CN113968861B - Compound with PI3K delta/BTK double-target-point activity and preparation method and application thereof - Google Patents

Abstract

The invention discloses a compound with PI3K delta/BTK double-target-point activity, a preparation method thereof and application thereof in preparing a pharmaceutical preparation for preventing or treating diseases caused by PI3K delta or BTK protein abnormality. In particular, the invention describes compounds of formula a. The main advantage of the combined application of the two inhibitors is that the therapeutic effect of 1+1 > 2 can be realized through complementary synergistic action, and the double-target inhibitor can not only ensure the therapeutic effect of the drug, but also reduce the risk of drug-drug interaction by reducing the administration dosage and further improve the safety, so that the compound with the PI3 Kdelta/BTK double-target activity provided by the invention can be widely applied to the preparation of pharmaceutical preparations for preventing or treating diseases caused by abnormal B cell receptors.

Description

Compound with PI3K delta/BTK double-target-point activity and preparation method and application thereof

Technical Field

The invention relates to the field of small molecule drugs, and particularly provides a compound with double target point inhibitory activity on Bruton Tyrosine Kinase (BTK) and phosphatidylinositol-3-kinase delta subtype (PI 3K delta), and synthesis and application of the compound.

Background

B-cell lymphoma is a heterogeneous group of lymphoproliferative disorders, a malignant tumor with a high degree of death, and can be divided into Hodgkin Lymphoma (HL) and non-hodgkin lymphoma (NHL). HL usually originates in lymph nodes with Reed-Sternberg tumor cells; NHL is frequently found in extranodal lymphoid tissues, and the incidence is generally higher, including Mantle Cell Lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), chronic Lymphocytic Leukemia (CLL), follicular Lymphoma (FL), marginal zone B-cell lymphoma (MZL), and the like. The current conventional treatment for B cell lymphoma is radiation, chemotherapy. The first-line treatment regimen for B-cell lymphoma is indicated in the NCCN clinical guidelines in the united states as R-CHOP therapy, a combination of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone. However, the toxicity associated with these therapies is poorly tolerated by elderly patients or patients who are themselves suffering from other diseases.

The pathogenesis of B cell lymphomas is closely related to the B Cell Receptor (BCR). In many B cell lymphomas, BCR is overexpressed, and the BCR signaling pathway is also overactive, resulting in inhibition of the normal differentiation and apoptosis processes of B cells and promotion of abnormal proliferation processes. Therefore, the BCR signaling pathway is crucial for the growth and survival of a variety of tumor cells, and different targeted drugs directed to this pathway can inhibit the progression of B-cell related diseases, including spleen tyrosine kinase (SYK), bruton Tyrosine Kinase (BTK), and phosphatidylinositol-3-kinase (PI 3K), among others.

PI3K is a lipid kinase and is involved in regulating and controlling physiological processes related to tumorigenesis and development. At present, 5 PI3K inhibitors are approved by FDA for marketing. In 7 months 2014, the first selective PI3K δ inhibitor Idelalisib was approved by FDA for marketing, with a selectivity over 40-fold higher than other subtypes, and was mainly used for the treatment of recurrent CLL, FL, or SLL. However, idelalisib has a black box warning with severe side effects such as hepatotoxicity, severe diarrhea, colitis, pneumonia and intestinal perforation. In view of the severe adverse effects of Idelalisib, 9 months 2017, FDA accelerated approval of the marketing of Copanlisib for the treatment of FL. Copalisib is a PI3K alpha/delta inhibitor, has similar treatment effect to Idelalisib, and has greatly improved safety. Duvelisib, approved to market in 2018 and 10 months, is a PI3K delta/gamma selective inhibitor, and the total remission rate can reach 78%. The Alpelisib is a PI3K alpha inhibitor, has high selectivity, is mainly used for treating the advanced metastatic breast cancer with PIK3CA mutation, fills the blank that the treatment effect of a patient with the PIK3CA mutated breast cancer is poor, and has a survival period which is three times longer than that of a placebo group. The newly approved PI3K delta/CK 1 epsilon dual-target inhibitor Umbralisib is even qualified by the FDA for orphan drug treatment of FL/MZL. At present, the research on the PI3K inhibitor in the world is still very hot, but a plurality of complex mechanisms exist in the PI3K target, each subtype has own unique function, so that the inhibitors aiming at different subtypes inhibit tumors through different mechanisms, and the toxicity is also superposed. Therefore, PI3K inhibitors with high subtype selectivity have a more promising prospect in terms of safety.

PI3K delta is only expressed in hematopoietic cells, and researches show that B cell functions of mice knocking out PI3K delta are seriously damaged, so that the PI3K delta is an effective target for treating B cell lymphoma. Meanwhile, the PI3K delta is also related to immune diseases, can regulate immune threshold and limit the immunity of endogenous microbiota, and is expected to be used for treating immune cell related diseases such as rheumatoid arthritis, multiple sclerosis, alzheimer's disease and the like. A more attractive second generation PI3K δ inhibitor, umbralisib, was recently marketed in the united states for MZL and FL patients who had previously received treatment. The tolerance and the safety of Umbralisib are better, and autoimmune toxicity is less. Therefore, the second generation PI3K inhibitor has higher selectivity, and reduces the side effect caused by the superimposed toxicity to a greater extent.

BTK kinase can promote cell proliferation, antibody secretion, switch-like recombination, and production of pro-inflammatory cytokines. At present, 5 BTK inhibitors are on the market all over the world, and clinical tests for treating pancreatic cancer, breast cancer, autoimmune diseases and the like are also in progress besides hematological tumors. Ibrutinib is the first BTK inhibitor to be successfully marketed, is approved by FDA in 2013, and still occupies the first position in BTK inhibitor sales. The acaraburtinib and zanuburtinib subsequently marketed are structurally similar to Ibrutinib, but with less off-target side effects. Orelabrutinib, which was recently approved in NMPA in China, has a higher selectivity, and clinical trials are continuing to develop more indications.

Due to the complex mechanism of the B cell signal pathway network and the existence of compensatory activation, most patients with recurrent or refractory B cell lymphoma usually cannot obtain long-term benefit from single-drug treatment and are easy to have drug resistance and side effects. The measures of combined medication or combined radiotherapy and chemotherapy are generally adopted clinically, so that the safety and the curative effect are improved. Although both PI3K inhibitors and BTK inhibitors belong to the BCR signaling pathway and are directed against B cell lymphomas, they are two relatively independent pathways, and inhibition of one kinase has little effect on activation of the other kinase, so that the combined administration of PI3K and BTK reduces compensatory activation in the signaling pathway, and can inhibit both targets at the same time with a lower drug dose, possibly with higher therapeutic effect. At present, clinical experiments on the combined application of PI3K and BTK inhibitors are ongoing, for example, the research on the combination of Copalisib and Ibrutinib for treating recurrent or refractory primary central nervous lymphoma (PCNSL) can achieve higher complete remission rate more quickly and remarkably prolong the recurrence time of tumors, which is in stage Ib/II. The results of synergistic administration of the PI3K inhibitor Umbralisib and the BTK inhibitor Ibrutinib to CLL/MCL tumor patients show good tolerability.

