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

Abstract

The invention discloses a class of ibrutinib acrylamide derivatives with antitumor activity and a synthesis method and application thereof, belonging to the field of medicinal chemistry. This type of ibrutinib acrylamide derivatives with anti-tumor activity also relates to the pharmaceutically acceptable salt of the compound; its synthetic route includes two routes: the synthetic route I is obtained in an aprotic solvent, an organic base and a catalyst It can be obtained by condensation of acid and amine under certain conditions; synthetic route II is obtained by using ionic liquid as solvent and under the action of catalyst. The invention also specifically discloses its application in anti-tumor drugs. Through activity screening, it has better anti-tumor activity, and it is expected to be widely used in the preparation of anti-blood tumor drugs.

Description

A class of ibrutinib acrylamide derivatives with antitumor activity and their synthesis Method and Application

technical field

The invention belongs to the technical field of medicinal chemistry, and specifically relates to a class of ibrutinib acrylamide derivatives with antitumor activity and a synthesis method and application thereof.

Background technique

Malignant tumors, as one of the largest public health problems in the world, have greatly endangered human health and will become the number one killer of human beings in the new century. Malignant tumors include solid tumors (such as lung cancer, liver cancer, gastric cancer, intestinal cancer, etc.) and hematological tumors (such as leukemia, lymphoma, etc.). Two of the more common blood cancers.

Ibrutinib is an oral first-in-class Bruton's tyrosine kinase (BTK) inhibitor drug for the treatment of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The drug irreversibly inhibits BTK by selectively covalently binding to the cysteine residue (Cys-481) in the active site of the target protein BTK, thereby effectively preventing the tumor from migrating from B cells to cells adapted to the tumor growth environment. lymphatic tissue.

A large number of studies have shown that aryl acrylic acid compounds have good antitumor activity, and the most representative compound is cinnamic acid. Cinnamic acid has the effect of inhibiting growth and proliferation and inducing differentiation of various tumor cells. Cinnamic acid can stimulate the activation of peroxidase proliferator-activated receptors (PPAR), inhibit the expression of oncogenes and proteases, destroy the invasion ability of tumor cells, and stimulate T cells to generate an immune response to tumor cells. At the same time, cinnamic acid can hinder the mevalonate pathway, thereby inhibiting the synthesis of tumor cell growth regulatory proteins and blocking tumor cell proliferation. Based on the fact that aryl acrylic compounds have anti-tumor activity, small molecular weight and low price, they are often combined with other structures with anti-tumor activity in the design of anti-tumor drugs in order to exert anti-tumor activity synergistically. The active fragment splicing strategy makes aryl acrylic compounds widely used in medicinal chemistry research.

Contents of the invention

In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an ibrutinib acrylamide derivative with antitumor activity and its synthesis method and application, which are used for the preparation of antitumor drugs.

In order to achieve the above object, the present invention adopts the following technical solutions to achieve:

The invention discloses a class of ibrutinib acrylamide derivatives with anti-tumor activity, which are the compound represented by formula I or its pharmaceutically acceptable salt, and the compound of formula I or its pharmaceutically acceptable salt Solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion, including racemic mixtures, of salts;

The structural formula of the compound of formula I is:

Wherein, _R is replaced by a hydrogen atom or a cyano group;

R is Wherein R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from a hydrogen atom, a halogen atom, a nitro group, a methoxy group, a trifluoromethyl group or a dimethylamino group.

Preferably, representative compounds are selected from the following compounds:

Preferably, the pharmaceutically acceptable salt is ibrutinib acrylamide derivatives and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, Salts of succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

The present invention also discloses a method for synthesizing the above-mentioned ibrutinib acrylamide derivatives with antitumor activity. The synthetic route I is to combine various substituted aryl acrylic compounds with 3- (4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine undergoes acid amide condensation to prepare ibrutinib acrylamide derivatives; synthetic route II is directly with each substituted aryl acrylic compound and 3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4 -D] pyrimidin-4-amine as raw material, ionic liquid as solvent, synthesized under the action of catalyst to prepare ibrutinib acrylamide derivatives.

