CN115611885A - N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivatives - Google Patents
N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivatives Download PDFInfo
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- CN115611885A CN115611885A CN202211044195.0A CN202211044195A CN115611885A CN 115611885 A CN115611885 A CN 115611885A CN 202211044195 A CN202211044195 A CN 202211044195A CN 115611885 A CN115611885 A CN 115611885A
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- amino
- substituted
- pyrimidin
- indole
- methyl
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- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
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Abstract
The invention discloses N-substituted-5- ((4-substituted pyrimidine-2-yl) amino) -1H-indole-2-formamide derivatives and a preparation method thereofIts preparation method and application, its structural formula is
The invention can be used for treating and preventing cancer or tumor-related diseases by inducing tumor cells to generate Nut 77-dependent cell megakaryocyte death (Methuosis) and inhibiting the proliferation of the tumor cells.
Description
Technical Field
The invention belongs to the technical field of chemical medicines, and particularly relates to an N-substituted-5- ((4-substituted pyrimidine-2-yl) amino) -1H-indole-2-formamide derivative and application thereof.
Background
In clinical treatment in the tumor field, cancer cells can acquire drug resistance through various ways, such as improving DNA repair capacity, increasing drug efflux, changing apoptosis ways and the like, which brings huge challenges to current chemotherapy and targeted therapy. Therefore, it is important to find alternative cell death pathways to kill anti-apoptotic cancer cells. Megalocytic death (methusas) is a non-apoptotic cell death mode in which a number of vacuoles are present in the cytoplasm as a result of dysregulation of the macropinocytic process, ultimately resulting in decreased metabolic activity and rupture of the cell membrane. Several classes of methahosis inducers have been reported, but most lack well-defined molecular mechanisms and effective in vivo pharmacological data.
The nuclear receptor Nur77 belongs to the NR4A subfamily of the nuclear receptor superfamily, and is widely involved in various physiological processes of organisms, such as cell growth and death, metabolism, inflammation and the like. In view of its important role in a variety of cellular activities and a variety of diseases, nur77 has become a promising drug target. In recent decades, the nuclear receptor Nur77 expression or the function abnormality thereof has been proved to be closely related to the proliferation, the metabolism, the metastasis and the like of various tumors; at present, a plurality of Nur77 small molecular regulators are reported to play excellent anticancer roles in vivo and in vitro through different action mechanisms. For example, nur77 antagonist CDIM derivatives (CDIM 5, CDIM 8) promote cancer cell apoptosis and inhibit cancer cell migration; nur77 agonists such as Cytosporone B, SK07, apaensin and Malayosine mediate Nur77 mitochondrial localization, regulate Nur77-Bcl2 interaction, promote cytochrome C release, further induce cancer cell apoptosis and inhibit tumor growth. The development of a Nur77 regulator with a novel action mechanism is one of the research hotspots in the anti-tumor field at present.
Disclosure of Invention
The invention aims to provide an N-substituted-5- ((4-substituted pyrimidine-2-yl) amino) -1H-indole-2-formamide derivative for inducing a workpiece tribochemical reaction.
The technical scheme of the invention is as follows:
the N-substituted-5- ((4-substituted pyrimidine-2-yl) amino) -1H-indole-2-formamide derivative has a structural formula
Wherein,
R 1 is 4-methyl-2- (methylamino) thiazolyl, 2, 5-dimethylthiazolyl, N-dimethylaniline, thienyl, 2-chlorothienyl, 2-chlorophenyl or 1, 3-bis (benzyloxy) phenyl;
R 2 <xnotran> N- , -1- , N- , -1- , (R) -2- ( ) -1- , (S) -2- ( ) -1- ,4- -1H- -1- , N-2- ( -2- ) , -1- ,4- -1- ,3- -1- ,4- -1- ,4- -1- , N- (1r,4r) -4- ,4- ( ) -1- ,3- ( ) -1- ,4- -1- ,4- -1- ,3,5- -1- ,4- ( ) -1- ,4- ( ) -1- ,4- -2- ,4- (2- ) -1- , N- (1- -4- ), 4- ( ) -1- , </xnotran> Morpholinyl, 2-dimethylmorpholinyl, N- (tetrahydro-2H-pyran-4-yl), thiomorpholinyl, N- (3-morpholinopropyl), N- (4-hydroxymorpholinopropyl)Phenylphenyl), N- (4-methoxyphenyl), N- (3-chloro-4-methoxyphenyl), methyl 2-hydroxy-4-aminobenzoate, N- (4-methoxybenzyl), N- (4-fluorobenzyl), N-phenylethyl, N- (4-hydroxyphenylethyl), 1, 4-diaza-1-carboxaldehyde, azepin-4-one, 4-amino-nonane-1-carboxylic acid tert-butyl ester, 4-methyl-1, 4-diaza-1-yl, azocinyl, N-cyclooctyl, N- (adamantan-1-yl), [1,4' -bipiperidine]-1' -yl, 4- (4-methylpiperazin-1-yl) piperidin-1-yl, 4-cyclopentylpiperazin-1-yl, 4- (4-methoxyphenyl) piperazin-1-yl or 4- (4-fluorophenyl) piperazin-1-yl.
In a preferred embodiment of the present invention, the compound has the formula
More preferably, the compound has a structural formula
A pharmaceutical composition comprises the above N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative or a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.
The use of the above N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative or a pharmaceutically acceptable salt thereof or the above pharmaceutical composition for the preparation of a composition for the treatment of cancer.
In a preferred embodiment of the present invention, the cancer is lung cancer, breast cancer, liver cancer, colorectal cancer, ovarian cancer, melanoma, esophageal cancer, cervical cancer or renal cancer.
The N-substituted-5- ((4-substituted pyrimidine-2-yl) amino) -1H-indole-2-formamide derivative or the medicinal salt thereof or the medicinal composition is applied as a giant cell death inducer.
The N-substituted-5- ((4-substituted pyrimidine-2-yl) amino) -1H-indole-2-formamide derivative or the medicinal salt thereof or the medicinal composition is applied as a Nur77 receptor regulator.
The invention has the beneficial effects that: the invention can be used for treating and preventing cancer or tumor-related diseases by inducing tumor cells to generate Nur 77-dependent cell megakaryocyte death (Methuosis) and inhibiting the proliferation of the tumor cells.
Drawings
FIG. 1 is a graph showing the results of experiments in examples 8 to 10 of the present invention.
FIG. 2 is a graph showing the results of experiments in examples 13 to 15 of the present invention.
FIG. 3 is a graph showing the results of experiments in examples 16 to 17 of the present invention.
FIG. 4 is a graph showing the results of the experiment in example 18 of the present invention.
FIG. 5 is a graph showing the results of an experiment conducted in example 19 of the present invention.
FIG. 6 is a second graph showing the experimental results of example 19 of the present invention.
FIG. 7 is a graph showing the results of an experiment in example 20 of the present invention.
FIG. 8 is a graph showing the second experimental result of example 20 of the present invention.
