CN106565782B - Phosphoryl pyrimidines, composition and purposes - Google Patents

Phosphoryl pyrimidines, composition and purposes Download PDF

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CN106565782B
CN106565782B CN201610884363.5A CN201610884363A CN106565782B CN 106565782 B CN106565782 B CN 106565782B CN 201610884363 A CN201610884363 A CN 201610884363A CN 106565782 B CN106565782 B CN 106565782B
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amine
phenyl
chloro
pyrimidinyl
acrylamide
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CN106565782A (en
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马晓东
葛阳
宋振东
黄珊珊
王长远
张建斌
唐泽耀
刘克辛
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Dalian Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings
    • C07F9/6512—Six-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to phosphoryl pyrimidines, composition and purposes, the phosphoryl pyrimidines are specially the compound shown in formula (I), and each substituent of formula (I) is defined in the description.The invention further relates to the compound shown in the formula (I) or its pharmaceutically acceptable salt, or tumor disease is treated by suppressing bruton's EGFR-TK containing its pharmaceutical composition, particularly it is used for the purposes for treating Burkitt's lymphoma, diffusivity large B cell lymphoid tumor, follicular lymphoma or chronic lymphocytic leukemia；

Description

Phosphoryl pyrimidine compound, composition and application
Technical Field
The invention relates to phosphoryl pyrimidine compounds, a composition and application, and belongs to the technical field of medicines.
Background
Protein Tyrosine Kinases (PTKs) regulate a series of physiological and biochemical processes such as growth, differentiation and apoptosis of cells by controlling signal transduction pathways of the cells. Receptor-type tyrosine kinases are a class of relatively large kinases that span the cell membrane, having a ligand-binding extracellular domain, a transmembrane domain, and an intracellular domain that functions as a kinase-phosphorylating specific tyrosine residues and thereby affecting cell proliferation. Abnormal expression of the kinase has been found in common human cancers (e.g., lung, breast, stomach, ovarian, lymphoma). Protein tyrosine kinase has become one of the important targets for research and development of antitumor drugs.
BTK is one of non-receptor protein tyrosine kinase Tec family members, is mainly expressed in hematopoietic cells (except T cells), and in a BCR signal pathway, BTK is activated depending on Syk and Lyn along with the activation of BCR, and the activated BTK can further phosphorylate PLC gamma 2 so as to cause the activation of downstream signals including MAPK, NF kappa B and the like. BTK plays an irreplaceable role in the production of B lymphocytes. BTK can control the development and differentiation of B cells by activating cell cycle forward regulatory factors and differentiation factors, and can also control the survival and proliferation of B cells by regulating the expression of pro-apoptotic and anti-apoptotic proteins. The sustained activation of BTK is a prerequisite for the development of Chronic Lymphocytic Leukemia (CLL), and the aberrant BCR-BTK signaling promotes survival of activated B-cell subtypes in diffuse large B-cell lymphoma (DLBCL). BTK small molecule inhibitors have good prospects for treating hematological malignancies and autoimmune disorders. Ibrutinib is an oral Bruton's Tyrosine Kinase (BTK) inhibitor developed by Pharmacyclics, california (US 7514444, CN101610676A), approved for marketing by the FDA in the united states for the treatment of Mantle Cell Lymphoma (MCL) and CLL. Several other compounds, such as CC-292(AVL-292) (US8563568, WO2014100748A1) are highly selective BTK inhibitors with IC's, which are covalently bound and administered orally50Less than 0.5nmol/L, exhibiting at least 1400-fold selectivity over the other kinases tested, the PhaseI study is currently being conducted (Evans, e.k., et al.j. pharmacol exp.ther.,2013,346, 219-228.); for another example, ONO-4059(Yasuhiro, T., et al. blood,2013,122,5151-5151.) is an analogue of ibrutinib, highly selective in inhibiting BTK, IC50At 23.9nmol/L, the PhaseI study was performed. In addition, CNX-774(Akinleye, A., et al, J. HematolOncol.,2013,6,59.) is also an orally effective highly selective BTK inhibitor, IC50Less than 1nmol/L, and patents such as: WO2010141406a2, US20140256759a1, CN 102083800A.
In view of the urgent need for cancer treatment, there is a need in the art to develop new drugs with better efficacy.
Disclosure of Invention
One of the purposes of the invention is to provide a phosphoryl pyrimidine compound or pharmaceutically acceptable salt thereof, wherein the compound has good antitumor activity.
The invention also aims to provide a pharmaceutical composition containing the phosphoryl pyrimidine compound or the pharmaceutically acceptable salt thereof.
The invention further aims to provide the phosphoryl pyrimidine compound or the pharmaceutically acceptable salt thereof, or the application of the composition.
To this end, in one aspect, the present invention provides a compound of formula (i) or a pharmaceutically acceptable salt thereof, wherein the compound of formula (i) has the following structure:
wherein,
x is selected from chlorine or fluorine;
l is selected from-CH2-or-O (CH)2)2CH2-；
R1Selected from hydrogen, methyl, methoxy or chlorine;
R2selected from methoxy, ethoxy, butoxy, isopropoxy,
R3Selected from methoxy, ethoxy, butoxy or isopropylAn oxy group.
