CN111253370B - N-polysubstituted pyridine-2-aminopyrimidine derivatives and application thereof - Google Patents
N-polysubstituted pyridine-2-aminopyrimidine derivatives and application thereof Download PDFInfo
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- CN111253370B CN111253370B CN202010214148.0A CN202010214148A CN111253370B CN 111253370 B CN111253370 B CN 111253370B CN 202010214148 A CN202010214148 A CN 202010214148A CN 111253370 B CN111253370 B CN 111253370B
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- C07—ORGANIC CHEMISTRY
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- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P35/02—Antineoplastic agents specific for leukemia
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
Abstract
The invention provides an N-polysubstituted pyridine-2-aminopyrimidine derivative and application thereof, wherein the derivative has a brand new skeleton, and experimental results show that most compounds show good CHK1 protein inhibition activity, have obvious in-vitro proliferation inhibition effect on blood tumor cell strains, are used as cell cycle checkpoint kinase 1(CHK1) inhibitors, and have good selectivity of targeting tumor cells in tumor treatment. The compound has the advantages of reasonable design of a synthetic route, easily obtained required raw materials, mild reaction conditions, high yield of each step, simple and convenient operation and suitability for industrial production. Therefore, the compound of the invention can be used as CHK1 inhibitor in tumor treatment. The structure of the general formula I of the derivative is as follows:
Description
Technical Field
The invention relates to the field of medicines, in particular to an N-polysubstituted pyridine-2-aminopyrimidine derivative and application thereof as a cell cycle checkpoint kinase 1(CHK1) inhibitor in antitumor medicines.
Background
Targeting the cell cycle has become a hotspot for anti-tumor studies. When the DNA of cells is damaged by cytotoxic drugs and radiotherapy, a DNA damage response channel in vivo is activated, so that the damaged DNA is repaired, wherein a cell cycle checkpoint kinase 1(CHK1) plays an important role in the channel. When DNA is damaged, it causes cell cycle arrest at the G1, S or G2/M phases, with the cell cycle checkpoint kinase 1(CHK1) being primarily responsible for the regulation of the S and G2/M checkpoints. In most tumor cells, the G1 checkpoint is deleted due to a defect in the p53 gene, so most tumor cells rely primarily on the S and G2/M phase checkpoints. If CHK1 protein is inhibited while DNA is damaged, only existing S and G2/M check points are eliminated, the self-repair of tumor cells is blocked, and the apoptosis of the tumor cells can be promoted. Because normal cells can be self-repaired and protected by means of p53, the CHK1 inhibitor is relatively insensitive, so that the CHK1 inhibitor has good selectivity of targeting tumor cells in tumor treatment. At present, 12 CHK1 small-molecule inhibitors are in clinical study, including GDC-0575, XCCS605B and LY-2880070 in phase I clinical study, and LY2606368 and CCT-245737 in phase II clinical study.
Disclosure of Invention
The invention aims to provide an N-polysubstituted pyridine-2-aminopyrimidine derivative, an optical isomer thereof or a pharmaceutically acceptable salt thereof.
In order to achieve the purpose, the N-polysubstituted pyridine-2-aminopyrimidine derivative provided by the invention has a structure shown in a general formula I:
and optical isomers thereof or pharmaceutically acceptable salts thereof,
wherein;
R1selected from trifluoromethyl, trifluoromethylpyrazole;
R2selected from H, methoxy, methylamino;
x is selected from O, NRa；
RaSelected from H, C1-3An alkyl group;
n is selected from 0, 1,2, 3 or 4;
R3is selected from-L1-NRbRc、
L1Is unsubstituted or substituted C2-4Alkyl, aryl, heteroaryl, and heteroaryl,
Said C2-4Alkyl groups may optionally be substituted with one or more groups selected from deuterium, halogen, amino, hydroxy, C1-4Alkyl, halo C1-4Alkyl substitution;
m and o are each independently selected from 0, 1,2 or 3, k is selected from 1,2, 3 or 4;
Rband RcEach independently selected from H, C1-4Alkyl, halo C1-4Alkyl radical, C3-8Cycloalkyl, halo C3-8A cycloalkyl group;
p and q are each independently selected from 1,2, 3, 4;
Rdselected from amino, hydroxyl;
more particularly still, preferred compounds of the structure of formula I of the present invention are selected from:
