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CN110143948B - CDK4/6 inhibitor, pharmaceutical composition, preparation method and application thereof - Google Patents

CDK4/6 inhibitor, pharmaceutical composition, preparation method and application thereof Download PDF

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CN110143948B
CN110143948B CN201910544001.5A CN201910544001A CN110143948B CN 110143948 B CN110143948 B CN 110143948B CN 201910544001 A CN201910544001 A CN 201910544001A CN 110143948 B CN110143948 B CN 110143948B
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谢军
姜春阳
李惠
张艳
李红昌
许全胜
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Shanghai Scienpharm Biotechnology Co ltd
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    • 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
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Abstract

The invention discloses a CDK4/6 inhibitor, a pharmaceutical composition, a preparation method and application thereof, and belongs to the field of medicines. The CDK4/6 inhibitor, pharmaceutically acceptable salt, hydrate and solvent thereof are subjected to condensation reaction with a corresponding active reagent in an inert solvent to obtain an intermediate of a formula (b), and deprotection is carried out under the action of acid or alkali to obtain the compound of the formula (I). In addition, the application of the CDK4/6 inhibitor shown in the formula (I), and pharmaceutically acceptable salts, hydrates, solvates, metabolic precursors or prodrugs thereof in preparing medicaments for preventing or treating tumors is also disclosed. The CDK4/6 inhibitor provided by the invention has excellent anti-CDK 4/6 activity, and can be used for preparing various antitumor medicinal preparations.
Figure DDA0002103440120000011

Description

CDK4/6 inhibitor, pharmaceutical composition, preparation method and application thereof
Technical Field
The invention relates to the field of pharmacy, and in particular relates to a CDK4/6 inhibitor, a pharmaceutical composition, a preparation method and application thereof.
Background
The occurrence of diseases such as cancer is related to the dysregulation of various oncogenes and cancer suppressor genes, and the functions and effects of almost all oncogenes and cancer suppressor genes play a role in the cell growth cycle. Thus, it can be said that cancer is a Cell Cycle Disease (CCD), and modulation or blocking of abnormal cell cycles is one of the approaches for cancer treatment.
Among the molecules that have been found to be involved in cell cycle regulation, cyclin-dependent protein kinases (Cdks), which are known for their activation by cyclins, play a key role in cell cycle regulation. There are three main types of cyclin-dependent protein kinases in eukaryotic cells.
CDKs are a group of serine (Ser)/threonine (Thr) protein kinases that act synergistically with cyclin, are important factors in cell cycle regulation, and participate in different phases of the cell cycle. In the cell cycle regulation process centered on CDKs, abnormalities in any of the links can cause cell cycle abnormalities, and may lead to the development of cancer.
CDKs have 21 subtypes which are found at present and can be combined with cyclins (cyclins) to form heterodimers, wherein CDKs are catalytic subunits, cyclins are regulatory subunits, different cyclin-CDK complexes regulate phosphorylation of different substrates through CDK activity, and further realize propulsion and transformation effects on different phases of a cell cycle. The activity of CDKs depends on the sequential expression of its positive regulatory subunit cyclin and the concentration of its negative regulatory subunit CDI (CDK inhibitor). Simultaneously, CDK activity is also regulated by phosphorylation and dephosphorylation, and oncogenes and oncosuppressors.
The functions of various isoforms of CDKs include, in addition to their effects on the cell cycle, the regulation of transcription, DNA repair, differentiation and apoptosis.
At least 9 CDKs, CDK 1-9, are present in mammals. In CDK subtype China involved in cell cycle, CDK4/6 plays a key role, cancer-related cell cycle mutations mainly exist in G1 phase and G1/S phase transformation processes, CDK4/6 is combined with CyclinD to form a kinase activity complex, bound transcription factor E2F is released through phosphorylation of cancer suppressor Rb product pRb, gene transcription related to S phase is started, and cell cycle is promoted to be transferred from G1 phase to S phase.
The specific activation of CDK4/6 is closely associated with proliferation of some tumor cells, with abnormalities in the Cyclin D-CDK4/6-INK4-Rb pathway in about 80% of human cancers. The change of the pathway leads to the accelerated progression of the G1 phase, and leads to the accelerated proliferation of tumor cells. Intervention in this pathway has become a cancer treatment strategy and CDK4/6 is a novel anti-tumor target.