Preferably, the compounds represented by the general formula (I) of the present invention have the structures represented by I-1 to I-30:
the structural compound shown above is a phosphoryl pyrimidine compound, and the screening of the antitumor activity of the invention shows that most of the compounds have stronger ability of inhibiting the proliferation of lymphocyte leukemia cells (Ramos and Raji), and part of the compounds show more excellent BTK resistance activity than Spebrutinib. As a molecule with novel structure, the compound has the potential of being developed into a novel high-efficiency BTK inhibitor, and has great application value for treating related tumor diseases, particularly Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia.
The structures represented by the foregoing I-1 to I-30 have the following names, respectively:
(I-1) N- [3- [ [ 5-chloro-2- [ [4- [ (diethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-2) N- [3- [ [ 5-chloro-2- [ [ 3-methoxy-4- [ (diethoxyphosphoryl) propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-3) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ (diethoxyphosphoryl) propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-4) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-morpholino) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-5) N- [3- [ [ 5-chloro-2- [ [ 2-methyl-4- [ [ (1-morpholino) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-6) N- [3- [ [ 5-chloro-2- [ [ 3-methoxy-4- [ [ (1-morpholino) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-7) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ [ (1-morpholino) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-8) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-9) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-10) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-ethylpiperazine) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-11) N- [3- [ [ 5-chloro-2- [ [4- [ (diisopropyloxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-12) N- [3- [ [ 5-chloro-2- [ [4- [ (dimethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-13) N- [3- [ [ 5-chloro-2- [ [4- [ (diethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-14) N- [3- [ [ 5-chloro-2- [ [ 2-methyl-4- [ (diethoxyphosphoryl) methyl ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-15) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ (diethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-16) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-morpholino) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-17) N- [3- [ [ 5-chloro-2- [ [ 3-methoxy-4- [ [ (1-morpholino) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-18) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ [ (1-morpholino) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-19) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-20) N- [3- [ [ 5-chloro-2- [ [4- [ (diisopropyloxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-21) N- [3- [ [ 5-chloro-2- [ [ 3-methoxy-4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-22) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-23) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-24) N- [3- [ [ 5-chloro-2- [ [4- [ (dimethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-25) N- [3- [ [ 5-fluoro-2- [ [4- [ (diethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-26) N- [3- [ [ 5-fluoro-2- [ [ 3-chloro-4- [ (diethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-27) N- [3- [ [ 5-fluoro-2- [ [4- [ (diethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-28) N- [3- [ [ 5-fluoro-2- [ [ 3-methoxy-4- [ (diethoxyphosphoryl) methyl ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-29) N- [3- [ [ 5-fluoro-2- [ [4- [ (dibutoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
(I-30) N- [3- [ [ 5-fluoro-2- [ [4- [ (dibutoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide;
in another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound represented by the general formula (i) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
The compounds of the invention are bases, wherein the desired salt form may be prepared by suitable methods known in the art, including treatment of the free base with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or treatment of the free base with organic acids such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid (pyrasidyl 1acid), e.g. glucuronic acid or galacturonic acid, α -hydroxy acid, e.g. citric acid or tartaric acid, amino acids such as aspartic acid or glutamic acid, aromatic acids such as benzoic acid or cinnamic acid, sulfonic acids, e.g. p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid and the like.
The pharmaceutical compositions of the invention will generally contain one compound of the invention. However, in some embodiments, the pharmaceutical compositions of the invention contain more than one compound of the invention. In addition, the pharmaceutical compositions of the present invention may optionally further comprise one or more other pharmaceutically active compounds.
The invention also provides the phosphoryl pyrimidine compound or the pharmaceutically acceptable salt thereof, and the application of the pharmaceutical composition in inhibiting tumor proliferation by inhibiting Bruton's tyrosine kinase. Specifically, the application is mainly the application in preparing the medicine for treating burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia.
The invention provides an application of a compound shown in the specification or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition in preparation of a Bruton's tyrosine kinase inhibitor.
The invention provides a compound shown in a general formula (I) or a pharmaceutically acceptable salt thereof, or application of a pharmaceutical composition in preparing a medicament for treating tumors. Preferably, the tumor is selected from burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia, further preferably chronic lymphocytic leukemia. More preferably, the use is primarily through inhibition of bruton's tyrosine kinase.
Detailed Description
The present invention is further described and explained below in conjunction with specific examples, which are not intended to limit the scope of the present invention.
The experimental method of the present invention, in which the specific conditions are not specified, is generally carried out under the conventional conditions or the conditions recommended by the manufacturers of the raw materials or the commercial products. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.
EXAMPLE 1 preparation of target molecules
The thin layer chromatography silica gel plate is HSGF254 of tobacco yellow sea or GF254 of Qingdao, the silica-amine plate used in Thin Layer Chromatography (TLC) is 0.15-0.2 mm, and the thin layer chromatography separation and purification product is 0.4-0.5 mm.
The raw materials used in the present invention are mainly purchased from chemical reagents of national medicine group, Beijing coupled technology, Inc., Aladdin chemical reagents, Inc., Darriy Chemicals, etc.
In the examples, the solution means an aqueous solution unless otherwise specified.
In the examples, the reaction temperature is, unless otherwise specified, from 20 ℃ to 30 ℃ at room temperature.
