CN114605418B - Ibrutinib acrylamide derivative with anti-tumor activity, and synthetic method and application thereof - Google Patents

Abstract

The invention discloses ibrutinib acrylamide derivatives with anti-tumor activity, and a synthesis method and application thereof, and belongs to the field of medicinal chemistry. The ibrutinib acrylamide derivatives with anti-tumor activity also relate to pharmaceutically acceptable salts of the compounds; the synthetic route comprises two routes: the synthetic route I is obtained by acid-amine condensation under the conditions of aprotic solvent, organic base and catalyst; the synthetic route II is synthesized by taking ionic liquid as a solvent under the action of a catalyst. The invention also specifically discloses application of the anti-tumor drug, and the anti-tumor drug has better anti-tumor activity through activity screening, and is particularly expected to be popularized and applied in preparing anti-blood tumor drugs.

Description

Ibrutinib acrylamide derivative with anti-tumor activity, and synthetic method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to ibrutinib acrylamide derivatives with anti-tumor activity, and a synthesis method and application thereof.

Background

Malignant tumors are one of the global major public health problems, greatly jeopardizing human health, and will become the first killer for new centuries of human beings. Malignant tumors comprise solid tumors (such as lung cancer, liver cancer, gastric cancer, intestinal cancer and the like) and hematological tumors (such as leukemia, lymphoma and the like), and the occurrence, development and treatment modes of the two diseases are quite different, wherein leukemia and lymphoma are two common hematological tumors.

Ibrutinib (ibutinib) is an oral Bruton's Tyrosine Kinase (BTK) inhibitor of the first new class of drugs for the treatment of Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). The drug can inhibit BTK irreversibly by selectively covalent binding with cysteine residue (Cys-481) at active site of target protein BTK, thereby effectively preventing tumor migration from B cells to lymphoid tissue suitable for tumor growth environment.

Numerous studies have shown that aryl acrylic compounds have good antitumor activity, with cinnamic acid being the most representative compound. Cinnamic acid has effects of inhibiting growth and proliferation, and inducing differentiation of various tumor cells. Cinnamic acid can stimulate activation of peroxisome proliferator-activated receptor (PPAR), inhibit expression of oncogene and protease, destroy invasion ability of tumor cells, and stimulate T cell to generate immune response to tumor cells. Meanwhile, cinnamic acid can block mevalonate pathway, thereby inhibiting synthesis of tumor cell growth regulating protein and blocking proliferation of tumor cells. Based on the characteristics of small molecular weight and low price of the aryl acrylic compound, the aryl acrylic compound has antitumor activity, and is often spliced with other structures with antitumor activity in the design of antitumor drugs so as to synergistically exert the antitumor activity, and the bioactive fragment splicing strategy enables the aryl acrylic compound to be widely applied to pharmaceutical chemistry research.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide ibrutinib acrylamide derivatives with anti-tumor activity, and a synthesis method and application thereof, which are used for preparing anti-tumor drugs.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

the invention discloses a ibrutinib acrylamide derivative with anti-tumor activity, which is a compound shown in a formula I or pharmaceutically acceptable salt thereof, and a solvent compound, enantiomer, diastereomer, tautomer or mixture of any proportion of the compound shown in the formula I or the pharmaceutically acceptable salt thereof, including racemic mixture;

the structural formula of the compound shown in the formula I is as follows:

wherein R is ₁ Is hydrogen or cyano;

r isWherein R is ₂ 、R ₃ 、R ₄ And R is ₅ Each independently selected from a hydrogen atom, a halogen atom, a nitro group, a methoxy group, a trifluoromethyl group, or a dimethylamino group.

Preferably, the representative compound is selected from the following compounds:

preferably, the pharmaceutically acceptable salt is a salt of ibrutinib acrylamide derivative with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

The invention also discloses a method for synthesizing the ibrutinib acrylamide derivative with anti-tumor activity, wherein the synthesis route I is that under the conditions of aprotic solvent, organic base and catalyst, each substituted aryl acrylic compound and 3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-D ] pyrimidine-4-amine undergo acid-amine condensation to prepare the ibrutinib acrylamide derivative; the synthetic route II is that each substituted aryl acrylic compound and 3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-D ] pyrimidine-4-amine are directly taken as raw materials, and ionic liquid is taken as a solvent, and the ibrutinib acrylamide derivative is synthesized under the action of a catalyst.

Preferably, the specific preparation process of scheme I is as follows:

1) Dissolving each substituted aryl acrylic compound, a catalyst and an organic alkali in an aprotic solvent, placing the aprotic solvent in a reactor, and stirring and activating the mixture at room temperature;

2) After complete activation, 3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-D ] pyrimidin-4-amine is dissolved in aprotic solvent and added to the reactor of step 1) for stirring reaction;

3) The reaction is tracked to be complete by thin layer chromatography, the reaction solution is decompressed and concentrated to remove the solvent, and the obtained crude product is washed, extracted, collected into an organic phase, separated, purified and dried to obtain the target product.

Further preferred catalysts in step 1) are 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N '-dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N, N' -diisopropylcarbodiimide, 4-dimethylaminopyridine or 1-hydroxybenzotriazole; the organic base is triethylamine, N-diisopropylethylamine or N-methylmorpholine; the aprotic solvent is dichloromethane, N-dimethylformamide or acetonitrile.

Preferably, the specific preparation process of synthetic route ii is as follows:

1) Adding each substituted aryl propylene compound, 3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-D ] pyrimidine-4-amine and silicomolybdic acid into a reactor, dissolving, introducing nitrogen for protection, and stirring at 25-60 ℃ for reaction;

2) And (3) tracking the reaction to completion by a thin layer chromatography, extracting the reaction liquid, concentrating under reduced pressure, recrystallizing, purifying and drying to obtain a target product.

It is further preferred that the molar ratio of 3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-D ] pyrimidin-4-amine and each substituted aryl acrylic compound in step 1) is 1 (1-1.4).

The invention also discloses application of the ibrutinib acrylamide derivative with anti-tumor activity in preparation of an anti-tumor drug.

Preferably, the tumor is a hematological neoplastic disease.

Further preferred, the hematological neoplastic disease is acute T cell leukemia.