3- (3-Aminopropoxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- (2-aminoethoxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((3-aminopropyl) amino) -5- ((4-methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((2-aminoethyl) amino) -5- ((4-methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S,4S) -4-aminocyclohexyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1R, 4R) -4-aminocyclohexyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S,4S) -4-aminocyclohexyl) oxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1R, 4R) -4-aminocyclohexyl) oxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S, 3S) -3-aminocyclobutyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1R, 3R) -3-aminocyclobutyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1R, 3R) -3-aminocyclopentyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S, 3S) -3-aminocyclopentyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1R, 3S) -3-aminocyclopentyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S, 3R) -3-aminocyclopentyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S,4S) -4-aminocyclohexyl) amino) -5- ((4-methoxy-5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S, 3S) -3-Aminocyclobutoxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1R, 3R) -3-Aminocyclobutoxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((2-aminoethyl) (methyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S,4S) -4-aminocyclohexyl) amino) -5- ((5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1R, 4R) -4-aminocyclohexyl) amino) -5- ((5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((2-amino-2-methylpropyl) amino) -5- ((4-methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((2-aminopropyl) amino) -5- ((4-methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -3- ((2- (methylamino) propyl) amino) -2-cyanopyridine
3- ((1-aminocyclopropyl) methyl) amino) -5- ((4-methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1-aminocyclobutyl) methyl) amino) -5- ((4-methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((2- (cyclopropylamino) ethyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((3-amino-3-methylbutyl) amino) -5- ((4-methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((3-aminopropyl) amino) -5- ((4-methylamino) -5- (1-trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- (3-Aminopropoxy) -5- ((4- (methylamino) -5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((2-aminoethyl) amino) -5- ((4-methylamino) -5- (1-trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((2-amino-2-methylpropyl) amino) -5- ((4-methylamino) -5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S,4S) -4-aminocyclohexyl) amino) -5- ((4- (methylamino) -5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1R, 4R) -4-aminocyclohexyl) amino) -5- ((4- (methylamino) -5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine
3- ((1S,4S) -4-aminocyclohexyl) oxy) -5- ((4- (methylamino) -5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine
The invention provides acceptable salts of compounds selected from inorganic and organic acids, for example, hydrochloride, hydrobromide, sulphate, phosphate, nitrate, formate, acetate, propionate, citrate, tartrate, maleate, fumarate, mandelate, oxalate, succinate, malate, gluconate, glucuronate, malonate, methanesulphonate, stearate and trifluoroacetate.
The invention also aims to provide application of the N-polysubstituted pyridine-2-aminopyrimidine derivative and the optical isomer or the pharmaceutically acceptable salt thereof in preparing antitumor drugs. The tumor is hematological tumor, breast cancer, lung cancer, prostate cancer, colon cancer, rectal cancer, renal cancer, pancreatic cancer, neuroblastoma, glioma, head and neck cancer, thyroid cancer, ovarian cancer, myeloma, gastrointestinal stromal tumor, liver cancer, bladder cancer, cervical cancer, and skin cancer. The medicine is prepared from N-polysubstituted pyridine-2-aminopyrimidine derivatives, optical isomers thereof or pharmaceutically acceptable salts thereof and pharmaceutically acceptable auxiliary materials. The drug is used as a cell cycle checkpoint kinase 1(CHK1) inhibitor, and has good selectivity of targeting tumor cells in tumor treatment. The N-polysubstituted pyridine-2-aminopyrimidine derivative provided by the invention has CHK1 inhibitory activity.
The medicine is used singly and/or combined with other chemotherapeutic drugs, radiotherapy, immunotherapy drugs and the like.
