CN114292270B - BTK inhibitor and preparation method and application thereof - Google Patents

BTK inhibitor and preparation method and application thereof Download PDF

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CN114292270B
CN114292270B CN202111542048.1A CN202111542048A CN114292270B CN 114292270 B CN114292270 B CN 114292270B CN 202111542048 A CN202111542048 A CN 202111542048A CN 114292270 B CN114292270 B CN 114292270B
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陈建军
黄俊力
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Southern Medical University
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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

BTK inhibitor and preparation method and application thereof
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 one of the members of the TEC non-receptor tyrosine kinase family, is expressed in cells of all hematopoietic lineages except T cells, and plays an important role in B Cell Receptor (BCR) and fcγ receptor (FcR) signaling pathways, regulating B cell production and activation. Abnormal activation of B cells plays a key role in the pathogenesis of B cell lymphomas and autoimmune diseases. BTK inhibitors are believed to have potential for the treatment of vascular malignant tumors and autoimmune diseases. So far, 5 BTK inhibitors have been approved for sale, all as irreversible BTK inhibitors. Ibutinib is the first BTK inhibitor on the market, and is a first-line drug for single-drug treatment of B-cell lymphomas (such as MCL, WM and CLL) due to its better activity and acceptable toxicity, but the development of drug resistance (mainly C481S mutation) and safety problems also limit its scope of use.
Targeting protein chimera (PROTAC) technology has become a very potential therapeutic intervention. PROTAC is a bifunctional molecule comprising a target protein ligand and an E3 ubiquitin ligase ligand, the middle of which is linked by a Linker. The protoc is capable of simultaneously binding a target Protein (POI) and E3 ubiquitin ligase to form a ternary complex, thereby causing ubiquitination and subsequent proteasome degradation of the target protein, thereby exerting its therapeutic effect. Compared with the traditional small molecule drug, the PROTAC is more efficient and safer, also expands the selection range of the target point of the patentable drug, and has very good development prospect. Searching for new targets, improving in vivo and in vitro activity, optimizing physicochemical properties and pharmacokinetic properties of the PROTAC molecule become great challenges for current PROTAC drug development.
The nitrogenous heterocyclic derivative is used as a Linker to be beneficial to more efficiently inducing the formation of ternary complex, improving the degradation activity and degradation efficiency of the compound, and simultaneously enabling PROTAC molecules to have better physicochemical properties and metabolic stability bioavailability. At present, a nitrogenous heterocyclic derivative is not used as an effective medicament of a Linker, so that development of the nitrogenous heterocyclic derivative is necessary.
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;
Figure BDA0003414628270000021
in formula (I), L is selected from
Figure BDA0003414628270000022
Figure BDA0003414628270000023
Preferably, in the L structure, when any one of the end groups is the same as that in formula (I)
Figure BDA0003414628270000024
When connected, the other end group of L is connected with +.>
Figure BDA0003414628270000025
Are connected.
Preferably, the compound comprises the structure shown below:
Figure BDA0003414628270000026
/>
Figure BDA0003414628270000031
in a second aspect, the present invention provides a process for the preparation of a compound of formula (1) according to the first aspect of the invention, comprising the steps of: mixing a compound shown in a formula (II) with a compound shown in a formula (III), boc-piperazine and 6-halogenated nicotinic acid tert-butyl ester, and reacting to obtain a compound shown in a formula (1);
Figure BDA0003414628270000032
in the formula (II), X is selected from halogen atoms;
Figure BDA0003414628270000033
preferably, in the formula (II), X is selected from fluorine, chlorine and bromine; further preferably, in the formula (II), X is fluorine.
In a third aspect, the present invention provides a process for the preparation of a compound of formula (2) -formula (5) according to the first aspect of the invention, comprising the steps of: mixing a compound shown in a formula (III), a compound shown in a formula (IV) and a compound shown in a formula (V) with 4-piperidinemethanol or azetidin-3-yl methanol, and reacting to obtain the compound shown in the formula (2) -formula (5);
Figure BDA0003414628270000041
in the formula (IV), X is selected from halogen atoms;
Figure BDA0003414628270000042
preferably, in the formula (IV), X is selected from fluorine, chlorine and bromine; further preferably, in the formula (iv), X is fluorine.
Preferably, when the structure of the compound shown in the formula (IV) is shown in the formula (II), the reaction raw material is 4-piperidinemethanol, so as to obtain a compound shown in the formula (2); when the structure of the compound shown in the formula (IV) is shown in the formula (II), the reaction raw material is azetidin-3-yl methanol, so that the compound shown in the formula (3) is obtained; when the structure of the compound shown in the formula (IV) is shown in the formula (VII), and the reaction raw material is 4-piperidinemethanol, obtaining the compound shown in the formula (4); when the structure of the compound shown in the formula (IV) is shown in the formula (VII), the reaction raw material is azetidin-3-yl methanol, so that the compound shown in the formula (5) is obtained;
Figure BDA0003414628270000043
in the formula (II) and the formula (VII), X is selected from halogen atoms.
