CN111171035B

CN111171035B - Preparation method and application of 4-phenoxyphenyl pyrazolopyrimidine amide derivative

Info

Publication number: CN111171035B
Application number: CN201911101531.9A
Authority: CN
Inventors: 赵桂森; 禚慧君; 杨庆滔; 景永奎; 冉凡胜; 包宇
Original assignee: Shandong University
Current assignee: Shandong Kangruijian Medical Technology Co ltd
Priority date: 2018-11-13
Filing date: 2019-11-12
Publication date: 2021-03-30
Anticipated expiration: 2039-11-12
Also published as: CN111171035A

Abstract

The invention provides a preparation method and application of a 4-phenoxyl phenyl pyrazolopyrimidine amide derivative. The compounds have a structure shown in a formula (I):

wherein X, Y is two linking groups, X is selected from benzyl, substituted benzyl, piperidyl, C_1～6A linear or branched alkyl group; y is- (CH)₂)_n-, n is any integer selected from 0 to 4; r is selected from hydroxyl, hydroxylamino and o-phenylenediamine; the structure of the formula (I) comprises a racemate and stereoisomers thereof. The compounds have BTK/HDAC double-target inhibition effect and growth inhibition activity on T cell leukemia cells and mantle cell lymphoma cells, and can be used for preparing antitumor drugs.

Description

Preparation method and application of 4-phenoxyphenyl pyrazolopyrimidine amide derivative

Technical Field

The invention relates to the field of organic compound synthesis and medical application, in particular to a preparation method and application of a 4-phenoxyl phenyl pyrazolopyrimidine amide derivative.

Background

Mantle Cell Lymphoma (MCL) is a highly malignant, poorly prognostic Non-Hodgkin lymphoma (NHL) characterized by a t (11; 14) (q 13; q32) chromosomal translocation leading to cyclin D1 overexpression. MCL is clinically very aggressive, and is not a curative lymphoma so far, and there is no standard first-line chemotherapy scheme at present, and compared with other B cell lymphomas, MCL is difficult to maintain long-term effect by chemotherapy. (see: Zhou Y, Wang H, Fang W, Romaguer JE, Zhang Y, Delalalle KB, Kwak L, Yi Q, Du XL, Wang M: index tresnds of mantle cell lymphoma in the United States between 1992and 2004 Cancer 2008,113(4):791.) in recent years, molecular targeted therapies have advanced to some extent, including mammalian target of rapamycin (mTOR) inhibitors, Brutons Type Kinase (BTK) inhibitors, Histone Deacylase (HDAC) inhibitors and cyclin-dependent kinase (CDK) inhibitors, with the advancement of the study of MCL pathogenesis. BTK is a regulator downstream of the B cell receptor signaling pathway and plays a crucial role in B cell development and maturation, and aberrant expression and activation of BTK is ubiquitous in MCL cells. (wine ES, Ingham RR, Castillo JJ: PCI-32765: a novel Bruton' S tyrosine kinase inhibitor for the treatment of lysine malignneces. expert Opinion on Investigational Drugs 2012,21(3):355-361.) Ibrutinib (Ibrutinib, IBN) was the first marketed BTK inhibitor to show clinically good therapeutic effect on MCL, FDA approved for the treatment of relapsing refractory MCL (Wang ML, Rule S, Martin P, Goy A, Auer R, Kahl BS, Jurczak W, Advani JERH, Roguera, Williams ME et al: Targetg K with irth mutant in fragment or fragment reductase RH, Medhuama et al shows a more gradual response to cysteine kinase, 507: 6. The III mutation of cysteine kinase inhibitor for patients with more extensive mutation of cysteine kinase-III, 507: 6. Ibrutinib kinase inhibitor for the treatment of hepatitis B + T-beta-alanine kinase, III expressed by the clinical effect of cysteine kinase inhibitors in vitro kinase, I-III-beta-alanine, how to increase the overall response rate and improve drug resistance of this part of patients is a hot spot of the current study on MCL treatment. (Woyach JA, Furman RR, Liu TM, Ozer HG, Zapatka M, Ruppert AS, Xue L, Li HH, Steggerda SM, Versel M: Resistance Mechanisms for the Bruton's disease Kinase Inhibitor Ibritininib. New England Journal of Medicine 2014 (24):2286-2294.) the abnormal expression and mutation of HDAC is closely related to the development and development of tumors, and the high expression of HDAC in lymphoma leads to histone hypoacetylation, therefore, inhibition of HDAC can block the cell cycle, thereby inducing apoptosis. (Noel MS, Friedberg JW, Barr PM: Novel Agents in Manual Cell lymphoma. best Practice & Research Clinical Haematology 2012,25(2):191-200.) Vorinostat (SAHA) is the first HDAC inhibitor to be marketed and is approved by the FDA for the treatment of cutaneous T-Cell lymphoma. Clinical experiments show that the treatment of the relapse-refractory MCL by using the vorinostat shows a certain treatment effect, but the effect of a single medicine is limited. (Kirschbium MH, Goldman BH, Zain JM, Cook JR, Rimsza LM, Forman SJ, Fisher RI: A Phase 2Study of Vorinostat for Treatment of delayed or Refractory Hodgkin Lymphoma: SWOG S0517.Leukemia & Lymphoma 2012, 53(2):259 and 262.) currently, MCL patients are treated clinically by combination therapy. The combination of HDAC inhibitors and BTK inhibitors has been reported to have a synergistic effect on MCL cell growth inhibition by inhibiting the NF- κ B pathway and down-regulating cyclin D1. (Hagiwara K, Kunishima S, Iida H, Miyata Y, Naoe T, Nagai H: The synergistic effect of BCR signaling inhibition combined with an HDAC inhibition on cell death in a mantle cell lymphoma cell line. Apoptosis 2015,20(7):975 + 985.) compared with combined administration, single molecule dual target drugs can solve The problems of drug interaction, poor patient compliance, etc. Therefore, designing and synthesizing compounds with double BTK/HDAC inhibition effects has important significance for improving MCL treatment.

Disclosure of Invention

The invention aims to provide a pyrazolopyrimidine compound and a preparation method and application thereof. The compound has certain BTK/HDAC double-target inhibition effect and growth inhibition activity on T cell leukemia Jurkat cells and mantle cell lymphoma Jeko-1 cells, and can be used for preparing antitumor drugs. The experimental data show that the compounds of the invention all show stronger inhibitory activity to BTK (the inhibitory rate is more than 80%) under the concentration of 1 mu M, and the compounds of the invention still show certain inhibitory activity to BTK under the concentration of 50nM, and more compounds have equivalent BTK inhibitory activity to IBN. Also, certain compounds of the invention exhibit potent inhibitory activity (IC) against HDAC₅₀Less than 1 μ M). And, the compounds of the invention are useful against T cell leukemia Jurkat cellsAlso has strong growth inhibitory activity. And, the compound of the present invention shows a strong antiproliferative activity against mantle cell lymphoma cells Jeko-1.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a compound having the structure according to formula (I):

wherein X, Y is two linking groups, X is selected from benzyl, substituted benzyl, piperidyl, C_1～6A linear or branched alkyl group; y is- (CH)₂)_n-, n is any integer selected from 0 to 4; r is selected from hydroxyl, hydroxylamino and o-phenylenediamine; the structure of the formula (I) comprises a racemate thereof and stereoisomers (R-configuration and S-configuration) thereof.

Preferably, Y is- (CH)₂)_n-, n is 0 or 1.

Preferably, Y is- (CH)₂)_nAnd when n is 0, X is benzyl or substituted benzyl.

Preferably, Y is- (CH)₂)_n-when n is 1, X is benzyl, substituted benzyl or piperidinyl;

as mentioned above, when X is benzyl or substituted benzyl, X is

a. b represents a linking position, wherein position b is linked to Y; r₁Selected from hydrogen, nitro, halogen and C_1～6Linear or branched alkoxy.

Wherein X is

When R is₁In the substitution position of

Preferably, X isFrom

Any one of (a); preferably, it is

Preferably, X is selected from

Preferably, X is

R₁When is H, X is selected from

Preferably, X is

R₁Is nitro, halogen or C_1～6When the alkoxy group is linear or branched, X is selected from

Preferably, X is

R₁Is nitro or C_1～6When the alkoxy group is linear or branched, X is

Preferably, R₁Is C_1～6Straight chain orWhen the alkoxy group is a branched chain, the alkoxy group is selected from the group consisting of methoxy, ethoxy, propoxy, and butoxy, preferably methoxy.

Preferably, R₁When halogen, the halogen is selected from F, Cl, Br and I, preferably Cl or Br.

When X is piperidinyl, X is selected from

a. b represents the attachment position, wherein the b position is attached to Y.

Wherein the content of the first and second substances,

are isomers of each other,

is racemic.

The invention discovers that when X is piperidyl and the two connection positions of a and b are in para-position, namely X is

However, such compounds do not achieve the technical effects of the present invention.

As noted above, the compounds of the present invention comprise the following structure:

wherein Y is- (CH)₂)_n-n is 0 or 1; r is selected from hydroxyl, hydroxylamino and o-phenylenediamine; r₁Selected from hydrogen, nitro, halogen and methoxy.

