A MYC inhibitor and its preparation method and application

Abstract

The present invention discloses a MYC inhibitor, its preparation method, and application. The MYC inhibitor is a compound represented by general formula (I) or (II), or a pharmaceutically acceptable salt or deuterated derivative thereof. These compounds have novel structures, significant in vitro and in vivo efficacy, can directly inhibit MYC function, interfere with the MYC-MAX interaction, and can be used to treat tumors and other MYC-related diseases, with broad prospects for clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a MYC inhibitor and a preparation method and application thereof. Background Art

[0002] MYC is a transcription factor encoded by the proto-oncogene MYC that regulates key processes such as cell proliferation, differentiation, metabolism, and apoptosis. Its overexpression is found in 70-80% of malignant tumors. Key oncogenic signaling pathways such as Wnt, Ras, and PI3K / Akt all require MYC to mediate their functions. The MYC family consists of three members: c-MYC (i.e., MYC), MYCL, and MYCN. They heterodimerize with MAX, inducing conformational changes and forming DNA-binding regions, thereby binding to the DNA E-box and initiating transcription of MYC-related genes. Because the MYC protein is a master regulator of tumor growth and immune evasion, and because it is widely expressed in all major types of tumors but rarely overexpressed in normal cells, it has long been considered one of the most valuable anti-tumor targets.

[0003] In the resting state, the expression level of MYC is strictly controlled by growth factor-dependent signals. Once MYC is overexpressed, it will lead to the occurrence and development of cancer and make the tumor dependent on MYC. Studies have shown that MYC can stimulate metabolic reprogramming by regulating key enzymes in the metabolic pathways of tumor cells, drive glucose and glutamine metabolism, fatty acid and nucleotide synthesis, and O-GlcNAc modification, and promote the growth and proliferation of tumor cells. At the same time, MYC can regulate the expression of cytokines and immune checkpoints, inhibit the expression of CD4 + It inhibits the activity of various immune cells such as T cells, macrophages and NK cells, thereby mediating tumor immune escape. In addition, overexpression of MYC can also induce tumor angiogenesis. Inhibiting MYC can restore cell cycle checkpoints, repair DNA, and reshape chromatin, thereby blocking tumor cell proliferation, leading to differentiation, aging and apoptosis. In MYC-dependent animal cancer models (including lymphoma, leukemia, osteosarcoma, hepatocellular carcinoma, renal cell carcinoma and lung cancer, etc.), sustained tumor regression can be observed after targeting MYC. Studies have confirmed that the anti-tumor efficacy of targeting MYC is not only related to direct effects on cancer cells, but also related to regulating tumor immunity and intervening in angiogenesis.

[0004] Although targeting MYC holds great promise for cancer treatment, the disordered nature of the MYC protein and its lack of a typical active pocket make the development of small molecule inhibitors directly targeting MYC challenging. To date, no compounds have entered clinical trials. Given the necessity of the MYC-MAX interaction for activating the former's transcriptional regulatory function, intervening in this interaction is an effective strategy for discovering small molecule inhibitors that directly target MYC. However, numerous compounds previously discovered, both domestically and internationally, that directly intervene in MYC function have significant drawbacks, including low in vitro activity, lack of in vivo efficacy, and poor safety. Therefore, the development of novel small molecule inhibitors that directly target MYC and possess in vivo efficacy is urgently needed. Summary of the Invention

[0005] To address the shortcomings of existing small-molecule inhibitors that directly interfere with MYC function in terms of anti-tumor efficacy, the present invention provides a novel MYC inhibitor, its preparation method, and its application. Multiple experiments have confirmed that the compounds of the present invention can directly inhibit MYC function, interfere with the MYC-MAX interaction, and exhibit significant anti-tumor activity.

[0006] The technical solutions of the present invention are as follows:

[0007] In a first aspect of the present invention, a MYC inhibitor is provided, which is a compound represented by general formula (I) or (II) or a pharmaceutically acceptable salt or deuterated derivative thereof:

[0008]

[0009] in:

[0010] n is each independently selected from an integer of 1 to 6;

[0011] R1 is independently selected from any one of hydrogen, C1-C6 alkyl, and C3-C8 cycloalkyl;

[0012] R2 is independently selected from any one of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, and C1-C6 haloalkoxy;

[0013] R3 is independently selected from any one of a C1-C10 alkylamino group, a substituted or unsubstituted C3-C20 nitrogen-containing heterocyclic group, a hydroxyl group, and a C1-C6 hydroxyalkyl group;

[0014] Ar is independently selected from any one of a substituted or unsubstituted C6-C14 aryl group and a substituted or unsubstituted C5-C14 heteroaryl group.

[0015] According to a specific embodiment of the present invention, n is independently selected from integers of 1 to 3.

[0016] According to a specific embodiment of the present invention, R1 is independently selected from any one of hydrogen and C1-C3 alkyl, preferably one of hydrogen and methyl.

[0017] According to a specific embodiment of the present invention, R2 is independently selected from any one of hydrogen, C1-C3 alkyl, and C1-C3 haloalkyl, preferably a trihalomethyl group.

[0018] According to a specific embodiment of the present invention, R3 is independently selected from any one of a C2-C6 dialkylamino group, a substituted or unsubstituted C4-C10 nitrogen-containing heterocyclic group, a hydroxyl group, and a C1-C3 hydroxyalkyl group; preferably, the C2-C6 dialkylamino group is a dimethylamino group; preferably, the C1-C3 hydroxyalkyl group is a hydroxyisopropyl group; preferably, the substituted or unsubstituted C4-C10 nitrogen-containing heterocyclic group is selected from any one of the following groups:

[0019]

[0020] According to a specific embodiment of the present invention, Ar is independently selected from any one of substituted or unsubstituted phenyl and pyridyl, and the substitution is at least one of halogen substitution and haloalkyl substitution.

[0021] According to a specific embodiment of the present invention, the MYC inhibitor is selected from any one of the following compounds or a pharmaceutically acceptable salt or deuterated substance thereof:

[0022]

[0023]

[0024]

[0025] The second aspect of the present invention provides a method for preparing the aforementioned MYC inhibitor, comprising:

[0026] The compound represented by formula (Ia) is subjected to a nucleophilic substitution reaction with the compound represented by formula (Ib) to obtain the compound represented by formula (Ic); the compound represented by formula (Ic) is subjected to a Sonagashira coupling reaction with the compound represented by formula (Id) to obtain the compound represented by formula (Ie); the compound represented by formula (Ie) is subjected to a hydrazinolysis reaction with the compound represented by formula (If) to obtain the compound represented by formula (I). The preparation route is as follows:

[0027]

[0028] or,

[0029] The compound represented by formula (Ia) is subjected to a Suzuki coupling reaction with the compound represented by formula (II-a) to obtain the compound represented by formula (II-b); the compound represented by formula (II-b) is reacted with trifluoromethanesulfonic anhydride to form a sulfonate to obtain the compound represented by formula (II-c); the compound represented by formula (II-c) is subjected to a Sonagashira coupling reaction with the compound represented by formula (Id) to obtain the compound represented by formula (II-d); the compound represented by formula (II-d) is subjected to a hydrazinolysis reaction with the compound represented by formula (If) to obtain the compound represented by formula (II). The preparation route is as follows:

[0030]

[0031] wherein X is independently selected from halogen; n, R1, R2, R3, and Ar are as defined above.

[0032] The third aspect of the present invention provides a pharmaceutical composition comprising the aforementioned MYC inhibitor as the sole or main active ingredient, and a pharmaceutically acceptable carrier or auxiliary ingredient, in any clinically or pharmaceutically acceptable dosage form.

[0033] The fourth aspect of the present invention provides the use of the aforementioned MYC inhibitor or pharmaceutical composition in the preparation of an anti-tumor drug; preferably, the tumor includes any one of a solid tumor and a hematological tumor.

[0034] The dosage of the compound of the present invention is 1 mg to 1000 mg / day, and may deviate from this range depending on the severity of the disease or the dosage form.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

[0036] Here, "halogen" refers to fluorine, chlorine, bromine or iodine.

[0037] The "C1-C6 alkyl group" refers to an alkyl group having 1 to 6 carbon atoms.

[0038] "C1-C6 haloalkyl" refers to an alkane having 1 to 6 carbon atoms in which at least one hydrogen atom is replaced by the same or different halogen groups as defined above, such as trifluoromethyl, difluoromethyl, monofluoromethyl, 2,2,2-trifluoroethyl, 1,1,-difluoroethyl, etc.

[0039] The "C1-C6 hydroxyalkyl group" refers to an alkane having 1 to 6 carbon atoms in which at least one hydrogen atom is replaced by a hydroxy group.

[0040] "C3-C8 cycloalkyl" refers to a saturated monocyclic or polycyclic group of 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0041] "C1-C6 alkoxy" refers to an alkane having 1 to 6 carbon atoms in which one hydrogen atom is replaced by an oxygen atom, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, neopentoxy, and n-hexoxy.

[0042] The "C1-C6 haloalkoxy group" refers to a C1-C6 alkoxy group as defined above in which at least one hydrogen atom is substituted by the same or different halogen groups as defined above.

[0043] The "C1-C10 alkylamino group" means that at least one hydrogen atom of an amino group is substituted with a C1-C10 alkyl group.