According to a fourth aspect of the present invention, there is provided a process for the preparation of a compound of formulae (6) to (7) according to the first aspect of the present invention, comprising the steps of: mixing a compound shown in a formula (III), a compound shown in a formula (IV) and a compound shown in a formula (VI) to react to obtain the compounds shown in the formulas (6) - (7);
Figure BDA0003414628270000051
in the formula (IV), X is selected from halogen atoms;
Figure BDA0003414628270000052
preferably, when the structure of the compound shown in the formula (IV) is shown in the formula (II), the compound shown in the formula (6) is obtained; when the structure of the compound shown in the formula (IV) is shown in the formula (VII), obtaining a compound shown in the formula (7);
Figure BDA0003414628270000053
in the formula (II) and the formula (VII), X is selected from halogen atoms.
In a fifth aspect the present invention provides a pharmaceutical composition comprising a compound according to the first aspect of the invention or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof.
In a sixth 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 or an autoimmune disease.
Preferably, the tumor comprises non-hodgkin's lymphoma, chronic lymphocytic leukemia, B-cell lymphoma, mantle cell lymphoma.
Preferably, the autoimmune disease is rheumatoid arthritis or psoriasis.
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 lymphoma cell proliferation inhibition effect; 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 Ramos cells or Mino cells, degrade BTK protein with C481S mutation in Hela cells, inhibit proliferation of human mantle cell lymphoma cells Mino, and has novel structure and high activity.
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 1 of example 1.
FIG. 2 is a nuclear magnetic resonance spectrum of compound 1 of example 1.
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of compound 2 of example 2.
FIG. 4 is a nuclear magnetic resonance spectrum of compound 2 of example 2.
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of compound 3 of example 3.
FIG. 6 is a nuclear magnetic resonance spectrum of compound 3 of example 3.
FIG. 7 is a nuclear magnetic resonance hydrogen spectrum of compound 4 of example 4.
FIG. 8 is a nuclear magnetic resonance spectrum of compound 4 of example 4.
FIG. 9 is a nuclear magnetic resonance hydrogen spectrum of compound 5 of example 5.
FIG. 10 is a nuclear magnetic resonance spectrum of compound 5 of example 5.
FIG. 11 is a nuclear magnetic resonance hydrogen spectrum of compound 6 of example 6.
FIG. 12 is a nuclear magnetic resonance spectrum of compound 6 of example 6.
FIG. 13 is a nuclear magnetic resonance hydrogen spectrum of compound 7 of example 7.
FIG. 14 is a nuclear magnetic resonance spectrum of compound 7 of example 7.
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 1
The reaction route for the BTK inhibitor of this example is as follows;
Figure BDA0003414628270000071
2- (2, 6-dioxopiperidin-3-yl) -5-fluoroisoindoline-1, 3-dione is denoted as compound 1a, the specific structure of which is shown below, and its specific preparation steps are shown in patent WO2017197056;
Figure BDA0003414628270000072
2- (2, 6-dioxopiperidin-3-yl) -5- (piperazin-1-yl) isoindoline-1, 3-dione hydrochloride was designated as compound 1b, which has the specific structure shown below,
Figure BDA0003414628270000073
the specific preparation steps of compound 1b are as follows:
500mg (1.81 mmol) of Compound 1a and 337.15mg (1.81 mmol) of Boc-piperazine were dissolved in 5mL of DMF and 615.67. Mu.L (3.62 mmol) of DIPEA (N, N-diisopropylethylamine) were added and heated to 80℃for reaction for 3h. After the reaction, 20mL of ethyl acetate was added to the reaction system, followed by washing with water and saturated brine for 3 times, drying over anhydrous sodium sulfate, and evaporating the solvent under reduced pressure to obtain a crude product. Purification by silica gel column chromatography (0% -5% methanol/dichloromethane, V/V) gave intermediate 4- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperazine-1-carboxylic acid tert-butyl ester (300 mg yield 37.46%). The intermediate (300 mg,0.68 mmol) was dissolved in 5mL hydrogen chloride/dioxane solution and reacted at room temperature for 1h, and a solid was precipitated. Filtration and drying gave 232mg of product compound 1b (yield 90.33%).
6- (4- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperazin-1-yl) nicotinic acid was designated as compound 1c, the specific structure of which is shown below,
Figure BDA0003414628270000081
the specific preparation procedure for compound 1c is as follows;
compound 1b (200 mg;0.53 mmol) and tert-butyl 6-chloronicotinate (112.7 mg;0.53 mmol) are dissolved in DMF and anhydrous sodium carbonate (111.9 mg,1.06 mmol) is added and reacted at room temperature for 3h. After the reaction is finished, 10mL of dichloromethane is added into a reaction system, the reaction system is washed with water and saturated saline water for 3 times in sequence, anhydrous sodium sulfate is dried, the solvent is removed by evaporation under reduced pressure to obtain a crude product, and the crude product is purified by silica gel column chromatography (0% -20% ethyl acetate/dichloromethane, V/V) to obtain 6- (4- (2, 6-dioxopiperidine-3-yl) -1, 3-dioxoisoindolin-5-yl) piperazin-1-yl) nicotinic acid tert-butyl ester. 5mL of trifluoroacetic acid/dichloromethane solution (1:3, V/V) was added to the product in ice bath, the mixture was slowly warmed to room temperature and stirred for 2h, and the solvent was distilled off under reduced pressure to give the product compound 1c (88 mg, total yield 35.96%).
