Biphenyl compounds and their use in the preparation of medicaments for the treatment of leukemia, lung cancer and breast cancer

Abstract

The application discloses a biphenyl compound and application thereof in preparation of drugs for treating leukemia, lung cancer and breast cancer, and belongs to the technical field of medicinal chemistry. The biphenyl compound is obtained by modifying a p-biphenyl compound with specific structures of R1, R2 and R3. The biphenyl compound can be combined with Hsp70 protein in vitro, in cells and in animal models, competitively dissociate Hsp70 / Bim protein dimers, thereby inducing leukemia cells, breast cancer cells and lung cancer to undergo apoptosis, so that the growth of leukemia cells, breast cancer cells and lung cancer cells is inhibited, and the leukemia cells, breast cancer cells and lung cancer cells are killed. The biphenyl compound is a kind of apoptosis inducer with very high activity, and can be used as a potential drug for treating or preventing leukemia, breast cancer and lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology and relates to a class of biphenyl compounds, particularly 4-chloro-3,5-dimethylphenyl biphenyls and their derivatives. Specifically, it relates to the dissociation of heat shock protein Hsp70 and Bim protein dimers by these compounds in vitro, in living cells and in vivo, thereby specifically inducing apoptosis in leukemia cells, lung cancer cells and breast cancer cells, and can be used as a therapeutic drug for leukemia, lung cancer and breast cancer. Background Technology

[0002] Hsp70, a molecular chaperone protein, undergoes conformational changes under various cellular environments and stress factors by recruiting different molecular chaperones and helper chaperones. It binds to and regulates a variety of client proteins, maintaining proteome stability through molecular machinery mechanisms. Its high expression is not only observed in normal cellular stress but is also associated with various cancers (including leukemia, breast cancer, and lung cancer) and resistance to various anticancer drugs. Recent studies suggest that the complexation of Hsp70 with certain helper chaperone proteins, rather than the overexpression of Hsp70 itself, is key to tumorigenesis. Heat shock proteins can specifically bind to and regulate certain client proteins through protein-protein interactions (PPIs) with different molecular chaperones and helper chaperones, activating tumorigenesis-related signaling pathways.

[0003] In recent years, the interaction between key apoptosis regulators, the Bcl-2 family proteins, and Hsp70 has been discovered. Bim, a member of the Bcl-2 family, has been found to be a helper chaperone protein of Hsp70, binding to an allosteric site in the NBD region of the Hsp70 protein, inhibiting Hsp70's ATPase activity, and affecting the conformation of its SBD region (substrate-binding region) and the folding of client proteins. In particular, the Hsp70 / Bim dimer level is significantly higher in leukemia cells, breast cancer cells, and lung cancer cells than in normal cells and other cancer cells. Downregulating the expression levels of Hsp70 and Bim in leukemia cells, breast cancer cells, and lung cancer cells using interfering RNA technology resulted in the downregulation of Hsp70's oncogenic client proteins such as Raf-1, AKT, c-Myc, and Cyclin D1, leading to significant apoptosis. However, in other normal cells and other cancer cells, such as cervical cancer (HeLa), these protein levels were not significantly affected, nor did they induce significant apoptosis.

[0004] In summary, the Hsp70 / Bim dimer protects leukemia, breast cancer, and lung cancer cells from apoptosis by stabilizing Hsp70's oncogenic client proteins such as Raf-1, AKT, and EIF4E, making it a potential therapeutic target for these diseases. The applicant's previously developed S1g-10 exhibited relatively specific anti-CML stem cell activity and good safety; however, the phenaenone scaffold has limited synthetic accessibility and few substitutable sites, making it difficult to obtain a large number of derivatives for new drug screening to further enhance its tumor-killing effect and improve the properties of ADMET. Summary of the Invention

[0005] To address the problems in the background technology, the applicant used a scaffold transition strategy, replacing the original phenadenone with biphenyl, a superior structure in medicinal chemistry, to obtain a new scaffold compound, a biphenyl derivative, with a chemical structure different from phenadenone. This provides a class of biphenyl compounds that, as Hsp70 / Bim protein-protein interaction inhibitors, can target and inhibit Hsp70 / Bim protein interactions with high affinity, thereby specifically inducing apoptosis in leukemia cells, breast cancer cells, and lung cancer cells, and can be used as drugs for the treatment of leukemia, lung cancer, and breast cancer.

