CN117777102A - BTK inhibitor and preparation method and application thereof - Google Patents
BTK inhibitor and preparation method and application thereof Download PDFInfo
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- CN117777102A CN117777102A CN202311656196.5A CN202311656196A CN117777102A CN 117777102 A CN117777102 A CN 117777102A CN 202311656196 A CN202311656196 A CN 202311656196A CN 117777102 A CN117777102 A CN 117777102A
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a BTK inhibitor and a preparation method and application thereof. The BTK inhibitor is a compound shown in the following formula or stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or eutectic crystal thereof;
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a BTK inhibitor and a preparation method and application thereof.
Background
Bruton's Tyrosine Kinase (BTK) is a member of the Tec family of tyrosine kinases and plays an important regulatory role in multiple signal pathways. In B cells, BTK plays a key role in the B cell receptor pathway (BCR), and plays an important role in regulating other pathways of B cells, such as fcγr, chemokine receptor (chemokine receptor), toll-like receptor (TLR) pathways, together with survival, activation, proliferation, differentiation and maturation of B cells. In addition to B cells, BTK is also expressed in multiple cells of the hematopoietic lineage, involved in the regulation of immune responses. Because of the broad expression and key regulatory role of BTK in B cells and other hematopoietic lineage cells, BTK is considered an effective target for the treatment of B cell and other hematological malignancies, autoimmune diseases.
Currently, 6 BTK inhibitors are marketed, but the scope of application of BTK inhibitors is limited by the generation of acquired drug resistance and safety issues. In recent years, targeted protein chimera (PROTAC) technology has made great progress in the field of drug discovery, and PROTACs utilize the ubiquitin-proteasome pathway to degrade pathogenic proteins by "hijacking" the E3 ubiquitin ligase. Because of its unique mechanism of action, "event driven", a new approach is provided to overcome the limitations of BTK inhibitors. To date, a number of BTK PROTAC have been reported, including non-covalent BTK protas and reversible covalent BTK protas, with the potential to overcome resistance and reduce adverse side effects of BTK inhibitors (such as ibutinib) and to exhibit high efficacy in the treatment of B cell malignancies. However, existing BTK protas have the disadvantages of insufficient degradation efficiency, poor oral bioavailability and the like, and in addition, the potential roles of BTK protas in autoimmune and inflammatory diseases have not been explored yet. Therefore, there is a need to further develop highly active, highly potent BTK protas and further expand their range of application in non-tumor indications such as autoimmune and inflammatory diseases.
Disclosure of Invention
In order to overcome the above problems of the prior art, it is an object of the present invention to provide a compound or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof; it is a second object of the present invention to provide a process for the preparation of such compounds; the third object of the present invention is to provide a pharmaceutical composition; it is a fourth object of the present invention to provide the use of such compounds or stereoisomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or co-crystals thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the present invention provides a compound of formula (i) or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof;
in the formula (I), L 1 Selected from the group consisting of
L 2 Selected from:
y is selected fromPreferably, Y is selected from->
R 1 Selected from the group consisting of
R 2 Selected from the group consisting ofPreferably, R 2 Selected from->
A is selected fromPreferably, A is selected from->
X is selected from halogen atoms, i.e. fluorine, chlorine, bromine or iodine, preferably X is fluorine.
Preferably, the compound comprises the structure shown below:
compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
In a second aspect the present invention provides a process for the preparation of a compound according to the first aspect of the invention.
Synthesis method 1 intermediates required for the synthesis of the compounds of the first aspect of the present invention, comprising the steps of:
the compound shown in the formula (1-1) and the compound shown in the formula (1-2) are subjected to substitution reaction or coupling reaction to obtain the compound shown in the formula (1-3), and further subjected to coupling or condensation reaction with the compound shown in the formula (1-4) to obtain the compound shown in the formula (1-5), and finally the Boc protection is removed to obtain the compound shown in the formula (1-6).
Synthetic method 2 is used to synthesize the compounds 1 to 9 according to the first aspect of the present invention, comprising the steps of:
the compounds shown in the first aspect of the invention are prepared by carrying out substitution reaction on the compounds shown in the formula 2-1 and the compounds shown in the formula 2-2 to obtain the compounds shown in the formula 2-3, then carrying out oxidation reaction to prepare the compounds shown in the formula 2-4, and finally carrying out reductive amination reaction on the compounds with the intermediates shown in the formula 1-6.
In a third aspect the present invention provides a pharmaceutical composition comprising a compound according to the first aspect of the present invention or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof.
In a fourth aspect, the present invention provides the use of a compound according to the first aspect of the present invention or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, in the manufacture of a medicament for the treatment and/or prophylaxis and/or delay and/or co-treatment of a disease associated with BTK activity or expression level.
Preferably, the disease is a tumor, an inflammatory or an autoimmune disease.
Preferably, the tumor comprises burkitt's lymphoma, mantle cell lymphoma, multiple myeloma.
Preferably, the inflammation is peritonitis, periodontitis.
Preferably, the autoimmune disease is rheumatoid arthritis.
The beneficial effects of the invention are as follows: the compound provided by the invention has novel structure, and test results show that the compound has excellent BTK protein degradation efficiency, and the effects of inhibiting the expression of the related gene of the broken bone and inhibiting periodontitis; the preparation method of the compound is simple, convenient, quick, green and safe, and the process route is mature; the compound or stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or eutectic thereof can be widely applied to preparing medicaments for treating diseases related to BTK activity or expression quantity.