3- (4-phenoxyphenyl) -1- (piperidin-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine is designated as compound 1d, which is specifically prepared as follows in Biochemistry,2018, 57 (26): 3564-3575;
Figure BDA0003414628270000082
5- (4- (5- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-carbonyl) pyridin-2-yl) piperazin-1-yl) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione was designated as compound 1, the specific structure of which is shown below,
Figure BDA0003414628270000091
the specific preparation steps of compound 1 are as follows;
compound 1c (30 mg,0.06 mmol) and compound 1d (23.12 mg,0.06 mmol) were dissolved in 2mL of DMF, 34.12mg (0.09 mmol) of HATU (2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate) were added, 30.52. Mu.L (0.18 mmol) of DIPEA and reacted at room temperature for 1h. After the reaction, 10mL of ethyl acetate was added to the reaction system, washed with water and saturated brine for 3 times, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to give a crude product, which was purified by column chromatography (0% -10% methanol/dichloromethane, V/V) to give the product compound 5 (8 mg, yield 22.28%). The nuclear magnetic data of Compound 1 are 1 HNMR(400MHz，Chloroform-d)δ8.45–8.36(m，2H)，7.77–7.71(m，2H)，7.66(d，J＝8.4Hz，2H)，7.41(t，J＝7.8Hz，2H)，7.34–7.31(m，1H)，7.23–7.15(m，3H)，7.13–7.07(m，3H)，6.72–6.66(m，1H)，5.16–5.04(m，1H)，4.97(dd，J＝12.1，5.4Hz，1H)，3.95–3.81(m，5H)，3.65–3.58(m，4H)，3.28–3.16(m，2H)，2.96–2.71(m，4H)，2.46–2.35(m，2H)，2.19–2.09(m，3H). 13 C NMR (101 mhz, chloroform-d) delta 171.27, 168.57, 167.57, 166.81, 157.72, 156.36, 155.37, 153.81, 148.08, 143.37, 134.61, 133.77, 129.89, 127.98, 123.92, 119.42, 119.20, 119.06, 112.27, 98.51, 89.28, 62.19, 54.38, 52.91, 48.92, 31.40, 31.15, 30.08, 22.67. Fig. 1 is a nuclear magnetic resonance spectrum of the compound 1 of example 1, and fig. 2 is a nuclear magnetic resonance spectrum of the compound 1 of example 1.
Example 2
The preparation reaction route of the BTK inhibitor of this example is as follows:
Figure BDA0003414628270000101
/>
2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione is denoted as compound 2a, the specific structure of which is shown below, and the specific preparation method thereof is the same as that of compound 1 a;
Figure BDA0003414628270000102
1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidine-4-carbaldehyde was designated as 2d and represented by the following structural formula,
Figure BDA0003414628270000103
the specific preparation steps of compound 2d are as follows:
compound 1a (200 mg,0.72 mmol) was dissolved in 5mL DMF and DIPEA (246.27. Mu.L, 1.45 mmol) was added and the temperature was raised to 80℃for 3h. After the reaction was completed, 20mL of ethyl acetate was added to the reaction system, followed by washing with water and saturated brine for 3 times, drying over anhydrous sodium sulfate, and evaporating the solvent under reduced pressure to obtain a crude product, which was purified by column chromatography (0% -5% methanol/dichloromethane, V/V) to obtain an intermediate product (300 mg, yield 37.46%). The product of the above step (200 mg,0.54 mmol) was dissolved in 5mL of dichloromethane, and dessert-martin oxidant (456.81 mg,1.08 mmol) was added under ice-bath and stirred at room temperature until the 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 the product compound 2d (180 mg, overall yield 33.9%).
1- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -2- (piperazin-1-yl) ethan-1-one hydrochloride was designated as compound 2H, which was prepared as follows,
Figure BDA0003414628270000111
the preparation procedure for compound 2h was as follows:
the first step: preparation of tert-butyl 4- (2- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -2-oxoethyl) piperazine-1-carboxylate.
Compound 1d (2.0 g,5.18 mmol) was dissolved in 20mL of dichloromethane with 2- (4- (tert-butoxycarbonyl) piperazin-1-yl) acetic acid (1.26 g,5.18 mmol), N, N, N ', N' -tetramethylchlorourea hexafluorophosphate (3.05 g,10.87 mmol), N-methylimidazole (1.44 mL,18.11 mmol) and stirred at room temperature for 30min. After the reaction is finished, water is added into the reaction system, an organic phase is separated, anhydrous sodium sulfate is dried, and a solvent is distilled off under reduced pressure to obtain a crude product. Separating the crude product by column chromatography (0% -5% methanol/dichloromethane, V/V) to obtain 4- (2- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3, 4-d)]Pyrimidin-1-yl) piperidin-1-yl) -2-oxoethyl-piperazine-1-carboxylic acid tert-butyl ester (1.5 g, yield 47.30%). The nuclear magnetic data are as follows: 1 H NMR(400MHz，DMSO-d6)δ8.25(s，1H)，7.69–7.62(m，2H)，7.44(t，J＝7.9Hz，2H)，7.25–7.07(m，5H)，4.98(d，J＝4.4Hz，1H)，3.63(d，J＝12.7Hz，2H)，3.25(d，J＝8.6Hz，2H)，2.45(q，J＝4.1Hz，5H)，2.42–2.38(m，4H)，2.02–1.91(m，4H)，1.40(s，9H)。
and a second step of: compound 2h was prepared.
The product of the above step was dissolved in 10mL of hydrogen chloride/dioxane solution, stirred at room temperature for 30min, and filtered to give compound 2h (1.2 g, yield 89.28%).