However, differences in PK properties between different drugs (e.g., drug half-life, drug distribution, and drug-drug interactions) limit the utility of some drug combinations, and multi-component administration may also present metabolic and drug resistance issues.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a compound with PI3K delta/BTK double-target-point activity, a pharmaceutically available salt thereof, a synthetic method thereof and application thereof in medicines for inhibiting B protein receptors.

The purpose of the invention is realized by the following technical scheme: a compound having PI3 Kdelta/BTK dual-target activity and pharmaceutically useful salts thereof, having a structure according to formula A:

wherein R has the following structure:

the invention also provides a preparation method of the compound with the PI3 Kdelta/BTK double-target activity, which comprises the following steps:

the first step is as follows: 3-iodine-1H-pyrazolo [3,4-d ] pyrimidine-4-amine and 3-fluoro-4-methoxy boric acid are reacted in a solvent system of N-methyl pyrrolidone and water to generate a compound with a B-1 structure under the action of sodium carbonate and 1,1' -bis diphenyl phosphine ferrocene palladium dichloride;

the second step is that: adding 3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine, triphenylphosphine oxide and triphenylphosphine, adding N, N-dimethylformamide for dissolving, placing the mixture under ice bath under the protection of nitrogen, and dropwise adding diisopropyl azodicarboxylate to obtain a compound with a B-2 structure;

the third step: intermediate B-2 was mixed with the starting N-Boc-protected alcohol, triphenylphosphine TPP was added, diisopropyl azodicarboxylate DIAD was added slowly dropwise in an ice bath, the reaction was stirred at room temperature, and the progress of the reaction was monitored by TLC. Adding water for quenching, extracting by dichloromethane, and separating by silica gel column chromatography to obtain a product C-1;

the fourth step: removing a protecting group of the obtained C-1 product in dilute hydrochloric acid to obtain C-2;

the fifth step: and carrying out condensation reaction on the intermediate C-2 and the acrylic acid substituted by R5 to obtain a target product A.

The reaction equation is as follows:

wherein the content of the first and second substances,

r3 is the following structure:

r4 is the following structure:

r5 is H, F or methyl;

the structural definition of R is consistent with the foregoing.

The compound with the PI3K delta/BTK double-target activity can be used alone or can be prepared into pharmaceutically acceptable salts by a conventional method, wherein the pharmaceutically acceptable salts are hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, oxalate, tartrate, methanesulfonate, p-toluenesulfonate, fumarate, taurate, citrate, succinate or mixed salts thereof.

The invention also provides a compound with PI3K delta/BTK double-target-point activity and an application of a pharmaceutically acceptable salt thereof in pharmacy, wherein the application specifically comprises the following steps: is used for preparing a pharmaceutical preparation for preventing or treating diseases caused by PI3K delta/BTK abnormality. The disease is lymphoma, including chronic lymphocytic leukemia, B cell lymphoma, mantle cell lymphoma, lymphoplasmacytic lymphoma, diffuse large B cell lymphoma, non-Hodgkin's lymphoma, follicular central lymphoma, marginal zone B cell lymphoma, or autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis, etc.

Compared with the prior art, the invention has the following advantages:

the main advantage of the combined application of the two inhibitors is that the therapeutic effect of 1+1 > 2 can be realized through complementary synergistic action, and the double-target inhibitor can not only ensure the therapeutic effect of the medicament, but also reduce the risk of medicament-medicament interaction by reducing the dosage of administration, and can further improve the safety, so that the compound with the PI3 Kdelta/BTK double-target activity provided by the invention can be widely applied to the preparation of pharmaceutical preparations for preventing or treating diseases caused by B cell receptor abnormality.

Drawings

Fig. 1 is a graph of plasma concentrations versus time for compounds CXY20, CXY23 and CXY 27.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.

Example 1N- (2- (1- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) -4-chloro-5-fluorophenyl) acrylamide

The first step is as follows: 3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine

3-iodo-1H-pyrazolo [3,4-d ] was sequentially added to the reaction flask]Pyrimidine-4-amine (20g, 77mmol), 3-fluoro-4-methoxyboric acid (33g, 192mmol) and sodium carbonate (1lg, 153mmol) were dissolved in a solvent (216 mL) of N-methylpyrrolidone and water 5, 1' -bis diphenylphosphino ferrocene dichloropalladium (3g, 4 mmol) was added under nitrogen protection, and the mixture was heated to 100 ℃ for reaction for 8 hours. Adding 2L of water into the reaction solution, heating and stirring for 30min, and performing suction filtration to obtain a black crude product. Adding N, N-dimethylformamide (400 ml) into the crude product, heating to 50 ℃, pulping for 1h, cooling, and performing suction filtration to obtain a brown product with the yield of 91%. ¹ H NMR(500MHz,DMSO)δ13.54(s,1H),8.21(s,1H),7.61–7.39(m,2H),7.33(d,J＝8.6Hz,1H),6.79(s,2H),3.91(s,3H)；ESI-MS:m/z＝260.01[M+H] ⁺ .

The second step is that: 3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidine-4-triphenylphosphamide

3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d is added into a reaction bottle in sequence]Pyrimidin-4-amine (20g, 77mmol), triphenylphosphine oxide (211g, 77mmol) and triphenylphosphine (30g, 116mmol)Anhydrous N, N-dimethylformamide (200 mL) was added to dissolve, the mixture was placed in an ice bath under nitrogen protection, and diisopropyl azodicarboxylate (23ml, 116mmol) was slowly added dropwise. After stirring at room temperature for 30min after dropping, ethyl acetate (400 mL. Times.2) and water (400 mL) were added for extraction, and the organic phases were combined, washed with saturated brine (400 mL), dried over anhydrous sodium sulfate, filtered and the solvent was concentrated to give a crude product. Separating by silica gel column chromatography, and drying in a vacuum drying oven at 60 deg.C for 24 hr to obtain white powdery solid 33g with yield of 83%. ¹ H NMR(500MHz,CDCl ₃ )δ8.62(d,J＝12.9Hz,1H),8.16(s,2H),7.86(dd,J＝12.2,7.8Hz,6H),7.57(t,J＝7.3Hz,3H),7.48(t,J＝7.5Hz,6H),7.06(t,J＝8.6Hz,1H),4.00(s,3H)；ESI-MS:m/z＝520.16[M+H] ⁺ .

