Benzofuran bispyrazoles and methods of making and using the same

Abstract

The application discloses a kind of benzofuran pyrazole compounds or its pharmaceutically acceptable salt or deuteride and preparation method and application thereof.A variety of biological experiments confirm that the compound prepared in the application has MYC inhibitory activity, and has significant antitumor activity and can enhance the response of tumor to immunotherapy, and can be used as MYC inhibitor for the treatment of tumor and other MYC related diseases, and has good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a kind of benzofuran pyrazole compounds and preparation method and application thereof. BACKGROUND

[0002] MYC is a major nuclear transcription factor located downstream of key oncogenic signaling pathways such as Wnt, Ras and PI3K / AKT, which regulates network-driven tumorigenesis by integrating multidimensional signal transduction. MYC expression in normal cells is strictly regulated, but its abnormal expression (such as gene amplification, upstream pathway mutation or epigenetic disorder) is closely related to the occurrence, development and maintenance of more than 70% of malignant tumors, and can manipulate almost all characteristics of tumors, such as genomic instability, proliferation, survival, angiogenesis, invasion and migration, immune escape and chemotherapy resistance. Studies have shown that inhibition of MYC can induce sustained regression of tumors and remodeling of tumor microenvironment. Therefore, MYC inhibitors have great application prospects in tumor treatment.

[0003] Although MYC is a core oncogenic hub in cells, it is often considered as an "undruggable" protein in traditional drug design due to its lack of a typical small molecule drug binding pocket as a naturally disordered protein. So far, there is no small molecule inhibitor with sufficient evidence to directly interfere with MYC function entering clinical research; the small molecule inhibitors found so far either have low activity or (and) poor pharmacokinetic properties, and most of them have no in vivo efficacy. In view of the great and unmet clinical treatment needs of MYC-related tumors, it is of great significance to develop small molecule inhibitors with in vitro and in vivo efficacy and directly interfering with MYC function, which will lay the foundation for discovering innovative anti-tumor drugs targeting MYC. SUMMARY

[0004] In view of the deficiencies of existing inhibitors directly targeting MYC in terms of anti-tumor effect, the purpose of the present application is to provide a class of benzofuran pyrazole MYC inhibitors and preparation method and application thereof. Through multiple biological experiments, it is confirmed that the compounds synthesized in the present application can directly inhibit MYC function and have significant anti-tumor activity.

[0005] The present application is realized by the following technical solutions:

[0006] On the one hand, the present application provides a kind to be with as shown in formula I benzofuran pyrazole compound or its pharmaceutically acceptable salt or deuteride,

[0007]

[0008] In the formula: X is independently selected from any one of -(CH2)n1-, C5-C14 heteroaryl, and C6-C14 aryl, which are substituted with at least one R1;

[0009] R1 is independently selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C20 nitrogen-containing heterocyclic group, R5C(=O)-, R5(CH2)n2-, R5C(=O)NH-, R5(CH2)n2-C(=O)NH-; n1 = 1-3, n2 = 1-3; when R1 is a C3-C20 nitrogen-containing heterocyclic group, it can be substituted by at least one R6;

[0010] R2 and R3 are each independently selected from hydrogen, C1-C6 alkyl, and C3-C8 cycloalkyl;

[0011] R4 is independently selected from any one of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, and C1-C6 haloalkoxy;

[0012] R5 is independently selected from any one of C1-C6 alkyl groups and C3-C20 nitrogen-containing heterocyclic groups; when R5 is a C3-C20 nitrogen-containing heterocyclic group, it can be substituted by at least one R7;

[0013] R6 and R7 are each independently selected from hydrogen, halogen, C1-C6 alkyl, and hydroxyl groups.

[0014] In certain preferred embodiments of the present invention, certain groups in benzofuran-bipyrazole compounds or their pharmaceutically acceptable salts or deuterated derivatives as shown in Formula I are defined as follows (undefined groups are as described in any embodiment of this application):

[0015] X is independently selected from -CH2-, -CH2CH2-, C5-C6 heteroaryl groups substituted with at least one R1. Any one of them;

[0016] R1 is independently selected from hydrogen, halogen, methyl, C3 hydroxyalkyl, trifluoromethyl, methoxy, trifluoromethoxy, C4-C6 nitrogen-containing heterocyclic group, R5C(=O)-, R5CH2-, R5CH2-C(=O)NH-; when R1 is a C4-C6 nitrogen-containing heterocyclic group, it can be substituted by at least one R6;

[0017] R2 and R3 are each independently selected from hydrogen and methyl, respectively;

[0018] R4 is independently selected from methyl and trifluoromethyl;

[0019] R5 is independently selected from C4-C6 nitrogen-containing heterocyclic groups and can be substituted by at least one R7;

[0020] R6 and R7 are respectively selected from hydrogen, halogen, methyl, and hydroxyl.

[0021] In certain preferred embodiments of the present invention, certain groups in benzofuran-bipyrazole compounds or their pharmaceutically acceptable salts or deuterated derivatives as shown in Formula I are defined as follows (undefined groups are as described in any embodiment of this application):

[0022] X is independently selected from -CH2-, -CH2CH2-, which are substituted by at least one R1. Any one of them;

[0023] R1 is independently selected from hydrogen, halogen, methyl, C3 hydroxyalkyl, methoxy, trifluoromethyl, trifluoromethoxy, C4-C6 nitrogen-containing heterocyclic group, R5C(=O)-, R5CH2-, R5CH2-C(=O)NH-; when R1 is a C4-C6 nitrogen-containing heterocyclic group, it can be substituted by at least one R6;

[0024] R2 is independently selected from hydrogen;

[0025] R3 is independently selected from either hydrogen or methyl;

[0026] R4 is independently selected from trifluoromethyl;

[0027] R5 is independently selected from C4-C6 nitrogen-containing heterocyclic groups and can be substituted by at least one R7;

[0028] R6 and R7 are independently selected from any one of hydrogen, halogen, methyl, and hydroxyl.

[0029] In certain preferred embodiments of the present invention, certain groups in benzofuran-bipyrazole compounds or their pharmaceutically acceptable salts or deuterated derivatives as shown in Formula I are defined as follows (undefined groups are as described in any embodiment of this application):

[0030] X is independently selected from those that are replaced by at least one R1. Any one of them;

[0031] R1 is independently selected from hydrogen, halogen, methyl, methoxy, trifluoromethyl, trifluoromethoxy, R5C(=O)-, R5CH2-, R5CH2-C(=O)NH-;

[0032] R2 is independently selected from hydrogen;

[0033] R3 is independently selected from either hydrogen or methyl;

[0034] R4 is independently selected from trifluoromethyl;

[0035] R5 is independently selected from C4-C6 nitrogen-containing heterocyclic groups; R5 may be substituted by at least one R7;

[0036] R7 is independently selected from any one of hydrogen, halogen, methyl, and hydroxyl.

[0037] In certain preferred embodiments of the present invention, certain groups in benzofuran-bipyrazole compounds or their pharmaceutically acceptable salts or deuterated derivatives as shown in Formula I are defined as follows (undefined groups are as described in any embodiment of this application):

[0038] X is independently selected from those that are replaced by at least one R1. Any one of them;

[0039] R1 is independently selected from hydrogen, halogen, methyl, methoxy, trifluoromethyl, trifluoromethoxy, R5C(=O)-, R5CH2-, R5CH2-C(=O)NH-;

[0040] R2 is independently selected from hydrogen;

[0041] R3 is independently selected from either hydrogen or methyl;

[0042] R4 is independently selected from trifluoromethyl;

[0043] R5 is independently selected from C4-C6 nitrogen-containing heterocyclic groups; R5 may be substituted by at least one R7;

[0044] R7 is independently selected from any one of hydrogen, halogen, methyl, and hydroxyl.

[0045] In certain preferred embodiments of the present invention, the benzofuran-bipyrazole compound as shown in Formula I, or its pharmaceutically acceptable salt or deuterated derivative, has any of the following compound structural formulas:

[0046]

[0047]

[0048] Furthermore, this invention also provides the use of the benzofuran-bipyrazole compounds shown in Formula I, or their pharmaceutically acceptable salts or deuterated derivatives, in the preparation of MYC inhibitors. The compounds prepared by this invention can directly inhibit MYC function and exhibit significant antitumor activity, and can be used as MYC inhibitors.

[0049] In another aspect, the present invention also provides a MYC inhibitor composition comprising an effective amount of a benzofuran-bipyrazole compound as shown in Formula I or a pharmaceutically acceptable salt or deuterated thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0050] In some preferred embodiments of the present invention, the MYC inhibitor composition further includes at least one other therapeutic agent, preferably a small molecule immune checkpoint inhibitor. The dosage form of the MYC inhibitor composition is any clinically or pharmaceutically acceptable dosage form, such as tablets, granules, capsules, injections, oral solutions, syrups, suppositories, transdermal formulations, and other pharmaceutically conventional formulations.

[0051] Finally, the present invention also provides the use of benzofuran bipyrazole compounds as shown in Formula I or pharmaceutically acceptable salts or deuterated derivatives thereof, or MYC inhibitor compositions, in the preparation of antitumor drugs.

[0052] The tumors described in this invention include solid tumors and hematologic tumors, including but not limited to breast cancer, sarcoma, lung cancer, prostate cancer, colon cancer, rectal cancer, kidney cancer, pancreatic cancer, neuroblastoma, glioma, head cancer, neck cancer, thyroid cancer, liver cancer, ovarian cancer, uterine cancer, endometrial cancer, gastric cancer, bladder cancer, gastrointestinal stromal tumor, nasopharyngeal carcinoma, leukemia, lymphoma, multiple myeloma, etc.

[0053] The dosage of the compound of this invention is 1mg-1000mg / day, but may deviate from this range depending on the severity of the condition or the dosage form.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] This invention provides benzofuran-bipyrazole compounds as shown in Formula I, or their pharmaceutically acceptable salts or deuterated derivatives. Through various biological experiments, it has been confirmed that the compounds prepared by this invention have MYC inhibitory activity, significant antitumor activity, and can enhance the tumor response to immunotherapy. They can be used as MYC inhibitors for the treatment of tumors and other MYC-related diseases, and have good application prospects. Attached Figure Description

[0056] Figure 1 To demonstrate the ability of compound 15 to interfere with the protein-protein interaction (PPI) of MYC / MAX in a co-immunoprecipitation (Co-IP) assay: (A) Protein levels of MYC and MAX were determined by Western blotting (WB); (B) Bar charts show the quantitative levels of the WB bands (results are expressed as mean ± standard deviation, n = 3). *** p < 0.001 **** p < 0.0001 vs. control group; #### p < 0.0001 (vs. positive control MYCi975 group).

[0057] Figure 2To illustrate the inhibitory effect of compound 15 on MYC / MAX-PPI in the AlphaLISA assay: (A) Dose-response curve of compound 15; (B) Dose-response curve of MYCi975.

[0058] Figure 3 To determine the MYC protein level at a specified temperature point after 1 h of treatment of PC-3 cells with compound 15 (10 μM) in a cell thermal displacement assay (CETSA): (A) MYC protein level determined by Western blotting; (B) Relationship between MYC protein level and temperature (results are expressed as mean ± standard deviation, n = 3).

[0059] Figure 4 The effect of the compound on MYC protein expression level: (A) MYC protein expression level was detected by Western blotting; (B) Bar chart showing the quantitative level of the Western blotting bands (results are expressed as mean ± standard deviation, n = 3). *** p < 0.001 **** p < 0.0001 (VS control group).

[0060] Figure 5 The ability of compound 15 to inhibit Luc2p expression (driven by the MYC response element Ebox): (A) Dose-response curve of compound 15; (B) Dose-response curve of MYCi975.

[0061] Figure 6 The in vivo antitumor efficacy of compound 15 in a mouse MYC-CaP prostate cancer model: (A, B) Tumor volume and body weight curves of experimental animals during treatment; (C, D) Final tumor size and weight of MYC-CaP tumor-bearing mice after 16 days of treatment (Results are expressed as mean ± standard deviation, n = 6). * p < 0.05 *** p < 0.001 (VS control group); (E) Hematoxylin-eosin (HE) stained sections of tumor tissue after treatment.

[0062] Figure 7 HE-stained sections of heart, liver, spleen, lung, and kidney tissues after treatment.

[0063] Figure 8 The in vivo antitumor efficacy of INCB086550, compound 15, and their combination in a mouse MYC-CaP prostate cancer model: (A, B) Tumor volume and body weight curves of experimental animals during treatment; (C, D) Final tumor size and weight of MYC-CaP tumor-bearing mice after treatment with INCB086550, 15, and their combination (Results are expressed as mean ± standard deviation, n = 6). ** p < 0.01 vs INCB086550 group; #p < 0.05 (VS Cpd.15); (E) HE-stained sections of tumor tissue after treatment.

[0064] Figure 9 HE-stained sections of heart, liver, spleen, lung, and kidney tissues after treatment with INCB086550, compound 15, and the combination of the two.