CDK4/6 has two major advantages as an anti-tumor target: (1) most of the cell proliferation was left with that of CDK4/6, but CDK4/6 inhibitors did not exhibit the cytotoxicity of "pan-CDK inhibitors", such as myelosuppression and intestinal response; (2) clinical tests show that if the cyclin D level of cells is increased or P12INK4a is inactivated, the sensitivity of the cells to drugs can be increased, and the targeting of the drugs is increased to a certain extent due to the phenomenon that tumor cells have relative to normal cells.
Currently, CDK inhibitor drugs that have been approved by the FDA for marketing include: palbociclib from Pfizer is approved by FDA on 3.2.2015 and Ribociclib from Novartis is approved by FDA on 13.3.2017, and the drug is used as CDK4/6 inhibitor for treating metastatic breast cancer. In addition, pharmaceutical companies such as celebrity, Astex, Tolero, G1 and the like continuously report a series of CDK4/6 inhibitors with good selectivity, which are used for treating diseases such as bone marrow diseases, blood tumors, breast tumors, lung cancer and the like, and are in different clinical test stages at present.
Through the search of the existing patent documents, the Chinese patent application with the application number of 201710110337.1 discloses the application of a CDK4/6 inhibitor and an aromatase inhibitor in combination in preparing a medicament for treating breast cancer, wherein the CDK4/6 inhibitor has the structural formula:
Figure BDA0002103440100000021
the Chinese patent application with the application number of 201711489600.9 discloses an anti-tumor drug composition which comprises an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient is prepared from gemcitabine and a formula
Figure BDA0002103440100000022
Illustrated CDK4 ^ er6 kinase inhibitor or pharmaceutically acceptable salt thereof, and the anticancer activity of the compound is improved by a combined medication mode.
However, the CDK4/6 kinase inhibitor reported in the prior art still has the defects of low anticancer activity, high toxicity and the like.
To achieve better cancer therapeutic efficacy and better meet clinical and market demands, we expect to develop a new generation of highly potent, low toxicity CDK4/6 inhibitors, and expect to improve the selectivity and effectiveness of the therapy.
Disclosure of Invention
The invention aims to provide a CDK4/6 inhibitor, a pharmaceutically acceptable salt, a hydrate, a solvate, a metabolic precursor or a prodrug thereof, a composition, a preparation method and application thereof. The CDK4/6 inhibitor, pharmaceutically acceptable salt, hydrate, solvate, metabolic precursor or prodrug thereof provided by the invention can be used for treating and/or preventing cancers.
In order to achieve the above purpose of the present invention, the present invention adopts the following technical scheme:
in a first aspect, the present invention provides a CDK4/6 inhibitor of formula (I), a pharmaceutically acceptable salt, hydrate, solvate, metabolic precursor or prodrug thereof:
Figure BDA0002103440100000031
wherein,
R1comprises a main component selected from-C1～6Alkyl, -C3～14Cycloalkyl, -C3～14Heterocycloalkyl, aryl, -C1～6alkane-OR2、-C1～6alkane-CO2R3At least one selected from the group consisting of; r2And R3Independent representative H, C1～6Alkyl, cycloalkyl, heterocycloalkyl, or aryl; if R is1Containing carboxyl groups, then R1Can be potassium salt, calcium salt, magnesium salt, aluminum salt, sodium salt and iron salt of carboxylic acid; all radicals being unsubstituted or optionally substituted by one or more F, Cl, Br, D, OH, CN, alkoxy groupsAnd (4) substitution.
In the invention, the pharmaceutically acceptable salt of the CDK4/6 inhibitor shown in the formula (I) can comprise a pharmaceutically acceptable inorganic acid salt or organic acid salt. The inorganic acid salt is preferably sulfate, sulfite, hydrochloride, hydrobromide, nitrate, phosphate, metaphosphate, pyrophosphate or perchlorate. The organic acid salt is preferably acetate, maleate, fumarate, succinate, citrate, p-toluenesulfonate, tartrate, formate, acetate, propionate, heptanoate, oxalate, benzoate, malonate, succinate, maleate, hydroxybutyrate, citrate, methanesulfonate, benzenesulfonate, lactate or mandelate.