In particular, the invention has the following advantages: 1. the compound provided by the invention can degrade BTK protein in Mino cells, and has the advantages of novel structure, high activity and short acting time. 2. The preparation method of the compound provided by the invention is simple, convenient, quick, green and safe, has mature process route and has the advantage of industrial mass production. 3. The compound provided by the invention has excellent BTK protein degradation efficiency and lymphoma cell proliferation inhibition effect, and the compound or stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or eutectic thereof can be widely applied to preparation of medicines for treating diseases related to BTK activity or expression quantity.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of compound 3;
FIG. 2 is a nuclear magnetic resonance spectrum of compound 3;
FIG. 3 is a graph showing that Compound 3 inhibits the activity of an osteoclast-associated gene;
FIG. 4 is the effect of compound 3 on treatment of a mouse periodontitis model; wherein, (A) Micro-CT three-dimensional scan image (left) and two-dimensional scan image (right); (B) CEJ-ABC measurement (. Mu.m); (C) BV/TV calculation results.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but are not intended to limit the practice and protection of the invention. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or instruments used did not identify the manufacturer and were considered conventional products available commercially.
The known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from the companies of taitan technology, an Naiji chemistry, shanghai de mer, chengdu Kelong chemical, shaoshan chemical technology, carbofuran technology, etc.; the thin layer chromatography silica gel plate uses a smoke table yellow sea HSGF254 or Qingdao GF254 silica gel plate, the specification of the silica gel plate used by the Thin Layer Chromatography (TLC) is 0.15mm-0.20mm, and the specification of the thin layer chromatography separation and purification product is 0.4mm-0.5mm; column chromatography uses yellow sea silica gel of 200-300 meshes as carrier; the structure of the compound was determined by Nuclear Magnetic Resonance (NMR). NMR shifts are given in units of (ppm). NMR was performed using a (Bruker Avance III) nuclear magnetic resonance apparatus in which the solvent was deuterated dimethyl sulfoxide (DMSO-de), deuterated chloroform (CDC 1) 3 ) Deuterated methanol (CD) 3 OD), internal standard is Tetramethylsilane (TMS).
Example 11 Synthesis of- (2, 6-Dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidine-4-carbaldehyde
The first step: 2- (2, 6-Dioxypiperidin-3-yl) -5- (4- (hydroxymethyl) piperidin-1-yl) isoindoline-1, 3-dione
2- (2, 6-Dioxypiperidin-3-yl) -5-fluoroisoindoline-1, 3-dione (1.81 mmol,1.0 equiv) and piperidin-4-ylmethanol (1.81 mmol,1.0 equiv) were dissolved in 5mL DMF, DIPEA (3.62 mmol,2.0 equiv) was added and the temperature was raised to 80℃for 3h. After the reaction is finished, 20mL of ethyl acetate is added into the reaction system, the mixture is washed with water and saturated saline water for 3 times in sequence, dried with anhydrous sodium sulfate, the solvent is distilled off under reduced pressure to obtain a crude product, and the crude product is purified by column chromatography (0% -5% methanol/dichloromethane, V/V) to obtain 2- (2, 6-dioxopiperidin-3-yl) -5- (4- (hydroxymethyl) piperidin-1-yl) isoindoline-1, 3-dione. The yield thereof was found to be 77.46%. MS (ESI) m/z 372.15[ M+H ]] + 。
And a second step of: 1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidine-4-carbaldehyde
2- (2, 6-Dioxypiperidin-3-yl) -5- (4- (hydroxymethyl) piperidin-1-yl) isoindoline-1, 3-dione (1.08 mmol,2.0 equiv.) was dissolved in 20mL of dichloromethane, and dessert-martin-oxidant (456.81 mg,1.08 mmol) was added under ice bath and stirred at room temperature until reaction was complete. After the reaction was completed, 2mL of a mixed solution of sodium thiosulfate and sodium bicarbonate (1:1, V/V) was added, stirred for 3 to 5 minutes, the organic phase was separated, the aqueous phase was washed with methylene chloride (10 mL. Times.3), the organic phases were combined, the organic phase was washed 3 times with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to give 1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidine-4-carbaldehyde. The yield thereof was found to be 64.9%. MS (ESI) m/z 370.14[ M+H ]] + 。
Example 21 Synthesis of- (2, 6-Dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) azetidine-3-carbaldehyde
The first step: 2- (2, 6-Dioxypiperidin-3-yl) -5- (3- (hydroxymethyl) azetidin-1-yl) isoindoline-1, 3-dione
The synthesis procedure is the same as in the first step of example 1. The yield thereof was found to be 85%. MS (ESI) m/z 344.12[ M+H ]] + 。
And a second step of: 1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) azetidine-3-carbaldehyde
The synthesis procedure is the same as in example 1. The yield thereof was found to be 69%. MS (ESI) m/z 342.120[ M+H ]] + 。
Example 34 Synthesis of tert-butyl- (4-amino-1H-pyrazol-1-yl) piperidine-1-carboxylate
The first step: 4- (4-Nitro-1H-pyrazol-1-yl) piperidine-1-carboxylic acid tert-butyl ester
3-nitropyrazole (8.84 mmol,1.0 equiv.) was reacted with 1-Boc-4-piperidinemethanol (17.69 mmol,2.0 equiv.) PPh 3 (17.69 mmol,2.0 equiv.) is dissolved in 30mL dry THF and the resulting reaction mixture is stirred at room temperature for 30min. After the reaction system was cooled to 0 ℃, DIAD (17.69 mmol,2.0 equiv) was slowly added, and after the completion of the dropwise addition, the reaction solution was slowly warmed to room temperature and stirred for 3 hours. TLC monitoringAfter completion of the reaction, the residue was purified by flash chromatography (EA: pe=1:5) after removal of the solvent by evaporation under reduced pressure to give tert-butyl 4- (4-nitro-1H-pyrazol-1-yl) piperidine-1-carboxylate. The yield thereof was found to be 63%. MS (ESI) m/z 297.31[ M+H ]] + 。