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

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

2) After the activation is complete, dissolve 3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine in non- A protic solvent is added to the reactor of step 1) to stir the reaction;

3) Thin-layer chromatography tracked the reaction until it was complete, concentrated the reaction solution under reduced pressure to remove the solvent, washed and extracted the obtained crude product, collected the organic phase, separated and purified, and dried to obtain the target product.

Further preferably, the catalyst in step 1) is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N'-di Cyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, 4-dimethylamino Pyridine or 1-hydroxybenzotriazole; organic bases are triethylamine, N,N-diisopropylethylamine, N-methylmorpholine; aprotic solvents are dichloromethane, N,N-dimethyl formamide or acetonitrile.

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

1) Each substituted arylpropene compound, 3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidine-4- Add amine and silicomolybdic acid into the reactor, dissolve, pass through the nitrogen protection, and stir the reaction at 25-60°C;

2) Thin-layer chromatography followed the reaction to completion, extracted the reaction solution, concentrated under reduced pressure, recrystallized and purified, and dried to obtain the target product.

Further preferably, 3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine in step 1) and The molar ratio of each substituted aryl acrylic compound is 1:(1-1.4).

The present invention also discloses the application of the above-mentioned ibrutinib acrylamide derivatives with antitumor activity in the preparation of antitumor drug preparations.

Preferably, the tumor is a hematological tumor disease.

Further preferably, the hematological tumor disease is acute T-cell leukemia.

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

The present invention provides a class of ibrutinib acrylamide derivatives with anti-tumor activity. Through in vitro anti-tumor activity experiments, it is found that preferred compounds 6, 10 and 14 of the ibrutinib acrylamide derivatives synthesized by the present invention The proliferation inhibition rates for Jurkat cells at a concentration of 1 μM were 24.13%, 23.14%, and 25.73%, respectively, which were superior to the positive control drug ibrutinib’s proliferation inhibition rate of 19.98%; The number of Jurkat cell proliferation was significantly reduced, the cells had obvious morphological changes, and death characteristics such as cell shrinkage appeared; for Daudi cells, the proliferation inhibitory ability of most ibrutinib cinnamic amides at a concentration of 10 μM was comparable to that of the positive control The drug is similar to ibrutinib, and the partial inhibition rate is above 90%. Observe the morphology of Daudi cells treated with different concentrations of compounds. With the increase of the concentration of the compound, the arrangement of the cells is gradually sparse, and cell death such as pyknosis and volume reduction occurs. feature. Anti-tumor activity experiments have verified that the ibrutinib acrylamide derivatives provided by the present invention have the potential to be used as new anti-tumor drugs in the preparation of anti-tumor drug preparations, especially in the preparation of hematological tumor drugs, further for acute T cell leukemia.

A class of synthetic methods of ibrutinib acrylamide derivatives with anti-tumor activity provided by the present invention, two synthetic methods utilize aryl acrylic compounds to 3-(4-phenoxyphenyl)-1-( Piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine (ibrutinib intermediate compound 19) was structurally modified to obtain a new class of ibrutinib acrylamide The use of ionic liquids as solvents in Synthetic Method II has not been reported yet. The preparation method provided by the present invention has high operational safety, mild reaction conditions and low cost, and the yield of the obtained ibrutinib acrylamide derivatives is relatively low. High, between 62.66%-83.74%, suitable for industrial production.

Description of drawings

Fig. 1 is the NMR spectrum figure of compound 1 in deuterated chloroform in the present invention;

Fig. 2 is the nuclear magnetic carbon spectrogram of compound 1 in deuterated chloroform in the present invention;

Fig. 3 is the NMR spectrum of compound 8 in deuterated chloroform in the present invention;

Fig. 4 is the nuclear magnetic carbon spectrogram of compound 8 in deuterated chloroform in the present invention;

Fig. 5 is the NMR spectrum of compound 10 in deuterated chloroform in the present invention;

Fig. 6 is the nuclear magnetic carbon spectrogram of compound 10 in deuterated chloroform in the present invention;

Fig. 7 is the NMR spectrum of compound 11 in deuterated chloroform in the present invention;

Fig. 8 is the nuclear magnetic carbon spectrogram of compound 11 in deuterated chloroform in the present invention;

Fig. 9 is a graph showing the cytotoxic effect of ibrutinib cinnamic amide compounds on U937 cells at concentrations of 1 μM and 10 μM in the present invention;

