CN113968860B - Reversible BTK inhibitor and synthesis method and application thereof - Google Patents
Reversible BTK inhibitor and synthesis method and application thereof Download PDFInfo
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Abstract
The invention discloses a reversible BTK inhibitor, a synthesis method and application thereof, which have a structure shown in a formula I, wherein X is independently hydrogen, hydroxyl, amino or halogen atom, and R is selected from the following structures:
Description
Technical Field
The invention relates to the field of small molecule medicines, in particular to a novel molecule with protein tyrosine kinase (BTK) inhibitory activity, and synthesis and application of the compound.
Background
According to the statistics of world health organization, the annual growth rate of lymphoma incidence is 5% -7%, the annual death number is over 20 ten thousand, at present, the annual growth rate of lymphoma incidence in China is 3% -5%, and the annual new incidence is about 10 ten thousand, so that the lymphoma has become the eighth high-incidence malignant tumor.
Bruton's tyrosine kinase, BTK), a membrane-bound protein, belongs to the Tec family of non-receptor tyrosine kinases, and is found in all hematopoietic cells except T cells and natural killer cells. BTK is an important signal molecule of B cell receptor pathway, expressed at each development stage of B cells, involved in regulating proliferation, differentiation and apoptosis of B cells, and plays an important role in survival and diffusion of malignant B cells. Therefore, BTK is a research hotspot for clinically treating B-cell tumors and B-cell immune diseases at present.
BTK inhibitors can inhibit lymphocyte proliferation by inhibiting the excessive activation of BCR signaling pathways, and show excellent therapeutic prospects for the treatment of B-cell malignancies and autoimmune diseases. In 2013, ibrutinib was approved by the FDA as the first effective BTK selective inhibitor, and breakthrough therapy was used for the treatment of chronic lymphocytic leukemia, mantle cell lymphoma, small lymphocytic lymphoma, fahrenheit macroglobulinemia, marginal zone lymphoma and graft-host disease, which is of epoch-making significance, making BTK a promising therapeutic target.
Although a number of covalently bound small molecule BTK inhibitors have been used clinically, such as ibrutinib, acartinib, zebutinib, etc., the increased number of patients receiving treatment and prolonged follow-up time, as well as resistance to ibrutinib caused by mutation at Cys481 residue, have triggered thinking and worry about the Michael acceptor moiety of covalent inhibitors. It is therefore generally believed that reversible inhibitors are more likely to have lower risk of toxicity in B cell malignancies and autoimmune disease patients than irreversible inhibitors.
CGI-1746 is the first reported compound to bind to BTK-specific inactive conformations, a reversible inhibitor of ATP competition with very high selectivity. CGI-1746 can inhibit two phosphorylation pathways of BTK at the same time, and the molecule can be combined in a binding pocket of BTK inactive conformation and has certain stability. GDC-0834 is inactive to receptors other than BTK and is a highly selective, non-covalent, reversible BTK inhibitor, which is IC in vitro 50 The value was 0.060. Mu.M. GDC-0834 shows efficacy in a rat collagen-induced arthritis (CIA) model, resulting in a dose-dependent decrease in ankle swelling and a reduction in morphological pathology, and is considered as a potential therapeutic drug for rheumatoid arthritis. ARQ-531 is a candidate drug for inhibiting BTK wild type and Cys481 mutant, and is an oral and reversible BTK inhibitor. In preclinical studies, ARQ-531 shows high oral bioavailability and good pharmacokinetic, metabolic properties. RN-486 is one ofHighly selective BTK inhibitors can inhibit BCR-mediated release of activation inducer CD 69. In addition, the partial reversible inhibitor can solve the problem of drug resistance caused by mutation of cysteine at Cys481 position in BTK kinase, for example, the compounds such as ARQ531, GNE-431 and the like have obvious inhibition effect on mutated BTK protein kinase. Reversible BTK inhibitors are shown below:
however, the development of reversible BTK inhibitors is slow compared to irreversible covalent inhibitors, which to date are still in the clinical trial phase. In view of the prospect of BTK for the treatment of autoimmune diseases, there is an urgent need to develop novel reversible inhibitors with good potency and pharmacokinetic properties.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides a small molecular compound with a pyrazolo [3,4-d ] pyrimidine structure and reversible BTK inhibitory activity, and pharmaceutically usable salts, a synthesis method and application thereof.