Preferably, in the structure of formula (II), according to R₁Formula (II) comprises the following structure:

Preferably, in the structure of formula (II), formula (II) includes the following structure according to the position of attachment of Y to the benzene ring:

Preferably, according to the above, the present invention comprises the following compounds:

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxybenzamide (compound Y1)

3- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxybenzamide (compound Y2)

2- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxybenzamide (compound Y3)

2- (4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N-hydroxyacetamide (compound Y4)

2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N-hydroxyacetamide (compound Y5)

(R)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N-hydroxyacetamide (compound Y6)

(S)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N-hydroxyacetamide (compound Y7)

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -3-bromo-N-hydroxybenzamide (compound Y8)

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -2-bromo-N-hydroxybenzamide (compound Y9)

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -3-chloro-N-hydroxybenzamide (compound Y10)

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxy-3-nitrobenzamide (compound Y11)

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxy-3-methoxybenzamide (compound Y12)

4- (2- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) acetamido) -N-hydroxybenzamide (compound Y13)

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) benzoic acid (compound Y14)

3- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) benzoic acid (compound Y15)

2- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) benzoic acid (compound Y16)

(4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) acetic acid (compound Y17)

2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) acetic acid (compound Y18)

(R)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) acetic acid (compound Y19)

(S)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) acetic acid (compound Y20)

4- (2- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) acetamido) benzoic acid (compound Y21)

(4- (2- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) acetylamino) phenyl) acetic acid (compound Y22)

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide (compound Y23)

3- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide (compound Y24)

2- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide (compound Y25)

2- (4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N- (2-aminophenyl) acetamide (compound Y26)

2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide (compound Y27)

(R)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide (Compound Y28)

(S)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide (compound Y29).

In a second aspect of the invention, the invention provides a process for the preparation of the above compound, which comprises preparing a compound of formula (I) starting from intermediate 1 by the following reaction scheme:

wherein X, Y, R is as defined above, R₂Is hydroxyl or halogen, preferably hydroxyl or Br.

Preferably, the method of the present invention comprises the steps of: taking the intermediate 1 as a starting material, and carrying out substitution reaction with N-bromosuccinimide in DMF under the heating condition to obtain an intermediate 2; the intermediate 2and 4-phenoxyphenylboronic acid are subjected to Suzuki reaction under the catalysis of tetrakis (triphenylphosphine) palladium to obtain an intermediate 3; intermediate 3 and

nucleophilic substitution reaction is carried out under alkaline condition to generate an intermediate 4; the intermediate 4 is subjected to ammonolysis reaction under alkaline conditions to obtain a compound shown in the formula (I), or the intermediate 4 is subjected to hydrolysis reaction under alkaline conditions to obtain a compound shown in the formula (I), or the intermediate 4 is hydrolyzed under alkaline conditions and then reacts with o-phenylenediamine to obtain a compound shown in the formula (I);

In a more specific embodiment of the invention, the compounds of the invention may be prepared by:

reagents and conditions in the reaction scheme: (i) NBS, DMF,80 ℃; (ii) 4-phenoxyphenylboronic acid, tetrakis (triphenylphosphine) palladium, potassium phosphate trihydrate, 1, 4-dioxane, 135 ℃; (iii) k₂CO₃KI, DMF, room temperature; (iv) hydroxylamine hydrochloride, KOH, anhydrous methanol, room temperature; (v) NaOH, methanol, 60 ℃; (vi) o-phenylenediamine, HBTU, Et₃N, anhydrous DMF.

According to the reaction route, taking the intermediate 1 as a starting material, and carrying out substitution reaction with N-bromosuccinimide in DMF under the heating condition to obtain an intermediate 2; the intermediate 2and 4-phenoxyphenylboronic acid are subjected to Suzuki reaction under the catalysis of tetrakis (triphenylphosphine) palladium to obtain an intermediate 3; intermediate 3 with the different intermediates (a: methyl 4- (bromomethyl) benzoate, b: methyl 3- (bromomethyl) benzoate, c: methyl 2- (bromomethyl) benzoate, d: methyl 2- (4- (bromomethyl) phenyl) acetate, e: methyl 3-hydroxypiperidine-1-carboxylate, f (S) -methyl 3-hydroxypiperidine-1-carboxylate, g (R) -methyl 3-hydroxypiperidine-1-carboxylate, h: methyl 3-bromo-4- (bromomethyl) benzoate, i: methyl 2-bromo-4- (bromomethyl) benzoate, j: methyl 3-chloro-4- (bromomethyl) benzoate, k: methyl 3-nitro-4- (bromomethyl) benzoate, l is methyl 3-methoxy-4- (bromomethyl) benzoate, m is methyl 4- (2-bromoacetamido) benzoate, n is methyl 2- (4- (2-bromoacetamido) phenyl) acetate, the structures of intermediates a-n are taken from the following text), and nucleophilic substitution reaction is carried out under alkaline conditions to generate an intermediate 4; intermediate 4 under basic conditions: the preparation method comprises the following steps of carrying out ammonolysis reaction to obtain a hydroximic acid derivative (a compound Y1-Y13), or carrying out hydrolysis reaction to obtain a carboxylic acid derivative (a compound Y14-Y22), or carrying out hydrolysis reaction to obtain a carboxylic acid derivative (a compound Y14-Y20), and then carrying out reaction with o-phenylenediamine to obtain an amide derivative (a compound Y23-Y29).

Preferably, in the above method, the preparation method of the hydroximic acid derivative (compound Y1-Y13) comprises the following steps:

(1) dissolving the intermediate 1 serving as a starting raw material in DMF, adding N-bromosuccinimide, stirring, heating in an oil bath at 80 ℃, changing the color of the solution from light yellow to red, reacting for 3 hours, detecting by TLC (thin layer chromatography), basically completely reacting, cooling to room temperature, pouring the reaction solution into ice water, stirring, precipitating a large amount of yellow solid, filtering, washing a filter cake with ice water, and drying to obtain an intermediate 2.

(2) Taking intermediate 2, 4-phenoxyphenylboronic acid, tetrakis (triphenylphosphine) palladium and K₃PO₄.3H₂And O, adding the mixture into a 250ml two-necked bottle, adding a solvent (1, 4-dioxane: water-4: 1) for dissolving, protecting with nitrogen, removing oxygen in the solution by ultrasonic, heating and refluxing the solution by using air in a nitrogen displacement device and an oil bath at 135 ℃ in an oil bath, reacting for 30 hours, detecting by TLC (thin layer chromatography), and basically completing the reaction. The reaction solution was cooled to room temperature, filtered through celite, and the filtrate was evaporated under reduced pressure to give a yellow solid, which was subjected to silica gel column chromatography (dichloromethane: methanol: 100:1) to give a pale intermediate 3.

(3) Taking intermediate 3, intermediates a-m and K₂CO₃KI was added to a 50ml reaction flask, and 15ml DMF was added to the flask to dissolve and stir the mixture, and the mixture was reacted at room temperature for 8 hours, and the reaction was completed by TLC. The reaction solution was poured into 100ml of ice water, stirred to precipitate a white solid, ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 200:1) to obtain an intermediate 4a to 4 m.

(4) Dissolving potassium hydroxide in absolute methanol, and performing ultrasonic assisted dissolution to obtain a solution a. And dissolving hydroxylamine hydrochloride in absolute methanol, and performing ultrasonic assisted dissolution and ice bath to obtain a solution b. Adding the solution a into a constant-pressure dropping funnel, slowly dropping into the solution b, reacting for 3 hours under ice bath condition, filtering precipitate (KCl) to retain filtrate, and drying the filtrate for 6 hours by using anhydrous sodium sulfate. And adding the filtrate into a reaction bottle, adding the intermediate 4a-4m, reacting for one hour, clarifying the solution, and detecting by TLC (thin layer chromatography) until the reaction is almost complete after continuing to react for 15 minutes. Evaporating most of solvent under reduced pressure, adding water, adjusting pH to 5-6 with 1M hydrochloric acid, precipitating solid, filtering, washing with ethyl acetate twice, recrystallizing the filter cake with methanol, and drying to obtain hydroximic acid derivative (compound Y1-Y13).

The synthetic route is as follows:

reagents and conditions in the reaction scheme: (i) NBS, DMF,80 ℃; (ii) 4-phenoxyphenylboronic acid, tetrakis (triphenylphosphine) palladium, potassium phosphate trihydrate, 1, 4-dioxane, 135 ℃; (iii) k₂CO₃KI, DMF, room temperature; (iv) hydroxylamine hydrochloride, KOH, anhydrous methanol, room temperature.

Preferably, in the above method, the preparation method of the carboxylic acid derivative (compound Y14-Y22) comprises the following steps:

(2) Taking intermediate 2, 4-phenoxyphenylboronic acid, tetrakis (triphenylphosphine) palladium and K₃PO₄.3H₂And O, adding the mixture into a 250ml two-necked bottle, adding a solvent (1, 4-dioxane: water-4: 1) for dissolving, protecting with nitrogen, removing oxygen in the solution by ultrasonic, heating and refluxing the solution by using air in a nitrogen displacement device and an oil bath at 135 ℃ in an oil bath, reacting for 30 hours, detecting by TLC (thin layer chromatography), and basically completing the reaction. Cooling the reaction solution to room temperature, filtering with diatomite, and evaporating the filtrate under reduced pressure to obtain yellowThe solid was chromatographed on silica gel (dichloromethane: methanol: 100:1) to give the pale intermediate 3.