Compared with the prior art, the invention has the following beneficial effects:

the ibrutinib acrylamide derivatives with anti-tumor activity provided by the invention are found by in vitro anti-tumor activity experiments, and the proliferation inhibition rates of the preferred compounds 6, 10 and 14 of the ibrutinib acrylamide derivatives synthesized by the invention on Jurkat cells at the concentration of 1 mu M are respectively 24.13%,23.14% and 25.73%, which are superior to the proliferation inhibition rate of ibrutinib as a positive control drug; cell morphology observation shows that the proliferation quantity of Jurkat cells treated by the compound is obviously reduced, the cells are obviously changed in morphology, and dead death characteristics such as cell shrinkage and the like appear; for Daudi cells, the proliferation inhibition capacity of most ibrutinib cinnamamide compounds at the concentration of 10 mu M is similar to that of ibrutinib serving as a positive control drug, and the partial inhibition rate is more than 90%; observing the morphology of Daudi cells treated by different concentrations of the compound, gradually thinning the cell arrangement along with the increase of the administration concentration of the compound, and generating death characteristics such as cell shrinkage, volume shrinkage and the like. The antitumor activity experiment proves that the ibrutinib acrylamide derivative provided by the invention has potential to be used as a novel antitumor drug to be applied to preparation of antitumor drugs, in particular to preparation of blood tumor drugs, and further to acute T-cell leukemia.

The invention provides a synthesis method of ibrutinib acrylamide derivatives with anti-tumor activity, which utilizes aryl acrylic compounds to carry out structural transformation on 3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-D ] pyrimidine-4-amine (ibrutinib intermediate compound 19) to obtain novel ibrutinib acrylamide derivatives, wherein the use of ionic liquid as a solvent in a synthesis method II is not yet reported, and the preparation method provided by the invention has the advantages of high operation safety, mild reaction conditions and low cost, and the yield of the prepared ibrutinib acrylamide derivatives is higher, is between 62.66 and 83.74 percent, and is suitable for industrial production.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of compound 1 in deuterated chloroform according to the present invention;

FIG. 2 is a nuclear magnetic resonance spectrum of compound 1 of the present invention in deuterated chloroform;

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of compound 8 of the present invention in deuterated chloroform;

FIG. 4 is a nuclear magnetic resonance spectrum of compound 8 of the present invention in deuterated chloroform;

FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of compound 10 of the present invention in deuterated chloroform;

FIG. 6 is a nuclear magnetic resonance spectrum of compound 10 of the present invention in deuterated chloroform;

FIG. 7 is a nuclear magnetic resonance hydrogen spectrum of compound 11 of the present invention in deuterated chloroform;

FIG. 8 is a nuclear magnetic resonance spectrum of compound 11 of the present invention in deuterated chloroform;

FIG. 9 is a graph showing the cytotoxic effect of ibrutinib cinnamamide compounds of the invention on U937 cells at concentrations of 1 μm and 10. Mu.M;

FIG. 10 is a graph showing the cytotoxic effect of ibrutinib cinnamamide compounds of the invention on Jurkat cells at concentrations of 1 μm and 10. Mu.M;

FIG. 11 is a graph showing the cytotoxic effect of ibrutinib cinnamamide compounds of the invention on Daudi cells at concentrations of 1 μm and 10. Mu.M;

FIG. 12 is a morphological image of the Jurkat cells of Compounds 6, 10, 14 of the invention with the positive control drug ibrutinib; wherein a is compound 6 (10 mu M), b is compound 6 (1 mu M), c is compound 10 (10 mu M), d is compound 10 (1 mu M), e is compound 14 (10 mu M), f is compound 14 (1 mu M), g is positive control drug ibrutinib (10 mu M), and h is positive control drug ibrutinib (1 mu M);

FIG. 13 is a morphological image of Daudi cells with the positive control drug ibrutinib and compounds 10, 13, 14 of the present invention; wherein a is compound 10 (10. Mu.M), b is compound 10 (1. Mu.M), c is compound 13 (10. Mu.M), d is compound 13 (1. Mu.M), e is compound 14 (10. Mu.M), f is compound 14 (1. Mu.M), g is the positive control drug ibrutinib (10. Mu.M), and h is the positive control drug ibrutinib (1. Mu.M).

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to fig. 1-13:

the structural formula of the ibrutinib acrylamide derivatives with anti-tumor activity provided by the invention is shown as formula I:

wherein R is ₁ Is hydrogen or cyano;

r isWherein R is ₂ 、R ₃ 、R ₄ And R is ₅ Each independently selected from a hydrogen atom, a halogen atom, a nitro group, a methoxy group, a trifluoromethyl group, or a dimethylamino group; the term "halogen" means fluorine, chlorine, bromine or iodine.

The ibrutinib acrylamide derivatives with anti-tumor activity provided by the invention also comprise a solvent compound, enantiomer, diastereoisomer, tautomer or mixture of any proportion of the compounds shown in the formula I or pharmaceutically acceptable salts thereof, including racemic mixtures.

The invention provides ibrutinib acrylamide derivatives with anti-tumor activity, which relate to pharmaceutically acceptable salts of the compounds, wherein the pharmaceutically acceptable salts are salts formed by ibrutinib acrylamide derivatives, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid; the pharmaceutically acceptable salt preferably has the following structural formula:

the synthetic route for synthesizing the ibrutinib acrylamide derivative with the anti-tumor activity is shown as follows:

the synthetic route I is that each substituted aryl acrylic compound and 3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-D ] pyrimidine-4-amine (compound 19) are subjected to acid-amine condensation under the conditions of aprotic solvent, organic base and catalyst; the synthetic route II is obtained by directly taking each substituted aryl acrylic compound and ibrutinib intermediate (compound 19) as raw materials, taking ionic liquid as a solvent, and synthesizing under the action of a catalyst;

wherein the structure of compound 19 is shown in the following figure:

the synthetic route I of the compound provided by the invention is shown as follows:

in order to realize the above synthetic route I, the synthetic steps of the invention are as follows:

(1) Dissolving a substituted aryl acrylic compound, a catalyst and an organic alkali in an aprotic solvent, placing the aprotic solvent in a reactor, and stirring and activating the mixture at room temperature for 1h;

wherein the catalyst is 2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethyl urea Hexafluorophosphate (HATU), N, N ' -Dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), N, N ' -Diisopropylcarbodiimide (DIC), 1H-benzotriazol-1-yloxytripyrrolidine hexafluorophosphate (PyBOP), N, N ' -Carbonyldiimidazole (CDI) or benzotriazole-N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HBTU), O- (6-chlorobenzotriazol-1-yl) -N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HCTU), 4-Dimethylaminopyridine (DMAP), 1-Hydroxybenzotriazole (HOBT) or 4-pyrrolidinylpyridine (4-PPY), more preferably 2- (7-azobenzotriazol) -N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HATU); the organic base is Triethylamine (TEA), N-Diisopropylethylamine (DIEA), N-methylmorpholine (NMM), morpholine or pyridine, more preferably N, N-Diisopropylethylamine (DIEA); the aprotic solvent is dichloromethane, N-dimethylformamide, N-dimethylacetamide or acetonitrile, and further preferably is dichloromethane;

(2) After the activation is completed, the compound 19 is dissolved in an aprotic solvent, and is added dropwise into the reactor in the step (1) for stirring reaction for 8-12h;

(3) The reaction is tracked to be complete by thin layer chromatography, the reaction solution is decompressed and concentrated to remove the solvent, the obtained crude product is washed by saturated sodium chloride aqueous solution, extracted by ethyl acetate, the organic phase is collected, separated and purified by column chromatography, and the target product is obtained by drying.

