CN112961159B - Aminopyrimidinopyrazole/pyrrole derivative and preparation method and application thereof - Google Patents

Aminopyrimidinopyrazole/pyrrole derivative and preparation method and application thereof Download PDF

Info

Publication number
CN112961159B
CN112961159B CN202110242165.XA CN202110242165A CN112961159B CN 112961159 B CN112961159 B CN 112961159B CN 202110242165 A CN202110242165 A CN 202110242165A CN 112961159 B CN112961159 B CN 112961159B
Authority
CN
China
Prior art keywords
compound
stereoisomer
enantiomer
pharmaceutically acceptable
acceptable salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110242165.XA
Other languages
Chinese (zh)
Other versions
CN112961159A (en
Inventor
叶庭洪
魏于全
曹丹
于艳
谢永美
欧阳亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
West China Hospital of Sichuan University
Original Assignee
West China Hospital of Sichuan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by West China Hospital of Sichuan University filed Critical West China Hospital of Sichuan University
Publication of CN112961159A publication Critical patent/CN112961159A/en
Application granted granted Critical
Publication of CN112961159B publication Critical patent/CN112961159B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Abstract

The invention relates to aminopyrimidine pyrazole/pyrrole derivatives, a preparation method and application thereof, belonging to the field of medicines. The invention provides a compound shown as a formula I, a stereoisomer thereof, a compound or a pharmaceutically acceptable salt of the stereoisomer thereof. Biological experiments prove that the compound can obviously inhibit the proliferation of various cancer cells such as breast cancer, lung cancer, gastric cancer, cholangiocarcinoma, urothelial cancer and the like, has a broad-spectrum anticancer effect, also has an inhibition effect on the proliferation of fibroblasts and hepatic stellate cells, can inhibit the growth of tumors in vivo, and provides a new choice for the development of anti-tumor, anti-lung and anti-hepatic fibrosis medicines.
Figure DDA0002962590890000011

