WO2021175316A1 - 氨基嘧啶并吡唑/吡咯类衍生物及其制备方法和用途 - Google Patents

氨基嘧啶并吡唑/吡咯类衍生物及其制备方法和用途 Download PDF

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WO2021175316A1
WO2021175316A1 PCT/CN2021/079338 CN2021079338W WO2021175316A1 WO 2021175316 A1 WO2021175316 A1 WO 2021175316A1 CN 2021079338 W CN2021079338 W CN 2021079338W WO 2021175316 A1 WO2021175316 A1 WO 2021175316A1
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compound
substituted
unsubstituted
cancer
enantiomer
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French (fr)
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叶庭洪
魏于全
曹丹
于艳
谢永美
欧阳亮
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四川大学华西医院
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    • 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

Definitions

  • the invention relates to aminopyrimidopyrazole/pyrrole derivatives and a preparation method and application thereof, and belongs to the field of medicine.
  • Receptor tyrosine kinase RTKs are a type of cell surface transmembrane protein receptor with endogenous protein tyrosine kinase activity. They usually consist of an extracellular domain that can bind to a specific ligand, a transmembrane region, and An intracellular kinase domain that can selectively bind to and phosphorylate substrates. Combining the ligand with the extracellular structural region of RTKs causes its structural changes to produce enzyme catalytic activity. It plays an important regulatory role in tumor angiogenesis, tumor cell survival, proliferation, differentiation, and migration.
  • fibroblast growth factor receptors FGFRs
  • epidermal growth factor receptors EGFRs
  • platelet-derived growth factor receptors Body platelet-derived growth factor receptors, PDGFRs
  • vascular endothelial growth factor receptors vascular endothelial growth factor receptors, VEGFRs
  • HGFRs hepatocyte growth factor receptors
  • Fibroblast growth factor receptors are receptor tyrosine kinases located on the cell membrane.
  • the FGF/FGFR signal pathway is abnormally activated in a variety of cancers such as gastric cancer, breast cancer, lung cancer, cholangiocarcinoma, urothelial cancer, and prostate cancer.
  • the FGFR signaling pathway is involved in the signal transduction process that regulates cell proliferation, differentiation, migration, survival, embryonic development, angiogenesis and organ formation.
  • the FGFR signaling pathway frequently changes in cancer, and preclinical models prove that the abnormal FGFR signaling pathway has the potential to promote the formation of cancer.
  • FGFR is located in the upstream of the cell signaling pathway, which activates downstream signaling pathways through adaptor proteins or directly combines with transcription factors to transmit signals. FGFR activating mutation or overexpression can make the FGFR signaling pathway continue to be overactivated, which is closely related to the cancer process.
  • FGF is also closely related to organ fibrosis. For example, studies have found that FGF is highly expressed in fibrotic lung tissue, serum, and alveolar perfusion washing fluid, and is positively correlated with the degree of pulmonary fibrosis. FGF-2 is derived from alveolar macrophages, fibroblasts, endothelial cells, etc., and can promote the synthesis and accumulation of collagen.
  • FGF-2 FGF-2 is significantly overexpressed in nephropathy caused by diabetes, and stimulates the proliferation of glomerular mesangial cells; promotes the regeneration of renal tubular cells and leads to cell fibrosis.
  • Inhibitor drugs targeting FGFR can inhibit the abnormal activation of the FGF/FGFR signaling pathway and have the potential to treat the above diseases.
  • FGFR inhibitor drugs have become one of the hot spots in drug research in recent years.
  • the purpose of the present invention is to provide aminopyrimidopyrazole/pyrrole derivatives and preparation methods and uses thereof.
  • the present invention provides a compound represented by formula I, its stereoisomers, a compound or its stereoisomers pharmaceutically acceptable salts:
  • R 1 is Ar or Ar is a substituted or unsubstituted aryl group
  • R 2 is -H, or R 2 is the same as the 1-position nitrogen atom to form an amide
  • X is CH or N.
  • R 1 is Ar is a substituted or unsubstituted aryl group.
  • Ar is a substituted or unsubstituted 5-10 membered aryl group.
  • aryl group satisfies at least one of the following:
  • the aryl group is a 5-6 membered monocyclic ring or a 9-10 membered condensed ring;
  • the aryl group contains 0 to 2 heteroatoms
  • the heteroatom is a nitrogen atom
  • the aryl group is selected from:
  • the aryl group is
  • substituted aryl group contains at least one substituent selected from the group consisting of substituted or unsubstituted alkoxy, halogen, substituted or unsubstituted amino, and substituted or unsubstituted alkyl.
  • 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.
  • the substituted aryl group contains at least one substituent selected from the following group: -OCH 3 , -Cl, -NH 2 .
  • Ar is selected from:
  • Ar is N
  • R 2 is -H or R 3 is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl.
  • R 3 is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl.
  • R 3 is selected from unsubstituted C1-C3 alkyl, substituted or unsubstituted C2-C4 alkenyl.
  • the substituted C2-C4 alkenyl group contains at least one substituent selected from the following group: -CN, C3-C6 cycloalkyl.
  • R 2 is selected from: -H,
  • R 2 is
  • the stereoisomer of the compound has the structure shown in Formula Ia.
  • the compound or its stereoisomer is selected from:
  • the pharmaceutically acceptable salt of the compound or its stereoisomer is hydrochloride.
  • the present invention provides a method for preparing stereoisomers of the compound, which includes the following steps:
  • Ha is a halogen
  • LG is a leaving group
  • Ha is -I
  • LG is -Boc
  • R 5 and R 6 are independently selected from H or alkyl, or R 5 and R 6 are connected to form an alicyclic ring;
  • compound 3 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • preparation method meets at least one of the following:
  • step a the molar ratio of compound 1: compound 2 or its enantiomer is 1: (1.0 ⁇ 1.5);
  • reaction solvent in step a is tetrahydrofuran
  • step a diisopropyl azodicarboxylate and triphenylphosphine are added;
  • the molar ratio of compound 1: diisopropyl azodicarboxylate in step a is 1: (2.0-3.0);
  • the molar ratio of compound 1: triphenylphosphine in step a is 1: (2.0-3.0);
  • Step a react in a protective atmosphere
  • reaction temperature in step a is 0-50°C;
  • step b the molar ratio of intermediate 1 or its enantiomer: compound 3 is 1: (1.0 ⁇ 1.5);
  • the reaction solvent in step b is a mixed solvent of 1,4-dioxane and water, and the volume ratio of 1,4-dioxane:water is (4-8):1;
  • the amount of palladium catalyst used in step b is 3-10 mmol%;
  • the palladium catalyst described in step b is selected from palladium acetate, [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride, [1,1'-bis(diphenylphosphine) two At least one of ferrocene] palladium dichloride dichloromethane complex and tris(dibenzylidene indeneacetone) dipalladium;
  • the molar ratio of intermediate 1 or its enantiomer: base in step b is 1: (2.0-3.0);
  • the base in step b is selected from at least one of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium phosphate, and cesium carbonate;
  • Step b reacts in a protective atmosphere
  • the reaction temperature in step b is 90 to 110°C.