The aim of the invention is realized by the following technical scheme: a small molecule compound of pyrazolo [3,4-d ] pyrimidine structure having reversible BTK inhibitory activity, having the structure:
wherein R is selected from the following structures:
x is independently hydrogen, hydroxy, amino or halogen.
Further, the small molecule compound with pyrazolo [3,4-d ] pyrimidine structure having reversible BTK inhibitory activity is selected from the following structures:
x is independently hydrogen or a fluorine atom.
More particularly, the small molecule compound with a pyrazolo [3,4-d ] pyrimidine structure having reversible BTK inhibitory activity is selected from the following structures:
all compounds showed inhibitory activity against BTK protein and BTK mutant strain (BTKC 481S) and activity against mutant strain was stronger than wild type. Among them, compound 11 is the best active, the inhibitory activity on BTK protein is in nM level; compound 6 has better activity on wild type and mutant.
The small molecular compound with the reversible BTK inhibitory activity and the pyrazolo [3,4-d ] pyrimidine structure can be singly used or can be prepared into pharmaceutically acceptable salts by a conventional method for use, wherein the pharmaceutically acceptable salts are hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, acetate, propionate, butyrate, oxalate, tartrate, methanesulfonate, p-toluenesulfonate, fumarate, taurine, citrate, succinate or mixed salts thereof.
The invention also provides a preparation method of a small molecular compound with a pyrazolo [3,4-d ] pyrimidine structure with reversible BTK inhibition activity, which specifically comprises the following steps:
step a, p-bromobenzoic acid or a substituent thereof is taken as an initial raw material, and is subjected to condensation reaction with 2-aminopyridine under the action of condensing agents EDC, HCl/HOBt and N, N-diisopropylethylamine to obtain N-pyridyl-4-bromobenzamide;
step b, coupling the obtained N-pyrimidinyl-4-bromobenzamide and bisboronic acid pinacol ester through a palace pump to obtain a corresponding borate product;
step c, carrying out Mitsunobu reaction on the raw material 4-amino-3-iodine-1H-pyrazolo [3,4-d ] pyrimidine and the raw material (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine to obtain (3R) -1-Boc-3- (4-amino-3-iodine-1H-pyrazolo [3,4-d ] pyrimidine-1-yl) piperidine;
step d, performing Suzuki reaction on the (3R) -1-Boc-3- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidine-1-yl) piperidine and the obtained boric acid ester product to obtain a coupling product;
step e, removing Boc protecting groups from the coupling product under the condition of 3M HCl-EA solution to obtain piperidine compounds;
and f, performing condensation or substitution reaction on the piperidine compound to obtain a target product.
The reaction equation is as follows:
wherein X, R is consistent with the foregoing.
The invention also provides application of the small molecule compound with the pyrazolo [3,4-d ] pyrimidine structure with reversible BTK inhibition activity and pharmaceutically acceptable salts thereof in pharmacy, wherein the application is specifically as follows: the preparation is used for preparing a pharmaceutical preparation for preventing or treating diseases caused by the excessive expression of BTK protein, wherein the diseases are lymphomas including chronic lymphocytic leukemia, B cell lymphomas, mantle cell lymphomas, lymphoplasmacytic lymphomas, diffuse large B cell lymphomas, non-Hodgkin lymphomas, follicular central lymphomas and marginal zone B cell lymphomas, or autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis and the like.
Compared with the prior art, the invention has the following advantages:
the reversible BTK inhibitor provided by the invention can competitively occupy the binding cavity of ATP and kinase to play a role in inhibition, and is independent of interaction with Cys481, the binding with the ATP hinge region is realized through weaker reversible acting forces such as hydrogen bond, van der Waals force, hydrophobic acting force and the like, and as the metabolism of the drug progresses, the drug and the enzyme are combined and separated to reach equilibrium. Thus, the side effects such as acquired drug resistance and the like are greatly reduced. Therefore, the pyrazolo [3,4-d ] pyrimidine compound with reversible BTK inhibitory activity and the pharmaceutically acceptable salt thereof can be applied to the preparation of medicaments for preventing or treating diseases caused by excessive growth of BTK protein, including B cell malignant tumor, asthma, rheumatoid arthritis and the like.
Detailed Description
The structure, the preparation method and the use of the pharmaceutical preparation for preventing or treating diseases caused by the overexpression of tubulin according to the present invention are further described below with reference to examples, but the present invention is not limited thereto.