[0044] "C3-C20 nitrogen-containing heterocyclic group" refers to a cycloalkyl group of 3 to 20 carbon atoms in which at least one carbon atom is substituted by a nitrogen atom, such as tetrahydropyrrole, piperidine, morpholine, piperazine, and the like.

[0045] "C6-C14 aryl" refers to an all-carbon monocyclic or fused polycyclic ring of 6 to 14 carbon atoms with a completely conjugated π electron system, such as a benzene ring, a naphthalene ring, an anthracene ring, and the like.

[0046] "C5-C14 heteroaryl" refers to a non-all-carbon monocyclic or fused polycyclic group of 5 to 14 ring atoms with a completely conjugated π electron system, such as pyridine, imidazole, thiophene, furan, thiazole, purine, indole, azaindole, etc.

[0047] 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-mentioned general formula (I) or (II), wherein the pharmaceutically acceptable salt is a hydrochloride, sulfate, phosphate, maleate, fumarate, citrate, methanesulfonate, tartrate, sodium salt or potassium salt.

[0048] The "pharmaceutically acceptable carriers or auxiliary ingredients" mentioned above refer to conventional drug carriers or pharmaceutical excipients in the pharmaceutical field, including conventional diluents, excipients (such as water, etc.), fillers (such as starch, etc.), binders (such as cellulose derivatives, gelatin, etc.), wetting agents (such as glycerol, etc.), disintegrants (such as agar, calcium carbonate, etc.), absorption accelerators (such as quaternary ammonium compounds, etc.), surfactants (such as cetyl alcohol, etc.), adsorption carriers (such as kaolin and bentonite, etc.), lubricants (such as talc, etc.), and flavoring agents, sweeteners, etc. may also be added if necessary.

[0049] "Any pharmaceutically acceptable dosage form" is suitable for administration by any appropriate route, such as oral (including buccal or sublingual administration), rectal administration, nasal administration, topical administration (including buccal, sublingual or transdermal administration), vaginal administration or parenteral administration (including subcutaneous injection, intramuscular injection, intravenous injection or intradermal injection). These preparations can be prepared by any method known in the art of pharmacy. For example, by mixing the active ingredient with a carrier or auxiliary ingredients.

[0050] The beneficial effects of the present invention are:

[0051] The present invention provides a novel structural compound with MYC inhibitory activity. Pharmacodynamic experiments show that it has significant in vitro and in vivo efficacy, can directly inhibit MYC function, interfere with MYC-MAX interaction, and can be used for the treatment of tumors and other MYC-related diseases, with broad prospects for clinical application. DETAILED DESCRIPTION

[0052] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0053] Taking compounds 2 (Route I), 22 (Route I), and 31 (Route II) as examples, the preparation methods of the compounds of the present invention are as follows:

[0054] In addition, compounds 1, 3-21, 23-30 were prepared according to Route I; compounds 32-48 were prepared according to Route II.

[0055]

[0056] In route I, the reactants and reaction conditions involved are: a is tetrahydropyrrole or morpholine, K2CO3, tetrahydrofuran (THF), N2 protection, 70°C;

[0057] b is trifluoroacetic anhydride (TFAA), CF3COONa, 125 °C;

[0058] c is I2, pyridine, CHCl3, room temperature (rt);

[0059] d is 4-chlorobenzyl chloride, K2CO3, KI, acetone, 60℃;

[0060] e is 50 or 51, Pd(PPh3)2Cl2, CuI, triethylamine (TEA), N2 protection, 90℃;

[0061] f is hydrazine hydrate (40%-50%), EtOH, N2 protection, 80℃;

[0062] g is methylhydrazine sulfate, NaOH, EtOH, N2 protection, 80℃.

[0063]

[0064] In route II: a is 4-chloro-3-(trifluoromethyl)phenylboronic acid, Pd(dppf)Cl2, Na2CO3, toluene, EtOH, H2O, N2 protection, 100°C;

[0065] b is trifluoromethanesulfonic anhydride, pyridine, dichloromethane (DCM), N2 protection, rt;

[0066] c is 51, Pd(PPh3)2Cl2, CuI, TEA, N2 protection, 90℃;

[0067] d is hydrazine hydrate (40%-50%), EtOH, N2 protection, 80℃.

[0068] Example 1: 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(tetrahydro-1H-pyrrol-1-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (2)

[0069] (1) Synthesis of 1-(prop-2-yn-1-yl)pyrrolidine (50)

[0070] To a round-bottom flask, 3-bromopropyne (500 mg, 4.20 mmol, 1.0 eq), tetrahydropyrrole (746 mg, 10.5 mmol, 2.5 eq), K2CO3 (1.40 g, 10.5 mmol, 2.5 eq), and THF (8 mL) were added sequentially. The system was fully purged with N2, and the reaction was stirred at 70°C. After TLC showed the reaction was complete, the organic solvent was evaporated under reduced pressure, and the mixture was washed sequentially with saturated NaHCO3 and saturated NaCl solutions, extracted with ethyl acetate (EA), and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain 278 mg of a colorless oil, which was used directly in the subsequent coupling reaction.

[0071] (2) Synthesis of 7-hydroxy-2-(trifluoromethyl)-4H-chromen-4-one (53)

[0072] 1-(2,4-Dihydroxyphenyl)ethanone 52 (8.00 g, 52.6 mmol, 1.0 eq), TFAA (55.3 g, 263 mmol, 5.0 eq), and CF3COONa (15.8 g, 116 mmol, 2.2 eq) were added sequentially to a sealed tube and stirred at 125°C. After TLC indicated completion of the reaction, the solution was transferred to a round-bottom flask and the pH was adjusted to 7-8 with saturated aqueous K2CO3. The mixture was extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 7.82 g of a white solid.

[0073] (3) Synthesis of 7-hydroxy-8-iodo-2-(trifluoromethyl)-4H-chromen-4-one (54)

[0074] To a round-bottom flask, intermediate 53 (5.20 g, 22.6 mmol, 1.0 eq), I (22.9 g, 90.4 mmol, 4.0 eq), pyridine (7.15 g, 90.4 mmol, 4.0 eq), and CHCl (100 mL) were added sequentially and stirred at room temperature. After TLC indicated completion of the reaction, saturated NaSO was added to quench the reaction. The mixture was washed sequentially with saturated NaHCO and saturated NaCl solutions, extracted with EA, and the organic phases were combined, dried over anhydrous NaSO, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to yield 7.54 g of a white solid.

[0075] (4) Synthesis of 7-(((4-chlorophenyl)methyl)oxy)-8-iodo-2-(trifluoromethyl)-4H-chromen-4-one (55)

[0076] To a round-bottom flask were added intermediate 54 (2.00 g, 5.60 mmol, 1.0 eq), 4-chlorobenzyl chloride (1.35 g, 8.40 mmol, 1.5 eq), K2CO3 (1.50 g, 11.2 mmol, 2.0 eq), KI (1.39 g, 8.40 mmol, 1.5 eq), and acetone (25 mL) in sequence, and the mixture was stirred at 60°C. After TLC indicated completion of the reaction, the mixture was washed sequentially with saturated NaHCO3 and saturated NaCl solutions, extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 2.41 g of a white solid.

[0077] (5) Synthesis of 7-(((4-chlorophenyl)methyl)oxy)-8-(3-(tetrahydro-1H-pyrrol-1-yl)prop-1-ynyl)-2-(trifluoromethyl)-4H-chromen-4-one (56)

[0078] To a round-bottom flask were added intermediate 55 (250 mg, 0.521 mmol, 1.0 eq), 1-(prop-2-yn-1-yl)pyrrolidine (170 mg, 1.56 mmol, 3.0 eq), Pd(PPh3)2Cl2 (73.0 mg, 0.104 mmol, 0.20 eq), CuI (19.8 mg, 0.104 mmol, 0.20 eq), and TEA (10 mL). The system was fully purged with N2, and the reaction was stirred at 90°C. After TLC showed completion of the reaction, the organic solvent was evaporated under reduced pressure, and the product was washed sequentially with saturated NaHCO3 and saturated NaCl solutions, extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 203 mg of a white solid.

[0079] (6) Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(tetrahydro-1H-pyrrol-1-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (2)

[0080] To a round-bottom flask were added intermediate 56 (200 mg, 0.434 mmol, 1.0 eq), hydrazine hydrate (40%-50%, calculated as N2H4) (0.896 g), and EtOH (2 mL) in sequence. The system was fully replaced with N2, and the reaction was stirred at 80°C. After TLC showed completion of the reaction, the organic solvent was evaporated under reduced pressure, and the mixture was washed sequentially with saturated NaHCO3 solution and saturated NaCl solution, extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 130 mg of a white solid.

[0081] 1 H NMR (400MHz, DMSO-d6) δ14.13(s,1H),7.72(d,J=8.4Hz,1H),7.60–7.47(m,4H),7.18(s,1H),7.03(d,J=8.4Hz ,1H),6.89(s,1H),5.33(s,2H),3.85(s,2H),2.61–2.55(m,4H),1.79–1.65(m,4H); ESI-HRMS:476.1337[M+H] + .