FIG. 9 is a graph showing the results of an experiment in example 21 of the present invention.
FIG. 10 is a graph showing the results of the experiment in example 22 of the present invention.
FIG. 11 is a graph showing the results of experiments in examples 23 to 26 of the present invention.
FIG. 12 is a second graph showing the experimental results of example 23 of the present invention.
FIG. 13 is a graph showing the results of an experiment in example 27 of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
The preparation method of the N-substituted-5- ((4-substituted pyrimidine-2-yl) amino) -1H-indole-2-formamide derivative comprises the following steps: the preparation method comprises the steps of taking 4-nitrophenylhydrazine (1) as a raw material, taking absolute ethyl alcohol as a solvent, dropwise adding ethyl pyruvate under a stirring state, heating to reflux after dropwise adding is finished, generating ethyl pyruvate p-nitrophenylhydrazone (2), heating the compound (2) under a polyphosphoric acid catalysis condition to obtain an intermediate 5-nitroindole-2 ethyl carboxylate (3), reducing nitro groups to amino groups to obtain 5-aminoindole-2 ethyl carboxylate (4), reacting the compound (4) with cyanamide and hydrochloric acid to generate guanidine hydrochloride (5), and mixing the guanidine hydrochloride with an ammonium nitrate solution to generate stable guanidine nitrate (6). And (3) carrying out reflux reaction on the synthesized nitrate (6) of the guanidine and 3- (dimethylamino) -1- (substituted or unsubstituted aryl) -2-propen-1-one in an ethanol solution containing sodium hydroxide to generate an intermediate ester (7), further hydrolyzing the compound (7) into an acid (8), and carrying out amide condensation on the compound (8) and different amines to obtain the target compound pyrimidine indole derivative (9). The specific synthetic route is as follows:
R 1 is 4-methyl-2- (methylamino) thiazolyl, 2, 5-dimethylthiazolyl, N-dimethylaniline, thienyl, 2-chlorothienyl, 2-chlorophenyl or 1, 3-bis (benzyloxy) phenyl;
R 2 is N-cyclopropyl, azetidin-1-yl, N-cyclobutyl, pyrrolidin-1-yl, (R) -2- (hydroxymethyl) pyrrolidin-1-yl, (S) -2- (hydroxymethyl) pyrrolidin-1-yl, 4-methyl-1H-imidazol-1-yl, N-2- (thiophen-2-yl) ethyl, piperidin-1-yl, 4-methylpiperidin-1-yl, 3-methylpiperidin-1-yl, 4-isopropylpiperidin-1-yl, 4-hydroxypiperidin-1-yl, N- (1r, 4r) -4-hydroxycyclohexyl, 4- (hydroxymethyl) piperidin-1-yl, 3- (hydroxymethyl) piperidin-1-yl, 4-methylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3, 5-dimethylpiperazin-1-yl, 4- (methylsulfonyl) piperazin-1-yl, 4- (tert-butyl formate) piperazin-1-yl, 4-tert-butyl formate-2-aminoethyl) piperazine-1-yl, 4- (2-ethyl) piperazin-1-ylButhyl ester, N- (1-methylpiperidin-4-yl), 4- (aminomethyl) piperidine-1-carboxylic acid tert-buthyl ester, morpholino ester, 2-dimethylmorpholino ester, N- (tetrahydro-2H-pyran-4-yl), thiomorpholino ester, N- (3-morpholinopropyl), N- (4-hydroxyphenyl), N- (4-methoxyphenyl), N- (3-chloro-4-methoxyphenyl), methyl 2-hydroxy-4-aminobenzoate, N- (4-methoxybenzyl), N- (4-fluorobenzyl), N-phenylethyl, N- (4-hydroxyphenylethyl), 1, 4-diaza-1-carboxalyl, azepin-4-onyl, 4-amino-nonane-1-carboxylic acid tert-buthyl ester, 4-methyl-1, 4-diaza-1-yl, azacyclooctyl, N-cyclooctyl, N- (adamantan-1-yl), [1,4' -bipiperidine]-1' -yl, 4- (4-methylpiperazin-1-yl) piperidin-1-yl, 4-cyclopentylpiperazin-1-yl, 4- (4-methoxyphenyl) piperazin-1-yl or 4- (4-fluorophenyl) piperazin-1-yl.
Specific compounds are shown in table 1:
TABLE 1 structural formulas of N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivatives of the present invention and corresponding nuclear magnetic characterization data
Example 1: preparation of N-cyclopropyl-5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-1)
In a dry 50mL reaction flask, 5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (69mg, 0.18mmol), EDCI (46mg, 0.24mmol), HOBt (24mg, 0.18mmol), DMF (10 mL) were added sequentially at room temperature. Stirring for 1h at room temperature. Cyclopropylamine (14mg, 0.24mmol) and N, N-diisopropylethylamine (31mg, 0.24mmol) were added thereto under stirring, and the reaction was carried out overnight at room temperature. The reaction was stopped by TLC monitoring, the reaction mixture was poured into 80mL of ice water, solid precipitated and filtered to give a filter cake (crude product). The resulting crude product was separated by silica gel column chromatography (eluent was petroleum ether: ethyl acetate =1, v/v) to give 37mg of N-cyclopropyl-5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide as a pale yellow solid in 47% yield.