Analytical data for the samples were determined by the following instrument:
the thermometer is uncorrected; bruker DRX400 nuclear magnetic resonance apparatus; agilent 5975 mass spectrometer; bruker Vector 22 IR spectrometer.
EXAMPLE 1 Synthesis of intermediate 1-1
1.31g (6.52 mmol) of p-bromobenzoic acid was weighed into a 10mL single-necked flask, 5mL of methylene chloride was added, 1.25g (6.52 mmol) of EDC. HCl was sequentially added under ice bath conditions, 2.81mL (16.14 mmol) of DIPEA was stirred for 5min, 0.50g (5.38 mmol) of 2-aminopyridine was added at 0℃and then left to react overnight at room temperature. The reaction was monitored by TLC and the dichloromethane was removed under reduced pressure, saturated brine (200 mL. Times.3) and ethyl acetate (150 mL) were added for extraction, the organic phases were combined, dried over anhydrous sodium sulfate, and dried by spin-drying followed by dry-stirring and column chromatography to give a brown powdered solid in about 89.1% yield. MS (ESI) m/z=276.99 [ M+H ]] + ； 1 H NMR(400MHz,CDCl 3 )δ8.93(s,1H),8.63(d,J＝1.3Hz,1H),8.46(d,J＝2.5Hz,1H),8.35(dd,J＝2.5,1.5Hz,1H),7.60–7.58(m,2H),7.55–7.53(m,2H).
EXAMPLE 2 Synthesis of intermediate 1-2
1.00g (3.61 mmol) of the product of example 1, 1.10g (4.33 mmol) of pinacol biborate and [1,1' -bis (diphenylphosphine) ferrocene were weighed out in sequence]Palladium dichloride dichloromethane Complex 79mg (0.11 mmol), potassium acetate 1.06g (10.83 mmol) in a 25mL three-necked flask, N 2 Protection, after which the syringe was added with tetrahydrofuran solution and refluxed overnight. After the reaction of the spot plate raw materials is finished, the reaction liquid is cooled to room temperature and is filtered by diatomite in a pumping way, and is washed by tetrahydrofuran for 2 to 3 times, the filtrate is concentrated and is added with water and is extracted by ethyl acetate, the organic layer is washed by water for 2 to 3 times, the crude product is obtained after decompression recovery, and the light brown powdery solid is obtained after separation and purification by column chromatography, and the yield is about 92.6 percent 1 H NMR(400MHz,DMSO-d 6 )δ10.89(s,1H),8.40(d,J＝4.8Hz,1H),8.19(d,J＝8.4Hz,1H),8.02(d,J＝8.2Hz,2H),7.88–7.82(m,1H),7.79(d,J＝8.2Hz,2H),7.24–7.15(m,1H),1.32(s,12H).
EXAMPLE 3 Synthesis of intermediate 2-1
1.00g (4.57 mol) of 3-fluoro-4-bromobenzoic acid was weighed into a single-necked flask, 5mL of methylene chloride was added, and after the flask was placed under ice-bath conditions, 1.74g (4.57 mmol) of HATU, 2.39mL (13.71 mmol) of DIPEA and 1.13g (3.81 mmol) of 2-aminopyridine were sequentially added, followed by transfer to room temperature for reaction overnight. After the reaction was completed by TLC, the solvent dichloromethane was removed under reduced pressure, saturated brine (200 ml×3) and ethyl acetate 250mL were added for extraction, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, dry-mixed and passed through a column, and PE wet-packed, EA: pe=1:4 eluted to give 1.12g of a pale brown powdery solid, the yield was about 74.6%. MS (ESI) m/z=294.98 [ M+H ]] + ； 1 H NMR(400MHz,DMSO-d6)δ10.99(s,1H),8.41(ddd,J＝4.8,1.9,0.8Hz,1H),8.17(d,J＝8.4Hz,1H),8.02(dd,J＝9.8,1.9Hz,1H),7.93–7.79(m,3H),7.25–7.14(m,1H).
EXAMPLE 4 Synthesis of intermediate 2-2
Operating in the same manner as example 2, intermediate 2-1 was substituted for intermediate 1-1 to give a white solid in a yield of about 91.6%; 1 H NMR(400MHz,DMSO-d 6 )δ10.93(d,J＝32.2Hz,1H),8.47–8.35(m,2H),8.18(d,J＝8.4Hz,1H),7.90–7.69(m,3H),7.24–7.14(m,1H),1.33(s,12H).