[0082] Example 2: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(3-(morpholin-4-yl)prop-1-ynyl)phenol (22)

[0083] (1) Synthesis of 4-propyne-1-morpholine (51)

[0084] To a round-bottom flask, 3-bromopropyne (500 mg, 4.20 mmol, 1.0 eq), morpholine (915 mg, 10.5 mmol, 2.5 eq), K2CO3 (1.40 g, 10.5 mmol, 2.5 eq), and THF (8 mL) were added sequentially. The system was fully purged with N2, and the reaction was stirred at 70°C. After TLC showed the reaction was complete, the organic solvent was evaporated under reduced pressure, and the mixture was washed sequentially with saturated NaHCO3 solution and saturated NaCl solution, extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain 324 mg of a colorless oil, which was used directly in the subsequent coupling reaction.

[0085] (2) Synthesis of 7-(((4-chlorophenyl)methyl)oxy)-8-(3-(morpholin-4-yl)prop-1-ynyl)-2-(trifluoromethyl)-4H-chromen-4-one (57)

[0086] To a round-bottom flask were added intermediate 55 (250 mg, 0.463 mmol, 1.0 eq), 4-propyne-1-morpholine (173 mg, 1.39 mmol, 3.0 eq), Pd(PPh3)2Cl2 (64.0 mg, 0.0926 mmol, 0.20 eq), CuI (17.6 mg, 0.0926 mmol, 0.20 eq), and TEA (10 mL). The system was fully purged with N2, and the reaction was stirred at 90°C. After TLC showed the reaction was complete, the organic solvent was evaporated under reduced pressure, and the product was washed with saturated NaHCO3 solution and saturated NaCl solution, extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 212 mg of a white solid.

[0087] (3) Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(3-(morpholin-4-yl)prop-1-ynyl)phenol (22)

[0088] To a round-bottom flask were added intermediate 57 (200 mg, 0.419 mmol, 1.0 eq), methylhydrazine sulfate (472 mg, 2.52 mmol, 6.0 eq), NaOH (201 mg, 5.03 mmol, 12 eq), and EtOH (6 mL). The system was fully purged with N2, and the reaction was stirred at 80°C. After TLC indicated completion of the reaction, the organic solvent was evaporated under reduced pressure, and the mixture was washed sequentially with saturated NaHCO3 solution and saturated NaCl solution, extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 101 mg of a white solid.

[0089] 1H NMR (500MHz, DMSO-d6) δ7.60–7.47(m,4H),7.39(d,J=8.0Hz,1H),7.02(d,J=8.0Hz,1H),6.95(s,1H),6.94(s ,1H),5.34(s,2H),3.86(s,3H),3.67(s,2H),3.60–3.54(m,4H),2.47–2.40(m,4H); ESI-HRMS:506.1435[M+H] + .

[0090] Example 3: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(dimethylamino)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (1)

[0091] Compound 1 was synthesized by referring to Example 1. Intermediate 55 and 1-dimethylamine-2-propyne were subjected to Sonagashira coupling according to the method of step (5) in Example 1, and then hydrazinolysis was performed according to the method of step (6). The prepared compound 1 was a white solid.

[0092] 1 H NMR(600MHz,DMSO-d6)δ14.09(s,1H),7.72(d,J=8.4Hz,1H),7.57–7.47(m,4H),7.16(s,1H),7.0 2(d,J=8.4Hz,1H),6.90(s,1H),5.32(s,2H),3.68(s,2H),2.24(s,6H); ESI-HRMS:450.1178[M+H] + .

[0093] Example 4: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(piperidin-1-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (3)

[0094] Compound 3 was synthesized by referring to Example 1, except that the tetrahydropyrrole in step (1) of Example 1 was replaced with piperidine. The resulting product was subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 3 was obtained as a white solid.

[0095] 1H NMR (400MHz, DMSO-d6) δ14.09(s,1H),7.71(d,J=8.4Hz,1H),7.58–7.46(m,4H),7.17(s,1H),7.02(d,J=8.4Hz,1H),6.8 7(s,1H),5.32(s,2H),3.72(s,2H),2.49–2.38(m,4H),1.55–1.45(m,4H),1.41–1.30(m,2H); ESI-HRMS:490.1488[M+H] + .

[0096] Example 5: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(morpholin-4-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (4)

[0097] Compound 4 was synthesized by referring to Example 1, except that the tetrahydropyrrole in step (1) of Example 1 was replaced with morpholine. The resulting product was then subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 4 was obtained as a white solid.

[0098] 1 H NMR (600MHz, DMSO-d6) δ14.08(s,1H),7.72(d,J=8.4Hz,1H),7.58–7.46(m,4H),7.18(s,1H),7.02(d,J=8.4Hz,1H ),6.92(s,1H),5.32(s,2H),3.76(s,2H),3.59(t,J=4.8Hz,4H),2.49(t,J=4.8Hz,4H); ESI-HRMS:492.1282[M+H] + .

[0099] Example 6: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(4-hydroxypiperidin-1-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (5)

[0100] Compound 5 was synthesized by referring to Example 1, except that the tetrahydropyrrole in step (1) of Example 1 was replaced with 4-hydroxypiperidine. The resulting product was then subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 5 was obtained as a white solid.

[0101] 1H NMR(600MHz,DMSO-d6)δ14.10(s,1H),7.71(d,J=8.4Hz,1H),7.57–7.47(m ,4H),7.18(s,1H),7.01(d,J=8.4Hz,1H),6.87(s,1H),5.32(s,2H),4.55(d ,J=3.6Hz,1H),3.73(s,2H),3.46–3.42(m,1H),2.82–2.73(m,2H),2.24–2. 14(m,2H),1.75–1.66(m,2H),1.45–1.37(m,2H); ESI-HRMS:506.1448[M+H] + .

[0102] Example 7: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(4-methylpiperazin-1-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (6)

[0103] Compound 6 was synthesized by referring to Example 1, except that the tetrahydropyrrole in step (1) of Example 1 was replaced with N-methylpiperazine. The resulting product was then subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 6 was obtained as a white solid.

[0104] 1 H NMR (600MHz, DMSO-d6) δ14.10(s,1H),7.72(d,J=8.4Hz,1H),7.58–7.46(m,4H),7.18(s,1H),7.01(d,J=8. 4Hz,1H),6.89(s,1H),5.32(s,2H),3.75(s,2H),2.54–2.24(m,8H),2.14(s,3H); ESI-HRMS:505.1593[M+H] + .

[0105] Example 8: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(4-(morpholin-4-yl)piperidin-1-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (7)

[0106] Compound 7 was synthesized with reference to Example 1. The tetrahydropyrrole in step (1) of Example 1 was replaced with 4-(4-piperidinyl)morpholine. The resulting product was then subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 7 was obtained as a white solid.

[0107] 1 H NMR(600MHz,DMSO-d6)δ14.12(s,1H),7.73(d,J=8.4Hz,1H),7.57–7.46(m, 4H),7.19(s,1H),7.02(d,J=8.4Hz,1H),6.87(s,1H),5.31(s,2H),3.72(s,2 H),3.52(t,J=4.8Hz,4H),2.97–2.90(m,2H),2.38(t,J=4.8Hz,4H),2.06–1 .98(m,3H),1.73–1.66(m,2H),1.42–1.33(m,2H); ESI-HRMS:575.2011[M+H] + .

[0108] Example 9: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(4-(oxetan-3-yl)piperazin-1-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (8)

[0109] Compound 8 was synthesized with reference to Example 1. The tetrahydropyrrole in step (1) of Example 1 was replaced with 1-(3-oxetanyl)piperazine. The resulting product was subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 8 was obtained as a white solid.

[0110] 1 H NMR(600MHz,DMSO-d6)δ14.10(s,1H),7.87(d,J=8.4Hz,1H),7.58–7.49(m,4H),7.44(s,1H),7.35(s,1H),7.09( d,J=8.4Hz,1H),5.35(s,2H),4.80–4.73(m,2H),4.71–4.59(m,4H),4.37–3.66(m,9H); ESI-HRMS:547.1703[M+H] + .

[0111] Example 10: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(6-aza-2-oxaspiro[3.3]hept-6-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (9)

[0112] Compound 9 was synthesized by referring to Example 1, except that the tetrahydropyrrole in step (1) of Example 1 was replaced with 2-oxa-6-aza-spiro[3,3]heptane. The resulting product was then subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 9 was obtained as a white solid.

[0113] 1 H NMR (400MHz, DMSO-d6) δ14.09(s,1H),7.70(d,J=8.4Hz,1H),7.59–7.46(m,4H),7.16(s,1H),7.01(d,J=8.4Hz ,1H),6.83(s,1H),5.32(s,2H),4.63–4.58(m,4H),3.71(s,2H),3.45–3.39(m,4H); ESI-HRMS:504.1278[M+H] + .

[0114] Example 11: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(3-(6-(((2-methylprop-2-yl)oxy)carbonyl)-2,6-diazaspiro[3.3]hept-2-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (10)

[0115] Compound 10 was synthesized with reference to Example 1. The tetrahydropyrrole in step (1) of Example 1 was replaced with tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate. The resulting product was then subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 10 was obtained as a white solid.

[0116] ESI-MS:603.2[M+H] + .

[0117] Example 12: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(4-hydroxybut-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (11)

[0118] Compound 11 was synthesized by referring to Example 1. Intermediate 55 and but-3-yn-1-ol were subjected to Sonagashira coupling according to the method of step (5) in Example 1, and then hydrazinolysis was performed according to the method of step (6). The prepared compound 11 was a white solid.