In the present invention, the following definitions are used:
“C1～6alkyl "means a straight or branched chain monovalent residue of 1 to 6 saturated and/or unsaturated carbon and hydrogen atoms, such as methyl (Me), ethyl (Et), propyl, isopropyl, butyl (Bu), isobutyl, tert-butyl (t-Bu), ethenyl, pentenyl, propenyl, butenyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like, which may be unsubstituted or substituted by one or more identical or different substituents selected from the group defined below.
"cycloalkyl" refers to a non-aromatic monovalent monocyclic, bicyclic, or tricyclic residue containing 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, or 14 carbon atoms, each of which may be saturated or unsaturated, and which may be unsubstituted or substituted with one or more of the same or different substituents selected from those defined herein.
"heterocycloalkyl" means a non-aromatic, monovalent monocyclic, bicyclic, or tricyclic residue containing 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, or 14 carbon atoms and including 1,2, 3, 4, 5, or 6 heteroatoms, which may or may not be the same, selected from nitrogen, oxygen, and sulfur. Each of which may be saturated or unsaturated and may be unsubstituted or substituted by one or more identical or different substituents selected from the group defined in the present invention.
"aryl" refers to phenyl and naphthyl. May be unsubstituted or substituted by one or more identical or different substituents selected from the group defined below, and may be substituted by one C3～14Cycloalkyl radical, C3～14A heterocycloalkyl or fused 5-to 6-membered aromatic heterocycle, which cycloalkyl, heterocycloalkyl or 5-6-membered aromatic heterocycle may itself be unsubstituted or substituted by one or more of the same or different substituents as defined herein.
The 5-6 membered aromatic heterocyclic ring is a five-membered or six-membered aromatic heterocyclic compound containing 1,2, 3 or 4 identical or different heteroatoms selected from nitrogen, oxygen and sulfur, and the five-membered or six-membered aromatic heterocyclic compound contains other atoms in the ring in addition to carbon atoms. May be unsubstituted or substituted by one or more identical or different substituents selected from the group defined below, and may be fused with a cycloalkyl, heterocycloalkyl, aryl or 5-6 membered aromatic heterocycle which may itself be unsubstituted or substituted by one or more identical or different substituents selected from the group defined herein.
The substituent is halogen, deuterium, carboxyl, ester group, C1～6Alkyl, alkoxy, acyl, acylamino, sulfonyl, mercapto, alkylthio, cycloalkyl, heterocycloalkyl, amino, alkylamino, dialkylamino, cyano, difluoromethyl, trifluoromethyl or C with the above groups1～4Alkyl group of (1).
Further, the CDK-4/6 inhibitor shown in the formula (I), and pharmaceutically acceptable salt, hydrate, solvate, metabolic precursor or prodrug thereof are any compound with the following number CDK 4/6-01-04:
Figure BDA0002103440100000041
Figure BDA0002103440100000051
in a second aspect, the present invention provides a method for preparing a CDK4/6 inhibitor of formula (I), a pharmaceutically acceptable salt, hydrate, solvate, metabolic precursor or prodrug thereof, comprising:
carrying out condensation reaction on a compound shown in the formula (a) and an active reagent in an inert solvent under the action of a basic reagent to obtain an intermediate shown in the formula (b), and then carrying out deprotection under the action of an acidic reagent or a basic reagent to obtain the compound shown in the formula (I):
Figure BDA0002103440100000052
wherein Pro is Boc, Ts acyl, sulfonyl protecting group.
Further, the inert solvent used in the above reaction includes, but is not limited to, N-Dimethylformamide (DMF), N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), Tetrahydrofuran (THF), dichloromethane, acetone, methyl butanone, methyl isobutyl ketone, chlorobenzene, dichlorobenzene, toluene, methanol, ethanol, preferably N, N-Dimethylformamide (DMF).
Further, the active reagents used in the above reaction include, but are not limited to, benzene sulfonate, methane sulfonate, and halogenated reagents.
Further, the above-mentioned basic reagents used include, but are not limited to, TEA, DIPEA, DBU, DMAP, dimethylamine, diethylamine, n-butylamine, aniline, sodium hydroxide, potassium hydroxide, lithium hydroxide, potassium carbonate, sodium bicarbonate, potassium bicarbonate, lithium hydroxide. Preferably DIPEA.
Further, the acidic reagent used in the above includes but is not limited to HCl, HBr, H2SO4、H2SO3、HNO3、H3PO4CHCOOH, AcOH, formic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid. HCl is preferred.