The invention provides a preparation method of N-polysubstituted pyridine-2-aminopyrimidine derivatives and optical isomers or pharmaceutically acceptable salts thereof, which is realized by the following steps:
the method comprises the following steps:
5-trifluoromethyl-2, 4-dichloropyrimidine is taken as a starting material, and N is obtained by ammoniation and methylamine substitution in sequence4-methyl-5- (trifluoromethyl) pyrimidine-2, 4-diamine intermediate;
under the alkaline condition, the 5-bromine-2-cyano-3-nitropyridine undergoes substitution reaction with different alcohols to obtain pyridine fragment intermediates with different substitutions, and then the pyridine fragment intermediates and the obtained N4-methyl-5- (trifluoromethyl) pyrimidine-2And 4-diamine intermediate reaction and deprotection to obtain the target compound:
the second method comprises the following steps:
carrying out substitution reaction on 5-bromo-2-cyano-3-fluoropyridine and different aliphatic amines under alkaline conditions to obtain different substituted pyridine fragment intermediates, and further carrying out substitution reaction on the pyridine fragment intermediates and the N obtained by the first method4-methyl-5- (trifluoromethyl) pyrimidine-2, 4-diamine intermediate reaction, and deprotection to obtain the target compound:
the third method comprises the following steps:
taking 2-chloro-5-trifluoromethyl pyrimidine as a starting material, and ammoniating to obtain a 5-trifluoromethyl-2-aminopyrimidine intermediate;
under the alkaline condition, the 5-bromo-2-cyano-3-fluoropyridine and different fatty amines undergo substitution reaction to obtain pyridine fragment intermediates with different substitutions, and then react with the obtained 5-trifluoromethyl-2-aminopyrimidine intermediates to remove protecting groups to obtain a target compound:
the method four comprises the following steps:
taking 5-trifluoromethyl-2, 4-dichloropyrimidine as a starting material, and sequentially carrying out ammoniation and methyl etherification to substitute the starting material to obtain 4-methoxy-5- (trifluoromethyl) pyridine-2-amine;
under the alkaline condition, the 5-bromo-2-cyano-3-fluoropyridine and different fatty amines undergo substitution reaction to obtain pyridine fragment intermediates with different substitutions, and then react with the obtained 4-methoxy-5- (trifluoromethyl) pyridine-2-amine to remove the protecting groups to obtain a target compound:
the method five comprises the following steps:
taking 5-bromo-2, 4-dichloropyrimidine as a starting material, sequentially carrying out methylamine and ammoniation, and carrying out Suzuki coupling to obtain N4-methyl-5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidine-2, 4-diamine, which in turn reacts with the obtained variously substituted pyridine fragment intermediates, deprotecting the groups to obtain the target compound:
the method six:
carrying out substitution reaction on 5-bromo-2-cyano-3-nitropyridine and different alcohols under alkaline conditions to obtain different substituted pyridine fragment intermediates, and further carrying out substitution reaction on the pyridine fragment intermediates and the N obtained by the fifth method4-methyl-5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidine-2, 4-diamine reaction, deprotection of the group to give the target compound:
the method comprises the following steps:
dissolving 1.2 equivalents of inorganic acid or organic acid in ethanol solution, slowly dripping into ethanol solution of the compound, adding appropriate amount of diethyl ether solution, vacuum filtering to obtain salt-forming compound, washing with diethyl ether, and drying.
Experiments prove that the N-polysubstituted pyridine-2-aminopyrimidine derivative with a brand-new framework has good CHK1 protein inhibition activity, and partial compounds have obvious in-vitro proliferation inhibition effect on hematological tumor cell strains such as MV4-11, Z138, MM1S and the like. The compound has the advantages of reasonable design of a synthetic route, easily obtained required raw materials, mild reaction conditions, high yield of each step, simple and convenient operation and suitability for industrial production. Therefore, the compound of the invention can be used as CHK1 inhibitor in tumor treatment.