EXAMPLE 5 Synthesis of intermediate 2-3
Weighing 4-amino-3-iodine-1H-pyrazolo [3,4-d]Pyrimidine 500mg (1.92 mmol), 1-t-butoxycarbonyl-3-hydroxypiperidine 774mg (3.83 mmol), triphenylphosphine 755mg (2.88 mmol) were added to a three-necked flask, N 2 The reaction was carried out under ice-bath conditions by adding anhydrous THF via syringe, slowly dropping DIAD in anhydrous THF, and transferring to room temperature for overnight reaction. After completion of the TLC plate starting material reaction, THF was removed under reduced pressure, dry-mixed and passed through a column, and the PE wet column was washed out with yellow DIAD, followed by gradient elution to give 708mg of white powdery solid, intermediate 2-3, in a yield of about 83.1%. MS (ESI) m/z=467.24 [ M+Na ]] + ； 1 H NMR(400MHz,DMSO-d 6 )δ8.21(s,1H),4.65–4.53(m,1H),3.88(t,J＝61.6Hz,2H),2.98(s,1H),2.19–2.08(m,1H),2.07–1.98(m,1H),1.87(s,1H),1.54(dt,J＝21.8,7.5Hz,2H),1.32(s,9H).
EXAMPLE 6 Synthesis of intermediates 1-3
Example 1 433 mg (1.35 mmol), example 5 500mg (1.13 mmol), pd (dppf) Cl2 25mg (0) were weighed out sequentially.03mmol)，K 2 CO 3 468mg (3.39 mmol) of the mixture was placed in a 10mL three-necked flask, and a mixed solution of 1,4 dioxane and water (3:1) was added under nitrogen and ice-bath conditions, followed by transfer to 105℃to start reflux reaction. After 8h, TLC monitors the reaction progress, after the reaction, the reaction solution is cooled to room temperature, pd (dppf) Cl2 is removed by diatomite suction filtration, and the reaction solution is washed 2 to 3 times by EA, then water (100 mL multiplied by 4) and 100mL ethyl acetate are added into the filtrate for extraction, and the organic phase is dried by anhydrous Na2SO4 and then eluted by gradient to obtain light brown powdery solid 1 to 3, and the yield is about 73.4%; MS (ESI) m/z=515.25 [ M+H ]] + ； 1 H NMR(400MHz,CDCl 3 )δ9.10(s,1H),8.44(d,J＝8.4Hz,1H),8.37(s,1H),8.32(d,J＝4.9Hz,1H),8.14(d,J＝8.3Hz,2H),7.87(d,J＝8.2Hz,2H),7.85–7.77(m,1H),7.13(dd,J＝7.3,5.0Hz,1H),4.87–4.73(m,1H),3.63(dd,J＝13.5,6.4Hz,2H),2.90(t,J＝11.5Hz,2H),2.26–2.17(m,2H),1.84–1.64(m,2H),1.46(s,12H).
EXAMPLE 7 Synthesis of intermediates 1-4
300mg (0.58 mmol) of example 6 was weighed into a single-necked flask, 2mL of ethyl acetate was added, 3mL of 3mol of HCl in ethyl acetate was added dropwise under ice bath, the reaction solution became turbid during the addition, and after the addition, the reaction solution was transferred to room temperature for 2 hours. After the TLC monitoring reaction is finished, the reaction liquid is filtered by filter paper, and a filter cake is the product hydrochloride. The filter cake was then placed at 100mL K at 0deg.C 2 CO 3 In aqueous solution, extracted with ethyl acetate (120 mL. Times.4), the organic phases combined and dried over anhydrous sodium sulfate to give 1-4 as a pale brown powdered solid in a yield of about 61.9%; MS (ESI) m/z=437.20 [ M+H ]] + ； 1 H NMR(400MHz,DMSO-d 6 )δ11.66(s,1H),9.75(d,J＝10.1Hz,1H),9.51(d,J＝10.9Hz,1H),8.64(s,1H),8.49(d,J＝4.8Hz,1H),8.36–8.26(m,2H),8.10(t,J＝7.7Hz,1H),7.86(d,J＝8.1Hz,1H),7.47–7.30(m,1H),5.26–5.10(m,1H),3.54(d,J＝10.7Hz,1H),3.50–3.38(m,2H),3.31(d,J＝11.5Hz,1H),3.12–2.93(m,1H),2.15(d,J＝5.2Hz,2H),1.05(t,J＝7.0Hz,1H).