[0065] Figure 10 Immunohistochemical (IHC) staining of tumor tissue: (A) Representative results of IHC staining for CD8, CD45, and Ki67 in the tumor microenvironment (TME) after treatment; (B) Bar graph showing quantitative results of IHC staining. * p < 0.05 ** p < 0.01, *** p < 0.001 (VS control group). Detailed Implementation

[0066] Unless otherwise defined, all technical terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains.

[0067] In this application, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0068] "C5-C14 heteroaryl" refers to a non-all-carbon monocyclic or fused polycyclic group with 5 to 14 ring atoms, possessing a fully conjugated π-electron system. Examples of heteroaryl groups include, but are not limited to, pyridyl, imidazolyl, thiophene, furanyl, and thiazolyl.

[0069] "C1-C6 alkyl" refers to alkyl groups with 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, etc.

[0070] "C6-C14 aryl" refers to an all-carbon monocyclic or fused polycyclic ring with 6 to 14 carbon atoms, possessing a fully conjugated π-electron system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracene, phenanthrene, fluorenyl, 2,3-dihydro-1H-isoindolyl, 2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, etc.

[0071] "C1-C6" hydroxyalkyl refers to an alkane with 1 to 6 carbon atoms in which at least one hydrogen atom is replaced by a hydroxyl group.

[0072] "C1-C6 haloalkyl" refers to an alkane with 1 to 6 carbon atoms in which at least one hydrogen atom is substituted by the same or different halogens as defined above.

[0073] "C1-C6 alkoxy" refers to an alkane with 1 to 6 carbon atoms in which one hydrogen atom is replaced by an oxygen atom.

[0074] "C1-C6 haloalkoxy" refers to a C1-C6 alkoxy group as defined above, in which at least one hydrogen atom is replaced by one or more halogen groups as defined above.

[0075] "C3-C20 nitrogen-containing heterocyclic groups" refer to cycloalkyl groups with 3 to 20 carbon atoms in which at least one carbon atom is replaced by a nitrogen atom, such as piperidine, morpholine, piperazine, etc.

[0076] "C3-C8 cycloalkyl" refers to saturated monocyclic or polycyclic groups with 3-8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0077] The compounds of the present invention or their pharmaceutically acceptable salts or deuterated derivatives have the same efficacy, wherein the pharmaceutically acceptable salt is a salt of the above general formula (I), wherein the pharmaceutically acceptable salt is a hydrochloride, sulfate, phosphate, maleate, fumarate, citrate, methanesulfonate, tartrate, sodium salt or potassium salt.

[0078] In this application, the term "pharmaceutical carrier" refers to conventional drug carriers in the pharmaceutical field, including conventional diluents, excipients (such as water), fillers (such as starch), binders (such as cellulose derivatives, gelatin, etc.), humectants (such as glycerin, etc.), disintegrants (such as agar, calcium carbonate, etc.), absorption enhancers (such as quaternary ammonium compounds, etc.), surfactants (such as hexadecyl alcohol, etc.), adsorbent carriers (such as kaolin and soap clay, etc.), lubricants (such as talc, etc.), and flavoring agents, sweeteners, etc. may be added when necessary.

[0079] In this application, the term "any pharmaceutically acceptable dosage form" applies to administration via any suitable route, such as oral (including sublingual or sublingual), rectal, nasal, topical (including sublingual, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) administration. These formulations can be prepared by any method known in the field of pharmaceutical science. For example, methods involving mixing the active ingredient with a carrier or excipient.

[0080] In this application, the term "effective amount" means an amount of at least one pharmaceutical agent or compound that, when taken orally, is sufficient to alleviate one or more symptoms of the disease or condition being treated to a certain extent. For example, an "effective amount" for treatment is the amount of a compound or composition disclosed herein that is clinically necessary to provide significant symptom relief.

[0081] Those skilled in the art will understand that, according to the conventions used in the art, the structural formulas of the descriptive groups described in this application... This refers to the fact that the corresponding group is connected to other fragments or groups in the compound through this site.

[0082] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0083] The reagents and raw materials used in this invention are all commercially available.

[0084] Taking compounds 15-17 (Route I), 21, 26, 29, 31 (Route II), and 34, 35 (Route III) as examples, the preparation methods of the compounds in this invention are as follows:

[0085] Furthermore, compounds 1-14 and 18-20 were prepared according to route I; compounds 22-25, 27, 28, 30, 32, and 33 were prepared according to route II; and compound 36 was prepared according to route III. It should be noted that the specific examples included below are for illustrative purposes only and should not be construed as limiting the scope of the invention. Moreover, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and such equivalent forms also fall within the scope defined by the appended claims.

[0086]

[0087] In Route I, the reactants and reaction conditions involved are as follows:

[0088] a represents CH3I, K2CO3, N,N-dimethylformamide (DMF), and rt;

[0089] b is 1-ethynyl-4-(trifluoromethyl)benzene, Pd(PPh3)2C12, CuI, triethylamine (TEA), DMF, N2 protection, 70℃;

[0090] c is (1) BBr3, dichloromethane (DCM), N2 protection, 0℃ to rt; (2) Cs2CO3, acetonitrile (CH3CN), N2 protection, 60℃;

[0091] d is hydrazine hydrate (40%-50%), ethanol (EtOH), N2 protection, 80℃; or methylhydrazine (40%), EtOH, N2 protection, 80℃.

[0092]

[0093] In Route II, the reactants and reaction conditions involved are as follows:

[0094] a represents piperidine, HOBT, HATU, DIPEA, DMF, N2 protection, and rt.

[0095] b represents morpholine, K2CO3, tetrahydrofuran (THF), N2 protection, reflux;

[0096] c represents (1-diazo-2-oxopropyl)phosphonate dimethyl ester, K2CO3, methanol (MeOH), rt;

[0097] d is 4-(prop-2-en-1-yl)morpholine, 42a, 44a or 46a, Pd(PPh3)2Cl2, CuI, TEA, PPh3, DMF / H2O, N2 protection, 90℃;

[0098] e is hydrazine hydrate (40%-50%), EtOH, N2 protection, 80℃.

[0099]

[0100] In Route III, the reactants and reaction conditions involved are as follows:

[0101] a is protected by SnCl2, EtOH, and N2 at 80℃;

[0102] b is acetyl chloride, TEA, DCM, N2 protection, 0℃ to rt;

[0103] c is hydrazine hydrate (40%-50%), EtOH, N2 protection, 80℃;

[0104] d is 2-chloroacetyl chloride, TEA, DCM, N2 protection, 0℃ to rt;

[0105] e represents morpholine, K2CO3, DMF, N2 protection, and rt;

[0106] Example 1: Synthesis of 5-(3-trifluoromethyl)-1H-pyrazol-5-yl)-2-(4-trifluoromethylphenyl)benzofuran-4-ol (compound 15)

[0107] (1) Synthesis of 8-iodo-7-methoxy-2-(trifluoromethyl)-4H-chromen-4-one (intermediate 38)

[0108]

[0109] 37 (600 mg, 1.69 mmol, 1.0 eq), CH3I (359 mg, 2.54 mmol, 1.5 eq), K2CO3 (350 mg, 2.54 mmol, 1.5 eq), and DMF (10 mL) were added sequentially to the reaction flask. The mixture was stirred at room temperature for 4 h under N2 protection. After the reaction was completed as monitored by TLC, the mixture was extracted with ethyl acetate and washed sequentially with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 600 mg of a white solid.

[0110] Yield 96%. 1 H NMR (600MHz, CDCl3): δ8.13 (d, J=9.0Hz, 1H), 6.94 (d, J=9.0Hz, 1H), 6.64 (s, 1H), 3.99 (s, 3H). ESI-MSm / z: 371[M+H] + .

[0111] (2) Synthesis of 7-methoxy-2-(trifluoromethyl)-8-(4-(trifluoromethyl)phenyl)ethynyl)-4H-chromen-4-one (intermediate 39k)

[0112]

[0113] Intermediate 38 (600 mg, 1.62 mmol, 1.0 eq), 1-ethynyl-4-(trifluoromethyl)benzene (552 mg, 3.24 mmol, 2.0 eq), Pd(PPh3)2Cl2 (56.9 mg, 0.0811 mmol, 0.05 eq), CuI (30.9 mg, 0.162 mmol, 0.10 eq), and a TEA / DMF mixed solvent (V / V = 1:4, 10 mL) were added sequentially to the reaction flask. The mixture was stirred at 70 °C for 8 h under N2 protection. After the reaction was completed as monitored by TLC, the mixture was extracted with ethyl acetate and washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 590 mg of a white solid.

[0114] Yield 65%. 1 H NMR (600MHz, CDCl3): δ8.12 (d, J=9.0Hz, 1H), 7.62 (d, J=8.4Hz, 2H), 7.58 (d, J=8 .4Hz, 2H), 7.04 (d, J=9.0Hz, 1H), 6.65 (s, 1H), 4.02 (s, 3H).ESI-MSm / z: 413[M+H] + .

[0115] (3) Synthesis of 2-(trifluoromethyl)-8-(4-(trifluoromethyl)phenyl)-4H-furano[2,3-h]benzopyran-4-one (intermediate 40k)

[0116]

[0117] Intermediate 39k (590 mg, 1.43 mmol, 1.0 eq) was added sequentially to the reaction flask. After N2 protection, anhydrous DCM (8 mL) was added to dissolve it. BBr3 (1.43 g, 5.73 mmol, 4.0 eq) was slowly added dropwise under ice bath, and the reaction was stirred at room temperature for 5 h. After the reaction was completed as monitored by TLC, saturated sodium bicarbonate solution was slowly added dropwise under ice bath to adjust the pH to 7-8. The mixture was extracted with ethyl acetate and washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was used directly in the next reaction step.

[0118] The crude product (300 mg, 0.754 mmol, 1.0 eq), Cs₂CO₃ (491 mg, 1.51 mmol, 2.0 eq), and CH₃CN (4 mL) were added sequentially to the reaction flask. The mixture was stirred at 60 °C for 5 h under N₂ protection. After the reaction was completed as monitored by TLC, the mixture was concentrated under reduced pressure to remove acetonitrile, extracted with ethyl acetate, and washed sequentially with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was then subjected to silica gel column chromatography to obtain 150 mg of a white solid.

[0119] Yield 42%. 1 H NMR (600MHz, CDCl3): δ8.10 (d, J=9.0Hz, 1H), 7.96 (d, J=8.4Hz, 2H), 7.69 (d, J=8 .4Hz, 2H), 7.60 (d, J=9.0Hz, 1H), 7.44 (s, 1H), 6.76 (s, 1H).ESI-MSm / z: 399[M+H] + .

[0120] (4) Synthesis of 5-(3-trifluoromethyl)-1H-pyrazol-5-yl)-2-(4-trifluoromethylphenyl)benzofuran-4-ol (compound 15)

[0121]

[0122] Intermediate 40k (150 mg, 0.377 mmol, 1.0 eq), hydrazine hydrate (40-50%) (350 mg, 10.9 mmol, 29 eq), and EtOH (2 mL) were added sequentially to a reaction flask. The mixture was stirred at 80 °C for 3 h under N2 protection. After the reaction was confirmed to be complete by TLC, the EtOH was removed by concentration under reduced pressure. The mixture was extracted with ethyl acetate and washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 115 mg of a white solid.

[0123] Yield 74%. 1 H NMR (600MHz, DMSO-d6): δ13.65 (s, 1H), 10.99 (s, 1H), 8.04 (d, J=7.8Hz, 2H), 7.86 (d, J=7.8Hz, 2H), 7.81 (s, 1H), 7.68 (d, J=9.0Hz, 1H), 7.27 (d, J=8.4Hz, 1H), 7.11 (s, 1H). 13 C NMR (151MHz, DMSO-d6): δ156.20, 152.61, 148.62, 141.84, 141.72 (q, J C-F =36.2Hz), 133.66, 128.96 (q, J C-F =31.7Hz), 126.59, 126.15, 125.47, 125.41(2C), 123.67, 122.51(q, J C-F =267.3Hz), 119.23, 110.06, 104.01, 103.02, 102.67.ESI-HRMSm / z: calcd for C 19 H 10 F6N2O2[M+H] + , 413.0719; found, 413.0712.

[0124] Example 2: Synthesis of 5-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-2-(4-(trifluoromethyl)phenyl)benzofuran-4-ol (compound 16)

[0125]

[0126] Intermediate 40k (100 mg, 0.251 mmol, 1.0 eq), methylhydrazine (40%) (87.0 mg, 1.88 mmol, 7.5 eq), and EtOH (2 mL) were added sequentially to a reaction flask. The mixture was stirred at 80 °C for 3 h under N2 protection. After the reaction was confirmed to be complete by TLC, the EtOH was removed by concentration under reduced pressure. The mixture was extracted with ethyl acetate and washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 80 mg of a white solid.