Further, the temperature of the reaction is 0-100 ℃, or 30-90 ℃, or 60-90 ℃, or 70-90 ℃.
In a third aspect, the invention provides an application of a CDK4/6 inhibitor represented by formula (I), a pharmaceutically acceptable salt, a hydrate, a solvate, a metabolic precursor or a prodrug thereof in preparing a medicament for preventing or treating tumors.
In a fourth aspect, the present invention provides a pharmaceutical composition comprising an inhibitor of CDK4/6, a pharmaceutically acceptable salt, hydrate, solvate, metabolic precursor or prodrug thereof, as defined above according to formula (I), and a pharmaceutically acceptable additive.
The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.
Unless otherwise indicated, the terms and abbreviations disclosed herein have their standard meanings.
The reagents and starting materials used in the present invention are commercially available.
Compared with the prior art, the invention has the following beneficial effects:
the CDK4/6 inhibitor, the pharmaceutically acceptable salt, the hydrate, the solvate, the metabolic precursor or the prodrug thereof have excellent activity of inhibiting CDK4/6, and are suitable for preparing various antitumor medicinal preparations.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic representation of the effect of CDK4/6 series of compounds on the volume of human gastric carcinoma MDA-MB-436 nude mouse transplants;
FIG. 2 is a graph showing the effect of CDK4/6 series compounds on relative tumor volume of human gastric carcinoma MDA-MB-436 nude mouse transplants;
FIG. 3 is a schematic diagram showing the experimental therapeutic effect of CDK4/6 series compounds on human gastric cancer MDA-MB-436 nude mouse transplantable tumor;
FIG. 4 is a CDK4/6-01 mass spectrometric detection profile;
FIG. 5 is a CDK4/6-02 mass spectrometric detection profile;
FIG. 6 is a CDK4/6-03 mass spectrometric detection profile;
FIG. 7 is a CDK4/6-04 mass spectrometric detection spectrum.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are further described in detail below with reference to examples:
example 1.3-acetyl-1-cyclopentyl 7- ((2-hydroxyethyl) (5- (piperazin-1-yl) pyridin-2-yl) amino) -4-methylquinolin-2 (1H) -one (CDK4/6-01)
Figure BDA0002103440100000071
A250 mL three-necked flask was charged with 10.0g (16.6mmol) of tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) amino) tert-butyl) pyridin-3-yl) piperazine-1-carboxylate, 80mL of N, N-dimethylformamide, 9.2g (66.6mmol) of potassium carbonate, 1.0g of potassium iodide, stirred, warmed to 50 to 60 ℃, added with 4.0g (23.9mmol) of 2-bromoethyl acetate, incubated at 90 ℃ for reaction, followed by LCMS, and if the reaction was not complete, appropriate amounts of cesium carbonate and 2-bromoethyl acetate were added until 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl acetate was obtained The tert-butyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) amino) tert-butyl) pyridin-3-yl) piperazine-1-carboxylate completely reacted, filtered, the filter cake was washed with an appropriate amount of DMF, ethyl acetate and water were added, stirring and liquid separation were performed, the aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, the organic phase was washed three times with water successively, and the organic phase was washed once with saturated saline. The organic phase was concentrated to dryness and purified by column chromatography to give 2.2g of tert-butyl 4- (6- ((2-acetoxyethyl) (6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2) yl) amino) pyridin-3-yl) piperazine-1-carboxylate.
The above tert-butyl 4- (6- ((2-acetoxyethyl) (6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2) yl) amino) pyridin-3-yl) piperazine-1-carboxylate was dissolved in 30mL of methanol, and 3.0g of concentrated hydrochloric acid was added thereto, followed by stirring and heating to 60 to 70 ℃ and LCMS follow-up reaction until the tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) amino) pyridin-3-yl) piperazine-1-carboxylate was obtained The butyl ester reacted completely, was concentrated to a large amount of solid which precipitated, filtered and dried to give 1.4g of 3-acetyl-1-cyclopentyl 7- ((2-hydroxyethyl) (5- (piperazin-1-yl) pyridin-2-yl) amino) -4-methylquinolin-2 (1H) -one in two-step yield: 17 percent.
LC-MS(m/z):490[M+H+]The mass spectrum detection spectrogram is shown in FIG. 4.