The compounds listed in table 1: azetidin-1-yl (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-2), N-cyclobutyl-5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-4), (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (pyrrolidin-1-yl) methanone (7-5), (R) - (2- (hydroxymethyl) pyrrolidin-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-6), (S) - (2- (hydroxymethyl) pyrrolidin-1-yl) (5- (4-methyl-2- ((4- (2-methyl-thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-6) Methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-11), (4-methyl-1H-imidazol-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-12), 5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -N- (2- (thiophen-2-yl) ethyl) -1H-indol-2-carboxamide (7-13), (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (piperidin-1-yl) methanone (7-14), (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (piperidin-4-methyl-piperidin-yl) (4-methyl-2-yl) 1-yl) methanone (7-15), (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (3-methylpiperidin-1-yl) methanone (7-16), (5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (3-methylpiperidin-1-yl) methanone (7-17), (4-isopropylpiperidin-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-18), (4-hydroxypiperidin-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-19), N1r- ((4-cyclohexyl) -4-hydroxy) -5- ((4-indol-2-yl) methanone (7-19), N1r- ((4-hydroxy) -4-cyclohexyl) -4- ((4-hydroxy-indol-2-yl) methanone (7-19), and (4-1-hydroxy-1-yl) amino) methyl-indol-2-yl) methanone (7-1-yl) and a mixture of a pharmaceutically acceptable salt thereof 4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-20), (4- (hydroxymethyl) piperidin-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-21), (3- (hydroxymethyl) piperidin-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-22), 5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -N- (1-methylpiperidin-4-yl) -1H-indole-2-carboxamide (7-23), (4-ethylpiperazin-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) Yl) amino) -1H-indol-2-yl) methanone (7-26), (3, 5-dimethylpiperazin-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-29), 5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (4- (methylsulfonyl) piperazin-1-yl) methanone (7-30), tert-butyl 4- (5- (4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carbonyl) piperazine-1-carboxylate (7-31), tert-butyl 4- (2- (5- ((4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-carbonyl) ethyl) piperazine-1-carboxylate (7-32), 5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -N- (1-methylpiperidin-4-yl) -1H-indole-2-carboxamide (7-33), tert-butyl 4- ((5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide) methyl) piperidine-1-carboxylate (7-34), (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (morpholino) methanone (7-35), (2, 2-dimethylmorpholino) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-37), 5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino N-morpholine-1H-indole-2-carboxamide (7-38), 5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -N- (tetrahydro-2H-pyran-4-yl) -1H-indole-2-carboxamide (7-39), (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (thiomorpholino) methanone (7-40), 5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -N- (3-morpholinopropyl) -1H-indole-2-carboxamide (7-41), N- (4-hydroxyphenyl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-47), N- (4-methoxyphenyl) -5- ((4- (4-methoxyphenyl) -4- ((4-methyl-thiazol-2-yl) pyrimidin-2-yl) amino) Methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-48), N- (3-chloro-4-methoxyphenyl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-49), methyl 2-hydroxy-4- (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide) (7-50), N- (4-methoxybenzyl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-51), N- (4-fluorobenzyl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-48), N- (4-fluorobenzyl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-yl) 52 5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -N-phenethyl-1H-indole-2-carboxamide (7-53), N- (4-hydroxybenzoethyl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-54), 4- (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carbonyl) -1, 4-diazane-1-carboxaldehyde (7-55), 1- (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carbonyl) azepin-4-one (7-56), tert-butyl 4- (5- (4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carbonyl) azepin-4-one (7-56), tert-butyl 4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-2-carbonyl Indole-2-carboxamido) azacycloalkane-1-carboxylate (7-57), (4-methyl-1, 4-diaza-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-58), azo-1-yl (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-62), N-cyclooctyl-5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-63), N- (adamantan-1-yl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-bis [ 4 '-piperidine ] -1, 4' -piperidin-2-yl ] - [ 4 '-piperidin-4- (4' -yl) pyrimidin-2-yl ] amino ] -1H-indole-2-carboxamide (7-bis [ 4, 64, 4 '-piperidine ] - [ 4' -piperidin-2-yl ] amino ] -1, 4-indole-carboxylic acid -methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-65), (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (4- (4-methylpiperazin-1-yl) piperidin-1-yl) methanone (7-66), (4-cyclopentylpiperazin-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-67), (4- (4-methoxyphenyl) piperazin-1-yl) (5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-68), (4- (4-fluorophenyl) piperazin-1-yl) (5- (4-methyl-propan-2-yl) (4- (4-methyl-propan-2-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-68), (4- (4-fluorophenyl) piperazin-1-yl) (5- (4-methyl-yl) piperazin-2-yl) propan-2-yl) ketone (4- (4-yl) propan-2-yl) ketone (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-69) was synthesized analogously to Compound 7-1.
Example 2: preparation of azetidin-1-yl (5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-3)
In a dry 50mL reaction vial, 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (66mg, 0.18mmol), EDCI (46mg, 0.24mmol), HOBt (24mg, 0.18mmol), DMF (10 mL) were added sequentially at room temperature. Stirring for 1h at room temperature. Then, as a catalyst, azetidine (14mg, 0.24mmol) and N, N-diisopropylethylamine (31mg, 0.24mmol) were added thereto under stirring, and the reaction was carried out overnight at room temperature. The reaction was stopped by TLC monitoring, the reaction mixture was poured into 80mL of ice water, solid precipitated and filtered to give a filter cake (crude product). The resulting crude product was separated by silica gel column chromatography (eluent petroleum ether: ethyl acetate =1, v/v) to give 44mg of azetidin-1-yl (5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone as a pale yellow solid in 56.1% yield.
Compounds listed in table 1: ( R) - (5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (2- (hydroxymethyl) pyrrolidin-1-yl) methanone (7-8), (5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (3-methylpiperidin-1-yl) methanone (7-17), (5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (4-methylpiperazin-1-yl) methanone (7-24), (5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (4-ethylpiperazin-1-yl) methanone (7-27), (5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (morpholinyl) methanone (7-36). ) Similar to the synthesis method of the compound 7-3.
Example 3: preparation of (R) - (5- ((4- (5-chlorothien-2-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (2- (hydroxymethyl) pyrrolidin-1-yl) methanone (7-7)
In a dry 50mL reaction flask, 5- ((4- (5-chlorothien-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (67mg, 0.18mmol), EDCI (46mg, 0.24mmol), HOBt (24mg, 0.18mmol), DMF (10 mL) were added sequentially at room temperature. Stirring for 1h at room temperature. D-prolinol (25mg, 0.24mmol) and N, N-diisopropylethylamine (31mg, 0.24mmol) were added as catalysts under stirring, and the reaction was carried out overnight at room temperature. TLC monitoring reaction is finished, reaction is stopped, reaction liquid is poured into 80mL of ice water, solid is separated out, and filtration is carried out to obtain a filter cake (crude product). The obtained crude product was separated by silica gel column chromatography (eluent was petroleum ether: ethyl acetate =1, v/v) to give 36mg of (R) - (5- ((4- (5-chlorothien-2-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (2- (hydroxymethyl) pyrrolidin-1-yl) methanone as a pale yellow solid, yield 42.2%.
Compounds listed in table 1: 5- ((4- (5-chlorothien-2-yl) pyrimidin-2-yl) amino) -N- (3-morpholinopropyl) -1H-indole-2-carboxamide (7-45) was synthesized analogously to Compound 7-7.
Example 4: preparation of (R) - (2- (hydroxymethyl) pyrrolidin-1-yl) (5- (4- (thiophen-2-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-9)
In a dry 50mL reaction flask, 5- ((4- (thiophen-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (61mg, 0.18mmol), EDCI (46mg, 0.24mmol), HOBt (24mg, 0.18mmol), and DMF (10 mL) were added sequentially at room temperature. Stirred at room temperature for 1h. D-prolinol (25mg, 0.24mmol) and N, N-diisopropylethylamine (31mg, 0.24mmol) were added as catalysts under stirring, and the reaction was carried out overnight at room temperature. TLC monitoring reaction is finished, reaction is stopped, reaction liquid is poured into 80mL of ice water, solid is separated out, and filtration is carried out to obtain a filter cake (crude product). The resulting crude product was separated by silica gel column chromatography (eluent was petroleum ether: ethyl acetate =1,v/v) to give 43mg of (R) - (2- (hydroxymethyl) pyrrolidin-1-yl) (5- (4- (thiophen-2-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone as a pale yellow solid in 49.7% yield.
Compounds listed in table 1: n- (3-Morpholinopropyl) -5- ((4- (thien-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (7-44), (4-methyl-1, 4-diaza-1-yl) (5- ((4- (thien-2-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) methanone (7-59) was synthesized analogously to Compound 7-9.