[0127] Yield 75%. 1 H NMR (600MHz, DMSO-d6): δ10.62 (s, 1H), 8.07 (d, J=7.8Hz, 2H), 7.87 (d, J=8.4Hz, 2H), 7.78 (s, 1H), 7.26 (q, J=8.4Hz, 2H), 6.75 (s, 1H), 3.78 (s, 3H). 13 CNMR (151MHz, DMSO-d6): δ156.95, 152.79, 149.54, 142.77, 139.76 (q, J C-F =37.8Hz), 133.68, 129.17, 129.02 (q, J C-F =33.2Hz), 126.60, 126.57, 125.49(2C), 124.57(q, J C-F =271.8Hz), 122.17(q, J) C-F =268.8Hz), 119.10, 110.43, 105.71, 103.78, 102.91, 38.21.ESI-HRMSm / z: calcd for C 20 H 12 F6N2O2[M+H] + , 427.0876; found, 427.0866.

[0128] Example 3: Synthesis of 5-(4-methoxy-2-(4-(trifluoromethyl)phenyl)benzofuran-5-yl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole (compound 17)

[0129]

[0130] Compound 16 (50.0 mg, 0.117 mmol, 1.0 eq), CH3I (25.0 mg, 0.176 mmol, 1.5 eq), K2CO3 (24.3 mg, 0.176 mmol, 1.5 eq), and DMF (1 mL) were added sequentially to a reaction flask. The mixture was stirred at room temperature for 4 h under N2 protection. After the reaction was completed as monitored by TLC, the mixture was extracted with ethyl acetate and washed sequentially with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 45 mg of a white solid.

[0131] Yield 87%. 1 H NMR (600MHz, DMSO-d6): δ8.21 (d, J=8.4Hz, 2H), 8.15 (s, 1H), 7.90 (d, J=8.4Hz, 2H), 7.47 (d, J=8.4Hz, 1H), 7.32 (d, J=8.4Hz, 1H), 6.78 (s, 1H), 4.16 (s, 3H), 3.74 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ157.66, 153.49, 151.76, 142.38, 139.82 (q, J C-F =37.8Hz), 133.50, 129.22 (q, J C-F =31.7Hz), 128.88, 126.49, 126.47, 125.73 (2C), 124.57 (q, J C-F =271.8Hz), 122.10(q, J) C-F =268.8Hz), 119.05, 113.46, 105.93, 105.74, 103.95, 60.36, 38.11.ESI-HRMS m / z: calcd for C 21 H 14 F6N2O2[M+H] + , 441.1032; found, 441.1018.

[0132] Example 4: Synthesis of 2-(4-chlorophenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (Compound 1)

[0133]

[0134] Compound 1 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 1-chloro-4-ethynylbenzene, and it was coupled with intermediate 38 via Sonogashira coupling. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 1 was a white solid.

[0135] Yield 82%. 1 H NMR (400MHz, DMSO-d6): δ13.66 (s, 1H), 10.94 (s, 1H), 7.86 (d, J=8.8Hz, 2H), 7.66 (d, J=2.0Hz, 1H), 7.64 (s, 1H), 7.59 (d, J=8.8Hz, 2H), 7.25 (d, J=8.4Hz, 1H), 7.11 (s, 1H). 13 C NMR (151MHz, DMSO-d6): δ155.99, 153.18, 148.45, 141.95, 141.55 (q, J C-F =34.7Hz), 133.69, 129.71(2C), 128.85, 126.59(2C), 125.59, 122.50(q, J C-F =268.8Hz), 119.37, 110.00, 103.92, 102.65, 101.31.ESI-HRMSm / z: calcd forC 18 H 10 ClF3N2O2[M+H] + , 379.0456; found, 379.0456.

[0136] Example 5: Synthesis of 2-(pyridin-4-yl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 2)

[0137]

[0138] Compound 2 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 4-ethynylpyridine, and it was coupled with intermediate 38 via Sonogashira. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 2 was a white solid.

[0139] Yield 58%. 1H NMR (600MHz, DMSO-d6): δ13.67 (s, 1H), 11.08 (s, 1H), 8.70 (d, J=5.4Hz, 2H), 7.91 (s, 1H), 7.79 (d, J=6.0Hz, 2H), 7.71 (d, J=8.4Hz, 1H), 7.30 (d, J=9.0Hz, 1H), 7.11 (s, 1H). 13 C NMR (151MHz, DMSO-d6): δ156.31, 151.59, 150.84 (2C), 148.79, 141.75, 141.58 (q, J C-F =36.2Hz), 136.83, 126.69, 122.49 (q, J C-F =268.8Hz), 119.08, 118.82(2C), 110.15, 104.59, 104.07, 102.73.ESI-HRMS m / z: calcd for C 17 H 10 F3N3O2[M+H] + , 346.0798; found, 346.0784.

[0140] Example 6: Synthesis of 2-(pyridin-3-yl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 3)

[0141]

[0142] Compound 3 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 3-ethynylpyridine, and it was coupled with intermediate 38 via Sonogashira. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 3 was a white solid.

[0143] Yield 46%. 1 H NMR (600MHz, DMSO-d6): δ13.69(br s, 1H), 11.11(br s, 1H), 9.05 (s, 1H), 8.60 (d, J=4.8Hz, 1H), 8.20 (dt, J=8.4, 1.8Hz, 1H), 7.90 (s, 1H), 7 .69 (d, J=8.4Hz, 1H), 7.55 (dd, J=7.8, 4.8Hz, 1H), 7.26 (d, J=8.4Hz, 1H), 7.10 (s, 1H). 13C NMR (151MHz, DMSO-d6): δ156.13, 151.65, 149.90, 148.50, 145.97, 141.87, 141.56 (q, J C-F =36.2Hz), 132.18, 126.13, 125.82, 124.60, 122.50 (q, J C-F =268.8Hz), 119.21, 110.08, 103.98, 102.69, 102.17.ESI-HRMS m / z: calcd for C 17 H 10 F3N3O2[M+H] + , 346.0798; found, 346.0792.

[0144] Example 7: Synthesis of 2-(2-methylthiazo-4-yl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 4)

[0145]

[0146] Compound 4 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 4-ethynyl-2-methylthiazole, and it was coupled with intermediate 38 via Sonogashira. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 4 was a white solid.

[0147] Yield 62%. 1 H NMR (600MHz, DMSO-d6): δ13.64(s, 1H), 10.89(s, 1H), 7.90(s, 1H), 7.64(d, J=8.4Hz, 1H), 7.56 (s, 1H), 7.22 (d, J=8.4Hz, 1H), 7.10 (s, 1H), 2.75 (s, 3H). 13 CNMR (151MHz, DMSO-d6): δ167.56, 155.82, 150.81, 148.53, 145.22, 141.94, 141.54 (q, J C-F =36.2Hz), 125.50, 122.52 (q, J C-F =268.8Hz), 118.85, 116.03, 109.86, 103.81, 102.59, 101.81, 19.30.ESI-HRMSm / z: calcd for C 16 H 10 F3N3O2S[M+H]+ , 366.0519; found, 366.0511.

[0148] Example 8: Synthesis of 2-(4-fluorophenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 5)

[0149]

[0150] Compound 5 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 1-ethynyl-4-fluorobenzene, and it was coupled with intermediate 38 via Sonogashira coupling. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 5 was a white solid.

[0151] Yield 75%. 1 H NMR (600MHz, DMSO-d6): δ13.62 (br s, 1H), 10.89 (br s, 1H), 7.90 (dd, J=8.4, 5.4Hz, 2H), 7.63 (d, J=9.0Hz, 1H), 7.58 (s, 1H), 7.37 (t, J=8.4Hz, 2H), 7.24 (d, J=8.4Hz, 1H), 7.10 (s, 1H). 13 C NMR (151MHz, DMSO-d6): δ163.51, 161.87, 155.92, 153.46, 148.36, 142.00, 141.54 (d, J C-F =39.3Hz), 127.15 (d, J) C-F =9.1Hz), 126.66 (d, J) C-F =3.0Hz), 125.28, 122.45 (q, J C-F =270.3Hz), 119.42, 116.77, 116.63, 109.95, 103.89, 102.63, 100.43.ESI-HRMSm / z: calcd for C 18 H 10 F4N2O2[M+H] + , 363.0752; found, 363.0751.

[0152] Example 9: Synthesis of 2-(3,4-difluorophenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 6)

[0153]

[0154] Compound 6 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 4-ethynyl-1,2-difluorobenzene, and it was coupled with intermediate 38 via Sonogashira coupling. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 6 was a white solid.

[0155] Yield 79%. 1 H NMR (600MHz, DMSO-d6): δ13.64 (s, 1H), 10.92 (s, 1H), 7.86 (t, J=9.6Hz, 1H), 7.68 (d, J=8. 4Hz, 1H), 7.64 (d, J=7.8Hz, 2H), 7.57 (q, J=9.0Hz, 1H), 7.23 (d, J=8.4Hz, 1H), 7.09 (s, 1H). 13 C NMR (151MHz, DMSO-d6): δ170.76, 155.98, 152.16, 150.35 (dd, J C-F =244.6, 12.1Hz), 149.98 (dd, J C-F =249.2, 13.6Hz), 148.47, 141.88, 141.56 (q, J C-F =36.2Hz), 125.71, 122.51 (q, J C-F =268.8Hz), 121.97(q, J) C-F =3.0Hz), 119.36, 118.97 (d, J C-F =18.1Hz), 114.03 (d, J) C-F =9.6Hz), 110.11, 103.92, 102.65, 101.75.ESI-HRMS m / z: calcd for C 18 H9F5N2O2[M+H] + , 381.0657; found, 381.0653.

[0156] Example 10: Synthesis of 2-(2,4-difluorophenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 7)

[0157]

[0158] Compound 7 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 1-ethynyl-2,4-difluorobenzene, and it was coupled with intermediate 38 via Sonogashira. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 7 was a white solid.

[0159] Yield 68%. 1 H NMR (600MHz, DMSO-d6): δ13.64(s, 1H), 10.92(s, 1H), 7.99(dt, J=8.4, 6.8Hz, 1H), 7.72(d , J=3.0Hz, 1H), 7.67 (d, J=8.4Hz, 1H), 7.53-7.44 (m, 1H), 7.31-7.22 (m, 2H), 7.10 (s, 1H). 13 C NMR (151MHz, DMSO-d6): δ162.46 (dd, J C-F =250.7, 13.6Hz), 159.30 (dd, J C-F =253.7, 12.1Hz), 155.29, 148.53, 147.51, 141.87, 141.56 (q, J C-F =36.2Hz), 128.34 (dd, J) C-F =9.1, 3.0Hz), 125.84, 122.51 (q, J C-F =268.8Hz), 119.15, 115.02 (dd, J C-F =12.1, 4.5Hz), 112.97 (dd, J C-F =21.1, 3.0Hz), 109.83, 105.60 (t, J C-F =25.7Hz), 104.86 (d, J) C-F =12.1Hz), 103.72, 102.65.ESI-HRMS m / z: calcd for C 18 H9F5N2O2[M+H] + , 381.0657; found, 381.0655.

[0160] Example 11: Synthesis of 2-(3,4-difluorophenyl)-5-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 8)

[0161]

[0162] Compound 8 was prepared according to Examples 1 and 2, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) of Example 1 was replaced with 4-ethynyl-1,2-difluorobenzene, and it was coupled with intermediate 38 via Sonogashira coupling. Then, it was demethylated and cyclized according to step (3), and finally subjected to methylhydrazine hydrolysis according to the method of Example 2. The prepared compound 8 was a white solid.

[0163] Yield 75%. 1 H NMR (600MHz, DMSO-d6): δ10.54 (s, 1H), 7.91 (t, J=10.2Hz, 1H), 7.72 (d, J=7.8Hz, 1H) , 7.62 (s, 1H), 7.59 (q, J=9.0Hz, 1H), 7.22 (q, J=8.4Hz, 2H), 6.74 (s, 1H), 3.77 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ156.74, 152.36, 150.37 (dd, J C-F =244.6, 12.1Hz), 150.02 (dd, J C-F =252.2, 12.1Hz), 149.37, 142.81, 139.75 (q, J C-F =36.2Hz), 128.72, 127.65 (q, J C-F =3.0Hz), 122.17(q, J C-F =268.8Hz), 122.04(q, J) C-F =3.0Hz), 119.24, 118.99 (d, J C-F =18.1Hz), 114.12 (d, J) C-F =19.6Hz), 110.46, 105.70, 103.71, 101.65, 38.18.ESI-HRMSm / z: calcd for C 19 H 11 F5N2O2[M+H] + , 395.0813; found, 395.0810.

[0164] Example 12: Synthesis of 2-(2,4-difluorophenyl)-5-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 9)

[0165]

[0166] Compound 9 was prepared according to Examples 1 and 2, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) of Example 1 was replaced with 1-ethynyl-2,4-difluorobenzene, coupled with intermediate 38 via Sonogashira, and then demethylated and cyclized according to step (3), followed by methylhydrazine hydrolysis according to Example 2. The prepared compound 9 was a white solid.

