compound S2

[0119]

[0120] Synthesis of compound 2-2:

[0121] Compounds 1-4 (76 mg, 0.216 mmol), 2-1 (81 mg, 0.238 mmol), and potassium carbonate (75 mg, 0.54 mmol) were dissolved in a mixed solution of 1,4-dioxane (2 mL) and water (0.5 mL). Tetra(triphenylphosphine)palladium (26 mg, 0.022 mmol) was added under nitrogen protection. The mixture was heated to reflux and stirred for 2 hours. The reaction was monitored by TLC until complete. After cooling to room temperature, the mixture was concentrated under reduced pressure. The mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography to give compound 2-2 (75 mg, 71%). 1 ¹H NMR (400MHz, deuterated chloroform) δ 7.85 (d, J = 8.4 Hz, 2H), 7.74–7.61 (m, 3H), 7.55–7.45 (m, 2H), 4.64 (s, 2H), 3.85–3.73 (m, 4H), 2.77 (s, 2H), 2.59–2.44 (m, 2H), 1.53 (s, 9H).

[0122] Synthesis of compounds 2-3:

[0123] Compound 2-2 (75 mg, 0.15 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete as detected by TLC, the solution was concentrated under reduced pressure to obtain crude compound 2-3, which was used directly in the next step without further processing.

[0124] Synthesis of compound S2:

[0125] Compounds 2-3 (60 mg, 0.15 mmol), 1-8 (27 mg, 0.165 mmol), and HATU (94 mg, 0.25 mmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (97 mg, 0.75 mmol) was added. The mixture was stirred overnight at room temperature. The reaction was monitored by TLC until complete. After cooling to room temperature, the solution was concentrated under reduced pressure. The solution was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The solution was filtered, concentrated under reduced pressure, and purified by column chromatography to give compound S2 (28 mg, 35%) as a white solid. 1H NMR(600MHz,DMSO-d6)δ8.74(t,J=5.6Hz,1H),8.19(s,1H),7.97–7.88(m,4H ),7.83(d,J=8.3Hz,2H),7.65(q,J=8.6Hz,2H),7.45(d,J=15.1Hz,1H),7.20– 7.05(m,1H),6.58–6.36(m,3H),4.90(d,J=120.1Hz,2H),3.99(d,J=60.2Hz, 2H), 3.53 (dd, J=12.7, 6.7Hz, 2H), 2.80 (d, J=42.1Hz, 2H), 2.66–2.52 (m, 2H).

[0126] Preparation Example 3: Preparation of Compound S3

[0127]

[0128] Synthesis of compound 3-2:

[0129] Compound 3-1 (3 g, 16.1 mmol) and N-bromosuccinimide (3.45 g, 19.4 mmol) were dissolved in acetonitrile (35 mL) and stirred at room temperature for 4 hours. The reaction was detected by TLC after completion. The mixture was cooled to 0 °C, ice water was added, and a solid precipitated out. The solid was filtered and dried to give compound 3-2 (3.98 g, 93%). 1 ¹H NMR (400 MHz, deuterated chloroform) δ 11.28 (s, 1H), 7.93–7.87 (m, 1H), 7.68 (d, J = 2.4 Hz, 1H), 3.98 (s, 3H).

[0130] Synthesis of compound 3-3:

[0131] Compound 3-2 (1.1 g, 4.14 mmol), ethyl bromoacetate (830 mg, 4.97 mmol), and potassium carbonate (1.71 g, 12.42 mmol) were dissolved in acetone (20 mL), heated to reflux, and stirred for 4 hours. The reaction was monitored by TLC until complete. After cooling to room temperature, the solution was concentrated under reduced pressure. Extraction with ethyl acetate was performed, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The solution was filtered, and the filtrate was concentrated under reduced pressure to give compound 3-3 (1.45 g, 99%). 1 ¹H NMR (400MHz, deuterated chloroform) δ 7.83 (d, J = 2.5Hz, 1H), 7.69 (d, J = 2.5Hz, 1H), 4.66 (s, 2H), 4.30 (q, J = 7.1Hz, 2H), 3.90 (s, 3H), 1.32 (t, J = 7.1Hz, 4H).

[0132] Synthesis of compounds 3-4:

[0133] Compound 3-3 (1.45 g, 4.1 mmol) was dissolved in a mixture of 1 M sodium hydroxide aqueous solution (50 mL) and ethanol (50 mL), and the mixture was heated to reflux for 6 hours. The reaction was completed by TLC. After cooling to 0 °C, hydrochloric acid solution was added to adjust the pH to acidic, and a solid precipitated out. The solid was then filtered to give compound 3-4 (1.09 g, 86%). 1 ¹H NMR (400MHz, deuterated methanol) δ 7.81 (d, J = 2.3 Hz, 1H), 7.77 (d, J = 2.4 Hz, 1H), 4.73 (s, 2H).

[0134] Synthesis of compounds 3-5:

[0135] Compounds 3-4 (1.09 g, 3.52 mmol) and sodium acetate (958 mg, 7.04 mmol) were dissolved in a mixed solution of acetic acid (5 mL) and acetic anhydride (15 mL), and the mixture was heated to 130 °C and reacted overnight. The reaction was monitored by TLC until complete. After cooling to room temperature, an appropriate amount of water was added, and the mixture was extracted three times with ethyl acetate and petroleum ether (1:1). The organic layers were combined, washed five times with sodium bicarbonate aqueous solution, and then washed with saturated brine. The mixture was dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give crude product 3-5.

[0136] Synthesis of compounds 3-6:

[0137] The crude product 3-5 was dissolved in a mixture of methanol (5 mL) and 1 M hydrochloric acid aqueous solution (2 mL), and heated to reflux for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, and an appropriate amount of ice water was added. A solid precipitated out, and the solid was obtained by filtration, yielding compound 3-6 (697 mg, 80%). 1 ¹H NMR (400MHz, deuterated chloroform) δ 7.75 (d, J = 1.8Hz, 1H), 7.71 (d, J = 1.8Hz, 1H), 4.76 (s, 2H).