[0006] This invention provides a class of biphenyl compounds having the following structure of general formula I:

[0007]

[0008] in:

[0009] R1 is selected from substituted or unsubstituted C. 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-8 cycloalkyl, C 3-8 Cycloalkenyl, wherein the substitution is selected from the following groups: C 1-3 Alkyl, C 2-3 alkenyl, C 2-3 Alkyne, phenyl, furanyl, thiophene, pyridyl, naphthyl, OH, I, Br, Cl, F, NO2, NHCH3, N(CH3)2, CN, CF3;

[0010] R2 is selected from C 1-8 Alkyl, substituted C 1-8 Alkyl or XR4;

[0011] The replaced C 1-8 Alkyl groups are C groups that are substituted with any of the following groups. 1-8 Alkyl groups: OH, OCH3, I, Br, Cl, NO2, NH 2、NHCH3, N(CH3)2, CH(OCH3)2, CHO, CN, COOH, COOCH3, COOC2H5, morpholino, thiomorpholino, or piperazine;

[0012] The X mentioned is selected from (CH2). m (CH2) m CONH(CH2) n or m and n are integers from 1 to 6;

[0013] R4 is selected from substituted or unsubstituted phenyl, naphthyl, benzofuranyl, thiophenyl, or indoleyl groups, wherein the substitution is selected from the following groups: C 1-4 Alkyl, C 2-4 alkenyl, C 2-4 Alkyne, phenyl, OH, I, Br, Cl, NO2, NHCH3, N(CH3)2, CN, CF3, COOH, SO3H, COOR5, CONHR5, SO3R5, SO2NHR5, or OR5, wherein R5 is selected from C 1-4 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group;

[0014] R3 is selected from -OH,C 1-4 Alkyl, C 2-4 alkenyl, C 2-4 Alkyne, phenyl, OH, I, Br, Cl, NO2, NHCH3, N(CH3)2, CN, CF3, COOH, CH2COOH, SO3H, COOR4, CONHR4.

[0015] Furthermore, R1 is 4-chloro-3,5-dimethylphenyl.

[0016] Furthermore, the biphenyl compounds are selected from compounds with the following structures:

[0017]

[0018] The present invention further provides a method for preparing the above-mentioned biphenyl compounds, comprising the following steps:

[0019] (1) 2,6-Difluoro-4-bromobenzonitrile reacts with benzyl alcohol at a molar ratio of 1:1.1-1:1.2, with NaH as the dehydrogenating agent, to prepare compound II; or, 2,6-difluoro-4-bromobenzonitrile reacts with dimethyl malonate at a molar ratio of 1:1.1-1:1.2, to prepare compound III; the reaction time is 3-12 h, the reaction temperature is 0-25 °C, and the reaction solvent is THF;

[0020]

[0021]

[0022] (2) Compound II or III or 2,6-difluoro-4-bromobenzonitrile is reacted with R2NH2 at a molar ratio of 1:1.1-1:20, with potassium carbonate as an acid-binding agent, to obtain compound IV or V or VI; the reaction time is 3-20 h, the reaction temperature is 20-110 °C, and the reaction solvent is 1,4-dioxane;

[0023]

[0024] (3) R1(OH)2 reacts with 4-hydroxyphenylboronic acid pinacol ester at a molar ratio of 1:1.1-1:20, with copper acetate as a catalyst and triethylamine as an acid binder, to obtain compound VII;

[0025]

[0026] (4) Compound IV or V or VI is coupled with VII or (4-cyclohexyloxy)phenylboronic acid or 4-phenoxyphenylboronic acid in a molar ratio of 1:1.1-1:20 to directly obtain or remove protection or introduce benzenesulfonate after removal protection, and finally obtain compound I.

[0027] Another object of the present invention is to provide stereoisomers, pharmaceutically acceptable salts, solvates, and in vivo hydrolyzable precursors of the compounds of Formula I described above.

[0028] Another aspect of the present invention provides a pharmaceutical composition comprising at least one of the aforementioned compounds.