And a second step of: 4- (4-amino-1H-pyrazol-1-yl) piperidine-1-carboxylic acid tert-butyl ester
Tert-butyl 4- (4-nitro-1H-pyrazol-1-yl) piperidine-1-carboxylate (0.35 mmol,1.0 equiv) was dissolved in a mixed solvent of ethanol and water (10:1), and iron powder (1.40 mmol,4.0 equiv) and NH were added 4 Cl (0.88 mmol,2.5 equiv), heating the reaction system to reflux and stirring for 2h, filtering the reaction system after the reaction is complete, washing a filter cake with cold ethanol, and spin-drying the filtrate to obtain an intermediate, namely the 4- (4-aminophenyl) piperazine-1-carboxylic acid tert-butyl ester. The yield thereof was found to be 73%. MS (ESI) m/z 267.29[ M+H ]] + 。
Example 43 Synthesis of tert-butyl- (4-amino-1H-pyrazol-1-yl) azetidine-1-carboxylate
The first step: 3- (4-Nitro-1H-pyrazol-1-yl) azetidine-1-carboxylic acid tert-butyl ester
The synthesis procedure is the same as in example 3. The yield thereof was found to be 58%. MS (ESI) m/z 269.23[ M+H ]] + 。
And a second step of: 3- (4-amino-1H-pyrazol-1-yl) azetidine-1-carboxylic acid tert-butyl ester
The synthesis method is the same asExample 3 the second step. The yield thereof was found to be 80%. MS (ESI) m/z 239.29[ M+H ]] + 。
Example 51 Synthesis of cyclopropyl-N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide
The first step: n- (4-bromo-2-methylbenzyl) -1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamide
(4-bromo-2-methylphenyl) methylamine (0.85 mmol,1.0 equiv), 1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxylic acid; 1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxylic acid (0.89 mmol,1.05 equiv) was dissolved in 5mL of LDMF, EDCI (1.3 mmol,1.5 equiv), HOBt (1.3 mmol,1.5 equiv), DIPEA (1.7 mmol,2.0 equiv) was added at room temperature, and the reaction mixture was stirred at room temperature for 2H. After the TLC monitoring reaction is completed, pouring the reaction system into ice water, precipitating solids, filtering, washing a filter cake with PE, drying to obtain a crude product, and purifying by a flash chromatography (EA: PE=1:3-1:1) to obtain N- (4-bromo-2-methylbenzyl) -1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamide. Yield: 67%. MS (ESI) m/z 514.62[ M+H ]] + 。
And a second step of: 1-cyclopropyl-N- (2-methyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl) -1H-1,2, 3-triazole-4-carboxamide
N- (4-bromo-2-methylbenzyl) -1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamide (0.7 mmol,1.0 equiv), pinacol biborate (0.85 mmol,1.2 equiv), pd (dppf) Cl 2 ·CH 2 Cl 2 (0.07 mmol,0.1 equiv), KOAc (2.11 mmol,3.0 equiv) in 10mL dioxane, reactantIs tied to N 2 The reaction was allowed to react overnight at 80℃under protection. After the completion of the reaction, TLC was monitored, the next step was directly performed.
And a third step of: n- (4- (2-chloro-5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamide
2, 4-dichloro-5-fluoropyrimidine (0.84 mmol,1.2 equiv.) and K were added to the reaction system 2 CO 3 (1.4 mmol,2.0 equiv) and 2mL of water with N 2 After the air in the reaction system was replaced, the reaction system was heated to 90℃and reacted overnight. After TLC monitored the reaction was complete, the reaction was filtered, the filter cake was washed with DCM, the solvent of the filtrate was evaporated under reduced pressure and purified by flash chromatography (EA: pe=1:3-1:1) to give N- (4- (2-chloro-5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamide. Yield: 46%. MS (ESI) m/z 387.22[ M+H ]] + 。
Fourth step: tert-butyl 4- (4- ((4- (4- ((1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) piperidine-1-carboxylate
N- (4- (2-chloro-5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamide and tert-butyl 4- (4-amino-1H-pyrazol-1-yl) piperidine-1-carboxylate (0.22 mmol,1.0 equiv) were dissolved in 10mL dioxane, pd was added 2 (dba) 3 (0.02mmol，0.1equiv)、S-PhOS(0.04mmol，0.2equiv)、Cs 2 CO 3 (0.44 mmol,2.0 equiv.) in N 2 Reflux reaction for 2h under protection. After TLC monitored completion of the reaction, the reaction mixture was filtered and the filter cake was washed with DCM, after evaporation of the solvent of the filtrate under reduced pressure, purification by flash chromatography (EA: dcm=1:5-1:1) afforded tert-butyl 4- (4- ((4- (4- ((1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamido) methyl) -3-methylphenyl) -5-fluoxastrobinPyridin-2-yl) amino) -1H-pyrazol-1-yl) piperidine-1-carboxylic acid ester. Yield: 52%. MS (ESI) m/z 617.41[ M+H ]] + 。
Fifth step: 1-cyclopropyl-N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide
Tert-butyl 4- (4- ((4- (4- ((1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) piperidine-1-carboxylate (0.2 mmol) was dissolved in 20% trifluoroacetic acid/DCM solution, the reaction system was stirred at room temperature for 30min, and the solvent was distilled off under reduced pressure to give 4- (tert-butyl) -N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) benzamide. Yield: 94%. MS (ESI) m/z 517.31[ M+H ]] + 。
Example 6 Synthesis of N- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamide
The first step: tert-butyl 3- (4- ((4- (4- ((1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) azetidine-1-carboxylate
The synthesis procedure is the same as in the fourth step of example 5. Yield: 55%. MS (ESI) m/z 589.39[ M+H ]] + 。
And a second step of: n- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1-cyclopropyl-1H-1, 2, 3-triazole-4-carboxamide
The synthesis method is the same as in the fifth step of example 5. Yield: 98%. MS (ESI) m/z 489.29[ M+H ]] + 。
Example 71 Synthesis of N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide
The first step: n- (4-bromo-2-methylbenzyl) -1- (tert-butyl) -1H-1,2, 3-triazole-4-carboxamide
The synthesis procedure is the same as in example 5. Yield: 74%. MS (ESI) m/z 351.16[ M+H ]] + 。
And a second step of: 1- (tert-butyl) -N- (2-methyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl) -1H-1,2, 3-triazole-4-carboxamide
The synthesis procedure is the same as in example 5, the second step.