[0138] Synthesis of compounds 3-7:

[0139] Compounds 1-2 (1.77 g, 10 mmol) were dissolved in tetrahydrofuran (20 mL), cooled to 0 °C, and sodium hydride (480 mg, 12 mmol) was carefully added and stirred for one hour. Compounds 3-6 (10 mmol, 2.47 g) dissolved in tetrahydrofuran (20 mL) were carefully added and stirred overnight. The reaction was monitored by TLC until complete, cooled to room temperature, and concentrated under reduced pressure. The mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, concentrated under reduced pressure, and purified by column chromatography to give compounds 3-7 (2.33 g, 86%). 1¹H NMR (400MHz, deuterated chloroform) δ 7.75 (s, 1H), 7.64 (d, J = 1.7Hz, 1H), 7.52 (d, J = 1.5Hz, 1H), 3.74 (d, J = 1.2Hz, 2H).

[0140] Synthesis of compounds 3-8:

[0141] The synthesis of compounds 3-8 is based on the synthesis of compounds 1-4. 1 ¹H NMR (600MHz, deuterated chloroform) δ 7.60 (d, J = 1.6Hz, 1H), 7.55 (s, 1H), 7.44 (d, J = 1.6Hz, 1H), 3.03 (t, J = 6.8Hz, 2H), 2.78 (t, J = 6.7Hz, 2H).

[0142] Synthesis of compound 3-10:

[0143] The synthesis of compounds 3-10 is based on the synthesis of compounds 1-6. 1 ¹H NMR (400MHz, deuterated chloroform) δ 7.47 (s, 1H), 7.39 (s, 1H), 4.63 (s, 2H), 3.75 (s, 2H), 2.68 (s, 2H), 1.50 (s, 9H).

[0144] Synthesis of compound 3-12:

[0145] The synthesis of compounds 3-12 is based on the synthesis of compounds 1-6. 1 ¹H NMR (400MHz, deuterated chloroform) δ 7.64 (d, J = 8.1 Hz, 2H), 7.55–7.46 (m, 4H), 4.66 (s, 2H), 4.01–3.53 (m, 6H), 2.76 (s, 2H), 2.16–1.87 (m, 4H), 1.50 (s, 9H).

[0146] Synthesis of compounds 3-13: Refer to the synthesis of compounds 1-7 for the synthesis of compounds 3-13.

[0147] Synthesis of compound S3:

[0148] The synthesis of compound S3 is the same as that of compound S1. It is a white solid. 1H NMR(600MHz,DMSO-d6)δ8.18(s,1H),7.91(d,J=8.3Hz,1H),7.88(s,1H),7.81( d,J=8.1Hz,2H),7.72(s,1H),7.54(d,J=8.1Hz,2H),7.44(d,J=15.1Hz,1H),7.1 8–7.06(m,1H),6.48(s,3H),5.05(s,1H),4.83(s,1H),3.97(d,J=63.0Hz,2H),3 .59(d,J=156.5Hz,4H),3.32(s,1H),2.79(d,J=40.9Hz,2H),2.13–1.96(m,4H).

[0149] Preparation Example 4: Preparation of Compound S4

[0150] Synthesis of compound S4: Same as in Example 3, except that 3-11 is replaced with

[0151] A white solid was obtained. 1 ¹H NMR (500 MHz, deuterated methanol) δ 8.11 (s, 1H), 7.90 (dd, J = 8.8, 2.2 Hz, 1H), 7.76–7.70 (m, 4H), 7.68 (s, 1H), 7.55 (d, J = 14.7 Hz, 2H), 7.12–6.97 (m, 1H), 6.63 (d, J = 8.8 Hz, 1H), 5.03–4.82 (m, 5H), 4.16–4.00 (m, 4H), 3.89 (s, 2H), 2.85 (d, J = 20.9 Hz, 2H), 1.34 (s, 6H).

[0152] Preparation Example 5: Preparation of Compound S5

[0153] Synthesis of compound S5: Same as in Example 3, except that 3-11 is replaced with

[0154] A white solid was obtained. 1¹H NMR (500 MHz, deuterated methanol) δ 8.10 (s, 1H), 7.90 (dd, J = 8.8, 2.3 Hz, 1H), 7.71 (t, J = 7.1 Hz, 2H), 7.68–7.59 (m, 3H), 7.55 (dd, J = 8.3, 7.0 Hz, 2H), 7.13–6.98 (m, 1H), 6.67–6.60 (m, 1H), 4.99–4.86 (m, 2H), 4.04 (t, J = 5.3 Hz, 2H), 3.94–3.58 (m, 5H), 2.84 (d, J = 19.5 Hz, 2H), 2.34–1.98 (m, 3H), 1.67–1.48 (m, 3H).

[0155] Preparation Example 6: Preparation of Compound S6:

[0156] Synthesis of compound S6: Same as in Example 3, except that 3-11 is replaced with

[0157] A white solid was obtained. 1 ¹H NMR (500 MHz, deuterated methanol) δ 8.10 (d, J = 1.7 Hz, 1H), 7.90 (dd, J = 8.8, 2.3 Hz, 1H), 7.64–7.59 (m, 2H), 7.58–7.53 (m, 1H), 7.49 (s, 1H), 7.38–7.34 (m, 2H), 7.11–6.99 (m, 1H), 6.63 (d, J = 8.8 Hz, 1H), 5.05–4.87 (m, 2H), 4.06 (s, 2H), 3.95–3.53 (m, 4H), 2.92–2.77 (m, 2H), 2.19–1.96 (m, 4H).

[0158] Preparation Example 7: Preparation of Compound S7

[0159]

[0160] Synthesis of Compound 7-3

[0161] Compounds 7-1 (9.28 g, 40 mmol) and 7-2 (6.16 g, 44 mmol) were dissolved in a mixture of N,N-dimethylformamide (40 mL) and water (20 mL). Potassium phosphate (25.44 g, 120 mmol) and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (1.46 g, 2 mmol) were added. Under nitrogen protection, the mixture was heated to 45 °C and stirred for 5 hours. The reaction was monitored by TLC until complete. After cooling to room temperature, the mixture was concentrated under reduced pressure. The mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography to give compound 7-3 (5.3 g, 54%). 1 ¹H NMR (400MHz, deuterated chloroform) δ 11.30 (s, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.54 (dd, J = 8.3, 5.7 Hz, 2H), 7.48 (d, J = 7.2 Hz, 1H), 7.11 (t, J = 8.6 Hz, 2H), 6.94 (t, J = 7.8 Hz, 1H), 3.96 (s, 3H).