The technical scheme adopted by the invention is as follows:
synthetic route, reagents and conditions for Compound (I) a) P (OEt)3、P(OMe)3P(OPr-i)3Or P (OBu-n)3,130℃,80–91％；b)(COCl)2,DMF,60℃；c)R2H,THF,0℃,92％；d)Fe-NH4Cl,MeOH-H2O, from 80 ℃ to rt, from 62 to 85%; e) NaHCO 23Acetonitrile at 0 deg.c for 10min, 85-93%; f) Fe-NH4Cl,MeOH-H2O, from 80 ℃ to rt, from 62 to 85%; g) DIPEA,1, 4-dioxane, 60 ℃, 49-72%; h) ArNH2,CF3COOH,100 ℃ and 5-21 percent; r in formula 2 and formula 32And R3Same, all are methoxy and ethoxyButoxy or isopropoxy; r in formula 5 and formula 62And R3In contrast, the remaining substituents are as defined above.
Synthesis of 8
Take 7(23.44mmol) and NaHCO3(4.5g,35.16mmol) is put in 50mL acetonitrile, acryloyl chloride (3.8g,23.44mmol) is slowly added, after reaction for 10min at 0 ℃, 400mL water is added after the reaction is finished, white solid is separated out, suction filtration and drying are carried out, the white solid is obtained, 19g,68mmol and ammonium chloride (7.3g,136mmol) are taken out and put in a reaction bottle, MeOH (25mL) and water (25mL) are added, iron powder (15g,272mmol) is added under stirring, the temperature is raised to 60 ℃ for reaction for 2h, suction filtration is carried out while the solution is hot, the aqueous phase is extracted by ethyl acetate (100mL multiplied by 3), the ethyl acetate layer is combined, saturated common salt is washed once, anhydrous sodium sulfate is dried, and dried by decompression and evaporation to obtain yellow white solid 8.
Synthesis of 10
9(23.44mmol) and DIPEA (4.5g,35.16mmol) are taken to be put into 50mL dioxane, 8(3.8g,23.44mmol) is slowly added, the temperature is raised to 60 ℃, after 5 hours of reaction, the reaction is finished, cooling is carried out, 400mL water is added, yellow white solid is separated out, suction filtration and drying are carried out, and off-white solid is obtained, and the reaction is directly carried out in the next step without purification.
Synthesis of object (I)
Taking 10(23.44mmol) and trifluoroacetic acid (4.5g,35.16mmol) to 2-BuOH (50ml), slowly adding substituted arylamine (3 or 6, 3.44mmol), heating to 100 ℃, reacting for 8 hours, cooling after the reaction is finished, pouring into saturated sodium bicarbonate solution, separating out a solid, carrying out suction filtration, washing with water, and carrying out silica gel column chromatography separation by drying to obtain the target molecule (I).
The target molecule was synthesized according to the above method, and the physicochemical data of the synthesized target molecule were as follows:
(I-1) N- [3- [ [ 5-chloro-2- [ [4- [ (diethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.15(s,1H),8.90(s,1H),8.10(s,1H),7.88(s,1H),7.57–7.40(m,3H),7.31(d,J＝5.2Hz,2H),6.69(d,J＝8.8Hz,2H),6.46(dd,J＝17.0,10.1Hz,1H),6.26(dd,J＝17.0,2.0Hz,1H),5.76(dd,J＝10.1,2.0Hz,1H),4.05–3.88(m,6H),1.92–1.79(m,4H),1.23(t,J＝7.0Hz,6H)；MS(ESI)m/z560.18[M+H]+。
(I-2) N- [3- [ [ 5-chloro-2- [ [ 3-methoxy-4- [ (diethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.17(s,1H),9.14(s,1H),8.92(s,1H),8.10(s,1H),7.88(s,1H),7.56–7.39(m,3H),7.31(d,J＝5.2Hz,1H),6.69(d,J＝8.8Hz,1H),6.50(s,1H),6.46(dd,J＝17.0,10.1Hz,1H),6.26(dd,J＝17.0,2.0Hz,1H),5.76(dd,J＝10.1,2.0Hz,1H),4.05–3.88(m,6H),3.75(s,3H),1.92–1.79(m,4H),1.23(t,J＝7.0Hz,6H)；MS(ESI)m/z 590.19[M+H]+。
(I-3) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ (diethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.16(s,1H),9.15(s,1H),8.91(s,1H),8.13(s,1H),7.88(s,1H),7.57–7.40(m,3H),7.31(d,J＝5.2Hz,1H),6.69(d,J＝8.8Hz,1H),6.50(s,1H),6.46(dd,J＝17.0,10.1Hz,1H),6.26(dd,J＝17.0,2.0Hz,1H),5.76(dd,J＝10.1,2.0Hz,1H),4.05–3.88(m,6H),1.92–1.79(m,4H),1.23(t,J＝7.0Hz,6H)；MS(ESI)m/z594.14[M+H]+。