And a third step of: 1- (tert-butyl) -N- (4- (2-chloro-5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide
The synthesis procedure is the same as in the third step of example 5. Yield: 42%. MS (ESI) m/z 403.25[ M+H ]] + 。
Fourth step: tert-butyl 4- (4- ((4- (4- ((1- (tert-butyl) -1H-1,2, 3-triazole-4-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) piperidine-1-carboxylate
The synthesis procedure is the same as in the fourth step of example 5. Yield: 55%. MS (ESI) m/z 633.45[ M+H ]] + 。
Fifth step: 1- (tert-butyl) -N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide
The synthesis method is the same as the fifth step yield of example 5: 95%. MS (ESI) m/z 533.38[ M+H ]] + 。
Example 8 Synthesis of N- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1- (tert-butyl) -1H-1,2, 3-triazole-4-carboxamide
The first step: tert-butyl 3- (4- ((4- (4- ((1- (tert-butyl) -1H-1,2, 3-triazole-4-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) azetidine-1-carboxylate
The synthesis procedure is the same as in the fourth step of example 5. Yield: 57%. MS (ESI) m/z 605.42[ M+H ]] + 。
And a second step of: n- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1- (tert-butyl) -1H-1,2, 3-triazole-4-carboxamide
The synthesis method is the same as the fifth step yield of example 5: 95%. MS (ESI) m/z 505.30[ M+H ]] + 。
Example 9 Synthesis of N- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -2- (tert-butyl) thiazole-5-carboxamide
The first step: tert-butyl 3- (4- ((4- (4- ((2- (tert-butyl) thiazole-5-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) azetidine-1-carboxylate
The synthesis procedure is the same as in the fourth step of example 5. Yield: 53%. MS (ESI) m/z 621.71[ M+H ]] + 。
And a second step of: n- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -2- (tert-butyl) thiazole-5-carboxamide
The synthesis method is the same as the fifth step yield of example 5: 98%. MS (ESI) m/z 521.37[ M+H ]] + 。
Example 10 Synthesis of 5- (tert-butyl) -N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-3-carboxamide
The first step: n- (4-bromo-2-methylbenzyl) -5- (tert-butyl) -1,2, 4-oxadiazole-3-carboxamide
The synthesis procedure is the same as in example 5. Yield: 72%. MS (ESI) m/z 352.17[ M+H ]] + 。
And a second step of: 5- (tert-butyl) -N- (2-methyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzyl) -1,2, 4-oxadiazole-3-carboxamide
The synthesis procedure is the same as in example 5, the second step.
And a third step of: 5- (tert-butyl) -N- (4- (2-chloro-5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-3-carboxamide
The synthesis procedure is the same as in the third step of example 5. Yield: 46%. MS (ESI) m/z 404.18[ M+H ]] + 。
Fourth step: tert-butyl 4- (4- ((4- (4- ((5- (tert-butyl) -1,2, 4-oxadiazole-3-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) piperidine-1-carboxylate
The synthesis procedure is the same as in the fourth step of example 5. Yield: 51%. MS (ESI) m/z 634.45[ M+H ]] + 。
Fifth step: 5- (tert-butyl) -N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-3-carboxamide
The synthesis method is the same as the fifth step yield of example 5: 95%. MS (ESI) m/z 534.60[ M+H ]] + 。
Example 11 Synthesis of N- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -5- (tert-butyl) -1,2, 4-oxadiazole-3-carboxamide
The first step: tert-butyl 3- (4- ((4- (4- ((5- (tert-butyl) -1,2, 4-oxadiazole-3-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) azetidine-1-carboxylic acid ester
The synthesis procedure is the same as in the fourth step of example 5. Yield: 51%. MS (ESI) m/z 606.31[ M+H ]] + 。
And a second step of: n- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -5- (tert-butyl) -1,2, 4-oxadiazole-3-carboxamide
The synthesis method is the same as the fifth step yield of example 5: 95%. MS (ESI) m/z 506.42[ M+H ]] + 。
Example 12 Synthesis of 3- (tert-butyl) -N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-5-carboxamide
The first step: n- (4-bromo-2-methylbenzyl) -3- (tert-butyl) -1,2, 4-oxadiazole-5-carboxamide
The synthesis procedure is the same as in example 5. Yield: 72%. MS (ESI) m/z 352.23[ M+H ]] + 。
And a second step of: 3- (tert-butyl) -N- (2-methyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzyl) -1,2, 4-oxadiazole-5-carboxamide
The synthesis procedure is the same as in example 5, the second step.