(3) Taking intermediate 3, intermediates a-g, m-n and K₂CO₃KI was added to a 50ml reaction flask, and 15ml DMF was added to the flask to dissolve and stir the mixture, and the mixture was reacted at room temperature for 8 hours, and the reaction was completed by TLC. The reaction mixture was poured into 100ml of ice water, stirred to precipitate a white solid, ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 200:1) to obtain intermediates 4a to 4g and 4m to 4 n.

(4) And adding 4a-4g of intermediate and 4M-4n into a 50ml reaction bottle, adding methanol for dissolving, adding 3M NaOH, heating for reacting for 8 hours at 60 ℃ in an oil bath, and detecting by TLC to basically complete the reaction. The solvent was evaporated under reduced pressure, water was added, pH was adjusted to 5-6 with 1M hydrochloric acid, a white solid was precipitated, filtered, washed twice with ice water, the filter cake was recrystallized from methanol, and dried to obtain a carboxylic acid derivative (compound Y14-Y22).

The synthetic route is as follows:

reagents and conditions in the reaction scheme: (i) NBS, DMF,80 ℃; (ii) 4-phenoxyphenylboronic acid, tetrakis (triphenylphosphine) palladium, potassium phosphate trihydrate, 1, 4-dioxane, 135 ℃; (iii) k₂CO₃KI, DMF, room temperature; (v) NaOH, methanol, 60 ℃.

Preferably, the preparation method of the amide derivative (compound Y23-Y29) in the above method comprises the following steps:

(3) Taking intermediate 3, intermediates a-g and K₂CO₃KI was added to a 50ml reaction flask, and 15ml DMF was added to the flask to dissolve and stir the mixture, and the mixture was reacted at room temperature for 8 hours, and the reaction was completed by TLC. The reaction solution was poured into 100ml of ice water, stirred, a white solid was precipitated, ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 200:1) to obtain intermediate 4a to 4 g.

(4) And adding 4a-4g of the intermediate into a 50ml reaction bottle, adding methanol for dissolving, adding 3M NaOH, heating in an oil bath at 60 ℃ for reacting for 8 hours, and detecting by TLC to basically complete the reaction. The solvent was evaporated under reduced pressure, water was added, pH was adjusted to 5-6 with 1M hydrochloric acid, a white solid was precipitated, filtered, washed twice with ice water, the filter cake was recrystallized from methanol, and dried to obtain a carboxylic acid derivative (compound Y14-Y20).

(5) And (3) adding a carboxylic acid derivative (compound Y14-Y20), TBTU and triethylamine into a 50ml reaction bottle, adding anhydrous DMF, stirring to dissolve, activating for 30 minutes under an ice bath condition, adding o-phenylenediamine, reacting overnight at room temperature, and detecting by TLC to basically complete the reaction. Pouring the reaction solution into ice water, separating out gray solid, adding ethyl acetate for extraction three times, combining organic phases, and saturating NH₄Washing with Cl, washing with saturated NaCl, removing the solvent by evaporation under reduced pressure, and performing silica gel column chromatography (dichloromethane: methanol 300:1) to obtain an amide derivative (compound Y23-Y29).

The synthetic route is as follows:

reagents and conditions: (i) NBS, DMF,80 ℃; (ii) 4-phenoxyphenylboronic acid, tetrakis (triphenylphosphine) palladium, potassium phosphate trihydrate, 1, 4-dioxane, 135 ℃; (iii) k₂CO₃KI, DMF, room temperature; (v) NaOH, methanol, 60 ℃; (vi) o-phenylenediamine, TBTU, Et₃N, anhydrous DMF, room temperature.

In a third aspect of the invention, the invention also provides a pharmaceutical composition comprising a compound as described above or a pharmaceutically acceptable salt thereof.

Pharmaceutical compositions of the compounds of the present invention may be administered in any manner selected from: oral, aerosol inhalation, rectal, nasal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, with oral, intramuscular, intraperitoneal or intravenous administration being preferred.

In a fourth aspect of the present invention, the present invention also provides a pharmaceutical preparation, which comprises the above compound or a pharmaceutically acceptable salt thereof or a composition containing the above compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant and/or carrier.

The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or mixed suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.

The pharmaceutical combination or pharmaceutical preparation of the present invention may further comprise a conventional carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, typically about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.

Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.

The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.

Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.

For parenteral administration, liquid dosage forms are generally prepared from the compound and a sterile carrier. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.

It will be appreciated that the optimum dosage and interval for administration of a compound of the invention will be determined by the nature of the compound and external conditions, such as the form, route and condition of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a nominal period of time, may be determined by methods known in the art.

In a fifth aspect of the invention, the invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof or a composition containing the above compound in the preparation of a BTK inhibitor medicament.

In a sixth aspect, the invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof or a composition containing the above compound in the manufacture of a medicament for inhibiting HDAC.

In a seventh aspect of the present invention, the present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof or a composition containing the above compound in the preparation of an antitumor drug.

Preferably, the tumor is a lymphoma or other hematological tumor including leukemia, myeloma, and the like.

In a seventh aspect of the invention, the invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof or a composition containing the above compound in the preparation of a medicament for treating lymphoma or leukemia.

The experimental data show that the compounds all show stronger inhibition activity (the inhibition rate is more than 80%) to BTK under the concentration of 1 mu M, and the compounds Y1, Y5-Y12, Y17-Y18, Y23 and Y27-Y29 still show certain inhibition activity to BTK under the concentration of 50nM, and the compounds Y5-Y6, Y27-Y28 and IBN have equivalent BTK inhibition activity. Compounds Y1, Y4 and Y23 showed strong inhibitory activity (IC) against HDAC₅₀Less than 1 μ M). The compounds Y1-Y4, Y8-Y13 and Y23-Y26 have strong growth inhibition activity on T cell leukemia Jurkat cells. The compounds Y1-Y4, Y8-Y13, Y23-Y24 and Y26 show stronger antiproliferative activity on mantle cell lymphoma cells Jeko-1. Therefore, the invention also provides the application of the pyrazolopyrimidine compound containing carboxylic acid and amide in preparing the antitumor drugsIn use, the tumor is leukemia and lymphoma.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Examples

Example 1 preparation of intermediate 2

4-amino pyrazolo [3,4-d]Pyrimidine (5g,37mmol) is dissolved in 30ml DMF, N-bromosuccinimide (7.9g, 44.4mmol) is added, stirring is carried out, oil bath heating at 80 ℃ is carried out, the solution color is changed from light yellow to red, reaction is carried out for 3 hours, TLC detection is carried out, the basic reaction is complete, cooling is carried out to room temperature, the reaction solution is poured into 300ml ice water, stirring is carried out, a large amount of yellow solid is separated out, filtering is carried out, a filter cake is washed by ice water, drying is carried out, 6.33g of light yellow solid is obtained, and the yield is 79.9. And Mp: 270 ℃ and 273 DEG C¹H NMR(400MHz,DMSO)δ 13.77(s,1H),8.18(s,1H).

Example 2 preparation of intermediate 3

Taking 3-bromo-1H-pyrazolo [3,4-d]Pyrimidin-4-amine (2g, 9.34mmol), 4-phenoxyphenylboronic acid (3.99g, 18.68mmol), tetrakis (triphenylphosphine) palladium (531mg, 0.46mmol), K₃PO₄·3H₂O (7.45g, 28.02mmol) was added to a 250ml two-necked flask, 100ml of a solvent (1, 4-dioxane: water ═ 4:1) was added to dissolve it, nitrogen gas was used for protection, oxygen in the solution was removed by ultrasonic wave, air in a nitrogen gas replacement device was used, the mixture was heated and refluxed in an oil bath at 135 ℃ and reacted for 30 hours, and then TLC detection was carried out, and the reaction was almost completed. Cooling the reaction solution to room temperature, filtering with diatomaceous earth, evaporating the filtrate under reduced pressure to obtain yellow solid, and performing silica gel column chromatography (dichloromethane): methanol 100:1) to give 2.23g of a pale yellow solid in 78.8% yield. And Mp: 251 deg.C 254 deg.C¹H NMR(400MHz,DMSO)δ13.57(s,1H),8.22 (s,1H),7.67(d,J＝7.8Hz,2H),7.44(t,J＝7.5Hz,2H),7.20(d,J＝7.4Hz,1H),7.14(t,J＝7.7Hz, 4H).

Example 3 preparation of intermediate 4

1. Preparation of intermediate 4a

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (250mg, 0.82mmol), methyl 4- (bromomethyl) benzoate (224mg, 0.98mmol), K₂CO₃(170mg, 1.23mmol) and KI (13mg, 0.08mmol) were put into a 50ml reaction flask, and 15ml of DMF was added thereto to dissolve and stir, followed by reaction at room temperature for 8 hours and completion of the reaction by TLC. The reaction solution was poured into 100ml of ice water, stirred to precipitate a white solid, 30ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 200:1) to obtain 287mg of a white solid, the yield of which was 77.6%.¹H NMR(400MHz,DMSO)δ8.28(s,1H),7.66(d,J＝6.8Hz,2H),7.45–7.41(m,2H),7.25–7.20(m, 4H),7.13(t,J＝7.7Hz,5H),5.53(s,2H),3.52(s,3H).