[0119] 1 H NMR (600MHz, DMSO-d6) δ14.04(s,1H),7.68(d,J=8.4Hz,1H),7.58–7.46(m,4H),7.20(s,1H),6.99(d,J=8.4Hz,1H),6. 78(s,1H),5.31(s,2H),4.86(t,J=5.4Hz,1H),3.81(q,J=6.0Hz,2H),3.00(t,J=6.6Hz,2H); ESI-HRMS:437.0868[M+H] + .

[0120] Example 13: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(4-(piperidin-1-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (12)

[0121] Compound 12 was synthesized with reference to Example 1. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with piperidine. The resulting product was subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 12 was obtained as a white solid.

[0122] 1 H NMR (600MHz, DMSO-d6) δ14.05(s,1H),7.68(d,J=8.4Hz,1H),7.56–7.46(m,4H),7.21(s,1H),6.98(d,J=8.4Hz,1H),6.75(s,1H),5.30( s,2H),3.00(t,J=7.2Hz,2H),2.67(t,J=7.2Hz,2H),2.46–2.34(m,4H),1.53–1.43(m,4H),1.40–1.33(m,2H); ESI-HRMS:504.1646[M+H] + .

[0123] Example 14: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(4-(morpholin-4-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (13)

[0124] Compound 13 was synthesized with reference to Example 1. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with morpholine. The resulting product was subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 13 was obtained as a white solid.

[0125] 1 H NMR (400MHz, DMSO-d6) δ14.04(s,1H),7.68(d,J=8.4Hz,1H),7.58–7.44(m,4H),7.20(s,1H),6.99(d,J=8.4Hz,1H),6.78(s,1H) ,5.31(s,2H),3.57(t,J=4.8Hz,4H),3.03(t,J=7.2Hz,2H),2.71(t,J=7.2Hz,2H),2.48–2.39(m,4H); ESI-HRMS:506.1441[M+H] + .

[0126] Example 15: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(4-(4-hydroxypiperidin-1-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (14)

[0127] Compound 14 was synthesized with reference to Example 1. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with 4-hydroxypiperidine. The resulting product was subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 14 was obtained as a white solid.

[0128] 1 H NMR(600MHz,DMSO-d6)δ14.03(s,1H),7.68(d,J=8.4Hz,1H),7.57–7.46(m,4H),7 .20(s,1H),6.98(d,J=8.4Hz,1H),6.76(s,1H),5.30(s,2H),4.64–4.45(m,1H),3. 50–3.41(m,1H),3.00(t,J=7.4Hz,2H),2.84–2.74(m,2H),2.69(t,J=7.4Hz,2H),2 .16–2.04(m,2H),1.75–1.67(m,2H),1.42–1.33(m,2H); ESI-HRMS:520.1591[M+H]+ .

[0129] Example 16: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(4-(4-methylpiperazin-1-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (15)

[0130] Compound 15 was synthesized with reference to Example 1. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with N-methylpiperazine. The resulting product was subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 15 was obtained as a white solid.

[0131] 1 H NMR (400MHz, DMSO-d6) δ14.04(s,1H),7.67(d,J=8.4Hz,1H),7.59–7.44(m,4H),7.19(s,1H),6.99(d,J=8.4Hz,1H),6.77(s ,1H),5.31(s,2H),3.01(t,J=7.4Hz,2H),2.70(t,J=7.4Hz,2H),2.48–2.24(m,8H),2.14(s,3H); ESI-HRMS:519.1755[M+H] + .

[0132] Example 17: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(4-(6-aza-2-oxaspiro[3.3]hept-6-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (16)

[0133] Compound 16 was synthesized with reference to Example 1. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with 2-oxa-6-aza-spiro[3,3]heptane. The resulting product was subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 16 was obtained as a white solid.

[0134] 1H NMR(600MHz,DMSO-d6)δ14.06(s,1H),7.67(d,J=8.4Hz,1H),7.56–7.46(m,4H),7.17(s,1H),6.98(d,J=8.4Hz,1H),6.7 3(s,1H),5.30(s,2H),4.57(s,4H),3.27(s,4H),2.81(t,J=7.0Hz,2H),2.71(t,J=7.0Hz,2H); ESI-HRMS:518.1438[M+H] + .

[0135] Example 18: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(5-hydroxypent-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (17)

[0136] Compound 17 was synthesized by referring to Example 1. Intermediate 55 and pent-4-yn-1-ol were subjected to Sonagashira coupling according to the method of step (5) in Example 1, and then hydrazinolysis was performed according to the method of step (6). The prepared compound 17 was a white solid.

[0137] 1 H NMR (600MHz, DMSO-d6) δ14.05(s,1H),7.67(d,J=8.4Hz,1H),7.57–7.45(m,4H),7.17(s,1H),6.99(d,J=8.4Hz,1H),6.74(s,1H) ,5.31(s,2H),4.60(t,J=5.4Hz,1H),3.51(q,J=6.0Hz,2H),2.89(t,J=7.6Hz,2H),1.92–1.85(m,2H); ESI-HRMS:451.1014[M+H] + .

[0138] Example 19: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(5-hydroxy-5-methylhex-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (18)

[0139] Compound 18 was synthesized by referring to Example 1. Intermediate 55 and 2-methylhex-5-yn-2-ol were subjected to Sonagashira coupling according to the method of step (5) in Example 1, and then hydrazinolysis was performed according to the method of step (6). The prepared compound 18 was a white solid.

[0140] 1H NMR(600MHz,DMSO-d6)δ14.04(s,1H),7.66(d,J=8.4Hz,1H),7.57–7.46(m,4H),7.18(s,1H),6.98(d,J=8.4Hz,1H), 6.72(s,1H),5.31(s,2H),4.39(s,1H),2.94–2.86(m,2H),1.88–1.81(m,2H),1.18(s,6H); ESI-HRMS:479.1339[M+H] + .

[0141] Example 20: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(5-(piperidin-1-yl)pent-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (19)

[0142] Compound 19 was synthesized with reference to Example 1. In Example 1, 3-bromopropyne in step (1) was replaced with 5-bromopent-1-yne, and tetrahydropyrrole was replaced with piperidine. The resulting product was subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 19 was obtained as a white solid.

[0143] 1 H NMR (600MHz, DMSO-d6) δ14.05(s,1H),7.67(d,J=8.4Hz,1H),7.56–7.45(m,4H),7.17(s,1H),6.98(d,J=8.4Hz,1H),6.73(s,1H),5.31 (s,2H),2.85(t,J=7.8Hz,2H),2.40–2.24(m,6H),1.91–1.84(m,2H),1.51–1.45(m,4H),1.40–1.33(m,2H); ESI-HRMS:518.1815[M+H] + .

[0144] Example 21: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(5-(morpholin-4-yl)pent-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (20)

[0145] Compound 20 was synthesized with reference to Example 1. In Example 1, 3-bromopropyne in step (1) was replaced with 5-bromopent-1-yne, and tetrahydropyrrole was replaced with morpholine. The resulting product was then subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 20 was obtained as a white solid.

[0146] 1 H NMR(600MHz,DMSO-d6)δ14.06(s,1H),7.68(d,J=8.4Hz,1H),7.56–7.46(m,4H),7.18(s,1H),6.99(d,J=8.4Hz,1H),6.74(s,1 H),5.31(s,2H),3.56(t,J=4.8Hz,4H),2.87(t,J=7.5Hz,2H),2.42–2.29(m,6H),1.92–1.86(m,2H); ESI-HRMS:520.1611[M+H] + .

[0147] Example 22: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-2-(5-(4-methylpiperazin-1-yl)pent-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (21)

[0148] Compound 21 was synthesized with reference to Example 1. In Example 1, 3-bromopropyne in step (1) was replaced with 5-bromopent-1-yne, and tetrahydropyrrole was replaced with N-methylpiperazine. The resulting product was then subjected to Sonagashira coupling with intermediate 55 according to the method of step (5), followed by hydrazinolysis according to the method of step (6). Compound 21 was obtained as a white solid.

[0149] 1 H NMR(600MHz,DMSO-d6)δ14.06(s,1H),7.67(d,J=8.4Hz,1H),7.58–7.44(m,4H),7.17(s,1H),6.98(d,J=8.4Hz,1H) ,6.73(s,1H),5.31(s,2H),2.85(t,J=7.6Hz,2H),2.50–2.09(m,13H),1.92–1.83(m,2H); ESI-HRMS:533.1913[M+H] + .

[0150] Example 23: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(3-(4-methylpiperazin-1-yl)prop-1-ynyl)phenol (23)

[0151] Compound 23 was synthesized by referring to Example 2, except that the morpholine in step (1) of Example 2 was replaced with N-methylpiperazine. The resulting product was then subjected to Sonagashira coupling with intermediate 55 according to the method of step (2), followed by methylhydrazine hydrolysis according to the method of step (3). Compound 23 was obtained as a white solid.

[0152] 1 H NMR (400MHz, DMSO-d6) δ7.73(d,J=8.0Hz,1H),7.58–7.46(m,4H),7.34(s,1H),6.96(d,J=8.0Hz,1H),6.85(s,1H),5.29(s,2H ),4.05(s,3H),3.73(s,2H),3.28–3.26(m,2H),2.70–2.65(m,2H),2.35–2.30(m,4H),2.13(s,3H); ESI-HRMS:519.1758[M+H] + .