And a third step of: 3- (tert-butyl) -N- (4- (2-chloro-5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-5-carboxamide
The synthesis procedure is the same as in the third step of example 5. Yield: 44%. MS (ESI) m/z 404.19[ M+H ]] + 。
Fourth step: tert-butyl 4- (4- ((4- (4- ((3- (tert-butyl) -1,2, 4-oxadiazole-5-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) piperidine-1-carboxylate
The synthesis procedure is the same as in the fourth step of example 5. Yield: 55%. MS (ESI) m/z 634.48[ M+H ]] + 。
Fifth step: 3- (tert-butyl) -N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-5-carboxamide
The synthesis method is the same as the fifth step yield of example 5: 97%. MS (ESI) m/z 534.65[ M+H ]] + 。
EXAMPLE 13 Synthesis of N- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -3- (tert-butyl) -1,2, 4-oxadiazole-5-carboxamide
The first step: tert-butyl 3- (4- ((4- (4- ((3- (tert-butyl) -1,2, 4-oxadiazole-5-carboxamido) methyl) -3-methylphenyl) -5-fluoropyrimidin-2-yl) amino) -1H-pyrazol-1-yl) azetidine-1-carboxylic acid ester
The synthesis procedure is the same as in the fourth step of example 5. Yield: 56%. MS (ESI) m/z 606.35[ M+H ]] + 。
And a second step of: n- (4- (2- ((1- (azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -3- (tert-butyl) -1,2, 4-oxadiazole-5-carboxamide
The synthesis method is the same as the fifth step yield of example 5: 98%. MS (ESI) m/z 506.43[ M+H ]] + 。
EXAMPLE 14 Synthesis of 1-cyclopropyl-N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) piperidin-4-yl) -1H-pyrazol-3-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide (Compound 1)
4- (tert-butyl) -N- (4- (5-fluoro-2- ((1- (piperidin-4-yl) -1H-pyrazol-4-yl) amino) pyrimidin-4-yl) -2-methylbenzyl) benzamide and 1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidine-4-carbaldehyde (0.05 mmol,1.0 eq) were dissolved in 5mL of a DCM/MeOH (1:1) mixed solutionTo this, TEA (0.05 mmol,1.0 equiv) was added, and the reaction system was stirred at room temperature for 10min. NaBH (OAc) was added in three portions 3 (0.25 mmol,5.0 equiv.) the reaction was stirred at room temperature. After completion of the TLC monitoring reaction, 10mL of saturated NaHCO was poured into the reaction system 3 The solution was stirred for 10min, allowed to stand for separation, the organic phase was separated, the aqueous phase (5 mL. Times.3) was extracted with DCM, the organic phases were combined, washed with saturated brine (10 mL. Times.3), and dried over Na 2 SO 4 Drying, evaporating the solvent under reduced pressure and purifying by flash chromatography (2-5% MeOH/DCM) to give 1-cyclopropyl-N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) piperidin-4-yl) -1H-pyrazol-3-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide (compound 21). Yield: 11%. Yellow solid. 1 H NMR(400MHz,DMSO-d 6 )δ11.07(s,1H),9.57(s,1H),9.06(t,J＝6.2Hz,1H),8.63(d,J＝1.4Hz,1H),8.53(d,J＝3.6Hz,1H),8.01(s,1H),7.87(s,1H),7.80(d,J＝8.1Hz,1H),7.65(d,J＝8.5Hz,1H),7.54(s,1H),7.40(d,J＝8.1Hz,1H),7.33(s,1H),7.25(d,J＝8.7Hz,1H),5.07(dd,J＝12.9,5.5Hz,1H),4.52(d,J＝6.0Hz,2H),4.16-3.96(m,4H),3.51-3.40(m,2H),3.04-2.82(m,5H),2.63-2.59(m,1H),2.56(d,J＝7.9Hz,1H),2.44(s,3H),2.23-2.15(m,2H),2.09-1.98(m,4H),1.97-1.88(m,2H),1.87-1.75(m,3H),1.58-1.42(m,1H),1.24(s,4H). 13 C NMR(101MHz,DMSO-d 6 )δ173.20,170.50,168.04,167.36,160.19,155.41,151.50,151.17,145.86,142.69,140.72,138.90,136.24,134.44,131.99,130.15,127.70,126.76,126.64,125.39,123.50,117.99,117.74,108.14,105.91,99.92,55.34,52.84,49.15,48.92,48.05,47.64,33.65,32.70,31.95,31.39,29.96,22.60,19.26,7.22.Purity:98.4％；HRMS(ESI):m/z calcd for C 45 H 48 FN 13 O 5 [M+H] + :870.3885,found:870.3951.
Example 15 Synthesis of 1-cyclopropyl-N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) azetidin-3-yl) -1H-pyrazol-3-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide (Compound 2)
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The synthesis was the same as in example 14. Yield: 9%. Yellow solid. 1 H NMR(400MHz,Chloroform-d)δ10.0(s,1H),8.3(s,1H),8.2(s,1H),8.0(s,1H),7.9-7.7(m,3H),7.6(t,J＝14.5Hz,3H),7.4(d,J＝8.1Hz,1H),7.2(s,1H),7.0(d,J＝8.7Hz,1H),5.0-4.9(m,2H),4.8-4.5(m,2H),4.1-3.7(m,5H),3.7-3.5(m,2H),3.0-2.6(m,6H),2.6-2.5(m,2H),2.4(s,3H),2.2-2.0(m,1H),1.9-1.8(m,2H),1.7-1.6(m,1H),1.4-1.3(m,1H),1.3-1.1(m,4H). 13 C NMR(101MHz,Chloroform-d)δ172.01,169.18,168.06,167.30,160.00,155.95,155.23,151.52,149.00,146.90,146.59,142.55,138.68,136.42,134.28,132.75,131.31,130.68,128.09,126.82,126.16,125.31,123.35,119.15,118.42,117.65,108.46,64.97,61.60,51.38,49.07,47.77,40.89,34.51,31.77,31.43,29.65,22.75,19.26,7.10,0.94.Purity:95.2％；HRMS(ESI):m/z calcd for C 43 H 44 FN 13 O 5 [M+H] + :842.3572,found:842.3675.
EXAMPLE 16 Synthesis of 1- (tert-butyl) -N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) piperidin-4-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide (Compound 3)
The synthesis was the same as in example 14. Yield: 12%. Yellow solid. 1 H NMR(400MHz,DMSO-d 6 )δ11.09(s,1H),9.60(s,1H),9.06(t,J＝6.1Hz,1H),8.72(s,1H),8.54(d,J＝3.6Hz,1H),8.01(s,1H),7.86(s,1H),7.81(d,J＝8.0Hz,1H),7.66(d,J＝8.4Hz,1H),7.54(s,1H),7.41(d,J＝8.1Hz,1H),7.34(s,1H),7.26(d,J＝8.6Hz,1H),5.07(dd,J＝12.9,5.4Hz,1H),4.53(d,J＝6.1Hz,2H),4.18-3.96(m,4H),3.46-3.42(m,1H),3.06-2.82(m,6H),2.63-2.59(m,1H),2.58-2.54(m,2H),2.44(s,3H),2.23-2.13(m,2H),1.99(s,4H),1.91(s,2H),1.85-1.79(m,2H),1.64(s,9H). 13 C NMR(101MHz,DMSO-d 6 )δ173.21,170.50,168.04,167.37,160.50,157.64,155.39,152.14,150.85,146.20,145.87,142.93,142.78,134.44,132.35,129.48,127.67,126.64,125.41,124.46,124.23,123.51,118.02,117.74,115.02,108.13,72.48,60.27,52.76,49.13,47.61,31.38,29.77,29.38,27.23,22.60,21.45,19.48,19.26,14.49.Purity:98.9％；HRMS(ESI):m/z calcd for C 46 H 52 FN 13 O 5 [M+H] + 886.4198, found:886.4282. Specific spectra are shown in FIGS. 1 and 2.