Example 5: preparation of (R) - (5- ((4- (2-chlorophenyl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (2- (hydroxymethyl) pyrrolidin-1-yl) methanone (7-10)
In a dry 50mL reaction vial, 5- ((4- (2-chlorophenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (66mg, 0.18mmol), EDCI (46mg, 0.24mmol), HOBt (24mg, 0.18mmol), DMF (10 mL) were added sequentially at room temperature. Stirring for 1h at room temperature. D-prolinol (25mg, 0.24mmol) and N, N-diisopropylethylamine (31mg, 0.24mmol) were added as a catalyst under stirring, and the reaction was allowed to proceed overnight at room temperature. TLC monitoring reaction is finished, reaction is stopped, reaction liquid is poured into 80mL of ice water, solid is separated out, and filtration is carried out to obtain a filter cake (crude product). The obtained crude product was subjected to silica gel column chromatography (eluent was petroleum ether: ethyl acetate =1, v/v) to give 24mg of (R) - (5- ((4- (2-chlorophenyl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (2- (hydroxymethyl) pyrrolidin-1-yl) methanone as a pale yellow solid, yield 26.9%.
Compounds listed in table 1: (5- ((4- (2-chlorophenyl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (4-methylpiperazin-1-yl) methanone (7-25), (5- ((4- (2-chlorophenyl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (4-ethylpiperazin-1-yl) methanone (7-28), and 5- ((4- (2-chlorophenyl) pyrimidin-2-yl) amino) -N- (3-morpholinopropyl) -1H-indole-2-carboxamide (7-43) were synthesized analogously to Compound 7-10.
Example 6: preparation of 5- ((4- (dimethylamino) phenyl) pyrimidin-2-yl) amino) -N- (3-morpholinopropyl) -1H-indole-2-carboxamide (7-42)
In a dry 50mL reaction flask, 5- ((4- (4- (dimethylamino) phenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (68mg, 0.18mmol), EDCI (46mg, 0.24mmol), HOBt (24mg, 0.18mmol), DMF (10 mL) were added sequentially at room temperature. Stirring for 1h at room temperature. N- (3-aminopropyl) morpholine (35mg, 0.24mmol) and N, N-diisopropylethylamine (31mg, 0.24mmol) were added to the reaction mixture under stirring, and the reaction mixture was allowed to stand overnight at room temperature. TLC monitoring reaction is finished, reaction is stopped, reaction liquid is poured into 80mL of ice water, solid is separated out, and filtration is carried out to obtain a filter cake (crude product). The obtained crude product was separated by silica gel column chromatography (eluent was petroleum ether: ethyl acetate =1, v/v) to give 30mg of 5- ((4- (dimethylamino) phenyl) pyrimidin-2-yl) amino) -N- (3-morpholinopropyl) -1H-indole-2-carboxamide as a pale yellow solid in a yield of 32.4%.
Compounds listed in table 1: (5- ((4- (dimethylamino) phenyl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (4-methyl-1, 4-diaza-1-yl) methanone (7-60) was synthesized analogously to Compound 7-42.
Example 7: preparation of 5- ((4- (3 ',5' -bis (benzyloxy) - [1,1' -biphenyl ] -4-yl) pyrimidin-2-yl) amino) -N- (3-morpholinopropyl) -1H-indole-2-carboxamide (7-46)
In a dry 50mL reaction flask, 5- ((4- (4- (dimethylamino) phenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (100mg, 0.18mmol), EDCI (46mg, 0.24mmol), HOBt (24mg, 0.18mmol), and DMF (10 mL) were added sequentially at room temperature. Stirred at room temperature for 1h. N- (3-aminopropyl) morpholine (35mg, 0.24mmol) and N, N-diisopropylethylamine (31mg, 0.24mmol) were added as a catalyst under stirring, and the reaction was allowed to proceed overnight at room temperature. The reaction was stopped by TLC monitoring, the reaction mixture was poured into 80mL of ice water, solid precipitated and filtered to give a filter cake (crude product). The obtained crude product was subjected to silica gel column chromatography (eluent was petroleum ether: ethyl acetate =1, v/v) to give 62mg of 5- ((4- (3 ',5' -bis (benzyloxy) - [1,1' -biphenyl ] -4-yl) pyrimidin-2-yl) amino) -N- (3-morpholinopropyl) -1H-indole-2-carboxamide as a pale yellow solid, yield 73.4%.
The compounds listed in table 1: (5- ((4- (3 ',5' -bis (benzyloxy) - [1,1' -biphenyl ] -4-yl) pyrimidin-2-yl) amino) -1H-indol-2-yl) (4-methyl-1, 4-diazo-1-yl) methanone (7-61) was synthesized analogously to Compound 7-46.
Example 8: binding kinetics of Compounds 7-41 to Nur77-LBD
(1) Surface Plasmon Resonance (SPR): the dissociation constant (Kd) of 7-41 from Nur77 was determined by Surface Plasmon Resonance (SPR). 50 μ g of purified Nur77-LBD protein was coupled to a CM5 sensor chip, and changes in the course of the binding and dissociation of the different concentrations of 7-41 (0.075, 0.134,0.238,0.422,0.751,1.335,2.373,4.219 and 7.5 μ M) to the chip surface protein were captured by SPR detector using a Biacore T200 instrument, and the binding map data was fitted using a kinetic fitting method. The experimental results are shown in FIG. 1B, and the compounds 7-41 can generate good combination with Nur77-LBD protein in vitro, K d Is 156nM
(2) Fluorescence titration experiment: binding of 7-41 to Nur77-LBD was analyzed by a fluorescence titration experiment. 200 mu L of purified Nur77-LBD protein solution with the concentration of 1.0 mu M is taken into a cuvette, and 7-41 solution is dropwise added to ensure that the action concentration is respectively as follows: 0,0.074,0.091,0.109,0.143,0.178,0.211,0.244,0.309,0.371,0.402,0.432,0.494,0.675,0.903,1.011,1.219,1.319,1.415, 1.607. Mu.M. And detecting the fluorescence intensity of the micromolecule-protein mixed solution with different concentrations in sequence by using a fluorescence spectrophotometer. The resulting data were plotted with Graphad prism 8.0 and dissociation constants were calculated (FIGS. 1C-1D). The dissociation constant Kd for binding of 7-41 to Nur77-LBD was finally fitted to 594.5nM.
(3) Dual luciferase reporter gene experiments: the dual luciferase reporter assay was used to analyze the binding of 7-41 to Nur 77-LBD. FIG. 1H shows the results of an experiment using the dual luciferase reporter system to detect binding of 7-41 to Nur 77-LBD. The results show that compounds 7-41 can inhibit the transcriptional activation function of Nur77.