[0167] Yield 62%. 1 H NMR (600MHz, DMSO-d6): δ10.62 (s, 1H), 8.02 (dt, J=8.4, 6.0Hz, 1H), 7.66 (d, J=3.0Hz, 1H), 7.54-7.47 (m, 1H), 7.29 (dt, J=8.4, 2.4Hz, 1H), 7.25 (d, J=8.4Hz, 1H), 7.24 (d, J=8.4Hz, 1H), 6.74 (s, 1H), 3.78 (s, 3H). 13 C NMR (151MHz, DMSO-d6): 162.51 (dd, J C-F =250.7, 12.1Hz), 159.33 (dd, J C-F =252.2, 12.1Hz), 156.05, 149.41, 147.64, 142.88, 139.74 (q, J C-F =36.2Hz), 128.87, 128.43 (dd, J C-F =9.1, 3.0Hz), 122.17 (q, J C-F =268.8Hz), 118.92, 115.00 (dd, J C-F =12.1, 4.5Hz), 113.00 (dd, JC- F =22.7, 3.0Hz), 110.21, 105.62 (t, Jc- F =28.7Hz), 104.67 (d, J) C-F =12.1Hz), 103.48(2C), 38.22.ESI-HRMSm / z: calcd for C 19 H 11 F5N2O2[M+H] + , 395.0813; found, 395.0806.

[0168] Example 13: Synthesis of 5-(2-(3,4-difluorophenyl)-4-methoxybenzofuran-5-yl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole (compound 10)

[0169]

[0170] Compound 10 was prepared according to Example 3, except that Compound 16 was replaced with Compound 8 for alkylation. The prepared Compound 10 was a white solid.

[0171] Yield 73%. 1 H NMR (600MHz, DMSO-d6): δ8.11-8.07 (m, 1H), 8.01 (s, 1H), 7.84 (d, J=8.4Hz, 1H), 7.63 (q, J=9. 0Hz, 1H), 7.43 (d, J=8.4Hz, 1H), 7.29 (d, J=8.4Hz, 1H), 6.77 (s, 1H), 4.13 (s, 3H), 3.74 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ157.46, 153.08, 151.54, 150.35 (dd, J C-F =252.2, 19.6Hz), 150, 15 (dd, J C-F =250.7, 19.6Hz), 142.41, 139.81 (q, J C-F =37.8Hz), 128.45, 127.40 (q, J C-F =3.0Hz), 122.14(q, J C-F =3.0Hz), 122.10(q, J C-F =267.3Hz), 119.16, 118.90 (d, J C-F =18.1Hz), 114.41 (d, J) C-F =19.6Hz), 113.45, 105.85, 105.71, 102.60, 60.27, 38.09.ESI-HRMS m / z: calcd for C 20 H 13 F5N2O2[M+H] + , 409.0970; found, 409.0960.

[0172] Example 14: Synthesis of 2-(3-chlorophenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 11)

[0173]

[0174] Compound 11 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 1-chloro-3-ethynylbenzene, and it was coupled with intermediate 38 via Sonogashira. Then, demethylation, cyclization and hydrazinolysis were carried out sequentially according to steps (3) and (4). The prepared compound 11 was a white solid.

[0175] Yield 83%. 1 H NMR (600MHz, DMSO-d6): δ13.69 (s, 1H), 10.97 (s, 1H), 7.90 (s, 1H), 7.86 (d, J=7.8Hz, 1H), 7.75 (s, 1H), 7.71 (d, J=8.4Hz, 1H), 7.60 (t, J=7.8Hz, 1H), 7.53 (d, J=8.4Hz, 1H), 7.31 (d, J=8.4Hz, 1H), 7.15 (s, 1H). 13 C NMR (151MHz, DMSO-d6): δ156.02, 152.63, 148.50, 141.89, 141.56 (q, J C-F =37.8Hz), 134.42, 131.98, 131.59, 128.95, 125.83, 124.31, 123.52, 122.51 (q, J C-F =267.3Hz), 119.30, 110.05, 103.99, 102.66, 102.04.ESI-HRMSm / z: calcd for C 18 H 10 CIF3N2O2[M+H] + , 379.0456; found, 379.0454.

[0176] Example 15: Synthesis of 2-(3,4-dichlorophenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 12)

[0177]

[0178] Compound 12 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 1,2-dichloro-4-ethynylbenzene, and it was coupled with intermediate 38 via Sonogashira coupling. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 12 was a white solid.

[0179] Yield 85%. 1H NMR (600MHz, DMSO-d6): δ13.62(br s, 1H), 11.01(br s, 1H), 8.05 (d, J=1.8Hz, 1H), 7.83 (dd, J=8.4, 1.8Hz, 1H), 7.78 (d, J=8.4Hz, 1H), 7.72 (s, 1H), 7.67 (d, J=8.4Hz, 1H), 7.25 (d, J=8.4Hz, 1H), 7.11 (s, 1H). 13 C NMR (151MHz, DMSO-d6): δ156.05, 151.72, 148.55, 141.84, 141.56 (q, J C-F =36.2Hz), 132.50, 131.87, 131.47, 130.51, 126.32, 126.02, 124.97, 122.50 (q, J C-F =267.3Hz), 119.31, 110.16, 103.98, 102.68, 102.61.ESI-HRMSm / z: calcd for C 18 H9Cl2F3N2O2[M+H] + , 413.0066; found, 413.0065.

[0180] Example 16: Synthesis of 2-(2,4-dichlorophenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 13)

[0181]

[0182] Compound 13 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 2,4-dichloro-1-ethynylbenzene, and it was coupled with intermediate 38 via Sonogashira coupling. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 13 was a white solid.

[0183] Yield 78%. 1 H NMR (600MHz, DMSO-d6): δ13.65(br s, 1H), 11.03(br s, 1H), 8.11 (s, 1H), 8.03 (d, J=9.0Hz, 1H), 7.84 (d, J=1.8Hz, 1H), 7.70 (d, J= 9.0Hz, 1H), 7.62 (dd, J=9.0, 2.4Hz, 1H), 7.26 (d, J=8.4Hz, 1H), 7.11 (s, 1H). 13C NMR (151MHz, DMSO-d6): δ155.30, 149.35, 148.75, 141.85, 141.56 (q, J C-F =36.2Hz), 133.97, 131.15, 130.89, 130.15, 128.54, 127.26, 126.31, 122.50 (q, J C-F =267.3Hz), 119.00, 109.79, 106.73, 103.71, 102.65.ESI-HRMSm / z: calcd for C 18 H9Cl2F3N2O2[M+H] + , 413.0066; found, 413.0065.

[0184] Example 17: Synthesis of 2-(3,4-dichlorophenyl)-5-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 14)

[0185]

[0186] Compound 14 was prepared according to Examples 1 and 2, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) of Example 1 was replaced with 1,2-dichloro-4-ethynylbenzene, coupled with intermediate 38 via Sonogashira, and then demethylated and cyclized according to step (3), followed by methylhydrazine hydrolysis according to Example 2. The prepared compound 14 was a white solid.

[0187] Yield 64%. 1 H NMR (600MHz, DMSO-d6): δ10.61 (s, 1H), 8.09 (d, J=1.8Hz, 1H), 7.85 (dd, J=8.4, 1.81H) , 7.78 (d, J=8.4Hz, 1H), 7.71 (s, 1H), 7.25 (q, J=8.4Hz, 2H), 6.76 (s, 1H), 3.76 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ156.81, 151.92, 149.49, 142.75, 139.74 (q, J C-F =36.2Hz), 132.53, 131.92, 131.53, 130.54, 129.06, 126.44, 125.07, 122.17 (q, J C-F=268.8Hz), 119.20, 110.52, 105.74, 103.78, 102.56, 38.20.ESI-HRMS m / z: calcd forC 19 H 11 Cl2F3N2O2[M+H] + , 427.0223; found, 427.0222.

[0188] Example 18: Synthesis of 2-(p-Tolyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (Compound 18)

[0189]

[0190] Compound 18 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 1-ethynyl-4-methylbenzene, and it was coupled with intermediate 38 via Sonogashira coupling. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 18 was a white solid.

[0191] Yield 86%. 1 H NMR (600MHz, DMSO-d6): δ13.68 (s, 1H), 10.86 (s, 1H), 7.78 (d, J=7.8Hz, 2H), 7.66 (d, J=9.0H z, 1H), 7.60 (s, 1H), 7.35 (d, J=7.8Hz, 2H), 7.27 (d, J=8.4Hz, 1H), 7.14 (s, 1H), 2.41 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ155.81, 154.57, 148.21, 142.03, 141.55 (q, J C-F =36.2Hz), 138.93, 130.19(2C), 127.30, 125.02, 124.84(2C), 122.53(q, J C-F =268.8Hz), 119.46, 109.80, 103.86, 102.58, 99.75, 21.39.ESI-HRMS m / z: calcd for C 19 H 13 F3N2O2[M+H] + , 359.1002; found, 359.0992.

[0192] Example 19: Synthesis of 2-(4-methoxyphenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 19)

[0193]

[0194] Compound 19 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 1-ethynyl-4-methoxybenzene, and it was coupled with intermediate 38 via Sonogashira coupling. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 19 was a white solid.

[0195] Yield 82%. 1 H NMR (600MHz, DMSO-d6): δ13.61 (s, 1H), 10.74 (s, 1H), 7.78 (d, J=9.0Hz, 2H), 7.59 (d, J=9.0H z, 1H), 7.46 (s, 1H), 7.21 (d, J=8.4Hz, 1H), 7.09 (d, J=4.8Hz, 2H), 7.08 (s, 1H), 3.83 (s, 3H). 13 C NMR (151MHz, DMSα-d6): δ160.21, 155.73, 154.57, 148.07, 142.07, 141.54 (q, J C-F =36.2Hz), 126.48(2C), 124.68, 122.66, 122.53(q, J C-F =268.8Hz), 119.60, 115.11(2C), 109.79, 103.81, 102.57, 98.72, 55.76.ESI-HRMS m / z: calcd for C 19 H 13 F3N2O3[M+H] + , 375.0951; found, 375.0954.

[0196] Example 20: Synthesis of 2-(4-(trifluoromethoxy)phenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (Compound 20)

[0197]

[0198] Compound 20 was prepared according to Example 1, except that 1-ethynyl-4-(trifluoromethyl)benzene in step (2) was replaced with 1-ethynyl-4-(trifluoromethoxy)benzene, and it was coupled with intermediate 38 via Sonogashira coupling. Then, demethylation, cyclization, and hydrazinolysis were performed sequentially according to steps (3) and (4). The prepared compound 20 was a white solid.

[0199] Yield 78%. 1 H NMR (600MHz, DMSO-d6): δ13.63 (br s, 1H), 10.90 (br s, 1H), 7.97 (d, J=8.4Hz, 2H), 7.67 (s, 1H), 7.66 (d, J=9.0Hz, 1H), 7.52 (d, J=7.8Hz, 2H), 7.26 (d, J=8.4Hz, 1H), 7.11 (s, 1H). 13 C NMR (151MHz, DMSO-d6): δ156.06, 152.92, 148.74, 148.46, 141.90, 141.57 (q, J C-F =36.2Hz), 129.25, 126.82(2C), 125.65, 122.51(q, J C-F =267.3Hz), 122.22(2C), 120.58(q, J C-F =256.7Hz), 119.34, 110.00, 103.94, 102.64, 101.56.ESI-HRMSm / z: calcd for C 19 H 10 F6N2O3[M+H] + , 429.0668; found, 429.0669.

[0200] Example 21: Synthesis of 2-(morpholinomethyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 21)

[0201] (1) Synthesis of 8-(morpholinomethyl)-2-(trifluoromethyl)-4H-furano[2,3-h]chromen-4-one (intermediate 47a)

[0202]

[0203] In a reaction flask, 37 (500 mg, 1.40 mmol, 1.0 eq), 4-propynyl-1-morpholine (264 mg, 2.11 mmol, 1.5 eq), Pd(PPh3)2C12 (98.6 mg, 0.140 mmol, 0.10 eq), CuI (53.5 mg, 0.281 mmol, 0.20 eq), TEA (213 mg, 2.11 mmol, 1.5 eq), PPh3 (73.7 mg, 0.281 mmol, 0.20 eq), and a mixed solvent of H2O / DMF (V / V = 1:9, 10 mL) were added sequentially. The mixture was stirred at 90 °C for 5 h under N2 protection. After the reaction was complete as shown by TLC, the mixture was extracted with ethyl acetate and washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 407 mg of a white solid.

[0204] Yield 82%. 1 H NMR (600MHz, CDCl3): δ8.04 (d, J=9.0Hz, 1H), 7.52 (d, J=9.0Hz, 1H), 6.97 (s, 1H ), 6.73 (s, 1H), 3.73-3.67 (m, 6H), 2.52 (t, J=4.2Hz, 4H).ESI-MSm / z: 354[M+H] + .