The synthetic route II of the compound provided by the invention is shown as follows:

in order to realize the synthetic route II, the synthetic steps of the invention are as follows:

(1) Adding a substituted aryl propylene compound, a compound 19 and a catalyst silicon molybdic acid into a reactor, dissolving by using ionic liquid 1-butyl-3-methylimidazole tetrafluoroborate, introducing nitrogen for protection, and stirring at 25-60 ℃ for reaction for 4-8h;

wherein the molar ratio of the compound 19 to each substituted aryl acrylic compound is 1 (1-1.4), and is further optimized to be 1 (1-1.2); the reaction temperature is more preferably 30 ℃, and the reaction time is more preferably 5 hours;

(2) And (3) tracking the reaction to completion by a thin layer chromatography, removing the catalyst and the ionic liquid from the reaction liquid by extraction and decompression concentration, then recrystallizing, purifying and drying to obtain a target product.

1. Specific examples of Synthesis of Compounds 1-15

The structural formula and numbering of representative compounds are as follows:

examples of the synthesis of the above compounds are given below, the structure of which is characterized by NMR.

Example 1

Compound 1: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3-phenylprop-2-en-1-one

74.1mg (0.5 mmol) of cinnamic acid was dissolved in 3mL of methylene chloride and placed in a reactor, to which were added HATU 380.2mg (1 mmol) and DIEA 260. Mu.L (1.5 mmol), followed by activation with stirring at room temperature for 1 hour. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of dichloromethane, added dropwise to the above reactor, and then reacted at room temperature for 8 hours with stirring, followed by TLC monitoring. After the reaction, the obtained reaction solution was concentrated under reduced pressure to remove dichloromethane, the obtained solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phase was collected, separated and purified by column chromatography (200 to 300 mesh silica gel powder as stationary phase, dichloromethane: methanol (V: V) =10:1 as mobile phase), and then dried to obtain 216.3mg of the objective compound 1 in 83.74% yield. The nuclear magnetic hydrogen spectrum of the compound 1 in deuterated chloroform is shown in fig. 1, and the nuclear magnetic carbon spectrum is shown in fig. 2.

¹ H NMR(400MHz,CDCl ₃ )δ8.42(s,1H),7.70(d,J＝8.2Hz,3H),7.60–7.37(m,7H),7.27–7.17(m,3H),7.13(d,J＝8.0Hz,2H),6.96(t,J＝19.3Hz,1H),5.95–5.74(m,1H),5.09–4.87(m,1.5H),4.61(d,J＝12.8Hz,0.5H),4.45–4.15(m,1H),3.92(t,J＝11.8Hz,0.5H),3.58–3.22(m,1H),3.11(t,J＝13.3Hz,0.5H),2.59–2.25(m,2H),2.21–2.03(m,1H),1.88–1.73(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.88,158.61,157.58,156.32,155.04,154.19,144.09,143.08,135.25,129.99,129.61,129.21,128.77,127.77,126.12,124.08,119.57,119.14,117.24,98.58,56.31,51.46,38.61,30.80,28.35.

Example 2

Compound 2: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (2-fluorophenyl) prop-2-en-1-one

99.7mg (0.6 mmol) of 2-fluorocinnamic acid, 193.3mg (0.5 mmol) of compound 19 and 8.6mg (5 mu mol) of silicomolybdic acid are sequentially placed in a 150mL reactor, 71.5mL of ionic liquid 1-butyl-3-methylimidazole tetrafluoroborate is added for complete dissolution, and N is introduced ₂ The reaction was stirred at 30℃for 5h. The reaction was followed to completion by thin layer chromatography and the protection device was removed. And (3) placing the reaction mixed system in a separating funnel, oscillating, standing for layering, and separating an ionic liquid layer from an ester layer, wherein the obtained ester layer is a crude product of the cinnamate derivative. Recrystallization drying is carried out by 50mL of methanol to obtain 212.1mg of target product compound 2, and the total yield is 79.35%.

¹ H NMR(400MHz,CDCl ₃ )δ8.39(s,1H),7.80–7.66(m,3H),7.59–7.39(m,4H),7.23–7.03(m,8H),6.04-5.79(m,1H),5.01–4.91(m,1.5H),4.57(d,J＝13.0Hz,0.5H),4.37–4.12(m,1H),3.92(t,J＝11.8Hz,0.5H),3.49(t,J＝12.6Hz,0.5H),3.30(t,J＝13.1Hz,0.5H),3.11(t,J＝12.3Hz,0.5H),2.69–2.27(m,2H),2.16–2.04(m,1H),1.88–1.74(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.96,161.32,158.64,157.44,156.42,155.03,154.18,144.10,143.29,129.96,128.77,128.09,127.56,126.09,124.67,124.32,123.17,119.59,119.15,118.84,115.47,98.67,56.50,51.48,38.63,30.79,28.36.

Example 3

Compound 3: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (4-fluorophenyl) prop-2-en-1-one

83.1mg (0.5 mmol) of 4-fluorocinnamic acid was dissolved in 3mL of methylene chloride, and then placed in a reactor, to which were added HATU 380.2mg (1 mmol) and TEA 210. Mu.L (1.5 mmol), followed by stirring and activation at room temperature for 1 hour. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of dichloromethane, added dropwise to the above reactor, and then reacted at room temperature for 8 hours with stirring, followed by TLC monitoring. After the reaction, the obtained reaction solution was concentrated under reduced pressure to remove dichloromethane, the obtained solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phase was collected, separated and purified by column chromatography (200 to 300 mesh silica gel powder as stationary phase, dichloromethane: methanol (V: V) =10:1 as mobile phase), and then dried to obtain 211.9mg of the objective compound 3 in 79.28% yield.