Example 9: kinetics of binding of Compounds 7-58 to Nur77-LBD
(1) Surface Plasmon Resonance (SPR): as a result of experiments in which Biacore T200 detected 7-58 (0.03, 0.08,0.23,0.56,1.00,1.33,1.78,2.37,3.16,4.22,5.63,7.50 and 10.00. Mu.M) binding to Nur77-LBD on the chip surface according to the method of example 8, as shown in FIG. 1F, compound 7-58 was able to produce good binding to Nur77-LBD protein in vitro, and K was d 46.9nM
(2) And (3) target fishing experiment: 7-58-alk and Biotin-N were activated intracellularly by click chemistry 3 The ligation was performed to construct 7-58-Biotin molecules. Cells were simultaneously interfered with 7-58-Biotin (10. Mu.M) and different concentrations of 7-58 (10,50. Mu.M), and Biotin was precipitated by immunoprecipitation experiments, and immunoblots showed changes in the amount of Nur77 protein that interacted with 7-58-Biotin.
As seen in the immunoblot experiment of FIG. 1G, nur77 was able to be pulled down by Avidin beads together with 7-58-Biotin, but not with Biotin. And Nur77 content of 7-58-Biotin pull-down was inhibited by 7-58 of unlabeled Biotin in a concentration-dependent manner. This indicates that compound 7-58 binds specifically to Nur77.
Example 10: characterization of other Compounds
(1) Surface Plasmon Resonance (SPR): according to example 8, the binding of various compounds (7-4, 7-15,7-40, 7-67) to Nur77-LBD was detected by SPR using a Biacore T200 instrument. The results are shown in FIGS. 1I-1L.
The results showed that Compound 7-4 bound Nur77-LBD with a dissociation constant (Kd) of 265nM; compound 7-15 binds Nur77-LBD with a dissociation constant (Kd) of 1497nM; the dissociation constant (Kd) of the compound 7-40 combined with Nur77-LBD is 1294nM; compounds 7-67 bound Nur77-LBD with a dissociation constant (Kd) of 596nM. Example 11: the vacuole induction effect of the N-substituted-5- ((4-substituted pyrimidine-2-yl) amino) -1H-indole-2-formamide derivative is preliminarily evaluated
Cell line: human non-small cell lung cancer cell line (A549), human normal liver cell (LO) 2 )
Preparation of compound solution: the compounds were dissolved in DMSO to prepare a 10mM stock solution for subsequent experiments, and the stock solution was stored in a refrigerator at-20 ℃.
A549 in DMEM Medium, LO 2 The culture was carried out using RPMI-1640 medium. Cells were plated in 6-well plates in log phase and cultured overnight. After the cells were treated with 0.05, 0.2, 1,2 μ M test compound solution (DMSO solution as a control) for 8h, the morphological changes of the tumor cells were observed using a microscope, and the vacuoles generated in the tumor cells were quantified. The vacuolation rate of the cells measured by experiments is shown in table 2, and the experimental results show that the N-substituted-5- ((4-substituted pyrimidine-2-yl) amino) -1H-indole-2-formamide derivative can remarkably induce tumor cells to generate vacuoles and has certain selectivity on normal human cells.
Example 12: evaluation of cell proliferation inhibitory Activity of N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative of the present invention
Logarithmic phase cells were seeded in 96-well plates and cultured overnight at 37 ℃. Adding to-be-detected compound solutions with different concentrations (20, 10,5, 2.5, 1.25 and 0.625 mu M) into each well, setting 3 multiple wells for each concentration, taking diluted DMSO solution in the same proportion as a control, continuously culturing for 48h, adding MTT with the working concentration of 0.5mg/mL, culturing for 3-4h at 37 ℃, discarding supernatant, adding 100 mu L DMSO into each well, and detecting the light absorption value at 492nm by using an enzyme-labeling instrument. Experimental data calculation of IC Using GraphPad Prism 8.0 50 Values such asAs shown in Table 2, the experimental results show that the series of compounds claimed by the invention have obvious inhibition effect on tumor cells A549 and simultaneously have LO (tumor necrosis factor) on normal human cells 2 Has certain selectivity.
Table 2 partial compounds of the invention on a549 cells and LO 2 Vacuolation rate and proliferation-inhibiting activity of cells
DMSO is negative control, momip is positive control.
Example 13: preferred compounds 7-41 selectively induce tumor cell vacuolation
Cell lines: human non-small cell lung cancer cell lines (H460, A549, H292, H1299, H157), human breast cancer cell lines (MDA-MB 231, HCC1937, MCF-7), human cervical cancer cells (HeLa), human liver cancer cell lines (HepG) 2 Huhu 7), a human esophageal cancer cell line (KYSE-150, eca-109), a human renal cancer cell line (ACHN), a human ovarian cancer cell line (OVCAR-3), human melanoma cell line (OCM-1A), human colorectal cancer cell line (HCT 116), and human normal liver cells (LO) 2 ) Human embryonic lung fibroblasts (MRC-5, BEAS-2B), human immortalized epidermal cells (HaCaT), human arterial vascular endothelial cells (Eahy.926).
The experimental procedure is as in example 11, where H460, H292, H157, LO 2 Using PRMI-1640 medium, A549, H1299, MDA-MB-231, MCF-7, HCC1937, heLa, hepG 2 DMEM medium was used for Huhu7, KYSE-150, eca-109, OVCAR-3, OCM-1A, ACHN, HCT116, haCaT, BEAS-2B, eayy.926 and MRC-5. The morphological changes of the tumor cells after 1. Mu.M treatment of 7-41 were observed by using a microscope, and the results are shown in FIG. 2A. Vacuoles were present in each cancer cell line after treatment 7-41, whereas almost no vacuoles were present in normal cell lines. EC of 7-41 induced H460 cytoplasmic vacuoles in terms of vacuole rate (fig. 2C, vacuole rate = cells appearing vacuole/total number of cells × 100%) 50 The value was 0.41. + -. 0.02. Mu.M. But has poorer vacuole induction capability and EC in human embryonic lung fibroblast MRC-5 compared with lung cancer cell H460,7-41 50 The value was 3.61. + -. 0.02. Mu.M, showing that the betterSelectivity of (2).
Example 14: preferred compounds 7-58 selectively induce KRAS mutant tumor cell vacuolation
Cell line: human colorectal cancer cell lines (HCT 116, SW620, HT-29, HCT-15, colo 205), human colon cells (CCD 18-Co).
The experiment was carried out in the same manner as in example 11, except that DMEM medium was used for HCT116, SW620, HT-29, HCT-15 and Colo205, and MEM medium was used for CCD 18-Co. The morphological changes of the tumor cells after 2. Mu.M treatment of 7-58 were observed by microscope and the results are shown in FIG. 2B. After 7-58 treatments, vacuoles were observed in all of the KRAS-mutated HCT116, SW620, HCT-15 cells, while almost none of the KRAS-non-mutated HT29, colo205, and CCD18-Co cells.
Example 15: preferred compounds 7-41 selectively inhibit tumor cell proliferation
The experimental procedure was as in example 12. The results are shown in FIG. 2E, and the experiments show that the compounds 7-41 have excellent inhibitory effect on various cancer cell lines, IC 50 1.3-15.1 μ M, and has greatly reduced inhibition effect on normal cell line and LO 2 IC of cells 50 IC at 19.23. Mu.M for MRC-5 cells 50 It was 66.81. Mu.M, showing better selectivity.