[0205] (2) Synthesis of 2-(morpholinomethyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 21)

[0206]

[0207] Intermediate 47a (340 mg, 0.963 mmol, 1.0 eq), hydrazine hydrate (40-50%) (895 mg, 27.9 mmol, 29 eq), and EtOH (5 mL) were added sequentially to a reaction flask. The mixture was stirred at 80 °C for 3 h under N2 protection. After the reaction was confirmed to be complete by TLC, the EtOH was removed by concentration under reduced pressure. The mixture was extracted with ethyl acetate and washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 150 mg of a white solid.

[0208] Yield 42%. 1H NMR (600MHz, DMSO-d6): δ13.58 (br s, 1H), 10.70 (br s, 1H), 7.59 (d, J=8.4Hz, 1H), 7.16 (d, J=9.0Hz, 1H), 7.10 (s, 1H), 7.08 (s, 1H), 3.65 (s, 2H), 3.60 (t, J=4.8Hz, 4H), 2.46 (t, J=4.8Hz, 4H). 13 C NMR (151MHz, DMSO-d6): δ156.16, 153.81, 148.06, 142.16, 141.52 (q, J C-F =31.7Hz), 124.57, 122.52 (q, J C-F =268.8Hz), 118.33, 109.37, 104.17, 103.82, 102.50, 66.60(2C), 55.10, 53.32(2C).ESI-HRMSm / z: calcd forC 17 H 16 F3N3O3[M+H] + , 368.1217; found, 368.1211.

[0209] Example 22: Synthesis of (4-(4-hydroxy-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-2-yl)phenyl)(piperidin-1-yl)methyl ketone (compound 26)

[0210] Synthesis of (1) (4-ethynylphenyl)(piperidin-1-yl)methyl ketone (intermediate 42a)

[0211]

[0212] 41 (500 mg, 3.42 mmol, 1.0 eq), piperidine (437 mg, 5.13 mmol, 1.5 eq), HOBT (462 mg, 3.42 mmol, 1.0 eq), HATU (1.30 g, 3.42 mmol, 1.0 eq), DIPEA (2.21 g, 17.1 mmol, 5.0 eq), and DMF (6 mL) were added sequentially to the reaction flask, and the mixture was stirred at room temperature for 6 h. After the TLC reaction was complete, the mixture was extracted with ethyl acetate and washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 670 mg of a white solid, with a yield of 92%.

[0213] (2) Synthesis of (4-(4-hydroxy-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-2-yl)phenyl)(piperidin-1-yl)methyl ketone (compound 26)

[0214]

[0215] Compound 26 was prepared according to Example 21, by replacing 4-propynyl-1-morpholine in step (1) with 42a, coupling and cyclizing it with intermediate 37 via Sonogashira, and then performing hydrazinolysis as described in step (2). The prepared compound 26 was a white solid.

[0216] Yield 65%. 1 H NMR (600MHz, DMSO-d6): δ13.62 (s, 1H), 10.95 (s, 1H), 7.89 (d, J=8.4Hz, 2H), 7.69 (s, 1H), 7.66 (d, J=8.4Hz, 1H), 7 .51 (d, J=7.8Hz, 2H), 7.26 (d, J=8.4Hz, 1H), 7.11 (s, 1H), 3.39-3.78 (m, 4H), 1.65-1.59 (m, 2H), 1.59-1.46 (m, 4H). 13 C NMR (151MHz, DMSO-d6): δ168.73, 156.04, 153.60, 148.44, 141.92, 141.54 (q, J C-F =37.8Hz), 136.94, 130.65, 128.12(2C), 125.64, 124.78(2C), 122.51(q, J C-F =267.3Hz), 119.35, 109.94, 103.95, 102.64, 101.49, 48.59, 42.86, 26.51, 25.71, 24.50.ESI-HRMSm / z: calcd forC 24 H 20 F3N3O3[M+H] + , 456.1530; found, 456.1525.

[0217] Example 23: Synthesis of 2-(4-(morpholinomethyl)phenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 29)

[0218] (1) Synthesis of 4-(4-ethynylbenzyl)morpholine (intermediate 44a)

[0219]

[0220] Add 43 (1.00 g, 5.16 mmol, 1.0 eq) to the reaction flask, dissolve in THF (12 mL), then add morpholine (1.12 g, 12.9 mmol, 2.5 eq) and K2CO3 (1.78 g, 12.9 mmol, 2.5 eq), and stir under reflux with N2 protection for 2 h. After TLC shows complete reaction, concentrate under reduced pressure to remove THF, extract with ethyl acetate, and wash successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. Combine the organic phases, dry to anhydrous sodium sulfate, concentrate under reduced pressure, and use the crude product directly for the next step.

[0221] (2) Synthesis of 2-(4-(morpholinomethyl)phenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 29)

[0222]

[0223] Compound 29 was prepared according to Example 21, by replacing 4-propynyl-1-morpholine in step (1) with 44a, coupling and cyclizing it with intermediate 37 via Sonogashira, and then performing hydrazinolysis as described in step (2). The prepared compound 29 was a white solid.

[0224] Yield 45%. 1 H NMR (600MHz, DMSO-d6): δ7.78 (d, J=7.8Hz, 2H,), 7.61 (d, J=8.4Hz, 1H), 7.58 (s, 1H), 7.44 (d, J=8.4 Hz, 2H), 7.19 (d, J=8.4Hz, 1H), 7.08 (s, 1H), 3.59 (t, J=4.8Hz, 4H), 3.51 (s, 2H), 2.41-2.35 (m, 4H). 13 C NMR (151MHz, DMSO-d6): δ155.90, 154.31, 148.33, 142.07, 141.51 (d, J C-F =43.8Hz), 131.97, 130.22, 129.11, 125.19, 124.79 (2C), 122.49 (d, J C-F =268.8Hz), 119.44, 109.86, 103.87 (2C), 102.63, 100.39, 67.86, 66.56, 65.48, 62.45, 53.56.ESI-HRMSm / z: calcd for C 23 H 20 F3N3O3[M+H] +, 444.1530; found, 444.1522.

[0225] Example 24: Synthesis of 2-(4-(piperidin-1-yl)phenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 31)

[0226] (1) Synthesis of 1-(4-ethynylphenyl)piperidine (intermediate 46a)

[0227]

[0228] Intermediate 45a (600 mg, 3.17 mmol, 1.0 eq), dimethyl (1-diazo-2-oxopropyl)phosphonate (731 mg, 3.81 mmol, 1.2 eq), K₂CO₃ (877 mg, 6.35 mmol, 2.0 eq), and MeOH (8 mL) were added sequentially to a reaction flask, and the mixture was stirred at room temperature for 4 h. After the reaction was completed as monitored by TLC, the MeOH was removed by concentration under reduced pressure, and the mixture was extracted with ethyl acetate. The extract was washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 320 mg of a white solid, with a yield of 55%.

[0229] (2) Synthesis of 2-(4-(piperidin-1-yl)phenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 31)

[0230]

[0231] Compound 31 was prepared according to Example 21, by replacing 4-propynyl-1-morpholine in step (1) with 46a, coupling and cyclizing it with intermediate 37 via Sonogashira, and then performing hydrazinolysis as described in step (2). The prepared compound 31 was a white solid.

[0232] Yield 66%. 1 H NMR (600MHz, DMSO-d6): δ13.59 (br s, 1H), 10.67 (br s, 1H), 7.65 (d, J=8.4Hz, 2H), 7.55 (d, J=8.4Hz, 1H), 7.35 (s, 1H), 7.18 (d, J=9.0Hz, 1H), 7.0 8 (s, 1H), 7.03 (d, J=8.4Hz, 2H), 3.26 (t, J=5.4Hz, 4H), 1.65-1.59 (m, 4H), 1.59-1.55 (m, 2H). 13C NMR (151MHz, DMSO-d6): δ155.57, 155.30, 151.88, 147.84, 142.15, 141.52 (q, J C-F =36.2Hz), 126.00(2C), 124.18, 122.54(q, J C-F =268.8Hz), 119.77, 119.36, 115.66(2C), 109.67, 103.72, 102.52, 97.36, 49.10(2C), 25.46(2C), 24.39.ESI-HRMS m / z: calcd forC 23 H 20 F3N3O2[M+H] + , 428.1580; found, 428.1568.

[0233] Example 25: Synthesis of 2-((4-methylpiperazin-1-yl)methyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 22)

[0234]

[0235] Compound 22 was prepared according to Example 21, except that 4-propyn-1-morpholine in step (1) was replaced with 1-methyl-4-(prop-2-yn-1-yl)piperazine, which was then coupled and cyclized with intermediate 37 via Sonogashira, followed by hydrazolysis as described in step (2). The prepared compound 22 was a white solid.

[0236] Yield 55%. 1 H NMR (600MHz, DMSO-d6): δ12.03(br s, 1H), 7.58 (d, J=9.0Hz, 1H), 7.12 (d, J=8.4Hz, 1H), 7.08 (s, 2H), 3.62 (s, 2H), 2.51-2.39 (m, 4H), 2.42-2.24 (m, 4H), 2.17 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ156.13, 154.03, 148.62, 142.57, 141.37 (q, J C-F =36.2Hz), 124.45, 122.54 (q, J C-F=268.8Hz), 118.48, 109.35, 104.05, 103.50, 102.27, 55.00(2C), 54.74, 52.60(2C), 45.98.ESI-HRMS m / z: calcd for C 18 H 19 F3N4O2[M+H] + , 381.1533; found, 381.1525.

[0237] Example 26: Synthesis of 2-(2-morpholinoethyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 23)

[0238]

[0239] Compound 23 was prepared according to Example 21, except that 4-propyn-1-morpholine in step (1) was replaced with 4-(but-3-yn-1-yl)morpholine, coupled with intermediate 37 via Sonogashira coupling and cyclization, and then subjected to hydrazolysis as described in step (2). The prepared compound 23 was a white solid.

[0240] Yield 65%. 1 H NMR (400MHz, DMSO-d6): δ13.44(br s, 1H), 10.75(br s, 1H), 7.53 (d, J=8.8Hz, 1H), 7.10 (d, J=8.4Hz, 1H), 7.06 (s, 1H), 6.95 (s, 1H), 3.58 ( t, J=4.4Hz, 4H), 2.94 (t, J=7.2Hz, 2H), 2.66 (t, J=7.6Hz, 2H), 2.45 (t, J=4.4Hz, 4H). 13 C NMR (151MHz, DMSO-d6): δ156.68, 155.76, 147.76, 142.39, 141.36 (q, J C-F =48.3Hz), 123.90, 122.52 (q, J C-F =267.3Hz), 118.78, 109.31, 103.58, 102.44, 101.28, 66.62(2C), 57.01, 53.55(2C), 25.82.ESI-HRMS m / z: calcd for C 18 H 18 F3N3O3[M+H] + , 382.1373; found, 382.1362.

[0241] Example 27: Synthesis of 2-(2-(4-methylpiperazin-1-yl)ethyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 24)

[0242]

[0243] Compound 24 was prepared according to Example 21, except that 4-propyn-1-morpholine in step (1) was replaced with 1-(but-3-yn-1-yl)-4-methylpiperazine, which was then coupled and cyclized with intermediate 37 via Sonogashira, followed by hydrazolysis as described in step (2). The prepared compound 24 was a white solid.

[0244] Yield 62%. 1 H NMR (600MHz, DMSO-d6): δ12.15(br s, 1H), 7.52 (d, J=9.0Hz, 1H), 7.09 (d, J=8.4Hz, 1H), 7.05 (s, 1H), 6.93 (s, 1H) , 2.92 (t, J=7.8Hz, 2H), 2.65 (t, J=7.8Hz, 2H), 2.48-2.20 (m, 8H), 2.16 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ156.67, 155.73, 148.03, 142.56, 141.33 (q, J C-F =36.2Hz), 123.83, 122.54 (q, J C-F =268.8Hz), 118.83, 109.29, 103.40, 102.29, 101.25, 56.14, 55.08(2C), 52.82(2C), 46.08, 26.12.ESI-HRMS m / z: calcd for C 19 H 21 F3N4O2[M+H] + , 395.1689; found, 395.1679.

[0245] Example 28: Synthesis of 2-(3-hydroxy-3-methylbutyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 25)

[0246]

[0247] Compound 25 was prepared according to Example 21, by replacing 4-propynyl-1-morpholine in step (1) with 2-methylhexane-5-yn-2-ol, coupling and cyclizing it with intermediate 37 via Sonogashira, and then performing hydrazinolysis as described in step (2). The prepared compound 25 was a white solid.