EXAMPLE 17 Synthesis of 1- (tert-butyl) -N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) azetidin-3-yl) -1H-pyrazol-4-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1H-1,2, 3-triazole-4-carboxamide (Compound 4)
The synthesis was the same as in example 14. Yield: 10%. Yellow solid. 1 H NMR(400MHz,DMSO-d 6 )δ11.09(s,1H),9.65(s,1H),9.06(t,J＝6.1Hz,1H),8.72(s,1H),8.55(d,J＝3.6Hz,1H),8.09(s,1H),7.83(d,J＝17.9Hz,2H),7.66(d,J＝8.6Hz,2H),7.41(d,J＝8.1Hz,1H),7.32(s,1H),7.24(d,J＝8.7Hz,1H),5.07(dd,J＝12.9,5.4Hz,1H),5.03-4.88(m,1H),4.52(d,J＝6.1Hz,2H),4.08-4.00(m,2H),3.80-3.64(m,1H),3.49-3.40(m,1H),3.02-2.81(m,4H),2.63-2.59(m,1H),2.58-2.54(m,1H),2.44(s,3H),2.35-2.27(m,1H),2.05-1.98(m,2H),1.91(s,2H),1.81-1.72(m,2H),1.64(s,9H). 13 C NMR(101MHz,DMSO-d 6 )δ173.20,172.40,170.50,168.02,167.35,160.49,156.55,155.30,151.24,148.75,142.79,141.69,140.76,140.22,136.22,134.44,132.08,130.30,127.67,126.60,125.40,124.49,124.07,117.99,117.80,108.13,61.49,60.26,60.16,49.14,34.17,31.38,29.77,29.57,22.60,21.45,21.16,19.33,14.49.Purity:97.2％；HRMS(ESI):m/z calcd for C 44 H 48 FN 13 O 5 [M+H] + :858.3885,found:858.3990.
Example 18 Synthesis of 2- (tert-butyl) -N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) azetidin-3-yl) -1H-pyrazol-3-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) thiazole-5-carboxamide (Compound 5)
The synthesis was the same as in example 14. Yield: 13%. Yellow solid. 1 H NMR(400MHz,Chloroform-d)δ9.0(s,1H),8.4-8.3(m,1H),8.0(s,1H),8.0(s,1H),7.9-7.9(m,2H),7.7-7.6(m,3H),7.5(d,J＝7.9Hz,1H),7.3-7.3(m,2H),7.0(d,J＝8.2Hz,1H),5.0-4.9(m,2H),4.7(d,J＝6.0Hz,2H),4.0-3.9(m,4H),3.7-3.5(m,2H),3.0-2.9(m,2H),2.9-2.7(m,3H),2.6-2.6(m,2H),2.5(s,3H),2.4-2.3(m,1H),2.2-2.1(m,2H),1.9-1.8(m,2H),1.7-1.7(m,1H),1.5(s,9H). 13 C NMR(101MHz,Chloroform-d)δ171.37,168.64,168.01,167.24,161.34,156.06,155.27,151.71,149.19,148.82,139.12,136.63,134.33,132.78,132.47,131.53,130.78,128.26,126.83,125.39,123.23,122.37,121.38,119.36,118.56,117.76,108.55,61.61,51.46,49.68,47.85,41.04,37.75,34.55,31.41,30.77,29.68,22.73,19.35,0.95.Purity:98.1％；HRMS(ESI):m/z calcd for C 45 H 48 FN 11 O 5 S[M+H] + :874.3545,found:874.3640.
EXAMPLE 19 Synthesis of 5- (tert-butyl) -N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) piperidin-4-yl) -1H-pyrazol-3-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-3-carboxamide (Compound 6)
The synthesis was the same as in example 14. Yield: 10%. Yellow solid. 1 H NMR(400MHz,Chloroform-d)δ9.27(s,1H),8.31(t,J＝2.8Hz,1H),7.93(d,J＝12.1Hz,3H),7.68(dd,J＝8.5,2.2Hz,1H),7.62(s,1H),7.49(s,1H),7.44(d,J＝8.0Hz,1H),7.36-7.22(m,3H),7.07(d,J＝8.8Hz,1H),5.01-4.90(m,1H),4.73(d,J＝5.8Hz,2H),4.12(t,J＝8.2Hz,1H),3.97(d,J＝12.9Hz,2H),2.99(q,J＝13.1,10.3Hz,4H),2.93-2.71(m,4H),2.46(s,3H),2.28(d,J＝6.9Hz,2H),2.21-2.02(m,9H),1.90(d,J＝13.3Hz,2H),1.82(s,1H),1.47(s,9H). 13 C NMR(101MHz,Chloroform-d)δ171.62,168.84,168.07,167.30,162.99,156.41,156.14,155.34,151.58,149.05,147.07,146.71,137.70,136.77,134.33,133.27,130.84,130.16,128.67,127.00,125.38,122.77,118.37,117.73,117.41,108.50,63.86,59.53,53.06,49.05,47.96,41.35,33.85,33.64,32.43,31.41,29.97,29.62,28.24,22.72,19.28,0.95.Purity:96.8％；HRMS(ESI):m/z calcd for C 46 H 51 FN 12 O 6 [M+H] + :887.4039,found:887.4088.