2. Preparation of intermediate 4b

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (250mg, 0.82mmol), methyl 3- (bromomethyl) benzoate (224mg, 0.98mmol), K₂CO₃(170mg, 1.23mmol) and KI (13mg, 0.08mmol) were put into a 50ml reaction flask, and 15ml of DMF was added thereto to dissolve and stir, followed by reaction at room temperature for 8 hours and completion of the reaction by TLC. The reaction mixture was poured into 100ml of ice water, stirred to precipitate a white solid, 30ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 200:1) to obtain 302mg of a white solid with a yield of 81.6%.

3. Preparation of intermediate 4c

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (250mg, 0.82mmol), methyl 2- (bromomethyl) benzoate (224mg, 0.98mmol), K₂CO₃(170mg, 1.23mmol), KI (13mg, 0.08mmol) were addedAnd (3) adding 15ml of DMF into a 50ml reaction bottle, dissolving and stirring, reacting at room temperature for 8 hours, and detecting the reaction completion by TLC. The reaction solution was poured into 100ml of ice water, stirred to precipitate a white solid, 30ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 200:1) to obtain 254mg of a pale yellow solid, with a yield of 68.6%.

4. Preparation of intermediate 4d

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (250mg, 0.82mmol), methyl 2- (4- (bromomethyl) phenyl) acetate (238mg, 0.98mmol), K₂CO₃(170mg, 1.23mmol) and KI (13mg, 0.08mmol) were put into a 50ml reaction flask, and 15ml of DMF was added thereto to dissolve and stir, followed by reaction at room temperature for 8 hours and completion of the reaction by TLC. The reaction solution was poured into 100ml of ice water, stirred to precipitate a white solid, 30ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 200:1) to obtain 284mg of a white solid with a yield of 74.5%.¹H NMR(400MHz,DMSO)δ8.28(s,1H),7.66(d,J＝8.6Hz,2H),7.43(t,J＝7.9Hz,2H),7.23(dd,J ＝14.8,7.8Hz,5H),7.18–7.10(m,4H),5.53(s,2H),3.63(s,2H),3.58(s,3H).

5. Preparation of intermediate 4e

3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine (303mg, 1mmol), methyl 3-hydroxypiperidine-1-carboxylate (238mg, 1.5mmol) and triphenylphosphine (789mg,3mmol) were added to a 50ml reaction flask, 20ml of anhydrous THF was added thereto, stirring was carried out in an ice bath for 20 minutes, 666mg of DIAD was added dropwise to the reaction solution, the reaction solution was clarified, the reaction was continued overnight, and the reaction was complete by TLC. Silica gel column chromatography (petroleum ether: ethyl acetate: 4:1) gave 320mg of a white solid in 72.1% yield.

6. Preparation of intermediate 4f

3- (4-Phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine (303mg, 1mmol), (S) -3-hydroxypiperidine-1-carboxylic acid methyl ester (238mg, 1.5mmol), triphenylphosphine (789mg,3mmol) were added to a 50ml reaction flask, 20ml of anhydrous THF was added, stirring was performed for 20 minutes under ice bath conditions, 666mg of DIAD was added dropwise to the reaction solution, the reaction solution became clear, the reaction was continued overnight, and the reaction was complete by TLC. Silica gel column chromatography (petroleum ether: ethyl acetate: 4:1) gave 330mg of a white solid in 74.5% yield.

7. Preparation of intermediate 4g

3- (4-Phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine (303mg, 1mmol), (R) -3-hydroxypiperidine-1-carboxylic acid methyl ester (238mg, 1.5mmol), triphenylphosphine (789mg,3mmol) were added to a 50ml reaction flask, 20ml of anhydrous THF was added, stirring was performed for 20 minutes under ice bath conditions, 666mg of DIAD was added dropwise to the reaction solution, the reaction solution became clear, the reaction was continued overnight, and the reaction was complete by TLC. Silica gel column chromatography (petroleum ether: ethyl acetate: 4:1) gave 302mg of a white solid in 68% yield.

8. Preparation of intermediate 4h

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (250mg, 0.82mmol), methyl 3-bromo-4- (bromomethyl) benzoate (302mg, 0.98mmol), K₂CO₃(170mg, 1.23mmol) and KI (13mg, 0.08mmol) were put into a 50ml reaction flask, and 15ml of DMF was added thereto to dissolve and stir, followed by reaction at room temperature for 8 hours and completion of the reaction by TLC. The reaction solution was poured into 100ml of ice water, stirred to precipitate a white solid, 30ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography (dichloromethane: methanol: 200:1) was performed to obtain 286mg of a pale yellow solid, which was 74.5% yield.

9. Preparation of intermediate 4i

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (250mg, 0.82mmol), methyl 2-bromo-4- (bromomethyl) benzoate (302mg, 0.98mmol), K₂CO₃(170mg, 1.23mmol) and KI (13mg, 0.08mmol) were put into a 50ml reaction flask, and 15ml of DMF was added thereto to dissolve and stir, followed by reaction at room temperature for 8 hours and completion of the reaction by TLC. Pouring the reaction solution into 100ml of ice water, stirring, precipitating a white solid, adding 30ml of ethyl acetate, extracting for three times by using a separating funnel, combining organic phases, washing with saturated saline water, evaporating the organic phases under reduced pressure, and performing silica gel column chromatography (dichloromethane: methanol: 200:1) to obtain 245mg of light yellow oily substanceThe rate was 56.5%.

10. Preparation of intermediate 4j

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (250mg, 0.82mmol), methyl 3-chloro-4- (bromomethyl) benzoate (258mg, 0.98mmol), K₂CO₃(170mg, 1.23mmol) and KI (13mg, 0.08mmol) were put into a 50ml reaction flask, and 15ml of DMF was added thereto to dissolve and stir, followed by reaction at room temperature for 8 hours and completion of the reaction by TLC. The reaction solution was poured into 100ml of ice water, stirred to precipitate a white solid, 30ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol ═ 200:1) to obtain 289mg of a white solid, with a yield of 72.6%.

11. Preparation of intermediate 4k

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (200mg, 0.66mmol), methyl 3-nitro-4- (bromomethyl) benzoate (217mg, 0.79mmol), K₂CO₃(137mg, 0.99mmol) and KI (10mg, 0.06mmol) were added to a 50ml reaction flask, and 15ml DMF was added to dissolve and stir, and the reaction was carried out at room temperature for 8 hours, and the reaction was completed by TLC. The reaction solution was poured into 100ml of ice water, stirred to precipitate a white solid, 25ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol 300:1) to obtain 213mg of a pale yellow solid, with a yield of 65.1%.

12. Preparation of intermediate 4l

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (200mg, 0.66mmol), methyl 3-methoxy-4- (bromomethyl) benzoate (205mg, 0.79mmol), K₂CO₃(137mg, 0.99mmol) and KI (10mg, 0.06mmol) were added to a 50ml reaction flask, and 15ml DMF was added to dissolve and stir, and the reaction was carried out at room temperature for 8 hours, and the reaction was completed by TLC. The reaction solution was poured into 100ml of ice water, stirred to precipitate a white solid, 25ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 200:1) to obtain 190mg of a white solid with a yield of 59.7%.

13. Preparation of intermediate 4m

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (200mg, 0.66mmol), methyl 4- (2-bromoacetamido) benzoate (215mg, 0.79mmol), K₂CO₃(137mg, 0.99mmol) and KI (10mg, 0.06mmol) were added to a 50ml reaction flask, and 15ml DMF was added to dissolve and stir, and the reaction was carried out at room temperature for 8 hours, and the reaction was completed by TLC. The reaction mixture was poured into 100ml of ice water, stirred to precipitate a white solid, 25ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 100:1) to obtain 201mg of a white solid with a yield of 61.3%. And Mp: 180 ℃ 183 DEG C¹H NMR(400MHz,DMSO)δ10.69(s,1H),8.28(d,J＝12.4Hz,2H),7.81(d,J＝7.9 Hz,1H),7.68(t,J＝6.8Hz,3H),7.50(d,J＝7.9Hz,1H),7.44(t,J＝7.8Hz,2H),7.20(d,J＝7.4Hz, 1H),7.15(dd,J＝10.8,8.7Hz,4H),5.26(s,2H),3.84(s,3H).

14. Preparation of intermediate 4n

Taking 3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine (200mg, 0.66mmol), methyl 2- (4- (2-bromoacetamido) phenyl) acetate (226mg, 0.79mmol), K₂CO₃(137mg, 0.99mmol) and KI (10mg, 0.06mmol) were added to a 50ml reaction flask, and 15ml DMF was added to dissolve and stir, and the reaction was carried out at room temperature for 8 hours, and the reaction was completed by TLC. The reaction solution was poured into 100ml of ice water, stirred to precipitate a white solid, 25ml of ethyl acetate was added, the mixture was extracted three times with a separatory funnel, the organic phases were combined, washed with saturated brine, the organic phase was distilled off under reduced pressure, and silica gel column chromatography was performed (dichloromethane: methanol: 200:1) to obtain 227mg of a pale yellow solid, which was 67.8% in yield.