Detailed Description
The present invention will be further described with reference to examples.
Preparation example 13 preparation of- (3-aminopropoxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine (Compound 1)
Step 1.5-trifluoromethyl-4-chloro-2-aminopyrimidine (intermediate 1-2) Synthesis
2, 4-dichloro-5-trifluoromethylpyrimidine (5.2g,24.07mmol) was dissolved in ammonia-saturated ethanol (25mL), stirred at room temperature for 2h, and the solvent was recovered under reduced pressure to give a residue, which was purified by silica gel column chromatography using PE: EA (5:1) as an eluent to give 1-2(2.3g,11.67mmol) as a white solid in yield: 48.5 percent.1H NMR(500MHz,CDCl3)δ8.57(s,1H),7.98(s,2H)；ESI-MS:m/z＝198.0[M+H]+。
Step 2.N4Synthesis of (E) -methyl-5- (trifluoromethyl) pyrimidine-2, 4-diamine (intermediate 1-3)
Dissolving the intermediate 1-2(1g,5.08mmol) in acetonitrile solution, dropwise adding methylamine alcohol solution (10mL), refluxing for 3h, recovering solvent under reduced pressure to obtain residue, purifying by silica gel column chromatography with PE: EA (2:1) as eluent to obtain white solid 1-3 with yield: 73.8 percent.1H NMR(500MHz,CDCl3)δ8.04(d,J＝1.0Hz,1H),5.30(s,1H),5.11(s,1H),2.99(d,J＝5.0Hz,1H)；ESI-MS:m/z＝193.0[M+H]+。
Step 3 Synthesis of (3- (3-N-tert-Butoxycarbonylaminopropoxy) -5-bromo-2-cyanopyridine (intermediate 1-5)
3-tert-Butoxycarbonylamino-1-propanol (800mg,4.55mmol) was dissolved in a solution of anhydrous THF (15mL), and 60% sodium hydride (218mg) was added in portions under ice bath and stirred at room temperature for half an hour. 5-bromo-2-cyano-3-nitropyridine (794.5mg,3.5mmol) was dissolved in anhydrous THF (5mL) under nitrogen, and the sodium salt prepared above was added dropwise to the solution and reacted at room temperature for 1 h. Adding saturated ammonium chloride solution to quench the reaction, recovering the solvent under reduced pressure to obtain a residue, and purifying by silica gel column chromatography with PE: EA (3:1) as an eluent, wherein the yield is as follows: 50.1 percent.1H NMR(500MHz,CDCl3)δ8.34(d,J＝2.0Hz,1H),7.52(d,J＝1.5Hz,1H),4.77(s,1H),4.17(t,J＝6.0Hz,2H),3.36(q,J＝6.5Hz,2H),2.13-2.06(m,2H),1.43(s,9H)；ESI-MS:m/z＝356.0[M+H]+。
Step 4.3- (3-Aminopropoxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine (Compound 1) Synthesis
Under the protection of nitrogen, a mixture of intermediates 1-3(122.8mg,0.64mmol), intermediates 1-5(250.0mg,0.70mmol), tetrakistriphenylphosphine palladium (37mg,0.032mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (37mg,0.06mmol) and cesium carbonate (312.1mg, 0.96mmol) was added with anhydrous dioxane (10mL), refluxed and stirred for 5h, suction filtered, and the solvent was recovered under reduced pressure to obtain a residue, which was purified by silica gel column chromatography using PE: EA (1:1) as an eluent to obtain a pale yellow solid. Removing protecting group with hydrochloric acid saturated ethyl acetate to obtain white solid compound 1. Yield: 70 percent.1H NMR(500MHz,DMSO-d6)δ8.43(br,2H),8.24(s,1H),7.43(s,1H),4.18(t,J＝6.5Hz,2H),2.96(d,J＝3.0Hz,3H),2.72(t,J＝6.5Hz,2H),1.89-1.80(m,2H)；ESI-MS:m/z＝368.1[M+H]+。
Preparation example 2 Synthesis of Compounds 2 to 6
The compounds 1-4 are taken as raw materials, N- (tert-butyloxycarbonyl) ethanolamine, cis-3-hydroxy-cyclobutyl-carbamic acid tert-butyl ester, trans-3-hydroxy-cyclobutyl-carbamic acid tert-butyl ester, cis-N-Boc-4-aminocyclohexanol and trans-N-Boc-4-aminocyclohexanol are respectively adopted to replace the compound 3-tert-butyloxycarbonyl-amino-1-propanol, and the compounds 2-6 are synthesized. The series of target molecules is shown in table 1 below.
TABLE 1
Preparation example 33 preparation of- ((3-aminopropyl) amino) -5- ((4-methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine (compound 7)
Synthesis of (3- (3-N-tert-butoxycarbonylaminopropanamino) -5-bromo-2-cyanopyridine (intermediate 1-7))