  • the preparation method also includes the following steps: compound 4 or its enantiomer and compound 5 undergo an amidation reaction to obtain compound 6 or its enantiomer:
  • Halo is halogen; preferably, Halo is -Cl.
  • the present invention provides the use of the compound, its stereoisomer, the compound or its pharmaceutically acceptable salt in the preparation of drugs for preventing and treating cancer.
  • the cancer is gastric cancer, breast cancer, lung cancer, cholangiocarcinoma, urothelial cancer, and prostate cancer.
  • the invention provides the use of the compound, its stereoisomer, the compound or its pharmaceutically acceptable salt in the preparation of a medicine for preventing and treating organ fibrosis.
  • the organ fibrosis is liver fibrosis or lung fibrosis.
  • the present invention provides the use of the compound, its stereoisomer, the compound or its pharmaceutically acceptable salt in the preparation of FGFR and/or VEGFR inhibitor drugs.
  • the FGFR inhibitor is FGFR1, FGFR2, FGFR3 and/or FGFR4 inhibitor.
  • the VEGFR inhibitor is Flt1, Flt4 and/or KDR inhibitor.
  • the present invention provides a pharmaceutical composition, which takes the compound, its stereoisomer, the compound or its pharmaceutically acceptable salt as the active ingredient, and adds pharmaceutically acceptable excipients or auxiliary materials. Ingredients, prepared preparations.
  • the formulation is an oral formulation.
  • the compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) naming system.
  • stereoisomers includes cis-trans isomers and optical isomers.
  • aryl refers to a group of a 4n+2 aromatic ring system containing or not containing heteroatoms in the aromatic ring system, wherein the heteroatoms are selected from nitrogen, oxygen and/or sulfur.
  • amide refers to a structure formed by replacing the hydroxyl group on the carboxyl group with an amino group or a substituted amino group.
  • alkyl is a linear or branched saturated hydrocarbon group.
  • C1-C6 alkyl groups include but are not limited to methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4) , Sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2- Butyl (C5), tert-amyl (C5) and n-hexyl (C6).
  • alkoxy refers to the group -OR, where 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-hexyloxy and 1,2-dimethylbutoxy.
  • alkenyl refers to a straight or branched chain hydrocarbon group containing at least one double bond.
  • 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.
  • cycloalkyl refers to a saturated cyclic hydrocarbon group with or without heteroatoms, wherein the heteroatoms are selected from sulfur, oxygen, phosphorus, and/or nitrogen.
  • alicyclic refers to a saturated or partially unsaturated cyclic hydrocarbon group.
  • pharmaceutically acceptable means that a certain carrier, carrier, diluent, excipient, and/or the formed salt is usually chemically or physically compatible with other ingredients constituting a certain pharmaceutical dosage form, and physiologically Compatible with the receptor.
  • pharmaceutically acceptable salt refers to the acid and/or basic salt formed by the compound of the present invention with inorganic and/or organic acids and bases, and also includes zwitterionic salts (internal salts), and also includes quaternary ammonium salts, For example, alkyl ammonium salts.
  • zwitterionic salts internal salts
  • quaternary ammonium salts For example, alkyl ammonium salts.
  • These salts can be obtained directly in the final isolation and purification of the compound. It can also be obtained by appropriately mixing the above-mentioned compound with a certain amount of acid or base (e.g., equivalent).
  • These salts may form a precipitate in the solution and be collected by filtration, or recovered after evaporation of the solvent, or prepared by freeze-drying after reaction in an aqueous medium.
  • the salt in the present invention can be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, butane Acid salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.
  • the method of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods 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.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or solubilizers, for example, starch , Lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, such as , Glycerin; (d) Disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, some complex silicates, and sodium carbonate; (e) Soothing solvents, such as paraffin; (f) absorption Accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetylene glyco
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the active compound or the release of the compound in such a composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the dosage form of the compound of the present invention for topical administration includes ointment, powder, patch, propellant and inhalant.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the pharmaceutically acceptable excipients in the present invention refer to substances contained in the dosage form in addition to the active ingredients.
  • the pharmaceutically acceptable auxiliary component of the present invention has certain physiological activity, but the addition of the component will not change the dominant position of the above-mentioned pharmaceutical composition in the course of disease treatment, but only exerts auxiliary functions. These auxiliary functions only It is the utilization of the known activity of the ingredient, and it is a commonly used adjuvant therapy in the medical field. If the aforementioned auxiliary components are used in combination with the pharmaceutical composition of the present invention, they should still fall within the protection scope of the present invention.
  • the present invention provides a class of aminopyrimidopyrazole/pyrrole compounds with novel structures, which have inhibitory effects on FGFR, VEGFR and other tyrosine kinases, and are potential multi-target anti-tumor drugs.
  • Biological experiments have proved that the compound of the present invention can not only significantly inhibit the proliferation of breast cancer, lung cancer, gastric cancer, cholangiocarcinoma, urothelial cancer and other cancer cells, but also has a broad-spectrum anti-cancer effect.
  • the proliferation of shaped cells also exhibits an inhibitory effect, and can inhibit the growth of tumors in vivo, providing new options for the development of anti-tumor and anti-fibrotic drugs.
  • Figure 1 is a graph of tumor growth in nude mice in biological experiments
  • Figure 2 is a diagram of tumor measurement in biological experiments
  • Figure 3 is a graph showing the statistical results of tumor weight in biological experiments
  • Figure 4 is a graph showing the weight change of nude mice in a biological experiment.