FIG. 2D shows IC of Compounds 7-58 inhibiting HCT116 cell proliferation 50 It was 1.153. Mu.M.
Example 16: preferably, the induction of vacuolation of cancer cells by Compound 7-41 is mediated by Nur77
Control plasmid, PX330-sgNur77 plasmid and Nur77 small interfering RNA were transfected into A549 and Hela cells, respectively. The vacuolation of A459 and Hela cells after compound 7-41 (1,2. Mu.M) treatment for 8h was observed by microscope, and the cell viability of A549 cells after different concentrations of 7-41 treatment for 48h was tested by MTT assay. Subsequently, nur77 in the cells was detected by immunoblotting (Western blotting). The results are shown in FIGS. 3A-3F.
FIGS. 3A-3B show Nur77 immunoblot analysis results and cell vacuolization in Hela cells transfected with different siRNAs and treated with 1 μm of 7-41. The results of fig. 3A show that Nur77siRNA effectively inhibited/knocked out the expression of Nur77 in cells; the results in FIG. 3B show that, in Nur 77-expressing cells, 7-41 were able to induce cell vacuolization; however, when the expression of Nur77 was knocked out, 7-41 lost vacuole induction.
FIGS. 3C-3E show Nur77 real-time fluorescent quantitative PCR results, immunoblot analysis results, and cell vacuolization phenomena in A549 cells transfected with different sgRNAs and treated with 1,2. Mu.M 7-41. The results of figures 3C-3D show that Nur77 sgRNA effectively inhibited/knocked out the expression of Nur77 in cells; the results in FIG. 3E show that, in Nur 77-expressing cells, 7-41 were able to induce cell vacuolization; however, when the expression of Nur77 was knocked out, 7-41 lost vacuole induction. Further fig. 3F shows a549 cell proliferation of a549 cells transfected with different sgrnas and treated for 48h at different concentrations of 7-41. The results show that the inhibition of A549 cell proliferation by 7-41 is obviously weakened after Nur77 expression is inhibited.
Example 17: preferably, the induction of vacuolation of cancer cells by Compounds 7-58 is mediated by Nur77
FIGS. 3G-3J show Nur77 immunoblot analysis results and cell vacuolization phenomena in HCT116 cells transfected with different siRNAs and treated with 2. Mu.M 7-58, according to the method in example 15. The results of fig. 3G show that Nur77siRNA effectively inhibits/knockdown expression of Nur77 in cells; the results of FIGS. 3H-3I show that, in Nur 77-expressing cells, 7-58 were able to induce cell vacuolization; however, when the expression of Nur77 was knocked out, 7-58 induced cell vacuolation ability was reduced; the results in figure 3J show that the ability of 7-58 to induce endocytosis of fluorescent dextran by cells is significantly diminished after the suppression of Nur77 expression.
Example 18: preferred compounds 7-41 and 7-58 induce non-apoptotic forms of cell death in tumor cells
PARP is poly ADP ribose polymerase which plays an important role in the process of DNA damage repair, and is a basic protein which can be cut by Caspase. PARP cleavage bands are a common indicator of apoptosis. Caspase inhibitor Z-VAD-FMK can block cell death path effectively.
FIGS. 4A-4B show the results of PARP immunoblot analysis in H460 cells treated at different concentrations 7-41 for different times. The results in FIG. 4A show that 2,5. Mu.M 7-41 did not cause cleavage of cellular PARP at different times, and the results in FIG. 4B show that 0.5,1,2,5. Mu.M 7-41 did not cause cleavage of cellular PARP for 24 h.
FIG. 4C shows the results of PARP immunoblot analysis in HCT116 cells treated at different concentrations 7-58 for different times. The results in FIG. 4C show that 1,2,5. Mu.M 7-58 effect for 12h caused little cellular PARP cleavage. FIG. 4D shows cytoplasmic vacuoles produced in H460 cells pretreated for 2H with 50. Mu.M of the apoptosis inhibitor Z-VAD-FMK and the necrosis inhibitor Necrostatin-1, treated for 8H with 1. Mu.M of 7-41. The results in FIG. 4D show that 7-41 induced cellular vacuolation was unaffected even in the presence of Z-VAD-FMK or Necrostatin-1.
FIG. 4E shows inhibition of cell proliferation after 7-41 treatment alone or co-treatment with 50 μ M Z-VAD-FMK for 48H. The results in FIG. 4E show that IC of 7-41 inhibits H460 cell proliferation 50 The values are: 1.5697. + -. 0.5923. Mu.M, IC when co-processed with Z-VAD-FMK 50 The values were 1.9810. + -. 0.1255. Mu.M, two groups of ICs 50 The values were not significantly different.
Example 19: preferred compounds 7-41 and 7-58 modulate the megalocytic pathway to induce cytoplasmic vacuole
Autophagy, endoplasmic reticulum stress, and megalocytic processes all may produce vacuoles in cells. During autophagy, vesicles with a double-layer membrane structure are formed. LC3 II can be used as an indicator protein of autophagy flux.
FIG. 5A shows the results of immunoblot analysis of LC3 II/I ratios in H460 cells after 24H treatment at different concentrations of 7-41. The results in FIG. 5A show that 0.5,1,2,5, 10. Mu.M 7-41 was not responsible for up-regulation of the LC3 II/I ratio in the cells for 24 h.
FIG. 5B shows cytoplasmic vacuoles produced in H460 cells pretreated for 2H with the autophagy inhibitor 3-MA (3 mM) and LY294002 (20 μ M), treated for 8H with 1 μ M7-41. The results in FIG. 5B show that the induction of cellular vacuolation by 7-41 was unaffected even in the presence of 3-MA or LY 294002.
FIG. 5C shows inhibition of cell proliferation after 2.5. Mu.M treatment of 7-41 alone or in combination with 20. Mu.M LY294002 for 48H. The results in FIG. 5C show that the effect of 7-41 in inhibiting H460 cell proliferation was not affected by LY 294002.
FIG. 5D shows cytoplasmic vacuoles produced in H460 cells pretreated with endoplasmic reticulum stress inhibitor 4-PBA (1 mM) for 2H, treated with 1. Mu.M 7-41 for 8H. The results in FIG. 5D show that 7-41 induced cell vacuolization was unaffected even in the presence of 4-PBA.
FIG. 5E shows inhibition of cell proliferation after 2.5. Mu.M treatment of 7-41 alone or in combination with 1mM 4-PBA for 48H. The results in FIG. 5E show that 7-41 inhibited H460 cell proliferation unaffected by 4-PBA.
FIG. 6A shows cytoplasmic vacuoles produced in H460 cells pretreated with the megalocytosis inhibitor EIPA (50. Mu.M) and CQ (20. Mu.M) for 2H, treated with 1. Mu.M for 7-41H. The results in FIG. 6A show that 7-41 completely lost vacuole-inducing ability in the presence of EIPA or CQ.