Example 4 preparation of the object Compounds Y1-Y13

Potassium hydroxide (2.83g, 50.4mmol) was dissolved in 7ml of anhydrous methanol and dissolved by sonication to give a solution a. Hydroxylamine hydrochloride (2.33g, 33.5mmol) was dissolved in 12ml of anhydrous methanol, and the solution was ultrasonically dissolved and subjected to ice-bath to obtain a solution b. Adding the solution a intoAnd slowly dropping the mixture into the solution b in a constant-pressure dropping funnel, reacting for 3 hours under an ice bath condition, filtering precipitate (KCl), reserving filtrate, and drying the filtrate for 6 hours by using anhydrous sodium sulfate. The filtrate was taken in a 50ml reaction flask, the intermediate 4a (120mg, 0.26mmol) from the previous step, example 3, was added and reacted for one hour, the solution became clear and after a further 15 minutes the reaction was essentially complete by TLC. Most of the solvent was evaporated under reduced pressure, 10ml of water was added, the pH was adjusted to 5-6 with 1M hydrochloric acid, the solid precipitated, filtered, washed twice with ethyl acetate, the filter cake was recrystallized from methanol, and dried to give Y1 as an off-white solid, 197mg, 80.8% yield. And Mp: 168 ℃ and 172 ℃. MS (ESI) M/z 451.1[ M-H ]]^-.¹H NMR(400MHz,DMSO)δ11.20(s,1H),9.09(s,1H),8.28(s,1H), 7.68(dd,J＝12.3,8.4Hz,4H),7.44(t,J＝7.8Hz,2H),7.33(d,J＝8.0Hz,2H),7.20(d,J＝7.4Hz,1H), 7.13(t,J＝8.7Hz,4H),5.60(s,2H).¹³C NMR(100MHz,DMSO)δ163.10,157.63,156.55,156.52, 155.66,155.46,153.84,143.16,139.32,129.53,129.46,127.15,126.86,126.51,123.20,118.39,118.37, 96.69,48.87.

According to the method for preparing the compound Y1, the intermediates 4b to 4m prepared in example 3 were used to prepare the compounds Y2 to Y13, respectively.

Pale pink solid, yield 72.5%. And Mp: 230 ℃ and 235 ℃. MS (ESI) M/z 451.4[ M-H ]]^-.¹H NMR(400MHz, DMSO)δ11.23(s,1H),9.03(s,1H),8.29(s,1H),7.66(dd,J＝19.0,10.3Hz,4H),7.42(dd,J＝13.4, 6.8Hz,4H),7.19(d,J＝7.4Hz,1H),7.13(t,J＝8.7Hz,4H),5.60(s,2H).¹³C NMR(100MHz,DMSO) δ163.36,157.63,156.55,155.67,155.45,153.84,143.15,136.89,132.56,129.74,129.52,129.45,129.17, 128.05,127.17,125.82,125.29,123.19,118.38,96.69,48.94.

Pale pink solid, yield 71.8%. And Mp: 190 ℃ and 194 ℃.¹H NMR(400MHz,DMSO)δ11.20(s,1H),9.22(s, 1H),8.24(s,1H),7.95(d,J＝7.6Hz,1H),7.69(d,J＝8.6Hz,2H),7.43(t,J＝7.8Hz,3H),7.40–7.35 (m,1H),7.20(d,J＝7.4Hz,1H),7.14(dd,J＝11.6,8.7Hz,4H),6.64(d,J＝7.5Hz,1H),5.96(s,2H). ¹³C NMR(100MHz,DMSO)δ168.83,158.75,157.64,156.75,156.52,155.29,144.33,138.87,136.24,

132.71,131.03,130.61,130.57,129.98,128.28,127.82,127.61,124.28,119.46,97.78,48.59.

White solid, yield 71.0%. And Mp: 180 ℃ and 184 ℃. MS (ESI) M/z 465.4[ M-H ]]^-.¹H NMR(400MHz,DMSO) δ10.63(s,1H),8.79(s,1H),8.28(s,1H),7.65(d,J＝8.6Hz,2H),7.45–7.41(m,2H),7.21(dd,J＝ 13.4,8.8Hz,5H),7.13(t,J＝7.7Hz,4H),5.51(s,2H),3.23(s,2H).¹³C NMR(100MHz,DMSO)δ 166.22,157.59,156.50,155.68,155.38,153.69,142.94,134.91,134.71,129.52,129.45,128.84,128.53, 127.23,127.00,126.64,123.18,118.37,96.68,48.96.

White solid, yield 59.5%. And Mp: 150 ℃ and 152 ℃.¹H NMR(400MHz,DMSO)δ10.51(s,1H),8.79(s,1H), 8.26(s,1H),7.66(d,J＝7.6Hz,2H),7.43(d,J＝7.0Hz,2H),7.24–7.08(m,5H),4.86(s,1H),2.98(s, 2H),2.84(d,J＝9.4Hz,1H),2.62(t,J＝10.1Hz,1H),2.24–2.12(m,1H),1.99(s,3H),1.77-1.71(m, 2H).¹³C NMR(100MHz,DMSO)δ166.16(s),158.64(s),157.53(s),156.81(s),156.07(s),154.25(s), 143.53(s),130.57(d,J＝7.1Hz),128.48(s),124.24(s),119.45(d,J＝5.6Hz),97.77(s),59.56(s), 57.92(s),53.31(d,J＝27.7Hz),29.57(s),24.49(s).

(R) -2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N-hydroxyacetamide (compound Y6)

White solid, yield 62.4%. And Mp: 151 ℃ and 154 ℃.¹H NMR(400MHz,DMSO)δ10.51(s,1H),8.79(s,1H), 8.26(s,1H),7.66(d,J＝7.6Hz,2H),7.43(d,J＝7.0Hz,2H),7.24–7.08(m,5H),4.86(s,1H),2.98(s, 2H),2.84(d,J＝9.4Hz,1H),2.62(t,J＝10.1Hz,1H),2.24–2.12(m,1H),1.99(s,3H),1.77-1.71(m, 2H).

(S) -2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N-hydroxyacetamide (compound Y7)

White solid, yield 49.5%. And Mp: 149 ℃ and 152 ℃.¹H NMR(400MHz,DMSO)δ10.49(s,1H),8.76(s,1H), 8.25(s,1H),7.65(d,J＝8.5Hz,2H),7.44(t,J＝7.9Hz,2H),7.30–7.09(m,5H),4.95–4.80(m,1H), 2.99(s,3H),2.92–2.80(m,1H),2.74–2.60(m,1H),2.25-2.16(m,1H),1.98(d,J＝3.0Hz,2H), 1.81-1.72(m,2H).

Pale pink solid, yield 74.1%. And Mp: 182 ℃ and 185 ℃.¹H NMR(400MHz,DMSO)δ9.14(s,1H),8.28(s,1H), 8.01(s,1H),7.67(d,J＝6.4Hz,3H),7.43(t,J＝7.9Hz,2H),7.20(d,J＝7.3Hz,1H),7.14(dd,J＝11.7, 8.6Hz,4H),6.98(d,J＝8.1Hz,1H),5.65(s,2H).¹³C NMR(100MHz,DMSO)δ158.73,157.70, 157.54,156.71,156.66,155.26,144.67,139.36,134.39,131.41,130.61,130.57,129.71,128.10,126.95, 124.30,122.39,119.46,99.89,97.78,50.10.

Off-white solid, yield 65.0%. And Mp: 226-230 ℃.¹H NMR(400MHz,DMSO)δ10.91(s,1H),9.24(s, 1H),8.30(s,1H),7.66(d,J＝8.6Hz,2H),7.61(s,1H),7.44(t,J＝7.9Hz,2H),7.32(d,J＝7.8Hz,1H), 7.27(d,J＝7.9Hz,1H),7.20(d,J＝7.4Hz,1H),7.14(t,J＝8.9Hz,4H),5.60(s,2H).¹³C NMR(100 MHz,DMSO)δ164.18,158.00,157.79,156.66,155.56,154.58,144.79,140.79,136.38,132.28,130.63, 130.55,130.13,127.85,127.23,124.34,120.25,119.52,119.46,97.72,49.34.

Light yellowA colored solid, 74.6% yield. And Mp: 190 ℃ and 193 ℃. MS (ESI) M/z 485.5[ M-H ]]^-.¹H NMR(400MHz, DMSO)δ11.32(s,1H),9.17(s,1H),8.28(s,1H),7.83(s,1H),7.65(t,J＝8.4Hz,3H),7.43(t,J＝7.8 Hz,2H),7.19(d,J＝7.3Hz,1H),7.16–7.10(m,4H),7.07(d,J＝8.0Hz,1H),5.68(s,2H).¹³C NMR (100MHz,DMSO)δ162.79,158.74,157.69,156.73,156.60,155.23,144.63,137.74,134.34,132.48, 131.98,130.61,130.57,130.27,129.96,128.16,126.42,124.28,119.46,97.75,47.70.