Description

Aminopyrimidinopyrazole/pyrrole derivative and preparation method and application thereof
Technical Field
The invention relates to aminopyrimidine pyrazole/pyrrole derivatives, a preparation method and application thereof, belonging to the field of medicines.
Background
Receptor tyrosine kinase RTKs are a class of cell surface transmembrane protein receptors with endogenous protein tyrosine kinase activity, generally consisting of an extracellular domain that can bind to a specific ligand, a transmembrane domain, and an intracellular kinase domain that can selectively bind to and phosphorylate a substrate. Binding of ligands to the extracellular domains of RTKs causes structural changes thereof resulting in enzymatic activity. It plays an important role in regulating and controlling tumor angiogenesis, tumor cell survival, proliferation, differentiation, migration and the like. More than 50 different members of the RTKs family have been found, including mainly Fibroblast Growth Factor Receptors (FGFRs), Epidermal Growth Factor Receptors (EGFRs), platelet-derived growth factor receptors (frgs), vascular endothelial growth factor receptors (vegfs), and Hepatocyte Growth Factor Receptors (HGFRs).
Fibroblast Growth Factor Receptors (FGFRs) are receptor tyrosine kinases located on cell membranes. FGF/FGFR signaling pathways are abnormally activated in various cancers such as gastric cancer, breast cancer, lung cancer, bile duct cancer, urothelial cancer, prostate cancer, and the like. FGFR signaling pathways are involved in signal transduction processes that regulate cell proliferation, differentiation, migration, survival, embryonic development, angiogenesis, and organogenesis. FGFR signaling pathways are frequently altered in cancer, and preclinical models demonstrate that abnormalities in FGFR signaling pathways have the potential to contribute to cancer development. FGFR is located upstream of cellular signaling pathways and signals by activation of downstream signaling pathways by adaptor proteins or direct binding to transcription factors. FGFR activation mutations or overexpression can continuously over-activate the FGFR signaling pathway and are closely related to cancer progression.
In recent years, FGFR has been closely related to organ fibrosis, for example, FGF is found to be highly expressed in fibrotic lung tissues, serum and alveolar perfusion wash and to be positively correlated with the degree of pulmonary fibrosis. FGF-2 is derived from macrophages, fibroblasts, endothelial cells and the like of alveoli, and can promote synthesis and accumulation of collagen. But elevated FGF-2 levels, increased FGFR-1 expression in epithelial, endothelial and smooth muscle/fibroblast-like cells, and increased FGFR-2 expression in mesenchymal cells were detected in idiopathic pulmonary fibrosis IPF patients, and studies have shown that FGF-2 is significantly overexpressed in diabetic nephropathy and stimulates mesangial cell proliferation; promoting tubular cell regeneration results in cell fibrosis. The FGFR-targeted inhibitor drug can inhibit abnormal activation of FGF/FGFR signaling pathway, has the potential of treating the diseases, and becomes one of hot spots of drug research in recent years.
Disclosure of Invention
The invention aims to provide aminopyrimidine pyrazole/pyrrole derivatives, and a preparation method and application thereof.
The invention provides a compound shown as a formula I, a stereoisomer thereof, a compound or a pharmaceutically acceptable salt of the stereoisomer thereof:
Figure BDA0002962590870000021
wherein R is1Is Ar or
Figure BDA0002962590870000022
Ar is a substituted or unsubstituted aryl group;
R2is-H, or R2Forming an amide with the 1-position nitrogen atom;
x is CH or N.
Further, R1Is composed of
Figure BDA0002962590870000023
Ar is a substituted or unsubstituted aryl group.
Further, Ar is substituted or unsubstituted 5-to 10-membered aryl.
Further, the aryl group satisfies at least one of:
the aryl is a 5-6-membered monocyclic ring or a 9-10-membered fused ring;
the aryl group contains 0-2 heteroatoms;
preferably, the heteroatom is a nitrogen atom;
preferably, said aryl group is selected from:
Figure BDA0002962590870000024
further preferably, said aryl is
Figure BDA0002962590870000025
Further, the substituted aryl group contains at least one substituent selected from the group consisting of: substituted or unsubstituted alkoxy, halogen, substituted or unsubstituted amino, substituted or unsubstituted alkyl.
Preferably, the substituted aryl group contains at least one substituent selected from the group consisting of: unsubstituted C1-C6 alkoxy, halogen, unsubstituted amino, C1-C6 alkyl substituted amino, unsubstituted C1-C6 alkyl.
Preferably, the substituted aryl group contains at least one substituent selected from the group consisting of: -OCH3、-Cl、-NH2。
Further, Ar is selected from:
Figure BDA0002962590870000031
preferably, Ar is
Figure BDA0002962590870000032
Further, R2is-H or
Figure BDA0002962590870000033
R3Selected from substituted or unsubstituted alkyl groups, substituted or unsubstituted alkenyl groups.
Preferably, R3Is selected from substituted or unsubstituted C1-C6 alkyl and substituted or unsubstituted C2-C6 alkenyl.
Preferably, R3Selected from unsubstituted C1-C3 alkyl, substituted or unsubstituted C2-C4 alkenyl.
Preferably, the substituted C2 to C4 alkenyl group contains at least one substituent selected from the group consisting of: -CN, C3-C6 cycloalkyl.
Preferably, R2Selected from: -H,
Figure BDA0002962590870000034
Further preferably, R2Is composed of
Figure BDA0002962590870000035
