CN115873018B - Benzopyrimidine and benzotriazine hematopoietic progenitor cell kinase 1 degradation agent and application thereof - Google Patents

Benzopyrimidine and benzotriazine hematopoietic progenitor cell kinase 1 degradation agent and application thereof Download PDF

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CN115873018B
CN115873018B CN202211448593.9A CN202211448593A CN115873018B CN 115873018 B CN115873018 B CN 115873018B CN 202211448593 A CN202211448593 A CN 202211448593A CN 115873018 B CN115873018 B CN 115873018B
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CN115873018A (en
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张阔军
毛杰
唐鹤
蒋晟
肖易倍
王淋
王凯振
周立昕
叶秀全
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China Pharmaceutical University
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Abstract

The invention discloses a benzopyrimidine and benzotriazine hematopoietic progenitor cell kinase 1 degradation agent and application thereof. The invention utilizes the advantage that PROTAC can realize selectivity which is difficult to realize by small molecules, and develops a class of benzimidazole and benzotriazine HPK1 PROTAC degradation agents. The PROTAC can degrade HPK1 protein with high efficiency and high selectivity, and has no degradation activity on other proteins in the same family. Compared with the HPK1 small molecule inhibitor, the anti-tumor immune response of the T cells can be stimulated more effectively, the effector cell factor is released, and the anti-tumor activity is stronger.

Description

Benzopyrimidine and benzotriazine hematopoietic progenitor cell kinase 1 degradation agent and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a benzopyrimidine and benzotriazine hematopoietic progenitor cell kinase 1 degradation agent and application thereof.
Background
Immunotherapy thoroughly changes the treatment mode of cancer and brings new hopes for patients. In particular, immune checkpoint inhibitors against the PD-1 and CTLA-4 pathways show significant clinical effects, but they are only effective against specific patient populations, specific types of tumors, with low tumor response rates (-30%), and most patients do not show long lasting symptomatic relief, develop resistance after a period of administration. Therefore, a new tumor immunotherapy target is needed to be discovered, the clinical effect of immune check points is improved, and the method has important significance for application and popularization of tumor immunotherapy.
Hematopoietic progenitor kinase 1 (HPK 1, MAP4K 1) is one of the members of the MAP4K family, a Ste20 silk/threonine protein kinase. There are 5 members of the MAP4K family, including MAP4K2, MAP4K3, MAP4K4, MAP4K5 and MAP4K6, in addition to HPK 1. HPK1 is expressed primarily in hematopoietic cells such as T cells, B cells, neutrophils, dendritic Cells (DCs), natural Killer (NK) cells, and macrophages. Numerous studies have shown that HPK1 is a negative regulator of T cell receptors and B cell receptors. After TCR activation, HPK1 in the cytoplasm is recruited to the plasma membrane, where its amino acid residues Y381, S171 and T165 are phosphorylated, resulting in a fully kinase-activated HPK1 kinase. The activated HPK1 phosphorylates the amino acid residue S376 of the linker protein SLP76, provides a binding site for the negative regulatory factor 14-3-3, promotes the degradation of SLP76 by a proteasome, and finally breaks down the stability of the TCR signal complex, thereby blocking the kinase signal pathway downstream for promoting T cell activation and proliferation. In addition to TCR signaling, HPK1 negatively regulates T cell signaling through prostaglandin E2 (PGE 2) receptors. In addition, HPK1 can also transmit immunosuppressive signals in B cells, NK cells, and dendritic cells under stimulation by growth factors, stress, inflammatory factors, and differentiation factors, among others. In addition, a recent study has shown that kinase activity of HPK1 inhibits immune function in a variety of cells, including CD4 + T cells, CD8 + T cells, NK cells and dendritic cells, and demonstrate that inhibition of the kinase activity of HPK1 is sufficient to induce an anti-tumor immune response. In addition, inhibiting the activity of HPK1 kinase can further promote T cell effector function and obviously increase the therapeutic effect of PD-L1 monoclonal antibody. HPK1 gene deletion, HPK1 inhibitor or HPK1 protac degradation agent can enhance CAR-T cell-based immunotherapy, in various preclinical hematologies andthe solid tumor mouse model shows better anti-tumor activity. Notably, neither the HPK1 gene knockout nor the inactivated kinase knockout mice showed a lethal inflammatory response, but these were significant in the absence of some other immune negative regulators, such as CTLA-4 and Cbl-b, indicating a higher safety profile for HPK1 inhibitors. In view of the above, HPK1 is a new target of tumor immunotherapy that is potentially effective, and research and development of HPK1 inhibitors are of great importance for improving the key problems currently faced by tumor immunotherapy.
Over a decade ago, developers discovered that HPK1 could be a potential tumor immunotherapy target, HPK1 inhibitors were favored by various pharmaceutical companies and scientific institutions, various structures of HPK1 inhibitors were reported, and it was not completely counted that 4 HPK1 inhibitors were currently in early clinical research stages, including CFI-402411 of Treadwell Therapeutics, BGB-15025 of baji, and NDI-101150 of PRJ1-3024 and Nimbus Therapeutics of zhuhai. At present, their structure has not been disclosed. Although a variety of HPK1 inhibitors have been reported, no related drugs are currently marketed. The challenges faced in the development of HPK1 inhibitors are mainly the difference in HPK1 family member functions, but the structural homology is very high, and the difficulty in designing highly selective inhibitors is great.
Compared with traditional medicines, PROTAC has the remarkable advantages that: the application range is wider, the activity is higher, and the target can be targeted to a target point which is not patent medicine; the selectivity, activity and safety are improved; overcomes drug resistance of tumor drugs, etc. The PROTAC can realize the selectivity which is difficult to realize by small molecules on certain targets, so that the development of the HPK1 PROTAC molecules is expected to solve the problems of low selectivity, incapacity of dose dependence of drug effect and the like of HPK1 inhibitors, and the HPK1 PROTAC molecules are rarely reported at present. Therefore, the research and development of the HPK1 PROTAC molecule are urgent, and the HPK1 PROTAC molecule has important scientific significance and development value.
Disclosure of Invention
Aiming at the problem of low selectivity of the HPK1 small molecule inhibitor, the invention utilizes the advantage of the selectivity which is difficult to realize by the small molecule of PROTAC, develops the PROTAC molecule of the targeted HPK1, and provides a benzimidazole and benzotriazine HPK1 PROTAC degradation agent. The PROTAC can degrade HPK1 protein with high efficiency and high selectivity, and has no degradation activity on other proteins in the same family. Compared with the HPK1 small molecule inhibitor, the anti-tumor immune response of the T cells can be stimulated more effectively, the effector cell factor is released, and the anti-tumor activity is stronger.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a compound of formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof,
in formula I:
R 1 hydrogen, halogen or cyano;
R 2 is unsubstituted or R 2-1 Substituted C 6-10 Aryl, unsubstituted or R 2-2 The substituted hetero atom is selected from one or more of ' N, O or S ', and the hetero atom number is 1-3 ' 5-10 membered heteroaryl;
R 2-1 is halogen, C 1-6 Alkoxy, unsubstituted or R 2-1-1 Substituted C 1-6 An alkyl group;
R 2-1-1 is hydroxy or halogen;
R 2-2 is halogen, amino or C 1-6 An alkyl group;
R 3 is hydrogen or amino;
q is N or C;
a is“/>"terminal" means attached to NH and "- - -" terminal means attached to L;
b is E3 ubiquitin ligase ligand CRBN or VHL;
l is“/>"terminal" means attached to A and "- -" terminal means attached to B;
m is an integer between 0 and 5;
n is an integer between 0 and 10;
o is an integer between 1 and 5;
x is C, O, NH,“/>The "terminal" means attached to L and the "- - -" terminal means attached to B.
In some embodiments, B isOr->Y is hydrogen, deuterium, fluorine or chlorine; w is CH 2 Or carbonyl; g is hydrogen or methyl; z is hydrogen or methyl.
In some embodiments, B is
In some embodiments, R 2 Is unsubstituted or R 2-1 Substituted phenyl, unsubstituted or R 2-2 Substituted pyridinyl, unsubstituted or R 2-2 Substituted pyridomorpholinyl.
In some embodiments, R 2 Is that
In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, is of the formula:
wherein A, L and B are as defined above.
In some embodiments, the compound of formula I is selected from compounds of any one of the following structures:
the use of a compound of formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, in the manufacture of a medicament for the treatment and/or prophylaxis of cancer.
In some embodiments, the cancer is one or more of bone cancer, lung cancer, stomach cancer, colon cancer, membranous adenocarcinoma, breast cancer, prostate cancer, lung cancer, brain cancer, ovarian cancer, cancer of the shoulders, cervical cancer, cancer of the kidneys, head and neck cancer, thyroid cancer, esophageal cancer, lymphatic cancer, leukemia, or skin cancer.
A pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, and a pharmaceutically acceptable carrier or adjuvant.
In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, is used in the pharmaceutical composition in an amount effective for treatment.
The invention provides an application of a pharmaceutical composition in preparing a medicament for treating and/or preventing cancer.
In some embodiments, the cancer is one or more of bone cancer, lung cancer, stomach cancer, colon cancer, membranous adenocarcinoma, breast cancer, prostate cancer, lung cancer, brain cancer, ovarian cancer, cancer of the shoulders, cervical cancer, cancer of the kidneys, head and neck cancer, thyroid cancer, esophageal cancer, lymphatic cancer, leukemia, or skin cancer.
The pharmaceutical excipients can be those which are widely used in the field of pharmaceutical production. Adjuvants are used primarily to provide a safe, stable and functional pharmaceutical composition, and may also provide means for allowing the subject to dissolve at a desired rate after administration, or for promoting effective absorption of the active ingredient after administration of the composition. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients can comprise one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, sizing agents, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption retarders, dissolution inhibitors, enhancing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.
The pharmaceutical compositions of the present invention may be prepared in accordance with the disclosure using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.