Yellow solid, yield 65.0%. And Mp: 187-192 ℃. MS (ESI) M/z 496.5[ M-H ]]^-.¹H NMR(400MHz,DMSO) δ11.55(s,1H),9.29(s,1H),8.46(s,1H),8.26(s,1H),8.00(d,J＝8.1Hz,1H),7.66(d,J＝8.6Hz,2H), 7.43(t,J＝7.9Hz,2H),7.20(d,J＝7.4Hz,1H),7.14(dd,J＝11.5,8.5Hz,4H),7.04(d,J＝8.1Hz,1H), 5.97(s,2H).¹³C NMR(100MHz,DMSO)δ162.11,158.76,157.74,156.69,155.42,148.00,144.88, 135.13,133.81,132.51,130.62,130.55,130.27,128.02,124.31,123.92,120.85,119.49,119.44,97.87, 47.30.

Pale yellow solid, yield 68.3%. And Mp: 198-205 ℃.¹H NMR(400MHz,DMSO)δ11.21(s,1H),9.05(s, 1H),8.27(s,1H),7.67(d,J＝8.6Hz,2H),7.44(d,J＝7.6Hz,2H),7.39(s,1H),7.24(d,J＝8.3Hz,1H), 7.19(d,J＝7.4Hz,1H),7.14(dd,J＝15.5,6.7Hz,4H),6.78(d,J＝7.8Hz,1H),5.55(s,2H),3.88(s, 3H).¹³C NMR(100MHz,DMSO)δ163.08,157.63,156.53,155.68,155.54,155.37,154.09,143.13, 132.76,130.17,129.52,127.48,127.27,127.01,123.19,118.58,118.38,108.67,96.66,55.17,43.96.

Pale pink solid, yield 52.5%. And Mp: 258 ℃ and 260 ℃.¹H NMR(400MHz,DMSO)δ11.13(s,1H),10.67(s, 1H),8.97(s,1H),8.26(s,1H),7.68(dt,J＝25.3,8.5Hz,6H),7.44(t,J＝7.8Hz,2H),7.20(d,J＝7.4 Hz,1H),7.15(dd,J＝10.9,8.7Hz,4H),5.26(s,2H).¹³C NMR(100MHz,DMSO)δ166.01,164.32, 158.66,157.68,156.71,156.40,155.85,144.18,141.55,130.62,130.47,128.29,128.21,128.10,124.32, 119.53,119.43,119.00,97.73,50.17.

Example 5 preparation of the object Compounds Y14-Y22

The intermediate 4a (160mg, 0.35mmol) prepared in the previous step, i.e. example 3, was taken and added to a 50ml reaction flask, dissolved in 20ml methanol, added with 4ml 3M NaOH, reacted at 60 ℃ in an oil bath for 8 hours with TLC detection of the basic completion of the reaction. The solvent was evaporated under reduced pressure, 10ml of water was added, the pH was adjusted to 5-6 with 1M hydrochloric acid, a white solid was precipitated, filtered, washed twice with ice water, the filter cake was recrystallized from methanol, and dried to give Y14 as a white solid, 140mg, 91.5% yield. And Mp: 306 ℃ and 309 ℃. MS (ESI) M/z 436.4 [ M-H]^-.¹H NMR(400MHz,DMSO)δ12.92(s,1H),8.29(s,1H),7.90(d,J＝8.2Hz,2H),7.67(d,J＝ 8.6Hz,2H),7.43(t,J＝7.9Hz,2H),7.37(d,J＝8.1Hz,2H),7.19(d,J＝7.4Hz,1H),7.13(t,J＝9.1Hz, 4H),5.64(s,2H).¹³C NMR(100MHz,DMSO)δ167.47,158.72,157.65,156.74,156.57,154.99,144.33, 142.49,130.60,130.55,130.12,128.20,128.10,124.27,119.47,119.44,97.76,49.94.

According to the method for producing the compound Y14, the compounds Y15-Y20 and Y21-Y22 were produced using the intermediates 4b-4g and 4m-4n produced in example 3, respectively.

White solid, yield 87.7%. And Mp: 283 ℃ and 285 ℃. MS (ESI) M/z 436.5[ M-H ]]^-.¹H NMR(400MHz,DMSO) δ13.02(s,1H),8.30(s,1H),7.86(d,J＝11.7Hz,2H),7.66(d,J＝8.5Hz,2H),7.55(d,J＝7.7Hz,1H), 7.48(d,J＝7.6Hz,1H),7.45–7.40(m,2H),7.19(d,J＝7.4Hz,1H),7.14(dd,J＝15.2,6.6Hz,4H), 5.63(s,2H).¹³C NMR(100MHz,DMSO)δ167.47,158.71,157.66,156.73,156.57,154.89,144.28, 138.15,132.63,131.54,130.60,130.52,129.44,129.02,128.90,128.19,124.28,119.47,97.75,49.89.

White solid, yield 86.6%. And Mp: 262 ℃ and 265 ℃.¹HNMR(400MHz,DMSO)δ8.24(s,1H),7.91(d,J＝ 7.4Hz,1H),7.69(d,J＝8.5Hz,2H),7.43(t,J＝7.9Hz,2H),7.35(t,J＝6.1Hz,2H),7.19(d,J＝7.3Hz, 1H),7.17–7.10(m,4H),6.56(d,J＝6.9Hz,1H),5.98(s,2H).¹³C NMR(100MHz,DMSO)δ158.74, 157.76,157.48,156.76,156.49,156.30,155.30,144.25,138.43,131.81,130.83,130.61,130.57,128.32, 127.68,127.53,127.13,124.27,119.46,97.76,48.76.

White solid, yield 86.6%. And Mp: 272 ℃ and 275 ℃. MS (ESI) M/z 450.4[ M-H ]]^-.¹H NMR(400MHz,DMSO) δ12.29(s,1H),8.28(s,1H),7.66(d,J＝8.5Hz,2H),7.43(t,J＝7.8Hz,2H),7.25(s,1H),7.24–7.18 (m,4H),7.13(t,J＝7.7Hz,4H),5.53(s,2H),3.52(s,2H).¹³C NMR(100MHz,DMSO)δ173.03, 158.65,157.53,156.75,156.43,154.75,144.06,135.92,134.88,130.60,130.54,130.07,128.29,128.08, 124.26,119.45,97.76,50.03.

White solid, yield 56.6%. And Mp: 140 ℃ to 142 DEG C¹H NMR(400MHz,DMSO)δ8.26(s,1H),7.66(d,J＝8.4 Hz,2H),7.44(t,J＝7.7Hz,2H),7.22–7.10(m,6H),4.93–4.84(m,1H),3.24(s,2H),3.12(d,J＝7.6 Hz,1H),2.95(d,J＝10.3Hz,1H),2.85(t,J＝10.4Hz,1H),2.42–2.33(m,1H),1.98(s,2H),1.84– 1.73(m,2H).¹³C NMR(100MHz,DMSO)δ172.58(s),158.63(s),157.53(s),156.79(s),156.10(s), 154.27(s),143.60(s),130.57(d,J＝6.1Hz),128.44(s),124.24(s),119.44(d,J＝5.8Hz),97.77(s), 56.91(s),53.14(s),52.28(s),29.48(s),24.19(s),21.66(s).

(R) -2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) acetic acid (compound Y19)

White solid, yield 66.4%. And Mp: 141 ℃ 143-¹H NMR(400MHz,DMSO)δ8.26(s,1H),7.66(d,J＝8.4 Hz,2H),7.44(t,J＝7.7Hz,2H),7.22–7.10(m,6H),4.93–4.84(m,1H),3.24(s,2H),3.12(d,J＝7.6 Hz,1H),2.95(d,J＝10.3Hz,1H),2.85(t,J＝10.4Hz,1H),2.42–2.33(m,1H),1.98(s,2H),1.84– 1.73(m,2H).

(S) -2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) acetic acid (compound Y20)

White solid, yield 59.2%. And Mp: 138 ℃ and 140 DEG C¹H NMR(400MHz,DMSO)δ8.26(s,1H),7.66(d,J＝8.4 Hz,2H),7.44(t,J＝7.7Hz,2H),7.22–7.10(m,6H),4.98–4.89(m,1H),3.25(s,2H),3.18(d,J＝7.6 Hz,1H),3.02(d,J＝10.3Hz,1H),2.87(t,J＝10.4Hz,1H),2.48–2.36(m,1H),1.98(s,2H),1.94– 1.75(m,2H).

White solid, yield 86.6%. And Mp: 240 ℃ and 244 ℃.¹H NMR(400MHz,DMSO)δ10.75(s,1H),8.26(s,1H), 7.90(d,J＝8.5Hz,2H),7.68(d,J＝8.4Hz,4H),7.44(t,J＝7.9Hz,2H),7.20(d,J＝7.3Hz,1H),7.18 –7.11(m,4H),5.27(s,2H).¹³C NMR(100MHz,DMSO)δ169.71,167.31,166.18,158.64,157.67, 156.71,156.39,155.85,155.31,130.96,130.62,130.46,124.32,119.53,119.42,118.99,97.68,48.42, 46.12,9.05.