[0162] Synthesis of compound 7-4

[0163] Compound 7-3 (3.1 g, 12.6 mmol) and N-bromosuccinimide (2.7 g, 15.1 mmol) were dissolved in acetonitrile (30 mL), and the mixture was heated to 60 °C and stirred at room temperature for 4 hours. The reaction was detected by TLC after completion. The mixture was cooled, ice water was added, and a solid precipitated out. The solid was filtered and dried to give compound 7-4 (3.8 g, 93%). 1 ¹H NMR (400MHz, deuterated chloroform) δ 11.24 (s, 1H), 7.96 (d, J = 2.5Hz, 1H), 7.58 (d, J = 2.1Hz, 1H), 7.51 (dd, J = 8.9, 5.4Hz, 2H), 7.11 (t, J = 8.8Hz, 2H), 3.97 (s, 3H).

[0164] Synthesis of compound S7

[0165] The subsequent steps are the same as in Preparation Example 3.

[0166] A white solid was obtained. 1¹H NMR (500 MHz, deuterated methanol) δ 8.10 (s, 1H), 7.90 (s, 3H), 7.79 (d, J = 8.2 Hz, 2H), 7.71 (s, 1H), 7.65 (s, 1H), 7.60–7.50 (m, 3H), 7.24 (t, J = 8.7 Hz, 2H), 7.05 (dd, J = 37.5, 15.3 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 5.00–4.86 (m, 2H), 4.14–4.02 (m, 2H), 3.76 (d, J = 112.2 Hz, 4H), 2.87 (d, J = 21.4 Hz, 2H), 2.08 (s, 4H).

[0167] Preparation Example 8: Preparation of Compound S8

[0168] Synthesis of compound S8: Same as in Example 7, except that 3-11 are replaced with

[0169] A white solid was obtained. 1 ¹H NMR (500MHz, deuterated methanol) δ 8.02 (d, J = 28.0 Hz, 1H), 7.89–7.74 (m, 3H), 7.68 (d, J = 8.0 Hz, 2H), 7.63–7.43 (m, 5H), 7.17 (q, J = 8.6 Hz, 2H), 6.96 (dd, J = 40.8, 15.2 Hz, 1H), 6.65–6.52 (m, 1H), 4.84–4.71 (m, 2H), 3.96 (s, 2H), 3.90–3.53 (m, 4H), 2.77 (s, 2H), 2.29–1.95 (m, 2H), 1.53 (dd, J = 46.5, 20.6 Hz, 3H).

[0170] Preparation Example 9: Preparation of Compound S9

[0171] Synthesis of compound S9: Same as in preparation example 7, except that 3-11 is replaced with 1-5.

[0172] A white solid was obtained. 1 ¹H NMR (500 MHz, deuterated chloroform) δ 8.23 ​​(s, 1H), 7.89–7.79 (m, 6H), 7.72–7.55 (m, 4H), 7.22 (t, J = 8.7 Hz, 2H), 6.81 (s, 1H), 6.53 (d, J = 8.6 Hz, 1H), 4.93 (s, 2H), 4.04 (d, J = 44.4 Hz, 2H), 3.78–3.74 (m, 4H), 3.10–3.02 (m, 4H), 2.91 (s, 2H).

[0173] Preparation Example 10: Preparation of Compound S10

[0174] Synthesis of compound S10: Same as in Example 7, except that 3-11 are replaced with

[0175] A white solid was obtained. 1 ¹H NMR (600 MHz, deuterated chloroform) δ 8.23 ​​(s, 1H), 7.83 (s, 2H), 7.73–7.62 (m, 2H), 7.58 (t, J = 7.7 Hz, 2H), 7.53 (s, 1H), 7.31–7.27 (m, 2H), 7.21 (t, J = 8.6 Hz, 2H), 4.90 (d, J = 38.0 Hz, 4H), 4.19–3.53 (m, 5H), 2.93 (d, J = 33.0 Hz, 2H), 2.08–1.83 (m, 3H).

[0176] Preparation Example 11: Preparation of Compound S11

[0177]

[0178] Synthesis of compound S11

[0179] Compounds 11-1 (223 mg, 1 mmol) and 11-2 (221 mg, 1.1 mmol) were dissolved in dichloromethane (5 mL), and pyridine (158 mg, 2 mmol) was added. The mixture was stirred at room temperature for 5 hours. After the reaction was complete as detected by TLC, the mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography to give compound 11-3 (330 mg, 85%). 1 ¹H NMR (400MHz, deuterated chloroform) δ 8.28–8.21 (m, 3H), 7.96 (d, J = 8.9 Hz, 1H), 7.67 (d, J = 6.6 Hz, 1H), 7.33 (d, J = 9.0 Hz, 2H), 5.44 (s, 1H), 4.41 (d, J = 6.0 Hz, 2H), 1.53 (s, 9H).

[0180] Compounds 11-3 (52 mg, 0.133 mmol) and 3-13a (52 mg, 0.12 mmol) were dissolved in tetrahydrofuran (2 mL), and N,N-diisopropylethylamine (46 mg, 0.36 mmol) was added. The mixture was stirred overnight at room temperature. After the reaction was complete as detected by TLC, the mixture was extracted with ethyl acetate. The organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (methanol:dichloromethane = 2.5%) to give compound 11-4 (50 mg, 61%).1 ¹H NMR (400 MHz, deuterated chloroform) δ 8.23 ​​(s, 1H), 8.18 (s, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.59 (d, J = 8.1 Hz, 2H), 7.49–7.42 (m, 4H), 4.59 (s, 2H), 4.40 (d, J = 5.4 Hz, 2H), 4–3.51 (m, 6H), 2.73 (s, 2H), 2.14–1.90 (m, 4H), 1.52 (s, 9H).