EXAMPLE 8 Synthesis of Compound I-1
8mg (0.09 mmol) of acetic acid was dissolved in methylene chloride, 14mg (0.08 mmol) of EDC. HCl, 21uL of triethylamine were sequentially added, and after stirring for 5 minutes, 1-4 30mg (0.08 mmol) of the intermediate was added and reacted at room temperature. After 3h of reaction, TLC monitors that the reaction is finished, the reaction liquid is concentrated to remove dichloromethane, 50mL of EA is added for dissolution, 70mL of water is added for extraction, then the organic layer is extracted by 1N of dilute HCl solution (70 mL multiplied by 2), 70mL of saturated NaCl solution is extracted once, the EA layer is dried by anhydrous Na2SO4 and then the solvent is spun off to obtain crude products, and white solid I-1 is obtained after gradient elution, and the yield is about 71.6%; MS (ESI) m/z=457.20 [ M+H ]] + ； 1 H NMR(400MHz,Acetone-d 6 )δ8.39(t,J＝7.5Hz,2H),8.32–8.22(m,3H),7.91(dt,J＝15.8,7.5Hz,3H),7.21–7.15(m,1H),3.80–3.69(m,1H),3.57(q,J＝7.0Hz,1H),3.17(q,J＝7.2Hz,2H),2.18–2.10(m,3H),1.95(s,3H),1.29(t,J＝7.2Hz,1H). 13 C NMR(101MHz,Acetone-d 6 )δ182.54,168.95,158.34,148.23,147.91,138.56,138.23,136.68,134.17,132.46,128.74,128.40,114.39,114.02,97.96,46.05,20.50.
Example 9 Synthesis of Compound I-2:
by operating in the same manner as in example 8, trifluoroacetic acid was used instead of acetic acid, light brown semisolid I-2 was obtained in a yield of about 52.4%; MS (ESI) m/z=511.18 [ M+H ]] + ； 1 H NMR(400MHz,CDCl 3 )δ8.96(s,1H),8.47–8.37(m,2H),8.33(d,J＝2.5Hz,1H),8.12(dd,J＝8.2,5.3Hz,2H),7.85(dd,J＝8.3,4.8Hz,2H),7.12(td,J＝6.5,1.4Hz,1H),4.94–4.79(m,1H),4.05(dd,J＝13.0,4.1Hz,1H),3.92–3.81(m,1H),3.73(dd,J＝13.1,10.6Hz,1H),3.36–3.15(m,2H),2.86–2.75(m,1H),2.40(dd,J＝12.0,3.5Hz,1H),1.82–1.62(m,1H). 13 C NMR(101MHz,CDCl 3 )δ182.54,168.95,158.34,148.23,147.91,138.56,138.23,136.68,134.17,132.46,128.74,128.40,114.39,114.02,97.96,46.05,20.50.
Example 10 Synthesis of Compound I-3:
weighing 50mg (0.12 mmol) of hydrochloride of the intermediate 4-9, placing in a tube sealer, adding trifluoroethanol for dissolution, adding 10 mu L (0.12 mmol) of propylene oxide under ice bath condition, then placing at 80 ℃ for reaction for 3h, after TLC (thin layer chromatography) spot plate reaction, removing the trifluoroethanol and the redundant propylene oxide under reduced pressure to obtain brown oily liquid I-3, wherein the yield is about 90.3%; MS (ESI) m/z=473.25 [ M+H ]] + ； 1 H NMR(400MHz,DMSO-d 6 )δ10.92(s,1H),8.42(dd,J＝4.8,1.1Hz,1H),8.29(s,1H),8.22(t,J＝7.9Hz,3H),7.92–7.84(m,1H),7.80(d,J＝8.3Hz,2H),7.24–7.15(m,1H),3.90–3.82(m,1H),3.40(d,J＝10.1Hz,1H),3.29(d,J＝7.0Hz,3H),2.65–2.46(m,3H),2.15–2.04(m,2H),1.90(dd,J＝9.3,5.7Hz,2H),1.60(dd,J＝24.5,13.2Hz,3H). 13 C NMR(101MHz,DMSO-d 6 )δ170.67,165.95,159.13,155.51,153.63,143.10,139.34,136.48,134.74,123.72,120.22,113.72,97.34,67.51,65.47,63.20,59.71,56.19,30.26,19.96.