Pink solid, yield 71.8%. And Mp: 280 ℃ and 282 ℃.¹H NMR(400MHz,DMSO)δ10.47(s,1H),8.26(s,1H), 7.68(d,J＝8.5Hz,2H),7.52(d,J＝8.4Hz,2H),7.44(t,J＝7.9Hz,2H),7.20(d,J＝8.0Hz,3H),7.18 –7.11(m,4H),5.22(s,2H),3.51(s,2H).¹³C NMR(100MHz,DMSO)δ173.25,165.51,158.66,157.64, 156.73,156.36,155.64,144.08,137.66,130.70,130.61,130.47,130.21,128.27,124.30,119.55,119.52, 119.42,97.72,50.09.

Example 6 preparation of the object Compounds Y23-Y29

The compound Y14(120mg, 0.27mmol), TBTU (103mg, 0.32mmol) and triethylamine (55mg, 0.54mmol) prepared in example 4 were taken and added to a 50ml reaction flask, 15ml of anhydrous DMF was added and dissolved with stirring, activated for 30 minutes in ice bath, o-phenylenediamine (35mg, 0.32mmol) was added and reacted overnight at room temperature with TLC detection for completion of the reaction. Pouring the reaction solution into 100ml of ice water, precipitating gray solid, adding 25ml of ethyl acetate for extraction three times, combining organic phases and saturating NH₄Washed with Cl, saturated NaCl, the solvent was evaporated under reduced pressure and chromatography on silica gel column (dichloromethane: methanol 300:1) afforded Y2356 mg as an off-white solid in 39.4% yield. And Mp: 197 ℃ and 200 ℃. MS (ESI) M/z 526.5[ M-H ]]^-.¹H NMR(400MHz,DMSO) δ9.60(s,1H),8.30(s,1H),7.92(d,J＝8.0Hz,2H),7.67(d,J＝8.6Hz,2H),7.47–7.36(m,4H),7.19 (d,J＝7.4Hz,1H),7.18–7.12(m,4H),7.11(s,1H),6.95(t,J＝8.1Hz,1H),6.76(d,J＝7.7Hz,1H), 6.58(t,J＝7.4Hz,1H),5.64(s,2H),4.88(s,2H).¹³C NMR(100MHz,DMSO)δ165.48,158.72,157.84, 156.75,156.55,154.96,144.27,143.56,140.90,134.48,130.61,130.54,128.57,128.24,127.93,127.10, 126.94,124.28,123.66,119.46,116.65,116.51,97.78,50.00,46.11.

According to the method for producing the compound Y23, the compounds Y24 to Y29 were produced using the compounds Y15 to Y20 produced in example 4, respectively.

Off-white solid, yield 34.6%. And Mp: 175 ℃ and 178 ℃.¹H NMR(600MHz,DMSO)δ9.69(s,1H),8.30(s,1H), 7.93(s,2H),7.67(d,J＝8.5Hz,2H),7.47(d,J＝4.4Hz,2H),7.43(t,J＝7.9Hz,2H),7.18(t,J＝7.4 Hz,1H),7.16–7.12(m,4H),7.11(s,1H),6.96(t,J＝7.5Hz,1H),6.77(d,J＝7.9Hz,1H),6.58(t,J＝ 7.3Hz,1H),5.64(s,2H),4.88(s,2H).¹³C NMR(100MHz,DMSO)δ165.53,158.71,157.63,156.74, 156.55,154.92,144.29,143.63,137.89,135.43,131.08,130.81,130.54,129.06,128.24,127.75,127.34, 127.18,127.00,124.28,123.63,119.46,116.67,116.54,97.77,50.06,46.10.

Grey solid, yield 33.6%. And Mp: 205 ℃ and 209 ℃.¹H NMR(400MHz,DMSO)δ9.84(s,1H),8.26(s,1H), 7.68(d,J＝8.5Hz,3H),7.47–7.39(m,5H),7.26(d,J＝7.6Hz,1H),7.20(d,J＝7.2Hz,1H),7.14– 7.10(m,4H),6.99(d,J＝7.1Hz,1H),6.78(d,J＝7.7Hz,1H),6.58(t,J＝7.4Hz,1H),5.83(s,2H), 5.07(s,2H).¹³C NMR(100MHz,DMSO)δ167.67,158.72,157.62,156.73,156.47,154.97,144.23, 143.55,136.35,135.71,130.60,128.63,128.22,127.87,127.13,127.06,124.28,123.37,119.46,119.40, 116.52,116.36,97.83,52.47,47.66,46.06,7.64.

Grey solid, yield 24.8%. And Mp: 209 ℃ and 212 ℃.¹H NMR(400MHz,DMSO)δ9.34(s,1H),8.28(s,1H), 7.66(d,J＝8.4Hz,2H),7.43(t,J＝7.8Hz,2H),7.28(dd,J＝16.8,8.1Hz,4H),7.19(d,J＝6.9Hz,1H), 7.13(t,J＝7.3Hz,5H),6.88(t,J＝7.5Hz,1H),6.69(d,J＝8.1Hz,1H),6.50(t,J＝7.5Hz,1H),5.53(s, 2H),4.81(s,2H),3.61(s,2H).¹³C NMR(100MHz,DMSO)δ169.42,158.67,157.58,156.77,156.45, 154.77,152.86,152.04,144.04,142.34,136.21,130.60,130.55,129.79,128.32,128.12,125.75,124.25, 123.74,119.45,116.61,116.30,97.77,46.08,42.72,9.04.

White solid, yield 36.6%. And Mp: 135 ℃ and 139 ℃.¹H NMR(400MHz,DMSO)δ9.22(s,1H),8.26(s,1H), 7.65(d,J＝8.6Hz,2H),7.44(t,J＝7.9Hz,2H),7.24-7.11(m,6H),6.91(t,J＝7.6Hz,1H),6.76(d,J ＝7.5Hz,1H),6.57(t,J＝7.4Hz,1H),5.02–4.93(m,1H),4.80(s,2H),3.25–3.09(m,3H),2.95(d,J ＝11.5Hz,1H),2.77–2.70(m,1H),2.35–2.23(m,1H),2.02(d,J＝4.1Hz,2H),1.84(d,J＝0.5Hz, 2H).

2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide (compound Y28)

White solid, yield 48.1%. And Mp: 137-139 ℃.¹H NMR(400MHz,DMSO)δ9.22(s,1H),8.26(s,1H), 7.65(d,J＝8.6Hz,2H),7.44(t,J＝7.9Hz,2H),7.24-7.11(m,6H),6.91(t,J＝7.6Hz,1H),6.76(d,J ＝7.5Hz,1H),6.57(t,J＝7.4Hz,1H),5.02–4.93(m,1H),4.80(s,2H),3.25–3.09(m,3H),2.95(d,J ＝11.5Hz,1H),2.77–2.70(m,1H),2.35–2.23(m,1H),2.02(d,J＝4.1Hz,2H),1.84(d,J＝0.5Hz, 2H).

2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide (compound Y29)

White solid, yield 32.5%. And Mp: 134 ℃ and 136 ℃.¹H NMR(400MHz,DMSO)δ9.22(s,1H),8.26(s,1H), 7.65(d,J＝8.6Hz,2H),7.44(t,J＝7.6Hz,2H),7.24-7.11(m,6H),6.91(t,J＝7.6Hz,1H),6.76(d,J ＝7.9Hz,1H),6.57(t,J＝7.0Hz,1H),5.01-4.80(m,1H),4.80(s,2H),3.20(d,J＝9.5Hz,2H),3.12 (dd,J＝10.4,3.5Hz,1H),2.94(d,J＝11.5Hz,1H),2.73(t,J＝10.6Hz,1H),2.27(td,J＝10.8,2.6Hz, 1H),2.02(d,J＝4.1Hz,2H),1.86-1.79(m,2H).

Examples of the experiments

BTK inhibitory Activity test, inhibitory Activity against HDAC, growth inhibition against Jurkat cells, and growth inhibitory Activity against JEKO-1 cells

1. Compound activity assay for inhibition of BTK kinase:

the experiment was carried out with the aid of Eurofins Pharma, british company.

TABLE 1 inhibitory Activity of the Compounds of interest on BTK

Note: ND: not determined; IBN: ibrutinib (Ibrutinib).

Under the condition of concentration of 1 mu M, the compounds all show stronger inhibition activity (the inhibition rate is more than 80%) to BTK, under the condition of concentration of 50nM, the compounds Y1, Y5-Y12, Y17-Y18, Y23 and Y27-Y29 still show certain inhibition activity to BTK, and the compounds Y5-Y6, Y27-Y28 and IBN have equivalent BTK inhibition activity.

2. Assay of the inhibitory Activity of Compounds on HDAC:

experimental materials and methods: Boc-Lys (acetyl) -AMC (HeLa cell nuclear extraction fluorogenic substrate) was purchased from Bachem AG, Switzerland, Tris-HCl, Trypsin and EDTA were purchased from Sigma-Aldrich, TSA was purchased from Allantin scientific and Biochemical Co., Ltd., glycerol, NaCl, 96-well flat-bottom fluorescent plate was purchased from Hongfei reagent.

Buffer solution: 15mM Tris-HCl (pH8.0), 250mM NaCl, 250. mu.M EDTA, 10% glycerol

HDAC enzyme solution: diluting with Buffer according to the test requirement.