[0029] Furthermore, the compound is the sole or primary active ingredient in the compositional drug.

[0030] Furthermore, the pharmaceutical composition comprises preferably less than 80%, more preferably less than 50% by weight of the compound of the present invention, and mixed with an inert pharmaceutically acceptable diluent, lubricant or carrier.

[0031] Furthermore, for tablets and lozenges, the diluent, lubricant, and carrier include: lactose, starch, talc, and stearic acid;

[0032] Furthermore, for capsules, the diluent, lubricant, and carrier include tartaric acid or lactose;

[0033] Furthermore, for the injection solution (liquid preparation), the diluent, lubricant, and carrier include: water, alcohol, glycerin, and vegetable oil;

[0034] Furthermore, for suppositories, the diluent, lubricant, and carrier include natural or hardened oils or waxes.

[0035] These pharmaceutical compositions can be administered orally, via the gastrointestinal tract, or topically through the skin or mucous membranes, or via intravenous or intramuscular injection.

[0036] These compositions can be solid or liquid and can be in all pharmaceutical forms commonly used in human medicine, such as simple or sugar-coated tablets, lozenges, drops, injectable formulations, granules, creams, ointments, gels, or gel capsules; they are prepared according to conventional methods. In this application, the active ingredient can be mixed with excipients commonly used in these pharmaceutical compositions, such as gum arabic, talc, starch, lactose, magnesium stearate, cocoa butter, animal or plant-derived lipids, paraffin derivatives, aqueous or non-aqueous carriers, glycols, various dispersants, wetting agents, emulsifiers, or preservatives.

[0037] The present invention further provides a method for preparing the above-mentioned pharmaceutical composition, the method comprising: mixing or compounding the components together, and forming the mixed components into tablets or suppositories, or filling the components into capsules, or dissolving the components to form an injection solution.

[0038] The present invention also provides the use of the compound or the composition in the preparation of medicaments for treating or preventing diseases that benefit from regulation of Hsp70 protein or Hsp70 / Bim protein dimer.

[0039] Furthermore, when the compounds of the present invention are used to treat diseases, they bind to the Hsp70 protein in the body and competitively dissociate the Hsp70 / Bim protein dimer, leading to a large number of apoptosis of leukemia cells, lung cancer cells, and breast cancer cells, thereby achieving the effects of inhibiting the growth of leukemia cells, lung cancer cells, and breast cancer cells and killing leukemia cells, lung cancer cells, and breast cancer cells.

[0040] Furthermore, the compounds of the present invention can be used alone or in combination for the treatment of leukemia, breast cancer, and lung cancer.

[0041] Furthermore, the leukemia includes K562, BV173, and KCL22 cell lines; the breast cancer includes MFC7 and T47D cell lines; and the lung cancer includes A549 and H23 cell lines.

[0042] Furthermore, the products of the present invention can be administered, in particular, alone or in combination with chemotherapy or radiotherapy, or in combination thereof, for example, in combination with other therapeutic agents, such other therapeutic agents may be commonly used antitumor drugs.

[0043] When using the compounds of the present invention to treat diseases, the patient is given a therapeutically effective amount of the compound of formula I, its stereoisomers, pharmaceutically acceptable salts, solvates, in vivo hydrolyzable precursors, or compositions comprising at least one of the foregoing, wherein the therapeutically effective amount is preferably 0.01-10 mg / kg, more preferably 0.3-7 mg / kg, more preferably 0.5-5 mg / kg, and even more preferably 2-5 mg / kg.

[0044] Based on the above findings, for patients weighing 30 kg to 100 kg, the compounds of the present invention can be formulated into unit-dose pharmaceutical compositions. A unit-dose pharmaceutical composition contains 0.3 mg to 1 g of the active pharmaceutical ingredient; preferably, a unit-dose pharmaceutical composition contains 9 mg to 700 mg. More preferably, a unit-dose pharmaceutical composition contains 15 mg to 500 mg of the active pharmaceutical ingredient; even more preferably, a unit-dose pharmaceutical composition contains 60 mg to 500 mg of the active pharmaceutical ingredient. The dosages described above are the daily dosage for the patient. However, this dosage formula is variable depending on the pharmaceutical compound used, the individual being treated, and the condition causing the problem.