¹ H NMR(400MHz,CDCl ₃ )δ8.39(s,1H),7.82–7.59(m,3H),7.46–7.39(m,4H),7.25–7.15(m,3H),7.15–7.00(m,4H),6.86(t,J＝18.6Hz,1H),6.00–5.76(m,1H),5.00–4.90(m,1.5H),4.58(d,J＝12.4Hz,0.5H),4.38–4.13(m,1H),3.92(t,J＝11.7Hz,0.5H),3.47(t,J＝12.4Hz,0.5H),3.36–3.21(m,0.5H),3.07(t,J＝12.1Hz,0.5H),2.59–2.26(m,2H),2.08(d,J＝13.6Hz,1H),1.89–1.73(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.78,161.12,158.54,157.34,156.34,155.09,154.29,144.11,142.36,131.22,130.10,128.59,127.65,126.08,123.99,119.56,119.14,118.42,115.43,98.56,56.26,51.57,38.64,30.75,28.38.

Example 4

Compound 4: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (3, 4-difluorophenyl) prop-2-en-1-one

92.1mg (0.5 mmol) of 3, 4-difluorocinnamic acid was dissolved in 3mL of methylene chloride, and the mixture was placed in a reactor, to which were added HATU 380.2mg (1 mmol) and NMM 170. Mu.L (1.5 mmol), and the mixture was stirred and activated at room temperature for 1 hour. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of dichloromethane, added dropwise to the above reactor, and then reacted at room temperature for 10 hours with stirring, followed by TLC monitoring. After the reaction, the obtained reaction solution was concentrated under reduced pressure to remove dichloromethane, the obtained solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phase was collected, separated and purified by column chromatography (200 to 300 mesh silica gel powder as stationary phase, dichloromethane: methanol (V: V) =10:1 as mobile phase), and dried to obtain 218.1mg of the objective compound 4 in 71.57% yield.

¹ H NMR(400MHz,CDCl ₃ )δ8.41(s,1H),7.61–7.40(m,3H),7.42(t,J＝8.0Hz,2H),7.25–7.13(m,3H),7.13–7.00(m,4H),6.83-6.75(m,2H),5.96–5.80(m,1H),5.02–4.89(m,1.5H),4.56(d,J＝12.8Hz,0.5H),4.39–4.10(m,1H),3.92(t,J＝11.6Hz,0.5H),3.48(t,J＝12.7Hz,0.5H),3.38–3.20(m,0.5H),3.10(t,J＝12.0Hz,0.5H),2.63–2.26(m,2H),2.10(d,J＝13.5Hz,1H),1.89–1.74(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.88,158.62,157.44,156.32,155.01,154.22,149.43,148.72,144.13,143.02,132.46,128.73 127.67,126.14,125.92,125.14,124.08,119.56,119.22,118.53,112.83,98.66,56.43,51.36,38.65,30.73,28.35.

Example 5

Compound 5: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (3, 4, 5-trifluorophenyl) prop-2-en-1-one

121.3mg (0.6 mmol) of 3,4, 5-trifluoro cinnamic acid was dissolved in 3mL of N, N-dimethylformamide, and then the mixture was placed in a reactor, to which were added 191.7mg (1 mmol) of EDCI, 135.1mg (1 mmol) of HOBT and 260. Mu.L (1.5 mmol) of DIEA, followed by stirring and activation at room temperature for 1 hour. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of N, N-dimethylformamide and added dropwise to the above reactor, followed by stirring at room temperature for 10 hours, followed by TLC monitoring. After the reaction is finished, the obtained reaction solution is washed by saturated sodium chloride aqueous solution, extracted by ethyl acetate, an organic phase is collected, column chromatography is used for separation and purification (200-300 meshes of silica gel powder is used as a stationary phase, methylene dichloride is used as methanol (V) =10:1 is used as a mobile phase), and 230.3mg of target product compound 5 is obtained by drying, and the yield is 80.72%.

¹ H NMR(400MHz,CDCl ₃ )δ8.40(s,1H),7.61–7.40(m,2H),7.42(t,J＝8.3Hz,2H),7.25–7.15(m,3H),7.12–7.01(m,3H),6.84-6.74(m,3H),5.99–5.80(m,1H),5.00–4.88(m,1.5H),4.55(d,J＝12.7Hz,0.5H),4.40–4.10(m,1H),3.91(t,J＝11.4Hz,0.5H),3.48(t,J＝12.6Hz,0.5H),3.40–3.21(m,0.5H),3.10(t,J＝12.2Hz,0.5H),2.62–2.26(m,2H),2.11(d,J＝13.5Hz,1H),1.86–1.75(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.82,159.53,158.61,157.43,156.23,154.94,154.13,144.12,143.29,139.33,134.02,128.63,127.77,126.10,124.09,119.65,119.14,118.56,108.42,98.66,56.53,51.53,38.55,30.68,28.33.

Example 6

Compound 6: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (4- (trifluoromethyl) phenyl) prop-2-en-1-one

129.7mg (0.6 mmol) of 4-trifluoromethyl cinnamic acid, 193.3mg (0.5 mmol) of compound 19 and 8.6mg (5 mu mol) of silicomolybdic acid are sequentially placed in a 150mL reactor, 71.5mL of ionic liquid 1-butyl-3-methylimidazole tetrafluoroborate is added for complete dissolution, and N is introduced ₂ Protection, reaction at 25℃for 8h. The reaction was followed to completion by thin layer chromatography and the protection device was removed. And (3) placing the reaction mixed system in a separating funnel, oscillating, standing for layering, and separating an ionic liquid layer from an ester layer, wherein the obtained ester layer is a crude product of the cinnamate derivative. Recrystallization drying with 50mL of methanol gave 218.5mg of the desired product compound 6 in a total yield of 74.76%.

¹ H NMR(400MHz,CDCl ₃ )δ8.40(s,1H),7.80–7.57(m,3H),7.47–7.39(m,4H),7.27–7.14(m,3H),7.15–7.01(m,4H),6.83(t,J＝18.7Hz,1H),6.05–5.79(m,1H),5.01–4.90(m,1.5H),4.56(d,J＝12.6Hz,0.5H),4.37–4.13(m,1H),3.96(t,J＝11.8Hz,0.5H),3.48(t,J＝12.0Hz,0.5H),3.35–3.24(m,0.5H),3.0(t,J＝12.0Hz,0.5H),2.63–2.29(m,2H),2.09(d,J＝13.5Hz,1H),1.88–1.73(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ166.03,158.63,157.34,156.35,155.09,154.21,144.01,143.26,138.52,130.29,129.03,128.79,127.78,126.09,125.06,124.69,123.98,119.52,119.24,118.84,98.50,56.39,51.46,38.57,30.81,28.30.