Further, stereoisomers of the compounds have the structure shown in formula Ia or formula Ib:
Figure BDA0002962590870000036
preferably, the structure of the stereoisomer of the compound is shown as the formula Ia.
Further, the compound or stereoisomer thereof is selected from:
Figure BDA0002962590870000041
further, the pharmaceutically acceptable salt of the compound or the stereoisomer thereof is hydrochloride.
The invention provides a preparation method of a stereoisomer of the compound, which comprises the following steps:
a. carrying out Mitsunobu reaction on the compound 1 and the compound 2 or an enantiomer thereof to obtain an intermediate 1 or an enantiomer thereof:
Figure BDA0002962590870000042
wherein Ha is halogen and LG is a leaving group;
preferably, Ha is-I;
preferably, LG is-Boc;
b. coupling the intermediate 1 or the enantiomer thereof with a compound 3 under the action of a palladium catalyst to obtain an intermediate 2 or an enantiomer thereof:
Figure BDA0002962590870000051
wherein R is5、R6Independently selected from H or alkyl, or, R5And R6Are connected to form an alicyclic ring;
preferably, compound 3 is
Figure BDA0002962590870000052
c. Removing the leaving group from the intermediate 2 or the enantiomer thereof to obtain a compound 4 or an enantiomer thereof:
Figure BDA0002962590870000053
further, the preparation method meets at least one of the following conditions:
compound 1 in step a: compound 2 or an enantiomer thereof in a molar ratio of 1: (1.0-1.5);
the reaction solvent in the step a is tetrahydrofuran;
b, adding diisopropyl azodicarboxylate and triphenylphosphine into the reaction system in the step a;
preferably, compound 1: the molar ratio of diisopropyl azodicarboxylate is 1: (2.0-3.0);
preferably, compound 1: the molar ratio of triphenylphosphine was 1: (2.0-3.0);
step a, reacting under a protective atmosphere;
the reaction temperature of the step a is 0-50 ℃;
intermediate 1 or its enantiomer in step b: the molar ratio of compound 3 is 1: (1.0-1.5);
the reaction solvent in the step b is a mixed solvent of 1, 4-dioxane and water, wherein the ratio of 1, 4-dioxane: the volume ratio of water is (4-8): 1;
based on the molar amount of the intermediate 1 or the enantiomer thereof, the using amount of the palladium catalyst in the step b is 3-10 mmol%;
the palladium catalyst in the step b is at least one selected from palladium acetate, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex and tris (dibenzylidene indeneacetone) dipalladium;
b, adding alkali into the reaction system of the step b;
preferably, in step b intermediate 1 or an enantiomer thereof: the molar ratio of the alkali is 1: (2.0-3.0);
preferably, the base in step b is selected from at least one of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium phosphate, cesium carbonate;
step b, reacting under a protective atmosphere;
the reaction temperature of the step b is 90-110 ℃.
Further, the preparation method also comprises the following steps: and carrying out amidation reaction on the compound 4 or the enantiomer thereof and the compound 5 to obtain a compound 6 or an enantiomer thereof:
Figure BDA0002962590870000061
wherein Halo is halogen; preferably, Halo is — Cl.
The invention provides application of the compound, the stereoisomer thereof, the compound or the stereoisomer thereof in preparing a medicament for preventing and treating cancer.
Preferably, the cancer is gastric cancer, breast cancer, lung cancer, bile duct cancer, urothelial cancer or prostate cancer.
The invention provides application of the compound, the stereoisomer thereof, the compound or the stereoisomer thereof in preparing a medicament for preventing and treating organ fibrosis.
Preferably, the organ fibrosis is liver fibrosis or lung fibrosis.
The invention provides application of the compound, the stereoisomer thereof, the compound or the stereoisomer thereof in preparing FGFR and/or VEGFR inhibitor medicaments in pharmacy.
Preferably, the FGFR inhibitor is an FGFR1, FGFR2, FGFR3 and/or FGFR4 inhibitor.
Preferably, the VEGFR inhibitor is a Flt1, Flt4, and/or KDR inhibitor.
The invention provides a pharmaceutical composition, which is a preparation prepared by taking the compound, the stereoisomer thereof, the pharmaceutically acceptable salt of the compound or the stereoisomer thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
Preferably, the formulation is an oral formulation.
Definition of terms:
the compounds and derivatives provided in the present invention may be named according to the IUPAC (international union of pure and applied chemistry) or CAS (chemical abstracts service, Columbus, OH) naming system.
The term "stereoisomer" includes cis-trans isomers, optical isomers.
The term "aryl" refers to a group of a 4n +2 aromatic ring system with or without heteroatoms in the aromatic ring system, wherein the heteroatoms are selected from nitrogen, oxygen and/or sulfur.
The term "amide" refers to a structure formed by replacing a hydroxyl group on a carboxyl group with an amino group or a substituted amino group.
The term "alkyl" is a radical of a straight or branched chain saturated hydrocarbon group. Examples of C1-C6 alkyl include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), t-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), t-pentyl (C5), and n-hexyl (C6).
The term "alkoxy" refers to the group-OR, wherein R is alkyl as defined above. Examples of C1-C6 alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1, 2-dimethylbutoxy.
The term "alkenyl" refers to a straight or branched chain hydrocarbon group containing at least one double bond. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, pent-3-enyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl.