The third step: 2-acetyl-4-chloro-5-fluorophenyl trifluoromethanesulfonate

1- (5-chloro-4-fluoro-2-hydroxyphenyl) ethan-1-one (3g, 11mmols) was added to a reaction flask, dissolved in anhydrous dichloromethane (30 mL), triethylamine (3mL, 24mmol) was added thereto, the reaction flask was placed in an ice bath, trifluoromethanesulfonic anhydride (3mL, 21mmol) was slowly added dropwise, and after stirring at room temperature for 1 hour, dichloromethane (100 mL. Times.2) and water (200 mL) were added thereto for extraction and liquid separation, the organic phases were combined, washed with saturated saline (200 mL), dried over anhydrous sodium sulfate, filtered and the solvent was concentrated, and the product was isolated by silica gel column chromatography to give 5g of a pale yellow liquid with a yield of 98%. ¹ H NMR(500MHz,CDCl ₃ )δ7.93(d,J＝7.8Hz,1H),7.21(d,J＝8.3Hz,1H),2.64(s,3H)；ESI-MS:m/z＝321.01[M+H] ⁺ .

The fourth step: 2-amino-4-fluoro-5-chloroacetophenone

2-acetyl-4-chloro-5-fluorophenyl trifluoromethanesulfonate (1g, 3mmol) and diphenylimine (785. Mu.L, 5 mmol) are sequentially added to a reaction flask, dissolved in toluene (8 mL), cesium carbonate (2g, 5mmol), 1 '-binaphthyl-2, 2' -bis (diphenylphosphine) (291mg, 0.5mmol) and tris (dibenzylideneacetone) dipalladium (286mg, 0.3mmol) are sequentially added to the reaction tube, nitrogen is used for protection, and after the mixture is reacted at 80 ℃ for 8 hours, the reaction solution is reactedSlowly adding 2N hydrochloric acid solution (20 mL), heating to 60 ℃, continuously heating and stirring for 1h, filtering the reaction solution through diatomite, adjusting the pH of the filtrate to be =8-10, extracting and separating liquid, drying an organic phase through sodium sulfate hydrate, and separating through silica gel column chromatography to obtain a yellow solid with the yield of 150 mg: 27%, SI-MS m/z =210.04[ m + Na ]] ⁺ .

The fifth step: 1- (2 ' -amino-4 ' -fluoro-5 ' -chloro-phenyl) -1-ethanol

Adding 2-amino-4-fluoro-5-chloroacetophenone (900mg, 5 mmol) into a reaction bottle, dissolving in anhydrous methanol (9 mL), placing the reaction bottle in an ice bath, slowly adding sodium borohydride (91mg, 2mmol), reacting at room temperature for 1h, adding water to quench the reaction, performing rotary evaporation to remove a methanol solvent, adding dichloromethane (150 mL multiplied by 2) and water (100 mL) to extract and separate, combining organic phases, drying with anhydrous sodium sulfate, filtering, concentrating the solvent, and purifying by silica gel column chromatography to obtain a white solid 901mg with the yield of 99%. ¹ H NMR(500MHz,CDCl ₃ )δ7.06(d,J＝8.0Hz,1H),6.43(d,J＝10.7Hz,1H),4.85(q,J＝6.6Hz,1H),4.52(d,J＝178.5Hz,2H),1.56(d,J＝6.6Hz,3H)；ESI-MS:m/z＝212.04[M+Na] ⁺ .

And a sixth step: 2- (1- (tert-butyldisilyloxy)) -4-chloro-5-fluoroaniline

1- (2 ' -amino-4 ' -fluoro-5 ' -chloro-phenyl) -1-ethanol (900mg, 5 mmol) was charged into a reaction flask, and dissolved in anhydrous dichloromethane (15 mL), and imidazole (647mg, 10mmol) and t-butyldimethylsilyl chloride (859mg, 6 mmol) were added thereto, followed by reaction at room temperature for 8 hours. Dichloromethane (100 mL. Times.2) and water (100 mL) were added to extract the separated layers, the organic layers were combined, washed with saturated brine (200 mL), the organic layer was dried over anhydrous sodium sulfate, the solvent was concentrated, and the product was purified by silica gel column chromatography to give 1g of a colorless transparent oil in 97% yield. ¹ H NMR(400MHz,CDCl ₃ )δ6.95(d,J＝8.1Hz,1H),6.43(d,J＝10.8Hz,1H),4.80(q,J＝6.5Hz,1H),4.52(s,2H),1.51(d,J＝6.6Hz,3H),0.92(s,9H),0.12(s,3H),0.02(s,3H)；ESI-MS:m/z＝304.12[M+H] ⁺ .

The seventh step: n- (2- (1- (tert-butyldisilyloxy)) ethyl) -4-chloro-5-fluorophenyl) acrylamide

Adding N- (2- (1- (tert-butyldisilyloxy)) ethyl) -4-chloro-5-fluorobenzene into a reaction bottleAnd (2) dissolving acrylamide (1g, 5 mmol) and acrylic acid (459 mu L,7 mmol) in anhydrous dichloromethane (30 mL), adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2g, 9 mmol), stirring for 30min in ice bath, adding dichloromethane (100 mL multiplied by 2) and water (100 mL) for extraction and liquid separation, combining organic phases, washing with saturated saline (200 mL), drying an organic layer with anhydrous sodium sulfate, concentrating a solvent, and purifying a product by silica gel column chromatography to obtain 1g of colorless transparent oily matter with the yield of 74%. ¹ H NMR(400MHz,CDCl ₃ )δ9.77(s,1H),8.44(d,J＝11.6Hz,1H),7.05(d,J＝7.7Hz,1H),6.44(d,J＝16.9Hz,1H),6.21(dd,J＝17.0,10.3Hz,1H),5.83(d,J＝10.4Hz,1H),4.89(q,J＝6.6Hz,1H),1.52(d,J＝6.6Hz,3H),0.93(s,9H),0.17(s,3H),0.07(s,3H)；ESI-MS:m/z＝358.13[M+H] ⁺

The eighth step: n- (4-chloro-5-fluoro-2- (1-hydroxyethyl) phenyl) acrylamide

N- (2- (1- (tert-butyldimethylsilyloxy)) ethyl) -4-chloro-5-fluorophenyl) acrylamide (1g, 3mmol) was added to a reaction flask, dissolved in anhydrous tetrahydrofuran (10 mL), and a tetrahydrofuran solution of tetrabutylammonium fluoride (3mL, 32mmol) was added dropwise to the flask under ice-cooling, and after completion of dropwise addition, the flask was stirred at room temperature for 3 hours. The mixture was concentrated to remove tetrahydrofuran, ethyl acetate (100 mL. Times.2) and water (100 mL) were added to extract and separate the layers, the organic phases were combined, washed with saturated brine (200 mL), the organic layer was dried over anhydrous sodium sulfate, the solvent was concentrated, and the product was purified by silica gel column chromatography to give 580mg of a white solid in 74% yield. ¹ H NMR(400MHz,CDCl ₃ )δ9.51(s,1H),8.28(d,J＝11.4Hz,1H),7.12(d,J＝7.8Hz,1H),6.38(d,J＝16.1Hz,1H),6.22(dd,J＝17.0,10.2Hz,1H),5.79(d,J＝9.3Hz,1H),4.95(q,J＝6.4Hz,1H),2.70(s,1H),1.57(d,J＝6.7Hz,3H)；ESI-MS:m/z＝244.05[M+H] ⁺ .