Example 7

Compound 7: preparation of N- (2- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) propyl) -N-methyl-3- (4-nitrophenyl) acrylamide

115.9mg (0.6 mmol) of 4-nitrocinnamic acid was dissolved in 3mL of N, N-dimethylformamide, and the mixture was placed in a reactor, followed by adding 206.3mg (1 mmol) of DCC and 12.2mg (0.1 mmol) of DMAP thereto, and stirring and activating at room temperature for 1 hour. 193.3mg (0.5 mmol) of compound 19 was dissolved in 5mL of N, N-dimethylformamide and added dropwise to the above reactor, followed by stirring at room temperature for 12 hours, followed by TLC monitoring. After the reaction is finished, the obtained reaction solution is washed by saturated sodium chloride aqueous solution, extracted by ethyl acetate, an organic phase is collected, column chromatography is used for separation and purification (200-300 meshes of silica gel powder is used as a stationary phase, methylene dichloride is used as methanol (V) =10:1 is used as a mobile phase), and 209.4mg of target product compound 7 is obtained by drying, and the yield is 76.21%.

¹ H NMR(400MHz,CDCl ₃ )δ8.42(s,1H),8.20–8.00(m,4H),7.60–7.37(m,5H),7.28–7.16(m,3H),7.14(d,J＝8.2Hz,2H),6.96(t,J＝19.2Hz,1H),5.96–5.76(m,1H),5.09–4.86(m,1.5H),4.62(d,J＝12.7Hz,0.5H),4.46–4.15(m,1H),3.93(t,J＝11.8Hz,0.5H),3.60–3.22(m,1H),3.11(t,J＝13.4Hz,0.5H),2.59–2.25(m,2H),2.21–2.03(m,1H),1.88–1.73(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.94,158.60,157.43,156.45,155.06,154.17,147.10,144.06,143.12,141.24,129.09,128.80,127.77,126.18,124.11,123.81,119.55,119.24,118.45,98.89,56.51,51.58,38.58,30.81,28.37.

Example 8

Compound 8: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (4- (dimethylamino) phenyl) prop-2-en-1-one

114.7mg (0.6 mmol) of 4- (dimethylamino) cinnamic acid was dissolved in 3mL of N, N-dimethylformamide, and the mixture was placed in a reactor, to which 126.2mg (1 mmol) of DIC, 135.1mg (1 mmol) of HOBT and 260. Mu.L (1.5 mmol) of DIEA were added, followed by stirring and activation at room temperature for 1 hour. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of N, N-dimethylformamide and added dropwise to the above reactor, followed by stirring at room temperature for 10 hours, followed by TLC monitoring. After the reaction is finished, the obtained reaction solution is washed by saturated sodium chloride aqueous solution, extracted by ethyl acetate, an organic phase is collected, column chromatography is used for separation and purification (200-300 meshes of silica gel powder is used as a stationary phase, methylene dichloride is used as methanol (V) =10:1 is used as a mobile phase), and 214.3mg of target product compound 8 is obtained by drying, and the yield is 76.59%. The nuclear magnetic hydrogen spectrum of the compound 8 in deuterated chloroform is shown in fig. 3, and the nuclear magnetic carbon spectrum is shown in fig. 4.

¹ H NMR(400MHz,CDCl ₃ )δ8.42(s,1H),7.71(d,J＝8.0Hz,3H),7.54-7.36(m,4H),7.21(t,J＝6.6Hz,3H),7.13(d,J＝8.0Hz,2H),6.83–6.64(m,3H),5.97–5.72(m,1H),5.08–4.90(m,1.5H),4.66(d,J＝12.7Hz,0.5H),4.47–4.17(m,1H),3.85(t,J＝10.0Hz,0.5H),3.50–3.21(m,1H),3.12–3.02(m,6.5H),2.48–2.30(m,2H),2.07(d,J＝13.7Hz,1H),1.90-1.75(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ166.61,158.53,157.80,156.36,155.50,154.34,151.35,143.93,143.60,129.98,129.34,127.84,126.21,124.04,123.18,119.53,119.16,118.85,111.61,98.58,56.35,51.52,40.21,38.61,30.62,28.27.

Example 9

Compound 9: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (4-methoxyphenyl) prop-2-en-1-one

98.0mg (0.55 mmol) of 4-methoxycinnamic acid was dissolved in 3mL of acetonitrile, and the mixture was placed in a reactor, to which were added HATU 380.2mg (1 mmol) and TEA 210. Mu.L (1.5 mmol), followed by stirring and activation at room temperature for 1 hour. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of acetonitrile, added dropwise to the above reactor, and then reacted at room temperature for 10 hours with stirring, followed by TLC monitoring. After the reaction, the obtained reaction solution was concentrated under reduced pressure to remove acetonitrile, the solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phase was collected, separated and purified by column chromatography (200 to 300 mesh silica gel powder as stationary phase, methylene chloride: methanol (V: V) =10:1 as mobile phase), and dried to obtain 217.7mg of the objective compound 9 in 79.64% yield.

¹ H NMR(400MHz,CDCl ₃ )δ8.39(s,1H),7.74–7.56(m,5H),7.42(t,J＝7.8Hz,2H),7.26–6.97(m,5H),6.93-6.74(m,3H),6.05–5.73(m,1H),5.06–4.89(m,1.5H),4.62(d,J＝12.3Hz,0.5H),4.44–4.11(m,1H),4.07-3.81(m,3.5H),3.51–3.22(m,1H),3.12(t,J＝13.4Hz,0.5H),2.56–2.20(m,2H),2.06(d,J＝12.9Hz,1H),1.89–1.74(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.93,159.83,158.63,157.44,156.26,155.01,154.13,144.13,143.43,130.27,128.85,127.85,127.46,126.05,124.09,119.54,119.14,118.32,114.22,98.56,56.32,55.76,51.48,38.57,30.77,28.26.