The pharmaceutical compositions of the present invention may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular). The pharmaceutical compositions of the invention may also be in controlled or delayed release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry formulations which may be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic formulations; aerosol: such as nasal sprays or inhalants; a liquid dosage form suitable for parenteral administration; suppositories and lozenges.
The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting the free form of such compounds with a sufficient amount of base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the free form of such compounds with a sufficient amount of acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogenphosphates, dihydrogenphosphates, sulfuric acid (forming sulfates or bisulphates), hydroiodic acid, phosphorous acid, and the like, and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like, salts of amino acids (such as arginine and the like), and salts of organic acids such as glucuronic acid.
The "pharmaceutically acceptable salts" of the present invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
The term "isomer" refers to compounds of the same chemical formula but having different arrangements of atoms.
The term "metabolite" refers to a pharmaceutically active product of a compound of formula I or a salt thereof produced by in vivo metabolism. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, glucuronidation, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes metabolites of the compounds of the present invention, including compounds produced by a method of contacting a compound of the present invention with a mammal for a period of time sufficient to obtain the metabolites thereof.
Identification of metabolites typically occurs by preparing a radiolabeled isotope of a compound of the invention, parenterally administering it to an animal, such as a rat, mouse, guinea pig, monkey, or human, in a detectable dose (e.g., greater than about 0.5 mg/kg), allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion product from urine, blood, or other biological samples. These products are easy to isolate because they are labeled (others are isolated by using antibodies that are capable of binding to epitopes present in the metabolite). The metabolite structures are determined in a conventional manner, for example by MS, LC/MS or NMR analysis. In general, the analysis of metabolites is performed in the same manner as conventional drug metabolism studies known to those skilled in the art. So long as it takes the place ofThe metabolite products are not otherwise undetectable in vivo, or they may be used in assays for the therapeutic dosing of the compounds of the invention. The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more of the atoms comprising the compounds. For example, compounds can be labeled with radioisotopes, such as tritium @, for example 3 H) Iodine-125% 125 I) Or C-14% 14 C) A. The invention relates to a method for producing a fibre-reinforced plastic composite All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
In addition to salt forms, the compounds provided herein exist in prodrug forms. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert to the compounds of the invention. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound of formula I) is a prodrug within the scope and spirit of the invention. For example, compounds containing a carboxyl group can form a physiologically hydrolyzable ester that acts as a prodrug by hydrolyzing in vivo to give the compound of formula I itself. The prodrugs are preferably administered orally, as hydrolysis occurs in many cases primarily under the influence of digestive enzymes. Parenteral administration may be used when the ester itself is active or hydrolysis occurs in the blood.
As will be appreciated by those skilled in the art, the present application describes "as used in the structural formula of a group" in accordance with convention used in the art ""and">"means that the corresponding group is attached to other fragments, groups in the compound of formula I through this site.
"substitution" in the present invention may be one or more, and when there are plural "substitution", the "substitution" may be the same or different.
The term "plurality" refers to 2,3, 4, or 5.
The term "halogen" refers to fluorine, chlorine, bromine or iodine.
The term "alkyl" refers to a straight or branched chain alkyl group having the indicated number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.
The term "aryl" refers to an aromatic group having the indicated number of carbon atoms, preferably a monocyclic, bicyclic or tricyclic aromatic group, and when bicyclic or tricyclic, each ring satisfies the shock rule. C of the invention 6-10 Aryl refers to an aromatic group containing 6 to 10 carbon atoms, such as phenyl or naphthyl.
The term "heteroaryl" refers to an aromatic group containing heteroatoms, preferably an aromatic 5-6 membered monocyclic, aromatic 9-10 membered bicyclic or 9-10 membered heteroaromatic saturated heterocycle containing 1,2 or 3 atoms independently selected from nitrogen, oxygen and sulfur. The 5-to 6-membered monocyclic ring includes, but is not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2, 3-triazolyl, 1,2, 4-triazolyl, furazanyl, 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl, thiadiazolyl, dithiazolyl, tetrazolyl, pyridyl, pyranyl, thiopyranyl, diazinyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazinyl, thiazinyl, dioxinyl, dithiinyl, 1,2, 3-triazinyl, 1,2, 4-triazinyl, 1,3, 5-triazinyl or tetrazinyl. The 9-to 10-membered bicyclic ring includes, but is not limited to, benzimidazolyl, indolyl, indazolyl, benzofuranyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzisozolyl, quinolinyl, isoquinolinyl, benzomorpholinyl, benzopiperidinyl, pyrazolomorpholinyl.
The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.
The reagents and materials used in the present invention are commercially available.
The invention has the advantages that:
(1) The HPK1 PROTAC molecule provided by the invention can efficiently and selectively degrade HPK1 protein, and has no degradation activity on proteins such as GLK in the same family.
(2) The HPK1 PROTAC molecules provided by the invention can more effectively stimulate the anti-tumor immune response of T cells
(3) The HPK1 PROTAC molecule provided by the invention has good therapeutic effect on cancers.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
EXAMPLE 1 Synthesis of Compound 4
a) 2, 6-tetramethylpiperidine (L87 g,1.33 mol) was dissolved in THF (1.4L), cooled to-78℃and a 2.5M n-BuLi/THF solution (400 mL,1.00 mol) was slowly added dropwise thereto, stirring was completed for 30 minutes, compound 1 (140 g,663 mmol) was dissolved in THF (1.12L), then the reaction solution was slowly added dropwise thereto, stirring was carried out at-78℃for 1 hour, and then ethyl formate (59 g,796 mol) was dissolved in THF (280 mL), and then the reaction solution was added dropwise thereto and stirring was carried out for 1 hour. After the reaction is completed, saturated NH is used 4 Cl solution (2L) was quenched and extracted with ethyl acetate (1 L×3), the combined organic layers were washed with saturated NaCl solution, dried over anhydrous Na 2 SO 4 Drying, suction filtration for salt removal, and column chromatography purification by vacuum concentration gave intermediate 2 (42 g, 27%). MS (ESI, M/z): 239 (M) + +1)。
b) Intermediate 2 (50 g,209 mmol), DIPEA (55 g,430 mmol), HNPMB 2 (63 g,240 mmol) was dissolved in 1, 4-dioxane (500 mL) and warmed to 100deg.C and stirred for 6h. The reaction solution was concentrated and purified by column chromatography to give intermediate 3 (90 g, 90%). MS (ESI, M/z): 476 (M) + +1)。
c) Intermediate 3 (90 g,189 mmol), cs 2 CO 3 (122 g,374 mmol) guanidine carbonate (44 g,242 mmol) was dissolved inIn DMA (360 mL), the reaction was stirred at 150℃for 30min. The reaction solution was cooled to room temperature, diluted with water and extracted with ethyl acetate (200 mL. Times.3), the combined organic layers were washed with saturated NaCl solution, and dried over anhydrous Na 2 SO 4 Drying, suction filtration for salt removal, and column chromatography purification by vacuum concentration gave intermediate 4 (31 g, 33%). MS (ESI, M/z): 497 (M) + +1)。
EXAMPLE 2 Synthesis of Compound 6
Guanidine carbonate (2.34 g,13.0 mmol) and DIPEA (4.42 mL,26.0 mmol) were dissolved in NMP (20 mL), heated to 160℃and a solution of Compound 5 (4.59 g,10.0 mmol) in NMP (5 mL) was slowly added to the hot reaction mixture. The reaction was stirred at 160℃for 2h, cooled to below 100℃and an ice-water mixture (100 mL) was added. The mixture was stirred at room temperature for 30min. The mixture was filtered and washed with water (20 mL) and ethanol (20 mL), and the filter cake was stirred with ethanol (20 mL) for 1h and then filtered again. The solid was washed with ethanol (20 mL), ethanol/toluene mixture (20 mL, 1:1) and toluene (20 mL) and the solid was dried to give product 6 (1.91 g, 40%). MS (ESI, M/z): 479 (M) + +1)。
EXAMPLE 3 Synthesis of Compound 8
Raw material 7 (2.17 g,5.8 mmol), guanidine hydrochloride (1.11 g,11.6 mmol), potassium tert-butoxide (5.2 g,46.4 mmol) were dissolved in THF (40 mL), refluxed for 2h, diluted with water after the reaction was complete, extracted with ethyl acetate (20 mL. Times.3), the combined organic layers were washed with saturated NaCl solution, and dried over anhydrous Na 2 SO 4 Drying, suction filtration of salt, concentration under reduced pressure gave intermediate 8 (672 mg, 29%) which was purified by column chromatography. MS (ESI, M/z): 396 (M) + +1)。
EXAMPLE 4 Synthesis of Compound 10
To a 30mL tube was added the starting material (268 mg,2.7 mmol), guanidine carbonate (4816 mg,2.7 mmol) was dissolved in DMA (13 mL), reacted at 100℃for 4h, the reaction solution was cooled and poured into 50mL of ice, stirred for 15min, and suction filtered to obtain an intermediate (553 mg, 91%). MS (ESI, M/z): 224 (M) + +1)。
Example 5 Synthesis of intermediates 13 and 15