The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group with or without heteroatoms, wherein the heteroatoms are selected from sulfur, oxygen, phosphorus, and/or nitrogen.
The term "alicyclic" refers to a saturated or partially unsaturated cyclic hydrocarbon group.
The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.
The term "pharmaceutically acceptable salts" refers to acid and/or base salts of the compounds of the present invention with inorganic and/or organic acids and bases, and also includes zwitterionic salts (inner salts), and also includes quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound may be obtained by appropriately (e.g., equivalent) mixing the above compound with a certain amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization. The salt in the invention can be hydrochloride, sulfate, citrate, benzene sulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.
The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or solubilizers, for example, starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.
In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.
The pharmaceutically acceptable auxiliary material of the invention refers to a substance contained in a dosage form except for an active ingredient.
The pharmaceutically acceptable auxiliary components have certain physiological activity, but the addition of the components does not change the dominant position of the pharmaceutical composition in the disease treatment process, but only plays auxiliary effects, and the auxiliary effects are only the utilization of the known activity of the components and are auxiliary treatment modes which are commonly used in the field of medicine. If the auxiliary components are used in combination with the pharmaceutical composition of the present invention, the protection scope of the present invention should still be included.
The invention provides an aminopyrimidine pyrazole/pyrrole compound with a novel structure, which has an inhibition effect on multiple tyrosine kinases such as FGFR, VEGFR and the like and is a potential multi-target antitumor drug. Biological experiments prove that the compound can obviously inhibit the proliferation of various cancer cells such as breast cancer, lung cancer, gastric cancer, cholangiocarcinoma, urothelial cancer and the like, has a broad-spectrum anticancer effect, also has an inhibition effect on the proliferation of fibroblasts and hepatic stellate cells, can inhibit the growth of tumors in vivo, and provides a new choice for the development of anti-tumor and anti-fibrosis drugs.
Drawings
FIG. 1 is a graph showing the growth of tumors in nude mice in a biological experiment;
FIG. 2 is a graph of tumor measurements in a biological experiment;
FIG. 3 is a graph of tumor weight statistics in biological experiments;
FIG. 4 is a graph showing the body weight change of nude mice in a biological experiment.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
EXAMPLE 1 preparation of the Compounds of the invention
Figure BDA0002962590870000091
Starting Compound 1(1.08g,8mmol) was dissolved in DMF (20mL), N-iodosuccinimide (4.5g,20mmol) was added, reacted at 80 ℃ for 5h and monitored by TLC. After the reaction is finished, quenching the reaction by using saturated aqueous solution of sodium hydrosulfite, pouring the reaction solution into water, precipitating yellow solid, filtering the solid and drying to obtain an intermediate 2 (white solid, 98%), MS m/z (ESI): 261.9[ M + H]+。
Figure BDA0002962590870000101
Intermediate 2(2.09g,8mmol) was dissolved in tetrahydrofuran 150mL of the reaction mixture was added with Compound 3(1.8g,9.6mmol), triphenylphosphine (4.2g,16mmol), and N2Protection, stirring at 0 ℃, then adding diisopropyl azodicarboxylate (3.1mL,16mmol) dropwise, after finishing adding dropwise, turning to room temperature after half an hour, reacting for 3h, and monitoring by TLC. After the reaction, the reaction solution was concentrated, washed with water 3 times, and the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography to obtain intermediate 4 (pale yellow solid, 70%), MS m/z (esi): 431.1[ M + H]+。
Figure BDA0002962590870000102
Compound 5(2.67g,8mmol), vinyl boronic acid pinacol ester compound 6(1.6mL,9.6mmol), DIEA (2.4mL,14.4mmol) were added to dry toluene (80mL), followed by addition of Pd2(dba)3(1 mmol%), tri-tert-butylphosphine tetrafluoroborate (1 mmol%), heating to 95 ℃ under the protection of nitrogen, reacting for 8h, and monitoring by TLC. After the reaction, the solvent was concentrated under reduced pressure, the residue was dissolved in ethyl acetate and then filtered through celite, the filtrate was washed three times with saturated NaCl solution, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and column chromatography (PE: EA ═ 4:1) was performed to obtain intermediate 7 (pale yellow oily, 53%), MS m/z (esi): 344.1[ M + H]+。
Figure BDA0002962590870000103
Intermediate 4(431mg,1mmol), intermediate 7(431mg,1.2mmol), Pd (dppf) Cl2(5 mmol%), Potassium carbonate (278mg,2mmol) and N in 25mL of a mixed solvent of 1, 4-dioxane and water (4:1)2After the displacement, the reaction was carried out at 100 ℃ for 10 hours and monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, filtered through celite, and the filtrate was concentrated and subjected to column chromatography (PE: EA ═ 2:1) to give compound 8 (pale yellow solid, 70%). MS m/z (ESI): 544.1[ M + H]+。
Figure BDA0002962590870000111