EXAMPLE 2 sodium N- (3-acetyl-1-cyclopentyl-4-methyl-2-oxo-1, 2-dihydroquinolin-7-yl) -N- (5- (piperazin-1-yl) pyridin-2-yl) glycinate (CDK4/6-02)
Figure BDA0002103440100000081
To a 250mL three-necked flask was added 6g (9.94mmol) of tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) amino) tert-butyl) pyridin-3-yl) piperazine-1-carboxylate, 4.14g (29.9mmol) of potassium carbonate, 40mL of N, N-dimethylformamide, 3.48g (20.8mmol) of ethyl bromoacetate, the temperature was raised to 60 to 90 ℃ and LCMS followed the reaction until 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [ 2), 3-d ] pyrimidin-2-yl) amino) tert-butyl) pyridine-3-yl) piperazine-1-carboxylic acid tert-butyl ester completely reacts, the temperature is reduced to room temperature, water and ethyl acetate are added, liquid separation is carried out through stirring, the water phase is extracted once by ethyl acetate, organic phases are combined, the organic phase is washed by water for three times in sequence, saturated salt is washed by water for three times, the organic phase is concentrated to be dry, purification by column chromatography gave 3.5g of tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) tert-butyl (2) ethoxy-2-oxoethyl) amino) pyridin-3-yl) piperazine-1-carboxylate.
Adding the above tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) tert-butyl (2) ethoxy-2-oxoethyl) amino) pyridin-3-yl) piperazine-1-carboxylate to 50ml of methanol, stirring, adding 5.0g of concentrated hydrochloric acid, heating to 60 to 70 ℃, and LCMS following reaction until 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) tert-butyl (2) ethoxy-2-oxoethyl) amino) After the reaction of pyridine-3-yl) piperazine-1-carboxylic acid tert-butyl ester is finished, adding NaOH aqueous solution until the reaction solution is alkaline, keeping the temperature for reaction for 1 hour, concentrating part of methanol, adding a proper amount of ethyl acetate, separating out solids, filtering, and drying in vacuum at 45 ℃ to obtain 2.2g of N- (3-acetyl-1-cyclopentyl-4-methyl-2-oxo-1, 2-dihydroquinolin-7-yl) -N- (5- (piperazin-1-yl) pyridin-2-yl) sodium glycinate, wherein the yield of the two steps is as follows: 42 percent.
LC-MS(m/z):504[M-Na++2H+]The mass spectrum detection spectrum is shown in FIG. 5.
Example 3.3-acetyl-1-cyclopentyl-7- (ethyl (5- (piperazin-1-yl) pyridin-2-yl) amino) -4-methylquinolin-2 (1H) -one (CDK4/6-03)
Figure BDA0002103440100000082
A250 ml three-necked flask was charged with 10.0g (16.6mmol) of tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) amino) tert-butyl) pyridin-3-yl) piperazine-1-carboxylate, 8.0g (57.9mmol) of potassium carbonate, 100ml of N, N-dimethylformamide, stirred, warmed to 40 to 50 ℃ and 1.5ml of iodoethane was added, the reaction was followed by LCMS, if the reaction was incomplete, iodoethane was added until 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) amino) tert-butyl) pyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester completely reacts, filtering, adding water and ethyl acetate into the filtrate, stirring, separating, extracting the water phase with ethyl acetate, mixing the organic phases, washing the organic phase with water twice, washing with saturated saline solution once, concentrating the organic phase to dryness, purification by column chromatography gave 6.5g of tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) tert-butyl (ethyl) amino) pyridin-3-yl) piperazine-1-carboxylate.
Adding the tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) tert-butyl (ethyl) amino) pyridin-3-yl) piperazine-1-carboxylate into 60ml of methanol, adding 7.5g of concentrated hydrochloric acid, stirring, heating to 60-70 ℃, and LCMS (liquid Crystal display System) tracking reaction until the tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) tert-butyl (ethyl) amino) pyridin-3-yl) piperazine-1-carboxylate The ester reaction was complete, concentrated to remove some of the methanol, ethyl acetate was added, stirred at room temperature for 1 hour, filtered and the filter cake was dried under vacuum at 45 ℃ to dryness to give 5.2g of 3-acetyl-1-cyclopentyl-7- (ethyl (5- (piperazin-1-yl) pyridin-2-yl) amino) -4-methylquinolin-2 (1H) -one in two steps: 67%.
