WO2022127261A1 - 一种化合物及其制备方法以及其在制备治疗抗癌药物中的应用 - Google Patents

一种化合物及其制备方法以及其在制备治疗抗癌药物中的应用 Download PDF

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WO2022127261A1
WO2022127261A1 PCT/CN2021/120046 CN2021120046W WO2022127261A1 WO 2022127261 A1 WO2022127261 A1 WO 2022127261A1 CN 2021120046 W CN2021120046 W CN 2021120046W WO 2022127261 A1 WO2022127261 A1 WO 2022127261A1
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compound
preparation
nmr
pharmaceutically acceptable
formula
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PCT/CN2021/120046
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French (fr)
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杨诚
杨光
周红刚
张亮
李建
伦东超
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天津济坤医药科技有限公司
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Priority to JP2022554428A priority Critical patent/JP7525630B2/ja
Priority to KR1020227032105A priority patent/KR20220142500A/ko
Priority to EP21905192.7A priority patent/EP4116302A4/en
Priority to CA3196572A priority patent/CA3196572A1/en
Priority to AU2021398802A priority patent/AU2021398802B2/en
Priority to US17/915,817 priority patent/US20230159537A1/en
Publication of WO2022127261A1 publication Critical patent/WO2022127261A1/zh

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the technical field of pharmacy, in particular to 2H-pyrazolo[3,4-d]pyrimidine derivatives and a preparation method thereof and their application in the preparation of anticancer drugs.
  • Fibroblast growth factor receptor is a member of the receptor tyrosine kinase (RTK) family and belongs to the receptor-type protein tyrosine kinase, which contains four highly conserved transmembrane tyrosine kinases: FGFR1, FGFR2 , FGFR3 and FGFR4.
  • the signaling pathway of FGFR is that ligand-receptor binding induces FGFR dimerization, which leads to cascade activation of downstream signaling pathways such as Ras-MAPK, PI3K-Akt, STAT and PLC ⁇ .
  • FGFRs play important roles in a variety of cellular functions, such as cell proliferation and differentiation, and biological processes including development, angiogenesis, homeostasis, and wound repair. According to research surveys (1-3) , 7.1% of cancer patients have abnormal FGFR. For example, FGFR1 amplification was highest in patients with squamous NSCLC at 20%, breast cancer at 10%, ovarian cancer at 5%, and bladder cancer at 3%.
  • FGFR2 mutations have been identified in gastric cancer (10%), endometrial cancer (12%), squamous NSCLC (5%) and triple-negative breast cancer (4%), while FGFR3 mutations in bladder cancer (50-60%) and myeloma (15-20%) are well known.
  • FGFR inhibitors as novel anticancer agents for the treatment of FGFR-related cancers.
  • TKIs multi-targeted tyrosine kinase inhibitors
  • FGFR abnormalities such as dovitinib (TKI-258), lucitanib (E-3810) (8) , nintedanib (BIBF-1120) (9) , and ponatinib (AP-24534) (10) .
  • FGFR inhibitors were non-selective (11) .
  • FGFR tyrosine kinase inhibitors with various scaffolds have been developed.
  • tyrosine kinase inhibitors are currently in various stages of clinical studies, such as AZD-4547 (Phase III) (12) , BGJ-398 (Phase II) (13) , LY-2874455 (Phase II) (14) and JNJ-42756493 (Phase II) (15) , these selective FGFR inhibitors are ATP-competitive inhibitors that bind to the active form of FGFR.
  • FGFR inhibitors are ATP-competitive inhibitors that bind to the active form of FGFR.
  • many medicinal chemistry laboratories have reported their development of FGFR inhibitors (16-22) .
  • the technical problems to be solved/objects achieved by the present invention at least include: overcoming the defects in the prior art, proposing 2H-pyrazolo[3,4-d]pyrimidine derivatives and their preparation methods, and their use in the preparation of anticancer drugs applications in .
  • the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:
  • Ar is any one of a substituted aromatic ring group and a substituted aromatic heterocyclic group
  • R is any one of H, alkyl, aryl, -CF and alkyl tertiary amine structures
  • Linker is any one of an alkyl group, an alkoxy group, a heteroatom substituent, and a substituted N heterocyclic ring.
  • the Ar 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
  • the R is H, CF 3 , (CH 3 ) 2 NCH 2 or CH 3 .
  • the compound is one of compounds 1 to 40:
  • the present invention also provides the preparation method of the compound described in the above-mentioned technical scheme or its pharmaceutically acceptable salt, and its synthetic route is:
  • the compound with the structure shown in formula a and the compound with the structure shown in formula b are subjected to Mitsunobu reaction to obtain the compound with the structure shown in formula c;
  • the temperature of the Mitsunobu reaction is room temperature, and the time is 4h;
  • the Suzuki coupling (Suzuki coupling) reaction is performed on the compound with the structure shown in the formula c and the compound with the structure shown in the formula d to obtain the compound with the structure shown in the formula e; the temperature of the Suzuki coupling reaction is 80 ° C;
  • Deprotection reaction of the compound with the structure represented by the formula e and trifluoroacetic acid is carried out to obtain the compound with the structure represented by the formula f; the temperature of the deprotection reaction is 0°C;
  • the acylation reaction of the compound with the structure shown in formula f is carried out to obtain the compound with the structure shown in the formula I; the temperature of the acylation reaction is room temperature;
  • the present invention also provides the application of the compound described in the above technical solution and a pharmaceutically acceptable salt thereof in the preparation of a tumor cell proliferation inhibitor, wherein the tumor cell is a related tumor cell with abnormal FGFR.
  • the tumor cells include NCI-H1581 or SNU-16.
  • the present invention also provides the use of the compounds described in the above technical solutions and their pharmaceutically acceptable salts in blocking the FGF/FGFR signaling pathway in tumor cells, which are related tumor cells with abnormal FGFR.
  • the tumor cells include NCI-H1581 or SNU-16.
  • the present invention also provides a pharmaceutical composition containing the compound described in the above technical solution and a pharmaceutically acceptable salt thereof, including one or more pharmaceutically acceptable excipients;
  • the dosage form of the pharmaceutical composition is any pharmaceutically acceptable dosage form.
  • the present invention also provides the application of the compound described in the above technical solution, a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in the above technical solution in preparing a medicine for treating and/or preventing cancer.
  • the cancer is a related cancer with FGFR abnormalities.
  • the present invention also provides the application of the pharmaceutical composition described in the above technical scheme for preparing an irreversible pan-fibroblast growth factor receptor inhibitor.
  • the present invention also provides a method for treating cancer, comprising the following steps:
  • the drugs for treating cancer include the compounds described in the above technical solutions, and pharmaceutically acceptable salts thereof.
  • the drug for treating cancer includes compound 10 or compound 36.
  • the administered dose is 50 mg/kg or 100 mg/kg;
  • the administered dose is based on the amount of the compound 10 or compound 36.
  • the mode of administration is oral.
  • the mode of administration is intraperitoneal injection.
  • pharmaceutically acceptable adjuvant includes pharmaceutically acceptable carriers, excipients, diluents, etc., which are compatible with the active pharmaceutical ingredients.
  • pharmaceutically acceptable carriers include pharmaceutically acceptable carriers, excipients, diluents, etc., which are compatible with the active pharmaceutical ingredients.
  • the preparation of pharmaceutical formulations using pharmaceutically acceptable excipients is well known to those of ordinary skill in the art.
  • the present invention combines the pharmaceutical composition and pharmaceutically acceptable adjuvants (such as carriers, excipients, diluents, etc. well known to those of ordinary skill in the art) to prepare various preparations, preferably solid preparations and liquid preparations
  • adjuvants such as carriers, excipients, diluents, etc. well known to those of ordinary skill in the art
  • various preparations preferably solid preparations and liquid preparations
  • Formulations such as tablets, pills, capsules, powders, suspensions, granules, syrups, emulsions, suspensions, etc., as well as various sustained-release dosage forms, are preferably in the form of oral administration.
  • Figure 1 shows that compound 10 inhibits the activation of FGFR1 and downstream signaling pathways in NCI-H1581 cells and inhibits the activation of FGFR2 and downstream signaling pathways in SNU-16 cells in a concentration-dependent manner, wherein A is the inhibition of FGFR1 and downstream signaling pathways in NCI-H1581 cells Activation of signaling pathways, B is to inhibit the activation of FGFR2 and downstream signaling pathways in SNU-16 cells;
  • Figure 2 shows that compound 36 concentration-dependently inhibits the activation of FGFR1 and downstream signaling pathways in NCI-H1581 cells and inhibits the activation of FGFR2 and downstream signaling pathways in SUN-16 cells, wherein A is the inhibition of FGFR1 and downstream signaling pathways in NCI-H1581 cells Activation of signaling pathways, B is to inhibit the activation of FGFR2 and downstream signaling pathways in SUN-16 cells;
  • Figure 3 shows the tumor volume curve graph (A) of compound 10 inhibiting tumor proliferation in the subcutaneous transplanted tumor model of NCI-H1581 tumor cells, the tumor tumor weight statistics graph (B) in mice after 26 days of treatment, and the body weight of mice during the treatment process.
  • Figure 4 is a graph showing the tumor volume change curve (A) of compound 36 inhibiting tumor proliferation in the subcutaneous transplanted tumor model of NCI-H1581 tumor cells (A), a statistical graph of tumor tumor weight in mice after 26 days of treatment (B), and the mice during the treatment process.
  • test reagents used in the following examples are conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified.
  • the specific preparation method is:
  • reaction was continued for 1 h at room temperature, and the completion of the reaction was monitored by TLC.
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 1-Boc-3-hydroxymethylpyrrolidine, and equimolar replaces 1 in the preparation method of compound (1-1).
  • -Boc-3-azetidine methanol the NMR test results of compound 2 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is N-Boc-4-hydroxypiperidine, and equimolar replaces 1-Boc in the preparation method of compound (1-1). -3-azetidine methanol, the NMR test results of compound 3 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is N-Boc-4-piperidinemethanol, and equimolar replaces 1-Boc in the preparation method of compound (1-1). -3-azetidine methanol, the NMR test results of compound 4 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is N-Boc-4-piperidineethanol, and equimolar replaces 1-Boc in the preparation method of compound (1-1). -3-azetidine methanol, the NMR test results of compound 5 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 1-Boc-3-hydroxymethylpiperidine, and equimolar replaces 1 in the preparation method of compound (1-1).
  • -Boc-3-azetidine methanol the NMR test results of compound 6 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is tert-butyl-4-(2-hydroxyethyl)piperazine-1-carboxylate, and equimolar substitution compound ( 1-Boc-3-azetidine methanol in the preparation method of 1-1), the nuclear magnetic test result of compound 7 is:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 1-BOC-4-(3-hydroxypropane)piperazine, and equimolar replaces the preparation method of compound (1-1) 1-Boc-3-azetidine methanol in the 1-Boc-3-azetidine methanol, the NMR test results of compound 8 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 1-BOC-4-(4-hydroxybutane)piperazine, which is equimolar instead of the preparation of compound (1-1).
  • 1-Boc-3-azetidine methanol in the method the nuclear magnetic test results of compound 9 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 1-BOC-4-(5-hydroxypentane)piperazine, which is equimolar instead of the preparation of compound (1-1).
  • 1-Boc-3-azetidine methanol in the method the NMR test results of compound 10 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 1-BOC-4-(6-hydroxyhexane)piperazine, which is equimolar instead of the preparation of compound (1-1).