(I-4) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-morpholino) ethoxyphosphoryl ] propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.21(s,1H),9.16(s,1H),8.89(s,1H),8.10(s,1H),7.89(s,1H),7.52(dd,J＝34.2,8.8Hz,3H),7.30(d,J＝4.9Hz,2H),6.69(d,J＝8.3Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.25(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.10–3.76(m,4H),3.56(d,J＝35.6Hz,4H),2.94(t,J＝38.4Hz,4H),2.05–1.68(m,4H),1.21(t,J＝7.0Hz,3H)；MS(ESI)m/z 601.21[M+H]+。
(I-5) N- [3- [ [ 5-chloro-2- [ [ 2-methyl-4- [ [ (1-morpholino) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.21(s,1H),9.16(s,1H),8.89(s,1H),8.10(s,1H),7.89(s,1H),7.53(dd,J＝34.2,8.8Hz,3H),7.30(d,J＝4.9Hz,1H),7.29(s,1H),6.69(d,J＝8.3Hz,1H),6.46(dd,J＝16.9,10.1Hz,1H),6.26(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.10–3.76(m,4H),3.56(d,J＝35.6Hz,4H),2.94(t,J＝38.4Hz,4H),2.35(s,3H),2.05–1.68(m,4H),1.21(t,J＝7.0Hz,3H)；MS(ESI)m/z 615.22[M+H]+。
(I-6) N- [3- [ [ 5-chloro-2- [ [ 3-methoxy-4- [ [ (1-morpholino) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.23(s,1H),9.14(s,1H),8.89(s,1H),8.11(s,1H),7.89(s,1H),7.52(dd,J＝34.2,8.8Hz,3H),7.30(d,J＝4.9Hz,1H),6.70(s,1H),6.69(d,J＝8.3Hz,1H),6.46(dd,J＝16.9,10.1Hz,1H),6.25(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.10–3.76(m,4H),3.75(s,3H),3.56(d,J＝35.6Hz,4H),2.94(t,J＝38.4Hz,4H),2.05–1.68(m,4H),1.21(t,J＝7.0Hz,3H)；MS(ESI)m/z 631.22[M+H]+。
(I-7) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ [ (1-morpholino) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.20(s,1H),9.15(s,1H),8.89(s,1H),8.12(s,1H),7.89(s,1H),7.54(dd,J＝34.2,8.8Hz,3H),7.30(d,J＝4.9Hz,1H),6.71(s,1H),6.69(d,J＝8.3Hz,1H),6.46(dd,J＝16.9,10.1Hz,1H),6.25(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.10–3.76(m,4H),3.56(d,J＝35.6Hz,4H),2.94(t,J＝38.4Hz,4H),2.05–1.68(m,4H),1.21(t,J＝7.0Hz,3H)；MS(ESI)m/z 635.17[M+H]+。
(I-8) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.21(s,1H),9.16(s,1H),8.89(s,1H),8.10(s,1H),7.89(s,1H),7.52(dd,J＝34.2,8.8Hz,3H),7.30(d,J＝4.9Hz,2H),6.69(d,J＝8.3Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.25(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.10–3.76(m,4H),3.56(d,J＝35.6Hz,4H),2.94(t,J＝38.4Hz,4H),2.26(s,3H),2.05–1.68(m,4H),1.21(t,J＝7.0Hz,3H)；MS(ESI)m/z 614.24[M+H]+。
(I-9) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] propoxy ] ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.20(s,1H),9.15(s,1H),8.89(s,1H),8.12(s,1H),7.89(s,1H),7.54(dd,J＝34.2,8.8Hz,3H),7.30(d,J＝4.9Hz,1H),6.71(s,1H),6.69(d,J＝8.3Hz,1H),6.46(dd,J＝16.9,10.1Hz,1H),6.25(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.10–3.76(m,4H),3.56(d,J＝35.6Hz,4H),2.94(t,J＝38.4Hz,4H),2.25(s,3H),2.05–1.68(m,4H),1.21(t,J＝7.0Hz,3H)；MS(ESI)m/z 648.21[M+H]+。
(I-10) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-ethylpiperazine) ethoxyphosphoryl ] propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.21(s,1H),9.16(s,1H),8.89(s,1H),8.10(s,1H),7.89(s,1H),7.52(dd,J＝34.2,8.8Hz,3H),7.30(d,J＝4.9Hz,2H),6.69(d,J＝8.3Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.25(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.10–3.76(m,4H),3.56(d,J＝35.6Hz,4H),2.94(t,J＝38.4Hz,4H),2.05–1.68(m,6H),1.21(t,J＝7.0Hz,6H)；MS(ESI)m/z 628.26[M+H]+。
(I-11) N- [3- [ [ 5-chloro-2- [ [4- [ (diisopropyloxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.15(s,1H),8.90(s,1H),8.10(s,1H),7.88(s,1H),7.57–7.40(m,3H),7.31(d,J＝5.2Hz,2H),6.69(d,J＝8.8Hz,2H),6.46(dd,J＝17.0,10.1Hz,1H),6.26(dd,J＝17.0,2.0Hz,1H),5.76(dd,J＝10.1,2.0Hz,1H),4.05–3.88(m,4H),1.92–1.79(m,4H),1.23(d,J＝7.0Hz,12H)；MS(ESI)m/z588.21[M+H]+。