a) Under argon, intermediate 4 (200 mg,0.4 mmol), 2-fluorophenylboronic acid pinacol ester (133 mg,0.6 mmol), pd (dppf) Cl 2 (29 mg,0.04 mmol) and Na 2 CO 3 (85 mg,0.8 mmol) was dissolved in 1, 4-dioxane (2 mL) and water (0.4 mL) and stirred at 100deg.C overnight. After completion of the reaction, the reaction mixture was cooled to room temperature, diluted with water, extracted with dichloromethane (10 mL. Times.3), the combined organic layers were washed with saturated NaCl solution, and dried over anhydrous Na 2 SO 4 Drying, suction filtration of salt, concentration under reduced pressure, and purification by column chromatography gave intermediate 11 (63 mg, 31%). MS (ESI, M/z): 513 (M) + +1)。
b) Under argon, intermediate 11 (313 mg,1 mmol), 2-trifluoro-1- (5-methoxy-7- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 4-dihydroisoquinolin-2 (1H) -yl) ethan-1-one (423 mg,1.1 mmol), K 3 PO 4 (316mg，1.5mmol)、Pd 2 (dba) 3 (92 mg,0.1 mmol), t-BuBrettPhos (96 mg,0.2 mmol), toluene (4 mL) and water (0.8 mL), and after completion of the reaction overnight at 110℃the filtrate was filtered off with suction and concentrated under reduced pressure and purified by column chromatography to give intermediate 12 (284 mg, 37%). MS (ESI, M/z): 770 (M) + +1)。
c) Intermediate 12 (6754 mg,1 mmol) was dissolved in methanol and K was added 2 CO 3 (276 mg,2 mmol) at room temperature for 2h, and after completion of the reaction, K was removed by suction filtration 2 CO 3 Spin-drying afforded intermediate 13 (620 mg, 92%). MS (ESI, M/z): 674 (M) + +1)。
d) Intermediate 13 (6754 mg,1 mmol) was dissolved in acetonitrile, ethyl bromoacetate (200 mg,1.2 mmol) was added, DIPEA reacted at room temperature for 2h, after completion of the reaction cooled to room temperature, ethyl acetate extracted (10 mL. Times.3), the combined organic layers were washed with saturated NaCl solution, and dried over anhydrous Na 2 SO 4 Drying, suction filtration for salt removal, and column chromatography purification by vacuum concentration gave intermediate 14 (292 mg, 85%). MS (ESI, M/z): 743.3 (M) + +1)。
e) Intermediate 14 (743 mg,1 mmol) was dissolved in tetrahydrofuran, 2M aqueous NaOH (1 mL) was added and reacted at room temperature for 2h, after completion of the reaction, pH was adjusted to acidity with 2M HCl, and the filter cake was obtained by suction filtration and dried under vacuum to obtain intermediate 15 (661mg, 92%). MS (ESI, M/z): 715 (M) + +1)。
Example 6 Synthesis of intermediates 18 and 20
The synthesis is described in reference to the synthesis method of example 5, and only the corresponding raw materials need to be replaced. Intermediate 18: MS (ESI, M/z): 715 (M) + +1). Intermediate 20: MS (ESI, M/z): 715 (M) + +1)。
Example 7 Synthesis of intermediates 22 and 24
The synthesis is described in reference to the synthesis method of example 5, and only the corresponding raw materials need to be replaced. Intermediate 22: MS (ESI, M/z): 715 (M) + +1). Intermediate 24: MS (ESI, M/z): 715 (M) + +1)。
Example 8 Synthesis of intermediate 26
The synthesis is described in reference to the synthesis method of example 5, and only the corresponding raw materials need to be replaced. Intermediate 26: MS (ESI, M/z): 715 (M) + +1)。
Example 9 Synthesis of intermediate 31
The synthesis is described in reference to the synthesis method of example 5, and only the corresponding raw materials need to be replaced. Intermediate 26: MS (ESI, M/z): 715 (M) + +1)。
Example 10 Synthesis of intermediates 34 and 36
The synthesis is described in reference to the synthesis method of example 5, and only the corresponding raw materials need to be replaced. Intermediate 26: MS (ESI, M/z): 715 (M) + +1)。
Example 11 Synthesis of intermediates 50, 52 and 53
a) Raw material 47 (2.0 g,7.25 mmol) was dissolved in DMF (10 mL), tert-butyl glycinate (1.14 g,8.69 mmol) and DIPEA (2.1 g,9.43 mmol) were added to the reaction system, reacted at 90℃for 5h, cooled to room temperature after completion of the reaction, diluted with water, extracted with dichloromethane (10 mL. Times.3), the organic phase was combined, washed with saturated NaCl solution, and anhydrous Na 2 SO 4 Drying, suction filtration to remove salt, and concentration under reduced pressure gave intermediate 48a (1.58 g, 56%) which was purified by column chromatography. MS (ESI, M/z): 410 (M) + +1). The intermediates 48b,51a-b,53a-e can be obtained by the same preparation method by only replacing the corresponding raw materials.
b) Intermediate 48a (387.4 mg,1 mmol) was dissolved in dioxymethane (2 mL), trifluoroacetic acid (148. Mu.L) was added, and reacted at 25℃for 8h, followed by direct spin-drying to give intermediate 49a (2910 mg, 88%). MS (ESI, M/z): 332 (M) + +1). The intermediates 49b,35a-b were prepared in the same manner, except that the corresponding starting materials were replaced.
c) Raw material 49a (331 mg,1 mmol) was dissolved in DMF, added5-Bromopentanamine (200 mg,1.2 mmol) and DIPEA (3838 mg,3 mmol) were added, reacted at room temperature for 2h, cooled to room temperature after completion of the reaction, diluted with water, extracted with dichloromethane (10 mL. Times.3), the organic phases were combined, washed with saturated NaCl solution, and dried Na 2 SO 4 Drying, suction filtration of salt, concentration under reduced pressure gave intermediate 50 (249 mg, 50%) which was purified by column chromatography. MS (ESI, M/z): 479 (M) + +1)。
d) Intermediate 51a (1 mmol) was dissolved in a solution of dichloromethane (1 mmol/5 mL) and methanol (1 mmol/1 mL), a solution of 4 MHz Cl in dioxane (0.5 mL) was added at 0deg.C, the reaction was allowed to proceed at room temperature for 4h, after completion of the TLC monitoring reaction, the reaction solution was dried by spin-drying, slurried with isopropyl ether, and suction filtered to obtain intermediate. MS (ESI, M/z): 317 (M) + +1). Intermediates 52a and 52b were prepared in the same manner, except that the corresponding starting materials were replaced.
Example 12 Synthesis of intermediate 55
Synthesis of intermediate 55 a: a) Bromoacetic acid (139 mg,1 mmol) was added to the reaction flask, and thionyl chloride (1 mL) was added thereto, and the reaction was stirred at 80℃for 5 hours. The reaction is cooled to room temperature completely, dried tetrahydrofuran is added for dissolution after spin drying, then raw material 54 (273 mg,1 mmol) is added for reaction at 50 ℃, after TLC monitoring the reaction is complete, the crude product of intermediate 55a can be obtained after cooling and suction filtration. MS (ESI, M/z): 394 (M) + +1)。
Synthesis of intermediate 55 b: intermediate 55b was prepared in the same manner, except that the corresponding starting materials were replaced. MS (ESI, M/z): 450 (M) + +1)。
Example 13 Synthesis of intermediates 58, 60 and 61
Synthesis of intermediate 58: a) Raw material 56 (1.57 g,5.71 mmol) was dissolved in DMF (57 mL) and K was added 2 CO 3 (1.19 g,8.58 mmol) and tert-butyl bromo-tetrapolyethylene glycol-propionate (2.2 g, 5).71 mmol), stirring at room temperature, reacting for 2h, quenching with water after the reaction is completed, extracting with dichloromethane (30 mL. Times.3), combining organic phases, washing with saturated NaCl solution, and anhydrous Na 2 SO 4 Drying, suction filtration to remove salt, concentration under reduced pressure, and purification by column chromatography gave intermediate 57 (3.07 g, 93%). MS (ESI, M/z): 579 (M) + +1). b) Raw material 57 (576 mg,1 mmol) was dissolved in DCM (5 mL), TFA (2 mmol) was added dropwise at 0deg.C, the reaction was stirred at room temperature for 3h and concentrated under reduced pressure to give crude intermediate 58 (470 mg, 90%). MS (ESI, M/z): 523 (M) + +1)。
Synthesis of intermediate 60: synthesis reference synthesis of intermediate 58 requires only the replacement of the corresponding starting materials. MS (ESI, M/z): 360 (M) + +1)。
Synthesis of intermediate 61: synthesis reference synthesis of intermediate 58 requires only the replacement of the corresponding starting materials. MS (ESI, M/z): 437 (M) + +1)。
Example 14 Synthesis of intermediates 64, 67 and 71
a) Under argon, starting material 62 (323 mg,1 mmol), 9-decyn-1-ol (185 mg,1.2 mmol), pd (PPh) 3 ) 2 Cl 2 (10 mol%), cuI (10 mol%) and DIPEA (1.29 g,10 mmol) were dissolved in THF and stirred at room temperature for 10h, quenched with water after completion of the reaction, extracted with ethyl acetate (10 mL. Times.3), the organic phases combined, washed with saturated NaCl solution, anhydrous Na 2 SO 4 Drying, suction filtration of salt, concentration under reduced pressure gave intermediate 63 (309 mg, 78%) which was purified by column chromatography. MS (ESI, M/z): 397 (M) + +1). Intermediates 66 and 69 were prepared in the same manner, except that the corresponding starting materials were replaced.
b) Raw material 63 (397 mg,1 mmol) was dissolved in acetone, and 2M Jones reagent (1 mL) was added dropwise at 0deg.C. The reaction was stirred at room temperature for 30min, monitored by TLC for completion, extracted with ethyl acetate, anhydrous Na 2 SO 4 Dried and concentrated under reduced pressure to give crude intermediate 64 (229 mg, 56%). MS (ESI, M/z): 411 (M) + +1). The intermediate is prepared by the same method only by replacing corresponding raw materials67 and 70.
c) Intermediate 70 (268 mg,1 mmol) was dissolved in THF (5 mL), 10% Pd/C (37 mg) was added and the mixture was purged three times with hydrogen balloon. The reaction was stirred at room temperature for 12h, filtered through celite, and the filtrate was concentrated under reduced pressure to give crude intermediate 71 (167 mg, 45%). MS (ESI, M/z): 373 (M) + +1)。
Example 15 Synthesis of intermediates 74 and 75
Synthesis of intermediate 74: a) Raw material 72 (444 mg,1 mmol), N, N-bis-Boc-4-aminobutyric acid (803 mg,1.2 mmol), HATU (560 mg,1.5 mmol) and DIPEA (3838 mg,3 mmol) were dissolved in THF and stirred at room temperature for 2h, ethyl acetate extraction after completion of the reaction, anhydrous Na 2 SO 4 Dried, concentrated under reduced pressure and purified by column chromatography to give intermediate 73 (693 mg, 95%). MS (ESI, M/z): 730 (M) + +1). b) Intermediate 73 (730 mg,1 mmol) was dissolved in dichloromethane (1 mmol/5 mL) and methanol (1 mmol/1 mL), a solution of 4 MHz dioxane (0.5 mL) was added at 0deg.C, the reaction was allowed to proceed at room temperature for 4h, after completion of TLC monitoring the reaction, the reaction mixture was spun dry, slurried with isopropyl ether, and suction filtered to give intermediate 74 (330 mg, 92%). MS (ESI, M/z): 530 (M) + +1)。
Synthesis of intermediate 75: the synthesis reference intermediate 74 is synthesized in the synthesis method step a, and only the corresponding raw materials need to be replaced. MS (ESI, M/z): 635 (M) + +1).