  • the starting material compound 1 (1.08 g, 8 mmol) was dissolved in DMF (20 mL), N-iodosuccinimide (4.5 g, 20 mmol) was added, and the reaction was carried out at 80° C. for 5 hours, followed by TLC monitoring. After the completion of the reaction, the reaction was quenched with a saturated aqueous solution of sodium hydroxide. The reaction solution was poured into water. A yellow solid precipitated out. The solid was filtered and dried to obtain Intermediate 2 (white solid, 98%), MS m/z (ESI) : 261.9[M+H] + .
  • Dissolve compound 8 (544mg, 1mmol) in methanol, add hydrochloric acid, stir at room temperature for 6h, precipitate precipitation, concentrate to obtain a solid, add ultra-dry dichloromethane, triethylamine (694 ⁇ L, 5mmol), add dropwise under ice bath conditions Acryloyl chloride (152 ⁇ L, 2 mmol), after the dripping, was transferred to room temperature and reacted for 30 minutes, and the reaction was monitored by TLC.
  • Ultra-clean workbench BHC-1000IIA/B3 Sujing Antai Biotechnology Company; Constant temperature water bath PolyScience9505: PolyScience Company; Sterilizer MLS-3780: SANYO Company; Oven: Binder Company; Ultra-pure water meter Milli-Q Integral 10: Millipore company; microplate reader Multiscan MK3, cell incubator, low-speed centrifuge Sorvall ST1: Thermofisher company; Centrifuge 5415C ultracentrifuge: Germany Eppendorf company; NUAIRE NU-425-600E biological safety cabinet: American Nuaire company; BCD-215YD type Ordinary refrigerator: China Haier Company; SANYO (-80°C) ultra-low temperature refrigerator: Japan Sanyo Electric Group; Rocker 51702 shaker: American Cole Parmer Company; 96-well cell culture plate: Costa Corning Company; ordinary optical microscope: Olympus Company; pipetting Gun: Thermo Company; PH meter: Coring Company; Autoclave: SANYO Company.
  • the cell line used in the experiment was purchased from ATCC, USA.
  • Various necessities for cell culture were purchased from GibcoBRL, including DMEM medium, RPMI 1640 medium, fetal bovine serum (FBS) and pancreatin.
  • Tetramethylazazole blue (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma Company in the United States.
  • the compound of the present invention is diluted with DMSO to 50 times the highest inhibitor concentration required in the final reaction.
  • This 96-well plate serves as the source plate.
  • Passage is usually once every 3 to 4 days; Passage of adherent growth cells: The cells adhere to the wall and grow to about 80% of the bottom of the bottle, remove the culture bottle from the incubator, aspirate the medium, and wash once with 0.25% pancreatin Then add 0.25% trypsin digestion solution for digestion. After observing the cell shrinkage and rounding, add complete medium to stop the digestion, and pipette to disperse and fall off the cells. Collect the cell suspension, centrifuge at 1500rpm/min for 3min, and pour the supernatant. , The cell pellet is resuspended in complete medium and pipetted evenly, and then divided into 3 to 5 bottles for culture. Generally, it is passaged once every 3 to 4 days.
  • the dosing group add 100 ⁇ L of the compound medium solution to each well according to the gradient (final concentrations are 1000, 333, 127, 42.3, 14.1, 4.7, 1.56, 0.53 nM), and each concentration has 3 replicate wells; negative control
  • 100 ⁇ L of blank medium containing 1 ⁇ DMSO was added to each well for a total of 6 multiple wells; in the blank control group, only 100 ⁇ L of medium was added to each well. Place the plate in a 37°C, 5% CO 2 cell culture incubator for 72 hours.
  • the invisible control group and the blank group add 20 ⁇ L of MTT solution (5mg/mL) to each well, continue to incubate for 2-4 hours.
  • SNU16 cells with good logarithmic growth phase were inoculated subcutaneously in Balb/C nude mice at an amount of 5 ⁇ 10 5 cells/100 ⁇ l/mouse. About 10 days after inoculation, the tumor grew to about 250 cm 2 , and randomized grouping was started. Group of 7 only.
  • the tumor is about 250cm 2 into groups and then administered orally for 10 consecutive days.
  • Evaluation index 10 days after the administration, the weight change of the nude mice, the growth curve of the tumor, and the size of the tumor were examined and compared.
  • the compounds of the present invention have inhibitory effects on FGFR and VEGFR (including Flt1, Flt4, KDR subtypes) and other tyrosine kinases. These kinases can survive, proliferate, differentiate, and migrate in tumor cells. It plays an important regulatory role in tumor angiogenesis and other processes, indicating that the compound of the present invention is a potential multi-target anti-tumor drug.
  • Table 5 The IC 50 value or inhibition rate of some compounds of the present invention in inhibiting the proliferation of gastric cancer cell lines
  • AZD4547 is a known and effective FGFR inhibitor.
  • Table 7 The IC 50 value or inhibition rate of some compounds of the present invention in inhibiting the proliferation of fibrotic cells
  • the tumor growth curve is shown in Figure 1, the tumor measurement is shown in Figure 2, the tumor weight statistics are shown in Figure 3, and the nude mouse body weight change curve is shown in Figure 4. It can be seen that both low dose and high dose of compound 9-15 of the present invention can significantly inhibit tumor growth and even eliminate tumors. Moreover, at the same dose (15mg/kg), compound 9-15 is significantly better than the positive control drug AZD4547 in inhibiting tumor growth.