[0181] Compound 11-4 (50 mg, 0.073 mmol) was dissolved in dichloromethane (1 mL) and trifluoroacetic acid (1 mL), and stirred overnight at room temperature. After the reaction was complete as detected by TLC, the mixture was extracted with ethyl acetate. The organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (methanol:dichloromethane = 5%) to give compound S11 (38 mg, 90%). 1 H NMR(500MHz,DMSO-d6)δ7.88(d,J=1.4Hz,1H),7.85–7.81(m,3H),7.73(d,J=1.5Hz,1H),7.59–7.54(m,2H),7.31(dd,J=8.4,2.4Hz,1H),7 .20(t,J=5.6Hz,1H),6.39(d,J=8.4Hz,1H),5.74(s,2H),4.64(s,2H),4.09(d,J=5.4Hz,2H),3.84–3.38(m,6H),2.74(s,2H),2.07(s,4H).

[0182] Preparation Example 12: Preparation of Compound S12

[0183]

[0184] Synthesis of compound S12: Same as in Preparation Example 11, except that 3-13a is replaced with 7-14.

[0185] A white solid was obtained. 1¹H NMR (500 MHz, deuterated methanol) δ 7.87–7.81 (m, 3H), 7.74 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 1.7 Hz, 1H), 7.60–7.57 (m, 2H), 7.53 (d, J = 8.3 Hz, 2H), 7.23–7.17 (m, 2H), 6.65 (d, J = 8.7 Hz, 1H), 4.59 (s, 2H), 4.28 (d, J = 23.6 Hz, 2H), 3.98–3.50 (m, 6H), 2.76 (s, 2H), 2.07 (bs, 4H).

[0186] Preparation Example 13: Preparation of Compound S13

[0187]

[0188] Synthesis of compound S13: Same as in preparation example 11, except that 11-3 is replaced with 13-2.

[0189] A white solid was obtained. 1 ¹H NMR (500 MHz, deuterated methanol) δ 8.51 (d, J = 19.3 Hz, 1H), 8.41 (s, 1H), 7.89–7.80 (m, 3H), 7.77–7.71 (m, 2H), 7.63 (d, J = 1.8 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.54–7.50 (m, 2H), 4.61 (s, 2H), 4.44 (s, 2H), 4.00–3.51 (m, 7H), 2.76 (t, J = 5.3 Hz, 2H), 2.07 (s, 4H).

[0190] Preparation Example 14: Preparation of Compound S14

[0191]

[0192] Compound 7-12 (5.9 g, 13.3 mmol) was dissolved in 100 mL of 1,4-dioxane, and potassium acetate (3.9 g, 39.8 mmol), Pd(dppf)Cl2 (485 mg, 0.66 mmol), and pinacol diboronate (4 g, 15.9 mmol) were added. The mixture was heated to reflux and stirred for 3 hours. The reaction was monitored by TLC until complete. After cooling to room temperature, the reaction solution was diluted with water and extracted three times with ethyl acetate. The organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound 14-1 (3.5 g, 47%). 1H NMR(400MHz,Chloroform-d)δ7.90(s,1H),7.86–7.78(m,3H),7.21–7.13(m,2H),4.61(s,2H),3.77(s,2H),2.76(s,2H),1.50(s,9H),1.38(s,12H).

[0193] Compound 14-1 (50 mg, 0.1 mmol) was dissolved in a mixed solution of 1,4-dioxane (0.9 mL) and water (0.3 mL), and Pd(PPh3)4 (12 mg, 0.01 mmol) and K2CO3 (35 mg, 0.25 mmol) were added. The mixture was refluxed at 100 °C for 1 hour. The reaction was monitored by TLC until complete. The reaction solution was cooled to room temperature, diluted with water, and extracted three times with ethyl acetate. The organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound 14-3 (50 mg, 83%). 1 ¹H NMR (400MHz, deuterated chloroform) δ 8.88 (s, 1H), 8.07 (dd, J = 8.1, 2.1 Hz, 1H), 7.88–7.77 (m, 3H), 7.61 (s, 1H), 7.55 (s, 1H), 7.22 (t, J = 8.6 Hz, 2H), 4.65 (s, 2H), 3.94 (t, J = 5.8 Hz, 2H), 3.81 (s, 2H), 2.81 (s, 2H), 2.22–2.08 (m, 4H), 1.51 (s, 9H).

[0194] Compound 14-3 (50 mg, 0.08 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 30 minutes, and the solvent was removed by vortexing to obtain compound 14-4. 14-4 was dissolved in DMF (1 mL), and compound 1-8 (16 mg, 0.1 mmol), HATU (47 mg, 0.12 mmol), and DIPEA (112 μL, 0.64 mmol) were added. The mixture was reacted overnight at room temperature. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound S14 (20 mg, 37%). 1¹H NMR (400MHz, deuterated chloroform) δ 8.88 (d, J = 1.8Hz, 1H), 8.24 (s, 1H), 8.08 (dd, J = 8.1, 2.3Hz, 1H), 7.88–7.78 (m, 3H), 7.71–7.60 (m, 3H), 7.57 (d, J = 1.8Hz, 1H), 7.22 (t, J = 8.6Hz) ,2H),6.88–6.70(m,1H),6.53(d,J=8.6Hz,1H),4.99–4.73(m,4H),4.15–3.97(m, 2H),3.94(t,J=5.9Hz,2H),3.81(t,J=5.9Hz,2H),2.91(s,2H),2.22–2.03(m,4H).

[0195] Preparation Example 15: Preparation of Compound S15

[0196] Synthesis of compound S15: Same as in Preparation Example 14, except that 14-2 is replaced with

[0197] A white solid was obtained. 1 ¹H NMR (500 MHz, deuterated methanol) δ 8.74 (d, J = 2.0 Hz, 1H), 8.09 (s, 3H), 8.03 (d, J = 8.2 Hz, 1H), 7.98–7.83 (m, 4H), 7.55 (d, J = 15.3 Hz, 1H), 7.24 (t, J = 8.4 Hz, 2H), 7.04 (dd, J = 33.8, 14.7 Hz, 1H), 6.63 (d, J = 7.9 Hz, 1H), 5.00–4.85 (m, 2H), 4.09–4.00 (m, 2H), 3.79 (d, J = 115.9 Hz, 4H), 2.92–2.78 (m, 2H), 2.23–1.96 (m, 4H).