[0153] Example 24: Synthesis of 3-(((4-chlorophenyl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(5-hydroxypent-1-ynyl)phenol (24)

[0154] Compound 24 was synthesized by referring to Example 2. Intermediate 55 and pent-4-yn-1-ol were subjected to Sonagashira coupling according to the method of step (2), and then methylhydrazine hydrolysis was performed according to the method of step (3). The prepared compound 24 was a white solid.

[0155] 1 H NMR (600MHz, DMSO-d6) δ7.69(d,J=8.4Hz,1H),7.56–7.45(m,4H),7.33(s,1H),6.93(d,J=8.4Hz,1H),6.69(s,1H),5.28(s,2H) ,4.53(t,J=5.4Hz,1H),4.05(s,3H),3.51(q,J=6.0Hz,2H),2.87(t,J=7.2Hz,2H),1.91–1.84(m,2H); ESI-HRMS:465.1200[M+H] + .

[0156] Example 25: Synthesis of 3-(((4-fluorophenyl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(3-(morpholin-4-yl)prop-1-ynyl)phenol (25)

[0157] Compound 25 was synthesized with reference to Examples 1 and 2. Intermediate 54 and 4-fluorobenzyl chloride were first subjected to a nucleophilic substitution reaction according to the method of step (4) in Example 1. The resulting product was then subjected to Sonagashira coupling with intermediate 51 according to the method of step (2) in Example 2. The product was then subjected to methylhydrazine hydrolysis according to the method of step (3) in Example 2 to obtain compound 25 as a white solid.

[0158] ESI-MS:490.2[M+H] + .

[0159] Example 26: Synthesis of 3-(((4-fluorophenyl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(3-(2-oxyylidenepiperidin-1-yl)prop-1-ynyl)phenol (26)

[0160] (1) Synthesis of 1-(prop-2-ynyl)piperidin-2-one

[0161] 2-Piperidone (1.04 g, 10.5 mmol, 2.5 eq) and 60% NaH (0.420 g, 10.5 mmol, 2.5 eq) were added to a three-necked flask in sequence. The system was fully purged with N2, and anhydrous THF (10 mL) was injected into the system. The mixture was stirred in an ice bath for 0.5 h. 3-Bromopropyne (500 mg, 4.20 mmol, 1.0 eq) was then injected into the system and stirred at room temperature. After TLC showed that the reaction was complete, the reaction was quenched with ice water and extracted with EA. The organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain 392 mg of a colorless oil, which was used directly in the subsequent coupling reaction.

[0162] (2) Synthesis of Compound 26

[0163] Compound 26 was synthesized with reference to Examples 1 and 2. Intermediate 54 and 4-fluorobenzyl chloride were subjected to a nucleophilic substitution reaction according to the method of step (4) in Example 1. The resulting product was then subjected to Sonagashira coupling with 1-(prop-2-ynyl)piperidin-2-one according to the method of step (2) in Example 2, followed by methylhydrazine hydrolysis according to the method of step (3) in Example 2 to obtain compound 26 as a white solid.

[0164] ESI-MS:502.2[M+H] + .

[0165] Example 27: Synthesis of 3-(((4-fluorophenyl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(3-(2-methyl-2,6-diazaspiro[3.4]octan-6-yl)prop-1-ynyl)phenol (27)

[0166] The synthesis of compound 27 was carried out with reference to Examples 1 and 2. The tetrahydropyrrole in step (1) of Example 1 was replaced with 2-methyl-6-(prop-2-ynyl)-2,6-diazaspiro[3.4]octane to obtain the corresponding alkynyl-containing fragment. Intermediate 54 was subjected to a nucleophilic substitution reaction with 4-fluorobenzyl chloride according to the method of step (4) of Example 1. The obtained product was then subjected to Sonagashira coupling with the above-mentioned alkynyl-containing fragment according to the method of step (2) of Example 2. The product was then subjected to methylhydrazine hydrolysis according to the method of step (3) of Example 2 to obtain the compound 27. The prepared compound 27 was obtained as a white solid.

[0167] ESI-MS:529.2[M+H] + .

[0168] Example 28: Synthesis of 3-(((6-chloropyridin-3-yl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(3-(morpholin-4-yl)prop-1-ynyl)phenol (28)

[0169] Compound 28 was synthesized with reference to Examples 1 and 2. Intermediate 54 and 2-chloro-5-chloromethylpyridine were subjected to a nucleophilic substitution reaction according to the method of step (4) in Example 1. The resulting product was then subjected to Sonagashira coupling with intermediate 51 according to the method of step (2) in Example 2, followed by methylhydrazine hydrolysis according to the method of step (3) in Example 2 to obtain compound 28 as a white solid.

[0170] ESI-MS:507.1[M+H] + .

[0171] Example 29: Synthesis of 3-(((6-chloropyridin-3-yl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(3-(4-methylpiperazin-1-yl)prop-1-ynyl)phenol (29)

[0172] The synthesis of compound 29 was carried out with reference to Examples 1 and 2. The tetrahydropyrrole in step (1) of Example 1 was replaced with N-methylpiperazine to produce the corresponding alkynyl-containing fragment. Intermediate 54 was subjected to a nucleophilic substitution reaction with 2-chloro-5-chloromethylpyridine according to the method of step (4) of Example 1. The resulting product was then subjected to Sonagashira coupling with the alkynyl-containing fragment according to the method of step (2) of Example 2. The product was then subjected to methylhydrazine hydrolysis according to the method of step (3) of Example 2 to produce compound 29. The resulting compound 29 was obtained as a white solid.

[0173] ESI-MS:520.2[M+H] + .

[0174] Example 30: Synthesis of 3-(((6-chloropyridin-3-yl)methyl)oxy)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)-2-(3-(7-methyl-2,7-diazaspiro[4.4]non-2-yl)prop-1-ynyl)phenol (30)

[0175] The synthesis of compound 30 was carried out with reference to Examples 1 and 2. The tetrahydropyrrole in step (1) of Example 1 was replaced with 2-methyl-2,7-diazaspiro[4.4]nonane to obtain the corresponding alkynyl-containing fragment. Intermediate 54 was subjected to a nucleophilic substitution reaction with 2-chloro-5-chloromethylpyridine according to the method of step (4) of Example 1. The resulting product was then subjected to Sonagashira coupling with the above-mentioned alkynyl-containing fragment according to the method of step (2) of Example 2, followed by methylhydrazine hydrolysis according to the method of step (3) of Example 2 to obtain the compound 30. The prepared compound 30 was obtained as a white solid.

[0176] ESI-MS:560.2[M+H] + .

[0177] Example 31: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-(morpholin-4-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (31)

[0178] (1) Synthesis of 8-(4-chloro-3-(trifluoromethyl)phenyl)-7-hydroxy-2-(trifluoromethyl)-4H-chromen-4-one (58)

[0179] To a round-bottom flask were added intermediate 54 (1.50 g, 4.21 mmol, 1.0 eq), 4-chloro-3-(trifluoromethyl)phenylboronic acid (944 mg, 4.21 mmol, 1.0 eq), Pd(dppf)Cl2 (309 mg, 0.421 mmol, 0.10 eq), Na2CO3 (893 mg, 8.43 mmol, 2.0 eq), and a mixed solvent (toluene:EtOH:water = 5:1:2, 16 mL). The atmosphere was fully purged with N2 and the reaction was stirred at 100°C. After TLC indicated completion of the reaction, the organic solvent was evaporated under reduced pressure, and the product was washed sequentially with saturated NH4Cl solution and saturated NaCl solution, extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 852 mg of a white solid.

[0180] (2) Synthesis of 8-(4-chloro-3-(trifluoromethyl)phenyl)-7-trifluoromethanesulfonate-2-(trifluoromethyl)-4H-chromen-4-one (59)

[0181] To a round-bottom flask were added intermediate 58 (800 mg, 1.96 mmol, 1.0 eq), pyridine (620 mg, 7.84 mmol, 4.0 eq), and DCM (8 mL). Trifluoromethanesulfonic anhydride (829 mg, 2.94 mmol, 1.5 eq) was added dropwise under ice-cooling. The atmosphere was fully purged with nitrogen and the reaction was stirred at room temperature. After TLC indicated completion of the reaction, water was added to quench the reaction. The pH was adjusted to 7-8 with saturated NaHCO₃ solution under ice-cooling, and the mixture was washed with saturated NaCl solution and extracted with EA. The organic phases were combined, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to afford 963 mg of a white solid.

[0182] (3) Synthesis of 8-(4-chloro-3-(trifluoromethyl)phenyl)-7-(3-(morpholin-4-yl)prop-1-ynyl)-2-(trifluoromethyl)-4H-chromen-4-one (60)

[0183] To a round-bottom flask were added intermediate 59 (300 mg, 0.556 mmol, 1.0 eq), intermediate 51 (209 mg, 1.67 mmol, 3.0 eq), Pd(PPh3)2Cl2 (78.0 mg, 0.111 mmol, 0.20 eq), CuI (21.2 mg, 0.111 mmol, 0.20 eq), and TEA (10 mL). The system was fully purged with N2, and the reaction was stirred at 90°C. After TLC showed completion of the reaction, the organic solvent was evaporated under reduced pressure, and the product was washed with saturated NaHCO3 solution and saturated NaCl solution, extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 241 mg of a white solid.