Example 11 Synthesis of Compound I-4:
by operating in the same manner as in example 10, glycidol was substituted for propylene oxide to obtain a brown oily liquid I-4 in a yield of about 91.4%; MS (ESI) m/z=489.23 [ M+H ]] + ； 1 H NMR(400MHz,DMSO-d 6 )δ8.42(s,1H),8.35(dd,J＝6.8,4.3Hz,1H),8.23(d,J＝8.3Hz,2H),7.87(dd,J＝10.8,4.6Hz,1H),7.81(t,J＝7.3Hz,2H),7.25–7.14(m,2H),3.85–3.72(m,1H),3.43(d,J＝4.0Hz,2H),3.09(t,J＝5.2Hz,2H),2.65–2.56(m,1H),2.17–2.09(m,2H),2.07–1.96(m,2H),1.87(dd,J＝9.1,5.9Hz,2H),1.53(dd,J＝24.3,12.8Hz,2H). 13 C NMR(101MHz,DMSO-d 6 )δ170.95,158.71,148.46,138.69,129.29,128.68,115.25,98.05,72.97,69.69,64.97,62.70,47.63,46.86,27.38,21.23.
Example 12 Synthesis of Compound I-5:
the same procedure used in example 8, boc-L-alanine instead of acetic acid, gave a pale brown solid I-5 in a yield of about 72.6%; MS (ESI) m/z=586.28 [ M+H ]] + ； 1 H NMR(400MHz,DMSO-d 6 )δ10.91(s,1H),8.42(s,1H),8.29(d,J＝8.3Hz,1H),8.22(t,J＝8.2Hz,2H),7.92–7.85(m,1H),7.80(d,J＝8.3Hz,2H),7.25–7.13(m,2H),4.54–4.44(m,1H),4.03(q,J＝7.1Hz,1H),3.68–3.56(m,2H),3.14(t,J＝6.6Hz,1H),2.17(dd,J＝13.5,10.3Hz,1H),1.99(dd,J＝10.6,4.2Hz,2H),1.91(d,J＝6.4Hz,3H),1.67–1.52(m,2H),1.36(s,9H).
Example 13 Synthesis of Compound I-6:
the same procedure as in example 8, L-alanine instead of acetic acid, gave pale brown semisolid I-6 in a yield of about 81.4%; MS (ESI) m/z=486.23 [ M+H ]] + ； 1 H NMR(400MHz,DMSO-d 6 )δ10.92(s,1H),8.78(s,1H),8.44(d,J＝3.9Hz,1H),8.31(d,J＝8.5Hz,1H),8.25(t,J＝7.9Hz,2H),7.68(d,J＝8.3Hz,2H),7.23–7.15(m,2H),4.57–4.42(m,1H),3.74(q,J＝5.8Hz,1H),3.69(d,J＝7.0Hz,1H),3.54(d,J＝10.2Hz,1H),3.13(t,J＝14.4,2H),2.17–1.86(m,2H),1.68–1.58(m,2H),1.27(td,J＝6.9,10.5Hz,3H). 13 C NMR(101MHz,DMSO-d 6 )δ173.69,164.58,158.46,154.32,152.74,148.65,142.51,139.98,137.26,134.70,131.47,128.93,117.46,115.82,101.42,64.58,51.46,47.35,43.76,29.73,20.97,18.36.
Example 14 Synthesis of Compound I-7:
operating in the same manner as in example 8, intermediate 7-9 was substituted for intermediate 1-6 and acetic acid was substituted for 2-methacrylic acid 1-7 to give white semi-solid I-7 in a yield of about 67.2%; MS (ESI) m/z=475.19 [ M+H ]] + ； 1 H NMR(400MHz,Acetone-d 6 )δ9.96(s,1H),8.37(d,J＝7.8Hz,2H),8.26(s,1H),8.11(d,J＝8.0Hz,1H),8.04(d,J＝10.8Hz,1H),7.84(dt,J＝21.5,7.8Hz,2H),7.21–7.14(m,1H),3.99–3.88(m,1H),3.63(t,J＝6.1Hz,1H),3.40(dd,J＝14.7,7.3Hz,1H),3.21(dd,J＝26.8,13.1Hz,2H),2.11(d,J＝8.7Hz,2H),2.06(s,3H),1.79(t,J＝6.1Hz,2H). 13 C NMR(101MHz,Acetone-d 6 )δ168.57,168.36,164.14,161.02,158.55,158.40,156.17,156.10,154.24,152.19,148.32,148.00,138.02,137.37,132.16,124.82,120.11,119.93,116.24,114.64,114.19,99.52,54.94,53.45,52.77,20.79,17.56.