[0198] Preparation Example S16: Preparation of Compound S16

[0199] Synthesis of compound S16: Same as in Preparation Example 14, except that 14-2 is replaced with

[0200] A white solid was obtained. 1¹H NMR (400MHz, deuterated chloroform) δ 9.07–9.01 (m, 2H), 8.22 (s, 1H), 8.15–8.03 (m, 2H), 7.85 (t, J = 6.5Hz, 2H), 7.72–7.55 (m, 2H), 7.21 (t, J = 8.6Hz, 2H), 6.87–6.68 (m, 1H), 6.51 (d, J = 8.7Hz, 1H), 5.02–4.62 (m, 4H), 4.15–3.91 (m, 4H), 3.82 (s, 2H), 2.91 (s, 2H), 2.21–2.04 (m, 4H).

[0201] Preparation Example S17: Preparation of Compound S17

[0202] Synthesis of compound S17: Same as in Example 14, except that 14-2 is replaced with

[0203] A white solid was obtained. 1 ¹H NMR (400MHz, deuterated chloroform) δ 9.08 (s, 2H), 8.23 ​​(s, 1H), 7.83 (dd, J = 8.5, 5.3Hz, 2H), 7.71–7.59 (m, 3H), 7.54 (d, J = 1.8Hz, 1H), 7.23 (t, J = 9.1Hz, 2H), 6.89–6.69 (m, 1H), 6.52 (d, J = 8.6Hz, 1H), 5.00–4.86 (m, 2H), 4.82 (s, 2H), 4.15–3.93 (m, 4H), 3.56 (t, J = 5.9Hz, 2H), 2.92 (s, 2H), 2.22–2.02 (m, 4H).

[0204] Preparation Example S18: Preparation of Compound S18

[0205] Synthesis of compound S18: Same as in Example 14, except that 14-2 is replaced with

[0206] A white solid was obtained. 1 ¹H NMR (400 MHz, deuterated chloroform) δ 8.89 (s, 2H), 8.62–8.54 (m, 2H), 8.24 (s, 1H), 7.94–7.86 (m, 2H), 7.74–7.58 (m, 2H), 7.22 (t, J = 8.5 Hz, 2H), 6.88–6.69 (m, 1H), 6.52 (d, J = 8.6 Hz, 1H), 5.06–4.53 (m, 4H), 4.22–3.58 (m, 6H), 2.93 (s, 2H), 2.09 (s, 4H).

[0207] Preparation Example S19: Preparation of Compound S19

[0208] Synthesis of compound S19: Same as in Example 14, except that 14-2 is replaced with

[0209] A white solid was obtained. 1 ¹H NMR (700MHz, deuterated chloroform) δ 8.25 (d, J = 19.7 Hz, 2H), 8.18–8.06 (m, 2H), 8.01 (d, J = 9.0 Hz, 1H), 7.92–7.83 (m, 2H), 7.74–7.59 (m, 2H), 7.22 (t, J = 8.3 Hz, 2H), 6.87–6.69 (m, 1H), 6.52 (d, J = 8.5 Hz, 1H), 5.00–4.86 (m, 2H), 4.81 (s, 2H), 4.16–3.94 (m, 6H), 2.92 (s, 2H), 2.31–2.10 (m, 4H).

[0210] Preparation Example S20: Preparation of Compound S20

[0211] Synthesis of compound S20: Same as in Example 14, except that 14-2 is replaced with

[0212] A white solid was obtained. 1 ¹H NMR (400MHz, deuterated chloroform) δ 8.25 (s, 2H), 8.17–8.05 (m, 3H), 7.92–7.84 (m, 2H), 7.66 (d, J = 14.8 Hz, 2H), 7.23 (t, J = 9.6 Hz, 2H), 6.88–6.70 (m, 1H), 6.52 (d, J = 8.6 Hz, 1H), 4.93 (d, J = 11.3 Hz, 2H), 4.70 (s, 2H), 4.17 (dt, J = 12.4, 6.3 Hz, 4H), 4.12–3.93 (m, 2H), 2.93 (s, 2H), 2.74 (t, J = 6.2 Hz, 2H), 2.69 (t, J = 6.4 Hz, 2H).

[0213] Preparation Example S21: Preparation of Compound S21

[0214] Synthesis of compound S21: Same as in Example 14, except that 14-2 is replaced with

[0215] A white solid was obtained. 1¹H NMR (400MHz, deuterated chloroform) δ 8.22 (d, J = 6.7Hz, 2H), 8.10 (d, J = 8.8Hz, 2H), 7.98 (d, J = 8.8Hz, 1H), 7.90–7.83 (m, 2H), 7.71–7.60 (m, 2H), 7.21 (t, J = 8.6Hz, 2H), 6.87–6.67 (m, 1H), 6.51 (d, J = 8.6Hz, 1H), 5.01–4.75 (m, 5H),4.45(d,J=14.0Hz,1H),4.03(d,J=33.8Hz,2H),3.52–3.43(m,1H),3.20(td,J=13.2,2.7Hz,1H),2 .91(s,2H),2.26–1.96(m,5H),1.90(td,J=12.9,4.0Hz,1H),1.69(td,J=13.3,4.3Hz,1H),1.48(s,3H).

[0216] Preparation Example S22: Preparation of Compound S22:

[0217] Synthesis of compound S22: Same as in Preparation Example 14, except that 14-2 is replaced with

[0218] A white solid was obtained. 1 ¹H NMR (400MHz, deuterated chloroform) δ 8.31–8.03 (m, 5H), 7.91–7.83 (m, 2H), 7.73–7.60 (m, 2H), 7.21 (t, J = 8.5Hz, 2H), 6.90–6.66 (m, 1H), 6.52 (d, J = 8.7Hz, 1H), 5.04–4.62 (m, 4H), 4.56 (d, J = 12.3Hz, 1H), 4.40 (dd, J = 33.7, 12.8Hz, 2H), 4.19–3.95 (m, 3H), 2.92 (s, 2H), 2.53–2.29 (m, 2H).