[0184] (4) Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-(morpholin-4-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (31)

[0185] Intermediate 60 (200 mg, 0.388 mmol, 1.0 eq), hydrazine hydrate (40%-50%, calculated as N2H4) (0.804 g), and EtOH (4 mL) were added sequentially to a round-bottom flask. The system was fully replaced with N2, and the reaction was stirred at 80°C. After TLC showed the reaction was complete, the organic solvent was evaporated under reduced pressure, and the mixture was washed sequentially with saturated NaHCO3 solution and saturated NaCl solution, extracted with EA, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 108 mg of a white solid.

[0186] 1 H NMR (600MHz, DMSO-d6) δ7.82(d,J=7.8Hz,1H),7.78(d,J=2.4Hz,1H),7.74(d,J=7.8Hz,1H),7.69–7.66( m,1H),7.22–7.14(m,2H),3.49–3.44(m,4H),3.32(s,2H),2.16–2.03(m,4H); ESI-HRMS:530.1046[M+H] + .

[0187] Example 32: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-(4-methylpiperazin-1-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (32)

[0188] Compound 32 was synthesized with reference to Examples 1 and 31. The tetrahydropyrrole in step (1) of Example 1 was replaced with N-methylpiperazine. The resulting product was then subjected to Sonagashira coupling with intermediate 59 according to the method of step (3) of Example 31, followed by hydrazinolysis according to the method of step (4). Compound 32 was obtained as a white solid.

[0189] 1H NMR(600MHz,DMSO-d6)δ11.75(s,1H),7.79–7.76(m,2H),7.70(d,J=7.8Hz,1H),7.68–7.65(m,1H) ),7.14(s,1H),7.12(d,J=8.4Hz,1H),3.29(s,2H),2.42–2.08(m,11H); ESI-HRMS:543.1369[M+H] + .

[0190] Example 33: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(piperidin-1-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (33)

[0191] Compound 33 was synthesized with reference to Examples 1 and 31. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with piperidine. The resulting product was subjected to Sonagashira coupling with intermediate 59 according to the method of step (3) in Example 31, followed by hydrazinolysis according to the method of step (4). Compound 33 was obtained as a white solid.

[0192] 1 H NMR(600MHz,DMSO-d6)δ7.82–7.76(m,2H),7.70–7.66(m,2H),7.13(s,1H),7.08(d,J=7.8Hz,1H),2.4 0(t,J=7.2Hz,2H),2.35–2.26(m,6H),1.46–1.42(m,4H),1.38–1.31(m,2H); ESI-HRMS:542.1408[M+H] + .

[0193] Example 34: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(morpholin-4-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (34)

[0194] Compound 34 was synthesized with reference to Examples 1 and 31. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with morpholine. The resulting product was subjected to Sonagashira coupling with intermediate 59 according to the method of step (3) in Example 31, followed by hydrazinolysis according to the method of step (4). Compound 34 was obtained as a white solid.

[0195] 1H NMR(600MHz,DMSO-d6)δ13.65(s,1H),9.54(s,1H),7.83–7.79(m,2H),7.71–7.66(m,2H),7.21–7.12(m,2H),3.5 0(t,J=4.8Hz,4H),2.39(t,J=7.2Hz,2H),2.26(t,J=4.8Hz,4H),2.22(t,J=7.2Hz,2H); ESI-HRMS:544.1201[M+H] + .

[0196] Example 35: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4-methylpiperazin-1-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (35)

[0197] Compound 35 was synthesized with reference to Examples 1 and 31. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with N-methylpiperazine. The resulting product was subjected to Sonagashira coupling with intermediate 59 according to the method of step (3) in Example 31, followed by hydrazinolysis according to the method of step (4). Compound 35 was obtained as a white solid.

[0198] 1 H NMR (600MHz, DMSO-d6) δ7.81(s,1H),7.78(d,J=7.8Hz,1H),7.69(d,J=9.0Hz,1H),7.66(d,J= 8.4Hz,1H),7.11(s,1H),7.06(d,J=8.4Hz,1H),2.44–2.20(m,15H); ESI-HRMS:557.1519[M+H] + .

[0199] Example 36: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4-(oxetan-3-yl)piperazin-1-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (36)

[0200] Compound 36 was synthesized with reference to Examples 1 and 31. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with 1-(3-oxetanyl)piperazine. The resulting product was subjected to Sonagashira coupling with intermediate 59 according to the method of step (3) in Example 31, followed by hydrazinolysis according to the method of step (4). Compound 36 was obtained as a white solid.

[0201] 1 H NMR (600MHz, DMSO-d6) δ7.83–7.78(m,2H),7.71–7.65(m,2H),7.19–7.11(m,2H),4.50(t,J=6.0 Hz,2H),4.39(t,J=6.0Hz,2H),3.37–3.34(m,1H),2.41–2.04(m,12H); ESI-HRMS:599.1627[M+H] + .

[0202] Example 37: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(6-aza-2-oxaspiro[3.3]hept-6-yl)but-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (37)

[0203] Compound 37 was synthesized with reference to Examples 1 and 31. In Example 1, 3-bromopropyne in step (1) was replaced with 4-bromobut-1-yne, and tetrahydropyrrole was replaced with 2-oxa-6-aza-spiro[3,3]heptane. The resulting product was subjected to Sonagashira coupling with intermediate 59 according to the method of step (3) in Example 31, followed by hydrazinolysis according to the method of step (4). Compound 37 was obtained as a white solid.

[0204] 1 H NMR(600MHz,DMSO-d6)δ7.83–7.77(m,2H),7.71–7.67(m,2H),7.14(s,1H),7.10(d,J =7.8Hz,1H),4.55(s,4H),3.21(s,4H),2.30–2.19(m,4H); ESI-HRMS:556.1220[M+H] + .

[0205] Example 38: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(5-hydroxy-5-methylhex-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (38)

[0206] Compound 38 was synthesized by referring to Example 31. Intermediate 59 and 2-methylhex-5-yn-2-ol were subjected to Sonagashira coupling according to the method of step (3) in Example 31, and then hydrazinolysis was carried out according to the method of step (4). The prepared compound 38 was a white solid.

[0207] 1H NMR(600MHz,DMSO-d6)δ13.77(s,1H),9.34(s,1H),7.83–7.78(m,2H),7.72–7.62(m,2H),7.18–7.0 7(m,2H),4.20(s,1H),2.27–2.21(m,2H),1.35–1.29(m,2H),0.98(s,6H); ESI-HRMS:517.1101[M+H] + .

[0208] Example 39: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-(morpholin-4-yl)prop-1-ynyl)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)phenol (39)

[0209] The synthesis of compound 39 was carried out by referring to Example 2, wherein intermediate 60 was subjected to methylhydrazine hydrolysis according to the method of step (3) in Example 2. The prepared compound 39 was a white solid.

[0210] 1 H NMR(400MHz,DMSO-d6)δ9.24(s,1H),7.85–7.78(m,2H),7.73–7.68(m,1H),7.35–7.17(m,2H),6.81(s, 1H),3.78(s,3H),3.47(t,J=4.4Hz,4H),3.33(s,2H),2.10(t,J=4.4Hz,4H); ESI-HRMS:544.1215[M+H] + .

[0211] Example 40: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-(4-methylpiperazin-1-yl)prop-1-ynyl)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)phenol (40)

[0212] Compound 40 was synthesized with reference to Examples 2 and 31. The morpholine in step (1) of Example 2 was replaced with N-methylpiperazine. The resulting product was subjected to Sonagashira coupling with intermediate 59 according to the method of step (3) of Example 31, followed by methylhydrazine hydrolysis according to the method of step (3) of Example 2. Compound 40 was obtained as a white solid.

[0213] 1H NMR(400MHz,DMSO-d6)δ9.21(s,1H),7.83–7.77(m,2H),7.70–7.65(m,1H),7.33–7.14(m, 2H),6.80(s,1H),3.77(s,3H),3.29(s,2H),2.32–2.06(m,11H); ESI-HRMS:557.1528[M+H] + .

[0214] Example 41: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(5-hydroxypent-1-ynyl)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)phenol (41)

[0215] Compound 41 was synthesized with reference to Examples 2 and 31. Intermediate 59 and pent-4-yn-1-ol were subjected to Sonagashira coupling according to the method of step (3) in Example 31, followed by methylhydrazine hydrolysis according to the method of step (3) in Example 2. Compound 41 was obtained as a white solid.

[0216] 1 H NMR(400MHz,DMSO-d6)δ9.21(s,1H),7.86–7.76(m,2H),7.73–7.68(m,1H),7.30–7.10(m,2H),6.80(s,1H),4.41(t,J =5.2Hz,1H),3.77(s,3H),3.25(q,J=6.0Hz,2H),2.27(t,J=7.2Hz,2H),1.46–1.37(m,2H); ESI-HRMS:503.0954[M+H] + .

[0217] Example 42: Synthesis of 2-(4-chloro-3-(trifluoromethyl)phenyl)-3-(5-hydroxy-5-methylhex-1-ynyl)-6-(2-methyl-5-(trifluoromethyl)pyrazol-3-yl)phenol (42)

[0218] Compound 42 was synthesized with reference to Examples 2 and 31. Intermediate 59 and 2-methylhex-5-yn-2-ol were subjected to Sonagashira coupling according to the method of step (3) in Example 31, followed by methylhydrazine hydrolysis according to the method of step (3) in Example 2. Compound 42 was obtained as a white solid.