EXAMPLE 16 Synthesis of Compound S1
a) Intermediate 18 (798 mg,1 mmol), intermediate 49a (390 mg,1.2 mmol), HATU (642 mg,1.5 mmol) and DIPEA (3838 mg,3 mmol) were dissolved in THF and reacted at room temperature with stirring for 2h, quenched with water after completion of the reaction, extracted with dichloromethane (10 mL. Times.3), the combined organic phases were washed with saturated NaCl solution, anhydrous Na 2 SO 4 Drying, suction filtering to remove salt, concentrating under reduced pressureColumn chromatography purification yielded intermediate 76 (777 mg, 70%). MS (ESI, M/z): 1129 (M) + +1)。
b) Intermediate 75 (1111 mg,1 mmol) was dissolved in dichloromethane (5 mL), TFA (5 mL) was added dropwise, and the mixture was stirred at room temperature for 2h, after completion of the reaction, concentrated under reduced pressure, and HPLC was performed to obtain Compound S1.
S1: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.41(s,1H),8.70(d,J＝1.4Hz,1H),8.20(d,J＝1.4Hz,1H),8.05(s,1H),7.58–7.53(m,2H),7.48(t,J＝7.8Hz,1H),7.41(d,J＝2.3Hz,1H),7.11(ddd,J＝12.1,7.8,1.1Hz,1H),6.00(t,J＝5.1Hz,1H),5.72(t,J＝3.7Hz,1H),5.55(d,J＝7.0Hz,1H),5.45(d,J＝7.0Hz,1H),5.27(t,J＝5.9Hz,1H),4.55(p,J＝5.7Hz,1H),4.32(ddd,J＝5.2,4.5,0.8Hz,2H),4.23(dd,J＝16.8,5.1Hz,1H),4.16(dd,J＝16.9,5.1Hz,1H),3.77(ddd,J＝11.9,7.9,5.6Hz,2H),3.71–3.57(m,2H),3.43(ddd,J＝11.9,7.9,5.6Hz,2H),2.68–2.51(m,2H),2.42(s,3H),2.19–2.03(m,2H),1.98(ddt,J＝11.2,7.9,5.6Hz,2H),1.84(ddt,J＝11.2,7.9,5.6Hz,2H).CMS(ESI,m/z):789(M + +1).
EXAMPLE 17 Synthesis of Compound S2
a) Intermediate 18 (798 mg,1 mmol), intermediate 55a (473 mg,1.2 mmol) and K 2 CO 3 (258 mg,2 mmol) was dissolved in acetonitrile, stirred at 80℃for 6h, quenched with water after completion of the reaction, extracted with ethyl acetate (10 mL. Times.3), the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous Na 2 SO 4 Drying, suction filtration of salt, concentration under reduced pressure, and purification by column chromatography gave intermediate 77a (777 mg, 70%). Intermediate 77b can be obtained by simply replacing the corresponding reaction substrate. MS (ESI, M/z): 1129 (M) + +1)。
b) Compound S2 was prepared according to step b of example 16.
S2: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.42(s,1H),8.71(d,J＝1.4Hz,1H),8.20(d,J＝1.4Hz,1H),8.03–7.95(m,2H),7.69–7.61(m,2H),7.59(d,J＝2.2Hz,1H),7.47(dd,J＝7.9,1.1Hz,1H),7.40(d,J＝2.3Hz,1H),5.72(t,J＝3.7Hz,1H),5.55(d,J＝7.0Hz,1H),5.45(d,J＝7.0Hz,1H),5.12(t,J＝5.9Hz,1H),4.37–4.27(m,3H),3.65(ddd,J＝5.2,4.4,3.7Hz,2H),3.19(d,J＝13.2Hz,1H),3.01–2.90(m,3H),2.64(ddd,J＝15.2,8.6,6.4Hz,1H),2.55–2.45(m,4H),2.38(s,3H),2.24–1.98(m,4H),1.88(ddt,J＝11.2,7.9,5.6Hz,2H).MS(ESI,m/z):789(M + +1).
EXAMPLE 18 Synthesis of Compound S3
Synthetic method referring to example 16, compound S3 can be prepared by simply replacing the corresponding starting materials.
S3: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.06(s,1H),8.71(d,J＝1.4Hz,1H),8.20(d,J＝1.4Hz,1H),8.03(d,J＝2.0Hz,1H),7.58(d,J＝5.1Hz,1H),7.53–7.45(m,2H),7.36(t,J＝4.4Hz,1H),7.08(ddd,J＝12.4,7.9,1.3Hz,2H),6.88(t,J＝3.8Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.13(t,J＝5.9Hz,1H),4.34–4.24(m,3H),3.65(td,J＝4.9,3.7Hz,2H),3.51(d,J＝13.4Hz,1H),3.38(dtd,J＝14.4,6.3,3.7Hz,1H),3.34–3.22(m,2H),3.20–3.10(m,2H),2.82(ddd,J＝12.1,8.0,5.6Hz,2H),2.69(ddd,J＝15.0,8.5,6.5Hz,1H),2.52(ddd,J＝15.3,8.6,6.4Hz,1H),2.48–2.38(m,2H),2.43(s,3H),2.17–2.00(m,4H),1.85(ddt,J＝11.4,7.9,5.7Hz,2H),1.69(dq,J＝13.0,6.5Hz,1H),1.63–1.50(m,1H),1.53–1.45(m,1H),1.48–1.36(m,3H),1.39–1.29(m,1H).MS(ESI,m/z):874(M + +1).
EXAMPLE 19 Synthesis of Compounds S4 and S5
Synthetic method referring to example 17, compounds S4 and S5 can be prepared by simply replacing the corresponding starting materials.
S4: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.06(s,1H),8.70(d,J＝1.6Hz,1H),8.15(d,J＝1.4Hz,1H),7.95(d,J＝2.0Hz,1H),7.59(d,J＝4.9Hz,1H),7.52–7.43(m,2H),7.09(ddd,J＝7.7,3.9,1.1Hz,2H),6.92(t,J＝3.8Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.13(t,J＝5.9Hz,1H),4.34–4.24(m,3H),3.65(td,J＝4.9,3.7Hz,2H),3.51(dtd,J＝14.5,6.3,3.8Hz,1H),3.29(dtd,J＝14.5,6.4,3.8Hz,1H),3.01(ddd,J＝11.9,8.0,5.7Hz,2H),2.69(ddd,J＝15.0,8.5,6.5Hz,1H),2.58–2.46(m,3H),2.44(dt,J＝11.9,6.3Hz,1H),2.40(s,2H),2.32(dt,J＝11.9,6.3Hz,1H),2.17–2.07(m,1H),2.11–1.98(m,3H),1.81–1.68(m,3H),1.66–1.53(m,1H),1.50(dq,J＝13.1,6.6Hz,1H),1.47–1.38(m,1H),1.40–1.33(m,1H),1.36–1.27(m,3H),1.30–1.16(m,1H).MS(ESI,m/z):832(M + +1).
S5: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.49(s,1H),8.70(d,J＝1.6Hz,1H),8.23(s,1H),8.15(d,J＝1.4Hz,1H),7.96(d,J＝2.0Hz,1H),7.58(d,J＝5.1Hz,1H),7.51(t,J＝7.9Hz,1H),7.10(ddd,J＝7.7,5.3,1.1Hz,2H),6.92(t,J＝3.8Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.15(t,J＝5.9Hz,1H),4.58(p,J＝5.7Hz,1H),4.34–4.28(m,2H),3.65(td,J＝4.9,3.7Hz,2H),3.42(dtd,J＝14.5,6.3,3.8Hz,1H),3.31(dtd,J＝14.5,6.3,3.7Hz,1H),3.05(ddd,J＝11.9,8.0,5.6Hz,2H),2.70(ddd,J＝15.2,8.6,6.3Hz,1H),2.58–2.48(m,3H),2.49–2.32(m,2H),2.40(s,3H),2.13–2.03(m,1H),2.02(ddd,J＝11.4,8.0,5.6Hz,2H),1.94(ddt,J＝11.3,8.6,6.2Hz,1H),1.79–1.65(m,3H),1.60(dp,J＝13.3,6.6Hz,1H),1.54–1.40(m,2H),1.43–1.15(m,12H).MS(ESI,m/z):887(M + +1).
EXAMPLE 20 Synthesis of Compound S6
Synthetic method referring to example 16, compound S6 can be prepared by simply replacing the corresponding starting materials.