[0219] Preparation Example S23: Preparation of Compound S23

[0220] Synthesis of compound S23: Same as in Preparation Example 14, except that 14-2 is replaced with

[0221] A white solid was obtained. 1¹H NMR (700MHz, deuterated dimethyl sulfoxide) δ 8.98 (d, J = 9.3 Hz, 1H), 8.63 (d, J = 8.8 Hz, 1H), 8.43 (s, 1H), 8.33 (d, J = 1.8 Hz, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.19 (d, J = 7.3 Hz, 1H), 8.04–7.99 (m, 2H), 7.92 (d, J = 8.6 Hz, 1H), 7.49–7.44 (m, 1H), 7.42 (t, J = 8.7 Hz, 2H), 7.14 (dd, J = 41.1, 15.3 Hz, 1H),6.52–6.41(m,3H),5.05(s,1H),4.85(s,1H),4.50–4.37(m,1H),4.02(d,J=71.2Hz,2H),3.11(td,J=10.9,5.0Hz,1H),2.89(s ,1H),2.82(d,J=10.9Hz,2H),2.43(dd,J=28.8,12.0Hz,1H),2.27(s,3H),2.24–2.19(m,1H),2.07–1.98(m,1H),1.89–1.81(m,1H).

[0222] Preparation Example S24: Preparation of Compound S24

[0223] Synthesis of compound S24: Same as in Example 14, except that 14-2 is replaced with

[0224] A white solid was obtained. 1 ¹H NMR (400MHz, deuterated chloroform) δ 8.31 (d, J = 8.9Hz, 1H), 8.24 (d, J = 14.8Hz, 2H), 8.11 (d, J = 8.8Hz, 2H), 7.86 (dd, J = 8.4, 5.4Hz, 2H), 7.72–7.58 (m, 2H), 7.22 (t, J = 8.5Hz, 2H), 6.88–6.67 (m, 1H), 6.52 ( d,J=8.6Hz,1H),6.06(d,J=4.2Hz,1H),5.03(t,J=9.9Hz,1H),4.97–4.82(m,5H),4.44–4.36( m,1H),4.30(dd,J=11.0,5.4Hz,1H),4.04(d,J=34.8Hz,2H),3.28–3.11(m,1H),2.91(s,2H).

[0225] Preparation Example S25: Preparation of Compound S25

[0226] Synthesis of compound S25: Same as in Example 14, except that 14-2 is replaced with

[0227] A white solid was obtained. 1 ¹H NMR (700 MHz, deuterated dimethyl sulfoxide) δ 8.60 (d, J = 8.9 Hz, 1H), 8.46 (s, 1H), 8.31 (s, 1H), 8.22–8.15 (m, 2H), 8.03 (dd, J = 8.4, 5.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 1H), 7.49–7.39 (m, 3H), 7.14 (dd, J = 44.7, 15.3 Hz, 1H), 6 .53–6.42(m,3H),5.18(t,J=5.4Hz,1H),4.94(d,J=139.8Hz,2H),4.52–4.43(m,2H),4.07(s,1H) ,3.97(s,2H),3.96(s,1H),3.90(d,J=10.1Hz,1H),3.66(d,J=5.4Hz,2H),2.86(d,J=51.6Hz,2H).

[0228] Preparation Example S26: Preparation of Compound S26

[0229] Synthesis of compound S26: Same as in Example 14, except that 14-2 is replaced with

[0230] A white solid was obtained. 1 ¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 8.60 (d, J = 8.9 Hz, 1H), 8.46 (s, 1H), 8.30 (s, 1H), 8.19 (s, 1H), 8.07–7.99 (m, 3H), 7.93 (d, J = 8.8 Hz, 1H), 7.50–7.37 (m, 3H), 7.14 (dd, J = 24.5, 14.9 Hz, 1H), 6.51–6.41 (m, 3H), 4.96 (d, J = 79.5 Hz, 2H), 4.03 (d, J = 41.3 Hz, 2H), 3.66–3.60 (m, 2H), 3.59–3.53 (m, 2H), 2.87 (d, J = 27.0 Hz, 2H).

[0231] Preparation Example S27: Preparation of Compound S27

[0232] Synthesis of compound S27: Same as in Example 14, except that 14-2 is replaced with

[0233] A white solid was obtained. 1¹H NMR (700 MHz, deuterated dimethyl sulfoxide) δ 8.86 (d, J = 8.8 Hz, 1H), 8.70 (s, 1H), 8.55 (s, 1H), 8.44 (s, 1H), 8.27 (dt, J = 11.4, 7.1 Hz, 3H), 8.18 (d, J = 8.7 Hz, 1H), 7.75–7.63 (m, 3H), 7.40 (dd, J = 43.8, 14.9 Hz, 1H), 6.74 (s, 3H), 5.20 (d, J = 138.4 Hz, 2H), 4.38–3.87 (m, 6H), 3.31–3.01 (m, 6H), 2.84 (s, 3H).

[0234] Preparation Example S28: Preparation of Compound S28

[0235] Synthesis of compound S28: Same as in Example 14, except that 14-2 is replaced with

[0236] A white solid was obtained. 1 ¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 9.22–9.09 (m, 1H), 8.63 (d, J = 8.9 Hz, 1H), 8.44 (s, 1H), 8.32 (d, J = 1.8 Hz, 1H), 8.26 (d, J = 8.8 Hz, 1H), 8.19 (s, 1H), 8.02 (dd, J = 8.5, 5.5 Hz, 2H), 7.93 ( d,J=8.8Hz,1H),7.50–7.36(m,3H),7.23–7.06(m,1H),6.48(s,3H),4.96(d,J=80.7Hz ,2H),4.02(d,J=41.3Hz,2H),3.63–3.50(m,5H),3.34(s,2H),2.86(d,J=27.7Hz,2H).

[0237] Preparation Example S29: Preparation of Compound S29

[0238] Synthesis of compound S29: Same as in Example 14, except that 14-2 is replaced with

[0239] A white solid was obtained. 1¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 9.12 (s, 1H), 8.60 (d, J = 8.9 Hz, 1H), 8.42 (d, J = 1.8 Hz, 1H), 8.29 (d, J = 1.8 Hz, 1H), 8.24 (d, J = 8.9 Hz, 1H), 8.15 (s, 1H), 8.03–7.96 (m, 2H), 7.89 (d, J = 8.8 Hz, 1H). 7.49–7.31(m,3H),7.20–7.01(m,1H),6.49–6.34(m,3H),5.02(s,1H),4.82(s,1H),3.99(d ,J=39.9Hz,2H),3.63–3.48(m,6H),3.45–3.39(m,2H),3.21(s,3H),2.84(d,J=26.9Hz,2H).