Dissolving 5-bromo-2-cyano-3-fluoropyridine (250mg,1.25mmol) and N-tert-butoxycarbonyl-1, 3-propanediamine (326mg,1.88mmol) in acetonitrile (10mL), dropwise adding triethylamine (0.5mL), refluxing for 12h, recovering the solvent under reduced pressure to obtain a residue, purifying by silica gel column chromatography with PE: EA (4:1) as an eluent, and obtaining the yield: 90 percent.1H NMR(500MHz,CDCl3)δ7.97(d,J＝1.5Hz,1H),7.18(d,J＝1.5Hz,1H),5.15(s,1H),4.72(s,1H),3.30-3.20(m,4H),1.82(p,J＝6.5Hz,2H),1.44(s,9H)；ESI-MS:m/z＝355.0[M+H]+。
Step 2.3 Synthesis of- ((3-aminopropyl) amino) -5- ((4-methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine (Compound 7)
Under the protection of nitrogen, anhydrous dioxane (10mL) is added into a mixture of intermediate 1-3(100.0mg,0.52mmol), intermediate 1-7(200.0mg,0.52mmol), tetratriphenylphosphine palladium (30.0mg,0.026mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethyl xanthene (30.0mg,0.052mmol) and cesium carbonate (254.0mg,0.78mmol), reflux stirring is carried out for 5h, suction filtration is carried out, a solvent is recovered under reduced pressure to obtain a residue, and the residue is purified by silica gel column chromatography, EA: CH2Cl2(1:2) as eluent to obtain white solid. Removing protective group with hydrochloric acid saturated ethyl acetate to obtain white solid compound 7. Yield: 66 percent.1H NMR(500MHz,DMSO-d6)δ8.24(s,1H),8.16(s,1H),7.91(s,1H),7.37(s,1H),6.61(s,1H),3.23-3.18(m,2H),2.96(d,J＝3.5Hz,4H),2.65(t,J＝6.0Hz,2H),1.70-1.61(m,2H)；ESI-MS:m/z＝367.1[M+H]+。
Preparation example 4 Synthesis of Compounds 8-24
The synthetic procedure was as in example 3, starting with 5-bromo-2-cyano-3-fluoropyridine and 3-N-tert-butoxycarbonyl-3-methyl-1, 3-butanediamine, N-tert-butoxycarbonyl-1, 2-ethylenediamine, N-dimethylformamide2-tert-butoxycarbonyl-N2-methyl-1, 2-propanediamine, tert-butyl 2- (methylamino) ethylcarbamate, 2-N-tert-butoxycarbonyl-1, 2-propanediamine, 2-N-tert-butoxycarbonyl-2-methyl-1, 2-propanediamine, 1-aminomethyl-1- (tert-butoxycarbonyl-amino) cyclopropane, 1-aminomethyl-1- (tert-butoxycarbonyl-amino) cyclobutane, N-tert-butoxycarbonyl-N-cyclopropylethylenediamine, cis-3-amino-1-tert-butyl-cyclobutylcarbamate, trans-3-amino-1-tert-butyl-carbamate, (1S,3R) -1- (tert-butoxycarbonyl-amino) -3-aminocyclopentane, (1R,3R) -1- (tert-butyloxycarbonyl-amino) -3-aminocyclopentane, (1S,3S) -1- (tert-butyloxycarbonyl-amino) -3-aminocyclopentane, (1R,3S) -1- (tert-butyloxycarbonyl-amino) -3-aminocyclopentane, N-tert-butyloxycarbonyl-trans-1, 4-cyclohexanediamine, N-tert-butyloxycarbonyl-cis-1, 4-cyclohexanediamine instead of N-tert-butyloxycarbonyl-1, 3-propanediamine to synthesize the corresponding intermediate, and further the intermediate is catalyzed by tetratriphenylphosphine palladium, 4, 5-bis (diphenylphosphino) -9, 9-dimethylxantheneReacting with intermediate 1-3, and removing protective group to obtain compound 8-24. The series of target molecules is shown in table 2 below.
TABLE 2
Preparation example preparation of 53- ((1S,4S) -4-aminocyclohexyl) amino) -5- ((5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine (compound 25)
Step 1.5-trifluoromethyl-2-aminopyrimidine (intermediate 1-9) Synthesis
Dissolving a compound 2-chloro-5-trifluoromethylpyrimidine (910mg,5mmol) in an ethanol (5mL), dropwise adding an ammonia saturated ethanol solution (15mL) under ice bath, reacting at room temperature for 2h, recovering the solvent under reduced pressure to obtain a residue, and purifying by silica gel column chromatography with PE: EA (4:1) as an eluent to obtain a white solid. ESI-MS: 164.0[ M + H ] M/z]+。
Step 2 Synthesis of (3- (4-tert-Butoxycarbonylaminocyclohexylamino) -5-bromo-2-cyanopyridine (intermediate 1-10)
Dissolving 5-bromo-2-cyano-3-fluoropyridine (200mg,1.0mmol) and N-tert-butoxycarbonyl-cis-1, 4-cyclohexanediamine (246mg,1.15mmol) in acetonitrile (10mL), dropwise adding triethylamine (0.5mL), refluxing for 12h, and recovering solvent under reduced pressure to obtain residual solventThe residue was purified by silica gel column chromatography using PE: EA (3:1) as an eluent, yield: 85 percent.1H NMR(500MHz,CDCl3)δ7.98(d,J＝2.0Hz,1H),7.17(d,J＝2.0Hz,1H),4.65(d,J＝6.5Hz,1H),4.60(s,1H),3.74-3.60(m,1H),3.56-3.44(m,1H),1.88-1.77(m,4H),1.75-1.55(m,4H),1.45(s,9H)；ESI-MS:m/z＝395.1[M+H]+。
Step 3.3- ((1S,4S) -4-aminocyclohexyl) amino) -5- ((5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine (compound 25) Synthesis