Fig. 10 is a diagram of the cytotoxic effect of ibrutinib cinnamate compounds on Jurkat cells at concentrations of 1 μM and 10 μM in the present invention;

Fig. 11 is a diagram of the cytotoxic effect of ibrutinib cinnamate compounds on Daudi cells at concentrations of 1 μM and 10 μM in the present invention;

Figure 12 is the morphological images of compounds 6, 10, 14 and the positive control drug ibrutinib on Jurkat cells in the present invention; wherein, a is compound 6 (10 μM), b is compound 6 (1 μM), and c is compound 10 (10 μM), d is compound 10 (1 μM), e is compound 14 (10 μM), f is compound 14 (1 μM), g is the positive control drug ibrutinib (10 μM), h is the positive control drug ibrutinib Ni (1μM);

Figure 13 is the morphological images of compounds 10, 13, 14 and the positive control drug ibrutinib on Daudi cells in the present invention; wherein, a is compound 10 (10 μM), b is compound 10 (1 μM), and c is compound 13 (10 μM), d is the compound 13 (1 μM), e is the compound 14 (10 μM), f is the compound 14 (1 μM), g is the positive control drug ibrutinib (10 μM), h is the positive control drug ibrutinib Nickel (1 μM).

Detailed ways

In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only It is an embodiment of a part of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

It should be noted that the terms "first" and "second" in the description and claims of the present invention and the above drawings are used to distinguish similar objects, but not necessarily used to describe a specific sequence or sequence. It is to be understood that the data so used are interchangeable under appropriate circumstances such that the embodiments of the invention described herein can be practiced in sequences other than those illustrated or described herein. Furthermore, the terms "comprising" and "having", as well as any variations thereof, are intended to cover a non-exclusive inclusion, for example, a process, method, system, product or device comprising a sequence of steps or elements is not necessarily limited to the expressly listed instead, may include other steps or elements not explicitly listed or inherent to the process, method, product or apparatus.

Below in conjunction with accompanying drawing 1-13, the present invention is described in further detail:

The structural formula of a class of ibrutinib acrylamide derivatives with anti-tumor activity provided by the present invention is shown in Formula I:

Wherein, _R is replaced by a hydrogen atom or a cyano group;

R is wherein R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from hydrogen atom, halogen atom, nitro, methoxy, trifluoromethyl or dimethylamino; the term "halogen" means fluorine, chlorine, bromine or iodine.

A class of ibrutinib acrylamide derivatives with anti-tumor activity provided by the present invention also includes the solvate, enantiomer, diastereoisomer, Tautomers or mixtures thereof in any proportion, including racemic mixtures.

A class of ibrutinib acrylamide derivatives with antitumor activity provided by the present invention relates to a pharmaceutically acceptable salt of the compound, and the pharmaceutically acceptable salt is an 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, The salt that toluenesulfonic acid, glutamic acid or aspartic acid form; Described pharmaceutically acceptable salt preferably has following structural formula:

The synthesis route provided by the present invention for synthesizing the above-mentioned ibrutinib acrylamide derivatives with anti-tumor activity is as follows:

Synthetic route I is under the conditions of aprotic solvent, organic base and catalyst, each substituted aryl acrylic compound and 3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyridine Azolo[3,4-D]pyrimidin-4-amine (compound 19) is obtained by acid amine condensation; synthetic route II is directly using substituted aryl acrylic compounds and ibrutinib intermediates (compound 19) as raw materials , the ionic liquid is used as a solvent and synthesized under the action of a catalyst;

Among them, the structure of compound 19 is shown in the figure below:

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

In order to realize the above-mentioned synthetic route I, the synthetic steps of the present invention are as follows:

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

Wherein, the catalyst is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N'-dicyclohexyl Carbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-diisopropylcarbodiimide (DIC ), 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), N,N'-carbonyldiimidazole (CDI), 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-azobenzotriazole azole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); the organic base is triethylamine (TEA), N,N-diisopropylethylamine (DIEA) , N-methylmorpholine (NMM), morpholine or pyridine, more preferably N, N-diisopropylethylamine (DIEA); the aprotic solvent is dichloromethane, N, N-dimethyl Formamide, N,N-dimethylacetamide or acetonitrile, more preferably dichloromethane;

(2) After the activation is complete, dissolve compound 19 in an aprotic solvent, add it dropwise to the reactor in step (1) and stir for 8-12 hours;

(3) Thin-layer chromatography tracks the reaction to completion, the reaction solution is concentrated under reduced pressure to remove the solvent, the resulting crude product is washed with saturated aqueous sodium chloride, extracted with ethyl acetate, the organic phase is collected, separated and purified by column chromatography, and dried to obtain the target product. product.