[0219] 1H NMR(500MHz,DMSO-d6)δ9.21(s,1H),7.85–7.79(m,2H),7.73–7.69(m,1H),7.31–7.11(m,2H),6.81(s,1H),4 .21(s,1H),3.78(s,3H),2.26(t,J=8.0Hz,2H),1.35(t,J=8.0Hz,2H),1.00(s,6H); ESI-HRMS:531.1277[M+H] + .

[0220] Example 43: Synthesis of 2-(3,5-bis(trifluoromethyl)phenyl)-3-(3-(morpholin-4-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (43)

[0221] Compound 43 was synthesized with reference to Example 2 and Example 31. 4-Chloro-3-(trifluoromethyl)phenylboronic acid in step (1) of Example 31 was replaced with 3,5-bis(trifluoromethyl)phenylboronic acid for Suzuki coupling. The resulting product was converted to a sulfonate ester by the method of step (2) of Example 31. The resulting sulfonate ester was then subjected to Sonagashira coupling with intermediate 51 by the method of step (3), followed by methylhydrazine hydrolysis by the method of step (3) of Example 2 to obtain compound 43. The prepared compound 43 was obtained as a white solid.

[0222] ESI-MS:578.1[M+H] + .

[0223] Example 44: Synthesis of 2-(3,5-bis(trifluoromethyl)phenyl)-3-(3-(1,1-dioxido-1,2-thiazin-2-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (44)

[0224] The synthesis of compound 44 was carried out with reference to Examples 2, 26, and 31. The 2-piperidone in step (1) of Example 26 was replaced with 1,4-butane sultam to produce the corresponding alkynyl-containing fragment. The 4-chloro-3-(trifluoromethyl)phenylboronic acid in step (1) of Example 31 was replaced with 3,5-bis(trifluoromethyl)phenylboronic acid to produce a Suzuki coupling reaction. The resulting product was converted to a sulfonate according to the method of step (2) of Example 31. The resulting sulfonate was then subjected to Sonagashira coupling with the alkynyl-containing fragment according to the method of step (3) of Example 31, followed by methylhydrazine hydrolysis according to the method of step (3) of Example 2 to produce the compound 44. The prepared compound 44 was obtained as a white solid.

[0225] ESI-MS:626.1[M+H]+ .

[0226] Example 45: Synthesis of 2-(3,5-bis(trifluoromethyl)phenyl)-3-(3-(2-(oxetan-3-yl)-2,7-diazaspiro[3.5]non-7-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (45)

[0227] The synthesis of compound 45 was carried out with reference to Example 2 and Example 31. The morpholine in step (1) of Example 2 was replaced with 2-(oxetan-3-yl)-2,7-diazaspiro[3.5]nonane to obtain the corresponding alkynyl-containing fragment. The 4-chloro-3-(trifluoromethyl)phenylboronic acid in step (1) of Example 31 was replaced with 3,5-bis(trifluoromethyl)phenylboronic acid to carry out Suzuki coupling. The product obtained from the reaction was converted into a sulfonate according to the method of step (2) of Example 31. The obtained sulfonate was then subjected to Sonagashira coupling with the above-mentioned alkynyl-containing fragment according to the method of step (3) of Example 31, and then subjected to methylhydrazine hydrolysis according to the method of step (3) of Example 2 to obtain the compound 45. The prepared compound 45 was obtained as a white solid.

[0228] ESI-MS:673.2[M+H] + .

[0229] Example 46: Synthesis of 2-(6-chloropyridin-3-yl)-3-(3-(morpholin-4-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (46)

[0230] Compound 46 was synthesized with reference to Example 2 and Example 31. 4-Chloro-3-(trifluoromethyl)phenylboronic acid in step (1) of Example 31 was replaced with 2-chloro-5-pyridineboronic acid for Suzuki coupling. The resulting product was converted to a sulfonate ester by the method of step (2) of Example 31. The resulting sulfonate ester was then subjected to Sonagashira coupling with intermediate 51 by the method of step (3) of Example 31, followed by methylhydrazine hydrolysis by the method of step (3) of Example 2 to obtain compound 46 as a white solid.

[0231] ESI-MS:477.1[M+H] + .

[0232] Example 47: Synthesis of 2-(6-chloropyridin-3-yl)-3-(3-(4-methylpiperazin-1-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (47)

[0233] The synthesis of compound 47 was carried out with reference to Examples 2 and 31. The morpholine in step (1) of Example 2 was replaced with N-methylpiperazine to obtain the corresponding alkynyl-containing fragment. The 4-chloro-3-(trifluoromethyl)phenylboronic acid in step (1) of Example 31 was replaced with 2-chloro-5-pyridineboronic acid to carry out Suzuki coupling. The product obtained from the reaction was converted into a sulfonate according to the method of step (2) of Example 31. The obtained sulfonate was then subjected to Sonagashira coupling with the above-mentioned alkynyl-containing fragment according to the method of step (3) of Example 31, and then subjected to methylhydrazine hydrolysis according to the method of step (3) of Example 2 to obtain the obtained compound 47. The prepared compound 47 was obtained as a white solid.

[0234] ESI-MS:490.2[M+H] + .

[0235] Example 48: Synthesis of 2-(6-chloropyridin-3-yl)-3-(3-(6-(oxetan-3-yl)-3,6-diazabicyclo[3.1.1]hept-3-yl)prop-1-ynyl)-6-(5-(trifluoromethyl)-2H-pyrazol-3-yl)phenol (48)

[0236] The synthesis of compound 48 was carried out with reference to Example 2 and Example 31, except that the morpholine in step (1) of Example 2 was replaced with 7-(oxetan-3-yl)-3,7-diazabicyclo[3.1.1]heptane to obtain the corresponding alkynyl-containing fragment. 4-Chloro-3-(trifluoromethyl)phenylboronic acid in step (1) of Example 31 was replaced with 2-chloro-5-pyridineboronic acid to carry out Suzuki coupling. The product obtained from the reaction was converted to a sulfonate according to the method of step (2) of Example 31. The obtained sulfonate was then subjected to Sonagashira coupling with the above-mentioned alkynyl-containing fragment according to the method of step (3) of Example 31, and then subjected to methylhydrazine hydrolysis according to the method of step (3) of Example 2 to obtain the compound 48. The prepared compound 48 was obtained as a white solid.

[0237] ESI-MS:544.2[M+H] + .

[0238] Example 49: Anti-tumor cell proliferation activity and toxicity to normal cells of the compounds of the present invention

[0239] In this example, the antiproliferative activity of the compounds of the present invention and MYCi975 against the prostate cancer cell line PC-3 (in which MYC is overexpressed) was evaluated using the MTT assay, using MYCi975, a recently reported representative small molecule inhibitor that directly targets MYC, as a positive control. Other compounds of the present invention exhibit similar beneficial effects to the compounds listed below, but this should not be construed as indicating that the compounds of the present invention exhibit only the following beneficial effects.

[0240] The test steps for anti-tumor cell proliferation activity are as follows: digest and collect tumor cells, inoculate them in a 96-well culture plate at a certain density, and place them in an incubator (37°C, 5% CO2) overnight. The cells were treated with compound solutions of different concentrations. After the compound acted for 72 hours, the culture medium was discarded, and the cells were gently washed 3 times with PBS. Subsequently, a certain volume of culture medium and MTT solution (5 mg / mL) were added to each well of the culture plate, and the culture was continued for a certain period of time. After the incubation was completed, the 96-well plate was centrifuged at 1500 rpm for 3 minutes, the supernatant of each well was discarded, 100 μL DMSO was added to each well, and the cells were shaken on a cell shaker for 10 minutes. After the crystals were fully dissolved, the absorbance OD value was measured at a wavelength of 490 nm using a multifunctional microplate reader to calculate the inhibition rate, IC 50 The values were obtained by fitting with GraphPad Prism 5 software.

[0241] Table 1 Anti-tumor cell proliferation activity of compounds

[0242]

[0243] In the table above, "++++" represents 0.1-1.0 μM; "+++" represents 1.0-10 μM; "++" represents 10-30 μM; and "+" represents 30-100 μM. Table 1 shows that all of the listed compounds exhibited significant anti-proliferative activity against PC-3 tumor cell lines, with activity superior to or comparable to that of MYCi975. Most of the compounds demonstrated superior anti-tumor cell proliferation activity compared to MYCi975, demonstrating promising application prospects.

[0244] We further evaluated the anti-proliferative activity of the compound on normal human umbilical vein endothelial cells (HUVEC) and human gastric epithelial cells (GES-1) using a similar test method for anti-proliferative activity of tumor cells. The results showed that the compound had no toxicity to the above normal cells, or IC 50 Much higher than its IC for tumor cells 50 , so the compound has excellent safety.

[0245] Example 50: In vivo efficacy of the compounds of the present invention

[0246] As previously mentioned, developing inhibitors directly targeting MYC with in vivo efficacy is currently a technical challenge. To further characterize the potential applications of the compounds of this invention, this example evaluated the in vivo antitumor efficacy of Compound 34 in a male BALB / c-nu nude mouse PC-3 tumor model (MYC overexpression). Other compounds of this invention exhibit similar beneficial effects to Compound 34, but this should not be construed as limiting the compounds of this invention to the following beneficial effects.