S6: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.06(s,1H),8.70(d,J＝1.4Hz,1H),8.20(d,J＝1.4Hz,1H),8.04(d,J＝2.0Hz,1H),7.58(d,J＝4.9Hz,1H),7.48(t,J＝7.9Hz,1H),7.23(s,1H),7.10(dd,J＝7.8,1.2Hz,1H),7.04(dd,J＝7.9,1.3Hz,1H),6.92(t,J＝3.8Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.12(t,J＝5.9Hz,1H),4.44(p,J＝5.6Hz,1H),4.34–4.28(m,2H),3.71(ddd,J＝11.9,7.9,5.7Hz,2H),3.65(td,J＝4.9,3.7Hz,2H),3.49–3.38(m,3H),3.27(dtd,J＝14.5,6.3,3.7Hz,1H),2.69(ddd,J＝15.0,8.5,6.5Hz,1H),2.52(ddd,J＝15.1,8.6,6.4Hz,1H),2.43(s,2H),2.28(t,J＝7.0Hz,2H),2.17–2.07(m,1H),2.10–1.99(m,3H),1.92(ddt,J＝11.2,7.9,5.7Hz,2H),1.70(dq,J＝13.0,6.6Hz,1H),1.58(dp,J＝7.8,6.7Hz,3H),1.44(dp,J＝13.7,6.8Hz,1H),1.33(dq,J＝13.3,6.7Hz,1H).MS(ESI,m/z):845(M + +1).
EXAMPLE 21 Synthesis of Compound S7
Synthetic method referring to example 21, compound S7 can be prepared by simply replacing the corresponding starting materials.
S7: 1 H NMR(500MHz,Chloroform-d)δ9.89(s,1H),9.54(s,1H),8.70(d,J＝1.5Hz,1H),8.20(d,J＝1.5Hz,1H),7.93(d,J＝2.0Hz,1H),7.73(dd,J＝7.8,1.3Hz,1H),7.63–7.55(m,2H),7.34–7.25(m,2H),6.74(d,J＝7.8Hz,1H),6.45(d,J＝7.8Hz,1H),5.78(t,J＝3.7Hz,1H),5.17(t,J＝6.0Hz,1H),4.36–4.27(m,4H),4.11(dt,J＝12.4,6.1Hz,1H),3.85–3.75(m,2H),3.79–3.69(m,2H),3.72–3.61(m,14H),3.65–3.60(m,1H),3.64–3.57(m,1H),3.60–3.47(m,1H),3.42(ddd,J＝11.9,8.0,5.7Hz,2H),2.68(ddd,J＝15.0,8.5,6.5Hz,1H),2.58(t,J＝7.1Hz,2H),2.53(ddd,J＝15.2,8.6,6.4Hz,1H),2.41(s,2H),2.19–2.08(m,1H),2.11–2.03(m,1H),2.02(ddd,J＝11.3,8.0,5.6Hz,2H),1.89(ddt,J＝11.3,8.1,5.7Hz,2H).MS(ESI,m/z):981(M + +1).
EXAMPLE 22 Synthesis of Compound S8
Synthetic method referring to example 21, compound S8 can be prepared by simply replacing the corresponding starting materials.
S8: 1 H NMR(500MHz,Chloroform-d)δ9.81(s,1H),9.06(s,1H),8.70(d,J＝1.4Hz,1H),8.20(d,J＝1.4Hz,1H),7.96–7.86(m,2H),7.61–7.50(m,2H),7.37(t,J＝7.9Hz,1H),7.27(s,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),4.38(d,J＝0.7Hz,2H),4.34–4.24(m,4H),3.65(td,J＝4.9,3.7Hz,2H),3.53(ddd,J＝11.9,8.0,5.7Hz,2H),3.44(ddd,J＝11.9,8.0,5.7Hz,2H),2.70(ddd,J＝15.2,8.6,6.3Hz,1H),2.56(ddd,J＝15.2,8.6,6.4Hz,1H),2.41(s,3H),2.40(dt,J＝12.3,6.1Hz,1H),2.35–2.24(m,2H),2.21(dt,J＝14.2,7.1Hz,1H),2.01–1.85(m,3H),1.87–1.74(m,3H),1.60(dp,J＝9.2,6.7Hz,3H),1.51–1.40(m,1H),1.43–1.20(m,6H).MS(ESI,m/z):868(M + +1).
EXAMPLE 23 Synthesis of Compounds S9 and S10
Synthetic method referring to example 16, compounds S9 and S10 can be prepared by simply replacing the corresponding starting materials.
S9: 1 H NMR(500MHz,Chloroform-d)δ9.81(s,1H),9.06(s,1H),8.70(d,J＝1.4Hz,1H),8.20(d,J＝1.4Hz,1H),8.04(d,J＝1.8Hz,1H),7.66–7.55(m,3H),7.49(ddt,J＝7.5,1.8,0.9Hz,1H),7.27(s,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),4.37(dd,J＝13.5,1.1Hz,1H),4.34–4.26(m,3H),4.16–4.04(m,2H),3.65(td,J＝4.8,3.7Hz,2H),3.58(ddd,J＝12.1,8.0,5.7Hz,2H),3.44(ddd,J＝11.9,7.9,5.7Hz,2H),2.67(ddd,J＝15.2,8.7,6.3Hz,1H),2.52(ddd,J＝15.2,8.6,6.3Hz,1H),2.45–2.16(m,8H),2.00(ddt,J＝11.4,8.1,5.7Hz,2H),1.85(dddt,J＝33.0,12.4,8.6,6.0Hz,3H),1.68–1.58(m,1H),1.61–1.51(m,2H),1.52–1.42(m,1H),1.45–1.32(m,1H),1.36–1.24(m,3H),1.28–1.20(m,2H).MS(ESI,m/z):868(M + +1).
S10: 1 H NMR(500MHz,Chloroform-d)δ9.81(s,1H),9.06(s,1H),8.70(d,J＝1.4Hz,1H),8.20(d,J＝1.4Hz,1H),7.93(d,J＝2.0Hz,1H),7.71(s,1H),7.60–7.49(m,3H),7.26(dq,J＝7.7,1.2Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),4.58(p,J＝5.7Hz,1H),4.39–4.28(m,5H),3.83(ddd,J＝11.9,8.1,5.7Hz,2H),3.65(td,J＝4.9,3.7Hz,2H),3.44(ddd,J＝12.1,8.1,5.7Hz,2H),2.66(ddd,J＝15.0,8.5,6.4Hz,1H),2.60–2.43(m,2H),2.40(s,3H),2.44–2.27(m,3H),2.07–1.77(m,8H).MS(ESI,m/z):812(M + +1).
EXAMPLE 24 Synthesis of Compound S11
Synthetic method referring to example 16, compound S11 can be prepared by simply replacing the corresponding starting materials.
S11: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.06(s,1H),8.70(d,J＝1.4Hz,1H),8.20(d,J＝1.4Hz,1H),8.03(d,J＝2.0Hz,1H),7.78(dd,J＝7.9,1.3Hz,1H),7.56(d,J＝4.9Hz,1H),7.51(t,J＝7.6Hz,1H),7.44(dq,J＝7.3,1.1Hz,1H),7.23(s,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.13(t,J＝5.9Hz,1H),4.38–4.28(m,3H),3.68–3.57(m,4H),3.44(ddd,J＝11.9,8.1,5.7Hz,2H),2.84(dtd,J＝14.8,6.4,1.0Hz,1H),2.68(ddd,J＝15.0,8.0,7.0Hz,1H),2.62–2.48(m,2H),2.42(s,2H),2.27(t,J＝7.0Hz,2H),2.16–2.00(m,4H),1.92(ddt,J＝11.4,8.1,5.7Hz,2H),1.74(dt,J＝13.1,6.6Hz,1H),1.73–1.64(m,1H),1.61(p,J＝6.9Hz,2H),1.51–1.34(m,3H).MS(ESI,m/z):830(M + +1).
EXAMPLE 25 Synthesis of Compound S12
Synthetic method referring to example 16, compound S12 can be prepared by simply replacing the corresponding starting materials.
S12: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.06(s,1H),8.80(dd,J＝7.8,1.2Hz,1H),8.70(d,J＝1.6Hz,1H),8.15(d,J＝1.4Hz,1H),8.04(d,J＝2.0Hz,1H),7.68(t,J＝7.8Hz,1H),7.52(d,J＝4.9Hz,1H),7.46(dd,J＝7.9,1.1Hz,1H),7.35(s,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.20(t,J＝5.9Hz,1H),4.35–4.27(m,3H),3.65(td,J＝4.9,3.7Hz,2H),2.99(ddd,J＝12.1,8.1,5.7Hz,2H),2.70(ddd,J＝15.0,8.4,6.2Hz,1H),2.55(ddd,J＝15.2,8.6,6.3Hz,1H),2.49–2.40(m,1H),2.32(s,3H),2.40–2.06(m,7H),1.90(ddt,J＝11.4,8.1,5.7Hz,2H),1.79–1.55(m,6H),1.54–1.44(m,1H),1.48–1.34(m,2H).MS(ESI,m/z):845(M + +1).
EXAMPLE 26 Synthesis of Compound S13
Synthetic method referring to example 17, compound S13 can be prepared by simply replacing the corresponding starting materials.
S13: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.06(s,1H),8.70(d,J＝1.6Hz,1H),8.15(d,J＝1.4Hz,1H),7.92(d,J＝2.0Hz,1H),7.56–7.44(m,4H),7.30(dd,J＝7.9,1.3Hz,1H),7.17(dd,J＝7.9,1.3Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),6.17(t,J＝5.1Hz,1H),5.78(t,J＝3.7Hz,1H),5.30(t,J＝5.9Hz,1H),4.45(p,J＝5.7Hz,1H),4.34–4.28(m,2H),4.20(dd,J＝17.7,5.0Hz,1H),4.02(dd,J＝17.6,5.1Hz,1H),3.65(td,J＝4.9,3.7Hz,2H),3.05(td,J＝6.1,4.4Hz,2H),2.91(ddd,J＝12.1,8.0,5.7Hz,2H),2.69(ddd,J＝14.9,8.4,6.4Hz,1H),2.61(ddd,J＝15.4,8.6,6.6Hz,1H),2.50–2.34(m,7H),2.18–2.08(m,1H),2.11–2.03(m,1H),2.02(ddd,J＝11.2,8.0,5.6Hz,2H),1.79(ddt,J＝11.4,8.1,5.7Hz,2H),1.67(dq,J＝12.8,6.4Hz,1H),1.56–1.28(m,5H).MS(ESI,m/z):874(M + +1).