Fluorogenic substrate solution: the substrate was dissolved in DMSO to prepare a 30mM stock solution, which was stored at-20 ℃ and diluted to 300. mu.M with HDAC Buffer at the time of use.

Stopping liquid: 10mg/mL Trypsin, 50mM Tris-HCl (pH8.0), 100mM NaCl, 2. mu.M TSA.

An enzyme-labeling instrument: the Varioskan Flash spectral scanning multifunctional reading instrument.

The experimental steps are as follows:

100% control group: mixing 10 μ L of HDACs enzyme solution with 50 μ L of HDAC Buffer, incubating at 37 deg.C for 5min, adding 40 μ L of substrate solution, further incubating at 37 deg.C for 30min, adding 100 μ L of stop solution, further incubating at 37 deg.C for 20min, and measuring the fluorescence intensity of reaction solution at 390nm/460nm with a microplate reader to obtain 100% group fluorescence.

Blank control group: mixing 40 μ L of the substrate solution with 60 μ L of HDAC Buffer, incubating at 37 deg.C for 30min, adding 100 μ L of stop solution, further incubating at 37 deg.C for 20min, and measuring the fluorescence intensity of the reaction solution at 390nm/460nm with microplate reader, wherein the value is the fluorescence intensity of the blank group.

Experimental groups: mixing 10 mu L of HDACs enzyme solution with 50 mu L of a compound to be detected diluted by HDAC Buffer, incubating at 37 ℃ for 5min, adding 40 mu L of substrate solution, continuing to incubate at 37 ℃ for 30min, adding 100 mu L of stop solution, continuing to incubate at 37 ℃ for 20min, and measuring the fluorescence intensity of the reaction solution at 390nm/460nm by using an enzyme labeling instrument, wherein the value is the fluorescence of the compound to be detected at the concentration.

Calculating the inhibition rate under different concentrations according to a formula, performing S-curve fitting by using origin software, and calculating the IC of the compound to be detected₅₀Values, as shown in table 2.

TABLE 2 inhibitory Activity of target Compounds on HDAC

Note: ND: not determined; SAHA: vorinostat (Vorinostat); IBN: ibrutinib (Ibrutinib).

The results in Table 2 indicate that compounds Y1, Y4, Y10, Y11, Y13 and Y23 exhibit strong inhibitory activity against HDAC (IC)₅₀Less than 1 μ M).

3. Compound inhibition activity on Jurkat and Jeko-1 cell growth assay:

experimental materials and methods: human mantle cell lymphoma cell lines Jeko-1 and Jurkat (American Type Culture Collection, ATCC), F-12 medium, fetal bovine serum (Gibco, USA). The drug was dissolved in DMSO at an initial concentration of 20 mM.

The experimental steps are as follows:

the cell counting method was used to examine the growth inhibitory effect of the compounds on Jurkat and Jeko-1 cells. A certain density (10 is multiplied by 10)⁴Per mL) The cell suspension is inoculated on a 24-well culture plate, 2 mL/well, and drugs with different concentrations are added at 37 ℃ and 5% CO₂After incubation for 72 hours in a saturated humidity incubator, taking 50 mu L of cell suspension and 50 mu L of trypan blue working solution for equal volume mixing, taking 10 mu L of mixed solution, using a blood counting plate to count the cell number of a control hole and a dosing hole under a microscope respectively, using a formula to calculate the cell growth inhibition rate, carrying out normalization treatment on the obtained value and a blank control group, and adopting GraphPad 5.0 to calculate GI software₅₀Values, as shown in table 3.

The survival rate (%) of the compound was equal to the number of cells in the administered group/the number of cells in the blank group × 100%

TABLE 3 inhibitory Activity of Compounds of interest on Jurkat and Jeko-1 cell growth

Experimental data show that the compounds Y1-Y4, Y8-Y13 and Y23-Y26 show stronger inhibitory activity on the growth of T cell leukemia Jurkat cells, and the activity is superior to that of ibrutinib. The compounds Y1-Y4, Y8-Y13, Y23-Y24 and Y26 show stronger antiproliferative activity on mantle cell lymphoma cells Jeko-1. Y1 and Y23 were shown to have greater growth inhibitory effects in both cells than either ibrutinib or vorinostat.

Claims

1. A compound having the structure shown in formula (I):

wherein X, Y is two linking groups, X is selected from substituted or unsubstituted benzyl, and the substituents are selected from nitro, halogen and C_1～6Linear or branched alkoxy;

a. b represents a linking position, wherein position b is linked to Y; piperidin-3-yl;

y is- (CH)₂)_n-, n is any integer selected from 0 to 4; r is selected from hydroxyl, hydroxylamino and o-phenylenediamine.

2. The compound of claim 1, wherein Y is- (CH)₂)_n-n is 0 or 1;

y is- (CH)₂)_n-when n is 0, X is selected from substituted or unsubstituted benzyl, said substituents being selected from nitro, halogen and C_1～6Linear or branched alkoxy;

a. b represents a linking position, wherein position b is linked to Y;

y is- (CH)₂)_nWhen n is 1, X is selected from substituted or unsubstituted benzyl, said substituents being selected from nitro, halogen and C_1～6Linear or branched alkoxy;

a. b represents a linking position, wherein position b is linked to Y; piperidin-3-yl.

3. The compound of claim 2,

when X is a substituted or unsubstituted benzyl group, X is

4. A compound according to claim 3, wherein X is selected from

5. A compound according to claim 3, wherein X is selected from

Any one of the above.

6. The compound of claim 5, wherein X is selected from the group consisting of

7. The compound of claim 5, wherein X is selected from the group consisting of

8. A compound of claim 5, wherein R is₁When is H, X is selected from

9. A compound of claim 8, wherein R is₁Is nitro or C_1～6When the alkoxy group is linear or branched, X is

10. A compound of claim 8, wherein R is₁Is C_1～6When the alkoxy is linear or branched, the alkoxy is selected from methoxy and ethoxyAlkyl, propoxy and butoxy.

11. The compound of claim 10, wherein R is₁Is methoxy.

12. A compound of claim 8, wherein R is₁When halogen, the halogen is selected from F, Cl, Br and I.

13. The compound of claim 12, wherein R is₁When halogen is used, the halogen is selected from Cl or Br.

14. A compound according to claim 2, wherein when X is piperidin-3-yl, X is selected from

15. The compound of claim 1 or 2, wherein the compound is selected from the following structures:

16. The compound of claim 15, wherein in the structure of formula (II), according to R₁Is selected from the following structures:

17. The compound of claim 15, wherein in the structure of formula (II), depending on the position of the bond between Y and the phenyl ring, formula (II) is selected from the following structures:

18. Compound according to any one of claims 1 or 2, characterized in that it is selected from the following compounds:

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxybenzamide;

3- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxybenzamide;

2- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxybenzamide;

2- (4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N-hydroxyacetamide;

2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N-hydroxyacetamide;

(R)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N-hydroxyacetamide;

(S)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N-hydroxyacetamide;

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -3-bromo-N-hydroxybenzamide;

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -2-bromo-N-hydroxybenzamide;

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -3-chloro-N-hydroxybenzamide;

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxy-3-nitrobenzamide;

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxy-3-methoxybenzamide;

4- (2- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) acetylamino) -N-hydroxybenzamide;

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) benzoic acid;

3- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) benzoic acid;

2- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) benzoic acid;

(4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) acetic acid;

2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) acetic acid;

(R)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) acetic acid;

(S)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) acetic acid;

4- (2- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) acetamido) benzoic acid;

(4- (2- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) acetamido) phenyl) acetic acid;

4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide;

3- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide;

2- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide;

2- (4- ((4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N- (2-aminophenyl) acetamide;

2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide;

(R)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide;

(S)2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide.

19. A process for the preparation of a compound as claimed in any one of claims 1 to 18, which comprises preparing a compound of formula (I) starting from intermediate 1 by the following reaction scheme:

wherein X, Y, R is as defined in any one of claims 1 to 18, R₂Is hydroxyl or halogen.

20. The method of claim 19, wherein R is₂Is hydroxyl or Br.

21. The method for preparing according to claim 19, characterized in that it comprises the following steps: taking the intermediate 1 as a starting material, and carrying out substitution reaction with N-bromosuccinimide in DMF under the heating condition to obtain an intermediate 2; the intermediate 2and 4-phenoxyphenylboronic acid are subjected to Suzuki reaction under the catalysis of tetrakis (triphenylphosphine) palladium to obtain an intermediate 3; intermediate 3 and

wherein X, Y, R is as defined in any one of claims 1 to 5, R₂Is hydroxyl or halogen.

22. The method of claim 21, wherein R is₂Is hydroxyl or Br.

23. A pharmaceutical composition comprising a compound of any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof.

24. A pharmaceutical formulation comprising a compound of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof or a composition of claim 23 and a pharmaceutically acceptable adjuvant and/or carrier.

25. Use of a compound of any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or a composition of claim 23, in the preparation of a BTK inhibitor medicament or an HDAC inhibitor medicament.

26. Use of a compound of any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or a composition of claim 23, in the preparation of an anti-neoplastic drug.

27. The use of claim 26, wherein the tumor is a lymphoma or leukemia.