FIG. 6B shows inhibition of cell proliferation after 2.5. Mu.M treatment of 7-41 alone or co-treatment with 50. Mu.M EIPA for 48H. The results in FIG. 6B show that the effect of 7-41 on the inhibition of H460 cell proliferation is attenuated by the intervention of EIPA.
FIGS. 6C-6E show that H460 cells were treated with 2. Mu.M of 7-41 or 7-58 for 12H, then with 2mg/mL of fluorescent Dextran (FITC-Dextran) for 2H, and the cells were followed by flow cytometry or confocal microscopy for the fluorescent signal generated by the macroendocytosis of Dextran by macrocytosis. The results in FIG. 6C show that the fluorescent signal released in the cells after the treatment of 7-41 was detected by flow-through enhancement; the results in FIG. 6D show that the fluorescent signal released in the cells after 7-58 treatment was significantly enhanced by flow detection; the results in FIG. 6E show that the fluorescence signal produced by endocytosis of FITC-Dextran increases after treatment with 7-41 or 7-58 as observed by confocal microscopy.
Example 20: compounds 7-41 and 7-58 induce apoptosis in cancer cells
Cytoplasmic vacuoles and the morphology of each organelle induced by compounds 7-41 and 7-58 were observed by transmission electron microscopy.
After the cells were treated with the compound, the medium was discarded, the cell surface was washed with PBS, and glutaraldehyde working solution (2.5% glutaraldehyde, PB system) was added and fixed at room temperature for 15min. After fixation, the cells were washed 3 times with PB buffer solution for 15min each time, and the cell sections were observed using a transmission electron microscope according to the electron microscope sample preparation procedure.
FIG. 7A shows the cell microstructure of 1 μ M compound 7-41 treated H460 cells 8H. FIG. 7B shows the cell microstructure after 12h treatment of HCT116 cells with 2. Mu.M of compounds 7-58. The results in FIGS. 7A-7B show that 7-41 and 7-58 induced cytoplasmic vacuoles were single-layered membrane structures with little content in the vacuoles, no mitochondrial rounding, no endoplasmic reticulum swelling, intact nuclei without shrinkage, consistent with the characteristics of megavesicle death (Methuosis).
FIG. 8 shows the results of co-localization of vacuoles to mitochondrial probes, endoplasmic reticulum probe, lysosomal probe, fluorescent yellow dye, late endosomal marker Rab7 and LAMP1 after 12H treatment of H460 cells with 2. Mu.M compound 7-41. The results in FIG. 8 show that neither the mitochondrial probe Mito-tracker nor the endoplasmic reticulum probe ER-tracker is incorporated into vacuoles, consistent with the fact that the megakaryosomes are derived from the cytoplasmic membrane; fluorescent Yellow dye (Lucifer Yellow) accumulates in vacuoles, and vacuolar membrane late endosomal markers Rab7 and LAMP1 mark, one of the hallmarks of macropinocytosis leading to cytoplasmic vacuoles; the lysosome probe Lyso-tracker can not overlap with vacuole, and also conforms to the characteristic that vacuole can not be fused with lysosome in macrovacuole type death.
Example 21: preferred compounds 7-41 induce Nur77 enucleation
The non-genotype functions of Nur77 regulated by 7-41 were analyzed by confocal microscopy and nuclear-cytoplasmic separation experiments.
(1) Immunofluorescence assay: figure 9A shows the results of immunofluorescence analysis of the Nur77 expression profiles in cells treated with compounds 7-41. The results in FIG. 9A show that Nur77 was distributed essentially intranucleally prior to 7-41 treatment, while Nur77 was distributed more extracuclearly after 1,3h in H460 cells treated with 2. Mu.M compound 7-41.
(2) Nuclear matter separation experiment: figure 9B shows the purification of nuclear components and proteomics by treatment with compounds 7-41 followed by nuclear-cytoplasmic separation experiments, respectively, and detection of Nur77 content in the nucleus and cytoplasm by immunoblotting. The results in FIG. 9B show that 2. Mu.M of compounds 7-41 increased expression in Nur77 cytoplasm after 1,3,6,8h treatment of H460 cells.
Figure 9C shows cytoplasmic vacuoles produced in H460 cells pretreated with Nur77 nuclear export inhibitor CDIM8 (20 μ M) for 2H, treated with 1 μ M7-41 for 8H. The results in FIG. 9C show that 7-41 induced a reduction in cytoplasmic vacuolation in the presence of CDIM 8.
FIGS. 9D-9E show inhibition of cell proliferation after treatment of H460 cells with 7-41 alone or with CDIM8 for 48H. The results in FIGS. 9D-9E show that the effect of 7-41 on the inhibition of H460 cell proliferation was attenuated by the intervention of CDIM 8.
Example 22: preferably, compounds 7-41 do not cause embryonic death or developmental malformations in zebrafish
FIGS. 10A-10B show the 7-41 toxicity to zebrafish embryo survival and development. The results in FIG. 10A show that 1.25, 2.5, 5. Mu.M 7-41 had little toxicity to zebrafish embryos, resulting in only about 20% of zebrafish embryo deaths at the 5. Mu.M acting concentration, with no significant difference compared to the DMSO control group; the results in FIG. 10B show that no deformity was observed in the zebrafish fish embryos 24,48,72h when 1.25, 2.5, 5. Mu.M 7-41 was applied to the zebrafish embryos.
Example 23: preferred compounds 7-41 can inhibit the growth of lung carcinoma H460 cell nude mouse xenografts
Selecting female BALB/c (nu/nu) nude mice (weight 18-20g, SPF level animal house feeding) to construct in-vitro mouse transplantation tumor model, injecting H460 cell suspension subcutaneously in right anterior axillary part of the nude mice until tumor volume reaches 100mm 3 . A control group (solvent), a low dose administration group (10 mg/kg) and a high dose administration group (25 mg/kg) were set, administered by intraperitoneal injection, once a day, and the body weight and tumor volume of nude mice were recorded for 14 days. After dosing, the nude mice were sacrificed by cervical dislocation, tumor was dissected, photographed and weighed. The results are shown in fig. 11A-11D, and the experimental results show that the compounds 7-41 can significantly inhibit the growth of H460 cell xenograft tumors, the inhibition rates are 56.83% and 88.84%, respectively, and the weight of nude mice is not significantly reduced during the administration period, and the toxicity is not obvious.
FIG. 12 shows the results of immunohistochemistry of the exfoliated tumor tissue and each organ after the H460 group mice were sacrificed. The results in fig. 12 show that the tumor tissues were sparse and the expression of the cell proliferation marker Ki67 was reduced after 7 to 41 continuous administration, and that 7 to 41 did not cause any significant toxic side effects on the mouse organs. No experimental animal deaths occurred throughout the experimental period.