[0248] Yield 70%. 1 H NMR (600MHz, DMSO-d6): δ13.56 (s, 1H), 10.54 (s, 1H), 7.53 (d, J = 8.4Hz, 1H), 7.10 (d, J = 9.0Hz, 1H), 7.06 (s, 1H), 6.89 (s, 1H,), 4.37 (s, 1H), 2.81 (t, J = 8.4Hz, 2H), 1.78 (t, J = 8.4Hz, 2H), 1.17 (s, 6H). 13 C NMR (151MHz, DMSO-d6): δ159.04, 155.80, 147.64, 142.27, 141.52 (q, J C-F =36.2Hz), 123.77, 22.54 (q, J C-F =268.8Hz), 118.79, 109.22, 103.55, 102.40, 100.16, 68.85, 41.61, 29.62(2C), 23.45.ESI-HRMSm / z: calcd for C 17 H 17 F3N2O3[M+H] + , 355.1264; found, 355.1256.

[0249] Example 29: Synthesis of (4-(4-hydroxy-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-2-yl)phenyl)(morpholino) methyl ketone (compound 27)

[0250]

[0251] Compound 27 was prepared according to Examples 21 and 22. In step (1) of Example 22, piperidine was replaced with morpholine for amide condensation. The product obtained from the reaction was coupled and cyclized with intermediate 37 using the method of Example 21, and then subjected to hydrazolysis using the method of step (2). The prepared compound 27 was a white solid.

[0252] Yield 54%. 1H NMR (600MHz, DMSO-d6): δ13.64 (s, 1H), 10.92 (s, 1H), 7.91 (d, J=8.4Hz, 2H), 7.71 (s, 1H), 7.66 (d, J=8.4Hz, 1H), 7.57 (d, J=8.4Hz, 2H), 7.26 (d, J=8.4Hz, 1H), 7.11 (s, 1H), 3.72-3.40 (m, 8H). 13 C NMR (151MHz, DMSO-d6): δ168.96, 156.07, 153.53, 148.46, 141.92, 141.56 (q, J C-F =37.8Hz), 135.94, 130.98, 128.55(2C), 125.70, 124.79(2C), 122.51(q, J C-F =267.3Hz), 119.36, 109.96, 103.97, 102.65, 101.68, 66.56(2C), 48.33, 42.81.ESI-HRMS m / z: calcd for C 23 H 18 F3N3O4[M+H] + , 458.1322; found, 458.1320.

[0253] Example 30: Synthesis of (4-(4-hydroxy-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-2-yl)phenyl)(4-methylpiperazin-1-yl)methyl ketone (compound 28)

[0254]

[0255] Compound 28 was prepared according to Examples 21 and 22. In step (1) of Example 22, piperidine was replaced with N-methylpiperazine for amide condensation. The product obtained from the reaction was coupled and cyclized with intermediate 37 using the method of Example 21, and then subjected to hydrazine hydrolysis using the method of step (2). The prepared compound 28 was a white solid.

[0256] Yield 50%. 1 H NMR (600MHz, DMSO-d6): δ7.89 (d, J=8.4Hz, 2H), 7.71 (s, 1H), 7.66 (d, J=8.4Hz, 1H), 7.53 (d, J=8. 4Hz, 2H), 7.25 (d, J=9.0Hz, 1H), 7.12 (s, 1H), 3.50-3.25 (m, 4H), 2.44-2.26 (m, 4H), 2.21 (s, 3H).13 C NMR (151MHz, DMSO-d6): δ168.84, 156.08, 153.49, 148.74, 142.23, 141.37 (q, J C-F =36.2Hz), 136.27, 130.91, 128.39(2C), 125.65, 124.77(2C), 122.49(q, J C-F =268.8Hz), 119.40, 109.97, 103.82, 102.58, 101.68, 49.06, 47.51, 46.00 (2C), 41.93.ESI-HRMS m / z: calcd for C 24 H21F3N4O3[M+H] + , 471.1639; found, 471.1625.

[0257] Example 31: Synthesis of 2-(4-(4-methylpiperazin-1-yl)methyl)phenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 30)

[0258]

[0259] Compound 30 was prepared according to Examples 21 and 23. In step (1) of Example 23, morpholine was replaced with N-methylpiperazine for nucleophilic substitution. The product obtained from the reaction was coupled and cyclized with intermediate 37 using the method of Example 21, and then subjected to hydrazine hydrolysis using the method of step (2). The prepared compound 30 was a white solid.

[0260] Yield 62%. 1 H NMR (600MHz, DMSO-d6): δ7.79 (d, J=8.4Hz, 2H), 7.62 (d, J=8.4Hz, 1H), 7.59 (s, 1H), 7.44 (d, J =8.4Hz, 2H), 7.22(d, J=8.4Hz, 1H), 7.10(s, 1H), 3.51(s, 2H), 2.50-2.27(m, 8H), 2.19(s, 3H). 13 C NMR (151MHz, DMSO-d6): δ155.91, 154.22, 149.03, 142.48, 141.34 (q, J C-F =37.8Hz), 139.51, 129.97(2C), 128.77, 125.10, 124.70(2C), 122.54(q, J C-F=268.8Hz), 119.57, 109.84, 103.51, 102.36, 100.42, 62.05, 55.00(2C), 52.74(2C), 45.89.ESI-HRMS m / z: calcd for C 24 H 23 F3N4O2[M+H] + , 457.1846; found, 457.1833.

[0261] Example 32: Synthesis of 2-(4-morpholinophenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 32)

[0262]

[0263] Compound 32 was prepared according to Examples 21 and 24. In Example 24, intermediate 45a in step (1) was replaced with 4-(4-morpholine)benzaldehyde and subjected to Seyferth-Gilbert carbon enrichment. The product obtained from this reaction was coupled with intermediate 37 via Sonogashira coupling and cyclization as described in Example 21, and then subjected to hydrazolysis as described in step (2). The prepared compound 32 was a white solid.

[0264] Yield 67%. 1 H NMR (600MHz, DMSO-d6): δ13.60 (s, 1H), 10.69 (s, 1H), 7.69 (d, J=9.0Hz, 2H), 7.57 (d, J=8.4Hz, 1H), 7.40 (s, 1 H), 7.19 (d, J=9.0Hz, 1H), 7.08 (d, J=4.2Hz, 2H), 7.06 (s, 1H), 3.76 (t, J=4.8Hz, 4H,), 3.21 (t, J=4.8Hz, 4H). 13 C NMR (151MHz, DMSO-d6): δ155.62, 155.05, 151.63, 147.96, 142.18, 141.51 (d, J C-F =39.3Hz), 131.97, 129.25, 125.95(2C), 124.32, 122.52(d, J C-F =265.8Hz), 120.47, 119.72, 115.34(2C), 103.74, 97.82, 66.43(2C), 48.13(2C).ESI-HRMS m / z: calcd for C 22 H 18 F3N3O3[M+H]+ , 430.1373; found, 430.1369.

[0265] Example 33: Synthesis of 2-(4-(4-methylpiperazin-1-yl)phenyl)-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-4-ol (compound 33)

[0266]

[0267] Compound 33 was prepared according to Examples 21 and 24. In Example 24, intermediate 45a in step (1) was replaced with 4-(4-methylpiperazine)benzaldehyde and subjected to Seyferth-Gilbert carbon enrichment. The product obtained from this reaction was coupled with intermediate 37 via Sonogashira coupling and cyclization as described in Example 21, and then subjected to hydrazolysis as described in step (2). The prepared compound 33 was a white solid.

[0268] Yield 52%. 1 H NMR (600MHz, DMSO-d6): δ13.56 (br s, 1H), 10.73 (br s, 1H), 7.67 (d, J=9.0Hz, 2H), 7.56 (d, J=9.0Hz, 1H), 7.37 (s, 1H), 7.19 (d, J=8.4Hz, 1H), 7 .08 (d, J=11.4Hz, 2H), 7.05 (s, 1H), 3.35-3.32 (m, 4H), 3.25 (t, J=5.4Hz, 4H), 2.26 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ155.60, 155.04, 151.27, 148.01, 142.34, 141.34 (d, J C-F =36.2Hz), 125.95(2C), 124.29, 122.50(d, J C-F =268.8Hz), 120.23, 119.75, 115.63(2C), 109.72, 103.68, 102.53, 97.83, 54.49(2C), 47.37(2C), 45.59.ESI-HRMSm / z: calcd for C 23 H 21 F3N4α2[M+H] + , 443.1689; found, 443.1677.

[0269] Example 34: Synthesis of N-(4-(4-hydroxy-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-2-yl)phenyl)acetamide (compound 34)

[0270] (1) Synthesis of 8-(4-aminophenyl)-2-(trifluoromethyl)-4H-furano[2,3-h]chromen-4-one (48)

[0271]

[0272] Intermediate 40O (200 mg, 0.533 mmol, 1.0 eq), anhydrous SnCl2 (202 mg, 1.07 mmol, 2.0 eq), and EtOH (4 mL) were added sequentially to a reaction flask. The mixture was stirred at 80 °C for 9 h under N2 protection. After the reaction was confirmed to be complete by TLC, the EtOH was removed by concentration under reduced pressure. The mixture was extracted with ethyl acetate and washed sequentially with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 100 mg of a white solid.

[0273] Yield 54%. 1 H NMR (600MHz, DMSO-d6): δ7.89 (d, J=9.0Hz, 1H), 7.79 (d, J=8.4Hz, 1H), 7.71 (d, J=9.0Hz, 2H), 7.47 (s, 1H), 7.10 (s, 1H), 6.67 (d, J=9.0Hz, 2H), 5.69 (s, 2H).ESI-MSm / z: 346[M+H] + .

[0274] (2) Synthesis of N-(4-(4-oxo-2-(trifluoromethyl)-4H-furano[2,3-h]chromen-8-yl)phenyl)acetamide (intermediate 49)

[0275]

[0276] Intermediate 48 (100 mg, 0.290 mmol, 1.0 eq), TEA (32.3 mg, 0.319 mmol, 1.1 eq), and DCM (2 mL) were added sequentially to a reaction flask. Acetyl chloride (59.2 mg, 0.754 mmol, 2.6 eq) was slowly added dropwise under ice bath conditions. The reaction was stirred at room temperature for 3 h under N2 protection. After TLC showed that the reaction was complete, the reaction was quenched with saturated sodium bicarbonate solution under ice bath conditions, extracted with DCM, and washed sequentially with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to give 90 mg of a white solid.

[0277] Yield 80%. 1 H NMR (600MHz, DMSO-d6): δ10.19 (s, 1H), 7.99 (s, 1H), 7.97 (d, J=9.0Hz, 2H), 7.86 (d, J=8.4H z, 1H), 7.80 (s, 1H), 7.75 (d, J=9.0Hz, 2H), 7.13 (s, 1H), 2.09 (s, 3H).ESI-MSm / z: 388[M+H] + .

[0278] (3) Synthesis of N-(4-(4-hydroxy-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-2-yl)phenyl)acetamide (compound 34)

[0279]

[0280] Intermediate 49 (90 mg, 0.233 mmol, 1.0 eq), hydrazine hydrate (40-50%) (216 mg, 6.74 mmol, 29 eq), and EtOH (1 mL) were added sequentially to a reaction flask. The mixture was stirred at 80 °C for 3 h under N2 protection. After the reaction was confirmed to be complete by TLC, the EtOH was removed by concentration under reduced pressure. The mixture was extracted with ethyl acetate and washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 65 mg of a white solid.

[0281] Yield 70%. 1 H NMR (600MHz, DMSO-d6): δ13.60 (br s, 1H), 10.80 (br s, 1H), 10.15 (s, 1H), 7.77 (d, J=8.4Hz, 2H), 7.74 (d, J=8.4Hz, 2H), 7.60 (d, J=8.4Hz, 1H), 7.50 (s, 1H), 7.21 (d, J=9.0Hz, 1H), 7.09 (s, 1H), 2.08 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ169.02, 155.77, 154.46, 148.18, 141.71, 141.53 (d, J C-F =36.2Hz), 140.31, 125.54(2C), 124.88, 124.61, 122.50(q, J C-F =268.8Hz), 119.73(2C), 119.53, 109.82, 103.82, 102.59, 99.29, 24.55.ESI-HRMSm / z: calcd for C20 H 14 F3N3O3[M+H] + , 402.1061; found, 402.1060.

[0282] Example 35: Synthesis of N-(4-(4-hydroxy-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-2-yl)phenyl)-2-morpholinoacetamide (compound 35)

[0283] (1) Synthesis of 2-chloro-N-(4-(4-oxo-2-(trifluoromethyl)-4H-furano[2,3-h]chromen-8-yl)phenyl)acetamide (intermediate 50)

[0284]

[0285] Intermediate 48 (500 mg, 1.45 mmol, 1.0 eq), TEA (161 mg, 1.59 mmol, 1.1 eq), and DCM (8 mL) were added sequentially to a reaction flask. 2-Chloroacetyl chloride (213 mg, 1.88 mmol, 1.3 eq) was slowly added dropwise under ice bath conditions. The reaction was stirred at room temperature for 8 h under N2 protection. After the reaction was complete as monitored by TLC, it was quenched with saturated sodium bicarbonate solution under ice bath conditions, extracted with DCM, and washed sequentially with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 400 mg of a white solid.