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Abstract

一种式I的氨基嘧啶并吡唑/吡咯类衍生物及其制备方法和用途。所述化合物能显著抑制乳腺癌、肺癌、胃癌、胆管癌、尿路上皮癌等多种癌细胞的增殖，且具有广谱的抗癌作用，对成纤维细胞和肝星状细胞的增殖也表现出抑制作用。

Description

氨基嘧啶并吡唑/吡咯类衍生物及其制备方法和用途 技术领域
本发明涉及氨基嘧啶并吡唑/吡咯类衍生物及其制备方法和用途，属于医药领域。
背景技术
受体酪氨酸激酶RTKs是一类具有内源性蛋白酪氨酸激酶活性的细胞表面跨膜蛋白受体,通常由一个可以与特定配体相结合的细胞外结构域、一个跨膜区及一个可以选择性地与底物结合并将其磷酸化的细胞内激酶域组成。将配体与RTKs的细胞外结构区域结合,引起其结构改变从而产生酶催化活性。其对肿瘤血管生成、肿瘤细胞存活、增殖、分化、迁移等发挥重要调控作用。现已发现有50多种不同的RTKs家族成员,其中主要包括成纤维细胞生长因子受体(fibroblast growth factor,FGFRs)、表皮生长因子受体(epidermal growth factor receptors,EGFRs)、血小板衍生生长因子受体(platelet-derivedgrowth factor receptors,PDGFRs)、血管内皮生长因子受体(vascular endothelial growth factor receptors,VEGFRs)和肝细胞生长因子受体(hepatocyte growthfactor receptors,HGFRs)。
成纤维生长因子受体(Fibroblastgrowthfactorreceptors,FGFRs)是一种位于细胞膜上的受体酪氨酸激酶。FGF/FGFR信号通路在胃癌、乳腺癌、肺癌、胆管癌、尿路上皮癌、前列腺癌等多种癌症中出现异常活化。FGFR信号通路参与了调控细胞增殖、分化、迁移、存活、胚胎发育、血管生成和器官形成的信号转导过程。FGFR信号通路在癌症中频繁出现改变，并且临床前模型证明FGFR信号通路异常具有促使癌症形成的潜力。FGFR处于细胞信号通路的上游，通过衔接蛋白活化下游信号通路或直接与转录因子结合传导信号。FGFR激活突变或过表达能使FGFR信号通路持续过度活化，与癌症进程密切相关。
近年研究表明FGFR与器官纤维化也有着密切的关系，例如，研究发现FGF在纤维化的肺组织、血清，肺泡灌注洗液中有高表达并与肺纤维化程度呈正相关。FGF-2来源于肺泡的巨噬细胞、成纤维细胞、内皮细胞等，可以促进胶原的合成及积聚。但在特发性肺纤维化IPF患者中检测到FGF-2水平升高，在上皮细胞、内皮细胞和平滑肌细胞/成纤维细胞样细胞中FGFR-1表达增加，在间质细胞中FGFR-2表达增加，并有研究表明，FGF-2在糖尿病引起的肾病中明显过表达，并刺激肾小球系膜细胞增殖；促进肾 小管细胞再生导致细胞纤维化。靶向FGFR的抑制剂药物可以抑制FGF/FGFR信号通路的异常激活，具有治疗以上疾病的潜力，FGFR抑制剂药物成为近年来药物研究的热点之一。
发明内容
本发明的目的在于提供氨基嘧啶并吡唑/吡咯类衍生物及其制备方法和用途。
本发明提供了式Ⅰ所示的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐：
Figure PCTCN2021079338-appb-000001
其中，R 1为Ar或
Figure PCTCN2021079338-appb-000002
Ar为取代或未取代的芳基；
R 2为-H，或者R 2同1-位氮原子形成酰胺；
X为CH或N。
进一步地，R 1为
Figure PCTCN2021079338-appb-000003
Ar为取代或未取代的芳基。
进一步地，Ar为取代或未取代的5～10元芳基。
进一步地，所述芳基满足以下至少一项：
所述的芳基为5～6元单环或9～10元稠环；
所述的芳基含有0～2个杂原子；
优选地，所述的杂原子为氮原子；
优选地，所述的芳基选自：
Figure PCTCN2021079338-appb-000004
进一步优选地，所述的芳基为
Figure PCTCN2021079338-appb-000005
进一步地，所述取代的芳基含有至少一个选自下组的取代基：取代或未取代的烷氧基、卤素、取代或未取代的氨基、取代或未取代的烷基。
优选地，所述取代的芳基含有至少一个选自下组的取代基：未取代的C1～C6烷氧 基、卤素、未取代的氨基、C1～C6烷基取代的氨基、未取代的C1～C6烷基。
优选地，所述取代的芳基含有至少一个选自下组的取代基：-OCH 3、-Cl、-NH 2。
进一步地，Ar选自：
Figure PCTCN2021079338-appb-000006
优选地，Ar为
Figure PCTCN2021079338-appb-000007
进一步地，R 2为-H或
Figure PCTCN2021079338-appb-000008
R 3选自取代或未取代的烷基、取代或未取代的烯基。
优选地，R 3选自取代或未取代的C1～C6烷基、取代或未取代的C2～C6烯基。
优选地，R 3选自未取代的C1～C3烷基、取代或未取代的C2～C4烯基。
优选地，所述取代的C2～C4烯基含有至少一个选自下组的取代基：-CN、C3～C6环烷基。
优选地，R 2选自：-H、
Figure PCTCN2021079338-appb-000009
进一步优选地，R 2为
Figure PCTCN2021079338-appb-000010
进一步地，所述化合物的立体异构体，其结构如式Ⅰa或式Ⅰb所示：
Figure PCTCN2021079338-appb-000011
优选地，所述化合物的立体异构体，其结构如式Ⅰa所示。
进一步地，所述化合物或其立体异构体选自：
Figure PCTCN2021079338-appb-000012
进一步地，所述化合物或其立体异构体在药学上可接受的盐为盐酸盐。
本发明提供了所述化合物的立体异构体的制备方法，包括如下步骤：
a、化合物1与化合物2或其对映异构体经过Mitsunobu反应，得到中间体1或其对映异构体：
Figure PCTCN2021079338-appb-000013
其中，Ha为卤素，LG为离去基团；
优选地，Ha为-I；
优选地，LG为-Boc；
b、中间体1或其对映异构体与化合物3在钯催化剂作用下偶联，得到中间体2或其对映异构体：
Figure PCTCN2021079338-appb-000014