Example 24: preferred compounds 7-41 inhibit the growth of breast cancer MDA-MB-231 cell xenograft tumors in nude mice
The experimental procedure was as in example 24. The right anterior axillary area of nude mice was injected subcutaneously with MDA-MB-231 cell suspension. Control group (solvent) and administration group (25 mg/kg) were set and administration was continued for 17 days. The results are shown in FIGS. 11E-11H, and the experimental results show that the compounds 7-41 can significantly inhibit the growth of MDA-MB-231 cell xenograft tumors, the inhibition rates are respectively 56.35%, and the compounds do not cause significant weight loss and obvious toxicity in nude mice during administration
Example 25: preferred compounds 7-41 can inhibit liver cancer HepG 2 Growth of cell nude mouse xenografts
The experimental procedure was as in example 24. Injecting HepG subcutaneously in the right anterior axilla of nude mice 2 A suspension of cells. Control group (solvent) and administration group (25 mg/kg) were set and administration was continued for 15 days. The results are shown in FIGS. 11I-11L, and the experimental results show that the compounds 7-41 can obviously inhibit HepG 2 The growth of the cell xenograft tumor has an inhibition rate of 60.29 percent, and does not cause the significant weight loss of the nude mice during the administration period and has no obvious toxicity.
Example 26: preferred compounds 7-58 inhibit the growth of colorectal cancer HCT-116 cell xenograft tumors in nude mice
The experimental procedure was as in example 24. Nude mice were grouped, inoculated with wild type human colon cancer cells (HCT-116/sh-ctr) and Nur 77-knocked-out human colon cancer cells (HCT-116/sh-Nur 77), respectively, and a control group (solvent) and an administration group (10 mg/kg) were set for continuous administration for 9 days. The results are shown in FIGS. 11M-11N, and the experimental results show that the compounds 7-58 can well inhibit the growth of wild type human colon cancer transplantable tumor in a mouse body, and the tumor proliferation inhibition activity depends on Nur77.
Example 27: preferred compounds 7-41 inhibit cisplatin-resistant A549 (A549/DDP) cell proliferation
(1) Real-time quantitative fluorescent PCR (RT-QPCR): total RNA of cisplatin-resistant A549 (A549/DDP) cells and non-resistant A549 (A549) cells is extracted, and RT-QPCR technology is utilized to detect the megalocytosis related genes such as: mRNA expression of RaC1, NHE1, RAB7A, CDC42, KEAP1, NFE2L, STX7, STX17, NR4A1 in two cells. The result is shown in fig. 13A, and the expression of the gene related to the megalobin drink in a549/DDP is significantly higher than that in the non-drug-resistant a549 cells.
(2) Confocal microscopy: FIG. 13B shows A549/DDP and A549 plated overnight, treated with 2mg/mL fluorescent Dextran (FITC-Dextran) for 2h, and confocal microscopy tracking the fluorescent signal of cells by macropinocytic endocytosis of Dextran. The results in fig. 13B show that under the same parameters, endocytic fluorescence produced by a549/DDP endocytic fluorescent dextran is significantly higher than the fluorescent signal in non-drug resistant a549 cells, indicating that a549/DDP cells have stronger macropotant activity compared to non-drug resistant a 549.
FIG. 13C shows cytoplasmic vacuoles generated by A549/DDP and A549 cells treated at 1. Mu.M 7-41 for 8 h. The results in FIG. 13C show that 7-41 produced a larger number of, and more voluminous, vacuoles in A549/DDP.
FIG. 13D shows inhibition of cell proliferation by Cisplatin (Cisplatin) and 7-41 after 48h treatment of A549/DDP and A549 cells. The results in FIG. 13D show that Cisplatin inhibits IC of A549/DDP and A549 cells 50 The values are respectively: 70.07 +/-2.59 and 9.79 +/-1.35 mu M, and the drug resistance index is about 7.16;7-41 IC inhibiting A549/DDP and A549 cells 50 The values are: 1.04. + -. 0.27 and 3.17. + -. 0.78. Mu.M.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, and all equivalent variations and modifications made within the scope of the present invention and the content of the description should be included in the scope of the present invention.
Claims (8)
- An N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative characterized by: the structural formula isWherein,
R 1 is 4-methyl-2- (methylamino) thiazolyl, 2, 5-dimethylthiazolyl, N-dimethylaniline, thienyl, 2-chlorothienyl, 2-chlorophenyl or 1, 3-bis (benzyloxy) phenyl;R 2 <xnotran> N- , -1- , N- , -1- , (R) -2- ( ) -1- , (S) -2- ( ) -1- ,4- -1H- -1- , N-2- ( -2- ) , -1- ,4- -1- ,3- -1- ,4- -1- ,4- -1- , N- (1r,4r) -4- ,4- ( ) -1- ,3- ( ) -1- ,4- -1- ,4- -1- ,3,5- -1- ,4- ( ) -1- ,4- ( ) -1- ,4- -2- ,4- (2- ) -1- , N- (1- -4- ), 4- ( ) -1- , </xnotran> Morpholinyl, 2-dimethylmorpholinyl, N- (tetrahydro-2H-pyran-4-yl), thiomorpholinyl, N- (3-morpholinopropyl), N- (4-hydroxyphenyl), N- (4-methoxyphenyl), N- (3-chloro-4-methoxyphenyl), methyl 2-hydroxy-4-aminobenzoate, N- (4-methoxybenzyl), N- (4-fluorobenzyl), N-phenethyl, N- (4-hydroxyphenylethyl), 1, 4-diaza-1-carboxaldehyde, azepin-4-one, 4-amino-nonane-1-carboxylic acid tert-butyl ester, 4-methyl-1, 4-diaza-1-yl, azacyclooctyl, N-cyclooctyl, N- (adamantan-1-yl), [1,4' -bipiperidine ] yl]-1' -yl, 4- (4-methylpiperazin-1-yl) piperidin-1-yl, 4-cyclopentylpiperazin-1-yl, 4- (4-methoxyphenyl) piperazin-1-yl or 4- (4-fluorophenyl) piperazin-1-yl. - 2. The N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative of claim 1 wherein: the structural formula is
- 3. The N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative of claim 2 wherein: the structural formula is
- 4. A pharmaceutical composition characterized by: comprising an N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative or a pharmaceutically acceptable salt according to any of claims 1 to 3 and a pharmaceutically acceptable carrier.
- 5. Use of an N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative according to any of claims 1 to 3 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 4 for the preparation of a composition for the treatment of cancer.
- 6. The use of claim 5, wherein: the cancer is lung cancer, breast cancer, liver cancer, colorectal cancer, ovarian cancer, melanoma, esophageal cancer, cervical cancer or renal cancer.
- 7. Use of an N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative according to any of claims 1 to 3 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 4 as a apoptosis-inducing agent.
- 8. Use of an N-substituted-5- ((4-substituted pyrimidin-2-yl) amino) -1H-indole-2-carboxamide derivative as claimed in any of claims 1 to 3 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as claimed in claim 4 as a Nur77 receptor modulator.
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| WO2011025167A2 (en) * | 2009-08-28 | 2011-03-03 | 동화약품주식회사 | Pharmaceutical composition for preventing or treating cancer comprising 2,6-substituted 3-nitropyridine derivative compound or pharmaceutically acceptable salt thereof |
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