(I-12) N- [3- [ [ 5-chloro-2- [ [4- [ (dimethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.15(s,1H),8.90(s,1H),8.10(s,1H),7.88(s,1H),7.57–7.40(m,3H),7.31(d,J＝5.2Hz,2H),6.69(d,J＝8.8Hz,2H),6.46(dd,J＝17.0,10.1Hz,1H),6.26(dd,J＝17.0,2.0Hz,1H),5.76(dd,J＝10.1,2.0Hz,1H),4.05–3.88(m,2H),3.35(s,6H),1.92–1.79(m,4H)；MS(ESI)m/z 532.15[M+H]+。
(I-13) N- [3- [ [ 5-chloro-2- [ [4- [ (diethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.19(s,1H),9.31(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.61–7.41(m,3H),7.40–7.22(m,2H),7.01(d,J＝6.7Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.26(dd,J＝17.0,1.8Hz,1H),5.75(dd,J＝10.1,1.8Hz,1H),3.99–3.81(m,4H),3.06(d,J＝21.1Hz,2H),1.15(t,J＝7.0Hz,6H)；MS(ESI)m/z516.16[M+H]+。
(I-14) N- [3- [ [ 5-chloro-2- [ [ 2-methyl-4- [ (diethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.19(s,1H),9.31(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.61–7.41(m,3H),7.40–7.22(m,2H),7.01(d,J＝6.7Hz,1H),6.46(dd,J＝16.9,10.1Hz,1H),6.26(dd,J＝17.0,1.8Hz,1H),5.75(dd,J＝10.1,1.8Hz,1H),3.99–3.81(m,4H),3.06(d,J＝21.1Hz,2H),2.35(s,3H),1.15(t,J＝7.0Hz,6H)；MS(ESI)m/z 530.17[M+H]+。
(I-15) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ (diethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.17(s,1H),9.32(s,1H),8.93(s,1H),8.15(s,1H),7.86(s,1H),7.61–7.41(m,3H),7.40–7.22(m,1H),7.01(d,J＝6.7Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.26(dd,J＝17.0,1.8Hz,1H),5.75(dd,J＝10.1,1.8Hz,1H),3.99–3.81(m,4H),3.06(d,J＝21.1Hz,2H),1.15(t,J＝7.0Hz,6H)；MS(ESI)m/z550.11[M+H]+。
(I-16) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-morpholino) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.29(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.63–7.41(m,3H),7.40–7.25(m,2H),7.03(d,J＝7.0Hz,2H),6.46(dd,J＝16.9 10.1Hz,1H),6.26(d,J＝16.8Hz,1H),5.75(d,J＝10.0Hz,1H),4.00–3.76(m,2H),3.40(d,J＝3.1Hz,4H),2.93(dd,J＝55.4,27.9Hz,6H),1.20(t,J＝7.0Hz,3H)；MS(ESI)m/z557.18[M+H]+。
(I-17) N- [3- [ [ 5-chloro-2- [ [ 3-methoxy-4- [ [ (1-morpholino) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.29(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.63–7.41(m,3H),7.40–7.25(m,2H),7.03(d,J＝7.0Hz,1H),6.46(dd,J＝16.9 10.1Hz,1H),6.26(d,J＝16.8Hz,1H),5.75(d,J＝10.0Hz,1H),4.00–3.76(m,2H),3.70(s,3H),3.40(d,J＝3.1Hz,4H),2.93(dd,J＝55.4,27.9Hz,6H),1.20(t,J＝7.0Hz,3H)；MS(ESI)m/z 587.19[M+H]+。
(I-18) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ [ (1-morpholino) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.29(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.63–7.41(m,3H),7.40–7.25(m,1H),7.03(d,J＝7.0Hz,2H),6.46(dd,J＝16.9 10.1Hz,1H),6.26(d,J＝16.8Hz,1H),5.75(d,J＝10.0Hz,1H),4.00–3.76(m,2H),3.40(d,J＝3.1Hz,4H),2.93(dd,J＝55.4,27.9Hz,6H),1.20(t,J＝7.0Hz,3H)；MS(ESI)m/z591.14[M+H]+。
(I-19) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.29(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.63–7.41(m,3H),7.40–7.25(m,2H),7.03(d,J＝7.0Hz,2H),6.46(dd,J＝16.9 10.1Hz,1H),6.26(d,J＝16.8Hz,1H),5.75(d,J＝10.0Hz,1H),4.00–3.76(m,2H),3.40(d,J＝3.1Hz,4H),2.93(dd,J＝55.4,27.9Hz,6H),2.27(s,3H),1.20(t,J＝7.0Hz,3H)；MS(ESI)m/z 570.21[M+H]+。
(I-20) N- [3- [ [ 5-chloro-2- [ [4- [ (diisopropyloxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.19(s,1H),9.31(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.61–7.41(m,3H),7.40–7.22(m,2H),7.01(d,J＝6.7Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.26(dd,J＝17.0,1.8Hz,1H),5.75(dd,J＝10.1,1.8Hz,1H),3.99–3.81(m,2H),3.06(d,J＝21.1Hz,2H),1.15(d,J＝7.0Hz,12H)；MS(ESI)m/z544.19[M+H]+。
(I-21) N- [3- [ [ 5-chloro-2- [ [ 3-methoxy-4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.29(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.63–7.41(m,3H),7.40–7.25(m,2H),7.03(d,J＝7.0Hz,1H),6.46(dd,J＝16.9 10.1Hz,1H),6.26(d,J＝16.8Hz,1H),5.75(d,J＝10.0Hz,1H),4.00–3.76(m,2H),3.70(s,3H),3.40(d,J＝3.1Hz,4H),2.93(dd,J＝55.4,27.9Hz,6H),2.25(s,3H),1.20(t,J＝7.0Hz,3H)；MS(ESI)m/z 600.22[M+H]+。