5- (4- ((4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -2-oxoethyl) piperidin-1-yl) methyl) piperidine-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione is designated as compound 2, having the following structural formula,
Figure BDA0003414628270000121
the specific preparation steps of compound 2 are as follows:
compound 2d (30 mg, 58.52. Mu.M) and compound 2h (21.62 mg, 58.62. Mu.M) were dissolved in 5mL of methylene chloride, triethylamine (9. Mu.L, 117.04. Mu.M) was added, and after stirring at room temperature for 5min, sodium triacetoxyborohydride (62.02 mg, 292.62. Mu.M) was added in portions and stirred at room temperature for 2h. After the reaction, a saturated sodium bicarbonate solution was added to the reaction system, the organic phase was separated, the inorganic phase was extracted with dichloromethane (3 ml×3), the organic phases were combined, the organic phase was washed 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 (0% -10% methanol/dichloromethane, V/V) to give product compound 2 (17.95 mg, yield 35.42%). The nuclear magnetic data are as follows: 1 H NMR(400MHz，Chloroform-d)δ8.40(s，1H)，7.65(t，J＝8.3Hz，3H)，7.43–7.34(m，2H)，7.28–7.26(m，1H)，7.21–7.11(m，3H)，7.11–6.99(m，3H)，5.08–4.97(m，1H)，4.95(dd，J＝11.9，5.4Hz，1H)，4.79–4.71(m，1H)，4.34–4.25(m，1H)，3.98–3.86(m，2H)，3.71(s，2H)，3.34–3.14(m，3H)，3.01–2.70(m，6H)，2.65–2.48(m，7H)，2.44–2.35(m，2H)，2.28–2.17(m，3H)，2.16–2.02(m，3H)，1.92–1.83(m，2H)，1.82–1.72(m，1H). 13 c NMR (101 mhz, chloroform-d) delta 172.02, 169.23, 168.05, 167.97, 167.28, 158.39, 157.90, 157.90, 157.90, 157.90, 157.90, 157.90, 157.90, 157.90, 157.90, 157.90, 157.90, 157.90, 157.90, 5292, 157.90, 67.00, 157.90, 157.90, 54.06, 53.43, 157.90, 49.06, 157.90, 157.90, 157.90, 33.10, 157.90, 157.90, 31.14, 30.03, 22.75. Fig. 3 is a nuclear magnetic resonance spectrum of the compound 2 of example 2, and fig. 4 is a nuclear magnetic resonance spectrum of the compound 2 of example 2.
Example 3
The preparation method of the BTK inhibitor is as follows;
1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) azetidine-3-aldehyde was designated as compound 2e, which has the following structural formula,
Figure BDA0003414628270000122
the specific preparation procedure of compound 2e was the same as that of compound 2d, starting from 1a and azetidin-3-ylmethanol hydrochloride, and was followed by two-step reaction to give compound 2e (175 mg, overall yield 59.00%).
5- (3- ((4- (2- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -2-oxoethyl) piperazin-1-yl) methyl) azetidin-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione is designated as compound 3, having the following structural formula,
Figure BDA0003414628270000131
the synthesis method of the compound 3 is the same as that of the compound 2, and the compound 2h and the compound 2e are taken as raw materials to obtain 18.35mg of a product compound 3, and the yield is 37.42%. The nuclear magnetic data are as follows: 1 H NMR(400MHz，Chloroform-d)δ8.40(s，1H)，7.66(d，J＝8.3Hz，2H)，7.49–7.35(m，3H)，7.22–7.13(m，4H)，7.09(d，J＝7.9Hz，2H)，6.58(d，J＝8.5Hz，1H)，5.08–4.88(m，2H)，4.76(d，J＝13.6Hz，1H)，4.43–4.35(m，2H)，4.33–4.24(m，1H)，3.98–3.90(m，2H)，3.34–3.17(m，3H)，3.02–2.67(m，6H)，2.65–2.51(m，6H)，2.45–2.21(m，3H)，2.17–2.06(m，3H)，2.00–1.78(m，2H). 13 c NMR (101 mhz, chloro form-d) delta 170.13, 167.83, 167.56, 166.83, 158.46, 157.80, 156.26, 151.26, 145.58, 143.67, 134.62, 133.75, 131.81, 129.90, 129.87, 127.72, 124.00, 119.46, 119.19, 119.04, 112.32, 61.08, 58.53, 56.94, 53.03, 52.80, 52.17, 48.92, 44.70, 31.39, 29.63, 27.50, 24.23, 22.62. Fig. 5 is a nuclear magnetic resonance spectrum of the compound 3 of example 3, and fig. 6 is a nuclear magnetic resonance spectrum of the compound 3 of example 3.
Example 4
The preparation method of the BTK inhibitor is as follows;
1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) piperidine-4-carbaldehyde was designated as compound 2f, which has the following structural formula,
Figure BDA0003414628270000132
the specific preparation steps of the compound 2f are the same as those of the compound 2d, and the compound 2a and 4-hydroxymethyl piperidine are taken as raw materials to obtain a product compound 2f (172 mg, total yield 62.17%) through two-step reaction.