  • 1-Boc-3-azetidine methanol in the method the NMR test results of compound 11 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 1-BOC-4-(7-hydroxyheptane)piperazine, which is equimolar instead of the preparation of compound (1-1).
  • 1-Boc-3-azetidine methanol in the method the nuclear magnetic test results of compound 12 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 3,4-methylenephenylboronic acid, and equimolar replaces 3,5 in the preparation method of compound (1-2).
  • -Dimethoxybenzeneboronic acid the NMR test results of compound 13 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is benzofuran-2-boronic acid, and equimolar replaces 3,5-dicarbonate in the preparation method of compound (1-2).
  • the NMR test results of methoxybenzeneboronic acid, compound 14 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is p-methoxyboronic acid, and equimolar replaces 3,5-dimethoxy in the preparation method of compound (1-2).
  • the NMR test results of phenylboronic acid, compound 15 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 4-acetamidophenylboronic acid, and equimolar replaces 3,5-dimethyl in the preparation method of compound (1-2).
  • the NMR test results of oxyphenylboronic acid, compound 16 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 4-trifluoromethoxybenzeneboronic acid, and equimolar replaces 3,5 in the preparation method of compound (1-2).
  • -Dimethoxyphenylboronic acid the NMR test results of compound 17 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 2-naphthalene boronic acid, and equimolar replaces 3,5-dimethoxy in the preparation method of compound (1-2).
  • the NMR test results of phenylboronic acid, compound 18 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 4-methylnaphthalene boronic acid, and equimolar replaces 3,5-dimethyl in the preparation method of compound (1-2).
  • the NMR test results of oxyphenylboronic acid, compound 19 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 4-bromonaphthalene boronic acid, and equimolar replaces 3,5-dimethoxyl in the preparation method of compound (1-2).
  • the NMR test results of phenylboronic acid, compound 20 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 2,5-dimethoxybenzeneboronic acid, and equimolar replaces 3 in the preparation method of compound (1-2), 5-dimethoxybenzeneboronic acid, the NMR test results of compound 21 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 3,4-dimethoxybenzeneboronic acid, and equimolar replaces 3 in the preparation method of compound (1-2), 5-Dimethoxybenzeneboronic acid, the NMR test results of compound 22 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 3,4,5-trimethoxybenzeneboronic acid, and equimolar replaces 3 in the preparation method of compound (1-2).
  • 5-dimethoxybenzeneboronic acid the NMR test results of compound 23 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 3-trifluoromethylbenzeneboronic acid, and equimolar replaces 3,5-
  • the NMR test results of dimethoxybenzeneboronic acid, compound 24 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 3-isopropoxyphenylboronic acid, and equimolar replaces 3,5-
  • the NMR test results of dimethoxybenzeneboronic acid, compound 25 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 3-benzyloxyphenylboronic acid, and equimolar replaces 3,5-dicarbonate in the preparation method of compound (1-2).
  • the NMR test results of methoxybenzeneboronic acid, compound 26 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 2-chloro-5-methoxybenzeneboronic acid, and equimolar replaces 3 in the preparation method of compound (1-2).
  • 5-dimethoxybenzeneboronic acid the NMR test results of compound 27 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is p-fluorophenylboronic acid, and equimolar replaces 3,5-dimethoxy in the preparation method of compound (1-2).
  • the NMR test results of phenylboronic acid, compound 28 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 3-fluoro-4-hydroxyphenylboronic acid, and equimolar replaces 3,5 in the preparation method of compound (1-2).
  • -Dimethoxybenzeneboronic acid the NMR test results of compound 29 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 6-methoxynaphthalene-2-boronic acid, and equimolar replaces 3 in the preparation method of compound (1-2), 5-dimethoxybenzeneboronic acid, the NMR test results of compound 30 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is p-nitrophenylboronic acid, and equimolar replaces 3,5-dimethoxyl in the preparation method of compound (1-2).
  • the NMR test results of phenylboronic acid, compound 31 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 3-fluoro-5-methoxybenzeneboronic acid, and equimolar replaces 3 in the preparation method of compound (1-2).
  • 5-dimethoxybenzeneboronic acid the NMR test results of compound 31 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 3,5-difluorophenylboronic acid, and equimolar replaces 3,5-difluorophenylboronic acid in the preparation method of compound (1-2).
  • the NMR test results of dimethoxybenzeneboronic acid, compound 33 are:
  • the specific preparation method is: similar to the preparation method of compound 1, except that one of the raw materials used is 3,5-ditrifluoromethylbenzeneboronic acid, and equimolar replaces 3 in the preparation method of compound (1-2).
  • 5-dimethoxybenzeneboronic acid the NMR test results of compound 34 are:
  • the specific preparation method is: similar to the preparation method of compound 1, the difference is that one of the raw materials used is 3-fluoro-5-trifluoromethylbenzeneboronic acid, and equimolar replaces the
  • the NMR test results of 3,5-dimethoxybenzeneboronic acid, compound 35 are:
  • the specific preparation method is: similar to the preparation method of compound 10, the difference is that one of the raw materials used is 4,4,4-trifluorobutenoic acid, and equimolar replaces propylene in the preparation method of compound (1-3)
  • the NMR test results of acid chloride, compound 36 are:
  • the specific preparation method is: similar to the preparation method of compound 34, except that one of the raw materials used is 4,4,4-trifluorobutenoic acid, and equimolar replaces propylene in the preparation method of compound (1-3) Acid chloride, the NMR test result of compound 37 is:
  • the specific preparation method is: similar to the preparation method of compound 35, except that one of the raw materials used is 4,4,4-trifluorobutenoic acid, and equimolar replaces propylene in the preparation method of compound (1-3)
  • the NMR test results of acid chloride, compound 38 are:
  • the specific preparation method is: similar to the preparation method of compound 10, the difference is that one of the raw materials used is trans-4-dimethylaminocrotonic acid, and equimolar replaces propylene in the preparation method of compound (1-3)
  • the NMR test results of acid chloride, compound 39 are:
  • the specific preparation method is: similar to the preparation method of compound 10, the difference is that one of the raw materials used is crotonic acid, and equimolar replaces the acryloyl chloride in the preparation method of compound (1-3), and the NMR test result of compound 40 is :
  • the present invention adopts homogeneous time-resolved fluorescence (HIRF) technology to evaluate the inhibitory effect of compounds on four subtypes of FGFR1, 2, 3 and 4.
  • HIRF time-resolved fluorescence
  • compounds 10 and 36 were selected, and the MTT colorimetric method was used to evaluate the proliferation inhibitory effects of the compounds 10 and 36 on various tumor cells.
  • Tumor cells in logarithmic growth phase were collected by trypsinization, resuspended in 1 mL of fresh medium, and then diluted with a small amount, and the number of cells was counted with a hemocytometer.
  • tumor cells were seeded into 96-well plates at a density of 3000-5000 cells/well, and then placed in a cell culture incubator for 24 hours. Then, the tumor cells were treated with compounds 10 and 36, and 8 concentrations were set, and three replicate wells were set for each concentration, and the cells were incubated for 96 h.
  • MTT solution 5 mg/mL was added to each well, beat and mixed, and then placed in a cell culture incubator for 3-4 hours. After incubation, carefully aspirate the liquid from the well plate, then add 100 ⁇ L of DMSO solution to each well. Set the microplate reader program, measure the absorbance values at 490nm and 570nm respectively, calculate the proliferation inhibition rate of each concentration according to the formula, and finally use Graphpad Prism 7 software for data processing to calculate IC50.
  • Example 43 Compounds 10 and 36 inhibit FGF/FGFR and its downstream signaling pathways
  • the old medium was aspirated, fresh medium containing compounds 10 and 36 at different concentrations was added, and the cells were incubated in a cell incubator for 12 h. After incubation, aspirate the supernatant, wash three times with pre-cooled 1X PBS solution, thoroughly remove the residual liquid, add 60 ⁇ L of RIPA lysis buffer to each well, gently scrape the cells with a cell scraper, and transfer the lysis buffer to In a 1.5mL EP tube, ice bath for 10min, centrifuge at 14000rpm for 10min, pipette the supernatant into a new 1.5mL EP tube, pipette a small amount for protein content determination, add a corresponding volume of 5X loading buffer to the rest, vortex to mix, 100 °C metal bath for 10min.
  • the corresponding loading amount was calculated according to the loading amount of 30 ⁇ g protein per well.
  • Use 8% SDS-PAGE gel electrophoresis to separate the protein transfer the separated protein to PDVF membrane, stain with Ponceau red, cut the PDVF membrane containing the protein according to the molecular weight of the target protein, wash the Ponceau red with 1X TBST solution, 5% nonfat dry milk was blocked for 30 min. Wash three times with 1X TBST solution, put the PDVF membrane containing the target protein band into the incubation box, add the corresponding primary antibody incubation solution, and incubate at 4°C overnight.
  • compounds 10 and 36 not only significantly inhibited the phosphorylation of FGFR1 in NCI-H1581 cells and FGFR2 in SNU-16 cells, but also inhibited the activation of downstream PLC ⁇ , AKT and ERK in a dose-dependent manner. Collectively, compounds 10 and 36 can strongly inhibit FGF/FGFR and downstream signaling pathways in vitro.
  • Example 44 Compounds 10 and 36 significantly inhibit tumor growth in mice
  • a mouse subcutaneous xenograft tumor model was established with human lung squamous cell carcinoma cells NCI-H1581 with high FGFR1 expression.
  • mice When the tumor volume grows to 50-100mm , the mice are randomly divided into three groups (control group, 50mg/kg, 100mg/kg), 6 mice in each group, compound 10 is administered orally every day, compound 36 is intraperitoneally every day After injection, the tumor volume and mouse body weight were measured. After 26 days of administration, the mice were treated, and the tumor tissue was dissected and fixed in formalin solution for use.
  • the present inventors optimized and discovered novel derivatives of 2H-pyrazolo[3,4-d]pyrimidines, compounds 10 and 36, as potent and selective inhibitors against FGFR1, FGFR2 and FGFR3, characterized irreversible binding.
  • the lead compound not only inhibited FGF/FGFR and its downstream signaling pathways at low concentrations, but also showed significant anti-proliferative effects on NCI-H1581 cancer cell line both in vitro and in vivo.
  • compounds 10 and 36 show low toxicity and have good PK properties and are currently being identified as potential drug candidates.

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Abstract

本发明涉及吡唑并[3,4-d]嘧啶类化合物，制备方法和在制备治疗抗肿瘤药物中的用途。该类化合物对FGFR1-3具有抑制作用，对NCI-H1581和SNU-16癌细胞系表现出抗增殖作用。

Description

一种化合物及其制备方法以及其在制备治疗抗癌药物中的应用
本申请要求于2020年12月16日提交中国专利局、申请号为202011483614.1、发明名称为“一种化合物及其制备方法以及其在制备治疗抗癌药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及制药技术领域，尤其涉及2H-吡唑并[3,4-d]嘧啶衍生物及其制备方法以及其在制备治疗抗癌药物中的应用。
背景技术
成纤维细胞生长因子受体(FGFR)是受体酪氨酸激酶(RTK)家族成员，属于受体型蛋白酪氨酸激酶，其包含四个高度保守的跨膜酪氨酸激酶：FGFR1、FGFR2、FGFR3和FGFR4。FGFR的信号传导途径为配体与受体的结合诱导FGFR二聚化，从而引起下游信号通路(如Ras-MAPK，PI3K-Akt，STAT和PLCγ)的级联激活。FGFR在多种细胞功能中发挥重要作用，例如细胞增殖和分化以及包括发育、血管新生、体内稳态和伤口修复在内的生物学过程。据研究调查 (1-3)，占比7.1％的癌症患者，FGFR都异常。例如，鳞状NSCLC患者中FGFR1扩增的比例最高，为20％，乳腺癌为10％，卵巢癌5％，膀胱癌3％。目前，已在胃癌(10％)，子宫内膜癌(12％)，鳞状NSCLC(5％)和三阴性乳腺癌(4％)中均发现了FGFR2的突变，而FGFR3的突变在膀胱癌(50-60％)和骨髓瘤(15-20％)中是众所周知的。异常的FGF19/FGFR4与肝细胞癌(HCC)密切相关。因此，开发新型FGFR选择性抑制剂来阻断FGF/FGFR信号通路为FGFR相关的癌症提出了有希望的治疗策略 (4-6)。
研究者们致力于研发FGFR抑制剂作为治疗FGFR相关的癌症的新型抗癌剂。首先，最初采用了几种多靶点酪氨酸激酶抑制剂(TKIs)来治疗具有FGFR异常的相关癌症 (7)，例如dovitinib(TKI-258)，lucitanib(E-3810) (8)，nintedanib(BIBF-1120) (9)，和ponatinib(AP-24534) (10)。这些早期 研发的FGFR抑制剂是非选择性的 (11)。后来，已经开发了几种带有各种支架的选择性FGFR酪氨酸激酶抑制剂。一些酪氨酸激酶抑制剂目前处于不同阶段的临床研究中，例如AZD-4547(III期) (12)，BGJ-398(II期) (13)，LY-2874455(II期) (14)和JNJ-42756493(II期) (15)，这些选择性FGFR抑制剂是与FGFR活性形式结合的ATP竞争性抑制剂。此外，还有很多药物化学实验室报道了他们开发的FGFR抑制剂 (16-22)。
发明内容
本发明要解决的技术问题/达到的目的至少包括：克服现有技术中的缺陷，提出2H-吡唑并[3,4-d]嘧啶衍生物及其制备方法以及其在制备治疗抗癌药物中的应用。
为实现上述目的，本发明公开了下述技术方案：
本发明提供了一种如式I所示的化合物或其在药学上可接受的盐：
Figure PCTCN2021120046-appb-000001
所述式I中，Ar为取代芳环基团和取代芳杂环基团中的任意一种；
R为H、烷基、芳基、-CF 3和烷基叔胺结构中的任意一种；
Linker为烷基、烷氧基、杂原子取代基、取代N杂环中的任意一种。
优选的，所述Ar为
Figure PCTCN2021120046-appb-000002
Figure PCTCN2021120046-appb-000003
Figure PCTCN2021120046-appb-000004
优选的，所述R为H、CF 3、(CH 3) 2NCH 2或CH 3。
优选的，所述Linker为
Figure PCTCN2021120046-appb-000005
Figure PCTCN2021120046-appb-000006
优选的，所述化合物为化合物1～40中的一种：
Figure PCTCN2021120046-appb-000007
Figure PCTCN2021120046-appb-000008
Figure PCTCN2021120046-appb-000009
Figure PCTCN2021120046-appb-000010
Figure PCTCN2021120046-appb-000011
Figure PCTCN2021120046-appb-000012
Figure PCTCN2021120046-appb-000013
本发明还提供了上述技术方案所述化合物或其药学上可接受的盐的制备方法，其合成路线为：
Figure PCTCN2021120046-appb-000014
优选的，包括以下步骤：
将具有式a所示结构化合物和具有式b所示结构化合物发生光延(Mitsunobu)反应，得到具有式c所示结构化合物；所述延光反应的温度为室温，时间为4h；
将所述具有式c所示结构化合物和具有式d所示结构化合物发生铃木偶联(Suzuki coupling)反应，得到具有式e所示结构化合物；所述铃木偶联反应的温度为80℃；
将所述具有式e所示结构化合物和三氟乙酸发生脱保护反应，得到具有式f所示结构化合物；所述脱保护反应的温度为0℃；
将所述具有式f所示结构化合物发生酰基化反应，得到具有式I所示结构化合物；所述酰基化反应的温度为室温；
Figure PCTCN2021120046-appb-000015
Ar-B(OH) 2式d、
Figure PCTCN2021120046-appb-000016
本发明还提供了上述技术方案所述的化合物、其药学上可接受的盐在制备肿瘤细胞增殖抑制剂中的应用，所述肿瘤细胞为具有FGFR异常的相关的肿瘤细胞。
优选的，所述肿瘤细胞包括NCI-H1581或SNU-16。
本发明还提供了上述技术方案所述的化合物、其药学上可接受的盐在阻断肿瘤细胞中FGF/FGFR信号通路中的应用，所述肿瘤细胞为具有FGFR异常的相关的肿瘤细胞。
优选的，所述肿瘤细胞包括NCI-H1581或SNU-16。
本发明还提供了一种含有上述技术方案所述的化合物、其药学上可接受的盐的药物组合物，包括一种或多种药学上可接受的赋形剂；
所述药物组合物的剂型为药学上可接受的任一剂型。
本发明还提供了上述技术方案所述的化合物、其药学上可接受的盐或上述技术方案所述的药物组合物在制备治疗和/或预防癌症的药物中的应用。
优选的，所述癌症为具有FGFR异常的相关癌症。
本发明还提供了上述技术方案所述的药物组合物在用于制备作为不可逆的泛成纤维细胞生长因子受体抑制剂中的应用。
本发明还提供了一种治疗癌症的方法，包括以下步骤：
通过口服或者腹腔注射的方式，将治疗癌症的药物对患者进行给药；
所述治疗癌症的药物包括上述技术方案所述的化合物、其药学上可接受的盐。
优选的，所述治疗癌症的药物包括化合物10或化合物36。
优选的，所述给药的剂量为50mg/kg或100mg/kg；
所述给药的剂量以所述化合物10或化合物36的用量计。
优选的，当所述治疗癌症的药物包括所述化合物10时，所述给药的方式为口服。
优选的，当所述治疗癌症的药物包括所述化合物36时，所述给药的方式为腹腔注射。
在本发明中，术语“药学上可接受的辅料”包括药学上可接受的载体、赋形剂、稀释剂等，它们与药物活性成分相容。运用药学上可接受的辅料制备药物制剂对本领域普通技术人员来说是公知的。
本发明将药物组合物和药学上可接受的辅剂(如本领域普通技术人员所熟知的载体、赋形剂、稀释剂等)组合在一起，配制成各种制剂，优选为固体制剂和液体制剂，如片剂、丸剂、胶囊、粉剂、混悬剂、颗粒剂、 糖浆剂、乳液剂、悬浮液等剂型以及各种缓释剂型，优选以口服给药形式。
附图说明
图1为化合物10浓度梯度依赖性地抑制NCI-H1581细胞中FGFR1以及下游信号通路的激活和抑制SNU-16细胞中FGFR2以及下游信号通路的激活，其中A为抑制NCI-H1581细胞中FGFR1以及下游信号通路的激活，B为抑制SNU-16细胞中FGFR2以及下游信号通路的激活；
图2为化合物36浓度梯度依赖性地抑制NCI-H1581细胞中FGFR1以及下游信号通路的激活和抑制SUN-16细胞中FGFR2以及下游信号通路的激活，其中A为抑制NCI-H1581细胞中FGFR1以及下游信号通路的激活，B为抑制SUN-16细胞中FGFR2以及下游信号通路的激活；
图3为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物10抑制肿瘤增殖的肿瘤体积曲线图(A)、处理26天后小鼠体内的肿瘤瘤重统计图(B)、处理过程中小鼠体重变化曲线图(C)和处理26天后小鼠体内肿瘤的照片图(D)；
图4为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物36抑制肿瘤增殖的肿瘤体积变化曲线图(A)、处理26天后小鼠体内的肿瘤瘤重统计图(B)、处理过程中小鼠体重变化曲线图(C)/处理26天后小鼠体内肿瘤的照片图(D)和免疫组化检测不同处理组织中FGFR1的磷酸化水平(E)。
具体实施方式
除有定义外，以下实施例中所用的技术术语具有与本发明创造所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂，如无特殊说明，均为常规生化试剂；所述实验方法，如无特殊说明，均为常规方法。
下面结合实施例来详细说明本发明创造。
实施例1：化合物1的制备
Figure PCTCN2021120046-appb-000017
具体制备方法为：
1)化合物(1-1)的制备：
Figure PCTCN2021120046-appb-000018
将3-溴-1H-吡唑并[3,4-D]嘧啶-4-胺(500mg，2.34mmol)溶解在干燥的THF(23mL)中，加入三苯基膦(2.08g，7.94mmol)，1-Boc-3-氮杂环丁烷甲醇。然后在0℃，氩气保护下，滴加偶氮二甲酸二异丙酯(1.60g，7.94mmol)。滴加完毕后，缓慢升至室温继续反应4h。反应液旋干，柱层析纯化(石油醚：乙酸乙酯＝5:1至2:1)得化合物1-1(黄色油状液体，685mg，69％)。
化合物1-1的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.06(s,1H),7.96–7.87(m,6H),7.64–7.45(m,9H),4.48(d,J＝7.3Hz,2H),3.97(t,J＝8.5Hz,2H),3.88–3.74(m,2H),3.19–2.98(m,1H),1.41(s,9H). 13C NMR(100MHz,CDCl 3)δ163.1,163.0,156.2,155.9,154.6,154.5,133.4,133.4,133.3,133.3,132.4,132.3,132.2,128.8,128.7,128.7,128.6,128.6,128.5,128.4,127.8,127.4,120.9,107.1,79.3,52.9,51.5,49.7,28.8,28.4,21.7,14.2.HRMS(ESI)calculated for C 32H 32BrN 6NaO 2P +:665.1400,found 665.1402.
2)化合物(1-2)的制备
Figure PCTCN2021120046-appb-000019
将化合物1-1(5.01mg，6.74mmol)，3,5-二甲氧基苯硼酸(1.59g，8.76mmol)溶解在1,4-二氧六环：水(9mL，v:v＝3:1)的混合溶液中，然后加入碳酸钾(1.87g，13.5mmol)，Pd(PPh 3) 4(778mg，0.674mmol)。体系氩气置换3次，油浴下升温至80℃，反应过夜，反应完成后用饱和氯化钠洗(270mL)，乙酸乙酯萃取(90mL)，有机相干燥浓缩，柱层析纯化(二氯甲烷：甲醇＝70:1至30:1)得化合物1-2(黄色油状液体，4.3g，60％)。
化合物1-2的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.06(d,J＝3.2Hz,1H),7.89–7.71(m,6H),7.56–7.40(m,11H),6.70–6.41(m,1H),4.58(dd,J＝7.3,3.2Hz,2H),4.11–3.96(m,2H),3.92–3.84(m,2H),3.76(d,J＝3.1Hz,6H),3.26–3.07(m,1H),1.41(d,J＝3.1Hz,9H). 13C NMR(100MHz,CDCl 3)δ163.9,163.9,163.5,163.4,160.3,156.3,156.3,155.2,154.7,146.1,135.6,134.2,133.5,133.4,133.4,133.3,132.3,132.2,132.0,132.0,132.0,1292,128.6,128.6,128.5,128.5,128.4,128.4,128.2,128.2,127.8,107.9,100.6,79.3,77.4,55.3,49.5,28.9,28.4,24.5.HRMS(ESI)calculated for C 40H 41N 6NaO 4P +:723.2819,found 723.2822.
3)化合物(1-3)的制备
Figure PCTCN2021120046-appb-000020
0℃下，将化合物1-2(450mg，0.587mmol)溶解于二氯甲烷中(5mL)， 然后缓慢滴加三氟乙酸(5mL)。30min后反应完全。反应液饱和NaHCO 3调碱，有机相浓缩干得棕黄色油状中间体。0℃下，将该中间体溶解于5mL二氯甲烷中，然后加入TEA(98.2μL，0.704mmol)，滴加丙烯酰氯(52.2μL，0.646mmol)。室温下继续反应1h，TLC监测反应完全，反应液用饱和NaCl洗，乙酸乙酯萃取，有机相干燥浓缩，柱层析分离纯化(二氯甲烷：甲醇＝30:1至10:1)得化合物1-3(黄色油状液体，78mg，72％)。
4)化合物1的制备：将化合物1-3(100mg，0.189mmol)中加入于醋酸：四氢呋喃：水(3mL，3:1:2)的混合溶液。室温下反应18h，TLC监测反应完全。反应液用饱和碳酸氢钠溶液调碱至PH 7～8(15mL)，乙酸乙酯萃取(10mL×2)，有机相干燥浓缩，柱层析纯化(二氯甲烷：甲醇＝15:1至7:1)得化合物5a(黄色固体，57.8mg，70％)。
化合物1的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.38(s,1H),6.80(d,J＝2.3Hz,2H),6.57(t,J＝2.3Hz,1H),6.33(dd,J＝17.0,1.9Hz,1H),6.17(dd,J＝17.0,10.3Hz,1H),5.76–5.57(m,3H),4.76–4.61(m,2H),4.38–4.14(m,3H),4.02(dd,J＝10.5,5.5Hz,1H),3.87(s,6H),3.49–3.25(m,1H). 13C NMR(100MHz,CDCl 3)δ165.7,161.6,157.8,156.2,154.7,144.7,134.8,127.4,127.2,125.7,106.3,101.2,98.4,55.6,53.5,50.8,49.4,32.7,29.1,27.9.HRMS(ESI)calculated for C 20H 22N 6NaO 3 +:417.1646,found 417.1648.
实施例2：化合物2的制备
Figure PCTCN2021120046-appb-000021
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-Boc-3-羟甲基吡咯烷，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物2的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.38(s,1H),6.81(d,J＝2.2Hz,2H),6.62–6.54(m,2H),6.25(dd,J＝16.9,2.0Hz,1H),5.76(s,2H),5.66(dd,J＝10.6,2.0Hz,1H),4.65(d,J＝13.3Hz,1H),4.49(t,J＝7.2Hz,2H),3.99(d,J＝13.7Hz,1H),3.86(s,6H),3.00(t,J＝12.6Hz,1H),2.64–2.52(m,1H),1.97–1.94(m,1H),1.91(s,2H). 13C NMR(100MHz,CDCl 3)δ164.5,161.5,158.0,156.0,154.6,144.6,134.9,128.5,127.5,106.4,101.3,98.3,55.6,50.0,49.1,45.7,45.2,39.9,37.7429.6,27.9.HRMS(ESI)calculated for C 21H 24N 6NaO 3 +:431.1802,found 431.1804.
实施例3：化合物3的制备
Figure PCTCN2021120046-appb-000022
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为N-Boc-4-羟基哌啶，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物3的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.38(s,1H),6.80(d,J＝2.2Hz,2H),6.67–6.51(m,2H),6.31(dd,J＝16.8,1.8Hz,1H),5.84–5.63(m,3H),5.05(tt,J＝11.3,4.2Hz,1H),4.85(d,J＝13.4Hz,1H),4.20(d,J＝13.9Hz,1H),3.86(s,6H),3.33(t,J＝12.7Hz,1H),3.02–2.85(m,1H),2.34(t,J＝11.8Hz,2H),2.11(d,J＝13.0Hz,2H). 13C NMR(101MHz,CDCl 3)δ161.5,157.8,155.8,154.7,154.3,144.1,135.1,132.1,131.6,106.3,101.1,100.0,98.3,79.6,55.7,55.6,52.9,45.5,29.7,28.4,26.8.HRMS(ESI)calculated for C 21H 24N 6NaO 3 +:431.1802,found 431.1805.
实施例4：化合物4的制备
Figure PCTCN2021120046-appb-000023
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为N-Boc-4-哌啶甲醇，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物4的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.34(s,1H),6.80(d,J＝2.3Hz,2H),6.57–6.49(m,2H),6.23(d,J＝16.8Hz,1H),5.89(s,2H),5.64(d,J＝10.6Hz,1H),4.65(d,J＝13.3Hz,1H),4.33(d,J＝7.1Hz,2H),3.97(d,J＝13.8Hz,1H),3.85(s,6H),3.00(t,J＝12.9Hz,1H),2.62(t,J＝12.9Hz,1H),2.33(d,J＝11.0Hz,1H),1.66(d,J＝16.3Hz,3H),1.33(td,J＝14.3,7.5Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.4,161.5,161.5,157.9,156.0,154.7,144.3,134.9,127.9,127.9,127.4,106.4,101.1,99.9,98.2,55.6,51.9,45.6,41.8,36.8,30.5,29.4.HRMS(ESI)calculated for C 22H 26N 6NaO 3 +:445.1959,found 445.1961.
实施例5：化合物5的制备
Figure PCTCN2021120046-appb-000024
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原 料之一为N-Boc-4-哌啶乙醇，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物5的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.38(d,J＝1.1Hz,1H),6.81(d,J＝2.3Hz,2H),6.57(d,J＝2.4Hz,1H),6.44–6.36(m,2H),5.68(dd,J＝9.6,2.7Hz,1H),4.50(dd,J＝12.8,6.9Hz,2H),3.87(s,7H),3.80–3.72(m,2H),3.72–3.65(m,1H),3.57–3.47(m,2H),3.01(d,J＝7.3Hz,1H),1.65(m,4H). 13C NMR(100MHz,CDCl 3)δ166.3,162.5,158.7,156.8,155.2,145.2,136.0,128.9,128.4,107.4,102.1,99.3,56.6,47.3,45.6,43.2,36.9,34.6,33.5,32.9,32.6,30.7,30.4.HRMS(ESI)calculated for C 23H 28N 6NaO 3 +:459.2115,found 459.2115.
实施例6：化合物6的制备
Figure PCTCN2021120046-appb-000025
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-Boc-3-羟甲基哌啶，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物6的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.39–8.33(m,1H),6.93–6.75(m,2H),6.63–6.46(m,2H),6.21(dd,J＝16.9,8.7Hz,1H),5.99(s,2H),5.61(dd,J＝32.0,10.5Hz,1H),4.53–4.27(m,3H),3.86(s,6H),3.07(dt,J＝23.7,12.4Hz,1H),2.81(dt,J＝44.0,12.0Hz,1H),2.35(s,1H),1.93(s,1H),1.58–1.29(m,2H),1.26(m,2H). 13C NMR(100MHz,CDCl 3)δ165.5,161.6,157.9,155.9,154.7,127.8,127.4,106.3,101.3,98.2,55.6,49.8,49.7,49.5,46.5,45.8,42.7,37.4,36.4,28.7,25.2,24.1.HRMS(ESI)calculated for C 22H 26N 6NaO 3 +:445.1959,found 445.1961.
实施例7：化合物7的制备
Figure PCTCN2021120046-appb-000026
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为叔丁基-4-(2-羟乙基)哌嗪-1-羧酸酯，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物7的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.36(s,1H),6.80(d,J＝2.3Hz,2H),6.60–6.47(m,2H),6.26(dd,J＝16.8,1.9Hz,1H),5.67(dd,J＝10.5,1.9Hz,1H),4.57(t,J＝6.7Hz,2H),3.85(s,6H),3.62(s,2H),3.49(s,2H),2.96(t,J＝6.7Hz,2H),2.56(t,J＝5.0Hz,4H). 13C NMR(100MHz,CDCl 3)δ165.3,161.5,157.4,155.1,154.5,144.6,134.8,127.9,127.4,106.3,101.2,98.2,95.6,56.7,55.6,52.6,50.9,45.6,44.4,41.8,31.9,29.1.HRMS(ESI)calculated for C 22H 27N 7NaO 3 +:460.2068,found 460.2072.
实施例8：化合物8的制备
Figure PCTCN2021120046-appb-000027
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原 料之一为1-BOC-4-(3-羟基丙烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物8的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.36(s,1H),6.81(d,J＝2.3Hz,2H),6.58–6.50(m,2H),5.88(s,2H),5.69(dd,J＝10.5,1.9Hz,1H),4.52(t,J＝6.9Hz,2H),3.86(s,6H),3.69(d,J＝6.9Hz,2H),3.57(s,2H),2.51(dt,J＝10.8,6.0Hz,6H),2.21(p,J＝7.0Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.3,160.3,154.4,133.4,133.3,132.2,132.1,132.0,129.0,128.6,128.6,128.4,128.1,127.3,127.3,107.9,100.5,55.4,55.3,53.1,52.5,45.2,26.4.HRMS(ESI)calculated for C 23H 29N 7NaO 3 +:474.2224,found 474.2226.
实施例9：化合物9的制备
Figure PCTCN2021120046-appb-000028
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-BOC-4-(4-羟基丁烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物9的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.40–8.36(m,1H),6.81(dd,J＝3.2,2.0Hz,2H),6.54(ddd,J＝17.4,8.7,4.1Hz,2H),6.28(dt,J＝16.9,2.5Hz,1H),5.72–5.66(m,1H),5.61(s,2H),4.46(s,2H),3.89–3.82(m,6H),3.67(s,2H),3.54(s,2H),2.41(s,6H),2.01(s,2H),1.56(s,2H). 13C NMR(100MHz,MeOD)δ169.4,166.0,152.2,151.70,147.6,145.9,132.7,128.5,126.6,106.2,106.1,101.5,101.4,96.5,56.6,54.7,53.5,51.1,40.5,39.1,38.5,35.2, 28.3,22.7.HRMS(ESI)calculated for C 24H 31N 7NaO 3 +:488.2381,found 488.2383.
实施例10：化合物10的制备
Figure PCTCN2021120046-appb-000029
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-BOC-4-(5-羟基戊烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物10的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.38(s,1H),6.82–6.80(m,2H),6.58–6.55(m,1H),6.53(d,J＝10.4Hz,1H),6.27(d,J＝16.7Hz,1H),5.68(d,J＝10.6Hz,1H),5.61(s,2H),4.43(s,2H),3.86(s,6H),3.67(s,2H),3.53(s,2H),2.42–2.38(m,4H),2.34–2.30(m,2H),2.02–1.96(m,2H),1.54(d,J＝7.4Hz,2H),1.39(d,J＝6.9Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.3,161.6,157.7,155.9,154.3,144.0,135.1,127.7,127.5,106.4,101.1,100.0,98.3,58.2,55.6,53.4,52.7,47.1,45.7,41.8,40.5,29.6,29.3,26.2,24.5.HRMS(ESI)calculated for C 25H 33N 7NaO 3 +:502.2537,found 502.2539.
实施例11：化合物11的制备
Figure PCTCN2021120046-appb-000030
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-BOC-4-(6-羟基己烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物11的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.34(d,J＝1.3Hz,1H),6.81(dd,J＝2.3,1.3Hz,2H),6.61–6.52(m,2H),6.29(dt,J＝16.9,1.6Hz,1H),6.00(s,2H),5.69(dt,J＝10.5,1.6Hz,1H),4.43(t,J＝7.3Hz,2H),3.86(d,J＝1.3Hz,6H),3.69(t,J＝5.0Hz,2H),3.56(t,J＝4.9Hz,2H),2.42(t,J＝5.0Hz,4H),2.33(t,J＝7.6Hz,2H),1.97(t,J＝7.2Hz,2H),1.54–1.45(m,2H),1.38(t,J＝4.3Hz,4H). 13C NMR(100MHz,CDCl 3)δ165.3,161.5,157.8,155.3,154.1,144.2,135.0,135.0,127.9,127.5,127.5,106.4,106.4,101.1,98.2,58.3,55.6,53.4,52.7,47.2,45.7,41.8,29.7,27.0,26.6,26.5.HRMS(ESI)calculated for C 26H 35N 7NaO 3 +:516.2694,found 516.2696.
实施例12：化合物12的制备
Figure PCTCN2021120046-appb-000031
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-BOC-4-(7-羟基庚烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物12的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.38(d,J＝2.8Hz,1H),6.82(t,J＝2.5Hz,2H),6.62–6.50(m,2H),6.33–6.23(m,1H),5.77–5.56(m,3H),4.42(s,2H),3.86(d,J＝2.8Hz,6H),3.70(s,2H),3.57(s,2H),2.50–2.37(m,4H),2.33(s,2H),1.96(s,2H),1.47(s,2H),1.33(d,J＝27.0Hz,6H). 13C NMR(101MHz,CDCl 3)δ165.6,161.8,158.1,156.1,154.5,144.3135.5,135.5,128.1,127.8,106.7,101.4,101.4,98.6,58.7,55.9,53.7,53.1,47.5,46.0,42.2,30.0,29.9,29.3,27.6,26.9,26.8.HRMS(ESI)calculated for C 27H 37N 7NaO 3 +:530.2850,found 530.2852.
实施例13：化合物13的制备
Figure PCTCN2021120046-appb-000032
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3，4-亚甲基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物13的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.36(s,1H),7.16(d,J＝2.7Hz,1H),6.97(d,J＝7.9Hz,1H),6.62–6.49(m,1H),6.28(dd,J＝16.9,2.0Hz,1H),6.06(s,2H),5.86–5.65(m,3H),4.42(t,J＝7.2Hz,2H),3.70(d,J＝7.5Hz,2H),3.55(t,J＝5.1Hz,2H),2.43(t,J＝4.9Hz,4H),2.35(t,J＝7.6Hz,2H),1.99(td,J＝16.9,15.0,9.4Hz,2H),1.79–1.14(m,6H). 13C NMR(100MHz,CDCl 3)δ165.2,157.8,155.7,154.2,148.6,148.5,143.8,127.4,127.1,122.1,109.0,108.9,101.6,101.5,98.2,58.1,53.3,52.7,46.9,45.6,41.7,29.5,26.1,24.5.HRMS(ESI)calculated for C 24H 29N 7NaO 3 +:4860.2224,found 4860.2226.
实施例14：化合物14的制备
Figure PCTCN2021120046-appb-000033
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为苯并呋喃-2-硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物14的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.39(s,1H),7.68(dd,J＝7.3,1.5Hz,1H),7.59–7.55(m,1H),7.40–7.32(m,3H),6.53(dd,J＝16.8,10.6Hz,1H),6.28(dd,J＝16.9,1.9Hz,1H),5.68(dd,J＝10.6,2.0Hz,1H),4.46(t,J＝7.2Hz,2H),3.69(s,2H),3.55(s,2H),2.44(s,4H),2.36(t,J＝7.6Hz,2H),2.01(p,J＝7.4Hz,2H),1.57(q,J＝7.6Hz,2H),1.44–1.36(m,2H). 13C NMR(100MHz,CDCl 3)δ165.3,163.8,157.6,156.0154.4,149.5,134.7,128.3,127.8,127.4,125.1,124.0,121.7,111.0,104.3,97.9,58.1,53.3,52.7,47.3,45.6,41.7,29.5,26.1,24.4.HRMS(ESI)calculated for C 25H 29N 7NaO 2 +:482.2269,found 482.2271.
实施例15：化合物15的制备
Figure PCTCN2021120046-appb-000034
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为对甲氧基硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物15的核磁测试结果为：
1H NMR(400MHz,MeOD)δ8.24(s,1H),7.61(d,J＝8.6Hz,2H),7.18–7.06(m,2H),6.82–6.64(m,1H),6.19(dd,J＝16.7,2.0Hz,1H),5.74(dd,J＝10.6,1.9Hz,1H),4.41(t,J＝6.8Hz,2H),3.88(s,3H),3.62(s,4H),2.48(s,4H),2.39(s,2H),1.96(q,J＝7.3Hz,2H),1.60–1.52(m,2H),1.35(q,J＝7.9Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.3,157.7,155.9,155.9,154.4,143.6,140.4,133.9,132.5,129.6,127.8,126.7,125.9,124.7,64.1,58.2,53.4,52.7,48.6,47.1,29.5,26.0,24.5,15.5.HRMS(ESI)calculated for C 24H 31N 7NaO 2 +:472.2426,found 472.2428.
实施例16：化合物16的制备
Figure PCTCN2021120046-appb-000035
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为4-乙酰氨基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物16的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.24(d,J＝1.3Hz,1H),8.01(s,1H),7.54–7.43(m,3H),6.72(dd,J＝16.9,10.7Hz,1H),6.18(dt,J＝16.8,1.7Hz,1H),5.73(dt,J＝10.6,1.7Hz,1H),4.43(t,J＝6.7Hz,2H),3.60(q,J＝6.7,4.6Hz,4H),2.46(d,J＝5.2Hz,4H),2.37(t,J＝7.6Hz,2H),2.16(d,J＝1.4Hz,3H),1.97(t,J＝7.4Hz,2H),1.57(dd,J＝11.0,4.7Hz,2H),1.34(p,J＝7.6Hz,2H). 13C NMR(100MHz,MeOD)δ170.6,165.9,158.4,155.4,153.8,144.4,139.14,133.2,129.6,127.3,127.3,123.7,120.4,120.1,100.0,97.6,57.6,52.8,52.3,45.0,41.3,28.9,25.3,23.9,22.4.HRMS(ESI)calculated for C 25H 32N 8NaO 2 +:472.2426,found 472.2428.
实施例17：化合物17的制备
Figure PCTCN2021120046-appb-000036
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为4-三氟甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物17的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.27(s,1H),7.70(t,J＝7.6Hz,1H),7.63(d,J＝10.6Hz,1H),7.49–7.42(m,1H),7.35(dd,J＝7.8,5.3Hz,1H),6.73(dd,J＝16.8,10.7Hz,1H),6.18(dd,J＝16.8,1.9Hz,1H),5.73(dd,J＝10.7,2.0Hz,1H),4.44(t,J＝6.8Hz,2H),3.61(d,J＝5.4Hz,4H),2.50–2.37(m,4H),2.35(d,J＝7.7Hz,2H),1.99(q,J＝7.1Hz,2H),1.59–1.55(m,2H),1.35(s,2H). 13C NMR(100MHz,CDCl 3)δ166.2,158.5,156.8,155.4,150.6,143.3,136.3,131.8,128.6,128.4,127.6,122.3,121.9,120.1,99.2,59.1,54.3,53.6,48.1,46.6,42.8,30.4,27.1,25.4.HRMS(ESI)calculated for C 24H 28F 3N 7NaO 2 +:526.2143,found 526.2145.
实施例18：化合物18的制备
Figure PCTCN2021120046-appb-000037
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为2-萘硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物18的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.41(s,1H),8.17(s,1H),8.03(d,J＝8.4Hz,1H),7.93(dd,J＝9.3,3.6Hz,2H),7.82(d,J＝8.4Hz,1H),7.58(dd,J＝6.2,3.2Hz,2H),6.51(dd,J＝16.8,10.5Hz,1H),6.27(d,J＝16.8Hz,1H),5.67(d,J＝10.7Hz,1H),4.49(t,J＝7.2Hz,2H),3.68(s,2H),3.51(d,J＝15.2Hz,2H),2.37(dd,J＝19.5,12.3Hz,6H),2.04(dd,J＝14.6,7.4Hz,2H),1.57(dd,J＝14.7,7.5Hz,2H),1.42(dd,J＝14.9,7.9Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.28,157.63,155.97,154.54,149.88,142.42,135.37,135.35,130.91,127.75,127.50,127.24,126.68,121.64,121.38,121.00,118.73,98.31,58.16,53.43,52.73,47.17,45.72,41.87,29.53,26.24,24.52.HRMS(ESI)calculated for C 27H 31N 7NaO+:492.2482,found 492.2484.
实施例19：化合物19的制备
Figure PCTCN2021120046-appb-000038
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为4-甲基萘硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物19的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.37(s,1H),8.10(d,J＝8.4Hz,1H),7.91(d,J＝8.3Hz,1H),7.63–7.41(m,4H),6.52(dd,J＝16.8,10.5Hz,1H),6.26(dd,J＝16.8,1.7Hz,1H),5.67(dd,J＝10.5,1.7Hz,1H),4.50(t,J＝7.1Hz,2H),3.66(s,2H),3.49(d,J＝16.9Hz,2H),2.78(s,3H),2.36(dd,J＝19.3,12.0Hz,6H),2.13–1.98(m,2H),1.57(dt,J＝14.9,7.6Hz,2H),1.42(dt,J＝15.0,7.6Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.27,159.72,157.75,155.67,154.23,144.16,134.49,130.52,127.93,127.88,127.43,120.40,120.11,115.46,114.30,98.54,98.31,63.68,58.15,53.31,52.69,47.03,41.74,29.71,29.54,26.11,24.50,24.04.HRMS(ESI)calculated for C 28H 33N 7NaO+:570.1587,found 570.1590.
实施例20：化合物20的制备
Figure PCTCN2021120046-appb-000039
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为4-溴萘硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物20的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.38(s,1H),8.11(d,J＝8.4Hz,1H),7.92(d,J＝8.3Hz,1H),7.64–7.42(m,4H),6.53(dd,J＝16.8,10.5Hz,1H),6.27(dd,J＝16.8,1.7Hz,1H),5.68(dd,J＝10.5,1.7Hz,1H),4.51(t,J＝7.1Hz,2H),3.67(s,2H),3.50(d,J＝17.0Hz,2H),2.37(dd,J＝19.3,12.0Hz,6H),2.13–1.99(m,2H),1.58(dt,J＝14.9,7.6Hz,2H),1.43(dt,J＝15.0,7.6Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.16,157.62,157.55,155.88,154.26,149.40,134.60,128.22,127.80,127.72,127.32,125.04,123.91,121.64,121.60,110.91,104.17,99.87,97.77,58.01,53.21,52.59,47.20,45.47,41.63,29.34,24.31.HRMS(ESI)calculated for C 27H 30BrN 7NaO+:570.1587,found 570.1590.
实施例21：化合物21的制备
Figure PCTCN2021120046-appb-000040
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为2,5-二甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物21的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.35(s,1H),7.07(s,1H),7.02(d,J＝8.6Hz,2H),6.56(dd,J＝16.8,10.6Hz,1H),6.28(d,J＝16.8Hz,1H),5.70(d,J＝7.6Hz,1H),4.44(t,J＝7.1Hz,2H),3.81(d,J＝11.0Hz,6H),3.69(s,2H),3.55(s,2H),2.47–2.32(m,6H),2.03(dd,J＝14.0,7.0Hz,2H),1.61–1.52(m,2H),1.41(dd,J＝14.7,7.6Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.28,158.41,155.55,154.40,153.90,150.49,140.79,127.83,127.47,123.11,117.10,116.10,113.43,100.37,58.17,56.76,55.85,53.35,52.72,47.06,45.66,41.80,29.55,26.21.HRMS(ESI)calculated for C 25H 33N 7NaO 3 +:502.2537,found 502.2539.
实施例22：化合物22的制备
Figure PCTCN2021120046-appb-000041
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3,4-二甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物22的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.39(s,1H),7.27(s,1H),6.89(s,2H),6.64–6.50(m,1H),6.29(d,J＝16.6Hz,1H),5.70(d,J＝10.2Hz,3H),4.43(d,J＝6.9Hz,2H),3.94(s,3H),3.91(s,3H),3.69(s,2H),3.56(s,2H),2.38(dd,J＝22.3,15.1Hz,6H),2.03–1.98(m,2H),1.57(d,J＝6.9Hz,2H),1.40(d,J＝7.0Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.34,165.29,157.82,155.94,155.91,155.87,154.22,154.05,153.99,144.18,138.75,128.59,127.92,127.42,105.53,98.33,61.03,58.18,56.33,53.39,52.70,47.06,29.61,26.19,24.53.HRMS(ESI)calculated for C 25H 33N 7NaO 3 +:502.2537,found 502.2539.
实施例23：化合物23的制备
Figure PCTCN2021120046-appb-000042
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3,4,5-三甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物23的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.30(s,1H),6.82(d,J＝1.3Hz,2H),6.47(ddd,J＝16.8,10.5,1.3Hz,1H),6.19(dt,J＝16.8,1.6Hz,1H),5.87(s,2H),5.60(dt,J＝10.5,1.6Hz,1H),4.36(t,J＝7.3Hz,2H),3.86(d,J＝1.3Hz,6H),3.83(d,J＝1.3Hz,3H),3.60(t,J＝5.0Hz,2H),3.47(t,J＝5.0Hz,2H),2.33(t,J＝5.1Hz,4H),2.27(d,J＝7.5Hz,2H),1.93(q,J＝7.6Hz,2H),1.49(t,J＝7.6Hz,2H),1.33(d,J＝7.6Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.2,158.0,155.8,154.2,153.9,144.1,138.8,128.6,128.6,128.4,127.7,127.5,105.6,98.3,61.0,58.2,56.3,53.4,52.7,47.0,45.7,41.9,29.7,29.6,26.3,24.5.HRMS(ESI)calculated for C 26H 35N 7NaO 4 +:532.2643,found 536.2645.
实施例24：化合物24的制备
Figure PCTCN2021120046-appb-000043
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-三氟甲基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物24的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.37(s,1H),7.68–7.54(m,3H),7.33(d,J＝6.5Hz,1H),6.59–6.47(m,1H),6.25(dd,J＝16.8,4.6Hz,1H),5.95–5.59(m,3H),4.43(d,J＝6.6Hz,2H),3.64(d,J＝9.2Hz,2H),3.52(s,2H),2.39(d,J＝5.7Hz,4H),2.32(d,J＝7.3Hz,2H),1.98(q,J＝7.1Hz,2H),1.56(s,2H),1.42–1.32(m,2H). 13C NMR(100MHz,CDCl 3)δ165.3,157.7,155.8,154.5,149.8,142.5,135.3,130.9,127.7,127.5,126.7,121.4,121.0,98.3,58.1,53.4,52.7,47.2,45.7,41.9,29.7,29.5,26.2,24.5.HRMS(ESI)calculated for C 24H 28F 3N 7NaO +:510.2205,found 510.2207.
实施例25：化合物25的制备
Figure PCTCN2021120046-appb-000044
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-异丙氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的 3，5-二甲氧基苯硼酸，化合物25的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.15(d,J＝1.2Hz,1H),7.36(t,J＝7.9Hz,1H),7.11(dd,J＝8.2,5.0Hz,2H),7.00–6.95(m,1H),6.70–6.60(m,1H),6.09(dt,J＝16.8,1.6Hz,1H),5.64(dt,J＝10.6,1.6Hz,1H),4.60(q,J＝6.0Hz,1H),4.32(t,J＝6.8Hz,2H),3.53(t,J＝6.3Hz,4H),2.38(s,4H),2.30(t,J＝7.6Hz,2H),1.87(d,J＝7.1Hz,2H),1.48(t,J＝7.8Hz,2H),1.25(dd,J＝6.0,1.3Hz,6H),1.22(d,J＝7.4Hz,2H). 13C NMR(100MHz,MeOD)δ166.0,158.6,158.5,155.4,153.7,144.7,134.0,130.2,127.3,127.3,120.1,116.4,115.5,100.0,97.6,69.9,57.6,52.8,52.3,46.4,45.0,41.3,28.9,25.2,23.9,21.0.HRMS(ESI)calculated for C 26H 35N 7NaO 2 +:500.2744,found 500.2747.
实施例26：化合物26的制备
Figure PCTCN2021120046-appb-000045
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-苄氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物26的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.37(s,1H),7.49–7.27(m,8H),7.11(d,J＝8.5Hz,1H),6.54(dd,J＝16.8,10.4Hz,1H),6.28(d,J＝16.8Hz,1H),5.69(d,J＝10.4Hz,1H),5.43(s,2H),5.17(s,2H),4.44(t,J＝7.4Hz,2H),3.62(d,J＝49.9Hz,4H),2.39(d,J＝27.3Hz,6H),2.03–1.93(m,2H),1.38(s,2H). 13C NMR(100MHz,CDCl 3)δ165.3,159.4,157.7,155.4,154.2,144.1,136.6,134.5,133.3,130.6,128.7,128.1,127.7,127.5,127.4,120.9, 116.0,114.5,98.2,70.0,58.1,53.3,52.7,47.1,45.7,41.8,29.5,26.2,24.5,21.2.HRMS(ESI)calculated for C 30H 35N 7NaO 2 +:548.2744,found 548.2747.
实施例27：化合物27的制备
Figure PCTCN2021120046-appb-000046
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为2-氯-5-甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物27的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.35(d,J＝4.2Hz,1H),7.43(s,1H),6.99(dq,J＝19.8,3.2Hz,2H),6.53(ddd,J＝15.6,10.7,3.9Hz,1H),6.25(dd,J＝16.9,3.9Hz,1H),5.80–5.22(m,3H),4.45(t,J＝6.5Hz,2H),3.82(d,J＝4.9Hz,3H),3.68–3.63(m,2H),3.51(s,2H),2.39(d,J＝5.4Hz,4H),2.31(q,J＝6.2,5.4Hz,2H),1.99(q,J＝6.8Hz,2H),1.53(q,J＝7.1Hz,2H),1.42–1.28(m,2H). 13C NMR(100MHz,CDCl 3)δ165.3,158.6,157.7,155.9,153.8,141.1,132.6,131.1,127.7,127.5,124.7,116.9,99.9,70.5,58.2,55.7,53.4,52.7,47.1,45.7,41.9,29.5,26.2,24.5.HRMS(ESI)calculated for C 24H 30ClN 7NaO 2 +:506.2042,found 506.2044.
实施例28：化合物28的制备
Figure PCTCN2021120046-appb-000047
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为对氟苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物28的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.31(s,1H),7.25(s,2H),6.54(dd,J＝16.8,10.5Hz,1H),6.29(dd,J＝16.8,1.7Hz,1H),6.24(s,2H),5.70(d,J＝10.5Hz,1H),4.35(t,J＝7.0Hz,2H),3.69(t,J＝29.6Hz,4H),2.44(d,J＝31.0Hz,6H),1.92(dd,J＝14.9,7.4Hz,2H),1.58(s,2H),1.35–1.30(m,2H). 13C NMR(100MHz,CDCl 3)δ165.21,158.54,157.68,155.61,153.68,141.11,132.50,131.04,127.66,127.44,124.68,116.84,99.80,58.07,55.65,53.26,52.63,47.07,45.59,41.74,29.40,26.09,24.40.HRMS(ESI)calculated for C 23H 28FN 7NaO +:460.2232,found 460.2230.
实施例29：化合物29的制备
Figure PCTCN2021120046-appb-000048
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-氟-4-羟基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物29的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.34(s,1H),7.72–7.62(m,2H),7.26–7.23(m,1H),6.55(dd,J＝16.8,10.5Hz,1H),6.28(dd,J＝16.8,1.9Hz,1H),5.69(dd,J＝10.5,1.9Hz,1H),4.44(t,J＝7.2Hz,2H),3.68(s,2H),3.51(d,J＝21.5Hz,2H),2.47–2.39(m,4H),2.36–2.31(m,2H),1.99(dt,J＝15.0,7.5Hz,2H),1.55(dd,J＝15.1,7.6Hz,2H),1.42–1.33(m,2H). 13C NMR(100MHz,CDCl 3)δ165.30,162.94,161.55,157.73,157.26,156.54,154.10,119.85,117.25,100.41,100.29,99.99,61.49,57.97,53.20,52.53,47.28,29.69,29.65,29.40,29.26,24.72,24.24..HRMS(ESI)calculated for C 23H 28FN 7NaO 2+:476.2181,found 476.2180.
实施例30：化合物30的制备
Figure PCTCN2021120046-appb-000049
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为6-甲氧基萘-2-硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物30的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.37(d,J＝5.6Hz,1H),8.08(s,1H),7.91(d,J＝8.4Hz,1H),7.83(d,J＝8.9Hz,1H),7.77(dd,J＝8.4,1.6Hz,1H),7.26–7.19(m,2H),6.51(dd,J＝16.8,10.5Hz,1H),6.31–6.16(m,1H),5.77(d,J＝66.8Hz,2H),5.67(dd,J＝10.5,1.9Hz,1H),4.47(t,J＝7.2Hz,2H),3.97(s,3H),3.69(s,2H),3.54(s,2H),2.38(dd,J＝21.6,14.2Hz,6H),2.02(dt,J＝14.8,7.4Hz,2H),1.63–1.53(m,2H),1.41(dt,J＝15.1,7.5Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.27,158.50,157.93,155.54,154.31,144.47,134.66,129.73,128.92,128.23,128.12,127.81,127.54,127.43,126.28,119.94,105.79,98.49,58.13,55.42,53.27,52.70,47.05,45.55,41.71,29.57,26.08,24.51.HRMS(ESI)calculated for C 28H 33N 7NaO 2 +:522.2588,found 522.2587.
实施例31：化合物31的制备
Figure PCTCN2021120046-appb-000050
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为对硝基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物31的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.45–8.36(m,3H),7.92(d,J＝8.8Hz,2H),6.54(dd,J＝16.8,10.5Hz,1H),6.27(dd,J＝16.9,2.0Hz,1H),5.90–5.63(m,3H),4.48(t,J＝7.2Hz,2H),3.67(s,2H),3.59–3.50(m,2H),2.42(t,J＝5.1Hz,4H),2.35(dd,J＝8.5,6.5Hz,2H),2.01(p,J＝7.6Hz,2H),1.63–1.55(m,2H),1.41(dt,J＝11.4,6.8Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.3,157.6,155.9,154.7,148.0,141.7,139.6,129.2,127.8,127.5,124.6,98.4,77.2,58.1,53.4,52.7,47.4,45.7,41.8,29.7,29.5,26.2,24.5.HRMS(ESI)calculated for C 23H 28N 8NaO 3 +:487.2177,found 487.2179.
实施例32：化合物32的制备
Figure PCTCN2021120046-appb-000051
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-氟-5-甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物31的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.38(s,1H),7.24(d,J＝2.1Hz,2H),6.99–6.87(m,1H),6.54(dd,J＝16.8,10.6Hz,1H),6.27(dd,J＝16.8,1.9Hz,1H),5.80–5.63(m,3H),4.44(t,J＝7.2Hz,2H),3.92(s,3H),3.66(d,J＝12.5Hz,2H),3.54(s,2H),2.45–2.33(m,6H),2.02–1.96(m,2H),1.55(dd,J＝15.0,7.5Hz,2H),1.37(dd,J＝15.2,7.2Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.31,164.85,164.72,162.35,162.23,157.51,155.78,154.50,127.78,127.48,111.59,111.33,104.53,104.28,98.15,58.13,53.38,52.69,47.23,45.66,41.82,29.49,26.17,24.51.HRMS(ESI)calculated for C 24H 30FN 7NaO 2 +:490.2337,found 490.2335.
实施例33：化合物33的制备
Figure PCTCN2021120046-appb-000052
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3,5-二氟苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物33的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.36(s,1H),7.01(dd,J＝8.4,2.2Hz,2H),6.73(dt,J＝10.5,2.3Hz,1H),6.55(dd,J＝16.8,10.6Hz,1H),6.28(dd,J＝16.8,1.9Hz,1H),5.83(s,2H),5.69(dd,J＝10.5,1.9Hz,1H),4.44(t,J＝7.2Hz,2H),3.66(m,J＝16.1Hz,2H),3.55(m,2H),2.37(dd,J＝23.0,15.7Hz,6H),1.98(dd,J＝14.9,7.5Hz,2H),1.56(dd,J＝14.8,7.4Hz,2H),1.45–1.36(m,2H). 13C NMR(100MHz,CDCl 3)δ165.28,162.81,161.77,161.65,157.58,155.96,154.41,127.74,127.49,110.07,110.04,107.79,107.57,102.38,102.13,98.25,58.15,55.81,53.35,52.74,47.12,29.51,24.50.HRMS(ESI)calculated for C 23H 27F 2N 7NaO +:478.2137,found 478.2139.
实施例34：化合物34的制备
Figure PCTCN2021120046-appb-000053
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3,5-二三氟甲基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物34的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.44(s,1H),8.20(s,2H),7.99(s,1H),6.61–6.51(m,1H),6.28(d,J＝17.0Hz,1H),5.69(d,J＝10.6Hz,1H),4.48(t,J＝7.0Hz,2H),3.64(s,4H),2.39(d,J＝26.1Hz,6H),2.05–1.99(m,2H),1.78(s,2H),1.38(dd,J＝23.0,12.9Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.3,157.5,156.0,154.8,140.9,135.5,133.2,132.9,132.6,128.5,127.8,127.5,124.4,122.5,121.7,98.4,58.1,53.4,52.7,47.3545.7,41.8,29.5,26.2,24.5.HRMS(ESI)calculated for C 25H 27F 6N 7NaO +:578.2073,found 578.2075.
实施例35：化合物35的制备
Figure PCTCN2021120046-appb-000054
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-氟-5-三氟甲基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物35的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.35(s,1H),7.73(s,1H),7.58(dd,J＝8.7,2.0Hz,1H),7.40–7.37(m,1H),6.48(dd,J＝16.8,10.5Hz,1H),6.20(dd,J＝16.8,1.9Hz,1H),5.61(dd,J＝10.5,1.9Hz,1H),4.39(t,J＝7.2Hz,2H),3.60(s,2H),3.47(s,2H),2.34(t,J＝5.1Hz,4H),2.30–2.24(m,2H),1.96–1.90(m,2H),1.52–1.47(m,2H),1.32(t,J＝7.6Hz,2H). 13C NMR(100MHz,CDCl 3)δ162.0,161.5,157.8,155.6,154.1,144.2,135.0,129.5,127.8,127.7,106.4,101.0,98.2,58.0,55.6,53.3,52.5,47.0,46.0,42.2,29.7,29.5,26.2,24.5.HRMS(ESI)calculated for C 24H 27F 4N 7NaO +:528.2105,found 528.2107.
实施例36：化合物36的制备
Figure PCTCN2021120046-appb-000055
具体制备方法为：与化合物10的制备方法类似，不同的是所使用的原料之一为4,4,4-三氟丁烯酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物36的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.32(s,1H),6.92(dt,J＝15.4,2.1Hz,1H),6.78(d,J＝2.3Hz,2H),6.73–6.64(m,1H),6.52(t,J＝2.3Hz,1H),4.40(t,J＝7.2Hz,2H),3.82(s,6H),3.65(t,J＝5.2Hz,2H),3.50(t,J＝5.0Hz,2H),2.40(q,J＝4.7Hz,4H),2.32(t,J＝7.5Hz,2H),1.96(p,J＝7.4Hz,2H),1.52(q,J＝7.6Hz,2H),1.36(q,J＝8.2Hz,2H). 13C NMR(100MHz,CDCl 3)δ162.3,161.8,158.1,155.8,154.3,144.4,135.3,129.7,129.4,128.0,127.9,121.4,106.6,101.3,98.5,58.3,55.8,53.5,52.7,47.3,46.2,42.5,29.9,29.8,26.4,24.7.HRMS(ESI)calculated for C 26H 32F 3N 7NaO 3 +:570.2411,found 570.2413.
实施例37：化合物37的制备
Figure PCTCN2021120046-appb-000056
具体制备方法为：与化合物34的制备方法类似，不同的是所使用的原料之一为4,4,4-三氟丁烯酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物37的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.43(d,J＝2.3Hz,1H),8.20(s,2H),7.99(s,1H),6.94(d,J＝15.5Hz,1H),6.72(dt,J＝14.9,6.8Hz,1H),4.48(t,J＝7.0Hz,2H),3.69(s,2H),3.54(s,2H),2.41(d,J＝31.2Hz,6H),2.02(q,J＝7.8Hz,2H),1.59(s,2H),1.44–1.26(m,2H). 13C NMR(100MHz,CDCl 3)δ164.1,162.1,161.6,157.4,155.9,154.7,141.3,136.6,127.7,121.0,118.9,118.7,113.4,113.2,98.2,58.0,53.3,52.5,47.2,45.9,42.2,29.7,29.5,26.1,24.5,14.1.HRMS(ESI)calculated for C 26H 26F 9N 7NaO +:646.1947,found 646.1947.
实施例38：化合物38的制备
Figure PCTCN2021120046-appb-000057
具体制备方法为：与化合物35的制备方法类似，不同的是所使用的原料之一为4,4,4-三氟丁烯酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物38的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.43(d,J＝2.3Hz,1H),8.20(s,2H),7.99(s,1H),6.94(d,J＝15.5Hz,1H),6.71(dq,J＝14.2,6.7Hz,1H),4.48(t,J＝7.0Hz,2H),3.69(s,2H),3.54(s,2H),2.41(d,J＝31.2Hz,6H),2.01(t,J＝7.6Hz,2H),1.59(s,2H),1.48–1.27(m,2H). 13C NMR(100MHz,CDCl 3)δ162.0,155.2,154.6,142.9,133.2,132.1,132.1,129.4,129.0,128.6,128.5,127.9,121.3,120.8,111.6,105.6,58.1,53.3,52.6,46.9,46.0,42.3,29.5,26.3,24.5,22.1.HRMS(ESI)calculated for C 26H 26F 9N 7NaO +:646.1947,found 646.1947.
实施例39：化合物39的制备
Figure PCTCN2021120046-appb-000058
具体制备方法为：与化合物10的制备方法类似，不同的是所使用的原料之一为反式-4-二甲基氨基巴豆酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物39的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.38(s,1H),6.81(d,J＝2.3Hz,2H),6.56(t,J＝2.3Hz,1H),5.60(s,2H),4.43(t,J＝7.2Hz,2H),3.86(s,6H),3.67(t,J＝5.1Hz,2H),3.59(t,J＝5.1Hz,2H),3.49(s,1H),2.45(t,J＝5.1Hz,4H),2.33(t,J＝7.4Hz,2H),1.96(d,J＝7.5Hz,2H),1.50–1.35(m,4H). 13C NMR(100MHz,CDCl 3)δ161.5,161.5,157.7,155.9,144.0,135.1,106.4,101.0,100.098.3,58.1,55.6,53.1,52.4,52.3,49.4,47.1,45.8,43.4,43.3,43.2,37.4,29.6,26.9,26.5.HRMS(ESI)calculated for C 25H 31N 7NaO 3 +:500.2831,found 500.2833.
实施例40：化合物40的制备
Figure PCTCN2021120046-appb-000059
具体制备方法为：与化合物10的制备方法类似，不同的是所使用的原料之一为巴豆酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物40的核磁测试结果为：
1H NMR(400MHz,CDCl 3)δ8.31(s,1H),6.88–6.73(m,3H),6.52(t,J＝2.3Hz,1H),6.20(dq,J＝15.0,1.7Hz,1H),4.39(t,J＝7.2Hz,2H),3.82(s,6H),3.62(d,J＝8.1Hz,2H),3.56–3.43(m,2H),2.36(t,J＝5.1Hz,4H),2.29(dd,J＝8.7,6.4Hz,2H),2.01–1.91(m,2H),1.83(dd,J＝6.9,1.7Hz,3H),1.52(t,J＝7.6Hz,2H),1.36(ddd,J＝15.3,9.1,5.4Hz,2H). 13C NMR(100MHz,CDCl 3)δ165.5,161.5,158.0,155.7,154.1,144.1,141.5,135.1,121.4,119.9,106.4,101.1,98.2,60.4,58.2,55.5,53.5,53.4,52.8,47.0,45.5,41.8,29.6,26.2,24.5,18.2.HRMS(ESI)calculated for C 26H 35N 7NaO 3 +:516.2694,found 516.2696.
实施例41酶活测试
本发明采用均相时间分辨荧光(HIRF)技术评价化合物对FGFR1,2,3和4四个亚型的抑制作用。首先构建FGFR1,2,3和4原核表达载体，鉴定正确后，大肠杆菌体系大批量表达纯化FGFR蛋白，测定蛋白浓度，分装保存。酶活测试开始前，选用cisbio HTRF KinEA(62TK0PEB)预先将酶活体系中需要的各种组分稀释到所需要的工作浓(使用酶活缓冲液进行稀释)。准备384孔板(66PL384025)，每孔依次加入5μL蛋白 溶液、1μL化合物、2μL底物，最后加入2μLATP启动反应。每个浓度设置3个平行实验，空白对照组将1μL化合物换成1μLDMSO，阳性对照组为阳性药AZD4547，其他条件保持不变。密封384孔板，室温下孵育1h左右。孵育完成后，向每孔中加入5μL澜系元素标记的磷酸化底物的抗体(供体)和5μL链酶亲和素标记的XL665(受体)终止反应，避光，室温下继续孵育1h左右。最后使用TECAN infinite M1000PRO多功能酶标仪检测荧光信号，计算抑制率，用GraphPad Prism 7.0拟合IC50值。
结果显示，化合物1-40均可不同程度的抑制FGFR1/2/3/4的蛋白活性。
表1.基于酶活结果的构效关系研究
Figure PCTCN2021120046-appb-000060
Figure PCTCN2021120046-appb-000061
Figure PCTCN2021120046-appb-000062
实施例42细胞增殖试验
本发明选取化合物10和36，采用MTT比色法评价该化合物10和36对多种肿瘤细胞的增殖抑制作用。胰酶消化收集对数生长期的肿瘤细胞，用1mL新鲜培养基重悬，然后取少量进行稀释，用血球计数板计算细胞数量。根据细胞生长速度不同，按照3000-5000个/孔的密度将肿瘤细胞接种到96孔板中，然后放置细胞培养箱中培养24h。随后用化合物10和36处理肿瘤细胞，设置8个浓度，每个浓度设置三个复孔，孵育96h。孵育完成后，每孔加入10μL MTT溶液(5mg/mL)，拍打混匀后继续放入细胞培养箱中培养3-4h。孵育完成后，小心地吸空孔板中液体，随后每孔加入100μL DMSO溶液。设置酶标仪程序，分别在490nm和570nm处的测定吸光光度值，根据公式计算每个浓度的增殖抑制率，最后用Graphpad Prism 7软件进行数据处理，计算IC50。
结果显示(表1)，化合物10和36显著抑制人肺鳞癌细胞NCI-H1581(FGFR1扩增)和人胃癌细胞SNU-16(FGFR2扩增)的增殖，对人肝癌细胞SK-hep-1(FGGR4扩增)具有一定程度的增殖抑制作用，而对FGFR表达正常的肿瘤细胞无明显抑制作用，表明化合物10和36具有很强的选择性。
表2.化合物36对特定癌细胞系的抗增殖活性
Figure PCTCN2021120046-appb-000063
实施例43化合物10和36抑制FGF/FGFR及其下游信号传导途径
为了进一步阐明化合物10和36对FGF/FGFR信号的影响，通过蛋白质免疫印迹实验验证其对FGFR和下游信号蛋白磷酸化的影响。胰酶消化收集处于对数生长期的人肺鳞癌细胞NCI-H1581和人胃癌细胞 SNU-16，用1mL新鲜培养基重悬，取少量体积进行稀释，用血球计数板计算细胞数量。按照3 x 105/个每孔的密度，取相应体积的肿瘤细胞悬浮液接种到6孔板中，放置细胞培养箱中培养24h。待细胞完全贴壁后，吸去旧培养基，加入含有不同浓度的化合物10和36的新鲜培养基，细胞培养箱中共孵育12h。孵育完成后，吸去上清，用预冷的1X PBS溶液洗涤三次，彻底吸除残液，每孔加入60μL RIPA裂解液，用细胞刮子将细胞轻轻地刮下来，将裂解液转移至1.5mL EP管中，冰浴10min，14000rpm离心10min，吸取上清至新的1.5mL EP管中，吸取少量进行蛋白含量测定，剩余加相应体积的5X上样缓冲液，涡旋混匀，100℃金属浴10min。根据测定的蛋白含量，按每孔30μg蛋白的上样量，计算相应的上样量。利用8％的SDS-PAGE凝胶电泳分离蛋白，将分离好的蛋白转至PDVF膜上，丽春红染色，根据目的蛋白的分子量裁剪含有蛋白的PDVF膜，1X TBST溶液洗净丽春红，5％的脱脂奶粉封闭30min。1X TBST溶液洗涤三次，将含有目标蛋白条带的PDVF膜放入孵育盒中，加入相应的一抗孵育液，4℃过夜孵育。一抗孵育完成后，1X TBST溶液洗涤三次，加入相应的二抗孵育液，常温孵育1.5h。孵育完成后，1X TBST溶液洗涤三次，用超敏HRP化学发光试剂盒显色。
如图1和2结果显示，化合物10和36不仅可以显著抑制NCI-H1581细胞中FGFR1和SNU-16细胞中FGFR2的磷酸化，而且可以剂量依赖的方式抑制下游PLCγ，AKT和ERK的激活。总的来说，化合物10和36可以在体外强烈抑制FGF/FGFR和下游信号传导途径。
实施例44化合物10和36明显抑制小鼠体内肿瘤生长
为了评估化合物10和36在体内的抗肿瘤功效，用FGFR1高表达的人肺鳞癌细胞NCI-H1581建立了小鼠皮下移植瘤模型。胰酶消化收集处于对数生长期的细胞，1X PBS洗涤三次，细胞沉淀用重悬液(1640培养基：Matrigel＝1:1)稀释至1×10 8/mL，按5×10 6个/只的数量将细胞接种至5-6周龄的雌性BALB/c Nude小鼠的前肢腋窝下端。待肿瘤体积长至50-100mm 3时，随机将小鼠分为三组(对照组，50mg/kg,100mg/kg),每 组6只小鼠，化合物10每天口服给药，化合物36每天腹腔注射给药，并量取肿瘤体积和小鼠体重，给药26天后处理小鼠，剖取肿瘤组织，固定于福尔马林溶液中备用。
图3和4结果显示，化合物10和36剂量依赖性地抑制NCI-H1581细胞在小鼠体内的增殖，高剂量组的抑制率达72％，抑制肿瘤组织内FGFR1的磷酸化激活，且没有明显的体重减轻现象，表明化合物10和36在体内对所有剂量均具有良好的耐受性。
总之，本发明优化并发现了2H-吡唑并[3,4-d]嘧啶的新型衍生物化合物10和36，作为对FGFR1，FGFR2和FGFR3的有效和选择性抑制剂，表征了对靶蛋白的不可逆结合作用。该先导化合物不仅可以在低浓度下抑制FGF/FGFR及其下游信号通路，而且在体内外均显示出对NCI-H1581癌细胞系的显著抗增殖作用。而且，化合物10和36显示出低毒性并且具有良好的PK特性，目前正被确认为潜在的候选药物。
以上所述仅为本发明创造的较佳实施例而已，并不用以限制本发明创造，凡在本发明创造的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明创造的保护范围之内。
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Claims (20)

  1. 一种如式I所示的化合物或其在药学上可接受的盐：
    Figure PCTCN2021120046-appb-100001
    所述式I中，Ar为取代芳环基团和取代芳杂环基团中的任意一种；
    R为H、烷基、芳基、-CF 3和烷基叔胺结构中的任意一种；
    Linker为烷基、烷氧基、杂原子取代基、取代N杂环中的任意一种。
  2. 如权利要求1所述的化合物或其在药学上可接受的盐，其特征在于，所述Ar为
    Figure PCTCN2021120046-appb-100002
    Figure PCTCN2021120046-appb-100003
  3. 如权利要求1所述的化合物或其在药学上可接受的盐，其特征在于，所述R为H、CF 3、(CH 3) 2NCH 2或CH 3。
  4. 如权利要求1所述的化合物或其在药学上可接受的盐，其特征在 于，所述Linker为
    Figure PCTCN2021120046-appb-100004
    Figure PCTCN2021120046-appb-100005
  5. 如权利要求1～4任一项所述的化合物或其在药学上可接受的盐，其特征在于，所述化合物为化合物1～40中的一种：
    Figure PCTCN2021120046-appb-100006
    Figure PCTCN2021120046-appb-100007
    Figure PCTCN2021120046-appb-100008
    Figure PCTCN2021120046-appb-100009
    Figure PCTCN2021120046-appb-100010
    Figure PCTCN2021120046-appb-100011
    Figure PCTCN2021120046-appb-100012
  6. 权利要求1～5任一项所述化合物或其药学上可接受的盐的制备方法，其合成路线为：
    Figure PCTCN2021120046-appb-100013
  7. 如权利要求6所述制备方法，其特征在于，包括以下步骤：
    将具有式a所示结构化合物和具有式b所示结构化合物发生光延反应，得到具有式c所示结构化合物；所述光延反应的温度为室温，时间为4h；
    将所述具有式c所示结构化合物和具有式d所示结构化合物发生铃木偶联反应，得到具有式e所示结构化合物；所述铃木偶联反应的温度为80℃；
    将所述具有式e所示结构化合物和三氟乙酸发生脱保护反应，得到具有式f所示结构化合物；所述脱保护反应的温度为0℃；
    将所述具有式f所示结构化合物发生酰基化反应，得到具有式I所示结构化合物；所述酰基化反应的温度为室温；
    Figure PCTCN2021120046-appb-100014
    Ar-B(OH) 2式d、
    Figure PCTCN2021120046-appb-100015
  8. 权利要求1～5任一项所述的化合物、其药学上可接受的盐在制备肿瘤细胞增殖抑制剂中的应用，其特征在于，所述肿瘤细胞为具有FGFR异常的相关的肿瘤细胞。
  9. 如权利要求8所述的应用，其特征在于，所述肿瘤细胞包括NCI-H1581、SNU-16。
  10. 权利要求1～5任一项所述的化合物、其药学上可接受的盐在阻断肿瘤细胞中FGF/FGFR信号通路中的应用，其特征在于，所述肿瘤细胞为具有FGFR异常的相关的肿瘤细胞。
  11. 如权利要求10所述的应用，其特征在于，所述肿瘤细胞包括NCI-H1581或SNU-16。
  12. 一种含有权利要求1～5任一项所述的化合物、其药学上可接受的盐的药物组合物，其特征在于，包括一种或多种药学上可接受的赋形剂；
    所述药物组合物的剂型为药学上可接受的任一剂型。
  13. 权利要求1～5任一项所述的化合物、其药学上可接受的盐或权利要求12所述的药物组合物在制备治疗和/或预防癌症的药物中的应用。
  14. 根据权利要求9所述的应用，其特征在于，所述癌症为具有FGFR异常的相关癌症。
  15. 权利要求12所述的药物组合物在用于制备作为不可逆的泛成纤维细胞生长因子受体抑制剂中的应用。
  16. 一种治疗癌症的方法，其特征在于，包括以下步骤：
    通过口服或者腹腔注射的方式，将治疗癌症的药物对患者进行给药；
    所述治疗癌症的药物包括权利要求1～5任一项所述的化合物、其药学上可接受的盐。
  17. 如权利要求16所述的方法，其特征在于，所述治疗癌症的药物包括化合物10或化合物36。
  18. 如权利要求17所述的方法，其特征在于，所述给药的剂量为50mg/kg或100mg/kg；
    所述给药的剂量以所述化合物10或化合物36的用量计。
  19. 如权利要求17或18所述的方法，其特征在于，当所述治疗癌症的药物包括所述化合物10时，所述给药的方式为口服。
  20. 如权利要求17或18所述的方法，其特征在于，当所述治疗癌症的药物包括所述化合物36时，所述给药的方式为腹腔注射。
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AU2021398802B2 (en) 2024-06-20
AU2021398802A1 (en) 2023-05-11
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