LC-MS(m/z):474[M+H+]The mass spectrum detection spectrogram is shown in FIG. 6.
Example 4.3-acetyl-1-cyclopentyl-7- (methyl (5- (piperazin-1-yl) pyridin-2-yl) amino) -4-methylquinolin-2 (1H) -one (CDK4/6-04)
Figure BDA0002103440100000091
A250 mL three-necked flask was charged with 10.0g (16.6mmol) of tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) amino) tert-butyl) pyridin-3-yl) piperazine-1-carboxylate, 80mL of N, N-dimethylformamide and 9.2g (66.6mmol) of potassium carbonate, stirred, warmed to 40 to 50 ℃, added with 6.8g (39.5mmol) of methyl benzenesulfonate, incubated at 60 ℃, and LCMS followed until 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) amino) tert-butyl) pyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester completely reacted, filtered, the filter cake was washed with an appropriate amount of DMF, ethyl acetate and water were added, stirring and liquid separation were performed, the aqueous phase was extracted once with ethyl acetate, the organic phases were combined, and the organic phase was washed successively with water and saturated brine. The organic phase was concentrated to dryness and purified by column chromatography to give 2.9g of tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) tert-butyl (methyl) amino) pyridin-3-yl) piperazine-1-carboxylate.
The tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) tert-butyl (methyl) amino) pyridin-3-yl) piperazine-1-carboxylate was dissolved in 20mL of methanol, 55g of concentrated hydrochloric acid was added, the mixture was stirred and warmed to 60 to 70 ℃, and the reaction was followed by LCMC until tert-butyl 4- (6- ((6- (1-butoxyvinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyrido [2,3-d ] pyrimidin-2-yl) amino) pyridin-3-yl) piperazine-1-carboxylate was tert-butyl The ester reaction was complete, concentrated to a large amount of solid which precipitated, filtered and dried to give 2.0g of 3-acetyl-1-cyclopentyl-7- (methyl (5- (piperazin-1-yl) pyridin-2-yl) amino) -4-methylquinolin-2 (1H) -one in two-step yield: 26 percent.
LC-MS(m/z):460[M+H+]The mass spectrum detection spectrum is shown in FIG. 7.
Example 5 in vitro anti-tumor Activity of CDK4/6 series of Compounds
1. Experimental procedure
Inoculating a certain amount of tumor cells into a 96-well culture plate according to the cell growth rate, after 72 hours of cells acted by a test object and a positive control, removing a culture solution, adding a pre-cooled 10% trichloroacetic acid (TCA) solution into each well to fix the cells, placing the cells in a refrigerator at 4 ℃ to fix the cells for 2 hours, washing each well of the culture plate for 5 times by deionized water to remove the trichloroacetic acid solution, drying the cells in the air, adding an SRB solution (4mg/ml) prepared by 1% acetic acid into each well, standing the cells for 20 minutes at room temperature, removing the liquid in each well, washing the liquid in each well for 5 times by 1% acetic acid, washing the unbound SRB dye, drying the air, adding a proper volume of 10mM Tris-base (Tris (hydroxymethyl aminomethane) solution with the pH of 10.5 into each well to dissolve the SRB solution, oscillating the solution on a plate oscillator for 10 minutes, and measuring the.
2. Data processing
According to the OD value measured by the microplate reader, the inhibition rate is calculated according to the following formula:
inhibition (%) × 100% (1-OD administration/OD control),
if the inhibition rate is less than or equal to 0 percent, the inhibition rate is recorded as 0 percent.
IC50 was calculated, the experiment was repeated three times, the mean and standard deviation were calculated, and the data are expressed as: mean ± standard deviation.
3. Results of the experiment
The CDK4/6 series compounds have strong proliferation inhibition effect on 12 human tumor cell strains from different tissue sources, the IC50 value of 72 hours of action is different from 4.72 +/-1.42 mu M to 31.54 +/-6.70 mu M, and the anti-tumor activity of the CDK4/6 series compounds is equivalent to that of a positive control Palbociclib (detailed in Table 1).