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

In order to realize the above-mentioned synthetic route II, the synthetic steps of the present invention are as follows:

(1) Add substituted arylpropene compound, compound 19 and catalyst silicomomolybdic acid into the reactor, dissolve it with ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate, pass nitrogen protection, and Stir and react for 4-8 hours at a temperature of 25-60°C;

Wherein, the molar ratio between the compound 19 and each substituted aryl acrylic compound is 1:(1-1.4), further optimized to 1:(1-1.2); the reaction temperature is more preferably 30°C, and the reaction time is more preferably 5h;

(2) Thin-layer chromatography followed the reaction to completion, and the reaction solution was extracted and concentrated under reduced pressure to remove the catalyst and ionic liquid, then recrystallized, purified, and dried to obtain the target product.

1. Specific examples of synthetic compound 1-15

The structural formulas and numbers of representative compounds are as follows:

The examples of the synthesis of the above compounds are given below, and the structures of the compounds are characterized by NMR.

Example 1

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

Dissolve 74.1mg (0.5mmol) of cinnamic acid in 3mL of dichloromethane and place it in a reactor, add HATU380.2mg (1mmol) and DIEA 260μL (1.5mmol), stir and activate at room temperature for 1 hour before use. 193.3mg (0.5mmol) of compound 19 was dissolved in 3mL of dichloromethane, added dropwise to the above reactor, and then stirred and reacted at room temperature for 8 hours, monitored by TLC. After the reaction, the resulting reaction solution was concentrated under reduced pressure to remove dichloromethane, the solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, the organic phase was collected, separated and purified by column chromatography (200-300 mesh silica gel powder was The stationary phase, dichloromethane:methanol (V:V)=10:1 is the mobile phase), and then dried to obtain 216.3 mg of the target compound 1 with a yield of 83.74%. The H NMR spectrum of compound 1 in deuterated chloroform is shown in Figure 1, and the C NMR spectrum is shown in Figure 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, 126.1 2,124.08,119.57,119.14,117.24,98.58,56.31 ,51.46,38.61,30.80,28.35.

Example 2

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

99.7 mg (0.6 mmol) of 2-fluorocinnamic acid, 193.3 mg (0.5 mmol) of compound 19 and 8.6 mg (5 μmol) of silicomomolybdic acid were placed in a 150 mL reactor in sequence, and 71.5 mL of ionic liquid 1-butyl-3- After methylimidazolium tetrafluoroborate was fully dissolved, N ₂ protection was introduced, and the reaction was stirred at 30° C. for 5 h. Thin-layer chromatography tracked the reaction until it was complete, and removed the protective device. The reaction mixture system was placed in a separatory funnel, oscillated, allowed to stand and separated into layers, and the ionic liquid layer was separated from the ester layer, and the obtained ester layer was a crude product of cinnamate ester derivatives. After recrystallization and drying with 50 mL of methanol, 212.1 mg of the target compound 2 was obtained, with a total yield of 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, 124.6 7, 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: 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl) Preparation of -3-(4-fluorophenyl)prop-2-en-1-one

Dissolve 83.1mg (0.5mmol) of 4-fluorocinnamic acid in 3mL of dichloromethane and place it in a reactor, add 380.2mg (1mmol) of HATU (1mmol) and 210μL (1.5mmol) of TEA to it, stir and activate at room temperature for 1 hour before use . 193.3mg (0.5mmol) of compound 19 was dissolved in 3mL of dichloromethane, added dropwise to the above reactor, and then stirred and reacted at room temperature for 8 hours, monitored by TLC. After the reaction, the resulting reaction solution was concentrated under reduced pressure to remove dichloromethane, the solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, the organic phase was collected, separated and purified by column chromatography (200-300 mesh silica gel powder was The stationary phase, dichloromethane:methanol (V:V)=10:1 is the mobile phase), and then dried to obtain 211.9 mg of the target compound 3 with a yield of 79.28%.