Dissolving compound 8(544mg,1mmol) in methanol, adding hydrochloric acid, stirring at room temperature for 6h to precipitate, concentrating to obtain solid, adding ultra-dry dichloromethane and triethylamine (694 μ L,5mmol), dropwise adding acryloyl chloride (152 μ L,2mmol) under ice bath condition, reacting at room temperature for 30min after dropwise adding, and monitoring by TLC. After the reaction is completed, the reaction solution is diluted by dichloromethane, washed by water for 3 times, an organic phase is dried by anhydrous sodium sulfate and then concentrated under reduced pressure, and thin layer chromatography is carried out to obtain a product, namely, a compound 9-1 (white solid, 50 percent), MS m/z (ESI): 489.1[ M + H]+。
The following compounds can be prepared in a similar manner:
TABLE 1 structural identification data for partial compounds of the invention
Figure BDA0002962590870000112
Figure BDA0002962590870000121
Figure BDA0002962590870000131
The beneficial effects of the invention are proved by biological experiments.
First, experimental instrument and material
Superclean bench BHC-1000 IIA/B3: sujing anti biotechnology; thermostatic water bath box polytscience 9505: polyscience, Inc.; sterilizing pot MLS-3780: SANYO corporation; oven: binder corporation; ultrapure water meter Milli-Q Integral 10: millipore Corp; microplate reader Multiscan MK3, cell incubator, low speed centrifuge Sorvall ST 1: thermofoisher company; centrifuge 5415C ultracentrifuge: eppendorf, Germany; NUAIRE NU-425-: nuaire, USA; BCD-215YD type general refrigerator: haier corporation, China; SANYO (-80 ℃) ultra-low temperature refrigerator: the japan ocean electrical group; rock 51702 table: cole Parmer, USA; 96-well cell culture plates: costa Corning corporation; ordinary optical microscopes: olympus corporation; a liquid transferring gun: thermo corporation; a pH meter: coring Corning Corp; an autoclave: SANYO corporation.
Cell lines used in the experiments were purchased from ATCC, USA. Various requirements for cell culture were purchased from GibcoBRL, including DMEM medium, RPMI 1640 medium, Fetal Bovine Serum (FBS) and pancreatin. Tetramethyltetrazolium blue (MTT), dimethyl sulfoxide (DMSO) was purchased from Sigma, USA.
Second, Experimental methods
1. Kinase assay
The compounds of the invention were diluted with DMSO to 50-fold the final desired maximum inhibitor concentration in the reaction. 100 μ L of compound dilutions were transferred to 96-well plates. Two blank wells of the same 96-well plate were loaded with 100. mu.L DMSO. The 96-well plate served as the source plate. 10 μ L of compound was transferred from the source plate to a 96-well plate as an intermediate plate. To each well of the intermediate plate 90. mu.L 1x kinase buffer was added. The compound in the middle plate was mixed for 10 minutes on a shaker. Transfer 5. mu.L of each well of the 96-well intermediate plate to a 384-well plate, and set the sub-well. The kinase was added to 1x kinase base buffer. FAM-labeled peptide and ATP were added to 1x kinase base buffer. Assay plates already contained 5 μ L of compound 10% DMSO solution. To each well of a 384 well assay plate was added 10 μ L of an L2.5x enzyme solution. Incubate at room temperature for 10 minutes. To each well of a 384 well assay plate was added 10 μ L of a 2.5x peptide solution. After incubation at 28 ℃ for a specified time, 25. mu.L of stop buffer was added to stop the reaction. Collecting data on the Caliper, and converting the data into IC50。
2. Cell culture
The tumor cells of the frozen preserved seeds are taken out from the liquid nitrogen, quickly placed in a thermostatic water bath at 37 ℃ for rewarming and thawing, and washed 1 time by using the culture medium under the aseptic condition. Then inoculating the whole culture medium into a culture flask at 37 deg.C and 5% CO2Culturing in an incubator, and replacing fresh cell culture solution the next day. Passage of suspension-grown cells: after the cells are cultured for 2-3 days, taking out the culture bottle from the incubator, and collecting the cellsAnd (3) centrifuging the suspension in a centrifuge tube at 1500rpm/min for 3min, pouring out supernatant, resuspending the cell precipitate with complete culture medium, uniformly blowing, and then separating into 3-5 bottles for culture. Generally, carrying out passage for 1 time in 3-4 days; passage of adherent growing cells: the cells grow to the wall of about 80% of the bottom of the bottle, the culture bottle is taken out of the incubator, the culture medium is sucked off, the cells are washed for 1 time by 0.25% of pancreatin, then 0.25% of pancreatin digestive juice is added for digestion, after the cells are observed to shrink and become round, the complete culture medium is added to stop digestion, the cells are blown to disperse and fall off, the cell suspension is collected, the cell suspension is centrifuged for 3min at 1500rpm/min, the supernatant is poured off, the cell sediment is resuspended by the complete culture medium and blown uniformly, and then the cells are cultured in 3-5 bottles. Passage is generally carried out for 1 time in 3-4 days.
3. Cell proliferation inhibition assay (MTT method)