TABLE 1 proliferation inhibitory effect of CDK4/6 series of compounds on human tumor cells
Figure BDA0002103440100000101
Figure BDA0002103440100000111
Example 6 in vivo antitumor Activity of CDK4/6 series of Compounds
1. Method of producing a composite material
Will be 1 × 107Injecting MDA-MB-436 cells into the left axilla of a nude mouse, after three generations, dissecting tumor masses of the MDA-MB-436 mice, placing the tumor masses into a glass dish containing physiological saline, peeling off surface blood vessels, cutting and removing necrotic areas, cutting the tumor masses into 3 with the diameter of 1-2mm, and inserting the tumor masses into the left axilla of the nude mouse by using a trocar.
After the tumor grows to the average volume of 80-100 mm3, randomly dividing 24 nude mice into 4 groups according to the tumor volume: solvent Control group (Control), CDK4/6-02 group (100mg/kg), CDK4/6-03 group (100mg/kg), CDK4/6-04 group (100mg/kg), the dose was adjusted to 200mg/kg on day 17 of administration, and 6 individuals were administered to each group. Each group was administered the test substance at the corresponding concentration by gavage 1 time per day at a dose capacity of 10 ml/kg.
Tumor volume was weighed and measured 2 times per week for a 22 day dosing cycle, body weight was measured on day 23, tumor volume was measured, nude mice were sacrificed and tumor masses were weighed, Relative Tumor Volume (RTV), relative tumor proliferation rate (T/C) and percent tumor Inhibition (IR) were calculated and statistically analyzed using SPSS 19.0.
2. Results
In the course of the experiment, no death of nude mice was observed in each group. At the end of the experiment, the weight average of the nude mice in each group was not significantly decreased compared to the Control group.
Compared with the tumor volume 646 + -55 mm3 of the Control group, the tumor volumes of the CDK 4/6-02100 mg/kg group, the CDK 4/6-03100 mg/kg group and the CDK 4/6-04100 mg/kg group were 397 + -80 mm3(P <0.05), 473 + -21 mm3(P <0.05) and 514 + -68 mm3 (see Table 2 and FIG. 1).
Compared with the RTV value of 7.57 +/-0.83 of the Control group, the RTVs of the CDK 4/6-02100 mg/kg group, the CDK 4/6-03100 mg/kg group and the CDK 4/6-04100 mg/kg group are respectively 4.25 +/-0.34 (P <0.01), 5.53 +/-0.49 and 5.83 +/-0.60; the T/C values were 56.23%, 73.05% and 77.01%, respectively (see Table 2 and FIG. 2).
Compared with 0.4807 +/-0.0308 g of tumor masses in a Control group, the tumor masses in a CDK 4/6-02100 mg/kg group, a CDK 4/6-03100 mg/kg group and a CDK 4/6-04100 mg/kg group respectively have 0.2231 +/-0.0568 g (P <0.01), 0.3340 +/-0.0307 g and 0.3659 +/-0.0735 g, and have the tumor inhibition rates of 53.59%, 30.52% and 23.89%. (see Table 3 and FIG. 3).
3. Conclusion
Under the experimental condition, the growth of the human gastric cancer MDA-MB-436 nude mouse transplanted tumor can be inhibited by the intragastric administration of CDK 4/6-02200 mg/kg, CDK 4/6-03200 mg/kg and CDK 4/6-04200 mg/kg (q.d., 22 days in total).
TABLE 2 CDK4/6 series of compounds affecting the body weight and tumor volume of human gastric cancer MDA-MB-436 nude mice transplanted tumors
Figure BDA0002103440100000121
Figure BDA0002103440100000122
t-test, compared to Control,.: p <0.001, x: p <0.01,: p < 0.05.
TABLE 3 CDK4/6 lineEffect of the Compounds listed on tumor weight of human gastric carcinoma MDA-MB-436 nude mice transplantable tumors
Figure BDA0002103440100000123
Figure BDA0002103440100000124
Figure BDA0002103440100000131
t-test, compared to Control,.: p <0.01,: p < 0.05.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims (2)

1. Application of a CDK4/6 inhibitor and pharmaceutically acceptable salts thereof in preparing medicines for preventing or treating colon cancer, liver cancer, breast cancer or ductal carcinoma of breast; the CDK4/6 inhibitor has the following structural formula:
Figure DEST_PATH_IMAGE001
。
2. use of a pharmaceutical composition for the manufacture of a medicament for the prevention or treatment of colon, liver, breast or ductal carcinoma, wherein said composition comprises a CDK4/6 inhibitor, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable additive; the CDK4/6 inhibitor has the following structural formula:
Figure 638374DEST_PATH_IMAGE001
。
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