The ninth step: n- (4-chloro-5-fluoro-2- (1- (3- (3-fluoro-4-methoxyphenyl) -4- ((triphenyl-15-phosphinylidene) amino) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) phenyl) acrylamide

Adding 3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d to a reaction bottle]Pyrimidine-4-triphenylphosphamide (213mg, 0.4mmol), N- (4-chloro-5-fluoro-2- (1-hydroxyethyl) phenyl) acrylamide (100mg, 0.4mmol), triphenylphosphine (161mg, 0.6mmol), dissolved in anhydrous tetrahydro-phosphineDiisopropyl azodicarboxylate (121 mu L,0.6 mmol) is slowly added dropwise in furan (4 mL) under ice bath, after stirring for 20min at room temperature, an appropriate amount of water is added to quench the reaction, tetrahydrofuran is removed by rotary evaporation, dichloromethane (100 mL. Times.2) and water (100 mL) are added to extract and separate the liquid, the organic phases are combined, the organic phase is washed with saturated saline (200 mL), the organic layer is dried with anhydrous sodium sulfate, the solvent is concentrated, and the product is purified by silica gel column chromatography to obtain 190mg of white solid. ESI-MS: m/z =745.19[ 2 ] M + H] ⁺ .

The tenth step: n- (2- (1- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) -4-chloro-5-fluorophenyl) acrylamide

Adding N- (4-chloro-5-fluoro-2- (1-hydroxyethyl) phenyl) acrylamide (190mg, 0.3 mmol), glacial acetic acid (2mL, 0.04mmol), water (2 mL) in the same volume ratio, heating to 70 ℃, reacting for 5 hours, adding saturated sodium bicarbonate solution, neutralizing the reaction solution, adjusting the pH to be neutral, extracting and separating by using dichloromethane (100 mL multiplied by 2) and water (100 mL), combining organic phases, washing by using saturated saline (200 mL), drying an organic layer by using anhydrous sodium sulfate, concentrating a solvent, and purifying a product by silica gel column chromatography to obtain 90mg of white powdery solid with the yield of 72%. ¹ H NMR(400MHz,CDCl ₃ )δ12.07(s,1H),8.31(s,1H),7.77(d,J＝10.8Hz,1H),7.42(d,J＝7.5Hz,1H),7.36(d,J＝11.6Hz,1H),7.31(d,J＝8.5Hz,1H),7.03(t,J＝8.5Hz,1H),6.68(dd,J＝17.0,10.2Hz,1H),6.53(d,J＝17.0Hz,1H),6.40(q,J＝7.2Hz,1H),6.30(s,1H),5.96(s,1H),5.79(d,J＝10.2Hz,1H),4.05(q,J＝7.1Hz,1H),3.88(s,3H),1.98(d,J＝4.2Hz,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ164.11,156.70,152.90,150.43,149.88,147.26,146.96,141.44,136.04,130.52,127.19,126.53,125.53,125.08,123.24,116.70,115.27,115.08,113.42,113.10,97.09,55.34,51.03,18.57；ESI-MS:m/z＝485.12[M+H] ⁺ .

Example 2N- (2- (1- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) phenyl) acrylamide

The first step is as follows: 1- (2-aminophenyl) ethanol

Reference example 1, step 5 reaction run. ¹ H NMR(500MHz,CDCl ₃ )δ7.11(t,J＝6.7Hz,2H),6.75(t,J＝7.5Hz,1H),6.70(d,J＝8.2Hz,1H),4.95(q,J＝6.6Hz,1H),3.18(s,3H),1.61(d,J＝6.6Hz,3H).

The second step is that: 2- (1- ((tert-butyldimethylsilyl) oxy) ethyl) aniline

Reference example 1, step 6. ¹ H NMR(400MHz,CDCl ₃ )δ6.94(t,J＝8.3Hz,1H),6.84(d,J＝7.4Hz,1H),6.55(dd,J＝14.2,7.6Hz,2H),4.75(q,J＝6.5Hz,1H),1.41(d,J＝6.6Hz,3H),0.80(d,J＝13.1Hz,9H),0.14–0.09(m,3H),0.08–0.01(m,3H)；ESI-MS:m/z＝252.17[M+H] ⁺ .

The third step: n- (2- (1- ((tert-butyldimethylsilyl) oxy) ethyl) phenyl) acrylamide

Reference example 1, step 7 reaction operation. ¹ H NMR(500MHz,CDCl ₃ )δ9.65(s,1H),8.42(d,J＝8.1Hz,1H),7.30(d,J＝2.7Hz,1H),7.02(d,J＝6.1Hz,2H),6.41(d,J＝18.0Hz,1H),6.23(dd,J＝17.0,10.3Hz,1H),5.76(d,J＝11.4Hz,1H),4.93(q,J＝6.6Hz,1H),1.51(d,J＝6.6Hz,3H),0.91(s,9H),0.14(s,3H),0.02(s,3H)；ESI-MS:m/z＝306.18[M+H] ⁺ .

The fourth step: n- (2- (1-hydroxyethyl) phenyl) acrylamide

Reference example 1 the 8 th reaction procedure. ¹ H NMR(500MHz,CDCl ₃ )δ9.40(s,1H),8.21(d,J＝7.8Hz,1H),7.30(d,J＝8.4Hz,1H),7.14(d,J＝7.3Hz,1H),7.08(t,J＝7.4Hz,1H),6.36(d,J＝16.9Hz,1H),6.25(dd,J＝17.0,10.2Hz,1H),5.74(d,J＝10.3Hz,1H),4.99(q,J＝6.6Hz,1H),3.06(s,1H),1.57(d,J＝6.7Hz,3H)；ESI-MS:m/z＝214.07[M+Na] ⁺ .

The fifth step: n- (2- (1- (3- (3-fluoro-4-methoxyphenyl) -4- ((triphenyl-15-phosphinylidene) amino) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) phenyl) acrylamide

Reference example 1, step 9. ESI-MS: m/z =693.25[ 2 ] M + H] ⁺ .

And a sixth step: n- (2- (1- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) phenyl) acrylamide

Reference example 1 the 10 th reaction procedure. ¹ H NMR(400MHz,CDCl ₃ )δ11.73(s,1H),8.38(s,1H),7.82(d,J＝7.8Hz,1H),7.50(d,J＝7.6Hz,1H),7.41(d,J＝11.7Hz,1H),7.34(t,J＝7.6Hz,2H),7.19(t,J＝7.3Hz,1H),7.07(t,J＝8.3Hz,1H),6.71(dd,J＝16.8,10.2Hz,1H),6.59–6.49(m,2H),5.76(d,J＝10.2Hz,1H),3.92(s,3H),2.04(d,J＝6.8Hz,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ165.17,157.88,153.85,150.95,148.15,147.69,142.96,136.46,131.89,130.74,129.71,127.34,126.76,125.84,125.72,124.26,116.30,116.09,114.04,98.12,56.33,52.89,19.62；ESI-MS:m/z＝433.17[M+H] ⁺ .