Under the protection of nitrogen, a mixture of intermediates 1 to 9(90.0mg,0.55mmol), intermediates 1 to 10(250.0mg,0.63mmol), tetrakistriphenylphosphine palladium (31.0mg,0.027mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (31.0mg,0.055mmol) and cesium carbonate (268.0mg,0.82mmol) was added with anhydrous dioxane (10mL), stirred under reflux for 5h, filtered under reduced pressure, the solvent was recovered under reduced pressure to obtain a residue, which was purified by silica gel column chromatography using PE: EA (1:1) as an eluent to obtain a white solid. Removing protective group with hydrochloric acid saturated ethyl acetate to obtain white solid compound 25. Yield: 60 percent.1H NMR(500MHz,DMSO-d6)δ8.96(s,2H),8.27(d,J＝1.5Hz,1H),7.88(s,1H),5.62(d,J＝7.0Hz,1H),3.44-3.36(m,1H),2.93-2.87(m,1H),1.86-1.77(m,2H),1.68-1.53(m,4H),1.51-1.43(m,2H)；ESI-MS:m/z＝378.1[M+H]+。
Preparation example preparation of 63- ((1R, 4R) -4-aminocyclohexyl) amino) -5- ((5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine (compound 26)
Synthesis procedure referring to example 5, N-t-butoxycarbonyl-trans-1, 4-cyclohexanediamine was used in place of N-t-butoxycarbonyl-cis-1, 4-cyclohexanediamine to give compound 26. Yield: 55 percent; ESI-MS: M/z 378.1[ M + H ]]+。
Preparation example 73 preparation of- ((1S,4S) -4-aminocyclohexyl) amino) -5- ((4-methoxy-5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine (compound 27)
Step 1.2 Synthesis of amino-4-methoxy-5-trifluoromethylpyrimidine (intermediate 1-12)
Dissolving compound 1-2(500mg,2.54mmol) in methanol (8mL), dropwise adding sodium methoxide (8mL) solution under ice bath, reacting at room temperature for 2h, recovering solvent under reduced pressure to obtain residue, and purifying by silica gel column chromatography with PE: EA (3:1) as eluent to obtain compound 1-12. Yield: 90 percent;1H NMR(500MHz,CDCl3)δ8.25(d,J＝1.0Hz,1H),5.48(s,2H),3.97(s,3H)；ESI-MS:m/z＝194.1[M+H]+。
step 2.3- ((1S,4S) -4-aminocyclohexyl) amino) -5- ((4-methoxy-5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine (compound 27) Synthesis
Synthetic procedure reference was made to procedure 4 of preparative example 1, substituting 2-amino-4-methoxy-5-trifluoromethylpyrimidine (intermediate 1-12) for N4-methyl-5- (trifluoromethyl) pyrimidine-2, 4-diamine (intermediate 1-3) to give compound 27. Yield: 50 percent; ESI-MS: M/z 408.1[ M + H ]]+。
Preparation example preparation of 83- ((3-aminopropyl) amino) -5- ((4-methylamino) -5- (1-trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine (compound 28)
Step 1.5-bromo-2-chloro-N-methylpyrimidin-4-amine (intermediate 1-14) Synthesis
Dissolving 5-bromo-2, 4-dichloropyrimidine (5.0g,22mmol) in methanol solution (42mL), slowly adding dropwise methylamine alcohol solution (8mL) under ice bath, reacting at room temperature for 1h, recovering solvent under reduced pressure to obtain residue, and purifying by silica gel column chromatography with PE: EA (5:1) as eluent to obtain compounds 1-14. Yield: 85 percent;1H NMR(500MHz,DMSO-d6)δ8.85(s,1H),7.75(s,1H),2.85(d,J＝4.0Hz,3H)；ESI-MS：m/z＝221.9[M+1]+。
step 2.5 bromo-N4Synthesis of (E) -methylpyrimidine-2, 4-diamine (intermediate 1-15)
Placing compounds 1-14(800mg,3.62mmol) in a sealed tube, adding ammonia saturated ethanol solution (15mL), reacting at 100 deg.C for 24h, cooling to room temperature, recovering solvent under reduced pressure to obtain residue, purifying by silica gel column chromatography with PE: EA (2:1) as eluent to obtain compounds 1-15 with yield: 78 percent;1H NMR(500MHz,CDCl3)δ7.86(s,1H),5.22(s,1H),4.85(s,2H),2.99(d,J＝6.0Hz,3H)；ESI-MS：m/z＝202.9[M+1]+。
step 3.N4Synthesis of (E) -methyl-5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidine-2, 4-diamine (intermediate 1-16)
Under the protection of nitrogen, compound 1-15(60mg,0.29mmol), 4-boronic acid pinacol ester-1- (trifluoromethyl) -1H-pyrazole (100mg,0.38mmol), [1, 1' -bis (diphenylphosphino) ferrocene]Palladium dichloride (6mg,0.0058mmol), potassium carbonate (81mg,0.59mmol) was dissolved in methylRefluxing and stirring the mixture in benzene solution for 12h, cooling to room temperature, filtering, recovering the solvent under reduced pressure to obtain a residue, and purifying by silica gel column chromatography with PE: EA (2:1) as an eluent to obtain compounds 1-16. Yield: 46.2 percent;1H NMR(500MHz,CDCl3)δ7.84(s,1H),7.80(s,1H),7.70(s,1H),4.95(s,1H),4.78(s,1H),2.95(d,J＝5.0Hz,1H)；ESI-MS：m/z＝259.1[M+1]+。