Example 15 Synthesis of Compound I-8:
operating in the same manner as example 8, intermediate 2-2 was substituted for intermediate 1-2 and trifluoroacetic acid was substituted for acetic acid to give white semi-solid I-8 in a yield of about 53.9%; MS (ESI) m/z=529.16 [ M+H ]] + ； 1 H NMR(400MHz,CDCl 3 )δ8.95(s,1H),8.39(t,J＝10.2Hz,2H),8.32(dd,J＝4.0,3.2Hz,2H),8.10(dd,J＝9.4,4.1Hz,3H),3.93–3.88(m,1H),3.66(t,J＝6.1Hz,2H),3.45(dd,J＝13.5,7.9Hz,2H),3.29(dd,J＝16.3,12.5Hz,2H),2.12(t,J＝7.5Hz,1H),2.03(t,J＝5.5Hz,1H). 13 C NMR(101MHz,CDCl 3 )δ163.65,158.23,155.64,153.54,151.62,145.64,141.46,129.10,126.14,124.07,118.38,114.29,112.86,111.04,99.49,65.08,52.68,51.29,30.65,24.39.
EXAMPLE 16 Synthesis of Compound I-9:
operating in the same manner as in example 10, intermediate 2-2 was substituted for intermediate 1-2 to give brown oily liquid I-9 in a yield of about 63.8%; MS (ESI) m/z=491.22 [ M+H] + ； 1 H NMR(400MHz,DMSO-d 6 )δ11.03(s,1H),8.44(d,J＝3.6Hz,1H),8.35–8.17(m,2H),8.14–8.00(m,2H),7.90(t,J＝7.2Hz,1H),7.72(t,J＝7.6Hz,1H),7.29–7.16(m,1H),4.03–3.90(m,1H),3.40(d,J＝10.0Hz,2H),3.11(dd,J＝17.6,8.7Hz,1H),2.71–2.60(m,2H),2.53–2.41(m,2H),2.04(d,J＝10.1Hz,1H),1.88(d,J＝16.9Hz,2H),1.65–1.52(m,1H),1.08(d,J＝5.9Hz,3H). 13 C NMR(101MHz,DMSO-d 6 )δ172.59,164.81,160.87,158.48,158.41,156.35,154.09,154.07,152.38,148.50,138.74,137.82,136.91,132.04,124.93,124.26,120.63,116.72,116.49,115.39,99.27,29.18,22.09.
Example 17 Synthesis of Compound I-10:
operating in the same manner as in example 10, intermediate 2-2 was substituted for intermediate 1-2 and glycidol was substituted for propylene oxide, to obtain brown oily liquid I-10 in a yield of about 63.8%; MS (ESI) m/z=507.23 [ M+H ]] + ； 1 H NMR(400MHz,DMSO-d 6 )δ8.44(s,1H),8.23(d,J＝8.3Hz,1H),8.06(dd,J＝11.9,5.7Hz,2H),7.94–7.81(m,2H),7.73(dt,J＝12.8,7.8Hz,1H),7.26–7.18(m,1H),4.06–3.89(m,1H),3.57(dd,J＝4.0,2.8Hz,4H),2.63(m,1H),2.29–2.15(m,2H),2.15–2.04(m,2H),1.90(dt,J＝9.3,5.7Hz,2H),1.60(dt,J＝24.5,13.2Hz,2H). 13 C NMR(101MHz,DMSO-d 6 )δ170.94,158.44,156.21,154.89,152.40,148.46,138.74,127.20,124.42,115.38,115.34,108.12,99.24,72.63,69.57,64.48,59.47,53.84,21.21.