其中，R 5、R 6独立地选自H或烷基，或者，R 5与R 6相连形成脂环；
优选地，化合物3为
Figure PCTCN2021079338-appb-000015
c、中间体2或其对映异构体脱去离去基团，即得化合物4或其对映异构体：
Figure PCTCN2021079338-appb-000016
进一步地，所述的制备方法满足以下至少一项：
步骤a中化合物1：化合物2或其对映异构体的摩尔比为1：(1.0～1.5)；
步骤a的反应溶剂为四氢呋喃；
步骤a的反应体系中加入了偶氮二甲酸二异丙酯和三苯基膦；
优选地，步骤a中化合物1：偶氮二甲酸二异丙酯的摩尔比为1：(2.0～3.0)；
优选地，步骤a中化合物1：三苯基膦的摩尔比为1：(2.0～3.0)；
步骤a在保护气氛下反应；
步骤a的反应温度为0～50℃；
步骤b中中间体1或其对映异构体：化合物3的摩尔比为1：(1.0～1.5)；
步骤b的反应溶剂为1,4-二氧六环和水的混合溶剂，1,4-二氧六环：水的体积比为(4～8)：1；
以中间体1或其对映异构体的摩尔量计，步骤b中钯催化剂的用量为3～10mmol％；
步骤b中所述的钯催化剂选自醋酸钯、[1,1'-双(二苯基膦基)二茂铁]二氯化钯、[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物、三(二亚苄基茚丙酮)二钯中至少一种；
步骤b的反应体系中加入了碱；
优选地，步骤b中中间体1或其对映异构体：碱的摩尔比为1：(2.0～3.0)；
优选地，步骤b中所述的碱选自碳酸钠、碳酸氢钠、碳酸钾、磷酸钾、碳酸铯中至少一种；
步骤b在保护气氛下反应；
步骤b的反应温度为90～110℃。
进一步地，所述的制备方法还包括如下步骤：化合物4或其对映异构体与化合物5经过酰胺化反应，即得化合物6或其对映异构体：
Figure PCTCN2021079338-appb-000017
其中，Halo为卤素；优选地，Halo为-Cl。
本发明提供了所述的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐在制备防治癌症的药物中的用途。
优选地，所述的癌症为胃癌、乳腺癌、肺癌、胆管癌、尿路上皮癌、前列腺癌。
本发明提供了所述的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐在制备防治器官纤维化的药物中的用途。
优选地，所述的器官纤维化为肝纤维化或肺纤维化。
本发明提供了所述的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐在制备FGFR和/或VEGFR抑制剂类药物中的用途。
优选地，所述的FGFR抑制剂为FGFR1、FGFR2、FGFR3和/或FGFR4抑制剂。
优选地，所述的VEGFR抑制剂为Flt1、Flt4和/或KDR抑制剂。
本发明提供了药物组合物，它是以所述的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐为活性成分，加入药学上可接受的辅料或者辅助性成分，制备而成的制剂。
优选地，所述的制剂为口服制剂。
术语定义：
本发明中提供的化合物和衍生物可以根据IUPAC(国际纯粹与应用化学联合会)或 CAS(化学文摘服务社，Columbus，OH)命名系统命名。
术语“立体异构体”包括顺反异构体、旋光异构体。
术语“芳基”是指在芳族环系中包含或不包含杂原子的4n+2芳族环系的基团，其中，杂原子选自氮、氧和/或硫。
术语“酰胺”是指羧基上的羟基被氨基或取代氨基所取代而形成的结构。
术语“烷基”是直链或支链的饱和烃基的基团。C1～C6烷基的实例包括但不限于甲基(C1)、乙基(C2)、正丙基(C3)、异丙基(C3)、正丁基(C4)、叔丁基(C4)、仲丁基(C4)、异丁基(C4)、正戊基(C5)、3-戊基(C5)、戊基(C5)、新戊基(C5)、3-甲基-2-丁基(C5)、叔戊基(C5)和正己基(C6)。
术语“烷氧基”是指基团-OR，其中R是上文所定义的烷基。C1～C6烷氧基的实例包括但不限于甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、叔丁氧基、仲丁氧基、正戊氧基、正己氧基和1,2-二甲基丁氧基。
术语“烯基”是指含有至少一个双键的直链或支链烃基。烯基的例子包括但不限于乙烯基、烯丙基、丁-1-烯基、丁-2-烯基、戊-1-烯基、戊-2-烯基、戊-3-烯基、己-1-烯基、己-2-烯基、己-3-烯基、己-4-烯基。
术语“环烷基”是指包含或不包含杂原子的饱和的环状烃基，其中，杂原子选自硫、氧、磷和/或氮。
术语“脂环”是指饱和或部分不饱和的环状烃基。
术语“药学上可接受的”是指某载体、运载物、稀释剂、辅料，和/或所形成的盐通常在化学上或物理上与构成某药物剂型的其它成分相兼容，并在生理上与受体相兼容。
术语“药学上可接受的盐”是指本发明化合物与无机和/或有机酸和碱形成的酸式和/或碱式盐，也包括两性离子盐(内盐)，还包括季铵盐，例如烷基铵盐。这些盐可以是在化合物的最后分离和纯化中直接得到。也可以是通过将上述化合物与一定数量的酸或碱适当(例如等当量)进行混合而得到。这些盐可能在溶液中形成沉淀而以过滤方法收集，或在溶剂蒸发后回收而得到，或在水介质中反应后冷冻干燥制得。本发明中所述盐可以是化合物的盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增溶剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明所述药学上可接受的辅料，是指除活性成分以外包含在剂型中的物质。
本发明所述药学上可接受的辅助性成分，它具有一定生理活性，但该成分的加入不会改变上述药物组合物在疾病治疗过程中的主导地位，而仅仅发挥辅助功效，这些辅助功效仅仅是对该成分已知活性的利用，是医药领域惯用的辅助治疗方式。若将上述辅助性成分与本发明药物组合物配合使用，仍然应属于本发明保护的范围。
本发明提供了一类结构新颖的氨基嘧啶并吡唑/吡咯类化合物，对FGFR、VEGFR等多种酪氨酸激酶具有抑制作用，是潜在的多靶点抗肿瘤药物。生物学实验证明，本发明化合物能够不仅可以显著抑制乳腺癌、肺癌、胃癌、胆管癌、尿路上皮癌等多种癌细胞的增殖，具有广谱的抗癌作用，对成纤维细胞和肝星状细胞的增殖也表现出抑制作用，而且能够在体内抑制肿瘤的生长，为抗肿瘤、抗纤维化药物的开发提供了新的选择。
附图说明
图1为生物学实验中裸鼠体内肿瘤生长曲线图；