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=12.1 Hz,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, 123.9 9,119.56,119.14,118.42,115.43,98.56,56.26 ,51.57,38.64,30.75,28.38.

Example 4

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

Dissolve 92.1mg (0.5mmol) of 3,4-difluorocinnamic acid in 3mL of dichloromethane and place it in a reactor, add 380.2mg (1mmol) of HATU (1mmol) and 170μL (1.5mmol) of NMM to it, stir and activate at room temperature for 1 Stand by after hours. 193.3 mg (0.5 mmol) of compound 19 was dissolved in 3 mL of dichloromethane, added dropwise to the above reactor, and then stirred and reacted at room temperature for 10 hours, monitored by TLC. After the reaction, the resulting reaction solution was concentrated under reduced pressure to remove dichloromethane, the solid residue was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, the organic phase was collected, separated and purified by column chromatography (200-300 mesh silica gel powder was The stationary phase, dichloromethane:methanol (V:V)=10:1 is the mobile phase), and dried to obtain 218.1 mg of the target compound 4 with a yield of 71.57%.

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=12.0 Hz,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, 125.9 2,125.14,124.08,119.56,119.22,118.53,112.83, 98.66, 56.43, 51.36, 38.65, 30.73, 28.35.

Example 5

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

121.3 mg (0.6 mmol) of 3,4,5-trifluorocinnamic acid was dissolved in 3 mL of N,N-dimethylformamide and placed in the reactor, EDCI 191.7 mg (1 mmol), HOBT 135.1 mg ( 1mmol) and DIEA 260μL (1.5mmol), stirred and activated at room temperature for 1 hour before use. 193.3mg (0.5mmol) of compound 19 was dissolved in 3mL of N,N-dimethylformamide, added dropwise to the above reactor, and then stirred and reacted at room temperature for 10 hours, monitored by TLC. After the reaction was finished, the resulting reaction solution was washed with saturated aqueous sodium chloride, extracted with ethyl acetate, collected the organic phase, and separated and purified by column chromatography (200-300 mesh silica gel powder was the stationary phase, dichloromethane:methanol (V:V )=10:1 is the mobile phase), dried to obtain 230.3 mg of the target compound 5, and the yield was 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=12.2 Hz,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, 124.0 9,119.65,119.14,118.56,108.42,98.66,56.53 ,51.53,38.55,30.68,28.33.

Example 6

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

Place 129.7mg (0.6mmol) of 4-trifluoromethylcinnamic acid, 193.3mg (0.5mmol) of compound 19 and 8.6mg (5μmol) of silicomomolybdic acid in a 150mL reactor, and add 71.5mL of ionic liquid 1-butyl After the -3-methylimidazolium tetrafluoroborate was fully dissolved, N ₂ was passed through for protection, and the reaction was carried out at 25°C for 8 hours. Thin-layer chromatography tracked the reaction until it was complete, and removed the protective device. The reaction mixture system was placed in a separatory funnel, oscillated, allowed to stand and separated into layers, and the ionic liquid layer was separated from the ester layer, and the obtained ester layer was a crude product of cinnamate ester derivatives. 50 mL of methanol was used for recrystallization and drying to obtain 218.5 mg of the target compound 6, with 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=12.0 Hz,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, 125.0 6,124.69,123.98,119.52,119.24,118.84,98.50 ,56.39,51.46,38.57,30.81,28.30.

Example 7

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

115.9mg (0.6mmol) of 4-nitrocinnamic acid was dissolved in 3mL of N,N-dimethylformamide and then placed in a reactor, DCC 206.3mg (1mmol) and DMAP 12.2mg (0.1mmol) were added thereto, Stir and activate at room temperature for 1 hour before use. 193.3mg (0.5mmol) of compound 19 was dissolved in 5mL of N,N-dimethylformamide, added dropwise to the above reactor, and then stirred and reacted at room temperature for 12 hours, monitored by TLC. After the reaction was finished, the resulting reaction solution was washed with saturated aqueous sodium chloride, extracted with ethyl acetate, collected the organic phase, and separated and purified by column chromatography (200-300 mesh silica gel powder was the stationary phase, dichloromethane:methanol (V:V )=10:1 is the mobile phase), drying to obtain 209.4 mg of the target compound 7, with a yield of 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, 124.1 1,123.81,119.55,119.24,118.45,98.89,56.51 ,51.58,38.58,30.81,28.37.