Example 10

Compound 10: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (3, 4-dimethoxyphenyl) prop-2-en-1-one

145.7mg (0.7 mmol) of 3, 4-dimethoxy cinnamic acid, 193.3mg (0.5 mmol) of Compound 19 and 8.6mg (5. Mu. Mol) of silicomolybdic acid were successively placed in a 150mL reactorAdding 71.5mL of ionic liquid 1-butyl-3-methylimidazole tetrafluoroborate for complete dissolution, and introducing N ₂ Protection, reaction at 30℃for 8h. The reaction was followed to completion by thin layer chromatography and the protection device was removed. And (3) placing the reaction mixed system in a separating funnel, oscillating, standing for layering, and separating an ionic liquid layer from an ester layer, wherein the obtained ester layer is a crude product of the cinnamate derivative. Recrystallization drying is carried out by 50mL of methanol to obtain 231.0mg of target product compound 10, and the total yield is 80.12%. The nuclear magnetic hydrogen spectrum of compound 10 in deuterated chloroform is shown in fig. 5, and the nuclear magnetic carbon spectrum is shown in fig. 6.

¹ H NMR(400MHz,CDCl ₃ )δ8.38(s,1H),7.71–7.59(m,3H),7.42(t,J＝7.7Hz,2H),7.24–6.97(m,7H),6.93–6.71(m,2H),6.04–5.76(m,1H),5.01–4.89(m,1.5H),4.61(s,0.5H),4.42–4.14(m,1H),4.01-3.84(m,6.5H),3.52–3.22(m,1H),3.04(s,0.5H),2.49–2.31(m,2H),2.07(d,J＝13.5Hz,1H),1.87–1.72(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ166.08,158.61,157.78,156.30,155.06,154.08,150.54,149.07,144.05,143.53,129.99,128.24,127.61,126.10,124.10,121.84,119.57,119.14,114.88,111.05,109.95,98.65,56.51,55.96,51.42,38.62,30.82,28.36.

Example 11

Compound 11: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (furan-2-yl) prop-2-en-1-one

82.9mg (0.6 mmol) of 3- (2-furyl) acrylic acid was dissolved in 3mL of acetonitrile and placed in a reactor, EDCI 191.7mg (1 mmol), HOBT 135.1mg (1 mmol) and TEA 210. Mu.L (1.5 mmol) were added thereto, and the mixture was stirred and activated at room temperature for 1 hour to prepare a product. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of acetonitrile, added dropwise to the above reactor, and then reacted at room temperature with stirring for 12 hours, followed by TLC monitoring. After the reaction, the obtained reaction solution was concentrated under reduced pressure to remove acetonitrile, the obtained solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phase (200 to 300 mesh silica gel powder as stationary phase, methylene chloride: methanol (V: V) =10:1 as mobile phase) was collected, and dried to obtain 187.2mg of the objective product compound 11 in a yield of 73.89%. The nuclear magnetic hydrogen spectrum of compound 11 in deuterated chloroform is shown in fig. 7, and the nuclear magnetic carbon spectrum is shown in fig. 8.

¹ H NMR(400MHz,CDCl ₃ )δ8.39(s,1H),7.68(d,J＝8.1Hz,2H),7.56–7.37(m,4H),7.24–7.15(m,3H),7.12(d,J＝8.0Hz,2H),6.84(dd,J＝41.1,15.2Hz,1H),6.52(dd,J＝35.5,14.0Hz,2H),6.09–5.77(m,1H),5.04–4.88(m,1.5H),4.60(d,J＝14.8Hz,0.5H),4.38–4.11(m,1H),4.03–3.87(m,0.5H),3.52–3.20(m,1H),3.05(t,J＝11.6Hz,0.5H),2.53–2.25(m,2H),2.16–2.01(m,1H),1.87–1.71(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.56,158.60,157.87,156.35,155.05,153.89,151.66,144.04,143.91,130.00,128.75,127.84,126.18,124.90,124.08,119.58,119.15,114.55,112.20,98.53,56.32,51.51,38.64,30.80,28.36.

Example 12

Compound 12: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (thiophen-2-yl) prop-2-en-1-one

92.5mg (0.6 mmol) of (E) -3- (thiophen-2-yl) acrylic acid was dissolved in 3mL of methylene chloride, and then, the mixture was placed in a reactor, EDCI 191.7mg (1 mmol), HOBT 135.1mg (1 mmol) and DIEA 260. Mu.L (1.5 mmol) were added thereto, and the mixture was stirred and activated at room temperature for 1 hour. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of dichloromethane, added dropwise to the above reactor, and then reacted at room temperature for 8 hours with stirring, followed by TLC monitoring. After the reaction, the obtained reaction solution was concentrated under reduced pressure to remove dichloromethane, the obtained solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phase was collected, separated and purified by column chromatography (200 to 300 mesh silica gel powder as stationary phase, dichloromethane: methanol (V: V) =10:1 as mobile phase), and dried to obtain 183.9mg of the objective compound 12 in 70.37% yield.

¹ H NMR(400MHz,CDCl ₃ )δ8.40(s,1H),7.70(d,J＝8.3Hz,2H),7.58–7.36(m,4H),7.25–7.16(m,4H),7.12(d,J＝8.2Hz,2H),6.53(dd,J＝35.6,14.0Hz,2H),6.10–5.78(m,1H),5.05–4.89(m,1.5H),4.60(d,J＝13.4Hz,0.5H),4.38–4.12(m,1H),3.96(t,J＝11.5Hz,0.5H),3.52–3.19(m,1H),3.06(t,J＝11.5Hz,0.5H),2.52–2.25(m,2H),2.16–2.01(m,1H),1.86–1.73(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.86,158.62,157.52,156.32,155.00,154.19,144.10,140.33,132.92,130.55,129.63,129.03,128.77,127.63,126.13,124.87,124.11,119.59,119.14,98.59,56.36,51.54,38.59,30.76,28.35.

Example 13

Compound 13: preparation of 4- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -4-oxo-2- (thiophen-2-yl) but-2-enenitrile

130.4mg (0.6 mmol) of (Z) -3-cyano-3- (thiophen-2-yl) acrylic acid potassium, 193.3mg (0.5 mmol) of compound 19 and 8.6mg (5 mu mol) of silicomolybdic acid are placed in a 150mL reactor in sequence, 71.5mL of ionic liquid 1-butyl-3-methylimidazole tetrafluoroborate is added for complete dissolution, and N is introduced ₂ Protection, reaction for 4h at 60 ℃. The reaction was followed to completion by thin layer chromatography and the protection device was removed. And (3) placing the reaction mixed system in a separating funnel, oscillating, standing for layering, and separating an ionic liquid layer from an ester layer, wherein the obtained ester layer is a crude product of the cinnamate derivative. Recrystallization drying is carried out by 50mL of methanol to obtain 171.6mg of target product compound 13, and the total yield is 62.66%.