[0247] Six-week-old male BALB / c-nu nude mice were randomly divided into a control group and a high (100 mg / kg), medium (75 mg / kg), and low (50 mg / kg) dose group of compound 34, for a total of four groups, with six mice in each group. The nude mice were injected subcutaneously into the right unilateral axilla to bear tumors. After tumor formation, the test compound was administered via intraperitoneal injection, while the control group was given normal saline for 21 consecutive days. After the start of treatment, the long and short diameters of the tumors were measured with a vernier caliper every two days to calculate the tumor volume, and the weight changes of the experimental animals were monitored. After the end of treatment, the mice were sacrificed by cervical dislocation, and the tumor tissue was removed, weighed, and photographed.

[0248] In vivo efficacy studies demonstrated that the compounds of the present invention exhibited significant tumor inhibition at high, medium, and low doses, with statistically significant differences and a clear dose-dependency. At a dose of 100 mg / kg, compound 34 exhibited a tumor inhibition rate of nearly 80%. Furthermore, at all doses, the experimental animals showed no weight loss, and HE staining of vital organs revealed no significant toxicity, further demonstrating the compound's safety.

[0249] Example 51: MYC protein degradation experiment of the compounds of the present invention

[0250] Literature indicates that compounds directly targeting MYC and interfering with the MYC-MAX interaction can induce MYC protein degradation. Based on this, to characterize the direct targeting of MYC and the interference with the MYC-MAX interaction by the compounds of the present invention, this example employed a Western blotting (WB) assay to evaluate the ability of compound 34 to induce intracellular MYC protein degradation. Other compounds of the present invention exhibit similar beneficial effects as compound 34, but this should not be construed as indicating that the compounds of the present invention exhibit only the following beneficial effects.

[0251] PC-3 cells in the logarithmic growth phase were harvested, seeded at a specific density into 6-well plates, and cultured overnight in an incubator. Compound 34 was added at a gradient concentration (1, 2, 5, and 10 μM) (a blank control) and cultured for an additional 24 hours. The cells were then digested, centrifuged, and harvested and washed with PBS. RIPA lysis buffer containing 1% PMSF and 2% phosphatase inhibitors was added, sonicated, and centrifuged. The supernatant was collected for BCA quantification. One-quarter volume of 5× SDS-PAGE buffer was added, mixed, and heated at 95°C for 5 minutes. The sample was then ready for Western blotting analysis. A 12% acrylamide gel was loaded, subjected to electrophoresis, transferred, blocked, and washed, then incubated with the primary antibody and incubated overnight at 4°C. The polyvinylidene fluoride membrane was removed, washed, and placed in an incubation chamber containing the secondary antibody for an additional 1 hour. Any residual secondary antibody incubation solution on the membrane was washed clean and evenly coated with developer for development. ImageJ software was used to calculate grayscale values and analyze MYC protein expression levels.

[0252] MYC protein degradation experiments showed that the compounds of the present invention could significantly induce intracellular MYC protein degradation (degradation rate greater than 50%) at all tested concentrations in a dose-dependent manner. Therefore, the compounds of the present invention can directly inhibit MYC function.

[0253] Example 52: Cellular Thermal Shift (CETSA) Experiment of the Compounds of the Invention

[0254] Compounds binding to proteins can affect their thermal stability, an effect that can be characterized using the CETSA assay. This example used CETSA to investigate the direct effect of compound 34 on MYC in cells. Other compounds of the present invention exhibit similar beneficial effects to compound 34, but this should not be construed as limiting the compounds of the present invention to the following beneficial effects.

[0255] PC-3 cells were treated with conventional trypsin to terminate digestion and collect cells. Compound 34 (final concentration 10 μM) was added to the experimental group, and the same volume of 0.5% DMSO was added to the control group. The cells were incubated at room temperature for 30 minutes, and the supernatant was discarded. Ethylphenyl polyethylene glycol lysis buffer containing 1% PMSF and 2% phosphatase inhibitors was added, ultrasonicated for 15 seconds, lysed on ice for 20 minutes, and centrifuged at 4°C for 20 minutes. Incubated at room temperature for 30 minutes, the samples were placed on ice. Gradient heating was performed separately, and the heated samples were immediately placed on ice, centrifuged, and the supernatant was taken. 1 / 4 volume of 5×SDS-PAGE buffer was added, and after thorough mixing, the samples were heated at 95°C for 5 minutes using a PCR instrument. After the samples cooled, they were used for WB detection. WB refers to Example 51.

[0256] The results of the CETSA experiment showed that the compounds of the present invention can reduce the thermal stability of MYC, and the thermal shift is obvious, which further reveals the direct effect of the compounds of the present invention on the MYC protein.

[0257] Example 53: Co-immunoprecipitation (Co-IP) experiment of the compounds of the present invention

[0258] To characterize the effects of the compounds of the present invention on the MYC-MAX interaction in cells, this example employed a Co-IP assay to investigate the effects of compound 34 on the MYC-MAX interaction at the cellular level. While other compounds of the present invention exhibit similar beneficial effects as compound 34, this should not be construed as limiting the compounds of the present invention to the following beneficial effects.

[0259] PC-3 cells in the logarithmic growth phase were harvested, seeded at a specific density into 6-well plates, and cultured overnight in an incubator. Compound 34 (final concentration 10 μM) was added to the experimental group, while the control group received the same volume of 0.5% DMSO. The cells were cultured for another 1 hour, digested, centrifuged, and harvested, then washed with PBS. RIPA lysis buffer containing 1% PMSF and 2% phosphatase inhibitors was added, mixed, and lysed on ice for 20 minutes. The supernatant was collected, the protein concentration of the cell lysate was determined by Bradford assay, and the supernatant was diluted to 2 mg / mL with PBS. 4 μL of immunoprecipitation antibody was added to 500 μL of cell lysate, and the cells were gently shaken at 4°C overnight. 20 μL of potein G magnetic bead suspension was added, and the cells were gently shaken at 4°C for another 12 hours. After shaking, the cells were separated on a magnetic rack for 10 seconds, the supernatant discarded, and the potein G magnetic beads washed with PBS. 200 μL of purified water and 50 μL of 5× SDS-PAGE buffer were added, mixed, and heated at 95°C for 5 minutes. After cooling, place on a magnetic stand and separate for 10 seconds, take the supernatant and use it for WB detection. For WB, refer to Example 51.

[0260] Co-IP results showed that the compounds of this invention significantly reduced the accumulation of MAX on magnetic beads, with a statistically significant difference compared to the control group (P < 0.0001), indicating that the compounds can clearly inhibit the MYC-MAX interaction in cells. Since the MYC-MAX interaction is essential for MYC function, the inhibition of the MYC-MAX interaction by the compounds indicates that they can directly inhibit MYC function.

[0261] In summary, the compounds prepared by the present invention have significant in vitro and in vivo pharmacological effects, can directly inhibit MYC function, interfere with MYC-MAX effects, and have good application prospects.

[0262] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A compound represented by general formula (I) or (II) or a pharmaceutically acceptable salt or deuterated derivative thereof: ； ； Formula (I) Formula (II) in: n is independently selected from an integer of 1 to 3; R1 is independently selected from any one of hydrogen and methyl; R2 is a trihalomethyl group; R3 are each independently selected from , any one of C2-C6 dialkylamino, C1-C6 hydroxyalkyl; Ar is independently selected from any one of substituted or unsubstituted phenyl and pyridyl, and the substitution is halogen substitution.

2. The compound according to claim 1 or a pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: It is selected from any one of the following compounds or pharmaceutically acceptable salts or deuterated derivatives thereof: 。 3. A method for preparing the compound according to claim 1 or 2 or a pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: The preparation method comprises: The compound represented by formula (Ia) is subjected to a nucleophilic substitution reaction with the compound represented by formula (Ib) to obtain the compound represented by formula (Ic); the compound represented by formula (Ic) is subjected to a Sonagashira coupling reaction with the compound represented by formula (Id) to obtain the compound represented by formula (Ie); the compound represented by formula (Ie) is subjected to a hydrazinolysis reaction with the compound represented by formula (If) to obtain the compound represented by formula (I). The preparation route is as follows: or, The compound represented by formula (Ia) is subjected to a Suzuki coupling reaction with the compound represented by formula (II-a) to obtain the compound represented by formula (II-b); the compound represented by formula (II-b) is reacted with trifluoromethanesulfonic anhydride to form a sulfonate to obtain the compound represented by formula (II-c); the compound represented by formula (II-c) is subjected to a Sonagashira coupling reaction with the compound represented by formula (Id) to obtain the compound represented by formula (II-d); the compound represented by formula (II-d) is subjected to a hydrazinolysis reaction with the compound represented by formula (If) to obtain the compound represented by formula (II). The preparation route is as follows: wherein X is independently selected from halogen; n, R1, R2, R3, and Ar are as defined in claim 1.

4. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the compound according to claim 1 or 2 or a pharmaceutically acceptable salt or deuterated substance thereof as the sole or main active ingredient, and a pharmaceutically acceptable carrier or auxiliary ingredient.

5. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt or deuterated substance thereof in the preparation of a MYC inhibitor.

6. The use according to claim 5, characterized in that Application of the MYC inhibitor in the preparation of drugs for treating tumors.

7. The use according to claim 6, characterized in that The tumor includes any one of a solid tumor and a blood tumor.

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