(I-22) N- [3- [ [ 5-chloro-2- [ [4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.29(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.63–7.41(m,3H),7.40–7.25(m,2H),7.03(d,J＝7.0Hz,2H),6.46(dd,J＝16.9 10.1Hz,1H),6.26(d,J＝16.8Hz,1H),5.75(d,J＝10.0Hz,1H),4.00–3.76(m,4H),3.40(d,J＝3.1Hz,4H),2.93(dd,J＝55.4,27.9Hz,6H),1.20(t,J＝7.0Hz,6H)；MS(ESI)m/z584.23[M+H]+。
(I-23) N- [3- [ [ 5-chloro-2- [ [ 3-chloro-4- [ [ (1-methylpiperazine) ethoxyphosphoryl ] methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.18(s,1H),9.29(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.63–7.41(m,3H),7.40–7.25(m,1H),7.03(d,J＝7.0Hz,2H),6.46(dd,J＝16.9 10.1Hz,1H),6.26(d,J＝16.8Hz,1H),5.75(d,J＝10.0Hz,1H),4.00–3.76(m,4H),3.40(d,J＝3.1Hz,4H),2.93(dd,J＝55.4,27.9Hz,6H),1.20(t,J＝7.0Hz,6H)；MS(ESI)m/z618.19[M+H]+。
(I-24) N- [3- [ [ 5-chloro-2- [ [4- [ (dimethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.19(s,1H),9.31(s,1H),8.95(s,1H),8.14(s,1H),7.86(s,1H),7.61–7.41(m,3H),7.40–7.22(m,2H),7.01(d,J＝6.7Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.26(dd,J＝17.0,1.8Hz,1H),5.75(dd,J＝10.1,1.8Hz,1H),3.36(s,6H),3.06(d,J＝21.1Hz,2H)；MS(ESI)m/z 488.13[M+H]+。
(I-25) N- [3- [ [ 5-fluoro-2- [ [4- [ (diethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.13(s,1H),9.38(s,1H),8.98(s,1H),8.06(d,J＝3.7Hz,1H),7.93(s,1H),7.51(dd,J＝13.1,9.1Hz,3H),7.42(d,J＝8.1Hz,1H),7.28(t,J＝8.1Hz,1H),6.75(d,J＝9.0Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.26(dd,J＝17.0,1.9Hz,1H),5.76(dd,J＝10.1,1.8Hz,1H),4.22–3.67(m,6H),1.92–1.79(m,4H),1.22(t,J＝7.0Hz,6H)；MS(ESI)m/z 544.21[M+H]+。
(I-26) N- [3- [ [ 5-fluoro-2- [ [ 3-chloro-4- [ (diethoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.13(s,1H),9.38(s,1H),8.98(s,1H),8.06(d,J＝3.7Hz,1H),7.93(s,1H),7.51(dd,J＝13.1,9.1Hz,3H),7.42(d,J＝8.1Hz,1H),6.75(d,J＝9.0Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.26(dd,J＝17.0,1.9Hz,1H),5.76(dd,J＝10.1,1.8Hz,1H),4.22–3.67(m,6H),3.69(s,3H),1.92–1.79(m,4H),1.22(t,J＝7.0Hz,6H)；MS(ESI)m/z 578.17[M+H]+。
(I-27) N- [3- [ [ 5-fluoro-2- [ [4- [ (diethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.16(s,1H),9.44(s,1H),9.17(s,1H),8.11(d,J＝2.7Hz,1H),7.93(s,1H),7.57(dd,J＝16.8,7.9Hz,3H),7.44(d,J＝7.5Hz,1H),7.29(t,J＝7.9Hz,1H),7.06(d,J＝6.9Hz,2H),6.47(dd,J＝16.8,10.2Hz,1H),6.26(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.09–3.77(m,4H),3.08(d,J＝21.0Hz,2H),1.16(t,J＝6.8Hz,6H)；MS(ESI)m/z 500.18[M+H]+。
(I-28) N- [3- [ [ 5-fluoro-2- [ [ 3-methoxy-4- [ (diethoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.16(s,1H),9.44(s,1H),9.17(s,1H),8.11(d,J＝2.7Hz,1H),7.93(s,1H),7.57(dd,J＝16.8,7.9Hz,3H),7.44(d,J＝7.5Hz,1H),7.29(t,J＝7.9Hz,1H),7.06(d,J＝6.9Hz,1H),6.47(dd,J＝16.8,10.2Hz,1H),6.26(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.09–3.77(m,4H),3.68(s,3H),3.08(d,J＝21.0Hz,2H),1.16(t,J＝6.8Hz,6H)；MS(ESI)m/z 530.20[M+H]+。
(I-29) N- [3- [ [ 5-fluoro-2- [ [4- [ (dibutoxyphosphoryl) propoxy ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.13(s,1H),9.38(s,1H),8.98(s,1H),8.06(d,J＝3.7Hz,1H),7.93(s,1H),7.51(dd,J＝13.1,9.1Hz,3H),7.42(d,J＝8.1Hz,1H),7.28(t,J＝8.1Hz,1H),6.75(d,J＝9.0Hz,2H),6.46(dd,J＝16.9,10.1Hz,1H),6.26(dd,J＝17.0,1.9Hz,1H),5.76(dd,J＝10.1,1.8Hz,1H),4.22–3.67(m,6H),1.92–1.79(m,4H),1.68–1.52(m,4H),1.48–1.25(m,4H),0.97(t,J＝7.0Hz,6H)；MS(ESI)m/z600.28[M+H]+。
(I-30) N- [3- [ [ 5-fluoro-2- [ [4- [ (dibutoxyphosphoryl) methyl ] phenyl ] amine-4-pyrimidinyl ] amine ] phenyl ] acrylamide