Example 3N- (2- (1- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) phenyl) methacrylamide

Refer to the procedure of example 2. ¹ H NMR(400MHz,CDCl ₃ )δ11.25(s,1H),8.40(s,1H),7.65(d,J＝7.8Hz,2H),7.49–7.41(m,2H),7.39(d,J＝5.9Hz,1H),7.36(s,1H),7.25–7.19(m,1H),7.08(t,J＝8.3Hz,1H),6.52(d,J＝6.8Hz,1H),6.29(s,1H),5.58(s,1H),4.97(dd,J＝14.7,6.9Hz,1H),3.93(s,2H),2.14(s,2H),2.08(s,2H)； ¹³ C NMR(101MHz,CDCl ₃ )δ165.17,157.88,153.85,150.95,148.15,147.69,142.96,136.46,131.89,130.74,129.71,127.34,126.76,125.84,125.72,124.26,116.30,116.09,114.04,98.12,56.33,52.89,29.73,19.62；ESI-MS:m/z＝447.09[M+H] ⁺ .

Example 4N- (3- (1- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) phenyl) acrylamide

Refer to the procedure of example 2. ¹ H NMR(400MHz,CDCl ₃ )δ9.36(s,1H),8.13(s,1H),8.00(s,1H),7.26–7.16(m,2H),7.10(d,J＝7.9Hz,1H),7.01(s,1H),6.80(d,J＝7.1Hz,1H),6.34(s,2H),5.60(s,1H),5.44(q,J＝6.6Hz,1H),3.92(s,3H),1.86(d,J＝4.8Hz,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ177.07,164.22,159.42,158.05,155.17,153.26,149.43,141.70,139.22,135.88,131.48,129.22,127.30,126.79,126.27,121.65,119.76,117.34,113.97,100.40,59.11,56.26,22.10；ESI-MS:m/z＝433.06[M+H] ⁺ .

Example 5N- (3- (1- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) phenyl) methacrylamide

Refer to the procedure of example 2. ¹ H NMR(400MHz,CDCl ₃ )δ8.29(s,1H),8.05(d,J＝18.0Hz,1H),7.80(s,1H),7.41(s,1H),7.23–7.03(m,2H),6.90(s,1H),5.80(s,2H),5.47(s,1H),5.43(s,1H),3.97(s,4H),2.03(s,4H),1.94(d,J＝6.4Hz,3H)； ¹³ C NMR(101MHz,CDCl3)δ165.17,157.88,153.85,150.95,148.15,147.69,142.96,136.46,131.89,130.74,129.71,127.34,126.76,125.84,125.72,124.26,116.30,116.09,114.04,98.12,56.33,52.89,29.73,19.62；ESI-MS:m/z＝447.07[M+H] ⁺ .

Example 6- (3- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one

The first step is as follows: 3- (3- (3-fluoro-4-methoxyphenyl) -4- ((triphenyl-l 5-phosphinylidene) amino) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylic acid tert-butyl ester

3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d is added into a reaction bottle in sequence]Pyrimidine-4-triphenyl phosphamide (2g, 3mmol), N-Boc-3-hydroxypiperidine (2g, 9mmol) and triphenylphosphine (2g, 6 mmol), and anhydrous tetrahydrofuran is added to dissolve the originalDiisopropyl azodicarboxylate (1mL, 6 mmol) was slowly added dropwise to the mixture in an ice bath, and the mixture was stirred at room temperature for 30min. After completion of the reaction, water was added to quench the reaction solution, tetrahydrofuran was removed by rotary evaporation, methylene chloride (200 mL. Times.2) and water (200 mL) were added to extract the reaction solution, the organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, the solvent was concentrated, and the mixture was separated by silica gel column chromatography to obtain 2g of a white solid. Yield: 94 percent. m/z =703.16[ m ] +H] ⁺ .

The second step is that: 1- (3- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one

To a reaction flask was added 3- (3- (3-fluoro-4-methoxyphenyl) -4- ((triphenyl-l 5-phosphinylidene) amino) -1H-pyrazolo [3, 4-d)]Pyrimidin-1-yl) piperidine-1-carboxylic acid tert-butyl ester (2g, 3mmol), 4M HCl in dioxane (3mL, 13mmol), was heated at 70 ℃ for 5h. Concentrating the solvent, adding methyl tert-butyl ether, pulping, performing suction filtration to obtain a white-like solid 2g, adding the white-like solid into a reaction bottle, simultaneously adding anhydrous dichloromethane (10 mL), triethylamine (553 uL, 4 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (766mg, 4 mmol), slowly dropwise adding acrylic acid (180 uL, 2 mmol) under ice bath, stirring at room temperature for 8h after dropwise adding, adding dichloromethane (100 mL multiplied by 2) and water (100 mL) for extraction, combining organic phases, washing with saturated saline (200 mL), drying with anhydrous sodium sulfate, filtering, concentrating the solvent, and separating the product by silica gel column chromatography to obtain a white solid 156mg with the yield of 30%. ¹ H NMR(400MHz,CDCl ₃ )δ8.34(d,J＝24.5Hz,1H),7.50–7.38(m,2H),7.13(t,J＝8.4Hz,1H),6.69–6.52(m,1H),6.30(dd,J＝16.2,11.7Hz,1H),5.79(s,2H),5.70(d,J＝15.9Hz,1H),4.90(s,1H),4.86–4.52(m,1H),4.12(dd,J＝61.7,13.1Hz,1H),3.97(s,3H),3.56(t,J＝12.5Hz,1H),2.89(t,J＝11.7Hz,1H),2.44–2.30(m,1H),2.26(s,1H),2.00(d,J＝13.8Hz,1H),1.72(d,J＝1.8Hz,1H)； ¹³ C NMR(101MHz,CDCl ₃ )δ165.75,157.75,155.72,155.58,153.89,151.42,148.58,143.22,128.16,127.59,124.44,116.49,116.30,113.97,98.39,56.40,50.02,45.94,42.17,25.29,23.92；ESI-MS:m/z＝397.17[M+H] ⁺ .

Example 7- (3- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -2-methylpropan-2-en-1-one

Refer to the procedure of example 6. ¹ H NMR(400MHz,CDCl ₃ )δ8.29(s,1H),7.41(dd,J＝14.7,10.2Hz,2H),7.11(t,J＝8.4Hz,1H),5.89(s,2H),5.15(s,1H),5.07(s,1H),4.83(s,1H),4.58(s,1H),4.09(d,J＝71.0Hz,1H),3.95(s,3H),3.52(s,1H),2.97(s 1H),2.31(s,1H),2.22(d,J＝9.9Hz,1H),2.00(s,1H),1.95(s,3H),1.69(s,1H)； ¹³ C NMR(101MHz,CDCl ₃ )δ171.49,157.90,155.77,153.87,151.40,148.50,143.19,140.40,126.02,124.44,116.59,116.30,115.48,113.95,98.41,56.39,53.39,45.55,41.34,26.96,20.58；ESI-MS:m/z＝411.19[M+H] ⁺ .