EXAMPLE 27 Synthesis of Compound S14
Synthetic method referring to example 17, compound S14 can be prepared by simply replacing the corresponding starting materials.
S14: 1 H NMR(500MHz,Chloroform-d)δ9.89(s,1H),9.06(s,1H),8.70(d,J＝1.6Hz,1H),8.15(d,J＝1.4Hz,1H),8.03(d,J＝2.0Hz,1H),7.64(dd,J＝7.7,1.3Hz,1H),7.61–7.53(m,2H),7.46(s,1H),7.24(dd,J＝7.7,1.3Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.14(t,J＝5.9Hz,1H),4.34–4.28(m,2H),4.28–4.14(m,2H),4.08(dt,J＝11.7,6.0Hz,1H),3.65(td,J＝4.9,3.7Hz,2H),2.99(ddd,J＝12.1,8.0,5.7Hz,2H),2.69(ddd,J＝15.2,8.4,6.5Hz,1H),2.53(ddd,J＝15.2,8.6,6.4Hz,1H),2.48–2.38(m,1H),2.42(s,3H),2.37–2.28(m,1H),2.23–2.00(m,6H),1.91–1.74(m,4H),1.57–1.45(m,1H),1.44–1.29(m,6H).MS(ESI,m/z):832(M + +1).
EXAMPLE 28 Synthesis of Compound S15
Synthetic method referring to example 16, compound S15 can be prepared by simply replacing the corresponding starting materials.
S15: 1 H NMR(500MHz,Chloroform-d)δ9.89(s,1H),9.06(s,1H),8.71(d,J＝1.4Hz,1H),8.20(d,J＝1.4Hz,1H),8.03(d,J＝2.0Hz,1H),7.73(dd,J＝7.9,1.3Hz,1H),7.64–7.54(m,2H),7.50(s,1H),7.43(t,J＝4.4Hz,1H),7.33(dd,J＝7.8,1.2Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.26(t,J＝5.9Hz,1H),4.35–4.26(m,3H),4.17(dt,J＝12.1,6.1Hz,1H),4.08(dt,J＝11.9,6.1Hz,1H),3.65(td,J＝4.9,3.7Hz,2H),3.50(d,J＝13.2Hz,1H),3.19–3.01(m,3H),2.83(ddd,J＝12.1,8.0,5.6Hz,2H),2.74–2.63(m,1H),2.57(ddd,J＝15.2,8.0,7.1Hz,1H),2.44(s,3H),2.49–2.39(m,2H),2.16–2.01(m,4H),1.83(ddt,J＝11.4,8.1,5.7Hz,2H),1.79–1.41(m,6H).MS(ESI,m/z):875(M + +1).
EXAMPLE 29 Synthesis of Compound S16
Synthetic method referring to example 17, compound S16 can be prepared by simply replacing the corresponding starting materials.
S16: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.12(s,1H),8.04(d,J＝2.0Hz,1H),7.63–7.57(m,3H),7.48(t,J＝7.9Hz,1H),7.37(s,1H),7.09(ddd,J＝7.8,4.3,1.3Hz,2H),6.92(t,J＝3.8Hz,1H),6.82–6.71(m,3H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.12(t,J＝5.9Hz,1H),4.34–4.28(m,2H),3.65(td,J＝4.8,3.7Hz,2H),3.41(dtd,J＝14.5,6.2,3.7Hz,1H),3.28(dtd,J＝14.5,6.3,3.7Hz,1H),3.24–3.15(m,4H),2.79–2.70(m,4H),2.73–2.64(m,1H),2.52(ddd,J＝15.3,8.6,6.5Hz,1H),2.42(s,3H),2.47–2.38(m,1H),2.32(dt,J＝12.0,6.4Hz,1H),2.17–2.01(m,2H),1.79–1.68(m,1H),1.66–1.52(m,2H),1.52–1.19(m,11H).MS(ESI,m/z):870(M + +1).
EXAMPLE 30 Synthesis of Compound S17
Synthetic method referring to example 16, compound S17 can be prepared by simply replacing the corresponding starting materials.
S17: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.12(s,1H),8.04(d,J＝2.0Hz,1H),7.64–7.57(m,3H),7.48(t,J＝7.9Hz,1H),7.40–7.31(m,2H),7.10(ddd,J＝7.7,4.9,1.2Hz,2H),6.88(t,J＝3.7Hz,1H),6.84–6.77(m,2H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.15(t,J＝5.9Hz,1H),4.34–4.28(m,2H),3.68–3.57(m,3H),3.53–3.42(m,2H),3.37(t,J＝5.3Hz,4H),3.26(dtd,J＝14.3,6.3,4.4Hz,1H),3.22–3.10(m,2H),2.82–2.71(m,4H),2.69(ddd,J＝15.2,8.4,6.5Hz,1H),2.52(ddd,J＝15.2,8.6,6.5Hz,1H),2.42(s,3H),2.16–2.01(m,2H),1.69(dq,J＝13.0,6.5Hz,1H),1.63–1.50(m,1H),1.53–1.41(m,2H),1.45–1.34(m,1H),1.38–1.28(m,1H).MS(ESI,m/z):885(M + +1).
EXAMPLE 31 Synthesis of Compound S18
Synthetic method referring to example 17, compound S18 can be prepared by simply replacing the corresponding starting materials.
S18: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.04(s,1H),7.94(d,J＝2.0Hz,1H),7.54–7.45(m,2H),7.13–7.02(m,3H),6.95–6.88(m,2H),6.86(dt,J＝8.4,1.0Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.09(t,J＝5.9Hz,1H),4.35–4.28(m,3H),3.95(dd,J＝13.6,1.1Hz,1H),3.83(s,3H),3.65(td,J＝4.9,3.8Hz,2H),3.51(dtd,J＝14.5,6.3,3.7Hz,1H),3.29(dtd,J＝14.5,6.3,3.7Hz,1H),2.95–2.80(m,3H),2.74–2.63(m,2H),2.59(dt,J＝12.0,6.4Hz,1H),2.57–2.46(m,2H),2.39(s,3H),2.17–2.01(m,2H),1.77–1.65(m,1H),1.64(dt,J＝13.1,6.5Hz,1H),1.56(tt,J＝13.0,6.5Hz,2H),1.45–1.28(m,2H).MS(ESI,m/z):829(M + +1).
EXAMPLE 32 Synthesis of Compound S19
Synthetic method referring to example 16, compound S19 can be prepared by simply replacing the corresponding starting materials.
S19: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.04(s,1H),8.03(d,J＝2.0Hz,1H),7.59(d,J＝4.9Hz,1H),7.50(t,J＝7.8Hz,1H),7.32(t,J＝4.3Hz,1H),7.23–7.12(m,3H),7.05(d,J＝8.4Hz,1H),6.91–6.84(m,2H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.15(t,J＝6.0Hz,1H),4.34–4.28(m,2H),3.89–3.82(m,1H),3.84(s,3H),3.65(td,J＝4.9,3.8Hz,2H),3.60(dd,J＝13.5,1.0Hz,1H),3.48–3.38(m,2H),3.32(dtd,J＝14.5,6.4,3.8Hz,1H),3.29–3.19(m,2H),3.07(dtd,J＝14.3,6.3,4.4Hz,1H),2.99(dd,J＝5.6,4.9Hz,2H),2.85(ddd,J＝15.4,5.7,4.8Hz,1H),2.76(dt,J＝15.4,5.2Hz,1H),2.69(ddd,J＝15.2,8.4,6.5Hz,1H),2.53(ddd,J＝15.2,8.5,6.4Hz,1H),2.43(s,3H),2.16–2.02(m,2H),1.76–1.64(m,1H),1.63–1.28(m,5H).MS(ESI,m/z):886(M + +1).
EXAMPLE 33 Synthesis of Compound S20
Synthetic method referring to example 17, compound S20 can be prepared by replacing the corresponding starting materials.
S20: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.49(s,1H),7.62–7.55(m,2H),7.48(t,J＝7.8Hz,1H),7.46–7.35(m,2H),7.33(t,J＝2.2Hz,1H),7.32–7.24(m,1H),7.11(dd,J＝7.8,1.1Hz,1H),7.10–7.01(m,2H),6.92(t,J＝3.7Hz,1H),6.85(dt,J＝8.4,1.0Hz,1H),5.65(d,J＝7.0Hz,1H),5.52(d,J＝7.0Hz,1H),5.13(t,J＝5.9Hz,1H),4.22–4.15(m,1H),3.83(s,3H),3.77(dd,J＝13.6,1.1Hz,1H),3.48(dtd,J＝14.5,6.3,3.7Hz,1H),3.32(dtd,J＝14.5,6.3,3.7Hz,1H),2.91–2.81(m,3H),2.77–2.59(m,3H),2.57–2.47(m,1H),2.51–2.42(m,1H),2.17–2.02(m,2H),1.76–1.49(m,4H),1.48–1.30(m,2H).MS(ESI,m/z):775(M + +1).
EXAMPLE 34 Synthesis of Compound S21
Synthetic method referring to example 16, compound S21 can be prepared by simply replacing the corresponding starting materials.