Cells in logarithmic growth phase were collected at 2.5X 10 per well3～1×104The number of cells was seeded in 96-well plates at 37 ℃ with 5% CO2Was cultured overnight for 24 hours in a cell culture chamber, and the test drugs were diluted with DMEM medium and added to 96-well plates in 8-gradient each containing 3 duplicate wells. Adding 100 μ L of culture medium solution of the compound into each well according to gradient (final concentration is 1000, 333, 127, 42.3, 14.1, 4.7, 1.56, 0.53nM respectively), and setting 3 multiple wells for each concentration; adding 100 microliter of blank culture medium containing 1 thousandth of DMSO into each hole of the negative control group, and repeating 6 holes; blank control group only 100. mu.L of medium was added to each well. The plates were placed at 37 ℃ in 5% CO2Culturing the cells in a cell culture incubator for 72 hours. After the drug-treated group, the stealth control group, and the blank group were incubated for 2 to 4 hours with 20. mu.L of MTT solution (5mg/mL) per well, after formazan was formed, the incubation was terminated, and after the supernatant was decanted, 150. mu.L of DMSO (50. mu.L of 20% SDS solution was directly added to suspension cells) per well was shaken on a shaker for 15 to 20 minutes. The absorbance (OD) of the cells per well was measured at a wavelength of 570nm using a microplate reader570) And taking the average value to record the result. Cell proliferation inhibition rate (control group OD)570Experimental group OD570) /(control group OD)570Blank OD570) X 100%. Finally, half maximal inhibitory concentrations were fitted using Graphpad Prism software.
4. In vivo experiments in animals
SNU16 cells in a good logarithmic growth phase were sampled at 5X 105cells/100. mu.l/mouse were inoculated subcutaneously into Balb/C nude mice, and tumors grew to 250cm approximately 10 days after inoculation2On the left and right, random grouping was started with 7 pieces per group.
1) The administration route is as follows: the oral administration is carried out by taking DMSO as a solvent, and PEG 40045% physiological saline as 50%.
2) Grouping: solvent group, AZD4547 group (15mg/kg), Compound 9-15 Low dose group (15mg/kg), Compound 9-15 high dose group (30mg/kg)
3) The administration time is as follows: the tumor was about 250cm2The administration was performed orally after divided into groups, and the administration was continued for 10 days.
4) Evaluation indexes are as follows: after 10 days of administration, nude mice were examined for weight change, tumor growth curve, and tumor size, and compared.
Third, experimental results
1. The table below lists the inhibition rates of FGFR1 kinase by some of the synthetic compounds of the invention.
Table 2 inhibitory rate of FGFR1 kinase by partial compound synthesized according to the present invention
Figure BDA0002962590870000151
Figure BDA0002962590870000161
As can be seen from Table 2, the compound with aminopyrimidinopyrazole/pyrrole skeleton provided by the invention has obvious inhibition effect on FGFR1 kinase.
On this basis, the preferred compounds with more than 80% inhibition of FGFR1 under low concentration conditions (0.1. mu.M), namely 9-1 and 9-15, were selected and further tested for IC's for inhibition of FGFR150Values, and inhibitory effect on FGFR2-4 and other tyrosine kinases. The results are as follows:
table 3 IC inhibition of FGFR1 by preferred compounds50Value of
Figure BDA0002962590870000162
TABLE 4 inhibition of other tyrosine kinases (IC) by preferred compounds50，nM)
Figure BDA0002962590870000163
Figure BDA0002962590870000171
As can be seen from tables 3 and 4, the compounds of the invention have inhibitory effects on a plurality of tyrosine kinases such as FGFR and VEGFR (including Flt1, Flt4 and KDR subtypes), and the kinases play important regulation and control roles in the processes of tumor cell survival, proliferation, differentiation, migration, tumor angiogenesis and the like, thereby indicating that the compounds of the invention are potential multi-target antitumor drugs.
2. The following table shows the inhibition of the proliferation of the gastric cancer cell line SNU16 by some of the compounds of the present invention.
TABLE 5 IC inhibition of proliferation of gastric cancer cell lines by partial compounds of the invention50Value or inhibition rate
Figure BDA0002962590870000172
Figure BDA0002962590870000181
Note: AZD4547 is a known potent FGFR inhibitor.
3. The following table lists the inhibition of proliferation of 4T1 murine breast cancer cells, A549 human non-small cell lung cancer cells by some compounds of the invention (results are expressed as IC)50Value, μ M; or the inhibition rate at a compound concentration of 10. mu.M).
TABLE 6 inhibition of growth of various tumor cells by a portion of the compounds of the invention
Compound number 4T1 A549
9-1 <3.3 <3.3
9-3 8.2 53％
9-7 40％ 45％
9-15 <3.3 <3.3
4. The following table lists the inhibition of proliferation of mouse embryonic fibroblasts NIH-3T3 and human hepatic stellate cells LX2 by a portion of the compounds of the invention.
TABLE 7 IC inhibition of fibroblast proliferation by a portion of the compounds of the invention50Value or inhibition rate
Figure BDA0002962590870000182
5. Results of in vivo animal experiments
The tumor growth curve is shown in FIG. 1, the tumor measurement is shown in FIG. 2, the tumor weight statistics are shown in FIG. 3, and the body weight change curve of nude mice is shown in FIG. 4. It can be seen that the low dose and the high dose of the compounds 9-15 of the present invention can significantly inhibit the growth of tumors and even eliminate tumors. Moreover, under the same dose (15mg/kg), the effect of the compound 9-15 in inhibiting the growth of tumors is obviously better than that of a positive control medicament AZD 4547.
It should be appreciated that the particular features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in any one or more embodiments. Furthermore, the various embodiments and features of the various embodiments described in this specification can be combined and combined by one skilled in the art without contradiction.