Example 8- (3- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -2-fluoroprop-2-en-1-one

Refer to the procedure of example 6. ¹ H NMR(400MHz,CDCl ₃ )δ8.33(s,1H),7.44(dd,J＝14.7,10.2Hz,2H),7.13(t,J＝8.4Hz,1H),5.99(s,1H),5.26(d,J＝3.3Hz,1H),5.12(s,1H),4.98–4.85(m,1H),4.59(d,J＝85.0Hz,1H),3.97(s,3H),3.89(d,J＝67.5Hz,4H),3.34(d,J＝95.9Hz,1H),2.76(d,J＝47.2Hz,1H),2.42–2.30(m,1H),2.25(d,J＝12.9Hz,1H),2.02(d,J＝14.5Hz,2H),1.77(d,J＝13.1Hz,1H)； ¹³ C NMR(101MHz,CDCl ₃ )δ171.24,161.59,161.29,157.84,155.60,153.88,151.41,148.65,143.36,125.86,124.42,116.48,116.29,113.97,98.40,60.44,56.39,42.56,30.26,21.10,14.21；ESI-MS:m/z＝415.16[M+H] ⁺ .

Example 9- (4- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one

Refer to the procedure of example 6. ¹ H NMR(400MHz,CDCl ₃ )δ8.27(s,1H),7.34(t,J＝10.7Hz,2H),7.04(t,J＝8.4Hz,1H),6.59–6.47(m,1H),6.21(d,J＝16.8Hz,1H),5.63(d,J＝10.6Hz,1H),4.95(dd,J＝9.8,5.7Hz,1H),4.74(d,J＝11.5Hz,1H),4.11(d,J＝12.2Hz,1H),3.88(s,3H),3.25(t,J＝9.0Hz,1H),2.84(t,J＝12.1Hz,1H),2.24(d,J＝11.9Hz,2H),2.02(d,J＝10.5Hz,2H)； ¹³ C NMR(101MHz,CDCl ₃ )δ174.77,165.51,158.00,155.67,153.85,151.36,148.48,146.57,143.03,127.74,124.43,116.44,113.99,98.46,56.38,53.85,45.01,41.25,31.77,31.07；ESI-MS:m/z＝397.09[M+H] ⁺ .

Example 10- (4- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -2-methylpropan-2-en-1-one

Refer to the procedure of example 6. ¹ H NMR(400MHz,DMSO)δ8.26(s,1H),7.75(d,J＝6.1Hz,1H),7.49(t,J＝10.0Hz,3H),7.36(t,J＝8.6Hz,1H),6.87(s,1H),5.68(s,1H),5.35(s,1H),4.78(s,1H),3.94(s,5H),2.53(s,6H),2.35(dd,J＝19.1,9.3Hz,3H),1.97(s,3H),1.89(s,4H)； ¹³ C NMR(101MHz,DMSO)δ168.28,158.61,155.82,154.10,153.24,150.81,147.96,142.27,140.80,126.43,125.31,119.12,116.41,116.22,114.81,97.88,56.58,54.03,45.42,28.53,27.73,19.63；ESI-MS:m/z＝425.20[M+H] ⁺ .

Example 11- (3- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidin-1-yl) prop-2-en-1-one

Refer to the procedure of example 6. ¹ H NMR(400MHz,CDCl ₃ )δ8.37(d,J＝2.8Hz,1H),7.42(dt,J＝14.3,7.1Hz,2H),7.12(t,J＝8.3Hz,1H),6.43–6.41(m,1H),5.79–5.69(m,2H),5.57(dd,J＝12.2,6.3Hz,1H),4.14–4.08(m,1H),3.97(s,3H),3.79(d,J＝9.1Hz,1H),3.31–3.20(m,1H),2.74–2.51(m,2H)； ¹³ C NMR(101MHz,CDCl ₃ )δ164.63,157.83,155.97,154.60,153.86,151.37,148.71,143.48,128.33,128.11,124.46,116.43,113.93,98.66,56.40,55.63,50.59,31.52,29.62；ESI-MS:m/z＝405.05[M+Na] ⁺ .

Example 12- (3- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidin-1-yl) -2-methylprop-2-en-1-one

Refer to the procedure of example 6. ¹ H NMR(400MHz,CDCl ₃ )δ8.37(s,1H),7.41(t,J＝16.0Hz,2H),7.13(d,J＝7.8Hz,1H),5.64(d,J＝27.7Hz,2H),5.18(t,J＝15.2Hz,2H),3.97(s,3H),3.72(s,2H),3.28(s,1H),2.26(s,3H),1.97(d,J＝15.2Hz,5H)； ¹³ C NMR(101MHz,CDCl ₃ )δ171.04,157.76,155.94,154.55,142.10,141.41,139.09,124.44,122.07,116.72,116.40,115.27,113.98,98.62,56.39,52.56,44.70,44.12,26.01,19.94；ESI-MS:m/z＝397.09[M+H] ⁺ .

Example 13N- (4- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) cyclohexyl) acrylamide

Refer to the procedure of example 6. ¹ H NMR(400MHz,DMSO)δ8.24(s,1H),7.46(t,J＝9.6Hz,2H),7.34(t,J＝8.5Hz,1H),6.43(dd,J＝16.9,10.2Hz,1H),6.11(d,J＝17.0Hz,1H),5.58(d,J＝10.0Hz,1H),4.75(s,1H),3.98(s,1H),3.92(s,3H),2.51(s,4H),2.25(d,J＝10.6Hz,2H),1.89(s,2H)； ¹³ C NMR(101MHz,DMSO)δ164.58,158.62,155.93,153.98,153.18,150.75,147.95,142.48,132.41,126.24,125.37,116.24,114.72,97.80,56.48,54.32,44.34,28.97,27.44；ESI-MS:m/z＝411.09[M+H] ⁺ .

Example 14N- (4- (4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) cyclohexyl) methacrylamide

Refer to the procedure of example 6. ¹ H NMR(400MHz,DMSO)δ8.26(s,1H),7.75(d,J＝6.1Hz,1H),7.49(t,J＝10.0Hz,3H),7.36(t,J＝8.6Hz,1H),6.87(s,1H),5.68(s,1H),5.35(s,1H),4.78(s,1H),3.94(s,5H),2.53(s,6H),2.35(dd,J＝19.1,9.3Hz,3H),1.97(s,3H),1.89(s,4H)； ¹³ C NMR(101MHz,DMSO)δ168.28,158.61,155.82,154.10,153.24,150.81,147.96,142.27,140.80,126.43,125.31,119.12,116.41,116.22,114.81,97.88,56.58,54.03,45.42,28.53,27.73,19.63；ESI-MS:m/z＝425.20[M+H] ⁺ .