¹ H NMR(400MHz,CDCl ₃ )δ8.39(s,1H),7.86(d,J＝8.3Hz,2H),7.62–7.33(m,6H),7.25–7.15(m,3H),7.12(d,J＝8.0Hz,2H),6.09–5.77(m,1H),5.05–4.68(m,1.5H),4.58(d,J＝13.2Hz,0.5H),4.3 6–4.11(m,1H),3.95(t,J＝11.5Hz,0.5H),3.51–3.21(m,1H),3.06(t,J＝11.6Hz,0.5H),2.54–2.25(m,2H),2.16–1.99(m,1H),1.86–1.71(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.87,158.63,157.52,156.28,155.04,154.19,144.12,140.05,136.66,130.58,128.77,128.34,127.75,127.19,126.13,124.52,124.12,119.62,119.14,118.87,98.57,56.36,51.54,38.61,30.79,28.38.

Example 14

Compound 14: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (pyridin-4-yl) prop-2-en-1-one

89.5mg (0.6 mmol) of 3- (pyridine-4-) acrylic acid was dissolved in 3mL of acetonitrile and placed in a reactor, HATU 380.2mg (1 mmol) and DIEA 260. Mu.L (1.5 mmol) were added thereto, and the mixture was stirred at room temperature for activation for 1 hour for use. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of acetonitrile, added dropwise to the above reactor, and then reacted at room temperature for 10 hours with stirring, followed by TLC monitoring. After the reaction, the obtained reaction solution was concentrated under reduced pressure to remove acetonitrile, the obtained solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phase was collected (200 to 300 mesh silica gel powder as stationary phase, methylene chloride: methanol (V: V) =10:1 as mobile phase), and dried to obtain 195.2mg of the objective compound 14 in 75.44% yield.

¹ H NMR(400MHz,CDCl ₃ )δ8.73-8.66(m,2H),8.41(s,1H),7.69(d,J＝8.3Hz,3H),7.60–7.37(m,4H),7.30–7.17(m,3H),7.14(d,J＝8.0Hz,2H),6.96(t,J＝19.4Hz,1H),5.95–5.74(m,1H),5.09–4.87(m,1.5H),4.61(d,J＝12.8Hz,0.5H),4.45–4.15(m,1H),3.92(t,J＝11.8Hz,0.5H),3.56–3.23(m,1H),3.11(t,J＝13.4Hz,0.5H),2.60–2.25(m,2H),2.20–2.03(m,1H),1.89–1.73(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.82,158.62,157.42,156.32,155.02,154.19,149.66,144.53,144.03,143.26,128.99,127.67,126.16,125.46,124.09,123.03,119.54,119.14,98.57,56.34,51.47,38.62,30.78,28.37.

Example 15

Compound 15: preparation of 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -3- (naphthalen-1-yl) prop-2-en-1-one

118.9mg (0.6 mmol) of (E) -3- (naphthalen-1-yl) acrylic acid are dissolved in 3mL of N, N-dimethylformamide, HATU 380.2mg (1 mmol) and DIEA 260. Mu.L (1.5 mmol) are added and the mixture is stirred at room temperature for activation for 1 hour. 193.3mg (0.5 mmol) of compound 19 was dissolved in 3mL of N, N-dimethylformamide and added dropwise to the above reactor, and the reaction was stirred at room temperature for 10 hours, monitored by TLC. After the reaction is finished, the obtained reaction solution is washed by saturated sodium chloride aqueous solution, extracted by ethyl acetate, an organic phase is collected, column chromatography is used for separation and purification (200-300 meshes of silica gel powder is used as a stationary phase, methylene dichloride is used as methanol (V) =10:1 is used as a mobile phase), and 222.3mg of target product compound 15 is obtained by drying, and the yield is 78.46%.

¹ H NMR(400MHz,CDCl ₃ )δ8.43(s,1H),8.00-7.90(m,4H),7.75–7.62(m,2H),7.60–7.37(m,5H),7.27–7.17(m,3H),7.10(d,J＝8.0Hz,2H),6.96(t,J＝18.9Hz,1H),5.96–5.74(m,1H),5.09–4.88(m,1.5H),4.61(d,J＝12.9Hz,0.5H),4.45–4.12(m,1H),3.93(t,J＝11.8Hz,0.5H),3.89–3.21(m,1H),3.12(t,J＝13.4Hz,0.5H),2.60–2.25(m,2H),2.20–2.03(m,1H),1.88–1.72(m,1H).

¹³ C NMR(101MHz,CDCl ₃ )δ165.89,158.61,157.44,156.30,155.01,154.27,144.09,143.91,133.67,132.89,132.04,129.07,128.77,128.31,127.77,126.97,126.37,126.08,125.94,124.04,123.83,122.95,119.57,119.14,118.83,98.55,56.33,51.47,38.62,30.77,28.39.

2. Compound antitumor cell proliferation assay and morphological influence of compound on tumor cells

The measurement principle is as follows: the ability of a compound to inhibit tumor cell proliferation can be measured using the CCK-8 method. CCK-8 is a detection reagent based on WST-8, which is widely applied to cell proliferation and cytotoxicity. WST-8 is reduced by a dehydrogenase in the mitochondria to orange yellow formazan (formazan) with high water solubility in the presence of the electron carrier dimethyl 1-methoxy-5-methylphenazinium sulfate. The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color, and for the same cells, the shade of color is proportional to the number of living cells, so that cell proliferation and toxicity assays can be directly performed using this property.

Experimental materials: CCK-8 kit (Shanghai Biyun biotechnology Co., ltd., C0038); positive control drug ibrutinib (Shanghai microphone Biochemical technologies Co., ltd.); u937 (human histiocyte lymphoma cells), jurkat (human T lymphocyte leukemia cells) and Daudi (human Burkitt's lymphoma cells), all three cells were purchased from ATCC pool in the United states.

Cell culture and administration: containing 5% CO at 37 DEG C ₂ In an incubator containing 10% fetal bovine serum in RPMI-1640 medium. The compound is prepared into a solution with mother liquor concentration of 1mM by using DMSO, and the solution is placed in a refrigerator with the temperature of-20 ℃ and diluted into corresponding concentration according to experimental requirements.