step 4.3- ((3-aminopropyl) amino) -5- ((4-methylamino) -5- (1-trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine (compound 28) Synthesis
Synthetic procedure reference was made to procedure 4 of preparation example 1; the yield is 40 percent; ESI-MS: 433.2[ M +1 ] M/z]+。
Preparation example 9 Synthesis of Compounds 29 to 32
Synthetic procedure referring to example 8, compounds 29-32 were obtained using N-tert-butoxycarbonyl-1, 2-ethylenediamine, 2-N-boc-2-methyl-1, 2-propanediamine, N-tert-butoxycarbonyl-trans-1, 4-cyclohexanediamine, N-tert-butoxycarbonyl-cis-1, 4-cyclohexanediamine instead of N-tert-butoxycarbonyl-1, 3-propanediamine, the series of target molecules being shown in table 3 below.
TABLE 3
Preparation example 103 preparation of (3-aminopropoxy) -5- ((4- (methylamino) -5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine (compound 33)
The synthesis step refers to step 4 of preparation example 1, and the yield is 60.0%; ESI-MS: m/z 434.1[ M +1 ]]+。
Preparation example 113- ((1S,4S) -4-aminocyclohexyl) oxy) -5- ((4- (methylamino) -5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine (Compound 34) was prepared
Step 1.3- ((1S,4S) -4-tert-Butoxycarbonylaminocyclohexyloxy) -5-bromo-2-cyanopyridine (intermediate 1-17) Synthesis
Synthetic procedure reference was made to example 1, step 3. ESI-MS: 396.1[ M +1 ] M/z]+。
Step 2.3- ((1S,4S) -4-aminocyclohexyl) oxy) -5- ((4- (methylamino) -5- (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine (Compound 34) Synthesis
Synthetic procedure reference was made to step 4 of preparative example 1, ESI-MS: 474.2[ M +1 ] M/z]+。
Preparation example 123- (3-Aminopropoxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine hydrochloride (Compound 35) preparation
Step 1.3- (3-Aminopropoxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine hydrochloride Synthesis (Compound 35)
Dissolving the compound 1(100mg,0.27mmol) in an ethanol solution (2mL), slowly dropping concentrated hydrochloric acid (0.03mL) into the ethanol solution of the compound 1, adding a proper amount of ether solution under stirring, performing suction filtration to obtain a crude product, washing with ether, and drying to obtain the compound 35.
Biological Activity test section
The compounds provided by the invention are tested for their inhibitory activity against cell cycle checkpoint kinase 1(CHK1) kinase.
The instrument comprises the following steps: enzyme-linked immunosorbent assay (ENVision)TM(PerkinElmer,USA)；
Materials: human recombinant CHK1, purchased from Sino Biological corporation, GST-fused CHK1 protein fragment (aa 1-476);
sample treatment: the samples were dissolved in DMSO and stored at low temperature, and the concentration of DMSO in the final system was controlled within a range that did not affect the detection activity.
The experimental steps are as follows: CHK1 and substrate were applied using HTRF Kinase buffer (1X Kinase buffer,5mM MgCl. sub.21mM DTT). mu.L of enzyme, 4. mu.L of substrate and 2. mu.L of test compound at different concentrations were added to 384 reaction plates (ProxiPlate. TM. -384Plus, Perkinelmer) in the form of 2% DMSO, 0.075 ng/. mu.L CHK1, 1. mu.M STK1, 50. mu.M ATP. After incubation for 1 hour at room temperature, the antibody was added for detection. And simultaneously setting a solvent control group and a blank control group which replace the compound to be detected with DMSO, and setting 3 multiple wells for each concentration of each sample. The activity of the sample is tested under a single concentration condition, e.g., 10. mu.M, selected for the primary screen. For samples that exhibit activity under certain conditions, e.g., an Inhibition% Inhibition greater than 50, the activity dose dependence, i.e., IC50 value, was tested by nonlinear fitting of sample activity to sample concentration, the software used was calculated as Graphpad Prism 5, the model used was fitted as sigmoidal dose-response (variable slope), and for most inhibitor screening models, the bottom and top of the fitted curve were set at 0 and 100. The test results are shown in table 4.