图2为生物学实验中肿瘤测量情况图；
图3为生物学实验中肿瘤重量统计结果图；
图4为生物学实验中裸鼠体重变化曲线图。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 本发明化合物的制备
Figure PCTCN2021079338-appb-000018
将原料化合物1(1.08g,8mmol)溶于DMF(20mL)中，加入N-碘代丁二酰亚胺(4.5g,20mmol)，80℃下反应5h后TLC监测。反应完毕后用保险粉的饱和水溶液淬灭反应，将反应液倒入水中，有黄色固体析出，将固体过滤后干燥，得中间体2(白色固体，98％)，MS m/z(ESI)：261.9[M+H] +。
Figure PCTCN2021079338-appb-000019
将中间体2(2.09g,8mmol)溶于四氢呋喃150mL中，加入化合物3(1.8g,9.6mmol)、三苯基膦(4.2g,16mmol)，N 2保护，于0℃搅拌，再滴加偶氮二甲酸二异丙酯(3.1mL,16mmol)，滴加完毕，半小时后转至室温反应3h后TLC监测。反应完毕后将反应液浓缩，水洗3次，有机相用无水硫酸钠干燥后减压浓缩，柱层析得中间体4(浅黄色固体，70％)，MS m/z(ESI)：431.1[M+H] +。
Figure PCTCN2021079338-appb-000020
将化合物5(2.67g,8mmol)、乙烯基硼酸频哪醇酯化合物6(1.6mL,9.6mmol)、DIEA(2.4mL,14.4mmol)加入干燥甲苯(80mL)中，再加入Pd 2(dba) 3(1mmol％)、三叔丁基膦四氟硼酸盐(1mmol％)，氮气保护下升温至95℃反应8h，TLC监测。反应完毕后减压浓缩溶剂，剩余物用乙酸乙酯溶解后经硅藻土过滤，滤液用饱和NaCl溶液洗涤三次，有机相用无水硫酸钠干燥后减压浓缩，柱层析(PE:EA＝4:1)得中间体7(浅黄色油状，53％)，MS m/z(ESI)：344.1[M+H] +。
Figure PCTCN2021079338-appb-000021
将中间体4(431mg,1mmol)、中间体7(431mg,1.2mmol)、Pd(dppf)Cl 2(5mmol％)，碳酸钾(278mg,2mmol)，加入1,4-二氧六环与水(4:1)的混合溶剂25mL中，N 2置换后于100℃下反应10h，TLC监测反应。反应完全后将反应液减压浓缩，剩余物用乙 酸乙酯溶解，经硅藻土过滤，滤液浓缩后，经柱层析(PE:EA＝2:1)得到化合物8(浅黄色固体，70％)。MS m/z(ESI)：544.1[M+H] +。
Figure PCTCN2021079338-appb-000022
将化合物8(544mg,1mmol)溶于甲醇中，加入盐酸，室温搅拌6h，有沉淀析出，浓缩得固体，加入超干二氯甲烷，三乙胺(694μL,5mmol),冰浴条件下滴加丙烯酰氯(152μL,2mmol)，滴加完毕后转至室温反应30min，TLC监测反应。反应完全后将反应液用二氯甲烷稀释，水洗3次，有机相用无水硫酸钠干燥后减压浓缩，薄层色谱层析得产物化合物9-1(白色固体，50％)，MS m/z(ESI)：489.1[M+H] +。
按照类似的方法可以制备得到如下化合物：
表1 本发明部分化合物的结构鉴定数据
Figure PCTCN2021079338-appb-000023
Figure PCTCN2021079338-appb-000024
Figure PCTCN2021079338-appb-000025
Figure PCTCN2021079338-appb-000026
以下通过生物学实验证明本发明的有益效果。
一、实验仪器及材料
超净工作台BHC-1000IIA/B3：苏净安泰生物技术公司；恒温水浴箱PolyScience9505：PolyScience公司；灭菌锅MLS-3780：SANYO公司；烘箱：Binder公司；超纯 水仪Milli-Q Integral 10：Millipore公司；酶标仪Multiscan MK3、细胞培养箱、低速离心机Sorvall ST1：Thermofisher公司；Centrifuge 5415C超速离心机：德国Eppendorf公司；NUAIRE NU-425-600E生物安全柜：美国Nuaire公司；BCD-215YD型普通冰箱：中国Haier公司；SANYO(-80℃)超低温冰箱：日本三洋电器集团；Rocker 51702摇床：美国Cole Parmer公司；96孔细胞培养板：Costa Corning公司；普通光学显微镜：Olympus公司；移液枪：Thermo公司；PH计：Coring公司；高压灭菌锅：SANYO公司。
实验中采用的细胞系购自美国ATCC公司。培养细胞所用各种必需品购自GibcoBRL公司，包括DMEM培养基、RPMI 1640培养基、胎牛血清(FBS)和胰酶。四甲基偶氮唑蓝(MTT)、二甲基亚砜(DMSO)购自美国Sigma公司。
二、实验方法
1、激酶测试
用DMSO将本发明化合物稀释至反应中最终所需最高抑制剂浓度的50倍。将100μL化合物稀释液转移到96孔板中。在同一96孔板的两个空白孔中加入100μL DMSO。该96孔板作为源板。将10μL化合物从源板转移到的96孔板中，作为中间板。向中间板的每个孔中加入90μLl1x激酶缓冲液。在振荡器上将中间板中的化合物混合10分钟。将96孔中间板的每孔取5μL转移至384孔板，设置副孔。在1x激酶碱缓冲液中加入激酶。在1x激酶碱缓冲液中加入FAM标记的肽和ATP。测定板中已含有5μL化合物10％DMSO溶液。向384孔测定板的每个孔中加入10μL l2.5x酶溶液。在室温下孵育10分钟。向384孔测定板的每个孔中加入10μL的2.5x肽溶液。在28℃下孵育特定时间后，加入25μL终止缓冲液以停止反应。在Caliper上收集数据，将数据进一步换算为IC 50。
2、细胞培养
从液氮中取出冻存保种的肿瘤细胞，迅速置于37℃恒温水浴复温融化，在无菌条件下用培养基洗涤1次。然后用完全培养基接种于培养瓶内，在37℃，5％CO 2培养箱中培养，第二天置换新鲜细胞培养液。悬浮生长细胞的传代：细胞培养2～3天后，从培养箱中取出培养瓶，收集细胞悬液于离心管中，1500rpm/min，离心3min，倒去上清液，细胞沉淀用完全培养基重悬并吹打均匀，然后分至3～5瓶培养。一般3～4天传代1次；贴壁生长细胞的传代：细胞贴壁长满至瓶底的80％左右，从培养箱中取出培养瓶，吸去培养基，用0.25％胰酶洗涤1次，然后加入0.25％的胰酶消化液消化，观察细胞收缩变圆后，加入完全培养基终止消化，并吹打使细胞分散脱落，收集细胞悬液，1500rpm/min，离心3min，倒去上清液，细胞沉淀用完全培养基重悬并吹打均匀，然后分至3～5瓶瓶 培养。一般3～4天传代1次。