Claims (20)

1. A compound represented by formula I, a stereoisomer thereof, a compound or a pharmaceutically acceptable salt of the stereoisomer thereof:
Figure FDA0003636748990000011
wherein R is1Is composed of
Figure FDA0003636748990000012
R2is-H or
Figure FDA0003636748990000013
R3Selected from substituted or unsubstituted C2-C4 alkenyl; the substituted C2-C4 alkenyl group contains at least one substituent selected from the group consisting of: -CN, C3-C6 cycloalkyl;
x is CH or N.
2. The compound, its stereoisomer, compound, or a pharmaceutically acceptable salt of its stereoisomer of claim 1, which is characterized by: r2Selected from: -H,
Figure FDA0003636748990000014
3. The compound, its stereoisomer, compound, or a pharmaceutically acceptable salt of its stereoisomer of claim 2, which is characterized by: r2Is composed of
Figure FDA0003636748990000015
4. The compound, its stereoisomer, compound, or a pharmaceutically acceptable salt of its stereoisomer of claim 1, which is characterized by: the structure is shown as formula Ia or formula Ib:
Figure FDA0003636748990000016
5. the compound, a stereoisomer thereof, a compound or a pharmaceutically acceptable salt thereof, according to claim 4, wherein: the structure is shown as formula Ia.
6. The compound, its stereoisomer, compound, or a pharmaceutically acceptable salt of its stereoisomer of claim 1, which is characterized by: selected from:
Figure FDA0003636748990000021
7. the compound, its stereoisomer, compound, or a pharmaceutically acceptable salt of its stereoisomer of claim 1, which is characterized by: the salt is a hydrochloride salt.
8. A compound, a stereoisomer thereof, a compound, or a pharmaceutically acceptable salt of the stereoisomer thereof, as shown below:
Figure FDA0003636748990000031
9. a process for the preparation of stereoisomers of compounds according to any of claims 1 to 7, characterized in that: the method comprises the following steps:
a. carrying out Mitsunobu reaction on the compound 1 and the compound 2 or an enantiomer thereof to obtain an intermediate 1 or an enantiomer thereof:
Figure FDA0003636748990000032
wherein Ha is halogen and LG is a leaving group;
b. coupling the intermediate 1 or the enantiomer thereof with a compound 3 under the action of a palladium catalyst to obtain an intermediate 2 or an enantiomer thereof:
Figure FDA0003636748990000033
wherein R is5、R6Independently selected from H or alkyl, or, compound 3 is
Figure FDA0003636748990000034
c. Removing the leaving group from the intermediate 2 or the enantiomer thereof to obtain a compound 4 or an enantiomer thereof:
Figure FDA0003636748990000041
10. the method of claim 9, wherein: at least one of the following is satisfied:
compound 1 in step a: compound 2 or an enantiomer thereof in a molar ratio of 1: (1.0-1.5);
the reaction solvent in the step a is tetrahydrofuran;
b, adding diisopropyl azodicarboxylate and triphenylphosphine into the reaction system in the step a; compound 1 in step a: the molar ratio of diisopropyl azodicarboxylate is 1: (2.0-3.0); compound 1 in step a: the molar ratio of triphenylphosphine was 1: (2.0-3.0);
step a, reacting under a protective atmosphere;
the reaction temperature of the step a is 0-50 ℃;
intermediate 1 or its enantiomer in step b: the molar ratio of compound 3 is 1: (1.0-1.5);
the reaction solvent in the step b is a mixed solvent of 1, 4-dioxane and water, wherein the ratio of 1, 4-dioxane: the volume ratio of water is (4-8): 1;
based on the molar amount of the intermediate 1 or the enantiomer thereof, the using amount of the palladium catalyst in the step b is 3-10 mmol%;
the palladium catalyst in the step b is at least one selected from palladium acetate, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex and tris (dibenzylidene indeneacetone) dipalladium;
b, adding alkali into the reaction system of the step b; intermediate 1 or its enantiomer in step b: the molar ratio of the alkali is 1: (2.0-3.0); the alkali in the step b is at least one selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium phosphate and cesium carbonate;
step b, reacting under a protective atmosphere;
the reaction temperature of the step b is 90-110 ℃.
11. The method of claim 9, wherein: step a, Ha is-I; LG is-Boc.
12. The method according to any one of claims 9 to 11, wherein: also comprises the following steps: and carrying out amidation reaction on the compound 4 or the enantiomer thereof and the compound 5 to obtain a compound 6 or an enantiomer thereof:
Figure FDA0003636748990000051
wherein Halo is halogen.
13. The method of claim 12, wherein: halo is-Cl.
14. Use of a compound, a stereoisomer thereof, a compound or a stereoisomer thereof according to any one of claims 1 to 8, in the manufacture of a medicament for the prevention or treatment of organ fibrosis.
15. Use according to claim 14, characterized in that: the organ fibrosis is liver fibrosis or pulmonary fibrosis.
16. Use of the compound, the stereoisomer thereof, the compound or the stereoisomer thereof according to any one of claims 1 to 8 in the preparation of FGFR and/or VEGFR inhibitor drugs.
17. The use according to claim 16, characterized in that: the FGFR inhibitor is an FGFR1, FGFR2, FGFR3 and/or FGFR4 inhibitor.
18. The use according to claim 16, characterized in that: the VEGFR inhibitors are Flt1, Flt4 and/or KDR inhibitors.
19. A pharmaceutical composition characterized by: the compound, the stereoisomer thereof, the compound or the pharmaceutically acceptable salt of the stereoisomer thereof as claimed in any one of claims 1 to 8 is used as an active ingredient, and pharmaceutically acceptable auxiliary materials are added to prepare the preparation.
20. The pharmaceutical composition of claim 19, wherein: the preparation is an oral preparation.
CN202110242165.XA 2020-03-05 2021-03-04 Aminopyrimidinopyrazole/pyrrole derivative and preparation method and application thereof Active CN112961159B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010147369 2020-03-05
CN2020101473690 2020-03-05