EXAMPLE 18 Synthesis of Compound I-11:
operating in the same manner as example 8, intermediate 2-2 was substituted for intermediate 1-2 and maleamic acid was substituted for acetic acid to give white solid I-11 in a yield of about 63.8%; MS (ESI) m/z=512.20 [ M+H ]] + ； 1 H NMR(400MHz,Acetone-d 6 )δ9.91(s,1H),8.37(d,J＝7.4Hz,2H),8.27(s,1H),8.12(d,J＝7.2Hz,1H),8.04(s,1H),7.93–7.73(m,2H),7.37(dd,J＝30.8,11.7Hz,1H),6.58(d,J＝13.1Hz,1H),6.04(d,J＝11.7Hz,1H),3.94(t,J＝7.8Hz,1H),3.78–3.63(m,1H),3.54(t,J＝7.0Hz,1H),2.48–2.34(m,2H),2.28(dd,J＝10.7,6.2Hz,1H),1.92–1.65(m,2H),1.12(d,J＝6.9Hz,1H). 13 C NMR(101MHz,Acetone-d 6 )δ164.11,162.26,158.37,156.21,155.83,152.13,148.34,141.97,138.00,137.54,124.65,123.94,120.12,119.92,116.00,115.69,114.46,106.30,99.45,59.68,56.84,29.19,28.42,17.84.
Example 19 in vitro enzyme inhibition assay
1. Compound formulation
1mg of the above compound was weighed and dissolved in 100% DMSO-d6 to prepare a 10mM stock solution, which was stored in a nitrogen cabinet in the absence of light.
2. Kinase reaction process
(1) 1 XKinase buffer was prepared.
(2) Preparing a compound concentration gradient: the initial test concentration of the test compound was 2400nM,7 concentrations, and multiple wells
And (5) detecting. 100% DMSO-d diluted to 100-fold final concentration in 384source plates 6 A solution. The final concentration of 250nl 100-fold compound was transferred to the plate of interest 3575 using a dispenser Echo 550.
(3) A2.5-fold final concentration of Kinase solution was prepared using a 1 XKinase buffer.
(4) Adding 10 μl of 2.5-fold final concentration kinase solution to each of the compound wells and positive control wells; in the negative control
Mu.l of 1 XKinase buffer was added to the wells.
(5) Centrifugation at 1000rpm for 30 seconds, the reaction plate was shaken and mixed well and incubated at room temperature for 10 minutes.
(6) A5/3-fold final concentration of a mixed solution of ATP and Kinase substrate 2 was prepared using a 1 XKinase buffer.
(7) The reaction was initiated by adding 15. Mu.l of a 5/3-fold final concentration of the mixed solution of ATP and substrate.
(8) The 384-well plate was centrifuged at 1000rpm for 30 seconds, and after shaking and mixing, incubated at room temperature for 30 minutes.
(9) The kinase reaction was stopped by adding 30. Mu.l of stop detection solution, centrifuging at 1000rpm for 30 seconds, and shaking and mixing.
(10) The conversion was read with Caliper EZ Reader.
The results are shown in Table 1.
TABLE 1 results of BTK inhibitory Activity of Compounds I-1 to I-10
a No Detection
As can be seen from Table 1, all compounds showed inhibitory activity against BTK protein and BTK mutant strain (BTKC 481S), and activity against mutant strain was stronger than that of wild type. In contrast, compound I-11 has the best activity, and the inhibition activity on BTK protein is in nM level; compound I-6 (IC) 50 ＝42.0nM,IC 50 C481S =31.0 nM) has better activity on both wild type and mutant.
Claims (6)
1. A small molecule compound having a pyrazolo [3,4-d ] pyrimidine structure, having the structure of formula I:
wherein R is selected from the following structures:
x is independently hydrogen, hydroxy, amino or halogen.
2. The compound of claim 1, wherein X is independently hydrogen, or a fluorine atom.
3. A compound according to claim 1, which is of the structure:
compound 1: the structure of the formula I, X is H, R is
Compound 2: the structure of the formula I, X is H, R is
Compound 3: the structure of the formula I, X is H, R is
Compound 4: the structure of the formula I, X is H, R is
Compound 5: the structure of the formula I, X is H, R is
Compound 6: the structure of the formula I, X is H, R is
Compound 7: the structure of formula I, X is F, R is
/>
Compound 8: the structure of formula I, X is F, R is
Compound 9: the structure of formula I, X is F, R is
Compound 10: the structure of formula I, X is F, R is
4. A small molecule compound of pyrazolo [3,4-d ] pyrimidine structure, characterized by being compound 11, having the structure of formula I:
wherein X is F, R is
5. A compound according to any one of claims 1 to 4 in a pharmaceutically acceptable salt.
6. The pharmaceutically acceptable salt of the compound of claim 5, 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.
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| 应对BTK突变所致耐药的分子设计策略;魏超等;《中国医药工业杂志》;20210810;第52卷(第8期);第987-997页 * |
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