EXAMPLE 20 Synthesis of 5- (tert-butyl) -N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) azetidin-3-yl) -1H-pyrazol-3-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-3-carboxamide (Compound 7)
The synthesis was the same as in example 14. Yield: 12%. Yellow solid. 1 H NMR(400MHz,Chloroform-d)δ8.33(s,1H),7.96(s,1H),7.94-7.87(m,2H),7.70-7.61(m,2H),7.53-7.40(m,3H),7.27(d,J＝6.9Hz,2H),7.03(d,J＝8.7Hz,1H),5.02-4.90(m,2H),4.76-4.66(m,2H),4.00-3.85(m,4H),3.67-3.54(m,2H),3.00-2.90(m,2H),2.88-2.72(m,3H),2.62-2.56(m,2H),2.47(s,3H),2.16-2.10(m,1H),1.91-1.83(m,2H),1.73-1.65(m,1H),1.40(s,9H). 13 C NMR(101MHz,Chloroform-d)δ178.13,171.72,168.95,168.01(d,J＝5.5Hz),167.27,156.07,155.26,153.17,151.66,149.10,146.68,145.85,137.22,136.80,134.31,133.36,131.53,131.00,128.82,126.96,125.37,123.18,119.33,118.48,117.72,108.52,64.97,61.58,51.46,49.06,47.83,41.60,34.58,32.62,31.41,29.67,28.29,24.79,22.73,19.35,0.95.Purity:97.2％；HRMS(ESI):m/z calcd for C 44 H 47 FN 12 O 6 [M+H] + :859.3726,found:859.3821.
EXAMPLE 21 Synthesis of 3- (tert-butyl) -N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) piperidin-4-yl) -1H-pyrazol-3-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-5-carboxamide (Compound 8)
The synthesis was the same as in example 14. Yield: 10%. Yellow solid. 1 H NMR(400MHz,Chloroform-d)δ9.7(s,1H),8.3(s,1H),8.0-7.9(m,3H),7.8-7.7(m,1H),7.7(d,J＝8.5Hz,1H),7.6(s,1H),7.6-7.5(m,1H),7.4(d,J＝8.0Hz,1H),7.3(s,1H),7.0(d,J＝8.7Hz,1H),4.9(dd,J＝12.1,5.4Hz,1H),4.7(d,J＝6.0Hz,2H),4.2-4.1(m,1H),4.0-3.9(m,2H),3.1-2.9(m,4H),2.9-2.7(m,4H),2.4(s,3H),2.3-2.2(m,2H),2.2-2.1(m,6H),2.1-2.0(m,2H),1.9-1.8(m,2H),1.8-1.7(m,1H),1.4(s,9H). 13 C NMR(101MHz,Chloroform-d)δ178.1,172.0,169.1,168.1(d,J＝6.1Hz),167.3,156.1,155.3,153.2,151.5,149.0,147.0,146.7,137.2,136.8,134.3,133.4(d,J＝5.6Hz),130.9(d,J＝5.5Hz),130.2,128.8,127.0(d,J＝7.6Hz),125.4,122.8,118.3,117.7,117.4,108.5,63.9,59.5,53.0,49.0,47.9,41.6,33.6,32.6,32.4,31.4,30.0,29.6,28.3,27.9,24.8,22.7,19.3,0.9.Purity:98.6％；HRMS(ESI):m/z calcd for C 46 H 51 FN 12 O 6 [M+H] + :887.4039,found:887.4149.
EXAMPLE 22 Synthesis of 3- (tert-butyl) -N- (4- (2- ((1- (1- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) methyl) azetidin-3-yl) -1H-pyrazol-3-yl) amino) -5-fluoropyrimidin-4-yl) -2-methylbenzyl) -1,2, 4-oxadiazole-5-carboxamide (Compound 9)
The synthesis was the same as in example 14. Yield is good:9%. Yellow solid. 1 H NMR(400MHz,DMSO-d 6 )δ11.08(s,1H),9.92-9.79(m,1H),9.63(s,1H),8.55(d,J＝3.4Hz,1H),8.09(s,1H),7.91-7.79(m,2H),7.67-7.59(m,2H),7.47(d,J＝8.1Hz,1H),7.30(s,1H),7.22(d,J＝8.8Hz,1H),5.07(dd,J＝13.0,5.3Hz,1H),5.02-4.92(m,1H),4.54(d,J＝5.8Hz,2H),4.02(d,J＝13.0Hz,2H),3.84-3.71(m,2H),3.00-2.82(m,3H),2.65-2.54(m,2H),2.44(s,3H),2.07-1.96(m,1H),1.84-1.72(m,2H),1.70-1.56(m,1H),1.37(s,9H),1.30-1.11(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ173.20,170.49,169.03,168.02,167.35,155.32,153.77,148.51,146.67,139.19,136.58,136.05,134.43,130.84,130.34,128.33,126.70,125.38,119.68,118.94,117.97,117.76,116.39,113.57,112.31,108.09,61.55,51.17,49.13,47.62,41.01,34.54,32.67,31.39,29.75,28.46,25.35,22.61,19.40.Purity:98.1％；HRMS(ESI):m/z calcd forC 44 H 47 FN 12 O 6 [M+H] + :859.3726,found:859.3821.
Biological test case
The instrument used for the test was derived from: BTK primary antibodies were purchased from Abcam, EPR20445; beta-action primary antibody was purchased from freude, FD0060; rabbit anti-purchased from freude, FDR007; murine anti-was purchased from freude, FDM007;1640 medium was purchased from Gibco;
test example 1 degradation of BTK Activity of degradation agent in Mino cells
Cell culture: mino (human mantle cell lymphoma cells) cell culture medium was RMPI1640+15% FBS+1% penicillin-streptomycin solution. Both cells were at 37℃and 5% CO 2 Culturing under the condition.
Western blotting: cells were uniformly seeded into 6-well or 12-well plates (1X 10) 6 Cells/wells) were treated with different concentrations of the compound. After the time required for incubation, cells were collected, centrifuged at 200g, the supernatant was discarded, whole cell lysates were collected using RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor), and protein concentration was determined by BCA method. Equal amounts of protein were electrophoresed by 10% SDS-PAGE and transferred to polyvinylidene fluoride transfer membranes (PVDF, 0.45 μm), blocked with skimmed milk powder or Bovine Serum Albumin (BSA), after whichIncubate with the target antibody overnight at 4 ℃. Then incubated with the indicated secondary antibodies for 2h. Chemiluminescent signals are quantified by enhanced chemiluminescent detection reagents of a gel imaging system. The target tape calculates the gray value by ImageJ software.