4- (4- ((4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -2-oxoethyl) piperidin-1-yl) methyl) piperidine-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione is designated as compound 4, which has the following structural formula,
Figure BDA0003414628270000141
the synthesis method of the compound 4 is the same as that of the compound 2, and the compound 2h and the compound 2f are taken as raw materials to obtain a product compound 4 10mg, yield 34.48%. The nuclear magnetic data are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.41(s,1H),7.68–7.62(m,2H),7.60–7.54(m,1H),7.43–7.34(m,3H),7.20–7.13(m,4H),7.11–7.06(m,2H),5.08–4.95(m,2H),4.80–4.72(m,1H),4.36–4.25(m,1H),3.80–3.68(m,2H),3.34–3.16(m,3H),2.93–2.81(m,5H),2.81–2.69(m,1H),2.63–2.50(m,7H),2.44–2.34(m,2H),2.30–2.24(m,2H),2.15–2.06(m,4H),1.95–1.86(m,2H),1.75–1.64(m,1H),1.53–1.40(m,2H). 13 c NMR (101 MHz, chloride-d) delta 171.73, 168.91, 168.03, 167.43, 166.62, 158.42, 157.87, 156.26, 155.42, 153.78, 150.82, 143.70, 135.35, 134.07, 129.90, 129.87, 127.75, 123.97, 123.57, 119.44, 119.02, 117.03, 115.10, 98.49, 64.35, 61.25, 54.00, 53.49, 53.03, 49.07, 44.73, 41.17, 32.93, 31.44, 30.92, 29.62, 22.66. Fig. 7 is a nuclear magnetic resonance spectrum of the compound 4 of example 4, and fig. 8 is a nuclear magnetic resonance spectrum of the compound 4 of example 4.
Example 5
The preparation method of the BTK inhibitor is as follows;
1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) azetidine-3-al was designated as compound 2g, having the following structural formula,
Figure BDA0003414628270000142
the specific preparation procedure of 2g of the compound was the same as that of 2d, and 2a and azetidin-3-ylmethanol hydrochloride were used as raw materials to obtain 2g of the compound (180 mg, total yield 55.84%) as a product through two-step reaction.
4- (3- ((4- (2- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -2-oxoethyl) piperazin-1-yl) methyl) azetidin-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione is designated as compound 5, having the following structural formula,
Figure BDA0003414628270000151
the synthesis method of the compound 5 is the same as that of the compound 2, and the compound 2h and the compound 2g are used as raw materials to obtain the product compound 5.6 mg, and the yield is 23.12%. 1 H NMR(400MHz，Chloroform-d)δ8.40(s，1H)，7.66(d，J＝8.3Hz，2H)，7.49–7.35(m，3H)，7.22–7.13(m，4H)，7.09(d，J＝7.9Hz，2H)，6.58(d，J＝8.5Hz，1H)，5.08–4.88(m，2H)，4.76(d，J＝13.6Hz，1H)，4.43–4.35(m，2H)，4.33–4.24(m，1H)，3.98–3.90(m，2H)，3.34–3.17(m，3H)，3.02–2.67(m，6H)，2.65–2.51(m，6H)，2.45–2.21(m，3H)，2.17–2.06(m，3H)，2.00–1.78(m，2H). 13 C NMR (101 mhz, chloro form-d) delta 170.13, 167.83, 167.56, 166.83, 158.46, 157.80, 156.26, 151.26, 145.58, 143.67, 134.62, 133.75, 131.81, 129.90, 129.87, 127.72, 124.00, 119.46, 119.19, 119.04, 112.32, 61.08, 58.53, 56.94, 53.03, 52.80, 52.17, 48.92, 44.70, 31.39, 29.63, 27.50, 24.23, 22.62. Fig. 9 is a nuclear magnetic resonance spectrum of the compound 5 of example 5, and fig. 10 is a nuclear magnetic resonance spectrum of the compound 5 of example 5.
Example 6
The preparation reaction route of the BTK inhibitor of this example is as follows:
Figure BDA0003414628270000161
(6- (piperidine-4-ethynyl) pyridin-3-yl) methanolic hydrochloride was designated 3a, having the following structural formula,
Figure BDA0003414628270000162
the specific preparation steps of compound 3a are as follows:
2-chloro-5-hydroxymethylpyridine (100 mg, 691.76. Mu. Mol), 1-Boc-4-ethynyl piperidine (159.26 mg, 760.94. Mu. Mol) were dissolved in DMF and ditriphenylphosphine palladium dichloride (48.56 mg, 69.18. Mu. Mol), cuprous iodide (26.35 mg, 138.35. Mu. Mol), triethylamine (287.68. Mu.L, 2.08 mmol), N were added 2 Heating to 80deg.C under protectionThe reaction was carried out for 3 hours. After the reaction was completed, 10mL of ethyl acetate was added to the system, and the organic phase was washed with water (20 ml×3), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain an intermediate product. Under ice bath, the product is dissolved in 3mL of 4mol/L hydrochloric acid/dioxane solution, stirred until the reaction is complete, and filtered to obtain the product compound 3a.
6- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) piperidin-4-yl) ethynyl) nicotinaldehyde is designated as 3b and has the following structural formula,
Figure BDA0003414628270000163
the specific preparation steps of compound 3b are as follows:
compound 3a (50 mg, 197.06. Mu. Mol) and compound 1a (49.48 mg, 179.15. Mu. Mol) were dissolved in 1mL of DMF, DIPEA (91.4. Mu.L, 537.44. Mu. Mol) was added, the temperature was raised to 80℃for 2h, 5mL of ethyl acetate was added to the system after the reaction was completed, the organic phase was washed with water (10 mL. Times.3), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product. The above product was dissolved in 3mL of methylene chloride, and dessert-martin oxidant (80.62 mg, 190.08. Mu. Mol) was added under ice bath and stirred at room temperature until the reaction was complete. After the reaction was completed, 3mL 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 (0% -3% methanol/dichloromethane, V/V) to give product compound 3b (15 mg, yield 51.7%).
6- ((1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) piperidin-4-yl) ethynyl) nicotinaldehyde was designated as 3c and has the following structural formula,
Figure BDA0003414628270000171
the synthesis method of the compound 3c is the same as that of 3b, and 2a and 3a are used as raw materials, and the compound 3c (18 mg, total yield 31%) is obtained through two-step reaction.
5- (4- ((5- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) methyl) piperidin-2-yl) ethynyl) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione is designated as compound 6, having the following structural formula,
Figure BDA0003414628270000172
the synthesis method of the compound 6 is the same as that of the compound 2, and the compound 3b and the compound 1d are used as raw materials to obtain a product (8 mg, yield 30.76%). The nuclear magnetic data are as follows: 1 H NMR(400MHz，Chloroform-d)δ8.53–8.49(m，1H)，8.40(s，1H)，7.75–7.62(m，4H)，7.44–7.36(m，3H)，7.32(d，J＝2.3Hz，1H)，7.21–7.14(m，3H)，7.12–7.05(m，3H)，4.96(dd，J＝12.1，5.3Hz，1H)，4.86–4.78(m，1H)，3.82–3.71(m，1H)，3.60(s，2H)，3.44–3.31(m，2H)，3.06–2.96(m，2H)，2.94–2.70(m，4H)，2.51–2.39(m，2H)，2.36–2.24(m，2H)，2.20–2.12(m，1H)，2.09–2.00(m，3H)，1.97–1.87(m，4H). 13 c NMR (101 mhz, chloro form-d) delta 171.37, 168.62, 167.91, 167.19, 158.33, 157.76, 156.37, 155.24, 153.74, 150.27, 136.73, 134.32, 129.91, 129.88, 126.62, 125.39, 123.90, 119.40, 119.09, 118.94, 117.91, 108.72, 98.47, 59.60, 52.63, 49.09, 46.43, 31.43, 31.20, 30.44, 29.63, 27.20, 22.71. Fig. 11 is a nuclear magnetic resonance spectrum of the compound 6 of example 6, and fig. 12 is a nuclear magnetic resonance spectrum of the compound 6 of example 6.
Example 7
The preparation method of the BTK inhibitor is as follows;
4- (4- ((5- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) methyl) piperidin-2-yl) ethynyl) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione is designated as compound 7, having the following structural formula,
Figure BDA0003414628270000181
the synthesis method of the compound 7 is the same as that of the compound 2, and the compound 3c and the compound 1d are used as raw materials to obtain a product (6.8 mg, yield 26.79%). The nuclear magnetic data are as follows: 1 H NMR(400MHz，Chloroform-d)δ8.55–8.48(m，1H)，8.40(s，1H)，7.73–7.68(m，1H)，7.68–7.64(m，2H)，7.61–7.57(m，1H)，7.43–7.37(m，4H)，7.24–7.14(m，4H)，7.12–7.07(m，2H)，5.04–4.94(m，1H)，4.86–4.76(m，1H)，3.66–3.61(m，1H)，3.60(s，2H)，3.31–3.22(m，2H)，3.06–3.00(m，2H)，2.99–2.91(m，1H)，2.90–2.87(m，1H)，2.86–2.80(m，1H)，2.79–2.73(m，1H)，2.51–2.40(m，2H)，2.36–2.26(m，2H)，2.18–2.11(m，3H)，2.07–1.99(m，5H). 13 c NMR (101 mhz, chloro form-d) delta 171.41, 168.63, 167.36, 166.53, 158.32, 157.78, 156.37, 155.25, 153.74, 150.66, 150.22, 143.48, 142.29, 136.69, 135.43, 134.08, 129.91, 129.88, 127.93, 126.62, 123.89, 123.60, 119.39, 119.09, 117.33, 115.43, 98.47, 92.04, 81.67, 59.65, 54.30, 52.65, 49.93, 49.09, 31.43, 31.22, 29.63, 26.94, 22.65. FIG. 13 is a nuclear magnetic resonance spectrum of the compound 7 of example 7, and FIG. 14 is a nuclear magnetic resonance spectrum of the compound 7 of example 7.
Comparative example 1
The structure of the BTK inhibitor of this example is as follows; the structure of MT802 is shown below, with specific synthetic method reference Biochemistry,2018, 57 (26): 3564-3575;
Figure BDA0003414628270000182
comparative example 2
The structure of the BTK inhibitor of this example is as follows; ibutinib has the structure shown below and is purchased from ambroxol;
Figure BDA0003414628270000191
performance testing
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; CCK8 was purchased from GLPBIO, GK10001.
1. Test of degradation agent for BTK Activity in Ramos cells and Mino cells
Cell culture: ramos (human Burkitt's lymphoma cells) cell culture medium was RMPI1640+10% FBS+1% penicillin-streptomycin solution; 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.