[0286] Yield 66%. 1 H NMR (600MHz, DMSO-d6): δ10.55 (s, 1H), 8.04 (d, J=8.4Hz, 2H), 7.98 (d, J=8.4Hz, 1H), 7.8 7 (d, J=8.4Hz, 1H), 7.85 (s, 1H), 7.77 (d, J=8.4Hz, 2H), 7.14 (s, 1H), 4.31 (s, 2H).ESI-MS m / z: 422[M+H] + .

[0287] (2) Synthesis of 2-morpholino-N-(4-(4-oxo-2-(trifluoromethyl)-4H-furano[2,3-h]chromen-8-yl)phenyl)acetamide (intermediate 51a)

[0288]

[0289] Intermediate 50 (200 mg, 0.475 mmol, 1.0 eq), morpholine (53.8 mg, 0.618 mmol, 1.3 eq), K₂CO₃ (131 mg, 0.950 mmol, 2.0 eq), and DMF (4 mL) were added sequentially to the reaction flask. The mixture was stirred at 60 °C for 4 h under N₂ protection. After the reaction was completed as monitored by TLC, the mixture was extracted with ethyl acetate and washed sequentially with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 119 mg of a white solid.

[0290] Yield 62%. 1 H NMR (600MHz, DMSO-d6): δ9.95 (s, 1H), 8.01 (d, J = 9.0Hz, 2H), 7.97 (d, J = 9.0Hz, 1H), 7.86 (d, J = 8.4Hz, 1H), 7.84 (d , J=9.0Hz, 2H), 7.80(s, 1H), 7.14(s, 1H), 3.62-3.53(m, 4H), 3.16(s, 2H), 2.55-2.38(m, 4H).ESI-MSm / z: 473[M+H] + .

[0291] (3) Synthesis of N-(4-(4-hydroxy-5-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-2-yl)phenyl)-2-morpholinoacetamide (compound 35)

[0292]

[0293] Compound 35 was prepared according to Example 34, except that step (3) 49 was replaced with 51a for hydrazinolysis. The prepared compound 35 was a white solid.

[0294] Yield 55%. 1 H NMR (600MHz, DMSO-d6): δ13.62 (br s, 1H), 10.80 (br s, 1H), 9.95 (s, 1H), 7.81 (d, J=9.6Hz, 2H), 7.79 (d, J=9.6Hz, 2H), 7.61 (d, J=9.0Hz, 1H), 7.52 (s, 1 H), 7.22 (d, J=8.4Hz, 1H), 7.09 (s, 1H), 3.65 (t, J=4.2Hz, 4H), 3.17 (s, 2H), 2.53 (t, J=4.8Hz, 4H). 13 C NMR (151MHz, DMSO-d6): δ169.14, 155.80, 154.32, 141.60, 141.40 (d, JC-F =36.2Hz), 139.24, 125.48 (2C), 125.20, 124.95, 122.37 (q, J C-F =268.8Hz), 120.45(2C), 119.53, 109.80, 103.71, 102.41, 99.46, 98.70, 66.46(2C), 62.31, 53.48(2C).ESI-HRMSm / z: calcd for C 24 H 21 F3N4O4[M+H] + , 487.1588; found.487.1581.

[0295] Example 36: Synthesis of N-(4-(7-hydroxy-6-(3-(trifluoromethyl)-1H-pyrazol-5-yl)benzofuran-2-yl)phenyl)-2-(4-methylpiperazin-1-yl)acetamide (compound 36)

[0296]

[0297] Compound 36 was prepared according to Examples 34 and 35, except that the morpholine in step (2) of Example 35 was replaced with N-methylpiperazine, nucleophilic substitution was performed with intermediate 50, and then hydrazinolysis was carried out according to step (3) of Example 34. The prepared compound 36 was a white solid.

[0298] Yield 66%. 1 H NMR (600MHz, DMSO-d6): δ9.90 (s, 1H), 7.80-7.77 (m, 4H), 7.60 (d, J=9.0Hz, 1H), 7.52 (s , 1H), 7.21 (d, J=8.4Hz, 1H), 7.09 (s, 1H), 3.15 (s, 2H), 2.63-2.36 (m, 8H), 2.20 (s, 3H). 13 C NMR (151MHz, DMSO-d6): δ168.96, 155.80, 154.27, 148.63, 142.44, 141.34 (q, J C-F =37.2Hz), 139.53, 125.45 (2C), 125.04, 124.90, 122.52 (q, Jc-F = 268.8Hz), 120.19 (2C), 119. 60, 109.85, 103.60, 102.43, 99.55, 62.20, 54.92(2C), 53.00(2C), 46.06.ESI-HRMSm / z: calcd for C 25 H24 F3N5O3[M+H] + , 500.1904; found, 500.1900.

[0299] Experimental Example 1: Antitumor Cell Proliferation Activity of the Compounds of the Present Invention

[0300] The prostate cancer cell line PC-3 (which overexpresses MYC) is a commonly used cell line for screening MYC inhibitors. In this experiment, MYCi975, a recently reported representative small-molecule inhibitor directly targeting the MYC protein (with in vivo efficacy and well-established MYC targeting), was used as a positive control. The MTT assay was used to evaluate the antiproliferative activity of the compounds of this invention and MYCi975 against PC-3. Other compounds of this invention have similar beneficial effects to those listed below, but this should not be construed as meaning that the compounds of this invention only possess the following beneficial effects.

[0301] The test procedure for anti-tumor cell proliferation activity is as follows: Collect PC-3 cells in the logarithmic growth phase, and then... 3 Cells were seeded per well in 96-well plates and incubated overnight at 37°C (5% CO2). After cell attachment, cells were treated with different concentrations of compound-containing culture media and cultured for 72 hours. MTT solution was then added to each well, and incubation continued for 4 hours. After incubation, the supernatant was discarded, and 100 μL of DMSO was added to each well. The absorbance was measured at 492 nm, and the IC50 was calculated using GraphPad Prism 9 software. 50 Values. The antiproliferative activity of the preferred compounds against different tumor cells and their cytotoxicity against normal cells were evaluated in the same manner as PC-3, and the results are shown in Table 1.

[0302] Table 1. Antitumor cell proliferation activity of the compounds

[0303]

[0304]

[0305] Table 1: "++++" represents 0.1-2.0μM; "+++" represents 2.0-10μM; "++" represents 10-40μM; "+" represents 40-100μM.

[0306] As shown in Table 1, all the listed compounds exhibited significant anti-proliferative activity against PC-3 cell lines, and their activity was comparable to or better than that of the positive control MYCi975; most of the compounds showed significantly better anti-PC-3 cell proliferation activity than MYCi975. Example 2: Broad-spectrum anti-tumor cell proliferation activity of the compounds of this invention.

[0307] Using a similar test method to the above-described tumor cell antiproliferative activity assay, the antiproliferative activity of compound 15 against multiple tumor cell lines (including solid tumor and hematologic malignancy cells) was further evaluated. Other compounds of the present invention have similar beneficial effects to the compounds listed below, but this should not be construed as meaning that the compounds of the present invention only have the following beneficial effects.

[0308] Table 2. Antiproliferative activity of compounds against other tumor cells

[0309]

[0310] In Table 2: "++++" represents 0.1-2.0 μM; "+++" represents 2.0-10 μM; "++" represents 10-40 μM; "+" represents 40-100 μM.

[0311] As shown in Table 2, the listed compounds exhibited significant anti-proliferative activity against 14 tumor cell lines other than PC-3 (their anti-tumor cell proliferation activity against all cell lines was superior to MYCi975, or comparable to the latter), indicating that the compounds have broad-spectrum anti-tumor function.

[0312] Experimental Example 3: Co-IP Experiment of the Compounds of the Present Invention

[0313] The interaction (PPI) between MYC and its chaperone protein MAX is crucial for activating MYC transcriptional function. To characterize the intervention effect of the compounds of this invention on MYC-MAX-PPI, this experimental example uses a Co-IP assay to examine the effect of compound 15 on MYC-MAX-PPI by detecting the level of MAX protein bound to the MYC / immunoprecipitation antibody / potein G magnetic bead ternary complex. Other compounds of this invention have similar beneficial effects to compound 15, but this should not be construed as meaning that the compounds of this invention only have the following beneficial effects.

[0314] PC-3 cells in logarithmic growth phase were collected and seeded into 10 cm dishes, then incubated overnight at 37°C (5% CO2). The experimental group received the test compound (final concentration 10 μM), while the control group received an equal volume of 0.5% DMSO. Cells were incubated for another 1 h, digested, centrifuged, collected, and washed with PBS. NP-40 lysis buffer (containing 1% PMSF and 2% phosphatase inhibitor) was added, mixed, and lysed on ice for 30 min. The cells were centrifuged, and the supernatant was collected. Protein concentration was determined using the BCA method, and the supernatant was diluted to the same concentration with lysis buffer. After pre-incubation with MYC primary antibody for 1 h, an appropriate amount of potein G magnetic bead suspension was added, and the cells were incubated overnight on a four-dimensional shaker at 4°C. After incubation, the magnetic beads were separated on a magnetic rack, the supernatant was discarded, and the cells were washed with PBS. SDS-PAGE buffer was added, mixed, and heated in a 95°C water bath for 7 min. After cooling, the cells were separated on a magnetic rack, and the supernatant was used for Western blotting.

[0315] Figure 1 The results showed that after co-incubating PC-3 cells with compound 15 and MYCi975 (both at a concentration of 10 μM), the levels of MYC and MAX proteins in the Input group did not change significantly; the MYC protein level in the IP group did not change significantly, while the MAX protein level decreased. The bar chart showed that after treatment with compound 15, the MAX level, which participates in the MYC / MAX-PPI pathway, decreased by more than 39%, and its effect was significantly better than that of the same concentration of MYCi975 (which reduced MAX levels by 16%).

[0316] Therefore, the compounds prepared in this invention can directly inhibit MYC function by interfering with MYC-MAX interactions.

[0317] Experimental Example 4: MYC / MAX ALphaLISA Experiment

[0318] This experimental example further characterizes the ability of the compound to interfere with MYC / MAX-PPI using MYC / MAX AlphaLISA assay.

[0319] Transfer the compound solutions of gradient concentrations (0.1 μL) to the assay plate; add MYC working solution (2.5 μL), centrifuge, and incubate the mixture at 25 °C for 10 min; add MAX working solution (2.5 mL), centrifuge, and incubate the assay plate at 25 °C for 1 h; add α donor and acceptor working solutions (5 μL), and incubate the test system at 25 °C for 1.5 h; detect the AlphaLISA signal to determine the inhibition rate.

[0320] Figure 2 This indicates that, in this experiment, the IC50 of compound 15 was... 50 The value is 3.06 μM, which is MYCi975 (IC). 50The activity of the compound prepared in this invention is 5 times that of MYC (15.7 μM), therefore, the compound can directly block the function of MYC by interfering with the PPI between MYC and MAX.

[0321] Experimental Example 5: CETSA test of the compound of the present invention

[0322] Small molecule compounds can affect the thermal stability of their targets upon binding, an effect that can be characterized by CETSA assays. This experiment used CETSA to investigate the direct effect of compound 15 on MYC within cells. Other compounds of this invention have similar beneficial effects to compound 15, but this should not be construed as meaning that the compounds of this invention only possess the following beneficial effects.

[0323] PC-3 cells in logarithmic growth phase were collected and seeded into 10 cm dishes, then incubated overnight in an incubator (37°C, 5% CO2). The experimental group was treated with compound 15 (final concentration 10 μM), while the control group was treated with an equal volume of 0.5% DMSO. Cells were incubated at room temperature for 1 h, digested, centrifuged, collected, and washed with PBS. The experimental and control groups were then treated with PBS (containing 1% PMSF and 2% phosphatase inhibitor) at the same concentration as the previously treated compound 15 or DMSO, respectively, and heated at the specified temperature. Afterward, the cells were subjected to three freeze-thaw cycles in liquid nitrogen, centrifuged, and the supernatant was collected. One-quarter volume of SDS-PAGE buffer was added, mixed, and heated in a 95°C water bath for 7 min. After cooling, the samples were ready for Western blotting experiments.

[0324] Figure 3 This indicates that compound 15 can reduce the thermal stability of MYC protein by acting directly on it intracellularly, with a significant thermal displacement—this experiment reveals the direct effect of the compound of the present invention on the MYC protein.

[0325] Experimental Example 6: Degradation of MYC protein by the compound of the present invention

[0326] MYC inhibitors can affect the stability of MYC protein by directly binding to MYC or inhibiting MYC / MAX-PPI, thereby inducing its degradation. Based on this, to further characterize the direct intervention effect of the compounds of this invention on MYC function, this experimental example uses a Western blotting (WB) experiment to evaluate the ability of compounds 12 and 15 to induce intracellular MYC protein degradation. Other compounds of this invention have similar beneficial effects to compounds 12 and 15, but this should not be construed as meaning that the compounds of this invention only have the following beneficial effects.