[0045] For the uses, methods, pharmaceuticals, and compositions mentioned in this application, the amount of compound used and the dosage form for administration can naturally vary depending on the compound used, the administration method, and the intended therapeutic purpose. Satisfactory results are generally obtained when the compounds of the present invention are administered at a daily dose of about 0.01-10 mg / kg body weight. These doses can be administered in fractionated doses 1 to 4 times daily or in a sustained-release form. For humans, the total daily dose ranges from 9 mg to 700 mg, more preferably from 15 mg to 500 mg, and suitable unit dosage forms for oral administration include 9 mg to 700 mg of the compound mixed with a solid or liquid pharmaceutically acceptable carrier, lubricant, or diluent. Attached Figure Description

[0046] Figure 1 This is a graph showing the trend of average body weight changes in three groups of mice during an acute toxicity experiment.

[0047] Figure 2 This is a comparison of mouse body weight and tumor tissue weight in an experiment where compounds 14-16,19 inhibited the growth of human leukemia cell line K562 (AB), breast cancer cell line MCF7 (CD), and lung cancer cell line A549 (EF) in a tumor-bearing mouse model. Detailed Implementation

[0048] In all the technical solutions described above in this invention, the term "alkyl / alkenyl / alkynyl" includes both straight-chain alkyl and branched-chain alkyl. When referring to a single alkyl group such as "propyl," it specifically refers to a straight-chain alkyl group; similarly, when referring to a single branched alkyl group such as "isopropyl," it specifically refers to a branched-chain alkyl group. For example, "C..."1-4 "alkyl" includes methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Similar rules apply to other groups used in this specification.

[0049] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the raw materials and reagents used can be purchased from chemical or biological reagent companies or prepared from disclosed methods.

[0050] Example 1: Preparation of 4'-(cyclohexyloxy)-3-((2-(dimethylamino)ethyl)amino)-5-fluoro-[1,1'-biphenyl]-4-carbamate (compound 1)

[0051]

[0052] (1) Synthesis of intermediate A1

[0053] N,N-dimethylethylenediamine (88 mg, 1.01 mmol) was added to a solution of 2,6-difluoro-4-bromobenzonitrile (200 mg, 0.92 mmol) in isopropanol (10 mL). The mixture was stirred at 50 °C for 16 hours. The evaporation was evaporated under reduced pressure, and the residue was purified by rapid chromatography (gradient: 0–16% EtOAc in petroleum) to give a white solid (149 mg, 57% yield).

[0054] (2) Synthesis of Compound 1

[0055] To a solution of A1 (236 mg, 0.83 mmol) in 1,4-dioxane / water = 10 (10 mL), K2CO3 (230 mg, 1.66 mmol), Pd2(dba)3 (76 mg, 0.083 mol), and 4-cyclohexyloxyphenylboronic acid (218 mg, 0.99 mmol) were added. The mixture was stirred at 100 °C under argon for 16 hours. The evaporation was carried out under reduced pressure, and the residue was purified by rapid chromatography (gradient: 0–5% MeOH in DCM) to give white solid 1 (180 mg, 57% yield). The NMR and mass spectrometry results of compound 1 are as follows: 1H NMR (400MHz, DMSO) δ7.64(d,J=8.5Hz,2H),7.00(d,J=8.4Hz,2H),6.81(d,J=11.0Hz,1H),6.77(s,1H),6.13(t,J=5.2Hz,1H),4.39(tt,J=8. 5,3.7Hz,1H),3.37(q,J=6.0Hz,2H),2.52(d,J=6.4Hz,2H),2.23(s,6H),1.98-1.83(m,2H),1.70(tt,J=8.4,4.4Hz,2H),1.51-1.19(m,6H). 13 C NMR(101MHz,DMSO)δ158.70,152.21,147.82,143.13,134.90,128.85,116.44,114.04,1 04.77,100.63,82.50,82.32,74.85,57.48,45.40,31.70,25.56,23.55.HRMS(ESI):m / z calcd for Chemical Formula:C 23 H 29 FN3O[M+H] + ,382.2295;found,382.2280.