[0240] Preparation Example S30: Preparation of Compound S30

[0241] Synthesis of compound S30: Same as in Example 14, except that 14-2 is replaced with

[0242] A white solid was obtained. 1 ¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 11.41 (s, 1H), 8.44 (s, 2H), 8.32 (d, J = 1.8 Hz, 1H), 8.18 (d, J = 2.1 Hz, 2H), 8.01 (td, J = 5.7, 2.0 Hz, 2H), 7.93 (d, J = 8.8 Hz, 1H), 7.50–7.32 (m, 3H), 7.22–7.05 (m, 1H), 6.54–6.38 (m, 3H), 4.94 (d, J = 80.4 Hz, 2H), 4.02 (d, J = 41.3 Hz, 2H), 2.97–2.74 (m, 6H).

[0243] Preparation Example S31: Preparation of Compound S31

[0244] Synthesis of compound S31: Same as in Example 14, except that 14-2 is replaced with

[0245] A white solid was obtained. 1¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 10.63 (s, ¹H), 8.42–8.31 (m, 2H), 8.25 (s, ¹H), 8.18–8.09 (m, 2H), 8.00–7.86 (m, 3H), 7.47–7.32 (m, 3H), 7.17–7.03 (m, ¹H), 6.49–6.38 (m, 3H), 4.9 0(d,J=79.9Hz,2H),4.55(dd,J=8.2,5.5Hz,1H),3.97(td,J=15.7,14.9,8.6Hz,3H), 3.82(q,J=7.1Hz,1H),2.81(d,J=28.0Hz,2H),2.28–2.14(m,1H),2.06–1.77(m,3H).

[0246] Experimental Example 1: Evaluation of the inhibitory activity of the compound on the proliferation of pancreatic cancer cells

[0247] The sulforhodamine B (SRB) protein staining method was used to evaluate the cell proliferation inhibitory activity of the compound.

[0248] 1. Pancreatic cancer cells MIAPaCa-2 in the logarithmic growth phase were seeded at an appropriate density in 96-well plates.

[0249] 2. Incubate overnight at 37℃ until cells adhere to the wall. Add 10 μL of different concentrations of the compound per well, with 3 auxiliary wells for each concentration. Set up a solvent control group and a cell-free blank control group.

[0250] 3. After culturing cells for 72 hours, discard the cell culture medium and add 100 μL of pre-cooled 10% trichloroacetic acid (TCA) solution to each well. Fix at 4°C for 1 hour. Discard the fixative, wash with distilled water, and dry in an oven.

[0251] 4. Add 100 μL of SRB dye to each well, stain at room temperature for 20 min, then discard the dye. Wash with 1% glacial acetic acid solution and dry in an oven.

[0252] 5. Add 150 μL of 10 mM Tris solution to each well to dissolve the SRB dye.

[0253] 6. Measure the OD value of each well at 560 nm using a microplate reader, and calculate the inhibition rate of the compound according to the following formula:

[0254] Inhibition rate % = (OD value of control group - OD value of treatment group) / OD value of control group × 100%.

[0255] Table 1. Inhibitory activity of compounds S19-S23, S25, and S26 on the proliferation of MIAPaCa-2 cells.

[0256]

[0257]

[0258] Cell proliferation inhibition activity assays showed that compounds S19-S23, S25, and S26 exhibited superior inhibitory activity against the in vitro proliferation of MIAPaCa-2 cells compared to KPT-9274. Among these, compounds S21 and S23 showed the highest IC50 values. 50 The values ​​were approximately 4-fold and 3-fold higher than those of KPT-9274, respectively. These results indicate that this class of compounds possesses good tumor cell proliferation inhibitory activity, and that its in vitro tumor cell proliferation inhibitory activity is superior to that of KPT-9274.

[0259] Experimental Example 2: Determining the effect of the compound on PAK4 and downstream signaling activities by immunoblotting.

[0260] 1. Seed U2-OS cells in logarithmic growth phase at an appropriate density in 6-well plates. After cell attachment, discard the original culture medium, add 2 mL of fresh culture medium, and add DMSO or other compounds of various concentrations. Incubate the cells at 37°C for 72 h. Discard the cell culture medium, wash three times with PBS, add an appropriate amount of 1×SDS (Sodium Dodecyl Sulfate) cell lysis buffer, and collect the sample into 600 μL EP tubes after complete cell lysis. Heat at 100°C for 20 min.

[0261] 2. Prepare a 10% SDS-PAGE gel (Sodium Dodecyl Sulfate Polyacrylamide Gel), add 1×TGS (25mM Tris, 250mM Glycine, 0.1% SDS) electrophoresis buffer, and take an appropriate amount of sample for gel electrophoresis. The initial voltage is 90V. When the sample enters the lower separating gel, adjust the voltage to 120V until the electrophoresis is complete.

[0262] 3. Transfer the protein from the gel to a nitrocellulose membrane using a transfer apparatus. Block the bands with a 5% skim milk powder solution (w / v) prepared with TBST solution (containing 1% Tween 20 in 10mM Tris-HCl and 150mM NaCl solution) at room temperature for 1 hour. Wash the bands with distilled water, cut the bands from each target protein location, and incubate them overnight at 4°C with primary antibody solutions of p-PAK4 (S474), p-β-Catenin (S675), and β-Actin, respectively.

[0263] 4. Recover the primary antibody and wash the bands three times with TBST solution on a shaker for 10 min each time. Incubate the bands with mouse and rabbit secondary antibody solutions prepared with the same TBST solution for 1 h, and wash three times with TBST solution. Develop and colorimetrically validate the compounds using a chromatograph. Initially screen the compounds using a 96-well plate system with a concentration of 1 μmol / L, serially diluted 3-fold, with duplicate wells for each concentration.

[0264] 5. Semi-quantitative analysis of downstream phosphorylation levels was performed using ImageJ software.

[0265] Table 2. Inhibition rates of compounds S19-S21, S23 and S25 on p-PAK4 and p-β-Catenin

[0266]

[0267]

[0268] Immunoblotting experiments showed that compounds S19-S21, S23, and S25 significantly inhibited the levels of downstream p-PAK4 and p-β-Catenin of PAK4. These results indicate that the compounds in this invention are novel PAK4 inhibitors that can effectively inhibit the phosphorylation of downstream PAK4 signals. Furthermore, the inhibitory activity of these compounds on downstream PAK4 signals is significantly superior to that of KPT-9274.