Example 8

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

Dissolve 114.7mg (0.6mmol) of 4-(dimethylamino)cinnamic acid in 3mL of N,N-dimethylformamide and place it in the reactor, add DIC 126.2mg (1mmol), HOBT 135.1mg (1mmol ) and 260 μL (1.5 mmol) of DIEA, stirred and activated at room temperature for 1 hour before use. 193.3mg (0.5mmol) of compound 19 was dissolved in 3mL of N,N-dimethylformamide, added dropwise to the above reactor, and then stirred and reacted at room temperature for 10 hours, monitored by TLC. After the reaction was finished, the resulting reaction solution was washed with saturated aqueous sodium chloride, extracted with ethyl acetate, collected the organic phase, and separated and purified by column chromatography (200-300 mesh silica gel powder was the stationary phase, dichloromethane:methanol (V:V )=10:1 is the mobile phase), dried to obtain 214.3 mg of the target product compound 8, and the yield was 76.59%. The H NMR spectrum of compound 8 in deuterated chloroform is shown in Figure 3, and the C NMR spectrum is shown in Figure 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, 123.1 8,119.53,119.16,118.85,111.61,98.58,56.35 ,51.52,40.21,38.61,30.62,28.27.

Example 9

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

Dissolve 98.0mg (0.55mmol) of 4-methoxycinnamic acid in 3mL of acetonitrile and place it in a reactor, add 380.2mg (1mmol) of HATU and 210μL (1.5mmol) of TEA, stir and activate at room temperature for 1 hour before use . 193.3 mg (0.5 mmol) of compound 19 was dissolved in 3 mL of acetonitrile, added dropwise to the above reactor, and then stirred and reacted at room temperature for 10 hours, monitored by TLC. 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, the organic phase was collected, separated and purified by column chromatography (200-300 mesh silica gel powder was used as the stationary phase, Dichloromethane:methanol (V:V)=10:1 is the mobile phase), and dried to obtain 217.7 mg of the target compound 9 with a yield of 79.64%.

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, 124.0 9,119.54,119.14,118.32,114.22,98.56,56.32 ,55.76,51.48,38.57,30.77,28.26.

Example 10

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

Place 145.7mg (0.7mmol) of 3,4-dimethoxycinnamic acid, 193.3mg (0.5mmol) of compound 19 and 8.6mg (5μmol) of silicomolybdic acid in a 150mL reactor, and add 71.5mL of ionic liquid 1- After butyl-3-methylimidazolium tetrafluoroborate was fully dissolved, N ₂ was passed through for protection, and the reaction was carried out at 30°C for 8 hours. Thin-layer chromatography tracked the reaction until it was complete, and removed the protective device. The reaction mixture system was placed in a separatory funnel, oscillated, allowed to stand and separated into layers, and the ionic liquid layer was separated from the ester layer, and the obtained ester layer was a crude product of cinnamate ester derivatives. After recrystallization and drying with 50 mL of methanol, 231.0 mg of the target compound 10 was obtained, with a total yield of 80.12%. The H NMR spectrum of compound 10 in deuterated chloroform is shown in Figure 5, and the C NMR spectrum is shown in Figure 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,6.5 H),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, 124.1 0,121.84,119.57,119.14,114.88,111.05,109.95 ,98.65,56.51,55.96,51.42,38.62,30.82,28.36.