[0327] PC-3 cells in logarithmic growth phase were collected at a concentration of 5 × 10⁻⁶ cells / cells. 5Cells were seeded per well in 6-well plates and incubated overnight at 37°C (5% CO2) for adhesion. Drug-treated wells were treated with 1, 5, and 10 μM culture medium containing compounds 12, 15, and MYCi975, respectively. Blank control wells were treated with an equal volume of complete culture medium. Cells were incubated for 24 h, washed, digested, and centrifuged. Cells were washed twice with PBS and centrifuged at 3000 rpm for 3 min to collect them. RIPA lysis buffer (containing 1% PMSF and 2% phosphatase inhibitor) was added, followed by sonication, centrifugation, and collection of the supernatant for BCA quantification. After balancing, 1 / 4 volume of SDS-PAGE buffer was added, mixed, and incubated at 95°C for 7 min. After cooling, the samples were ready for Western blotting.

[0328] Figure 4 This indicates that the compounds of this invention can significantly induce intracellular MYC protein degradation (MYC degradation rate greater than 50%) at concentrations of 5 μM and 10 μM, and their efficacy is significantly better than that of MYCi975. Therefore, the compounds of this invention can directly affect the function of this protein by inducing MYC degradation.

[0329] Experiment 7: Luciferase Reporter Gene Detection Experiment

[0330] Small molecule inhibitors can affect the stability of the MYC / MAX dimer by inducing MYC degradation, binding to MYC, or inhibiting MYC / MAX-PPI, thereby interfering with the dimer's interaction with the DNA-Ebox and ultimately inhibiting the transcription of MYC-related genes. In this study, based on the construction of a stable HEK293T cell line expressing the MYC-responsive element (E-box-driven Luc2p luciferase reporter gene), the effect of compound 15 on E-box-Luc2p expression was evaluated to investigate its MYC transcriptional repression function. Other compounds of this invention have similar beneficial effects to compound 15, but this should not be construed as meaning that the compounds of this invention only have the following beneficial effects.

[0331] HEK293T cells transfected with the luciferase reporter gene (E-box-Luc2p) were used at a rate of 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and incubated overnight. After cell attachment, the test compound was added, and the plates were cultured for another 24 hours. 40 μL of Lysis buffer (containing 1% PMSF and 2% phosphatase inhibitor) was added, mixed, and incubated at room temperature with shaking for 15 min. 40 μL of One-Glo luciferase assay reagent was added, and the plates were shaken in the dark and incubated for 5 min to stabilize the luminescence signal. The absorbance was measured at 560 nm, and the IC50 was calculated using GraphPadPrism 9 software. 50 value.

[0332] Figure 5This indicates that compound 15 has a significant inhibitory effect on E-box-driven luc2p expression in the HEK293T stable cell line (IC50). 50 =0.739μM), the activity level was higher than that of the positive control MYCi975 (IC50). 50 =3.67 μM) is 5 times that of MYC. Therefore, the compound of the present invention can directly inhibit MYC function by interfering with MYC gene transcription.

[0333] Experimental Example 8: In vivo efficacy of the compounds of the present invention

[0334] To further characterize the application prospects of the compounds of this invention, this experimental example used a male FVB mouse MYC-CaP prostate cancer model to evaluate the in vivo antitumor efficacy of compound 15. Other compounds of this invention have similar beneficial effects to compound 15, but this should not be construed as meaning that the compounds of this invention only possess the following beneficial effects.

[0335] Six-week-old male FVB mice were used and, after acclimatization, were subcutaneously injected with 1×10⁻⁶ ppm of the drug into the right unilateral axilla. 6 MYC-CaP cells bearing tumors were fed until the average tumor volume was 30 mm. 3 Grouping was performed. To assess the in vivo efficacy of the selected compounds, tumor-bearing mice were randomly divided into five groups: a control group, high-dose (50 mpk) groups of compound MYCi975, high-dose (50 mpk), medium-dose (30 mpk), and low-dose (10 mpk) groups of compound 15, with six mice in each group. After grouping, MYCi975 was administered intraperitoneally once daily, and compound 15 was administered intraperitoneally once every two days. The control group received an equal volume of physiological saline. Treatment lasted for 16 days. After the start of the experiment, the long and short diameters of the tumor were measured every 2-3 days, and the tumor volume was calculated (tumor volume = 1 / 2 × long diameter × short diameter). 2 The weight changes of the mice were recorded. After the experiment, the mice were euthanized by cervical dislocation, and the tumor tissue was removed, weighed, and photographed. Tumor tissue and heart, liver, spleen, lungs, and kidneys of three mice in each group were taken, fixed in 4% paraformaldehyde, prepared into paraffin sections, and stained with hematoxylin and eosin (HE).

[0336] Figure 6 This indicates that the compounds of the present invention exhibit significant therapeutic effects on prostate cancer allografts, with a clear dose-dependent effect, and their in vivo efficacy is significantly superior to the previously reported representative MYC inhibitor MYCi975; at doses of 30 mpk and 50 mpk, administered once every two days, tumor inhibition rates reached 57% and 70%, respectively. Furthermore, no significant weight loss was observed in any of the treatment groups. Figure 6 (B) and signs of organ toxicity ( Figure 7It exhibits good tolerability. Notably, the compounds of this invention reduce the frequency of administration; at the same dosage, administration every two days is significantly more effective than once-daily administration of MYCi975.

[0337] Experimental Example 9: Antitumor effect of the compound of the present invention in combination with a small molecule immune checkpoint inhibitor

[0338] Tumor immunotherapy, represented by immune checkpoint inhibitors, has brought about a major revolution in the clinical treatment of cancer. However, the overall low response rate of tumors to immunotherapy remains a bottleneck problem. Exploring the combination of immune checkpoint inhibitors with other anti-tumor drugs to enhance the tumor response rate to immunotherapy is an effective strategy to address this clinical problem. Currently, no studies have investigated the anti-tumor effects of combining small molecule MYC inhibitors with small molecule immune checkpoint inhibitors.

[0339] This invention investigated the synergistic antitumor effect of the compound in combination with the small molecule PD-1 / PD-L1 immune checkpoint inhibitor INCB086550. Other compounds of this invention have similar beneficial effects to compound 15, but this should not be construed as meaning that the compounds of this invention only possess the following beneficial effects.

[0340] Tumor-bearing mice were randomly divided into a control group, an INCB086550 inhibitor group (20 mpk), a compound 15 administration group (30 mpk), and a combination group [INCB086550 (20 mpk) and compound 15 (30 mpk)], with 6 animals in each group. After grouping, INCB086550 inhibitor was administered by gavage (once a day), compound 15 was administered by intraperitoneal injection (once every two days), and the control group received an equal volume of physiological saline. Treatment lasted for 20 days. After the start of the experiment, the long and short diameters of the tumor were measured every 2-3 days, the tumor volume was calculated, and the weight changes of the mice were recorded. After the experiment, the mice were euthanized by cervical dislocation, the tumor tissue was dissected, weighed, and photographed. Tumor tissue, heart, liver, spleen, lung, and kidney were taken from three mice in each group, fixed in 4% paraformaldehyde, prepared paraffin sections, and stained with hematoxylin and eosin (HE) and eosin (IHC).

[0341] Figure 8 This indicates that the combination of compound 15 and the small molecule PD-1 / PD-L1 immune checkpoint inhibitor INCB086550 has a synergistic anti-tumor effect, which is significantly better than the anti-tumor effect of INCB086550 or compound 15 monotherapy. It significantly enhances the tumor response to INCB086550, and no significant weight loss was observed in the combination group. Figure 8 (B) Furthermore, no significant signs of organ toxicity (e.g., HE staining) were found in any of the treatment groups. Figure 9 ).

[0342] IHC analysis of tumor tissues showed increased infiltration of CD8 and CD45 immune cells in the tumor microenvironment in the combination therapy group, while the expression level of the proliferation antigen Ki67 decreased (e.g., Figure 10 This provides a reasonable explanation for the synergistic antitumor efficacy of the combination therapy. This indicates that the compounds of this invention also have the potential for application in combination with immune checkpoint inhibitors, which could enhance the tumor's response to immunotherapy.

[0343] In summary, the compounds prepared in this invention exhibit excellent in vitro and in vivo antitumor efficacy, possess broad-spectrum antitumor functions, and their direct inhibitory effect on the function of the "drug-resistant" protein MYC has been confirmed by various biological experiments. Furthermore, they can enhance the tumor's response to immunotherapy and have promising application prospects.

Claims

1. A compound having a benzofuran-bipyrazole class as shown in Formula I, or a pharmaceutically acceptable salt or deuterated thereof, In the formula: X is independently selected from -CH2-, -CH2CH2-, C5-C6 heteroaryl groups substituted with at least one R1. Any one of them; R1 is independently selected from hydrogen, halogen, methyl, C3 hydroxyalkyl, trifluoromethyl, methoxy, trifluoromethoxy, C4-C6 nitrogen-containing heterocyclic group, R5C(=O)-, R5CH2-, R5C(=O)NH-, R5CH2-C(=O)NH-; when R1 is a C4-C6 nitrogen-containing heterocyclic group, it can be substituted by at least one R6; R2 and R3 are each independently selected from hydrogen and methyl, respectively; R4 is independently selected from methyl and trifluoromethyl; R5 is independently selected from methyl, C4-C6 nitrogen-containing heterocyclic groups, and may be substituted by at least one R7; R6 and R7 are respectively selected from hydrogen, halogen, methyl, and hydroxyl.

2. The benzofuran-bipyrazole compound according to claim 1, or its pharmaceutically acceptable salt or deuterated derivative, characterized in that: X is independently selected from -CH2-, -CH2CH2-, which are substituted by at least one R1. Any one of them; R1 is independently selected from hydrogen, halogen, methyl, C3 hydroxyalkyl, methoxy, trifluoromethyl, trifluoromethoxy, C4-C6 nitrogen-containing heterocyclic group, R5C(=O)-, R5CH2-, R5CH2-C(=O)NH-; when R1 is a C4-C6 nitrogen-containing heterocyclic group, it can be substituted by at least one R6; R2 is independently selected from hydrogen; R3 is independently selected from either hydrogen or methyl; R4 is independently selected from trifluoromethyl; R5 is independently selected from C4-C6 nitrogen-containing heterocyclic groups and can be substituted by at least one R7; R6 and R7 are independently selected from any one of hydrogen, halogen, methyl, and hydroxyl.

3. The benzofuran-bipyrazole compound according to claim 2, or its pharmaceutically acceptable salt or deuterated derivative, characterized in that: X is independently selected from those that are replaced by at least one R1. Any one of them; R1 is independently selected from hydrogen, halogen, methyl, methoxy, trifluoromethyl, trifluoromethoxy, R5C(=O)-, R5CH2-, R5CH2-C(=O)NH-; R2 is independently selected from hydrogen; R3 is independently selected from either hydrogen or methyl; R4 is independently selected from trifluoromethyl; R5 is independently selected from C4-C6 nitrogen-containing heterocyclic groups; R5 may be substituted by at least one R7; R7 is independently selected from any one of hydrogen, halogen, methyl, and hydroxyl.

4. The benzofuran-bipyrazole compound according to claim 3, or its pharmaceutically acceptable salt or deuterated derivative, characterized in that: X is independently selected from those that are replaced by at least one R1. Any one of them; R1 is independently selected from hydrogen, halogen, methyl, methoxy, trifluoromethyl, trifluoromethoxy, R5C(=O)-, R5CH2-, R5CH2-C(=O)NH-; R2 is independently selected from hydrogen; R3 is independently selected from either hydrogen or methyl; R4 is independently selected from trifluoromethyl; R5 is independently selected from C4-C6 nitrogen-containing heterocyclic groups; R5 may be substituted by at least one R7; R7 is independently selected from any one of hydrogen, halogen, methyl, and hydroxyl.

5. The benzofuran-bipyrazole compound according to claim 1, or its pharmaceutically acceptable salt or deuterated derivative, characterized in that, Its structural formula is any one of the following compound structural formulas: 。 6. The use of the benzofuran bipyrazole compound or its pharmaceutically acceptable salt or deuterated derivative as described in any one of claims 1-5 in the preparation of MYC inhibitors.

7. A MYC inhibitor composition comprising an effective amount of a benzofuran-bipyrazole compound as described in any one of claims 1-5 or a pharmaceutically acceptable salt or deuterated thereof, and at least one pharmaceutically acceptable carrier or excipient.

8. The MYC inhibitor composition according to claim 7, characterized in that, It also includes at least one other therapeutic agent, said other therapeutic agent being a small molecule immune checkpoint inhibitor.

9. The use of the benzofuran bipyrazole compound of any one of claims 1-5 or its pharmaceutically acceptable salt or deuterated derivative, or the MYC inhibitor composition of claim 7 or 8, in the preparation of an MYC-mediated antitumor drug, wherein the tumor includes breast cancer, colorectal cancer, prostate cancer, lung cancer, neuroblastoma, ovarian cancer, liver cancer, rhabdomyosarcoma, skin cancer, osteosarcoma, multiple myeloma, or leukemia.

Images