And (3) paving: in a 6-well plate, 2X 10 wells are added per well 6 After plating of individual cells, solutions of the test compounds of different concentrations were added to each well at 37℃with 5% CO 2 Culturing in incubator for 24 hr.
Preparing a protein sample: after the culture, collecting cells, adding RIPA lysate containing 1% broad-spectrum protease inhibitor, phosphatase inhibitor and PMSF, performing lysis on ice for 15min, centrifuging at 13000 Xg and 4 ℃ for 10min, collecting a supernatant protein sample, quantifying protein by using a BCA kit, preparing a mixed solution with the diluted protein sample and 1 Xloading Buffer at a concentration of 1mg/mL, heating and denaturing for 5min at 100 ℃, and preserving at 4 ℃.
Western Blot experiments:
1) And (3) glue preparation: SDS-PAGE gels of different concentrations were prepared.
2) Loading: the prepared protein samples were added to SDS-PAGE gel loading wells at 10. Mu.L/well.
3) Electrophoresis: the voltage of the protein sample in the concentrated gel is 80V, after the protein enters the separation gel, the voltage is adjusted to 120V to continue electrophoresis, and when bromophenol blue reaches the bottom of the separation gel, electrophoresis is stopped.
4) Transferring: and (3) completely placing the gel after electrophoresis in a glass vessel containing electric transfer liquid, and mounting filter paper, PVDF membrane and gel in a transfer membrane clamp according to the positive electrode-sponge-double-layer filter paper-PVDF membrane-gel-double-layer filter paper-sponge-negative electrode, and transferring the membrane for 75min under constant current of 300mA in an ice bath.
5) Closing: after the film transfer is completed, the PVDF film is taken out, placed into 5% skim milk powder sealing liquid, placed into a shaking table (70 rpm) and sealed for 90min at room temperature.
6) Incubation resistance: after the end of the blocking, the PVDF membrane was washed 5min X5 times with TBST, the primary antibody diluted in a certain proportion was added, and incubated overnight at 4 ℃.
7) Secondary antibody incubation: after the primary antibody incubation is finished, the primary antibody is sucked away, the PVDF membrane is washed for 5min multiplied by 5 times by TBST liquid, and the secondary antibody diluted in proportion is added according to the species of the primary antibody (rabbit antibody or mouse antibody) and incubated for 1h at room temperature.
8) Developing: after the secondary antibody incubation was completed, the secondary antibody was aspirated, and the PVDF membrane was washed 5min×5 times with TBST. And (3) during color development, uniformly coating ECL developer on the PVDF film, and placing the PVDF film in an imaging analysis system for development and photographing.
And (3) data processing: and processing a picture obtained by a Western Blot experiment by using Image J software, calculating a gray value, and calculating the relative abundance of the BTK through the gray values of the BTK and the internal reference. And calculating DC of the compound in GraphPad Prism 7 software according to the concentration of the compound and the relative abundance of BTK 50 . Table 1 shows the results of the test for the activity of compounds 1-7 in Ramos and Mino cells for degradation of BTK. "DC 50 "means the dose at which 50% of the protein is degraded.
Table 1: examples 1-7 and comparative examples 1-2 results of the test for the degradation of BTK Activity in Ramos and Mino cells
Figure BDA0003414628270000201
The test results in Table 1 show that the nitrogen-containing ring derivatives of examples 1 to 7 are BTK degrading agents as Linker capable of efficiently degrading BTK protein in Ramos cells or Mino cells, and are superior in effect to MT802 of comparative example 1 and Ibrutinib of comparative example 2.
2. Compound degradation BTK C481S activity assay
Cell culture: hela cells (cervical cancer cells) were cultured in DMEM+10% FBS+1% penicillin-streptomycin solution at 37deg.C, 5% CO 2 Cultivation under conditionsAnd (5) nourishing.
Expression of BTK C481S protein:
1) Plasmid extraction: mu.L of a bacterial solution of Escherichia coli containing BTK C481S plasmid (purchased from Changsha Youze Biotechnology Co., ltd.) was added to 5mL of a prepared sterile LB liquid medium (containing 0.5% kanamycin), and the mixture was placed on a constant temperature shaker, and shaking was carried out at 37℃and 220rpm for 16 hours, after the medium was cloudy, 1.0-5.0mL of the bacterial solution was placed in an EP tube, 10kg was centrifuged for 1min, and the supernatant was discarded. Using
Figure BDA0003414628270000211
Plasmid Mini Kit I plasmids were extracted and plasmid concentrations were determined.
2) And (3) paving: in a 6-well plate, 1X 10 wells are added per well 6 Individual cells were placed in an incubator (37 ℃,5% co) 2 ) Culturing until the cells adhere to the wall.
3) Transfection: after the cells were attached, the medium was changed to Opti-MEM medium, and the prepared plasmid/Lipo 3000 mixed solution (2. Mu.g plasmid/well) was added and placed in an incubator for culturing for 6 hours. After transfection, the medium was changed to DMEM complete medium, and the culture was continued for 96 hours with different concentrations of compound.
After the completion of the culture, proteins were extracted as in test example 1, western Blot experiments were performed to test DCs of the compounds 50 Method the degradation agent in performance test 1 was tested for BTK degradation activity in Ramos cells and Mino cells. Table 2 shows the results of the activity test of examples 2-5 and comparative examples 1-2 on degradation of BTK C481S. "DC 50 "means the dose at which 50% of the protein is degraded.