1H NMR(400MHz,DMSO-d6):δ10.16(s,1H),9.44(s,1H),9.17(s,1H),8.11(d,J＝2.7Hz,1H),7.93(s,1H),7.57(dd,J＝16.8,7.9Hz,3H),7.44(d,J＝7.5Hz,1H),7.29(t,J＝7.9Hz,1H),7.06(d,J＝6.9Hz,2H),6.47(dd,J＝16.8,10.2Hz,1H),6.26(d,J＝16.8Hz,1H),5.76(d,J＝10.0Hz,1H),4.09–3.77(m,4H),3.08(d,J＝21.0Hz,2H),1.68–1.52(m,4H),1.48–1.25(m,4H),0.95(t,J＝6.8Hz,6H)；MS(ESI)m/z 556.25[M+H]+。
Method for salifying target molecule
The preparation method of the inorganic acid salt comprises the following steps: dissolving a target molecule (1mmol) in 10mL of anhydrous methanol, slowly dropwise adding a 5mL of anhydrous methanol solution of inorganic acid (1mmol) in ice bath, stirring for 30 minutes at the temperature after dropwise adding, and then evaporating the methanol at normal temperature to obtain the inorganic acid salt of the target molecule. By the method, hydrochloride (I-4-1), hydrobromide (I-4-2), sulfate (I-4-3) and methanesulfonate (I-4-4) of the compound I-4 are prepared;
the preparation method of the organic acid salt comprises the following steps: dissolving a target molecule (1mmol) in 10mL of anhydrous methanol, slowly dropwise adding 5mL of dry ether of organic acid (1mmol) in ice bath, stirring for 30 minutes at the temperature after dropwise adding, and then evaporating the solvent at normal temperature to obtain the organic acid salt of the target molecule. By this method, maleate salt (I-4-5), succinate salt (I-4-6) and fumarate salt (I-4-7) of compound I-4 were prepared.
Preparation of a mixture of two target molecules
And (3) putting the two target molecules with equal molar weight (1mmol) into anhydrous methanol (5mL), stirring for 10 minutes at room temperature, and evaporating the solvent at room temperature to obtain a mixture of the target molecules. Three target molecule mixtures of (I-1) - (I-4), (I-4) - (I-13) and (I-1) - (I-13) were prepared by this method.
Example 2 evaluation of biological Activity of target molecule
1. Method for testing in vitro inhibitory activity of receptor tyrosine kinase
Preparation of kinase assay buffer
① Kinase Detection Buffer (Kinase Detection Buffer) was dissolved at room temperature and observed for precipitation.
② if precipitation occurs, it is incubated at 37 ℃ for 15 minutes (Kinase Detection Buffer) and shaken often to dissolve the precipitate or, alternatively, the supernatant is carefully aspirated off to remove the precipitate.
Preparation of kinase assay reagent
① Kinase Detection buffers (Kinase Detection buffer) and (KinaseDetection Substrate) were equilibrated at room temperature before use.
② Kinase Detection reagent is prepared by pouring the whole amount of Kinase Detection Buffer (Kinase Detection Buffer) into a brown bottle containing a Kinase Detection Substrate (Kinase Detection Substrate) to dissolve the Substrate of the lyophilized powder.
③ mix well by gentle shaking, vortexing or inversion to form a homogeneous solution, and the substrate should be dissolved within 1 minute.
④ the kinase detection reagent should be used immediately after preparation or stored at-20 deg.C, and we believe that the prepared reagent has no loss of activity of the cyclic signal after several times of freeze thawing.
Standard Curve for conversion of ATP to ADP was generated
① UltraPure ATP and ADP provided in the kit were diluted with 1 Xkinase reaction buffer (kinase reaction buffer) to make 900. mu.L of 50. mu.M ATP and 500. mu.L of 50. mu.M ADP.
② the 50. mu.M ATP and 50. mu.M ADP solutions prepared in the previous step were mixed in a 384-well plate A1-A12 as shown in Table 1, simulating the concentration of ATP and ADP for each conversion percentage, and mixed well.
TABLE 1 preparation of 50. mu.M series of ATP + ADP standards
③ mu.L of ADP-Glo was added per wellTMReagents to terminate the kinase reaction. Incubate at room temperature for 40 minutes.