[0056] Example 2: Preparation of Compound 2

[0057]

[0058] (1) Synthesis of intermediate A2

[0059] At 0 °C, benzyl alcohol (545 mg, 5.05 mmol) was slowly added to a suspension of NaH (238 mg, 5.96 mmol) in 20 mL of THF (15 mL). The mixture was stirred at room temperature (30 min). Then, 2,6-difluoro-4-bromobenzonitrile (1 g, 4.59 mmol) was added, and the mixture was stirred at room temperature for 16 h. Inorganic matter was removed by filtration, the evaporator was evaporated under reduced pressure, and the residue was purified by rapid chromatography (gradient: 0–16% EtOAc in petroleum) to give a white solid A2 (488 mg, 35% yield).

[0060] (2) Synthesis of intermediate B1

[0061] To a solution of A2 (459 mg, 1.51 mmol) in 1,4-dioxane (10 mL), K2CO3 (418 mg, 3.03 mmol) and N,N-dimethylethylenediamine (147 mg, 1.67 mmol) were added. The mixture was stirred at 110 °C for 16 hours. The evaporation was evaporated under reduced pressure, and the residue was purified by rapid chromatography (gradient: 0–5% MeOH in DCM) to give a white solid B1 (360 mg, 64% yield).

[0062] (3) Synthesis of intermediate C1

[0063] To a solution of B1 (309 mg, 0.83 mmol) in 1,4-dioxane / water = 10 (10 mL), K2CO3 (230 mg, 1.66 mmol), Pd2(dba)3 (76 mg, 0.083 mol), and 4-cyclohexyloxyphenylboronic acid (218 mg, 0.99 mmol) were added. The mixture was stirred at 100 °C under argon for 16 hours. The evaporation was carried out under reduced pressure, and the residue was purified by rapid chromatography (gradient: 0–5% MeOH in DCM) to give a white solid C1 (253 mg, 65% yield).

[0064] (4) Synthesis of Compound 2

[0065] Palladium-carbon (10 mg, 0.09 mmol) was added to a solution of C1 (81 mg, 0.17 mmol) in MeOH (10 mL). The mixture was bubbled under a hydrogen atmosphere (1 atm) and stirred at room temperature for 16 hours. The mixture was filtered and concentrated to give a crude product. The crude product was purified by thin-layer chromatography (DCM / MeOH = 15) to give a white solid 2 (58 mg, 90% yield).

[0066] The NMR and mass spectrometry results for compound 2 are as follows: 1 H NMR (400MHz, DMSO) δ7.51(d,J=8.3Hz,2H),7.00(d,J=8.4Hz,2H),6.35(d,J=17.4Hz,2H),5.53(t,J=5.1Hz,1H),4.38(tt,J=8.4,3.7H z,1H),3.27(q,J=6.0Hz,2H),2.51(d,J=12.3Hz,2H),2.21(s,6H),1.99-1.85(m,2H),1.72(dt,J=14.1,4.5Hz,2H),1.55-1.23(m,6H). 13C NMR (101MHz, DMSO) δ161.81,158.15,151.92,146.90,132.35,128.43,116.67,116.47, 101.52,99.87,83.09,74.87,57.64,45.44,40.64,31.75,25.58,23.59.HRMS(ESI):m / z calcd for ChemicalFormula:C 23 H 28 N3O2[MH] - ,378.2187;found,378.2184.

[0067] Example 3: Preparation of compounds 3-4

[0068]

[0069] (1) Synthesis of intermediate A3

[0070] Similar to A2 in Example 2, simply replace benzyl alcohol with dimethyl malonate to obtain a white solid (590 mg, 39% yield).

[0071] (2) Synthesis of intermediate B2

[0072] Similar to B1 in Example 2, a white solid (343 mg, 57% yield) was obtained.

[0073] (3) Synthesis of intermediate C2

[0074] Similar to C1 in Example 2, white solid (225 mg, 55% yield).