Example 11

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

Dissolve 82.9mg (0.6mmol) of 3-(2-furyl)acrylic acid in 3mL of acetonitrile and put it in a reactor, add EDCI 191.7mg (1mmol), HOBT 135.1mg (1mmol) and TEA 210μL (1.5mmol) , stirred and activated at room temperature for 1 hour and then set aside. 193.3 mg (0.5 mmol) of compound 19 was dissolved in 3 mL of acetonitrile, added dropwise to the above reactor, and stirred at room temperature for 12 hours, monitored by TLC. After the reaction, the obtained reaction solution was concentrated under reduced pressure to remove acetonitrile, and the solid residue obtained was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phase was collected (200-300 mesh silica gel powder as the stationary phase, dichloromethane:methanol (V:V)=10:1 is the mobile phase), and dried to obtain 187.2 mg of the target compound 11 with a yield of 73.89%. The H NMR spectrum of compound 11 in deuterated chloroform is shown in Figure 7, and the C NMR spectrum is shown in Figure 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, 124.0 8,119.58,119.15,114.55,112.20,98.53,56.32 ,51.51,38.64,30.80,28.36.

Example 12

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

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

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=13.4 Hz,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, 126.1 3,124.87,124.11,119.59,119.14,98.59,56.36 ,51.54,38.59,30.76,28.35.

Example 13

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

130.4 mg (0.6 mmol) (Z)-3-cyano-3-(thiophen-2-yl) potassium acrylate, 193.3 mg (0.5 mmol) compound 19 and 8.6 mg (5 μmol) silicomolybdic acid were placed in 150 mL for reaction After adding 71.5mL of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate into the container and fully dissolving it, N ₂ protection was introduced, and the reaction was carried out at 60°C for 4h. Thin-layer chromatography tracked the reaction until it was complete, and removed the protective device. The reaction mixture system was placed in a separatory funnel, oscillated, allowed to stand and separated into layers, and the ionic liquid layer was separated from the ester layer, and the obtained ester layer was a crude product of cinnamate ester derivatives. After recrystallization and drying with 50 mL of methanol, 171.6 mg of the target compound 13 was obtained, with a total yield of 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, 126.1 3,124.52,124.12,119.62,119.14,118.87,98.57 ,56.36,51.54,38.61,30.79,28.38.

Example 14

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

Dissolve 89.5mg (0.6mmol) of 3-(pyridine-4-)acrylic acid in 3mL of acetonitrile and place it in a reactor, add 380.2mg (1mmol) of HATU (1mmol) and 260μL (1.5mmol) of DIEA to it, stir and activate at room temperature for 1 hour Backup. 193.3 mg (0.5 mmol) of compound 19 was dissolved in 3 mL of acetonitrile, added dropwise to the above reactor, and then stirred and reacted at room temperature for 10 hours, monitored by TLC. After the reaction, the obtained reaction solution was concentrated under reduced pressure to remove acetonitrile, and the solid residue obtained was washed with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phase was collected (200-300 mesh silica gel powder as the stationary phase, dichloromethane:methanol (V:V)=10:1 is the mobile phase), and dried to obtain 195.2 mg of the target compound 14 with a yield of 75.44%.

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, 124.0 9,123.03,119.54,119.14,98.57,56.34,51.47 ,38.62,30.78,28.37.

Example 15

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

118.9mg (0.6mmol) (E)-3-(naphthalene-1-yl)acrylic acid was dissolved in 3mL N,N-dimethylformamide, added HATU 380.2mg (1mmol) and DIEA 260μL (1.5mmol), at room temperature Stir and activate for 1 hour and set aside. 193.3mg (0.5mmol) of compound 19 was dissolved in 3mL of N,N-dimethylformamide, added dropwise to the above reactor, stirred and reacted at room temperature for 10 hours, monitored by TLC. After the reaction was finished, the resulting reaction solution was washed with saturated aqueous sodium chloride, extracted with ethyl acetate, collected the organic phase, and separated and purified by column chromatography (200-300 mesh silica gel powder was the stationary phase, dichloromethane:methanol (V:V )=10:1 is the mobile phase), dried to obtain 222.3 mg of the target product compound 15, and the yield was 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, 127.7 7,126.97,126.37,126.08,125.94,124.04,123.83 ,122.95,119.57,119.14,118.83,98.55,56.33,51.47,38.62,30.77,28.39.

2. Determination of anti-tumor cell proliferation of compounds and their morphological effects on tumor cells

Measuring principle: the compound's ability to inhibit tumor cell proliferation can be measured by the CCK-8 method. CCK-8 is a widely used detection reagent for cell proliferation and cytotoxicity based on WST-8. In the presence of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate, WST-8 is reduced to highly water-soluble orange-yellow formazan by dehydrogenase in mitochondria. The more cells proliferate, the faster the color will be; the greater the cytotoxicity, the lighter the color. For the same cells, the color depth is proportional to the number of living cells, so this feature can be used to directly analyze cell proliferation and toxicity Determination.