④ mu.L of Kinase Detection Reagent (Kinase Detection Reagent) was added per well to convert ADP to ATP and luciferase and luciferin were introduced to detect ATP.
⑤ were incubated at room temperature for 30-60 minutes, fluorescence was measured with a multifunctional microplate reader and fluorescence values were recorded.
⑥ Standard curves for conversion of ATP to ADP were plotted.
Determination of IC of kinase inhibitors50Value of
① 1 Xkinase reaction buffer (kinase reaction buffer), 2.5X 50 ng/. mu.L kinase and 2.5X 0.5. mu.g/. mu.L substrate and 125. mu.M ATP were prepared according to the Promega kit instructions.
② mu.L of 1 Xkinase reaction buffer (kinase reaction buffer), 2. mu.L of 2.5X 0.5. mu.g/. mu.L substrate and 125. mu.M ATP are added to the enzyme-free control wells 1. mu.L of 1 Xkinase reaction buffer, 2. mu.L of 2.5X 50 ng/. mu.L kinase, 2. mu.L of 2.5X 0.5. mu.g/. mu.L substrate and 125. mu.M ATP are added to the negative control wells 1. mu.L of 1 Xkinase reaction buffer, 2. mu.L of 5 Xtest drug, 2. mu.L of 2.5X 50 ng/. mu.L kinase, 2. mu.L of 2.5X 0.5. mu.g/. mu.L substrate and 125. mu.M ATP are added to the test wells.
③ the plates were mixed and incubated for 60 minutes.
④ mu.L of ADP-Glo was added per wellTMReagents to terminate the kinase reaction. Incubate at room temperature for 40 minutes.
⑤ mu.L of Kinase Detection Reagent (Kinase Detection Reagent) was added to each well to convert ADP to ATP and luciferase and luciferin were introduced to detect ATP, incubated at room temperature for 30-60 minutes, fluorescence measured with a multifunctional plate reader and the fluorescence value recorded.
⑥ results analysis, the results are shown in Table 2.
2. Cell growth experiment (CCK-8 detection method)
Cell inoculation: cells in logarithmic growth phase were collected and cell suspension concentration was adjusted to 5 × 10 per well3Each cell, 100 μ L per well volume, was seeded into 96-well plates, each set of 4 replicates (marginal wells filled with sterile PBS);
cell culture: after cell attachment, 0% FBS RPMI-1640 was starved for 8h, and the control group was cultured with 10% FBS RPMI-1640 at 37 ℃ and 5% CO2Continuously culturing in an incubator (culturing for different times according to experimental requirements);
color generation: adding 10 μ L CCK-8 solution (5mg/mL) into the three groups of cells after culturing for 72h, terminating the culture after 4h, and oscillating on a shaking table at low speed for 10min to fully dissolve crystals;
color comparison: the absorbance value (OD value) of each well is measured on an enzyme linked immunosorbent assay detector, the wavelength of 570nm is selected, the blank well of cell-free RPMl-1640 culture solution is adjusted to zero, and the absorbance value of each well is measured. The experiment was repeated three times
The results are recorded: cell growth inhibition rate (control absorbance value-experimental absorbance value)/control absorbance value x 100%, cell proliferation rate (experimental absorbance value/control absorbance value) x 100;
drawing a cell growth curve: the cell growth curve was plotted with time as abscissa and inhibition/proliferation rate as ordinate.
Inhibitor concentrations were plotted in GraphPad Prism mapping software in GraphPad software to determine log [ inhibitor [ ]]Variable slope model estimation of IC versus response50。
The test results are shown in table 2, and table 2 shows the activity effect of the obtained compounds in inhibiting BTK tyrosine kinase and inhibiting proliferation of tumor cells.
TABLE 2
a:IC50: ramos, Raji are typical B-lymphocytic leukemia cells, highly expressed BTK kinase.
The biological activity results show that the molecule has stronger inhibition effect on BTK kinase, most compounds reach the activity level of nanomolar level, and nearly half of the effective inhibition concentration IC of the compounds50Values of less than 10nmol are comparable to ibrutinib and Spebrutinib. The results of anti-cell proliferation activity revealed that most of the compounds had very potent inhibitory effects on lymphocytic leukemia cells (Ramos and Raji), wherein compounds I-1, I-3, I-4, I-7, I-10, I-4-2, I-4-6, (I-1) - (I-4), I-13, I-14, I-25, I-26 and I-27 also showed superior activity to Spebrutinib, and in particular the effects of I-1, I-3, I-4, I-7 and I-10 were unexpected by those skilled in the art. The molecules are predicted to have the potential of being developed into novel high-efficiency BTK inhibitors, and have great application value in treating related tumor diseases, particularly Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (6)

1. A compound having a structure represented by the following formula (I-4), (I-7) or (I-10), or a pharmaceutically acceptable salt thereof:
2. a pharmaceutical composition comprising an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
3. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 2, in the manufacture of a medicament for the treatment of a tumour.
4. The use of claim 3, wherein the tumor is selected from one or more of Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, or chronic lymphocytic leukemia.
5. The use of claim 4, wherein the tumor is chronic lymphocytic leukemia.
6. Use according to claim 3, wherein the use is primarily by inhibition of Bruton's tyrosine kinase.
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