[0075] (4) Synthesis of compounds 3 and 4

[0076] C2 (200 mg, 0.41 mmol) was dissolved in a 6N HCl aqueous solution (approximately 8 eq), and the solution was heated to 100 °C for 12 hours. The mixture was cooled to room temperature and allowed to stand for half an hour. The mixture was poured into water and extracted with EtOAc. The organic layer was washed twice with water and dried over anhydrous Na2SO4, then filtered and concentrated. The crude product was purified by thin-layer chromatography (DCM / MeOH = 20) to give white solid 3 (30 mg, 20% yield). The aqueous layer was filtered and dried to obtain white solid 4 (40 mg, 24% yield). The NMR and mass spectrometry results for compounds 3 and 4 are as follows:

[0077] 4'-(cyclohexyloxy)-3-((2-(dimethylamino)ethyl)amino)-5-methyl-[1,1'-biphenyl]-4-carbamate (compound 3): 1H NMR (400MHz, DMSO) δ7.60(d,J=8.2Hz,2H),7.01(d,J=8.2Hz,2H),6.79(d,J=32.5Hz,2H),5.75-5.50(m,1H),4.39( s,1H),3.22(s,2H),2.54(s,2H),2.39(s,3H),2.23(s,6H),1.94(d,J=10.2Hz,2H),1.72(s,2H),1.58-1.15(m,6H). 13 C NMR (101MHz, DMSO) δ158.19,151.46,145.65,142.83,131.93,128.67,117.68,116.48,116. 09,106.30,94.20,88.77,74.82,57.56,45.40,31.74,25.58,23.57,20.97.HRMS(ESI):m / z calcd forChemical Formula:C 24 H 30 N3O[MH] - ,376.2394; found,376.2393.

[0078] 2-(4-cyano-4'-(cyclohexyloxy)-5-((2-(dimethylamino)ethyl)amino)-[1,1'-biphenyl]-3-yl)acetic acid (compound 4): 1 H NMR (400MHz, DMSO) δ7.58(d,J=8.3Hz,2H),7.00(d,J=8.2Hz,2H),6.84(s,1H),6.75(s,1H),5.55(d,J=5.1Hz,1H),4.38(td, J=8.7,4.2Hz,1H),3.31(q,J=5.9Hz,4H),2.38(s,2H),2.22(s,6H),1.97-1.82(m,2H),1.79-1.60(m,2H),1.52-1.25(m,6H). 13 C NMR (101MHz, DMSO) δ172.38,158.08,151.23,145.15,132.13,128.61,117.73,116.68,116.48, 106.29,95.26,81.98,74.82,57.66,45.48,43.17,39.36,31.75,25.59,23.58.HRMS(ESI):m / z calcd for Chemical Formula:C 25 H30 N3O3[MH] - ,420.2293; found,420.2281.

[0079] Example 4: Preparation of Compound 5

[0080]

[0081] (1) Synthesis of intermediate D1

[0082] TEA (2.23 g, 22.07 mmol), Na₂SO₄ (appropriate amount), pinacol 4-hydroxyphenylboronic acid (1.78 g, 8.09 mmol), and Cu(OAc)₂ (2 g, 11.03 mmol) were added to a solution of m-tolylboronic acid (1 g, 7.36 mmol) in DCM (50 mL). The mixture was stirred at room temperature for 16 hours. The evaporation was evaporated under reduced pressure, and the residue was purified by rapid chromatography (gradient: 0–5% EtOAc in petroleum) to give a colorless oil (114 mg, 5% yield).

[0083] (2) Synthesis of intermediate B1

[0084] See Example 2.

[0085] (3) Synthesis of intermediate C3

[0086] Intermediate C3 is similar to C1. Replacing the starting material 4-cyclohexyloxyphenylboronic acid with D1 yields a white solid (44 mg, 43% yield).

[0087] (4) Synthesis of compound 5

[0088] Similar to compound 2, replacing C1 with C3 yields compound 5 (26 mg, 80% yield). The NMR and mass spectrometry results for compound 5 are as follows: 1 H NMR (400MHz, DMSO) δ7.64-7.58(m,2H),7.31(d,J=7.8Hz,1H),7.28(d,J=6.8Hz,1H),7.09-7.04(m,2H),6.90(t,J=2.1Hz,1H),6.86( dd,J=8.2,2.5Hz,1H),6.42-6.35(m,2H),5.60(t,J=5.0Hz,1H),3.28(d,J=5.9Hz,2H),2.57-2.53(m,2H),2.31(s,3H),2.24(s,6H). 13C NMR (101MHz, DMSO) δ161.76,157.76,156.66,151.95,146.52,140.37,135.21,130.32,128.93,12 5.06,120.05,119.04,116.59,101.70,100.27,83.44,57.55,49.07,45.38,21.39.HRMS(ESI):m / z calcd for Chemical Formula:C 24 H 24 N3O2[MH] - ,386.1874; found,386.1973.