3、细胞增殖抑制实验(MTT法)
收集对数生长期的细胞，以每孔2.5×10 3～1×10 4个的数量接种于96孔板中，在37℃、5％CO 2的细胞培养箱中培养过夜24小时，采用DMEM培养基稀释待测药物并加入到96孔板中，每种药物8个梯度，每个梯度含3个复孔。加药组中，按梯度(终浓度分别为1000、333、127、42.3、14.1、4.7、1.56、0.53nM)每孔加入100μL化合物的培养基溶液，每个浓度设3个复孔；阴性对照组每孔中加入含1‰DMSO的空白培养基100μL，共6个复孔；空白对照组每孔中只加入100μL培养基。将板置于37℃、5％CO 2细胞培养孵箱内培养72小时。药物处理组、隐形对照组和空白组每孔加入20μL MTT溶液(5mg/mL)，继续培养2-4小时，待甲瓒形成后，终止培养，倾去上清液后每孔加150μL DMSO(对于悬浮细胞则直接加入50μL 20％SDS溶液)，在摇床上摇15～20分钟。用酶标仪在570nm波长下检测每孔细胞吸光度(OD  570)，取其平均值记录结果。细胞增殖抑制率＝(对照组OD 570-实验组OD 570)/(对照组OD  570-空白OD 570)×100％。最后，使用Graphpad Prism软件拟合半数抑制浓度。
4、动物体内实验
取对数生长期良好的SNU16细胞，以5×10 5cells/100μl/只的量接种于Balb/C裸鼠皮下，大约接种后10天，肿瘤长到250cm 2左右，开始进行随机分组，每组7只。
1)给药途径：口服给药，溶剂为DMSO 5％，PEG400 45％生理盐水50％。
2)分组：溶剂组，AZD4547组(15mg/kg)，化合物9-15低剂量组(15mg/kg)，化合物9-15高剂量组(30mg/kg)
3)给药时间：肿瘤约为250cm 2分组后进行口服给药，连续给药10天。
4)评价指标：在给药10天后，考察裸鼠体重变化，肿瘤的生长曲线，肿瘤的大小，并进行比较。
三、实验结果
1、下表列举了本发明合成的部分化合物对FGFR1激酶的抑制率。
表2 本发明合成的部分化合物对FGFR1激酶的抑制率
Figure PCTCN2021079338-appb-000027
Figure PCTCN2021079338-appb-000028
从表2可以看出，本发明提供的具有氨基嘧啶并吡唑/吡咯骨架的化合物，对FGFR1激酶具有明显的抑制作用。
在此基础上，挑选在低浓度条件下(0.1μM)对FGFR1抑制率大于80％的优选化合物，即9-1和9-15，进一步测试其抑制FGFR1的IC 50值，以及对FGFR2-4和其它酪氨酸激酶的抑制作用。结果如下：
表3 优选化合物抑制FGFR1的IC 50值
Figure PCTCN2021079338-appb-000029
表4 优选化合物对其它酪氨酸激酶的抑制作用(IC 50，nM)
Figure PCTCN2021079338-appb-000030
从表3、表4可以看出，本发明化合物对FGFR和VEGFR(包括Flt1、Flt4、KDR亚型)等多种酪氨酸激酶具有抑制作用，这些激酶在肿瘤细胞存活、增殖、分化、迁移和肿瘤血管生成等过程中发挥着重要的调控作用，表明本发明化合物是潜在的多靶点抗肿瘤药物。
2、下表列举了本发明部分化合物对胃癌细胞株SNU16增殖的抑制情况。
表5 本发明部分化合物抑制胃癌细胞株增殖的IC 50值或抑制率
Figure PCTCN2021079338-appb-000031
Figure PCTCN2021079338-appb-000032
注：AZD4547是已知有效的FGFR抑制剂。
3、下表列举了本发明部分化合物对4T1鼠源乳腺癌细胞，A549人非小细胞肺癌细胞增殖的抑制情况(结果表示为IC 50值，μM；或化合物浓度为10μM时的抑制率)。
表6 本发明部分化合物对多种肿瘤细胞生长的抑制情况
化合物编号 4T1 A549
9-1 <3.3 <3.3
9-3 8.2 53％
9-7 40％ 45％
9-15 <3.3 <3.3
4、下表列举了本发明部分化合物对小鼠胚胎成纤维细胞NIH-3T3和人肝星状细胞LX2增殖的抑制情况。
表7 本发明部分化合物抑制纤维化细胞增殖的IC 50值或抑制率
Figure PCTCN2021079338-appb-000033
Figure PCTCN2021079338-appb-000034
5、体内动物实验结果
肿瘤生长曲线见图1，肿瘤测量情况见图2，肿瘤重量统计结果见图3，裸鼠体重变化曲线见图4。可以看出，本发明化合物9-15低剂量和高剂量均能显著抑制肿瘤的生长，甚至使肿瘤消除。而且，在同等剂量下(15mg/kg)，化合物9-15抑制肿瘤的生长的效果明显优于阳性对照药物AZD4547。
需要说明的是，本说明书中描述的具体特征、结构、材料或者特点可以在任一个或多个实施例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例以及不同实施例的特征进行结合和组合。

Claims (17)

  1. 式Ⅰ所示的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐：
    Figure PCTCN2021079338-appb-100001
    其中，R 1为Ar或
    Figure PCTCN2021079338-appb-100002
    Ar为取代或未取代的芳基；
    R 2为-H，或者R 2同1-位氮原子形成酰胺；
    X为CH或N。
  2. 如权利要求1所述的化合物，其特征是：R 1为
    Figure PCTCN2021079338-appb-100003
    Ar为取代或未取代的芳基。
  3. 如权利要求1或2所述的化合物，其特征是：Ar为取代或未取代的5～10元芳基。
  4. 如权利要求3所述的化合物，其特征是：所述芳基满足以下至少一项：
    所述的芳基为5～6元单环或9～10元稠环；
    所述的芳基含有0～2个杂原子；
    优选地，所述的杂原子为氮原子；
    优选地，所述的芳基选自：
    Figure PCTCN2021079338-appb-100004
    进一步优选地，所述的芳基为