IC of representative Compounds for CHK1 kinase in Table 450(nM)
A<100nM；100nM<B<500nM；
The results in table 4 show that most compounds exhibit good CHK1 inhibitory activity, indicating the potential of this class of compounds for the treatment of cancer.
The compounds provided by the invention have cell proliferation inhibition activity.
Cell lines: MV-4-11 (human acute myelomonocytic leukemia), Z-138 (human mantle cell line lymphoma cell), MM1S (human myeloma cell)
The experimental steps are as follows: cell viability was measured using the MTS method. Cells grown in logarithmic growth phase were aspirated, gently blown and counted. Inoculating 90uL of the mixture in a 96-well plate at the corresponding cell density, adding 10 uL of the mixture, setting a concentration gradient for each mixture, setting three wells for each concentration, adding each concentration to the corresponding well, respectively, and setting the final concentration of DMSO to be 0.2% in a solution containing 4.5% CO2The cells were cultured in an incubator at 37 ℃ for 3 days, and 20. mu.L of MTS was added thereto. After 3 hours incubation at 37 ℃, the IC was obtained by using spectra max 340 to measure the 490nm (L1) light absorption value, referencing the wavelength to 690nm (L2), plotting the (L1-L2) values against the different inhibitor concentrations, and fitting the percent (%) percentage versus compound OD value-BLANK OD value/DMSO OD value-BLANK OD 100% with graphpad50. The results are shown in tables 5 and 6.
TABLE 5 proliferation inhibitory Activity of Compound 8 and Compound 24 on various tumor cells
TABLE 6 proliferation inhibitory Activity of Compound 23 on various tumor cells
The results in tables 5 and 6 show that the selected representative compounds have significant proliferation inhibitory activity against MV-4-11, Z-138, MM1S cell line.
Claims (7)
1. An N-polysubstituted pyridine-2-aminopyrimidine derivative is characterized in that the structure of the general formula I is as follows:
and optical isomers thereof or pharmaceutically acceptable salts thereof,
wherein;
R1is selected from trifluoromethyl;
R2 selected from methylamino;
x is selected from O, NRa；
RaIs selected from H;
n is selected from 0;
R3is selected from
；
p and q are each independently selected from 2;
Rdselected from amino groups.
2. An N-polysubstituted pyridine-2-aminopyrimidine derivative, characterized in that the derivative is selected from the group consisting of:
3-（（1S，4S) -4-aminocyclohexyl) amino) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine,
3-（（1S，4S) -4-aminocyclohexyl) oxy) -5- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -2-cyanopyridine,
3-（（1S，4S) -4-aminocyclohexyl) amino) -5- ((4- (methylamino) -5-, (1- (trifluoromethyl) -1H-pyrazol-4-yl) pyrimidin-2-yl) amino) -2-cyanopyridine,
and optical isomers thereof or pharmaceutically acceptable salts thereof.
3. An N-polysubstituted pyridine-2-aminopyrimidine derivative according to any of the claims 1-2, characterized in that the pharmaceutically acceptable salts of the compounds are selected from inorganic or organic acids.
4. An N-polysubstituted pyridine-2-aminopyrimidine derivative according to claim 3 characterized in that the salt is selected from the group consisting of hydrochloride, sulfate, phosphate, formate, acetate, propionate, citrate, tartrate, maleate, fumarate, mandelate, oxalate, succinate, malate, gluconate, methanesulfonate and trifluoroacetate.
5. The use of the N-polysubstituted pyridine-2-aminopyrimidine derivatives and the optical isomers thereof or the pharmaceutically acceptable salts thereof according to any one of claims 1 to 3 in the preparation of antitumor drugs.
6. The use of claim 5, wherein the tumor is a hematological tumor.
7. The use of claim 5, wherein the medicament is prepared from N-polysubstituted pyridine-2-aminopyrimidine derivatives, optical isomers or pharmaceutically acceptable salts thereof and pharmaceutically acceptable auxiliary materials.
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