Table 1 results of test for degradation Activity of Compounds 1 to 9 and comparative example 1 on BTK
"Degradation%" refers to the proportion of compound degrading BTK at a concentration of 100 nM; "DC 50，4h "means the dose at which 50% of the protein is degraded within 4 hours. "t 1/2,20nM "means the time required to degrade 50% of the BTK protein at a concentration of 20 nM; "-" indicates no measurement. Comparative example 1 is from example 3 in patent CN 114292270B.
Test example 2 modulation of mRNA levels of an osteoclast-associated Gene by Compound 3
Cell culture: bone marrow cells were extracted using 6-8 week old C57BL/6 mice (BoneMarrowMyeloidcell, BMM). After mice were sacrificed and soaked with alcohol for 1min, femur and tibia were removed and soft tissues were stripped, bone marrow cavities were exposed and rinsed with sterile PBS. The cells were collected and the erythrocytes were lysed. The cells obtained were cultured overnight in a medium containing 30ng/ml m-CSF to remove bone marrow stromal cells and non-adherent cells (i.e., BMM cells) were collected for subsequent use. BMM cells were cultured in alpha-MEM medium (10% FBS+1% penicillin-streptomycin solution) at 37℃with 5% CO 2 Culturing under the condition.
Real-time quantitative PCR (RT-qPCR): in 48-well plates, 1X 10 cells were seeded per well 4 BMM cells were pretreated with different concentrations of the compound for 2h, and then stimulated with RANKL (100 ng/mL) and m-CSF (30 ng/mL) for 72h. The medium was aspirated and retained and the cells were collected, and total RNA was isolated from the cultured BMM cells using Trizol reagent according to the instructions of the reagent manufacturer. RNA quality was assessed by A260/A280 ratio. Reverse transcription was performed using hyperscript tmiii 1st Stand cDNA Synthesis Kit. Solid-borne on a LightCycler480 fluorescent quantitative PCR apparatus using S6 Universal SYBR qPCR mixAnd (5) time PCR. The sequences of all primers used are shown in Table 2. Gene expression level 2 -ΔΔCT And (5) calculating a method.
TABLE 2 oligonucleotide primer sequences for qualitative quantitative real-time PCR (qPCR)
The test results are shown in fig. 3, compound 3 significantly down-regulates the osteoclast-associated gene TRAP, dcstamp, CTSK, and NFATc1.
Test example 3 Effect of Compounds on mouse periodontitis model (Compound 3 is taken as an example)
Experimental protocol: c57BL/6 mice were randomly divided into Normal (Normal), model (Model), compound 3 high dose (30 mg/kg, i.p.) and low dose (10 mg/kg, i.p.) control groups, and ybutinib (30 mg/kg, i.p.) control groups, and after one day of advanced dosing, 5-0 sutures soaked with LPS were ligated around the second molar on the right side of the mouse's upper jaw, and on days 1, 4, 7 after molding, the mouse's upper jaw group was taken and bone resorption was observed by Micro-CT after day 10. And scanning the right maxilla by Micro-CT, and performing three-dimensional reconstruction to obtain a 3D image. The distance between the enamel cementum boundary (CEJ) to the alveolar ridge crest (ABC) at the distal root buccal site of the right side of the upper jaw (CEJ-ABC) was measured with Burker DataViewer software to detect changes in alveolar bone height. The right maxillary first molar and the second molar were measured and analyzed between: bone volume fraction (bone volume fraction, BV/TV).
The experimental results are shown in fig. 4, and with ibutinib as a control, compound 3 significantly relieves alveolar bone resorption in mice, and the bone volume fraction of the high dose administration group is comparable to the CEJ-ABC value.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A compound of formula (i) or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof;
wherein L is 1 Selected from the group consisting of
L 2 Selected from:
y is selected fromPreferably, Y is selected from->
R 1 Selected from the group consisting of
R 2 Selected from the group consisting ofPreferably, R 2 Selected from->
A is selected fromPreferably, A is selected from->
X is selected from halogen atoms, i.e. fluorine, chlorine, bromine or iodine, preferably X is fluorine.
2. The compound of claim 1, or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein: the compounds include the structures shown below:
3. a process for the preparation of a compound characterized by:
the compound shown in the formula (1-1) and the compound shown in the formula (1-2) are subjected to substitution reaction or coupling reaction to obtain a compound shown in the formula (1-3), further subjected to coupling or condensation reaction with the compound shown in the formula (1-4) to obtain a compound shown in the formula (1-5), and finally subjected to Boc protection removal to obtain a compound shown in the formula (1-6);
4. a process for the preparation of a compound characterized by:
carrying out substitution reaction on a compound shown in a formula 2-1 and a compound shown in a formula 2-2 to obtain a compound shown in a formula 2-3, preparing a compound shown in a formula 2-4 through oxidation reaction, and finally carrying out reductive amination reaction on the compound and an intermediate shown in a formula 1-6 to prepare compounds 1-9 in the formula 2;
5. a pharmaceutical composition characterized by: the pharmaceutical composition comprising a compound of any one of claims 1-2, or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof.
6. Use of a compound according to any one of claims 1-2, or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, for the manufacture of a medicament for the treatment and/or prophylaxis and/or delay and/or co-treatment of a disease associated with BTK activity or expression level.
7. The use according to claim 6, characterized in that: the disease is tumor or autoimmune disease.
8. The use according to claim 7, characterized in that: the tumors include non-hodgkin lymphoma, chronic lymphocytic leukemia, B-cell lymphoma, mantle cell lymphoma.
9. The use according to claim 7, characterized in that: the autoimmune disease is rheumatoid arthritis or periodontitis.
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