    Figure PCTCN2021079338-appb-100005
  5. 如权利要求1～4任意一项所述的化合物，其特征是：所述取代的芳基含有至少一个选自下组的取代基：取代或未取代的烷氧基、卤素、取代或未取代的氨基、取代或未取代的烷基；
    优选地，所述取代的芳基含有至少一个选自下组的取代基：未取代的C1～C6烷氧基、卤素、未取代的氨基、C1～C6烷基取代的氨基、未取代的C1～C6烷基；
    优选地，所述取代的芳基含有至少一个选自下组的取代基：-OCH 3、-Cl、-NH 2。
  6. 如权利要求1～5任意一项所述的化合物，其特征是：Ar选自：
    Figure PCTCN2021079338-appb-100006
    优选地，Ar为
    Figure PCTCN2021079338-appb-100007
  7. 如权利要求1所述的化合物，其特征是：R 2为-H或
    Figure PCTCN2021079338-appb-100008
    R 3选自取代或未取代的烷基、取代或未取代的烯基；
    优选地，R 3选自取代或未取代的C1～C6烷基、取代或未取代的C2～C6烯基；
    优选地，R 3选自未取代的C1～C3烷基、取代或未取代的C2～C4烯基；
    优选地，所述取代的C2～C4烯基含有至少一个选自下组的取代基：-CN、C3～C6环烷基；
    优选地，R 2选自：-H、
    Figure PCTCN2021079338-appb-100009
    进一步优选地，R 2为
    Figure PCTCN2021079338-appb-100010
  8. 如权利要求1所述化合物的立体异构体，其特征是：结构如式Ⅰa或式Ⅰb所示：
    Figure PCTCN2021079338-appb-100011
    优选地，结构如式Ⅰa所示。
  9. 如权利要求1～8任意一项所述的化合物或其立体异构体，其特征是：选自：
    Figure PCTCN2021079338-appb-100012
  10. 如权利要求1所述化合物或其立体异构体在药学上可接受的盐，其特征是：所述的盐为盐酸盐。
  11. 权利要求1～10任意一项所述化合物的立体异构体的制备方法，其特征是：包括如下步骤：
    a、化合物1与化合物2或其对映异构体经过Mitsunobu反应，得到中间体1或其对映异构体：
    Figure PCTCN2021079338-appb-100013
    其中，Ha为卤素，LG为离去基团；
    优选地，Ha为-I；
    优选地，LG为-Boc；
    b、中间体1或其对映异构体与化合物3在钯催化剂作用下偶联，得到中间体2或其对映异构体：
    Figure PCTCN2021079338-appb-100014
    其中，R 5、R 6独立地选自H或烷基，或者，R 5与R 6相连形成脂环；
    优选地，化合物3为
    Figure PCTCN2021079338-appb-100015
    c、中间体2或其对映异构体脱去离去基团，即得化合物4或其对映异构体：
    Figure PCTCN2021079338-appb-100016
  12. 如权利要求11所述的制备方法，其特征是：满足以下至少一项：
    步骤a中化合物1：化合物2或其对映异构体的摩尔比为1：(1.0～1.5)；
    步骤a的反应溶剂为四氢呋喃；
    步骤a的反应体系中加入了偶氮二甲酸二异丙酯和三苯基膦；
    优选地，步骤a中化合物1：偶氮二甲酸二异丙酯的摩尔比为1：(2.0～3.0)；
    优选地，步骤a中化合物1：三苯基膦的摩尔比为1：(2.0～3.0)；
    步骤a在保护气氛下反应；
    步骤a的反应温度为0～50℃；
    步骤b中中间体1或其对映异构体：化合物3的摩尔比为1：(1.0～1.5)；
    步骤b的反应溶剂为1,4-二氧六环和水的混合溶剂，1,4-二氧六环：水的体积比为(4～8)：1；
    以中间体1或其对映异构体的摩尔量计，步骤b中钯催化剂的用量为3～10mmol％；
    步骤b中所述的钯催化剂选自醋酸钯、[1,1'-双(二苯基膦基)二茂铁]二氯化钯、[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物、三(二亚苄基茚丙酮)二钯中至少一种；
    步骤b的反应体系中加入了碱；
    优选地，步骤b中中间体1或其对映异构体：碱的摩尔比为1：(2.0～3.0)；
    优选地，步骤b中所述的碱选自碳酸钠、碳酸氢钠、碳酸钾、磷酸钾、碳酸铯中至少一种；
    步骤b在保护气氛下反应；
    步骤b的反应温度为90～110℃。
  13. 如权利要求11或12所述的制备方法，其特征是：还包括如下步骤：化合物4或其对映异构体与化合物5经过酰胺化反应，即得化合物6或其对映异构体：
    Figure PCTCN2021079338-appb-100017
    其中，Halo为卤素；优选地，Halo为-Cl。
  14. 权利要求1～10任意一项所述的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐在制备防治癌症的药物中的用途；优选地，所述的癌症为胃癌、乳腺癌、肺癌、胆管癌、尿路上皮癌、前列腺癌。
  15. 权利要求1～10任意一项所述的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐在制备防治器官纤维化的药物中的用途；优选地，所述的器官纤维化为肝纤维化或肺纤维化。
  16. 权利要求1～10任意一项所述的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐在制备FGFR和/或VEGFR抑制剂类药物中的用途；优选地，所述的FGFR抑制剂为FGFR1、FGFR2、FGFR3和/或FGFR4抑制剂；优选地，所述的VEGFR抑制剂为Flt1、Flt4和/或KDR抑制剂。
  17. 药物组合物，其特征是：它是以权利要求1～10任意一项所述的化合物、其立体异构体、化合物或其立体异构体在药学上可接受的盐为活性成分，加入药学上可接受的辅料或者辅助性成分，制备而成的制剂；优选地，所述的制剂为口服制剂。
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