The experimental method comprises the following steps: counting cells in good logarithmic phase, and making into 5×10 ⁴ cell/mL suspension, cell suspension was seeded into 96-well plates at 100. Mu.L per well, placed at 37℃in 5% CO ₂ After 24h of incubation, 10. Mu.L of medium containing different concentrations of compound was then added to the 96-well plate, and the compound concentration gradient was set to 1. Mu.M and 10. Mu.M. Each group was provided with 3 duplicate wells, and a blank control group (no drug added) and a positive control group (ibrutinib). Placed at 37 ℃ and 5% CO ₂ After incubation in a constant temperature incubator for 24 hours, 10. Mu.L of CCK-8 solution was added to each well, and the incubation in the incubator was continued for 1 hour. Finally, the absorbance (OD) of the 96-well plate is detected by an enzyme-labeled instrument at the wavelength of 450nm, and the morphological influence of the compound on tumor cells is observed under an optical microscope. The cell proliferation inhibition rate was calculated according to the following formula.

Cell proliferation inhibition (%) = (1-average OD of experimental group/average OD of blank control group) ×100%

The results of the anti-tumor cell proliferation capacity of the ibrutinib cinnamamide compound prepared by the invention are shown in table 1:

table 1: anti-tumor cell proliferation inhibition rate of ibrutinib cinnamamide compound

As can be seen from Table 1, the ibrutinib cinnamamide compounds prepared by the invention have proliferation inhibition activities to different degrees on U937 cells, jurkat cells and Daudi cells, and have excellent inhibition rate at the concentration of 10 mu M and concentration-dependent inhibition activity. For U937 cells, the proliferation inhibition capacity of compounds 1-15 was lower than that of the positive control drug ibrutinib (fig. 9); for Jurkat cells, the proliferation inhibition rates of compounds 6, 10 and 14 were 24.13%,23.14% and 25.73%, respectively, at a concentration of 1 μm, which is superior to the proliferation inhibition rate of the positive control drug Ibrutinib (Ibrutinib) of 19.98% (fig. 10). Cell morphology observation shows that the proliferation number of Jurkat cells treated by the compound is obviously reduced, the cells are also obviously changed in morphology, and dead death characteristics such as cell shrinkage and the like appear (figure 12); for Daudi cells, at the concentration of 10 mu M, the proliferation inhibition capacity of most of ibrutinib cinnamamide compounds is similar to that of ibrutinib serving as a positive control drug, and the partial inhibition rate is more than 90%; whereas at low concentrations, compounds 10, 13 and 14 were superior in proliferation inhibition capacity (FIG. 11). The morphology of Daudi cells treated with different concentrations of compound was observed, and as the concentration of compound administered increased, the cell arrangement was gradually sparse, and dead characteristics such as cell shrinkage and volume shrinkage were observed (fig. 13).

The experimental results show that the ibrutinib cinnamamide compound provided by the invention can inhibit proliferation and migration of different types of tumor cells, and has stronger inhibition capability on Jurkat cells and Daudi cells, so that the ibrutinib cinnamamide compound can be applied to preparation of anti-acute T cell leukemia and human Burkitt's lymphoma drugs.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (9)

1. The ibrutinib acrylamide derivatives with antitumor activity are characterized by being compounds shown in a formula I or pharmaceutically acceptable salts thereof;

the structural formula of the compound shown in the formula I is as follows:

wherein R is ₁ Is cyano-substituted;

r isOr->。

2. The ibrutinib acrylamide derivatives with antitumor activity according to claim 1, wherein the structural formula of the representative compounds is as follows:

。

3. the ibrutinib acrylamide derivatives having antitumor activity according to claim 1, wherein the pharmaceutically acceptable salt is a salt of ibrutinib acrylamide derivative with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

4. The method for synthesizing the ibrutinib acrylamide derivatives with anti-tumor activity according to any one of claims 1-3, wherein the synthetic route I is characterized in that each substituted aryl acrylic compound and 3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-D ] pyrimidine-4-amine are subjected to acid-amine condensation under the conditions of aprotic solvent, organic base and catalyst to prepare ibrutinib acrylamide derivatives; the synthetic route II is that each substituted aryl acrylic compound and 3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-D ] pyrimidine-4-amine are directly taken as raw materials, and ionic liquid is taken as a solvent to prepare the ibrutinib acrylamide derivative through synthesis under the action of a catalyst;

wherein each substituted aryl acrylic compound has the structural formula ofThe method comprises the steps of carrying out a first treatment on the surface of the Wherein R is ₁ Is cyano-substituted; r is->Or->。

5. The method according to claim 4, wherein the specific preparation process of the synthetic route I is as follows:

1) Dissolving each substituted aryl acrylic compound, a catalyst and an organic alkali in an aprotic solvent, placing the aprotic solvent in a reactor, and stirring and activating the mixture at room temperature;

2) After complete activation, 3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-D ] pyrimidin-4-amine is dissolved in aprotic solvent and added to the reactor of step 1) for stirring reaction;

3) The reaction is tracked to be complete by thin layer chromatography, the reaction solution is decompressed and concentrated to remove the solvent, and the obtained crude product is washed, extracted, collected into an organic phase, separated, purified and dried to obtain the target product.

6. The process according to claim 5, wherein the catalyst in step 1) is 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N '-dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N, N' -diisopropylcarbodiimide, 4-dimethylaminopyridine or 1-hydroxybenzotriazole; the organic base is triethylamine, N-diisopropylethylamine or N-methylmorpholine; the aprotic solvent is dichloromethane, N-dimethylformamide or acetonitrile.

7. The method according to claim 4, wherein the specific preparation process of the synthetic route II is as follows:

1) Adding each substituted aryl acrylic compound, 3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-D ] pyrimidine-4-amine and silicomolybdic acid into a reactor, dissolving, introducing nitrogen for protection, and stirring at 25-60 ℃ for reaction;

2) And (3) tracking the reaction to completion by a thin layer chromatography, extracting the reaction liquid, concentrating under reduced pressure, recrystallizing, purifying and drying to obtain a target product.

8. The method according to claim 7, wherein the molar ratio of 3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-D ] pyrimidin-4-amine and each substituted aryl acrylic compound in step 1) is 1 (1-1.4).

9. The use of ibrutinib acrylamide derivatives with anti-tumor activity in preparation of anti-tumor drug preparations according to any one of claims 1-3, wherein the tumor is a hematological tumor disease, and the hematological tumor disease is acute T-cell leukemia.