Example 15- (4- ((4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) piperidin-1-yl) prop-2-en-1-one

Refer to the procedure of example 6. ¹ H NMR(400MHz,CDCl ₃ )δ8.36(s,1H),7.55–7.39(m,2H),7.13(t,J＝8.4Hz,1H),6.56(dd,J＝16.8,10.6Hz,1H),6.24(d,J＝16.8Hz,1H),5.81(s,2H),5.66(d,J＝10.5Hz,1H),4.67(d,J＝12.5Hz,1H),4.34(d,J＝7.0Hz,2H),3.97(s,3H),3.03(t,J＝12.1Hz,1H),2.69–2.58(m,1H),2.34(s,2H),1.70(s,2H),1.37–1.27(m,2H)； ¹³ C NMR(101MHz,CDCl ₃ )δ165.43,157.88,156.00,154.80,151.42,148.52,143.13,127.85,127.52,124.44,116.54,114.00,98.16,56.40,51.94,45.62,41.82,36.86,30.47,29.35；ESI-MS:m/z＝411.09[M+H] ⁺ .

Example 16- (2- ((4-amino-3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyrrolidin-1-yl) prop-2-en-1-one

Refer to the procedure of example 6. ¹ H NMR(400MHz,CDCl ₃ )δ8.30(d,J＝8.4Hz,1H),7.42–7.30(m,2H),7.05(dd,J＝17.9,8.4Hz,1H),6.61(dd,J＝16.6,10.3Hz,1H),6.38–6.31(m,2H),5.74(d,J＝10.4Hz,1H),5.62(dd,J＝8.8,3.5Hz,1H),4.61(dd,J＝15.4,5.5Hz,1H),4.36(t,J＝6.6Hz,1H),3.90(d,J＝3.8Hz,3H),3.43(dd,J＝7.9,5.1Hz,1H),3.24(td,J＝12.5,6.3Hz,1H),2.51–2.45(m,1H),2.30(t,J＝6.7Hz,1H),2.20(s,2H),1.96(d,J＝7.5Hz,1H)； ¹³ C NMR(101MHz,CDCl ₃ )δ163.95,163.55,156.68,155.30,155.02,129.88,128.64,127.89,127.01,126.85,123.42,115.44,113.00,97.18,55.34,46.82,43.78,34.43,26.41,22.77；ESI-MS:m/z＝397.09[M+H] ⁺ .

EXAMPLE 17 preparation of the hydrochloride salt (exemplified by the Compound of example 12)

Dissolving a proper amount of the compound in ethanol, and slowly introducing HCl gas to saturation at room temperature. The mixture was cooled in an ice bath to precipitate white crystals slowly as the hydrochloride of the compound.

Example 18 kinase inhibition assay

1. Measurement of PI3K delta kinase inhibitory Activity

The inhibitory activity of PI3K delta kinase is detected by adopting an ADP-Glo method:

the experimental process comprises the following steps: add 2.5. Mu.L of 2-fold final concentration kinase solution to each well, centrifuge at 1000rpm for 30s, mix well with shaking, and incubate at room temperature for 10min. Then 2.5. Mu.L of a mixed solution of ATP and the substrate at a final concentration of 2 times was added thereto, and the mixture was centrifuged for 30 seconds, shaken and mixed well, and then reacted at room temperature. Add 5. Mu.L ADP-Glo Reagent, centrifuge for 30s, mix well with shaking and incubate at room temperature for 180min. 10 μ L of Kinase Detection Reagent was added, centrifuged, shaken and mixed well and incubated at room temperature for 30min. And finally, reading the luminescence value RLU by using a microplate reader.

2. Determination of BTK kinase inhibitory Activity

The inhibitory activity of BTK kinase was measured by the Mobility shift assay method:

the experimental process comprises the following steps: mu.L of a 2.5-fold final concentration kinase solution was added to each well, centrifuged at 1000rpm for 30s, the reaction plate was shaken and mixed, and incubated at room temperature for 10min. Add 15. Mu.L of a mixed solution of ATP and substrate at 25/15 times the final concentration, centrifuge for 30s, shake and mix well, and start the reaction at room temperature. Add 30. Mu.L of termination detection solution to stop the kinase reaction, centrifuge at 1000rpm for 30s, shake and mix. The conversion was read with a microplate reader.

3. The results are as follows:

TABLE 1 inhibitory Activity of Compounds of interest on PI3K delta and BTK kinases

( Note: NA indicates no investigated activity; * Data indicating published activity of the positive drug, not observed )

The series of compounds have good inhibition activity on PI3K delta, the highest inhibition activity can reach 9.0nM, the highest inhibition activity on BTK can also reach 19.5nM, and the compounds CXY20, CXY23 and CXY27 have good PI3K delta/BTK kinase dual inhibition activity.

Example 19 measurement of inhibitory Activity at cellular level

Cell inoculation: the suspension cells were spun down, resuspended in culture medium, and then counted using a cytometer. The cell suspension is diluted in the medium to the desired density. According to the plate map, 100 u L cells were inoculated into 96 hole culture dish. Medium only was used as background control (Min) and incubated overnight at 37 ℃.

Sample preparation: 200 Xsolutions were prepared in DMSO and the compounds were diluted to 3-fold final concentration with medium. Adding 50. Mu.L of sample to the cells, 37 ℃,5% CO ₂ And (5) incubating for 72h.

And (4) measuring the result: the plates were allowed to equilibrate to room temperature before measurement, 40. Mu.L per well

The contents were mixed on an orbital shaker for 2min to induce cell lysis. And (5) incubating at room temperature for 60min, and reading a luminescence value by a microplate reader.

The results were as follows:

TABLE 2 inhibitory Activity of Compounds of interest 20,23 and 27 on tumor cells

It can be seen that: although CXY20, CXY23 and CXY27 had lower inhibitory activity than the positive drug Ibrutinib at the kinase level (about 40 times lower as seen in table 1), the cell inhibitory activity of this experiment was similar to, or even higher than, that of the positive drug. In minio cells, compound CXY20, 23 did not show better inhibitory activity, but compound CXY27 was slightly more effective than the single positive control; in both JeKo-1 and H9 cell lines, compound CXY27 showed superior inhibitory effects to those of the positive drug administered alone and in synergy, and its IC ₅₀ 1.6. Mu.M and 10.8. Mu.M, respectively. This indicates that the dual-target inhibitor designed by us really has the effect of synergistically inhibiting the two targets.

While CXY20 compound has a good kinase inhibitory activity, it is not remarkable in cell activity, and may be caused by its poor physicochemical properties and poor membrane permeability. Therefore, we further examined the metabolism of the target compound in rats.

EXAMPLE 20 determination of in vivo metabolic Properties of drugs

Sprague-Dawley male rats 9, divided into 3 groups of 3 rats, fasted for more than 8h before administration. Compounds CXY20, 23 and 27 were each taken 10mg, and DMSO, HS15 and physiological saline were added to prepare a 1mg/mL solution. Groups 3 rats were individually gavaged with 10mg/kg compound (groups A-C). 0.2-0.25 mL of blood is collected from the eye sockets before (T0) administration and 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h and 24h after administration, the blood is added into a centrifuge tube containing 20 mu L of anticoagulant EDTA-2Na, the blood plasma is obtained after centrifugation (4000 rpm, 10min) within 30min, and 50 mu L of blood plasma is collected and stored in a refrigerator at the temperature of-20 ℃.