Table 2: results of the test for the Activity of examples 2-5 and comparative examples 1-2 at degradation of BTK C481S
Compounds of formula (I) DC 50 (nM)
Example 2 116.6
Example 3 34.48
Example 4 75.95
Example 5 117.7
Comparative example 1 333.7
Comparative example 2 >1000
The above results show that the nitrogen-containing ring derivatives of examples 2-5 prepared in the present application are BTK degrading agents as Linker capable of efficiently degrading the C481S mutated BTK protein in Hela cells, and the effect is superior to that of ibutinib and MT802 of the comparative example.
3. Compounds inhibition of Mino cell proliferation activity assay
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.
And (3) paving: in a 96-well plate, 50. Mu.L of cell suspension (10000 cells) was added to each well, and the mixture was placed in an incubator for overnight culture. Adding the medicine: the next day, 50 μl of compounds of different concentrations were added to each well, 3 duplicate wells were set for each concentration, 3 zeroing wells and 3 blank wells were additionally set, and after dosing, 96 well plates were placed in the incubator for 72h.
Cell viability assay: after 72h, 10 μl CCK8 was added to each well and incubation was continued in an incubator for 4h. After 4 hours, absorbance was measured using an enzyme-labeled instrument.
And (3) data processing: experimental data were processed using Graphpad Prism 8 software to calculate IC's for compounds to inhibit cell proliferation 50 Values. Table 3 shows the results of the test for inhibiting Mino cell proliferation activity of examples 2-5 and comparative examples 1-2.
Table 3: test results of examples 2-5 and comparative examples 1-2 for inhibiting Mino cell proliferation Activity
Compounds of formula (I) IC 50 (nM)
Example 2 9.092
Example 3 0.9438
Example 4 10.42
Example 5 6.047
Comparative example 1 4971
Comparative example 2 5065
The test results in table 3 show that the nitrogenous ring derivative is a BTK degradation agent serving as a Linker, can significantly inhibit proliferation of human mantle cell lymphoma cells Mino, and has better effect than that of MT802 and ibutinib of comparative examples.
The foregoing examples are illustrative of the present invention and are not intended to be limiting, but rather, the invention is intended to be limited to the specific embodiments shown, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the invention are intended to be equivalent substitutes and modifications within the scope of the invention.

Claims (10)

1. A compound of formula (i) or a pharmaceutically acceptable salt thereof;
Figure QLYQS_1
（Ⅰ）；
in formula (I), L is selected from
Figure QLYQS_2
、/>
Figure QLYQS_3
、/>
Figure QLYQS_4
、/>
Figure QLYQS_5
。
2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: the compounds include the structures shown below:
Figure QLYQS_6
（1）、/>
Figure QLYQS_7
（2）、
Figure QLYQS_8
（3）、/>
Figure QLYQS_9
（4）、
Figure QLYQS_10
（5）、/>
Figure QLYQS_11
（6）、
Figure QLYQS_12
（7）。
3. a process for the preparation of a compound characterized by: the method comprises the following steps: mixing a compound shown in a formula (II) with a compound shown in a formula (III), boc-piperazine and 6-halogenated nicotinic acid tert-butyl ester, and reacting to obtain a compound shown in a formula (1);
Figure QLYQS_13
（Ⅱ）；/>
Figure QLYQS_14
（Ⅲ）；
in the formula (II), X is selected from halogen atoms;
Figure QLYQS_15
（1）。
4. a process for the preparation of a compound characterized by: the method comprises the following steps: mixing a compound shown in a formula (III), a compound shown in a formula (IV) and a compound shown in a formula (V) with 4-piperidinemethanol or azetidin-3-yl methanol, and reacting to obtain the compound shown in the formula (2) -formula (5);
Figure QLYQS_16
（Ⅲ）；/>
Figure QLYQS_17
（Ⅳ）；/>
Figure QLYQS_18
（Ⅴ）；
in the formula (IV), X is selected from halogen atoms;
Figure QLYQS_19
（2）、/>
Figure QLYQS_20
（3）、
Figure QLYQS_21
（4）、/>
Figure QLYQS_22
（5）。
5. a process for the preparation of a compound characterized by: the method comprises the following steps: mixing a compound shown in a formula (III), a compound shown in a formula (IV) and a compound shown in a formula (VI) to react to obtain the compounds shown in the formulas (6) - (7);
Figure QLYQS_23
（Ⅲ）；/>
Figure QLYQS_24
（Ⅳ）；/>
Figure QLYQS_25
（Ⅵ）；
in the formula (IV), X is selected from halogen atoms;
Figure QLYQS_26
（6）、/>
Figure QLYQS_27
（7）。
6. a pharmaceutical composition characterized by: the pharmaceutical composition comprising a compound according to any one of claims 1-2 or a pharmaceutically acceptable salt thereof.
7. Use of a compound according to any one of claims 1-2, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prophylaxis and/or delay of progression and/or co-treatment of a disease associated with BTK activity or expression level.
8. The use according to claim 7, characterized in that: the disease is tumor or autoimmune disease.
9. The use according to claim 8, characterized in that: the tumors include non-hodgkin lymphoma, chronic lymphocytic leukemia, B-cell lymphoma, mantle cell lymphoma.
10. The use according to claim 8, characterized in that: the autoimmune disease is rheumatoid arthritis or psoriasis.
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