[0269] Experimental Example 3: Solubility of hydrochloride salts of compounds S21 and S23 in water by high performance liquid chromatography

[0270] Compounds S21, S23 and KPT-9274 were dissolved in a small amount of methanol, and an appropriate amount of 4 mol / L hydrochloric acid methanol solution was added. The mixture was stirred for one hour, and the methanol was removed by evaporation. This process was repeated three times to prepare hydrochloric acid salts of S21, S23 and KPT-9274.

[0271] Weigh an appropriate amount of the compound and dissolve it in 1 mL of methanol or water. Dilute the solution 5, 10, 50, and 100 times in sequence. Detect the five concentrations of the solution sequentially by high performance liquid chromatography under the same injection volume conditions to establish a linear relationship between concentration and peak area.

[0272] Weigh an appropriate amount of the compound and prepare 1 mL of saturated aqueous solution. Filter the solution through a filter membrane and detect the peak area of ​​the filtrate by high performance liquid chromatography. Substitute the peak area into the linear equation to calculate the solubility of the compound in water.

[0273] Figure 1 This is a linear relationship graph of KPT-9274 (hydrochloride) solution concentration and peak area.

[0274] Calculations show that the solubility of KPT-9274 (hydrochloride) in water is <0.002 mg / mL.

[0275] Figure 2 This is a linear relationship graph of the concentration and peak area of ​​compound S21 (hydrochloride) solution.

[0276] The solubility of compound S21 (hydrochloride) in water was calculated to be 0.252 ± 0.014 mg / mL.

[0277] Figure 3 This is a linear relationship graph of the concentration and peak area of ​​compound S23 (hydrochloride) solution.

[0278] The solubility of compound S23 (hydrochloride) in water was calculated to be 33.98 ± 1.36 mg / mL.

[0279] Both compounds S21 and S23 exhibit excellent water solubility. Compared with KPT-9274, the solubility of the two compounds in water increased by 126 times and 16,990 times, respectively. These data indicate that compounds S21 and S23 have excellent physicochemical properties and are expected to be used in the research and treatment of PAK4-related diseases.

[0280] In summary, drugs targeting PAK4 have promising market prospects, but no drugs have yet been marketed, highlighting the urgent need to develop more novel PAK4 inhibitors to meet clinical demands. This invention, based on the structure of ATG-019 (KPT-9274), a Phase I clinical trial compound from Karyopharm, designs and synthesizes a novel class of tricyclic PAK4 inhibitors. These PAK4 inhibitors exhibit excellent cell proliferation inhibitory activity against MIAPaCa-2 cells and show good inhibitory effects on PAK4 phosphorylation and downstream β-catenin phosphorylation, representing a completely new type of PAK4 inhibitor. Furthermore, some compounds demonstrate significantly superior cell proliferation inhibitory activity, PAK4 downstream signaling inhibitory activity, and water solubility compared to the Phase I clinical compound KPT-9274. Therefore, the compounds described in this patent hold promise as effective in vivo PAK4 inhibitors, providing new treatment options for cancer patients.

[0281] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound, characterized in that, The compound is a compound of Formula I or a pharmaceutically acceptable salt thereof. Equation I in, A is selected from the following group: , , , , where R 0 Selected from the following group: hydrogen, C1-C6 alkyl; V, W, X, and Y are each independently selected from the following groups: N, CR 4 , where R 4 Selected from the following group: hydrogen, halogen, hydroxyl, C1-C6 alkoxy, amino, cyano, C1-C6 alkyl; Z is selected from the following group: , ; R 1 Selected from the group consisting of: hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted 4-8 membered heterocyclic alkyl containing 1, 2 or 3 heteroatoms selected from N, O, S, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O, S; wherein, the substitution independently refers to substitution by one or more substituents selected from the group consisting of: halogen, hydroxyl, cyano, C1-C6 alkoxy, amino, C1-C6 alkyl; R 2 Selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 ynyl, substituted or unsubstituted 4-8 membered heterocyclic alkyl containing 1, 2 or 3 heteroatoms selected from N, O, and S, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O, and S. ; wherein, n is selected from the group consisting of: 0, 1, 2, 3, and the substitution independently refers to being substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, amino, cyano, mercapto, difluoromethyl, oxo (=O), C1-C6 alkoxy, C1-C6 alkyl, and the heterocyclic alkyl is selected from the group consisting of: monocyclic, fused, spirocyclic, and bridged ring. R 3 Selected from the following groups: hydrogen, amino.

2. The compound according to claim 1, characterized in that, A is selected from the following group: , , , , where R 0 For H.

3. The compound according to claim 1, characterized in that, Selected from the following group: , , , , , , , ; R 4 Selected from the following group: hydrogen, halogens.

4. The compound according to claim 1, characterized in that, R 2 Selected from the following group: substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted 4-8 membered heterocyclic alkyl groups containing 1, 2, or 3 heteroatoms selected from N, O, and S, substituted or unsubstituted C3-C8 cycloalkyl groups. Wherein, n is selected from the group consisting of 0, 1, 2, 3, and the substitution independently refers to being substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, mercapto, difluoromethyl, oxo (=O), C1-C6 alkoxy, C1-C6 alkyl, and the heterocyclic alkyl is selected from the group consisting of monocyclic, fused, spirocyclic, and bridged ring.

5. The compound according to claim 1, characterized in that, R 1 Selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C6-C10 aryl groups; wherein the substitution independently refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C6 alkyl.

6. The compound according to claim 1, characterized in that, The compounds are selected from the group consisting of: 。 7. A pharmaceutical composition, characterized in that, The compound comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compound of claim 1.

8. Use of the compound according to claim 1, characterized in that, Used to prepare a medicine for the prevention and / or treatment of diseases selected from the group consisting of: cancer, infectious diseases, and neurological diseases.

9. Use of the compound according to claim 1, characterized in that, Used to prepare a drug for inhibiting PAK4 kinase activity.

10. Use of the compound according to claim 1, characterized in that, Used to prepare a drug for the prevention and / or treatment of diseases related to PAK4 kinase activity.

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