Sample treatment and determination methods: compounds 20,23 and 27 were formulated as 1mg/mL stock solutions and diluted in gradient to working solutions at concentrations of 1000, 500, 250, 100, 50, 25, 10, 5, 2.5ng/mL for use. mu.L of chromatographic acetonitrile containing loratadine was added to 50. Mu.L of plasma samples, vortexed for 2-3min, centrifuged (14000rpm, 10min), and 200. Mu.L of supernatant was taken in a lined tube to test all plasma samples.

Chromatographic conditions are as follows: sample introduction volume: 1 mu L of the solution; the type of the chromatographic column: poroshell 120EC-C18 (2.1X 50mm,1.9 μm); sample chamber temperature: 8 ℃; temperature of the column oven: at 37 ℃; mobile phase a was water (0.1% formic acid in water) and mobile phase B was acetonitrile, the flow rate was 0.4mL/min, gradient elution.

A. And (3) preparing a standard curve for the three groups of samples B and C respectively, and preparing low, medium and high 3 concentration (the concentration of the compound is 5, 100 and 500ng/mL respectively) follow-up quality control samples for detection. And finally, calculating the concentration of the drug to be detected in the plasma sample of the rat after administration according to the standard curve. Pharmacokinetic parameters were calculated using a fitting with a non-compartmental model in Phoenix WinNonlin software (version 6.3, pharsight Inc., USA).

The blood concentration of the test compound within 24 hours after oral administration (Dose: 10 mg/kg) of SD rats was measured, the blood taking time was 0.25,0.5,1,2,4,6,8,12,24h, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software, and the results are shown in Table 3:

TABLE 3 Primary pharmacokinetic parameters for Compounds CXY20, 23 and 27

(data obtained from non-identical experiments)

By measuring the plasma levels of compounds CXY20, 23 and 27 in rats, the pharmacokinetic properties associated with the absorption, distribution and elimination of these compounds can be obtained. The compounds CXY20, 23 and 27 reach C quickly between 0.25 and 0.33h _max Peak concentrations of 80.89 + -15.76, 316.51 + -138.39, 365.65 + -128.00 ng/mL ^-1 ，AUC _0-∞ Respectively 175.13 +/-79.73, 393.48 +/-77.98，551.42±262.85h·ng·mL ^-1 Indicating that the exposure levels of compounds 23 and 27 were generally higher than compound 20. The apparent volume distributions of the compounds 20,23 and 27 were 675.97. + -. 635.07, 166.56. + -. 12.97, 161.03. + -. 135.33 L.kg ^-1 Indicating that the compound 20 is widely distributed in vivo. The half-life of the target compound is between 4 and 6 hours, and the plasma clearance rates are respectively 64.12 +/-23.13, 26.03 +/-4.68 and 22.07 +/-12.73 L.h ^-1 ·kg ^-1 . The pharmacokinetic parameters of the target compound all vary greatly from individual to individual.

The peak concentrations of the three target compounds reached quickly in 0.25-0.33 h, which is consistent with the positive drug Ibrutinib, indicating that these compounds absorb at a fast rate, thus leading to two discussions: (1) the time point of the absorption phase is designed to be too loose, and the sampling points of 5min and 45min are increased to fit the curve of the absorption phase. (2) The compound has fast absorption and can release medicine stably via slow release preparation.

From the drug absorption point of view, the exposure level of compound 27 was higher and similar to the positive drug Ibrutinib. And C of Compound 20 _max Lower, absorption is poorer, and in view of higher kinase and cytostatic activity, the absorption of the compound can be increased by changing the drug property, the drug dosage form or the administration way, and the like, so as to achieve better drug effect, which will be part of research in the later period. From a drug elimination perspective, compound 27 eliminated more slowly than Ibrutinib. Thus, by this single dose experiment, it was initially judged that compound 27 was exposed to higher levels, eliminated at a slower rate, had a longer duration of drug action, and had potential for further development.

Claims (8)

1. A compound having PI3K δ/BTK dual-target activity, having the structure of formula a:

wherein R has the following structure:

2. the pharmaceutically acceptable salt of a compound having dual PI3K δ/BTK target activity according to claim 1, wherein the pharmaceutically acceptable salt is a hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, oxalate, tartrate, methanesulfonate, p-toluenesulfonate, fumarate, taurate, citrate, succinate, or a mixed salt thereof.

3. The method of preparing a compound having PI3K δ/BTK dual-target activity according to claim 1, comprising the steps of:

the first step is as follows: 3-iodine-1H-pyrazolo [3,4-d ] pyrimidine-4-amine and 3-fluoro-4-methoxy boric acid are reacted in a solvent system of N-methyl pyrrolidone and water to generate a compound with a B-1 structure under the action of sodium carbonate and 1,1' -bis diphenylphosphino ferrocene palladium dichloride;

the second step: adding 3- (3-fluoro-4-methoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine, triphenylphosphine oxide and triphenylphosphine, adding N, N-dimethylformamide for dissolving, placing under an ice bath under the protection of nitrogen, and dropwise adding diisopropyl azodicarboxylate to obtain a compound with a B-2 structure;

the third step: intermediate B-2 with alcohol R ₃ OH is mixed, triphenylphosphine TPP is added, and azodicarbonyl is added dropwise under ice bathDiisopropyl formate, stirring for reaction, monitoring the reaction process by TLC, adding water for quenching, extracting by dichloromethane, and separating by silica gel column chromatography to obtain a product C-1;

wherein R3 is one of the following structures:

the fourth step: removing a protecting group of the obtained C-1 product in dilute hydrochloric acid to obtain C-2;

wherein R4 is one of the following structures:

the fifth step: carrying out condensation reaction on the intermediate C-2 and acrylic acid substituted by R5 to obtain a compound with a structure shown in a formula A;

r5 is H, F or methyl, and the acrylic acid substituted by R5 is

4. Use of a compound having PI3K δ/BTK dual-target activity according to claim 1 for the preparation of a pharmaceutical preparation for the prevention or treatment of a disease caused by an abnormality in PI3K δ or BTK protein.

5. Use of a compound having a PI3K δ/BTK dual-target activity according to claim 2 in the preparation of a pharmaceutical preparation of a pharmaceutically acceptable salt for the prevention or treatment of a disease caused by an abnormality of PI3K δ or BTK protein.

6. The use according to claim 4 or 5, wherein the disease is lymphoma or is an autoimmune disease.

7. The use of claim 6, wherein said lymphoma is selected from the group consisting of chronic lymphocytic leukemia, B-cell lymphoma, mantle cell lymphoma, lymphoplasmacytic lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, follicular central lymphoma, marginal zone B-cell lymphoma.

8. The use of claim 6, wherein said autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis.

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