S21: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.62(s,1H),7.62–7.55(m,2H),7.46(t,J＝7.9Hz,1H),7.46–7.35(m,2H),7.36–7.25(m,3H),7.13–7.02(m,3H),6.88(t,J＝3.7Hz,1H),6.81(dt,J＝8.4,1.0Hz,1H),5.65(d,J＝7.1Hz,1H),5.53(d,J＝7.0Hz,1H),5.04(t,J＝5.9Hz,1H),3.88(dd,J＝13.5,0.9Hz,1H),3.82(s,3H),3.72(dd,J＝13.6,0.9Hz,1H),3.60(dtd,J＝14.5,6.3,3.8Hz,1H),3.41(d,J＝13.2Hz,1H),3.34–3.21(m,2H),3.24–3.14(m,1H),3.10(dtd,J＝14.3,6.3,4.4Hz,1H),2.99(t,J＝5.3Hz,2H),2.90–2.81(m,1H),2.73–2.60(m,2H),2.48(ddd,J＝15.1,8.7,6.3Hz,1H),2.21(ddt,J＝11.2,8.6,6.1Hz,1H),2.05(ddt,J＝11.2,8.6,6.0Hz,1H),1.69(dq,J＝13.0,6.5Hz,1H),1.63–1.51(m,1H),1.53–1.45(m,1H),1.49–1.42(m,1H),1.46–1.28(m,2H).MS(ESI,m/z):832(M + +1).
EXAMPLE 35 Synthesis of Compound S22
Synthetic method referring to example 16, compound S22 can be prepared by simply replacing the corresponding starting materials.
S22: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.75(d,J＝2.2Hz,1H),8.70(d,J＝1.6Hz,1H),8.24–8.14(m,3H),8.06(s,1H),7.99(dd,J＝8.6,2.0Hz,1H),7.53(t,J＝7.8Hz,1H),7.36(t,J＝4.4Hz,1H),7.30–7.23(m,2H),7.10(dd,J＝7.8,1.2Hz,1H),6.88(t,J＝3.7Hz,1H),5.60(t,J＝3.7Hz,1H),5.13(t,J＝5.9Hz,1H),4.37–4.27(m,3H),3.65(ddd,J＝5.2,4.6,3.8Hz,2H),3.60–3.47(m,2H),3.25(dtd,J＝14.5,6.3,3.8Hz,1H),3.16(d,J＝13.2Hz,1H),3.08(td,J＝6.3,4.4Hz,2H),2.83(ddd,J＝12.1,8.0,5.7Hz,2H),2.69(ddd,J＝15.2,8.5,6.5Hz,1H),2.57–2.47(m,1H),2.44(ddd,J＝12.1,8.1,5.7Hz,2H),2.39(s,3H),2.17–2.01(m,4H),1.84(ddt,J＝11.4,8.1,5.7Hz,2H),1.76–1.64(m,1H),1.63–1.42(m,3H),1.42–1.28(m,2H).MS(ESI,m/z):841(M + +1).
EXAMPLE 36 Synthesis of Compound S23
Synthetic method referring to example 17, compound S23 can be prepared by simply replacing the corresponding starting materials.
S23: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),9.74(d,J＝1.8Hz,1H),8.70(d,J＝1.6Hz,1H),8.24–8.17(m,3H),8.08–8.01(m,2H),7.50(t,J＝7.9Hz,1H),7.26(s,1H),7.20(ddd,J＝16.1,7.9,1.3Hz,2H),6.92(t,J＝3.8Hz,1H),5.60(t,J＝3.7Hz,1H),5.16(t,J＝5.9Hz,1H),4.32(td,J＝4.9,0.8Hz,2H),4.27(p,J＝5.7Hz,1H),3.65(ddd,J＝5.2,4.6,3.8Hz,2H),3.46(dtd,J＝14.5,6.3,3.8Hz,1H),3.23(dtd,J＝14.5,6.3,3.8Hz,1H),3.01(ddd,J＝11.9,8.0,5.6Hz,2H),2.68(ddd,J＝15.0,8.6,6.3Hz,1H),2.52(ddd,J＝15.2,8.8,6.4Hz,1H),2.43(dt,J＝11.9,6.3Hz,1H),2.39(s,3H),2.32(dt,J＝11.9,6.3Hz,1H),2.27–2.17(m,3H),2.12–2.01(m,3H),1.85–1.78(m,1H),1.81–1.75(m,1H),1.78–1.68(m,1H),1.67–1.55(m,1H),1.58–1.27(m,5H),1.30–1.16(m,1H).MS(ESI,m/z):798(M + +1).
EXAMPLE 37 Synthesis of Compound S24
Synthetic method referring to example 17, compound S24 can be prepared by simply replacing the corresponding starting materials.
S24: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),7.69(t,J＝2.1Hz,1H),7.62(ddd,J＝8.1,5.0,1.1Hz,1H),7.54–7.45(m,2H),7.48–7.37(m,2H),7.27(ddd,J＝8.3,6.4,2.4Hz,1H),7.10(ddd,J＝7.7,4.5,1.2Hz,2H),7.04(d,J＝8.4Hz,1H),6.98–6.89(m,2H),6.86(dt,J＝8.6,1.0Hz,1H),6.08(d,J＝7.0Hz,1H),5.63(d,J＝7.1Hz,1H),5.17(t,J＝5.9Hz,1H),4.19(dd,J＝13.6,0.9Hz,1H),3.86(s,3H),3.77(dd,J＝13.6,1.1Hz,1H),3.52(dtd,J＝14.5,6.3,3.7Hz,1H),3.33(dtd,J＝14.5,6.3,3.7Hz,1H),2.91–2.82(m,2H),2.73–2.43(m,6H),2.22(ddt,J＝11.3,8.6,6.1Hz,1H),2.08(ddt,J＝11.3,8.6,6.2Hz,1H),1.71(ddq,J＝25.9,13.0,6.6Hz,2H),1.66–1.54(m,1H),1.52–1.35(m,2H),1.38–1.31(m,1H),1.34–1.20(m,4H).MS(ESI,m/z):786(M + +1).
EXAMPLE 38 Synthesis of Compound S25
Synthetic method referring to example 17, compound S25 can be prepared by simply replacing the corresponding starting materials.
S25: 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),7.79–7.70(m,2H),7.62(ddd,J＝8.3,5.1,1.3Hz,1H),7.54–7.43(m,2H),7.46–7.35(m,3H),7.28(ddd,J＝8.3,6.9,2.2Hz,1H),7.21(dd,J＝7.9,1.3Hz,1H),7.09–7.00(m,2H),6.99(t,J＝3.7Hz,1H),6.81(dt,J＝8.4,1.0Hz,1H),6.08(d,J＝7.0Hz,1H),5.63(d,J＝7.1Hz,1H),5.14(t,J＝5.9Hz,1H),3.81(dd,J＝13.6,1.1Hz,1H),3.80(s,3H),3.52(dd,J＝13.6,0.9Hz,1H),3.38(dtd,J＝14.3,6.1,3.7Hz,1H),3.33–3.19(m,3H),3.09(dtd,J＝14.4,6.2,4.3Hz,1H),3.03–2.94(m,3H),2.82–2.68(m,2H),2.63(dt,J＝15.4,5.3Hz,1H),2.53(ddd,J＝15.2,8.6,6.3Hz,1H),2.10(ddt,J＝11.3,8.8,6.1Hz,1H),1.94(ddt,J＝11.3,8.8,6.1Hz,1H),1.72–1.41(m,4H).MS(ESI,m/z):801(M + +1).
EXAMPLE 39 Synthesis of Compound S26
Synthetic method referring to example 16, compound S26 can be prepared by simply replacing the corresponding starting materials.
S26: 1 H NMR(500MHz,Chloroform-d)δ9.32(s,1H),8.74(s,1H),7.76(dd,J＝5.0,2.1Hz,1H),7.66(d,J＝9.0Hz,1H),7.52(dddd,J＝8.0,5.0,2.1,1.2Hz,1H),7.51–7.44(m,2H),7.46–7.34(m,3H),7.35–7.28(m,2H),7.29–7.22(m,1H),7.18(d,J＝8.6Hz,1H),7.13(s,1H),7.05(d,J＝8.4Hz,1H),6.84(dt,J＝8.4,1.0Hz,1H),6.58(d,J＝7.9Hz,1H),6.39(d,J＝7.9Hz,1H),5.11(dqt,J＝8.1,6.0,1.0Hz,1H),4.66(d,J＝8.8Hz,1H),4.51(dd,J＝5.9,4.9Hz,1H),4.36(ttd,J＝5.8,4.7,3.3Hz,1H),3.86(dd,J＝13.6,1.1Hz,1H),3.84(s,3H),3.68–3.58(m,2H),3.56(d,J＝5.7Hz,1H),3.46–3.38(m,2H),3.31(dtd,J＝14.3,6.3,4.4Hz,1H),3.19(d,J＝13.2Hz,1H),3.09(dtd,J＝14.3,6.3,4.4Hz,1H),3.03–2.94(m,2H),2.85–2.76(m,1H),2.69(dt,J＝15.6,5.3Hz,1H),2.52(s,3H),2.39–2.24(m,2H),2.18(ddd,J＝13.2,5.9,4.8Hz,1H),2.06(ddd,J＝13.2,5.9,4.9Hz,1H),1.87(tdd,J＝13.3,7.1,6.2Hz,1H),1.72(tdd,J＝13.4,7.0,6.3Hz,1H),1.53(d,J＝6.2Hz,3H),0.96(s,9H).MS(ESI,m/z):1003(M + +1).
EXAMPLE 40 Synthesis of Compound S27
Synthetic method referring to example 17, compound S27 can be prepared by simply replacing the corresponding starting materials.