Publications (2)

Publication Number Publication Date
CN112961159A CN112961159A (en) 2021-06-15
CN112961159B true CN112961159B (en) 2022-07-01

Family

ID=76276770

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110242165.XA Active CN112961159B (en) 2020-03-05 2021-03-04 Aminopyrimidinopyrazole/pyrrole derivative and preparation method and application thereof

Country Status (2)

Country Link
CN (1) CN112961159B (en)
WO (1) WO2021175316A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230295167A1 (en) * 2020-10-05 2023-09-21 Dana-Farber Cancer Institute, Inc. Potent and selective inhibitors of her2

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010085824A (en) * 1998-09-18 2001-09-07 스타르크, 카르크 Pyrrolopyrimidines as protein kinase inhibitors
FR2868422B1 (en) * 2004-03-31 2006-07-14 Aventis Pharma Sa NOVEL PYRROLO (2,3-B) PYRIDINE DERIVATIVES, THEIR PREPARATION AND THEIR PHARMACEUTICAL USE AS INHIBITORS OF KINASES
US8426428B2 (en) * 2008-12-05 2013-04-23 Principia Biopharma, Inc. EGFR kinase knockdown via electrophilically enhanced inhibitors
JP2012526810A (en) * 2009-05-13 2012-11-01 イントラ−セルラー・セラピーズ・インコーポレイテッド Organic compounds
US8377946B1 (en) * 2011-12-30 2013-02-19 Pharmacyclics, Inc. Pyrazolo[3,4-d]pyrimidine and pyrrolo[2,3-d]pyrimidine compounds as kinase inhibitors
CN103183674B (en) * 2011-12-31 2017-09-12 江苏先声药业有限公司 One class fused heterocyclic derivative and its application
WO2013118817A1 (en) * 2012-02-07 2013-08-15 大鵬薬品工業株式会社 Quinolyl pyrrolopyrimidine compound or salt thereof
TN2016000094A1 (en) * 2013-09-30 2017-07-05 Pharmacyclics Llc Inhibitors of bruton's tyrosine kinase.
CN103896953A (en) * 2014-04-10 2014-07-02 广东众生药业股份有限公司 2, 4-dihydroxyl pyrrole [2, 3-d] pyrimidine derivative and preparation method and use thereof
EP3174539A4 (en) * 2014-08-01 2017-12-13 Pharmacyclics, LLC Inhibitors of bruton's tyrosine kinase
CN106928231B (en) * 2015-12-31 2021-06-01 合肥中科普瑞昇生物医药科技有限公司 Novel EGFR wild type and mutant kinase inhibitors
CN107513068A (en) * 2016-06-16 2017-12-26 中国科学院上海药物研究所 A kind of new compound and its preparation and application with FGFR inhibitory activity
CN109970740A (en) * 2017-12-27 2019-07-05 广东众生药业股份有限公司 4- amidino-pyridine and azepine ring derivatives and its preparation method and application

Also Published As

Publication number Publication date
WO2021175316A1 (en) 2021-09-10
CN112961159A (en) 2021-06-15

Similar Documents

Publication Publication Date Title
JP5452811B2 (en) Compounds for inhibiting mitotic progression
JP5938400B2 (en) Pyrrolyl-substituted dihydroindol-2-one derivatives, preparation methods and uses thereof
JP6963598B2 (en) Thienopyrimidine compounds, their production methods, pharmaceutical compositions and their applications
JP2020529968A (en) Substituted 5- and 6-membered heterocyclic compounds, their preparation methods, drug combinations and their use
CN112961159B (en) Aminopyrimidinopyrazole/pyrrole derivative and preparation method and application thereof
CN109575045B (en) Thienopyrimidine compound, preparation method thereof, medicinal composition and application thereof
CN113773316B (en) TNIK inhibitor and preparation method and application thereof
EP1918277B1 (en) Azulene compounds
CN111205227A (en) 3-vinyl indazole derivative and preparation method and application thereof
CN112961158B (en) Aminopyrimidinopyrazole/pyrrole derivative and preparation method and application thereof
CN110467616B (en) Preparation and application of triazolopyrazine compound containing heteroaryl substituted pyridazinone structure
CN112694474B (en) Indazole derivative and preparation method and application thereof
CN114181208B (en) Tri-fused ring AhR inhibitor and application thereof
CN106536522B (en) Heterocyclic compounds for the treatment of psoriasis
CN112794820B (en) Indazole derivative and preparation method and application thereof
WO2021232367A1 (en) 3-vinyl indazole derivative, preparation method therefor, and use thereof
CN110028439B (en) Phthalimide derivative and preparation method and application thereof
CN111233774A (en) Amino pyrimidine compound
CN114805350B (en) Benzo heterocycle-pyridone derivative, and preparation method and application thereof
KR101713027B1 (en) Composition for preventing or treating cancer comprising phenyloxazol derivatives
CN117677614A (en) Heterocyclic compound with AKT kinase inhibition activity, preparation method and medical application thereof
CN111848641A (en) 4-phenoxy thiophene [3,2-d ] pyrimido alpha-acylamino thioamide compound and preparation method and application thereof
JP5476650B2 (en) New DIF-1 derivative
CN116768903A (en) Pyrrolo pyrimidine derivative, preparation method thereof, pharmaceutical composition and application
CN117700397A (en) 2-oxo indoline derivative and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant