A compound capable of regulating fibroblast growth factor receptor 2 protein levels, and methods of making and using the same

Abstract

This invention provides a compound that can regulate the level of fibroblast growth factor receptor 2 (FGFR2) protein, its preparation method, and its uses, belonging to the pharmaceutical field. The compound of this invention is the compound shown in Formula I, its salt, its stereoisomer, its solvate, its hydrate, or its prodrug. The compound of this invention exhibits good inhibitory activity against FGFR2 protein expression with high selectivity; it can also selectively inhibit the proliferation and migration of FGFR2-overexpressing cells, demonstrating good antitumor activity; furthermore, the compound of this invention has good safety during use, and its synthetic raw materials are readily available and the synthesis method is easy to implement, providing a new option for the development and application of antitumor drugs.

Description

A compound that can regulate the level of fibroblast growth factor receptor 2 protein, its preparation method and uses Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a compound that can regulate the level of fibroblast growth factor receptor 2 protein, its preparation method, and its uses. Background Technology

[0002] Fibroblast growth factor (FGF) and its receptor (FGFR) are a class of receptor tyrosine kinases whose distribution on various cell membranes is crucial for biological processes such as cell growth, differentiation, and migration. However, abnormal FGF / FGFR regulation and signal transduction disorders can lead to various diseases. In recent years, FGFR gene abnormalities and protein overexpression have been reported in various types of tumors. FGFR abnormalities are associated with tumorigenesis and disease progression, and in some tumors, they are even considered a trigger for tumor progression. Several marketed pan-FGFR inhibitors target FGF23 / FGFR1, causing hyperphosphatemia, and FGF19 / FGFR4, causing severe diarrhea and other toxic side effects, leading to reduced dosages or even discontinuation of treatment, thus affecting patient outcomes.

[0003] Selective targeting of FGFR2 can not only avoid the problems associated with these toxic side effects, but also improve the precision of treatment by targeting specific tumor cells. However, research on FGFR2-targeted therapies is currently limited, and developing highly selective FGFR2 drugs remains a pressing clinical challenge in the field of cancer treatment. Therefore, researching FGFR2-targeted drugs with low toxicity and reduced drug resistance, with the aim of improving patient treatment outcomes and quality of life, is of significant clinical importance. Summary of the Invention

[0004] The purpose of this invention is to provide a compound that can regulate the level of fibroblast growth factor receptor 2 protein, as well as its preparation method and uses.

[0005] This invention provides compounds of Formula I, their salts, their stereoisomers, their solvates, their hydrates, or their prodrugs:

[0006]

[0007] in,

[0008] Linker is selected from aliphatic alkyl chains, aliphatic alkyl acyl chains, amino aliphatic alkyl acyl chains, or aliphatic alkyl diacyl chains with a length of 0-6 carbon atoms; the carbon atoms in the alkyl backbone are not replaced or are replaced by heteroatoms; the heteroatoms are O or N;

[0009] The ligands are selected from hydrophobic tags containing norbornene, hydrophobic tags containing adamantane, CRBN ligands, VHL ligands, and RNF126 ligands.

[0010] Furthermore,

[0011] Linker is selected from none.

[0012] X is selected from NH or O;

[0013] n is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0014] Furthermore,

[0015] ligands selected from

[0016] R1, R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, and halogen.

[0017] Preferably, R1, R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C6 alkyl, trifluoromethyl, C1-C6 alkoxy, and halogen.

[0018] Furthermore,

[0019] ligands selected from

[0020] Furthermore, the compound is as shown in Formula II:

[0021]

[0022] in,

[0023] Linker is selected from none.

[0024] X is selected from NH or O;

[0025] n is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0026] Furthermore, the compound is as shown in Formula III:

[0027]

[0028] in,

[0029] Linker is selected from none.

[0030] X is selected from NH or O;

[0031] n is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0032] Furthermore, the chemical

[0033]

[0034]

[0035]

[0036] The present invention also provides the use of the aforementioned compounds, their salts, their stereoisomers, their solvates, their hydrates or their prodrugs in the preparation of FGFR2 inhibitors.

[0037] The present invention also provides the use of the aforementioned compounds, their salts, their stereoisomers, their solvates, their hydrates or their prodrugs in the preparation of medicaments for the prevention and / or treatment of tumors;

[0038] Preferably, the tumor is brain cancer, glioblastoma, leukemia, lymphoma, Bannayan-Zonana syndrome, Cowden's disease, Lhermitte-Duclos disease, breast cancer, inflammatory breast cancer, Wilms' tumor, Ewing sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, colon cancer, gastric cancer, bladder cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, kidney cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, osteosarcoma, giant cell tumor of bone, or thyroid cancer.

[0039] The present invention also provides a medicament for the prevention and / or treatment of tumors, which is a formulation prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients to the aforementioned compounds, their salts, their stereoisomers, their solvates, their hydrates or their prodrugs as active ingredients.

[0040] Alternatively, a pharmaceutical composition for the prevention and / or treatment of tumors, characterized in that it comprises the aforementioned compound, its salt, its stereoisomer, its solvate, its hydrate, or its prodrug.

[0041] The compounds and derivatives provided in this invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0042] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0043] "Substitution" refers to the replacement of hydrogen atoms in a molecule with other different atoms or molecules; "halogenation" refers to the replacement of hydrogen atoms in a molecule with halogens.

[0044] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a ~C b Alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, "C1 to C6 alkyl" refers to an alkyl group containing 1 to 6 carbon atoms; "C1 to C6 alkoxy" refers to an alkoxy group containing 1 to 6 carbon atoms.

[0045] "Alkyl" refers to a saturated hydrocarbon chain with a specified number of carbon atoms. For example, C1-C6 alkyl refers to an alkyl group with 1 to 6 carbon atoms, that is, alkyl groups with 1, 2, 3, 4, 5, or 6 carbon atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl, etc.

[0046] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0047] This invention provides a compound that can regulate the level of fibroblast growth factor receptor 2 protein. The compound of this invention has a good inhibitory effect on FGFR2 protein expression and is highly selective. The compound of this invention can also selectively inhibit the proliferation of cells with high FGFR2 expression and has good anti-tumor activity. At the same time, the raw materials for the synthesis of the compound of this invention are readily available and the synthesis method is easy to implement, providing a new option for the development and application of anti-tumor drugs.

[0048] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0049] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0050] Figure 1 shows the results of Western blotting assay to evaluate the total FGFR2 content of KATOIII cells after treatment with some candidate compounds (100 nM and 1 μM) for 24 hours: A is the result of compounds B1-B5; B is the result of compounds J1-J7.

[0051] Figure 2 shows the results of Western blot analysis of the total FGFR2 content in KATOIII cells after treatment with another set of candidate compounds (100 nM and 1 μM) for 24 hours: A is the result of compounds C1-C3; B is the result of compounds C4-C9; C is the result of compounds C10-C15; D is the result of compounds V1-V5; and E is the result of compounds R1-R8.

[0052] Figure 3 shows the effect of compound C14 on FGFR2 protein expression in KATOIII cells: AB represents the time-dependent degradation (100 nM) and quantitative analysis of FGFR2 relative levels by C14; CD represents the concentration-dependent degradation of FGFR2 by C14 (0–300 nM) in KATOIII cells after 24 hours of treatment and quantitative analysis of FGFR2 relative levels; E represents the concentration-dependent degradation of FGFR2 by C14 (0–300 nM) in KATOIII cells after 72 hours of treatment.

[0053] Figure 4 shows the effects of compound C14 on the expression of FGFR1 / 3 / 4 proteins in non-FGFR2 high-expression cell lines: AB represents the effect of C14 on FGFR1 degradation in BXPC3 cells and the quantitative analysis of the relative FGFR level; CD represents the effect of C14 on FGFR3 degradation in MCF-7 cells and the quantitative analysis of the relative FGFR level; EF represents the effect of C14 on FGFR4 degradation in Huh-7 cells and the quantitative analysis of the relative FGFR level.

[0054] Figure 5 illustrates the mechanism by which C14 induces the degradation of FGFR2.

[0055] Figure 6 shows the levels of P-FGFR, P-PLCγ, and P-FRS2α in KATOIII cells after 24 hours of culture with 0.1, 1, and 10 nM C14 and LY2874455. Detailed Implementation

[0056] The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are synthesized by methods described in the literature or obtained by purchasing commercially available products.

[0057] The raw materials and intermediates involved in this invention can be commercially available products or synthesized using conventional methods in the field and similar methods described in patents (such as patent application number 201180048148.7) or documents.

[0058] The raw materials, intermediates, and compound synthesis routes involved in this invention are shown below:

[0059] Synthesis Route 1:

[0060]

[0061] a) 1 undergoes a nucleophilic substitution reaction with 2 under alkaline conditions to obtain intermediate 3, wherein the base is cesium carbonate (Cs2CO3); the reaction temperature is 60-80℃; the solvent for the reaction is acetonitrile; and the molar ratio of 1, 2, and the base is 1:1-2:1-5.

[0062] Synthesis Route 2:

[0063]

[0064] b) 4 undergoes a substitution reaction with methanesulfonyl chloride under alkaline conditions to obtain intermediate 5; the solvent for the reaction is dichloromethane (DCM) or dichloroethane; the reaction temperature is 0–25 °C; the base is triethylamine (TEA); the molar ratio of 4, methanesulfonyl chloride, and triethylamine is 1:1.2:1.5.

[0065] Synthesis Route 3:

[0066]

[0067] c) 6 undergoes a substitution reaction with tert-butyldimethylchlorosilane (TBDMSCl) under alkaline conditions to obtain intermediate 7; the base of the reaction is 1H-imidazolium; the reaction temperature is 0–25 °C; the solvent of the reaction is N,N-dimethylformamide (DMF); the molar ratio of 6, tert-butyldimethylchlorosilane, and base is 1:1.5–2:1.5–2;

[0068] d) 7 reacts with N-iodosuccinimide (NIS) to give intermediate 8; the solvent for the reaction is dichloromethane (DCM) or dichloroethane; the reaction temperature is 5-25°C; the molar ratio of 7 to N-iodosuccinimide is 1:1.1;

[0069] e) 8 reacts with 3,4-dihydro-2H-pyran (DHP) under acidic conditions to generate intermediate 9; the solvent for the reaction is a mixed solvent of dichloromethane (DCM) and tetrahydrofuran (THF) in a volume ratio of 1:1; the reaction temperature is 0–25°C; the acid is methanesulfonic acid; the molar ratio of 8, 3,4-dihydro-2H-pyran, and the acid is 1:1.5:0.2;

[0070] f) Step 1: 9 reacts with vinylboronic acid pinacol ester under alkaline conditions and with a catalyst; the solvent for the reaction is DMF; the base for the reaction is N,N-diisopropylethylamine (DIEA); the reaction temperature is 100°C; the catalyst is Pd[P(t-Bu)3]2; the molar ratio of 9, vinylboronic acid pinacol ester, base, and catalyst is 1:1.2:1.5:0.05;

[0071] Step 2: The mixture generated in Step 1 reacts with 3 under alkaline conditions and with a catalyst; the solvent for the reaction is DMF and water; the base for the reaction is Na2CO3; the reaction temperature is 90℃; the catalyst is PdCl2(dppf)2(II)DCM complex; the molar ratio of the mixture, 3, base, and catalyst is 1:1.2:1.5:0.05;

[0072] Step 3: The preliminarily purified product generated in Step 2 reacts in TBAF solution; the solvent for the reaction is THF; the concentration of TBAF solution in the reaction is 1 minTHF; the reaction temperature is 5–25°C.

[0073] a') The preparation conditions for this step are the same as those for step a).

[0074] g) The raw materials react under acidic conditions; the solvent for the reaction is methanol or ethanol; the reaction temperature is 0–25°C; the acid is trifluoroacetic acid or hydrochloric acid; the molar ratio of the raw materials to the acid is 1:3–10.

[0075] Synthesis Route 4:

[0076]

[0077] h) An amine undergoes amide condensation with an acid under alkaline conditions; the base in the reaction is DIEA; the condensing agent in the reaction is HATU; the solvent in the reaction is DMF; the reaction temperature is 0–25°C; and the molar ratio of the amine, acid, condensing agent, and base is 1:1:1.5:3.

[0078] i) The raw materials react under acidic conditions; the solvent for the reaction is dichloromethane or dichloroethane; the reaction temperature is 0–25°C; the acid is trifluoroacetic acid (TFA) or hydrochloric acid; the molar ratio of the raw materials to the acid is 1:10.

[0079] Synthesis Route 5:

[0080]

[0081] In this synthetic route, the synthesis conditions for steps h) and g) are the same as those for steps h) and g) above.

[0082] Synthesis Route 6:

[0083]

[0084] In this synthetic route, the synthesis conditions for steps i) and h) are the same as those for steps i) and h) above.

[0085] j) The raw materials react under alkaline and catalytic conditions; the solvent for the reaction is DMSO; the reaction temperature is 90℃; the alkali is NaHCO3; the catalyst is KI; the molar ratio of the raw materials, alkali, and catalyst is 1:2:0.2.

[0086] k) The raw material and linker react under alkaline conditions; the solvent for the reaction is DMSO; the reaction temperature is 90°C; the base is DIEA; the molar ratio of the raw material, linker, and base is 1:1.2:3.

[0087] Synthesis Route 7:

[0088]

[0089] In this synthetic route, the synthesis conditions for steps h) and i) are the same as those for steps h) and i) above.

[0090] Synthesis Route 8:

[0091]

[0092] The RNF126 selections in R1-R8 are shown below:

[0093]

[0094] In this synthetic route, the synthesis conditions in step h) are the same as those in step h) above.

[0095] In the synthesis method of this invention, room temperature refers to 25°C.

[0096] Example 1: Preparation of compound B1

[0097] Step 1: L1 (100 mg, 0.226 mmol), tert-butyl malonate (38.2 μL), HATU (128.65 mg, 0.338 mmol), and DIEA (0.5 mL) were dissolved in 10 mL of LDM and reacted at RT for 6 hours. The reaction was detected by TLC. After the reaction was complete, the reactants were quenched with water, and the reaction solution was diluted with EA and the layers were separated. The aqueous layer was extracted three times with EA, the organic layers were combined, washed twice with water, washed once with saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give a white solid 12a (92 mg, yield 69.7%).

[0098] Step 2: Dissolve 12a in DCM (6 mL) (avoiding the presence of alcohols in the system), cool in an ice bath, add 6 mL of LTFA, and react overnight at RT. Monitor the reaction by TLC. After the reaction is complete, concentrate the reaction solution directly under reduced pressure to obtain compound 13a, which is then used directly for the next reaction without purification.

[0099] Step 3: Dissolve the unpurified product from the previous step in DMF, add 1.1 times the 12a equivalent of 5-norbornene-2-methylamine, 1.4 times the 12a equivalent of HATU, and 3 times the 12a equivalent of DIEA, and react at RT for 6 hours. Detect the reaction by TLC. After the reaction is complete, quench the reactants with water, add EA to dilute the reaction solution, and separate the layers. Extract the aqueous layer three times with EA, combine the organic layers, wash twice with water, wash once with saturated brine, dry with Na2SO4, filter, and concentrate under reduced pressure. Purify the residue by column chromatography to give a white solid B1 (49.8 mg, 50% yield). 1HNMR(400MHz,DMSO)δ12.93(s,1H),8.63(s,2H),8.30–8.17(m,1H),8.00(s,2H),7.81(s,1H),7.4 3(d,J=9.0Hz,1H),7.16(s,1H),7.13–7.04(m,2H),6.99(d,J=16.9Hz,1H),6.13–5.96(m,2H),5.91 (dd,J=5.9,2.9Hz,1H),4.21(t,J=5.9Hz,2H),3.58(s,1H),3.50(q,J=6.4Hz,2H),3.06(d,J=4.1Hz ,2H),2.92–2.59(m,4H),2.21–2.08(m,1H),1.76(d,J=6.7Hz,3H),1.21(s,1H),1.20–1.05(m,2H).

[0100] Example 2, Preparation of compound B2

[0101] The reaction is similar to that used to synthesize compound B1, by replacing mono-tert-butyl malonate with mono-tert-butyl succinate. 1 HNMR (400MHz, DMSO) δ12.92(s,1H),8.62(s,2H),8.05(t,J=5.8Hz,1H),7.98(s,1H),7.82(s,1H),7.79(d,J=5.6Hz,1 H),7.42(d,J=9.0Hz,1H),7.17(d,J=2.4Hz,1H),7.11(d,J=16.9Hz,1H),7.06(dd,J=8.9,2.2Hz,1H),7.00(d,J=16.9H z,1H),6.25–5.97(m,2H),5.97–5.80(m,1H),4.18(t,J=6.1Hz,2H),3.46(q,J=5.9Hz,2H),3.29–2.90(m,1H),2.89–2 .55(m,4H),2.35(d,J=3.4Hz,4H),2.21–2.04(m,1H),1.75(d,J=6.7Hz,3H),1.25(d,J=8.2Hz,1H),1.22–0.92(m,2H).

[0102] Example 3: Preparation of compound B3

[0103] The reaction is similar to that used to synthesize compound B1, by replacing tert-butyl malonate with tert-butyl 1,5-glutarate. 1HNMR (400MHz, DMSO) δ13.04(s,1H),9.61(s,1H),8.61(s,2H),8.11(t,J=5.6Hz,1H),7.98(s,1H),7.80(s,1H),7.43 (d,J=9.0Hz,1H),7.15(d,J=2.3Hz,1H),7.10(s,1H),7.04(d,J=2.2Hz,1H),6.98(d,J=16.9Hz,1H),6.07(q,J=4.8H z,2H),6.04–5.87(m,1H),4.20(t,J=6.2Hz,2H),3.57(h,J=6.6Hz,4H),3.08(q,J=7.3Hz,4H),2.77(dt,J=12.6,6.1 Hz,1H),2.69(d,J=21.5Hz,2H),2.63–2.53(m,1H),2.49(s,1H),2.08(dt,J=13.8,7.4Hz,4H),1.74(d,J=6.5Hz,3H).

[0104] Example 4: Preparation of compound B4

[0105] The reaction is similar to that used to synthesize compound B1, by replacing tert-butyl malonate with 6-(tert-butoxy)-6-oxohexanoic acid. 1 HNMR (400MHz, DMSO) δ12.92(s,1H),8.62(s,2H),7.98(t,J=5.8Hz,1H),7.95(s,1H),7.82(s,1H),7.72(t,J=5.7Hz,1H),7.42(d, J=9.0Hz,1H),7.17(d,J=2.3Hz,1H),7.10(d,J=15.0Hz,1H),7.06(d,J=7.1Hz,1H),7.00(d,J=16.9Hz,1H),6.17–5.97(m,2H),5.9 2(dd,J=5.8,2.9Hz,1H),4.19(t,J=6.2Hz,2H),3.48(q,J=6.0Hz,2H),3.24–2.85(m,1H),2.85–2.53(m,4H),2.10(t,J=6.4Hz,2H) ,2.06(d,J=6.5Hz,2H),1.76(d,J=6.7Hz,3H),1.72–1.65(m,1H),1.48(p,J=3.6Hz,4H),1.34–1.17(m,2H),1.14(d,J=8.1Hz,1H).

[0106] Example 5: Preparation of compound B5

[0107] The reaction is similar to that used to synthesize compound B1, by replacing tert-butyl malonate with 7-(tert-butoxy)-7-oxoheptanoic acid. 1 HNMR (400MHz, DMSO) δ12.92(s,1H),8.61(s,2H),7.97(t,J=5.7Hz,1H),7.94(s,1H),7.82(s,1H),7.69(t,J=5.7Hz,1H),7.4 2(d,J=9.0Hz,1H),7.16(d,J=2.3Hz,1H),7.10(d,J=16.7Hz,1H),7.07–7.03(m,1H),7.00(d,J=16.8Hz,1H),6.16–5.97(m,2 H),5.92(dd,J=5.7,2.9Hz,1H),4.19(t,J=6.2Hz,2H),3.48(q,J=6.0Hz,2H),3.25–2.84(m,1H),2.84–2.53(m,4H),2.06(dt ,J=19.1,7.4Hz,4H),1.75(d,J=6.6Hz,3H),1.72–1.65(m,1H),1.49(h,J=7.9Hz,4H),1.33–1.16(m,4H),1.16–1.06(m,1H).

[0108] Example 6: Preparation of compound J1

[0109] L1 (52 mg, 0.12 mmol), 1-adamantanecarboxylic acid (23.3 mg, 0.13 mmol), HATU (67 mg, 0.176 mmol), and DIEA (0.5 mL) were dissolved in 10 mL of LDM and reacted at RT for 6 hours. The reaction was detected by TLC. After the reaction was complete, the reactants were quenched with water, the reaction solution was diluted with EA, and the layers were separated. The aqueous layer was extracted three times with EA, the organic layers were combined, washed twice with water, washed once with saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give a white solid J1 (44 mg, yield 62%). 1 HNMR (400MHz, DMSO) δ12.91(s,1H),8.60(s,2H),7.89(s,1H),7.81(s,1H),7.52(s,1H),7.42(d,J=8.9Hz,1H),7.13(d,J=21.2H z,1H),7.06(s,1H),7.01(t,J=15.0Hz,2H),6.19–5.94(m,1H),4.20(s,2H),3.47(s,2H),1.92(s,3H),1.75(s,9H),1.62(s,6H).

[0110] Example 7: Preparation of compound J2

[0111] The reaction is similar to that used to synthesize compound J1, but 1-adamantanecarboxylic acid is replaced with 1-adamantaneacetic acid. 1 HNMR (400MHz, DMSO) δ12.90 (s, 1H), 8.61 (s, 2H), 7.94 (s, 1H), 7.83 (d, J = 5.7Hz, 1H), 7. 80(s,1H),7.42(d,J=9.1Hz,1H),7.15(s,1H),7.13–7.06(m,1H),7.05(s,1H),6.98(d, J=16.9Hz,1H),6.07(q,J=6.6Hz,1H),4.20(t,J=6.2Hz,2H),3.51(q,J=5.9Hz,2H),1.8 4(s,2H),1.82(s,3H),1.75(d,J=6.6Hz,3H),1.56(d,J=15.5Hz,6H),1.51–1.39(m,6H).

[0112] Example 8: Preparation of compound J3

[0113] Step 1: L1 (93 mg, 0.21 mmol), Boc-glycine (40.5 mg, 0.231 mmol), HATU (120 mg, 0.315 mmol), and DIEA (0.5 mL) were dissolved in 10 mL of DMF and reacted at RT for 6 hours. The reaction was detected by TLC. After the reaction was complete, the reactants were quenched with water, and the reaction solution was diluted with EA and the layers were separated. The aqueous layer was extracted three times with EA, the organic layers were combined, washed twice with water, washed once with saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give a white solid 14a (78.2 mg, yield 62%).

[0114] Step 2: Dissolve 14a in DCM (6 mL) (avoiding the presence of alcohols in the system), cool in an ice bath, add 3 mL of HCl in EtOH solution (2 M), and react overnight at RT. Monitor the reaction by TLC. After the reaction is complete, concentrate the reaction solution directly under reduced pressure to obtain compound 15a, which is then used directly for the next reaction without purification.

[0115] Step 3: Dissolve the unpurified product from the previous step in DMF, add 1.1 times the 14a equivalent of 1-adamantaneacetic acid, 1.4 times the 14a equivalent of HATU, and 3 times the 14a equivalent of DIEA, and react at RT for 6 hours. Detect the reaction by TLC. After the reaction is complete, quench the reactants with water, add EA to dilute the reaction solution, and separate the layers. Extract the aqueous layer three times with EA, combine the organic layers, wash twice with water, wash once with saturated brine, dry with Na₂SO₄, filter, and concentrate under reduced pressure. Purify the residue by column chromatography to give a white solid J3 (43 mg, yield 48%). 1 HNMR (400MHz, DMSO) δ12.91(s,1H),8.62(s,2H),7.98(d,J=6.8Hz,2H),7.93(t,J=5.9Hz,1H),7 .82(s,1H),7.42(d,J=9.0Hz,1H),7.25–7.15(m,1H),7.11(d,J=16.9Hz,1H),7.06(dd,J=8.9,2. 3Hz,1H),7.01(d,J=16.8Hz,1H),6.08(q,J=6.6Hz,1H),4.20(t,J=6.2Hz,2H),3.68(d,J=5.7Hz, 2H),3.50(q,J=6.0Hz,2H),1.89(s,2H),1.84(s,3H),1.75(d,J=6.7Hz,3H),1.64–1.46(m,12H).

[0116] Example 9: Preparation of compound J4

[0117] The reaction is similar to that used to synthesize compound J3, by replacing Boc-glycine with Boc-β-alanine. 1 HNMR(400MHz,DMSO)δ12.92(s,1H),8.62(s,2H),8.09(t,J=5.3Hz,1H),7.97(s,1H),7.82(s,1H) ,7.68(t,J=5.7Hz,1H),7.42(d,J=9.0Hz,1H),7.16(d,J=2.3Hz,1H),7.13–7.03(m,2H),6.99(d, J=16.8Hz,1H),6.18–6.00(m,1H),4.19(t,J=6.1Hz,2H),3.48(q,J=6.2Hz,2H),3.25(q,J=6.6Hz ,2H),2.27(t,J=7.2Hz,2H),1.82(s,3H),1.77(d,J=5.0Hz,3H),1.75(s,2H),1.65–1.39(m,12H).

[0118] Example 10: Preparation of Compound J5

[0119] The reaction is similar to that used to synthesize compound J3, by replacing Boc-glycine with Boc-4-aminobutyric acid. 1 HNMR(400MHz,DMSO)δ12.92(s,1H),8.61(s,2H),7.98(d,J=20.7Hz,2H),7.81(s,1H),7.66(s,1H),7.42(s,1H),7.16(s,1 H),7.06(s,3H),6.08(s,1H),4.20(s,2H),3.01(s,2H),2.10(s,2H),1.84(s,3H),1.76(s,3H),1.50(s,12H),1.18(s,2H).

[0120] Example 11: Preparation of compound J6

[0121] The reaction is similar to that used to synthesize compound J3, by replacing Boc-glycine with Boc-5-aminovaleric acid. 1 HNMR (400MHz, DMSO) δ12.97(s,1H),8.66(s,2H),8.03(t,J=5.9Hz,1H),8.00(s,1H),7.87(s,1H),7.66(t ,J=5.6Hz,1H),7.47(d,J=9.0Hz,1H),7.28–7.19(m,1H),7.19–7.09(m,2H),7.05(d,J=16.9Hz,1H),6.13( q,J=6.6Hz,1H),4.24(t,J=6.2Hz,2H),3.53(q,J=5.9Hz,2H),3.06(q,J=6.6Hz,2H),2.15(t,J=7.4Hz,2H) ,1.90(s,3H),1.83(s,2H),1.80(d,J=6.6Hz,3H),1.66(s,2H),1.63–1.49(m,12H),1.41(p,J=7.3Hz,2H).

[0122] Example 12, Preparation of Compound J7

[0123] The reaction is similar to that used to synthesize compound J3, by replacing Boc-glycine with Boc-6-aminohexanoic acid. 1HNMR (400MHz, DMSO) δ12.91(s,1H),8.62(s,2H),7.96(d,J=5.6Hz,1H),7.94(s,1H),7.81(s,1H),7.57(t,J=5.7H z,1H),7.42(d,J=9.0Hz,1H),7.17(d,J=2.3Hz,1H),7.14–7.03(m,2H),6.99(d,J=16.9Hz,1H),6.10(q,J=6.6Hz,1 H),4.18(t,J=6.2Hz,2H),3.47(q,J=6.1Hz,2H),2.98(q,J=6.6Hz,2H),2.07(t,J=7.5Hz,2H),1.86(s,3H),1.77(d ,J=2.8Hz,3H),1.76(s,2H),1.61(d,J=12.1Hz,2H),1.58–1.42(m,12H),1.36(p,J=7.0Hz,2H),1.26–1.20(m,2H).

[0124] Example 13, Preparation of Compound C1

[0125] Step 1: Dissolve 16 (186.1 mg, 0.68 mmol), NaHCO3 (114 mg, 1.36 mmol), and KI (6 mg, catalytic amount) in DMSO. After reacting at 90 °C for 0.5 hours, add tert-butyl bromoacetate (119 mL), and continue the reaction for 4 hours. Monitor the reaction by TLC. After the reaction is complete, quench the reaction solution with water, add EA to dilute the reaction solution, and separate the layers after cooling to room temperature. Extract the aqueous layer three times with EA, combine the organic phases, wash three times with water, wash once with saturated brine, dry with Na2SO4, filter, and concentrate under reduced pressure. Purify the residue by column chromatography to give a white solid 17a (218 mg, yield 82%).

[0126] Step 2: Dissolve 17a in DCM (6 mL) (avoiding the presence of alcohols in the system), cool in an ice bath, add 6 mL of LTFA, and react overnight at RT. Monitor the reaction by TLC. After the reaction is complete, concentrate the reaction solution directly under reduced pressure to obtain compound 18a, which is then used directly for the next reaction without purification.

[0127] Step 3: Dissolve the unpurified product from the previous step in DMF, add 1.1 times the 17a equivalent of L1, 1.4 times the 17a equivalent of HATU, and 3 times the 17a equivalent of DIEA, and react at RT for 6 hours. Detect the reaction by TLC. After the reaction is complete, quench the reactants with water, add EA to dilute the reaction solution, and separate the layers. Extract the aqueous layer three times with EA, combine the organic layers, wash twice with water, wash once with saturated brine, dry with Na2SO4, filter, and concentrate under reduced pressure. Purify the residue by column chromatography to give a pale yellow solid C1 (257 mg, yield 61%). 1 HNMR (400MHz, DMSO) δ12.96(s,1H),11.16(s,1H),8.65(s,2H),8.26(t,J=5.8Hz,1H),8.02(s,1H),7.87( s,1H),7.82(dd,J=8.5,7.3Hz,1H),7.48(dd,J=8.3,4.3Hz,2H),7.39(d,J=8.6Hz,1H),7.22(s,1H),7.17 –7.09(m,2H),7.03(d,J=16.9Hz,1H),6.19–6.08(m,1H),5.16(dd,J=12.9,5.4Hz,1H),4.86(s,2H),4.30 (t,J=6.1Hz,2H),3.65(q,J=6.0Hz,2H),2.99–2.62(m,2H),2.60(t,J=4.7Hz,2H),1.80(d,J=6.6Hz,3H).

[0128] Example 14, Preparation of compound C2

[0129] The reaction is similar to that used to synthesize compound C1, by replacing tert-butyl bromoacetate with tert-butyl 4-bromobutyrate. 1HNMR (400MHz, DMSO) δ13.09(s,1H),11.17(s,1H),9.68(s,2H),8.66(s,2H),8.24(t,J=5.7Hz,1H),8.05(s ,1H),7.86(s,1H),7.77(t,J=8.0Hz,1H),7.55–7.45(m,2H),7.44–7.33(m,1H),7.11(t,J=5.5Hz,2H),7.0 2(d,J=16.9Hz,1H),6.11(q,J=6.7Hz,1H),5.13(dd,J=12.7,5.5Hz,1H),4.26(dt,J=19.8,6.3Hz,4H),3.6 2(td,J=6.8,3.1Hz,4H),2.66(s,2H),2.39(t,J=7.3Hz,2H),2.06(t,J=6.9Hz,2H),1.79(d,J=6.6Hz,3H).

[0130] Example 15, Preparation of compound C3

[0131] The reaction is similar to that used to synthesize compound C1, by replacing tert-butyl bromoacetate with tert-butyl 6-bromohexanoate. 1 HNMR (400MHz, DMSO) δ12.90(s,1H),11.10(s,1H),8.62(s,2H),7.98(t,J=5.7Hz,1H),7.95(s,1H),7.82(s,1H),7.66(ddd,J=8.7 ,7.3,2.0Hz,1H),7.42(d,J=9.0Hz,1H),7.40–7.33(m,2H),7.14(s,1H),7.11–7.04(m,2H),6.98(d,J=16.9Hz,1H),6.08(q,J=6. 6Hz,1H),5.07(dd,J=12.7,5.4Hz,1H),4.20(t,J=6.1Hz,2H),4.12(t,J=6.5Hz,2H),3.49(q,J=6.0Hz,2H),2.97–2.68(m,2H),2. 67–2.52(m,2H),2.12(t,J=7.3Hz,2H),1.76(d,J=6.6Hz,3H),1.72(d,J=7.5Hz,2H),1.58(p,J=7.4Hz,2H),1.42(q,J=8.2Hz,2H).

[0132] Example 16, Preparation of compound C4

[0133] Step 1: Dissolve 19 (197 mg, 0.71 mmol), glycine tert-butyl ester (0.13 mL), and DIEA (0.5 mL) in DMSO. Then react at 90 °C for 4 hours. Monitor the reaction by TLC. After the reaction is complete, quench the reaction solution with water, add EA to dilute the reaction solution, and separate the layers after the reaction solution cools to room temperature. Extract the aqueous layer three times with EA, combine the organic phases, wash three times with water, wash once with saturated brine, dry with Na₂SO₄, filter, and concentrate under reduced pressure. Purify the residue by column chromatography to give a yellow-green solid 20a (56 mg, yield 24%).

[0134] Step 2: Dissolve 20a in DCM (6 mL) (avoiding the presence of alcohols in the system), cool in an ice bath, add 6 mL of LTFA, and react overnight at RT. Monitor the reaction by TLC. After the reaction is complete, concentrate the reaction solution directly under reduced pressure to obtain compound 21a, which is then used directly for the next reaction without purification.

[0135] Step 3: Dissolve the unpurified product from the previous step in DMF (6 mL), add 1.1 times the 20a equivalent of L1, 1.4 times the 20a equivalent of HATU, and 3 times the 20a equivalent of DIEA, and react at RT for 6 hours. Detect the reaction by TLC. After the reaction is complete, quench the reactants with water, add EA to dilute the reaction solution, and separate the layers. Extract the aqueous layer three times with EA, combine the organic layers, wash twice with water, wash once with saturated brine, dry with Na2SO4, filter, and concentrate under reduced pressure. Purify the residue by column chromatography to give a yellow solid C1 (57 mg, yield 45%). 1 HNMR (400MHz, DMSO) δ12.93(s,1H),11.10(s,1H),8.61(s,2H),8.30(t,J=5.7Hz,1H),7.96(s,1H),7.83(s,1H ),7.56(t,J=7.8Hz,1H),7.44(d,J=9.0Hz,1H),7.17(s,1H),7.14–7.06(m,2H),7.03(d,J=8.3Hz,2H),6.98(d ,J=3.5Hz,1H),6.85(d,J=8.5Hz,1H),6.09(q,J=6.6Hz,1H),5.08(dd,J=13.0,5.4Hz,1H),4.24(t,J=5.8Hz,2 H),3.97(d,J=5.7Hz,2H),3.58(t,J=6.1Hz,2H),2.98–2.61(m,2H),2.61–2.52(m,2H),1.76(d,J=6.6Hz,3H).

[0136] Example 17, Preparation of Compound C5

[0137] The reaction is similar to that used to synthesize compound C4, by replacing glycine tert-butyl ester with tert-butyl 3-aminopropionate. 1 HNMR (400MHz, DMSO) δ12.96(s,1H),11.10(s,1H),8.61(d,J=7.0Hz,2H),8.25(t,J=5.9Hz,1H),7.96(s,1H),7.82(s,1H ),7.53(t,J=7.8Hz,1H),7.44(d,J=9.0Hz,1H),7.14(s,1H),7.12(d,J=8.6Hz,1H),7.08(s,1H),7.06(s,1H),7.03–6.91 (m,2H),6.74(t,J=6.3Hz,1H),6.08(d,J=6.6Hz,1H),5.04(dd,J=13.1,5.5Hz,1H),4.20(t,J=6.4Hz,2H),3.61–3.56(m ,2H),3.51(t,J=6.4Hz,2H),3.11(q,J=7.3Hz,2H),2.58(d,J=17.7Hz,2H),2.46(d,J=8.6Hz,2H),1.76(d,J=6.8Hz,3H).

[0138] Example 18, Preparation of compound C6

[0139] The reaction is similar to that used to synthesize compound C4, by replacing glycine tert-butyl ester with 4-aminobutyric acid tert-butyl ester. 1HNMR (400MHz, DMSO) δ12.91(s,1H),11.09(s,1H),8.62(s,2H),8.07(t,J=5.7Hz,1H),7.96(s,1H),7.81(s,1H),7.51(t,J=8.1Hz, 1H),7.42(d,J=8.9Hz,1H),7.13(s,1H),7.10–7.07(m,1H),7.05(d,J=2.1Hz,2H),6.99(d,J=1.3Hz,1H),6.95(dd,J=7.0,2.0Hz,1 H),6.60(t,J=6.1Hz,1H),6.12–6.02(m,1H),5.03(dd,J=12.7,5.4Hz,1H),4.21(t,J=6.1Hz,2H),3.50(q,J=6.0Hz,2H),3.28(q,J =6.5Hz,2H),3.07–2.71(m,2H),2.57(dd,J=17.6,12.8Hz,2H),2.20(t,J=7.2Hz,2H),1.81(q,J=7.2Hz,2H),1.76(d,J=6.6Hz,3H).

[0140] Example 19, Preparation of compound C7

[0141] The reaction is similar to that used to synthesize compound C4, by replacing glycine tert-butyl ester with 5-aminovalerate tert-butyl ester. 1HNMR (400MHz, DMSO) δ12.91(s,1H),11.11(s,1H),8.60(s,2H),8.01(t,J=5.7Hz,1H),7.95(s,1H),7.83(s,1H),7.43(d,J=6.4Hz,1H), 7.41(d,J=5.8Hz,1H),7.14(s,1H),7.10(d,J=16.6Hz,1H),7.06(d,J=2.2Hz,1H),7.00(d,J=16.8Hz,1H),6.97–6.88(m,2H),6.48(t,J= 5.8Hz,1H),6.07(q,J=6.6Hz,1H),5.05(dd,J=13.0,5.4Hz,1H),4.22(t,J=6.2Hz,2H),3.51(q,J=6.2Hz,2H),3.23(q,J=6.4Hz,2H),2. 89(ddd,J=17.8,14.5,5.4Hz,2H),2.67–2.52(m,2H),2.15(t,J=6.9Hz,2H),1.75(d,J=6.6Hz,3H),1.56(ddt,J=19.7,14.0,7.4Hz,4H).

[0142] Example 20: Preparation of compound C8

[0143] The reaction is similar to that used to synthesize compound C4, by replacing glycine tert-butyl ester with 6-aminohexanoate tert-butyl ester. 1HNMR (400MHz, DMSO) δ12.93(s,1H),11.10(s,1H),8.61(s,2H),8.01(t,J=5.7Hz,1H),7.95(s,1H),7.82(s,1H),7.44(d,J=8.6Hz,1H),7 .41(d,J=2.7Hz,1H),7.12(d,J=2.3Hz,1H),7.09(d,J=13.5Hz,1H),7.05(d,J=7.3Hz,1H),7.00(s,1H),6.94(dd,J=11.1,7.7Hz,2H),6.4 2(t,J=5.9Hz,1H),6.06(q,J=6.5Hz,1H),5.03(dd,J=12.7,5.4Hz,1H),4.20(t,J=6.1Hz,2H),3.57(q,J=6.4Hz,2H),3.20(q,J=6.8Hz,2H ),3.08(q,J=7.4Hz,2H),2.97–2.75(m,2H),2.65–2.52(m,2H),2.10(t,J=7.3Hz,2H),1.75(d,J=6.6Hz,3H),1.53(qt,J=7.5,4.9Hz,4H).

[0144] Example 21, Preparation of compound C9

[0145] 19 (68.5 mg, 0.248 mmol), L1 (100 mg, 0.2256 mmol), and DIEA (0.5 mL) were dissolved in DMSO. The reaction was then carried out at 90 °C for 6 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was quenched with water, and EA was added to dilute the reaction solution. After the reaction solution cooled to room temperature, the layers were separated. The aqueous layer was extracted three times with EA, the organic phases were combined, washed three times with water and once with saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give a yellow-green solid C9 (67 mg, yield 43%). 1HNMR(400MHz,DMSO)δ12.99(s,1H),10.92(s,1H),8.64(s,2H),8.08(s,1H),7.91(s,1H),7.61(t,J=7.8Hz,1 H),7.47(d,J=8.9Hz,1H),7.20(d,J=2.3Hz,1H),7.16(s,1H),7.12(s,1H),7.10(s,1H),7.08(s,1H),7.02(d, J=16.9Hz,1H),6.83(t,J=6.2Hz,1H),6.12(q,J=6.6Hz,1H),5.11(dd,J=12.7,5.4Hz,1H),4.45(t,J=5.9Hz,2 H), 3.83 (q, J = 6.2Hz, 2H), 2.90 (ddt, J = 22.3, 13.9, 6.3Hz, 2H), 2.60 (t, J = 12.3Hz, 2H), 1.80 (d, J = 6.6Hz, 3H).

[0146] Example 22, Preparation of compound C10

[0147] The reaction is similar to that used to synthesize compound C4; simply replace 22 with 19. 1 HNMR(400MHz,DMSO)δ13.02(s,1H),11.08(s,1H),9.53(s,1H),8.62(s,2H ),8.39(s,1H),8.01(s,1H),7.82(s,1H),7.58(s,1H),7.47(s,1H),7.16(s ,1H),7.08(s,2H),6.99(s,1H),6.87(s,2H),6.09(s,1H),5.03(s,1H),4.2 4(s,2H),3.90(s,2H),3.10(s,2H),2.87(s,2H),1.97(s,2H),1.76(s,3H).

[0148] Example 23, Preparation of compound C11

[0149] The reaction is similar to that used to synthesize compound C4, by replacing glycine tert-butyl ester with 3-aminopropionate tert-butyl ester and 19 with 22. 1HNMR (400MHz, DMSO) δ12.97(s,1H),11.06(s,1H),8.60(s,2H),8.23(t,J=5.7Hz,1H),7.97(s,1H),7.82(s,1H),7.49(d,J=8.4 Hz,1H),7.43(d,J=9.0Hz,1H),7.21(t,J=5.7Hz,1H),7.14(s,1H),7.09(d,J=12.1Hz,1H),7.07–7.05(m,1H),7.00(s,1H),6.98 (d,J=2.1Hz,1H),6.87(dd,J=8.4,2.2Hz,1H),6.07(q,J=6.6Hz,1H),5.01(dd,J=12.7,5.4Hz,1H),4.21(t,J=6.1Hz,2H),3.58( d,J=6.6Hz,2H),3.09(d,J=7.4Hz,2H),2.94–2.78(m,2H),2.57(d,J=19.0Hz,2H),2.44(t,J=6.9Hz,2H),1.74(d,J=6.6Hz,3H).

[0150] Example 24: Preparation of compound C12

[0151] The reaction is similar to that used to synthesize compound C4. Glycine tert-butyl ester is replaced with tert-butyl 4-bromobutyrate, and 19 is replaced with 22. 1 HNMR (400MHz, DMSO) δ12.93(s,1H),11.06(s,1H),8.61(s,2H),8.09(t,J=5.8Hz,1H),7.97(s,1H),7.82(s,1H),7.51(d,J=8.3Hz,1H ),7.43(d,J=9.0Hz,1H),7.20–7.14(m,1H),7.13(s,1H),7.09(d,J=12.9Hz,1H),7.06(d,J=5.3Hz,1H),6.98(d,J=16.8Hz,1H),6.94( s,1H),6.82(dd,J=8.6,2.1Hz,1H),6.07(q,J=6.6Hz,1H),5.03(dd,J=12.9,5.4Hz,1H),4.22(t,J=6.2Hz,2H),3.51(q,J=6.0Hz,2H) ,3.18(d,J=5.1Hz,2H),3.14(t,J=6.5Hz,2H),2.66–2.53(m,2H),2.23(t,J=7.3Hz,2H),1.82(q,J=6.2Hz,2H),1.75(d,J=6.6Hz,3H).

[0152] Example 25: Preparation of compound C13

[0153] The reaction is similar to that used to synthesize compound C4. Glycine tert-butyl ester is replaced with 5-aminovalerate tert-butyl ester, and 19 is replaced with 22. 1 HNMR(400MHz,DMSO)δ12.94(s,1H),11.08(s,1H),8.60(s,2H),8.04(s,1H),7.96(s,1H),7.83(s,1H ),7.55–7.35(m,2H),7.14(s,2H),7.07(d,J=14.0Hz,2H),7.00(d,J=16.8Hz,1H),6.92(s,1H),6.74 (d,J=8.5Hz,1H),6.06(s,1H),5.03(d,J=12.4Hz,1H),4.22(s,2H),3.11(s,2H),2.89(s,2H),2.59( d,J=19.7Hz,2H),2.16(s,2H),1.74(d,J=6.7Hz,2H),1.59(d,J=28.7Hz,3H),1.22(d,J=25.8Hz,4H).

[0154] Example 26: Preparation of compound C14

[0155] The reaction is similar to that used to synthesize compound C4. Glycine tert-butyl ester is replaced with 6-aminohexanoate tert-butyl ester, and 19 is replaced with 22. 1HNMR (400MHz, DMSO) δ12.92(s,1H),11.09(s,1H),8.61(s,2H),2.05–1.94(m,1H),7.99(t,J=5.8Hz,1H),7.94(s,1H),7.83(s ,1H),7.44(dd,J=23.6,8.6Hz,2H),7.12(d,J=4.5Hz,1H),7.07(d,J=3.2Hz,2H),7.04(d,J=3.3Hz,2H),7.00(s,1H),6.89(s,1 H),6.73(d,J=8.5Hz,1H),6.06(q,J=6.5Hz,1H),5.03(dd,J=13.0,5.4Hz,1H),4.20(t,J=6.1Hz,2H),3.48(q,J=6.1Hz,2H),3 .07(q,J=6.5Hz,2H),2.56(d,J=27.7Hz,4H),2.08(s,2H),1.74(d,J=6.5Hz,3H),1.53(q,J=7.6Hz,4H),1.31(p,J=7.7Hz,2H).

[0156] Example 27, Preparation of compound C15

[0157] The reaction is similar to that used to synthesize compound C9; simply replace 19 with 22. 1 HNMR (400MHz, DMSO) δ12.93(s,1H),11.08(s,1H),8.60(s,2H),8.02(d,J=13.9Hz,1H),7.86(d,J=3.2Hz ,1H),7.59(d,J=8.4Hz,1H),7.43(d,J=9.0Hz,1H),7.28(t,J=6.1Hz,1H),7.16(s,1H),7.09(d,J=9.4Hz ,1H),7.06(s,1H),7.04(s,1H),7.00(s,1H),6.98–6.89(m,1H),6.09(q,J=6.6Hz,1H),5.05(d,J=11.6H z,1H),4.48–4.29(m,2H),3.85–3.67(m,2H),2.87(t,J=13.5Hz,2H),1.98(s,2H),1.76(d,J=6.6Hz,3H).

[0158] Example 28, Preparation of Compound V1

[0159] Step 1: L1 (100 mg, 0.226 mmol), tert-butyl malonate (38.2 μL), HATU (128.65 mg, 0.338 mmol), and DIEA (0.5 mL) were dissolved in DMF (6 mL) and reacted at RT for 6 hours. The reaction was detected by TLC. After the reaction was complete, the reactants were quenched with water, the reaction solution was diluted with EA, and the layers were separated. The aqueous layer was extracted three times with EA, the organic layers were combined, washed twice with water, washed once with saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give a white solid 25a (92 mg, yield 69.7%).

[0160] Step 2: Dissolve 25a in DCM (6 mL) (avoiding the presence of alcohols in the system), cool in an ice bath, add 6 mL of LTFA, and react overnight at RT. Monitor the reaction by TLC. After the reaction is complete, concentrate the reaction solution directly under reduced pressure to obtain compound 26a, which is then used directly for the next reaction without purification.

[0161] Step 3: The unpurified product from the previous step was dissolved in DMF, and 1.1 times the 25a equivalent of VHL ligand ((2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide), 1.4 times the 25a equivalent of HATU, and 3 times the 25a equivalent of DIEA were added. The mixture was reacted at RT for 6 hours. The reaction was detected by TLC. After the reaction was complete, the reactants were quenched with water, the reaction solution was diluted with EA, and the layers were separated. The aqueous layer was extracted three times with EA, the organic layers were combined, washed twice with water, washed once with saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give a white solid V1 (59 mg, yield 40%). 1HNMR (400MHz, DMSO) δ12.90(s,1H),8.62(s,2H),8.28–8.11(m,2H),8.02(d,J=2.2Hz,1H),8.01(s,1H),7.81(s,1H),7.43(d,J=3.6Hz,1H),7.4 1(d,J=3.6Hz,1H),7.38(d,J=8.0Hz,1H),7.30(d,J=7.9Hz,1H),7.17(s,1H),7.11(d,J=2.4Hz,1H),7.08(d,J=2.5Hz,1H),7.06(d,J=2.3Hz,1H ),7.02(s,1H),6.97(s,1H),6.10(q,J=6.5Hz,1H),4.21(t,J=5.3Hz,2H),4.01(d,J=5.9Hz,1H),3.60(p,J=6.4Hz,2H),3.49(d,J=7.1Hz,2H),3 .23(s,2H),3.12(d,J=7.5Hz,2H),2.92(q,J=7.3Hz,1H),2.29(s,1H),1 .77(d,J=6.6Hz,3H),1.56(d,J=31.8Hz,2H),1.26(s,3H),1.24(s,9H).

[0162] Example 29, Preparation of Compound V2

[0163] The reaction is similar to that used to synthesize compound V1, by replacing mono-tert-butyl malonate with mono-tert-butyl succinate. 1HNMR(400MHz,DMSO)δ13.01(s,1H),9.03(s,1H),8.68(s,2H),8.65(s,1H),8.14(s,1H),8.05(s,1H),8.00(d,J=9.1Hz,1H),7.88(s,1H) ,7.51(s,1H),7.48(d,J=5.0Hz,2H),7.43(d,J=8.1Hz,2H),7.23(s,1H),7.17(d,J=16.5Hz,1H),7.13(d,J=9.6Hz,1H),7.07(d,J=16.6H z,1H),6.15(d,J=7.7Hz,1H),5.22(s,1H),4.69–4.50(m,2H),4.45(d,J=28.2Hz,2H),4.31(s,1H),4.28–4.17(m,2H),3.72(s,2H),3.67 –3.58(m,1H),3.52(s,2H),3.15(d,J=8.6Hz,1H),2.56(s,2H),2.43(s,3H),2.04(d,J=54.9Hz,2H),1.81(d,J=6.5Hz,3H),1.32(s,9H).

[0164] Example 30: Preparation of Compound V3

[0165] The reaction is similar to that used to synthesize compound V1, by replacing tert-butyl malonate with tert-butyl 1,5-glutarate. 1HNMR(400MHz,DMSO)δ12.94(s,1H),8.96(s,1H),8.61(s,2H),8.58(d,J=6.1H z,1H),8.01(t,J=5.8Hz,1H),7.97(s,1H),7.88(d,J=9.3Hz,1H),7.82(s,1H) ,7.43(d,J=4.8Hz,1H),7.42(s,1H),7.41(s,1H),7.38(s,1H),7.36(s,1H),7 .17(d,J=2.3Hz,1H),7.07(d,J=8.6Hz,2H),7.00(d,J=16.8Hz,1H),6.09(q,J= 6.5Hz,1H),5.18(d,J=3.6Hz,1H),4.54(d,J=9.2Hz,1H),4.44(q,J=7.5Hz,2H ),4.36(s,1H),4.20(q,J=4.7Hz,2H),3.67(d,J=3.0Hz,2H),3.58(p,J=6.6Hz, 2H),3.51–3.47(m,2H),3.10(q,J=7.4Hz,1H),2.43(s,3H),2.29–2.15(m,2H) ,2.10(t,J=7.6Hz,2H),2.05–1.85(m,2H),1.75(d,J=6.6Hz,3H),1.25(s,9H).

[0166] Example 31, Preparation of Compound V4

[0167] The reaction is similar to that used to synthesize compound V1, by replacing tert-butyl malonate with 6-(tert-butoxy)-6-oxohexanoic acid. 1HNMR (400MHz, DMSO) δ12.92(s,1H),8.97(d,J=5.4Hz,1H),8.62(s,2H),8.57(t,J=6.2Hz,1H),7.97(d,J=8.6Hz,2H),7.83(d,J=12.6Hz,2H) ,7.44–7.42(m,1H),7.40(d,J=3.9Hz,2H),7.38(s,1H),7.36(s,1H),7 .17(s,1H),7.12–7.03(m,2H),7.00(d,J=16.9Hz,1H),6.10(q,J=6.6H z,1H),5.15(d,J=3.5Hz,1H),4.54(d,J=9.4Hz,1H),4.47–4.41(m,2H),4.36(s,1H),4.20(d,J=6.5Hz,2H),3.66(s,2H),3.58(q,J=6.9Hz,2 H),3.48(s,2H),3.13–3.08(m,1H),2.26(d,J=13.6Hz,2H),2.12–2.07(m,2H),1.76(d,J=6.6Hz,3H),1.49(s,4H),1.26(s,3H),1.24(s,9H).

[0168] Example 32, Preparation of Compound V5

[0169] The reaction is similar to that used to synthesize compound V1, by replacing tert-butyl malonate with 7-(tert-butoxy)-7-oxoheptanoic acid. 1HNMR (400MHz, DMSO) δ12.98(s,1H),9.03(s,1H),8.68(s,2H),8.63(d,J=6.4Hz,1H),8.03(d,J=5.7Hz,2H),7.90(d,J=10.6Hz,2H),7.50(s,1H), 7.47(s,2H),7.43(d,J=8.1Hz,2H),7.23(s,1H),7.19–7.10(m,2H),7.0 6(d,J=16.9Hz,1H),6.15(q,J=6.6Hz,1H),5.21(d,J=3.6Hz,1H),4.61(d ,J=9.4Hz,1H),4.56–4.47(m,2H),4.43(s,1H),4.25(t,J=6.7Hz,2H),3 .73(s,2H),3.54(q,J=6.3Hz,2H),3.17(d,J=7.7Hz,1H),2.56(d,J=2.9H z,2H),2.32(dt,J=14.9,7.7Hz,2H),2.15(tq,J=14.1,7.4Hz,4H),1.82( d,J=6.6Hz,3H),1.55(dt,J=12.8,7.0Hz,4H),1.31(s,3H),1.29(s,9H).

[0170] Example 33, Preparation of compound R1

[0171] The reaction is similar to that used to synthesize compound J1, but 1-adamantanecarboxylic acid is replaced with 3-benzoylacrylic acid. 1 HNMR(400MHz,DMSO)δ12.91(s,1H),8.80(t,J=5.8Hz,1H),8.59(s,2H),8.00(s,1H),7.99(d,J=2.4Hz, 2H),7.86(s,1H),7.80(d,J=15.4Hz,1H),7.66(t,J=7.4Hz,1H),7.54(t,J=7.6Hz,2H),7.42(d,J=8.9Hz ,1H),7.15(s,1H),7.11(d,J=16.8Hz,1H),7.06(dd,J=9.1,2.2Hz,1H),7.03(d,J=6.6Hz,1H),6.99(d,J =8.0Hz,1H),6.06(q,J=6.6Hz,1H),4.30(t,J=6.0Hz,2H),3.67(d,J=6.2Hz,2H),1.73(d,J=6.6Hz,3H).

[0172] Example 34, Preparation of compound R2

[0173] The reaction is similar to that used to synthesize compound J1, but 1-adamantanecarboxylic acid is replaced with 3-(4-methoxybenzoyl)acrylic acid. 1 HNMR (400MHz, DMSO) δ12.91 (s, 1H), 8.76 (t, J = 5.8Hz, 1H), 8.58 (s, 2H), 8.00 (s, 1H), 7.98 ( s,2H),7.85(s,1H),7.81(d,J=15.2Hz,1H),7.42(d,J=9.0Hz,1H),7.14(s,1H),7.10(d,J= 16.8Hz,1H),7.07–7.03(m,2H),7.01(d,J=7.2Hz,2H),6.96(d,J=4.8Hz,1H),6.05(q,J=6. 6Hz, 1H), 4.30 (t, J = 6.0Hz, 2H), 3.80 (s, 3H), 3.66 (d, J = 6.3Hz, 2H), 1.72 (d, J = 6.7Hz, 3H).

[0174] Example 35: Preparation of compound R3

[0175] The reaction is similar to that used to synthesize compound J1, by replacing 1-adamantanecarboxylic acid with (E)-4-(4-fluorophenyl)-4-oxo-2-butenoic acid. 1 HNMR (400MHz, DMSO) δ12.92 (s, 1H), 8.81 (t, J = 5.9 Hz, 1H), 8.58 (s, 2H), 8.08 (dd, J = 8. 5,5.4Hz,2H),7.98(s,1H),7.86(s,1H),7.80(d,J=15.4Hz,1H),7.42(d,J=9.1Hz,1H) ,7.39–7.27(m,2H),7.13(s,2H),7.10–7.06(m,1H),7.04(s,1H),7.01(d,J=6.2Hz,1H ),6.04(q,J=6.4Hz,1H),4.31(t,J=5.8Hz,2H),3.76–3.61(m,2H),1.79–1.65(m,3H).

[0176] Example 36: Preparation of compound R4

[0177] The reaction is similar to that used to synthesize compound J1, but 1-adamantanecarboxylic acid is replaced with 3-(4-chlorobenzoyl)acrylic acid. 1HNMR(400MHz,DMSO)δ12.92(s,1H),8.82(t,J=5.8Hz,1H),8.58(s,2H),8.00(s,1H),7.98(d, J=2.1Hz,2H),7.86(s,1H),7.78(d,J=15.3Hz,1H),7.56(dd,J=8.6,2.1Hz,2H),7.42(d,J=9.0 Hz,1H),7.13(d,J=2.7Hz,1H),7.08(s,1H),7.07–7.04(m,1H),7.04(s,1H),7.02–6.96(m,1H) ,6.10–6.00(m,1H),4.31(t,J=6.0Hz,2H),3.68(dd,J=8.9,3.9Hz,2H),1.72(d,J=6.4Hz,3H).

[0178] Example 37, Preparation of compound R5

[0179] The reaction is similar to that used to synthesize compound J1, but 1-adamantanecarboxylic acid is replaced with 3-(4-bromobenzoyl)acrylic acid. 1 HNMR(400MHz,DMSO)δ12.92(s,1H),8.81(t,J=5.8Hz,1H),8.58(s,2H),7.99(s,1H),7.9 1(d,J=8.5Hz,2H),7.86(s,1H),7.77(d,J=15.4Hz,1H),7.71(s,1H),7.69(s,1H),7.42( d,J=9.0Hz,1H),7.13(s,1H),7.09(s,1H),7.07–7.02(m,2H),6.99(d,J=12.0Hz,1H),6. 04(q,J=6.7Hz,1H),4.31(t,J=6.0Hz,2H),3.68(q,J=6.3Hz,2H),1.72(d,J=6.6Hz,3H).

[0180] Example 38, Preparation of compound R6

[0181] The reaction is similar to that used to synthesize compound J1, but 1-adamantanecarboxylic acid is replaced with trans-4-trifluoromethylcinnamic acid. 1HNMR (400MHz, DMSO) δ12.93 (s, 1H), 8.57 (s, 2H), 8.46 (t, J = 5.8Hz, 1H), 7.99 (s, 1H), 7. 86(s,1H),7.82–7.64(m,4H),7.54(d,J=15.8Hz,1H),7.42(d,J=9.0Hz,1H),7.13(s,1H) ,7.09(s,1H),7.05(d,J=9.3Hz,1H),7.00(d,J=16.8Hz,1H),6.82(d,J=15.9Hz,1H),6. 03(q,J=6.6Hz,1H),4.31(t,J=6.0Hz,2H),3.67(q,J=6.1Hz,2H),1.70(d,J=6.6Hz,3H).

[0182] Example 39, Preparation of Compound R7

[0183] The reaction is similar to that used to synthesize compound J1, by replacing 1-adamantanecarboxylic acid with 3-(trifluoromethyl)cinnamic acid. 1 HNMR(400MHz,DMSO)δ12.93(s,1H),8.58(s,2H),8.37(t,J=5.8Hz,1H),7.98(s,1H),7.91(s,1H),7.88 (d,J=6.9Hz,2H),7.69(d,J=7.9Hz,1H),7.62(d,J=7.7Hz,1H),7.57(d,J=15.9Hz,1H),7.43(d,J=9.0H z,1H),7.16–7.12(m,1H),7.09(s,1H),7.06(dd,J=9.0,2.2Hz,1H),7.00(d,J=16.9Hz,1H),6.82(d,J= 15.8Hz, 1H), 6.05 (q, J = 6.6Hz, 1H), 4.31 (t, J = 6.0Hz, 2H), 3.67 (q, J = 6.2Hz, 2H), 1.72 (d, J = 6.6Hz, 3H).

[0184] Example 40: Preparation of compound R8

[0185] The reaction is similar to that used to synthesize compound J1, by replacing 1-adamantanecarboxylic acid with trans-3,5-bis(trifluoromethyl)cinnamic acid. 1HNMR(400MHz,DMSO)δ12.91(s,1H),8.57(s,2H),8.37(t,J=5.7Hz,1H),8.26(s,2H),8.04 (s,1H),7.98(s,1H),7.87(s,1H),7.67(d,J=15.8Hz,1H),7.43(d,J=9.0Hz,1H),7.13(s,1 H),7.09(s,1H),7.06(dd,J=9.1,2.3Hz,1H),7.01(d,J=5.5Hz,1H),6.97(d,J=4.6Hz,1H), 6.05(q,J=6.6Hz,1H),4.31(t,J=5.9Hz,2H),3.68(q,J=6.4Hz,2H),1.72(d,J=6.7Hz,3H).

[0186] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0187] Example 1: Inhibitory activity of the candidate compounds in this invention against KATOIII cell proliferation and against FGFR2 kinase.

[0188] 1. Inhibitory activity against KATOIII cell proliferation

[0189] KATOIII cell line was purchased from the American Type Culture Collection (ATCC); DMEM medium and fetal bovine serum (FBS) were purchased from GIBICO, USA; penicillin and streptomycin were purchased from Hyclone, USA; culture dishes and 96-well plates were purchased from Sepp, Inc.; centrifuge tubes of various sizes were purchased from BD, Inc.; MTT reagent was purchased from Donjindo, Japan; and other reagents were purchased from Sigma, USA.

[0190] The inhibitory activity of each candidate compound on the proliferation of KATOIII cell line was tested. Cells in the logarithmic growth phase were collected and seeded into 96-well plates, then cultured overnight at 37°C and 5% CO2. The next day, cell adhesion and growth were observed. A series of serum-containing fresh medium solutions with different concentrations of the test compounds were prepared in deep-well plates. 100 μL of medium containing different concentrations of the test compound was added to each well, with two replicates for each concentration. A negative control group containing only 0.1% DMSO was also included. After 3 days of drug treatment, 20 μL of 5 mg / mL LMT solution was added to each well, and the cells were cultured for another 3 hours. Then, 10% SDS solution was added and the cells were incubated overnight. The absorbance was measured at 490 nm the next day. The inhibition rate of the compounds on tumor cell proliferation was calculated based on the absorbance of the control and experimental groups. The proliferation inhibition curves were then fitted using GraphPad Prism 9 software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The results are shown in Tables 1 and 2.

[0191] 2. Inhibitory activity against FGFR2 kinase

[0192] The present invention uses the Lanthascreen Assay method to test the FGFR2 kinase inhibitory activity, which is briefly described below:

[0193] 2.1 Prepare 1× kinase buffer: 1x kinase buffer (containing 50 mM HEPES pH 7.5, 10 mM MgCl2, 4 mM MTT, 0.01% Tween-20, and 0.01% BSA).

[0194] 2.2 Compound Preparation: Dilute the compound to 100-fold with pure DMSO to the desired maximum inhibitor concentration. For a concentration of 10 nM for this test, prepare a 1000 nM compound DMSO solution in this step. Transfer the compound from the EP tube to the wells of a 96-well plate, then transfer 15 μl to the next well (30 μl) for serial dilution, repeating this process for a total of 6 concentrations. Add 40 μl of pure DMSO to two empty wells in the same 96-well plate, without a composite control or enzyme control. Label this plate as the source plate. Repeat the transfer of 200 nl from each well from the source plate to the 384-well detection plate.

[0195] 2.3 Kinase Reaction Procedure: Preparation of 2x Kinase Solution: Prepare a 2x kinase solution for each reagent at twice the final concentration in 2x kinase buffer. Then, add 10 μl of kinase solution (with 10 μl of 1x kinase buffer) to each well of the assay plate, except for the control well (which does not contain enzyme). Shake the plate and incubate at room temperature for the specified time. Preparation of 2x Substrate Solution: Prepare substrate solutions of luciferin GT and ATP at twice the final concentration of each reagent required in the experiment in 1x kinase reaction buffer. Add 10 μl of substrate solution to each well of the assay plate to start the reaction. Shake the plate, cover it, and incubate at room temperature for the specified time.

[0196] 2.4 Kinase assay: Prepare a test solution with a final concentration of 2x in antibody dilution buffer. Add 20 μl of the test solution to each well of the test plate to stop the reaction. Briefly mix with a centrifuge, incubate at room temperature for the specified time, and then read the fluorescence on a plate reader.

[0197] 2.5 Data Readout: Collect prospective data for excitation at 340nm and emission at 520nm and 495nm.

[0198] 2.6 Curve Fitting: Calculate the ratio of RFU520nm / RFU495nm; convert the ratio to a percentage inhibition value: Inhibition percentage = (max - sampleRatio) / (max - min) * 100. "min" represents the ratio controlled without enzyme, and "max" represents the ratio controlled by DMSO; data are presented in MS Excel, XLFitexcel plugin version 5.4.0.8. The curve fitted by the equation for calculating the half-maximum inhibitory concentration is: Y = Bottom + (Top - Bottom) / (1 + (IC50 - 0.5%) 50 / X)^HillSlope.

[0199] The inhibition rates of the compounds against FGFR2 kinase at 10 nM are shown in Table 2.

[0200] Compound 4 in Tables 1 and 2 is LY2874455.

[0201] Table 1. Antiproliferative effects of some compounds on KATOIII cell line (MTT assay)

[0202]

[0203] Table 2. Antiproliferative effects (MTT assay) and inhibitory effects on FGFR2 kinase of some compounds on KATOIII cell line.

[0204]

[0205]

[0206] In Table 2, NT indicates no detection.

[0207] As shown in Tables 1 and 2, multiple tested compounds, including C5, C6, C8, C9, and C14, exhibited strong inhibitory activity against the KATOIII cell line, with an IC50 value of [missing information]. 50 Value ≤ 10nM.

[0208] Experimental Example 2: Effects of candidate compounds on FGFR2 protein expression in KATOIII cells

[0209] KATOIII cell line was purchased from the American Type Culture Collection (ATCC); DMEM medium and fetal bovine serum (FBS) were purchased from GIBICO, USA; penicillin and streptomycin were purchased from Hyclone, USA; culture dishes and 96-well plates were purchased from Sepp, Inc.; centrifuge tubes of various sizes were purchased from BD, Inc.; and other reagents were purchased from Sigma, Inc. (USA).

[0210] Tumor cells in the logarithmic growth phase were injected at a rate of 3 × 10⁻⁶. 3 Cells were seeded at a concentration of [number] cells / well in 6-well plates. The following day, after cell attachment, the supernatant was aspirated, and 2 mL of prepared culture medium containing different concentrations of the compound was added. Cells were treated with medium containing 0.1% DMSO as a negative control. After 24 hours of drug treatment, the cell suspension and adherent cells were collected and lysed with RIPA buffer for 1 hour. The cell lysates were then centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected and protein concentration was determined using the BCA method. Equal amounts of protein samples were separated on an SDS-PAGE gel and transferred to a PVDF membrane. The membrane was then incubated with the appropriate primary and secondary antibodies. Finally, under light-protected conditions, the bands were immersed in developing solution and exposed to obtain Western blot images. These images can be quantitatively analyzed using ImageJ software.

[0211] As shown in Figures 1 and 2, compounds of the C11-C14 class can cause different degrees of degradation at two different concentrations. Among them, C14 has the strongest degradation ability, and it can be found that the degradation effect at 1 μM is worse than that at 100 nM. At the concentrations of 1 μM and 100 nM, 47% and 80% of FGFR2 protein were degraded, respectively, indicating that C14 may have already exhibited a hook effect at 1 μM.

[0212] Experimental Example 3: Effect of compound C14 on FGFR2 protein expression in KATOIII cells

[0213] The reagents and methods used in this experiment are the same as those in Experiment 2.

[0214] The degradation effect of compound C14 on FGFR2 protein in KATOIII cells was investigated. The degradation effect of 100 nM C14 on FGFR2 protein at specified time points was observed (Figures 3A-3B). The results showed that significant protein degradation was observed after 24 hours, and the degradation effect became more pronounced with prolonged treatment to 48 hours, indicating that the drug can reduce FGFR2 expression in a time-dependent manner. Simultaneously, the degradation effect of C14 at concentration gradients on FGFR2 protein after 24 hours was observed. It was found that C14 exhibited a significant concentration-dependent degradation effect, almost completely degrading FGFR2 at 33.3 nM. max Approximately 86%, DC 50 The concentration was 0.645 nM, and a hook effect appeared at 300 nM (Figures 3C-3D). Notably, protein levels recovered slightly with prolonged drug treatment time (72 h, 96 h), and the drug concentration-dependent hook effect appeared earlier at 72 h (Figure 3E).

[0215] Experiment 4: Effect of compound C14 on FGFR1 / 3 / 4 protein expression in non-FGFR2 high-expression cell lines

[0216] The reagents and methods used in this experiment are the same as those in Experiment 2.

[0217] To verify the effect of C14 on FGFR1 / 3 / 4 protein expression, FGFR1 / 3 / 4 expression was investigated in non-FGFR2-overexpressing cell lines. Under the same treatment time (24 h) and concentration conditions (0–300 nM) as KATOIII, C14 had almost no effect on cell lines overexpressing FGFR3 (MCF-7) and FGFR4 (Huh-7), but only slightly degraded FGFR1 overexpressed in the BXPC3 cell line (Figure 4), with only about 30% of FGFR1 protein degraded at 300 nM. These results indicate that in C14-treated cells, the main downregulated protein is FGFR2, while FGFR1 / 3 / 4 (DC...) is downregulated... 50 The >300 nM) was retained to some extent, which confirms that C14 has a significant and highly selective degradation effect on FGFR2.

[0218] Example 5: Compound C14 degrades FGFR2 protein via the ubiquitin-proteasome system. The experimental reagents and methods in this example are the same as in Example 2.

[0219] C14, as a PROTAC molecule, degrades the target protein FGFR2 via the ubiquitin-proteasome system (UPS). To investigate the potential mechanism by which compound C14 degrades FGFR2, cells were treated with C14 followed by the addition of the parental inhibitor LY2874455 and the proteasome inhibitors bortezomib and MG132. The degrading agents competed with these inhibitors, affecting the degradation activity. As shown in Figure 5, by treating cells with C14 followed by the addition of the parental inhibitor LY2874455 and the proteasome inhibitors bortezomib and MG132, the degrading agents competed with them, affecting the degradation activity. Therefore, this invention investigated the mechanism by which C14 leads to FGFR2 degradation. The addition of the parental inhibitor LY2874455 indeed competed with C14 for the binding site of the FGFR2 protein, resulting in a decrease in the degradation activity of C14. The use of the proteasome inhibitors bortezomib and MG132 significantly limited the degradation activity of C14, while the addition of the lysosomal inhibitor chloroquine had no effect. This study indicates that the degradation of FGFR2 by C14 is related to the ubiquitin-proteasome pathway, rather than the lysosomal pathway.

[0220] Experimental Example 6: Compound C14 affects the downstream signaling pathway of FGFR2 protein.

[0221] The reagents and methods used in this experiment are the same as those in Experiment 2.

[0222] Immunoblot experiments were used to verify the effect of C14 on the phosphorylation of FGFR2 and its downstream key proteins PLCγ and FRS2. As shown in Figure 6, in untreated KATOIII cells, FGFR exhibited significant autophosphorylation at the A-cycle tyrosine residue (Tyr656 / 657). Treatment with 1 nM C14 in this invention rapidly inhibited A-cycle phosphorylation of FGFR2 protein, with a better effect than treatment with 1 nM of the parental inhibitor LY2874455. Furthermore, compared with cells treated with 1 nM and 10 nM concentrations of the parental inhibitor LY2874455, cells treated with the same concentration of C14 showed FGFR2 protein degradation and lower levels of PLCγ and FRS2 phosphorylation.

[0223] In summary, this invention provides a compound that can regulate the level of fibroblast growth factor receptor 2 protein. The compound exhibits good inhibitory effects on FGFR2 protein expression with high selectivity. Furthermore, the compound can selectively inhibit the proliferation of cells with high FGFR2 expression, demonstrating good anti-tumor activity. At the same time, the raw materials for the synthesis of the compound are readily available, and the synthesis method is easy to implement, providing a new option for the development and application of anti-tumor drugs.

Claims

1. A compound or a salt thereof, characterized in that: The compound is shown in Formula II: Formula II, where Linker is selected from none, X is selected from NH or O; n is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

2. A compound or a salt thereof, characterized in that: The compound is shown in Formula III: Formula III, where Linker is selected from none, X is selected from NH or O; n is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

3. A compound or a salt thereof, characterized in that: The structure of the compound is one of the following compounds: 。 4. Use of the compound or salt thereof according to any one of claims 1 to 3 in the preparation of FGFR2 inhibitors.

5. Use of the compound or salt thereof according to any one of claims 1 to 3 in the preparation of a medicament for the prevention and / or treatment of tumors associated with FGFR2.

6. The use according to claim 5, characterized in that: The tumors mentioned are brain cancer, leukemia, lymphoma, Bannayan-Zonana syndrome, Cowden's disease, Lhermitte-Duclos disease, breast cancer, colon cancer, stomach cancer, bladder cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, giant cell tumor of bone, or thyroid cancer.

7. The use according to claim 5, characterized in that: The tumors are glioblastoma, ependymoma, and neuroblastoma.

8. The use according to claim 5, characterized in that: The tumor is inflammatory breast cancer.

9. The use according to claim 5, characterized in that: The tumors are Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma.

10. The use according to claim 5, characterized in that: The tumor is a Wilms' tumor.

11. A drug for the prevention and / or treatment of tumors, characterized in that: It is a formulation prepared by using the compound or its salt as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients, as described in any one of claims 1 to 3.

12. A pharmaceutical composition for the prevention and / or treatment of tumors, characterized in that: It includes the compound or salt thereof as described in any one of claims 1 to 3.

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