S27: 1 H NMR(500MHz,Chloroform-d)δ9.31(s,1H),8.74(s,1H),7.76(dd,J＝5.0,2.1Hz,1H),7.58(d,J＝9.0Hz,1H),7.56–7.46(m,3H),7.46–7.34(m,2H),7.38–7.31(m,2H),7.25(ddd,J＝7.9,7.0,1.8Hz,1H),7.18(t,J＝4.3Hz,2H),7.04(d,J＝8.4Hz,1H),6.82(dt,J＝8.4,1.0Hz,1H),6.58(d,J＝7.9Hz,1H),6.39(d,J＝7.9Hz,1H),5.11(dqt,J＝8.1,6.1,1.0Hz,1H),4.58(d,J＝9.0Hz,1H),4.52(dd,J＝5.9,4.9Hz,1H),4.41–4.32(m,1H),4.21(dd,J＝13.5,0.9Hz,1H),3.85–3.76(m,5H),3.64(dd,J＝10.8,4.4Hz,1H),3.59(d,J＝5.7Hz,1H),3.43(dd,J＝10.9,3.4Hz,1H),2.91–2.82(m,2H),2.80(ddd,J＝15.4,5.9,4.8Hz,1H),2.71–2.59(m,2H),2.49(s,3H),2.49(dt,J＝11.9,6.4Hz,2H),2.33–2.19(m,2H),2.07(ddd,J＝13.2,5.9,4.9Hz,1H),1.87(ddd,J＝13.4,6.0,4.9Hz,1H),1.69–1.46(m,7H),1.39–1.29(m,1H),1.32–1.20(m,3H),0.97(s,9H).MS(ESI,m/z):988(M + +1).
EXAMPLE 41 Synthesis of Compound S28
Synthetic method referring to example 17, compound S28 can be prepared by simply replacing the corresponding starting materials.
S28: 1 H NMR(500MHz,Chloroform-d)δ9.51(s,1H),8.73(s,1H),8.11(d,J＝2.0Hz,1H),7.87(d,J＝5.1Hz,1H),7.58(d,J＝9.0Hz,1H),7.44–7.37(m,2H),7.36–7.29(m,2H),7.22–7.15(m,2H),7.04(d,J＝8.4Hz,1H),6.88(dt,J＝8.4,1.0Hz,1H),6.74(d,J＝7.9Hz,1H),6.45(d,J＝7.9Hz,1H),5.78(t,J＝3.7Hz,1H),5.11(dqt,J＝8.1,6.1,1.0Hz,1H),4.66(d,J＝9.0Hz,1H),4.46–4.37(m,1H),4.34–4.27(m,3H),4.21(dd,J＝5.8,5.0Hz,1H),3.84(s,2H),3.72(dd,J＝13.6,1.1Hz,1H),3.68–3.61(m,3H),3.43(dd,J＝10.9,3.4Hz,1H),2.96–2.82(m,2H),2.81–2.72(m,2H),2.74–2.60(m,2H),2.58–2.49(m,1H),2.54(s,3H),2.39(s,2H),2.22(td,J＝7.1,1.8Hz,2H),2.05–1.91(m,2H),1.74(dh,J＝13.0,6.5Hz,1H),1.64(dq,J＝12.9,6.9Hz,1H),1.61–1.48(m,5H),1.43–1.18(m,6H),0.97(s,9H).MS(ESI,m/z):1042(M + +1).
EXAMPLE 42 test of the Compounds of the invention for HPK1 kinase Activity
HPK1 kinase domain (1-307 aa) was solubilized in the kinase buffer (20mM HEPES pH7.5,10mM MgCl) 2 1mM EGTA,0.5mM TCEP,0.01%Brij-35,0.05% BSA) was added to a 96-well plate at a final concentration of 20nM, then a gradient of a test compound of the present invention (10 concentration points were set for each compound), HPK1 kinase was pre-incubated with the compound of the present invention for 30min, then 20. Mu.L of a substrate solution (SLP 76-SH at a final concentration of 0.5 mg/mL) was added to each well 2 Domain and 10 μm ATP) initiates the enzymatic reaction. Incubation was performed for 1h at room temperature, and the inhibition of HPK1 kinase activity by the compounds of the invention was then determined at each concentration according to the instructions of Luminescent Kinase Assay Kit (Beyotime Biotechnology Company). Calculation of the inhibitory Activity of the Compounds of the invention against HPK1 kinase IC using GraphPad fitting 50 The values were determined by merck, compound A, B and C as positive controls. The experimental results are shown in table 1.
TABLE 1 kinase inhibitory Activity of the inventive Compounds against HPK1 protein (IC 50 nM)
Numbering of compounds Enzyme inhibition Activity Numbering of compounds Enzyme inhibition Activity
S1 20.5 S17 32.5
S2 22.3 S18 25.3
S3 9.9 S19 37.3
S4 9.5 S20 7.3
S5 9.6 S21 8.9
S6 30.5 S22 32.6
S7 40.8 S23 37.5
S8 28.3 S24 8.9
S9 29.6 S25 7.6
S10 28.7 S26 22.3
S11 28.3 S27 26.2
S12 10.2 S28 19.6
S13 9.9 A 7.7
S14 10.5 B 21.8
S15 12.3 C 36.8
S16 33.2
Compared with the positive control compounds A and B, the compound provided by the invention has basically equivalent positive control activity, which shows that the compound provided by the invention basically maintains the HPK1 inhibitory activity and has better combination with HPK1 protein.
EXAMPLE 43 assay of the degradation Activity of some of the compounds of the invention on HPK1 protein
(1) Collecting cells: jurkat cells were cultured for 12h with adherence, and a portion of the compounds of the invention (0.1. Mu.M, 1. Mu.M, 10. Mu.M) was incubated with the cells for 12h.
(2) Extracting protein: the original culture medium is discarded, the cells are washed three times by precooled PBS, a proper amount of lysate is added, the mixture is evenly mixed and placed on ice for being cracked for 30min, the mixture is centrifuged for 5min at 12000rpm at 4 ℃, and the supernatant after centrifugation is transferred into a new centrifuge tube for preparation for quantification.
(3) Protein quantification: protein is quantified by adopting BCA, and BCA working solution is obtained after the solution A and the solution B are evenly mixed according to the proportion of 50:1. 100. Mu.L of BCA working solution, 9. Mu.L of triple distilled water and 1. Mu.L of protein sample were added to a 96-well plate, and incubated at 37℃for 30min after mixing, absorbance was measured with a full-wavelength microplate reader, and protein concentration was calculated from a standard curve. The quantified protein was mixed with a 5×loading buffer at a ratio of 4:1, denatured in boiling water for 8min, placed on ice, cooled and stored at-20℃until use.
(4) Preparing SDS-PAGE gel, concentrating gel at 90V, separating gel at 120V and at 4deg.C, transferring membrane at 100V for 1 hr, immediately placing protein membrane into 5% BSA solution, slowly shaking on shaking table, and sealing at room temperature for 1 hr.
(5) Referring to the instructions of HPK1 anti-body (4472 SCST), GLK (D1L 4G) Rabbit mAb (92427 SCST), rabbit mAb (92711 TCST), β -actin Mouse Monoclonal Antibody (BE 0021 easybio), the primary Antibody was diluted in the appropriate proportions, incubated overnight with slow shaking at 4℃and washed 3 more times with PBST for 10 min/time.
(6) According to the primary antibody selection of the Goat Anti-Rabbit IgG (H & L) -HRP Conj ugated (BE 0101 easy) or Goat Anti-Mouse IgG (H & L) -HRP Conj ugated (BE 0102 easy), reference to the second antibody instructions, the second antibody was diluted in the appropriate proportion, slowly shaken at room temperature for incubation for 1H, washed 3 times with PBST, 10 min/time.
(7) ECL luminescence was added, the results were observed by a developing instrument, western results were quantified using Image J software, and degradation rates (HPK 1 and GLK) were calculated by comparison with a control group without compound. The experimental results are shown in table 2.
TABLE 2 kinase inhibitory Activity of the inventive Compounds against HPK1 protein (IC 50 nM)
Experimental results show that the compound has good degradation activity on HPK1 protein and is dose-dependent, and the degradation rate of the compound on GLK protein is low at 1.0 mu M, which indicates that the compound can efficiently and selectively reduce HPK1 protein.
Example 44T cytokine Release assay
HPK1 kinase inhibits T cell effector cytokine release, and thus it was examined whether the compounds of the present invention could enhance T cell cytokine release capacity by ELISA method. Human PBMC cells were thawed and suspended in RPMI 1640 (Gibco) medium containing 10% bovine placental serum and cells were seeded in 96-well plates (1X 10) 5 /hole). The compound of the invention was added in a gradient dilution to a 96-well plate at 37℃with 5% CO 2 Incubate in incubator for 1h. PBMCs cells were activated by addition of 100. Mu.Lof anti-CD3/CD28 antibody (final concentration 1. Mu.g/mL) and then incubated at 37℃with 5% CO 2 The supernatant was diluted with 8-fold PBS and 100. Mu.L of the diluted supernatant was used to measure the release of cytokine IL-2 at each concentration of the compounds of the invention using a human IL-2 ELISA kit (4 abio). Calculation of EC of the compounds of the invention using GraphPad fitting 50 Values. The experimental results are shown in table 3.
TABLE 3 results of assays for the IL-2 cytokine Activity of the inventive compounds to stimulate PBMC release
Numbering of compounds EC 50 (nM)
S13 32nM
S20 25nM
A 140nM
The compounds S13 and S20 of the invention are capable of significantly enhancing the release of IL-2 by PBMC cells and are superior to the positive control compound A. This demonstrates that the compounds of the invention are capable of enhancing T cell effector cytokine release by degrading the HPK1 protein, thereby restoring or enhancing an anti-tumor immune response.

Claims (6)

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof,
in formula I:
R 1 hydrogen, halogen or cyano;
R 2 is that
R 3 Is hydrogen or amino;
q is N or C;
a is Terminal represents an association with NH, terminal represents an association with L;
b isY is hydrogen, deuterium, fluorine or chlorine; w is CH 2 Or carbonyl; g is hydrogen or methyl;
z is hydrogen or methyl;
l is Terminal represents a bond to A, "- -" terminal represents a bond to B;
m is an integer between 0 and 5, n is an integer between 0 and 10, and o is an integer between 1 and 5;
x is C, O, NH, The terminal is attached to L and the "- -" terminal is attached to B.
2. A compound or pharmaceutically acceptable salt thereof according to claim 1,
b is
3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula I has the formula:
4. the compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula I is selected from the group consisting of compounds of any one of the following structures:
5. use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of diseases which are associated with HPK1 kinase.
6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or adjuvant.
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