Experimental materials: CCK-8 kit (Shanghai Biyuntian Biotechnology Co., Ltd., C0038); positive control drug ibrutinib (Shanghai Macklin Biochemical Technology Co., Ltd.); U937 (human histiocytic lymphoma cells), Jurkat (human T lymphocytic leukemia cells) and Daudi (human Burkitt's lymphoma cells), all three cells were purchased from the US ATCC bank.

Cell culture and administration: Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37 °C in an incubator with 5% _CO2 in humidified air. The compound was prepared with DMSO to make a solution with a mother solution concentration of 1 mM, placed in a -20°C refrigerator, and diluted to a corresponding concentration according to the needs of the experiment.

Experimental method: Take cells in good logarithmic growth phase, count them, make a suspension of 5×10 ⁴ cells/mL, inoculate the cell suspension in a 96-well plate, 100 μL per well, and place at 37°C, 5% CO After culturing for 24 hours under the condition of ₂ , 10 μL of medium containing different concentrations of compounds was added to the 96-well plate, and the compound concentration gradient was set to 1 μM and 10 μM. Three replicate wells were set up for each group, and a blank control group (no drug addition) and a positive control group (ibrutinib) were set up. Place in a constant temperature incubator at 37°C with 5% CO ₂ , and after incubation for 24 hours, add 10 μL of CCK-8 solution to each well, and continue to incubate in the incubator for 1 hour. Finally, the absorbance value (OD value) of the 96-well plate was detected by a microplate reader at a wavelength of 450 nm, and the morphological influence of the compound on the tumor cells was observed under an optical microscope. The cell proliferation inhibition rate was calculated according to the following formula.

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

The results of the anti-tumor cell proliferation ability of the ibrutinib cinnamic amide compounds prepared by the present invention are as shown in Table 1:

Table 1: Anti-tumor cell proliferation inhibition rate of ibrutinib cinnamic amide compounds

It can be seen from Table 1 that the ibrutinib cinnamic amide compounds prepared by the present invention showed different degrees of proliferation inhibitory activity on U937 cells, Jurkat cells, and Daudi cells, and the inhibitory rate at a concentration of 10 μM was better, showing Concentration-dependent inhibitory activity. For U937 cells, the proliferation inhibitory ability of compounds 1-15 was lower than that of the positive control drug ibrutinib (Figure 9); for Jurkat cells, at a concentration of 1 μM, the proliferation inhibitory rates of compounds 6, 10 and 14 were 24.13% , 23.14% and 25.73%, better than the positive control drug Ibrutinib (Ibrutinib) growth inhibition rate of 19.98% (Figure 10). Observation of cell morphology found that the proliferation of Jurkat cells treated with the above compounds was significantly reduced, and the cells also underwent obvious morphological changes, and death characteristics such as cell shrinkage appeared (Figure 12); for Daudi cells, at a concentration of 10 μM, The growth inhibitory ability of most ibrutinib cinnamic amide compounds is similar to that of the positive control drug ibrutinib, and the partial inhibition rate is over 90%; while at low concentrations, the proliferation inhibitory ability of compounds 10, 13 and 14 is better (Figure 11). Observing the morphology of Daudi cells treated with different concentrations of the compound, as the concentration of the compound was increased, the arrangement of the cells was gradually sparse, and death characteristics such as cell pyknosis and volume reduction appeared (Figure 13).

The above experimental results show that the ibrutinib cinnamic amide compound provided by the present invention can inhibit the proliferation and migration of different types of tumor cells, and has a stronger inhibitory ability to Jurkat cells and Daudi cells, so it can be applied to anti-acute T-cell leukemia and Preparation of Drugs for Human Burkitt's Lymphoma.

The above content is only to illustrate the technical ideas of the present invention, and cannot limit the protection scope of the present invention. Any changes made on the basis of the technical solutions according to the technical ideas proposed in the present invention shall fall within the scope of the claims of the present invention. within the scope of protection.

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.