[0089] Example 5: Preparation of compounds 6, 8, 12, 14-16

[0090] Similar to the preparation method of compound 5, compounds 6, 8, 12, and 14-16 were synthesized. The yields and characterization data of compounds 6, 8, 12, and 14-16 are as follows:

[0091] Compound 6: 61 mg, yield 93%. 1 H NMR (400MHz, DMSO) δ7.65-7.55(m,2H),7.07-7.02(m,2H),6.82(s,1H),6.68(s,2H),6.39(d,J=10.7H z,2H),5.57(t,J=5.1Hz,1H),3.29(d,J=5.8Hz,2H),2.53(d,J=6.3Hz,2H),2.26(s,6H),2.23(s,6H). 13 C NMR (101MHz, DMSO) δ161.84,157.84,156.65,151.96,146.53,139.95,135.13,128.87,125.88 ,119.01,117.15,116.57,101.76,100.20,83.47,57.60,45.42,40.60,21.33.HRMS(ESI):m / z calcd forChemical Formula:C 25 H 26 N3O2[MH] - ,400.2030; found,400.2024.

[0092] Compound 8: 55 mg, yield 88%. 1H NMR (400MHz, DMSO) δ7.61(d,J=8.4Hz,2H),7.25-7.22(m,1H),7.03(d,J=8.4Hz,2H),6.90(d,J=1.7Hz,2H),6.3 9(d,J=13.8Hz,2H),5.57(t,J=5.1Hz,1H),3.29(q,J=6.0Hz,2H),2.58-2.51(m,2H),2.23(s,6H),1.27(s,18H). 13 CNMR(101MHz,DMSO)δ161.92,158.14,155.80,153.05,151.94,146.50,134.74,128.84,118.30,1 18.13,114.07,101.74,100.06,83.42,74.00,57.55,45.37,35.15,31.59,25.42.HRMS(ESI):m / z calcd for Chemical Formula:C 31 H 38 N3O2[MH] - ,484.2969; found,484.2965.

[0093] Compound 12: 74 mg, yield 90%. 1 H NMR (400MHz, DMSO) δ7.61(d,J=8.3Hz,2H),7.07(d,J=8.3Hz,2H),6.94(s,2H),6.38(d ,J=8.5Hz,2H),5.57(t,J=5.1Hz,1H),3.28(q,J=5.9Hz,4H),2.32(s,6H),2.21(s,6H). 13 C NMR (101MHz, DMSO) δ161.88,157.56,154.64,151.98,146.45,137.99,135.43,128.96,128.93 ,119.68,119.03,116.57,101.75,100.19,83.48,57.64,45.47,40.26,20.80.HRMS(ESI):m / z calcd for Chemical Formula:C 25 H 25 ClN3O2[MH]-,434.1640; found,434.1636.

[0094] Compound 14: 61 mg, yield 84%. 1¹H NMR (400 MHz, DMSO) δ 7.61 (d, J = 8.3 Hz, 2H), 7.07 (d, J = 8.3 Hz, 2H), 6.94 (s, 2H), 6.38 (d, J = 14.2 Hz, 2H), 5.67 (t, J = 5.0 Hz, 1H), 3.26-3.16 (m, 2H), 2.67 (t, J = 6.2 Hz, 2H), 2.26 (s, 6H), 1.08 (s, 4H), 0.99 (t, J = 7.1 Hz, 6H). 13 ¹³C NMR (101 MHz, DMSO) δ 161.79, 157.54, 154.66, 152.09, 146.45, 137.98, 135.44, 128.94, 119.64, 119.03, 116.50, 101.75, 100.30, 83.58, 74.00, 50.97, 46.80, 25.41, 20.79, 12.31. HRMS (ESI): m / z calcd for Chemical Formula: C 27 H 31 ClN₃O₂ [M+H]⁺ + , 464.2104; found, 464.2103.

[0095] Compound 15: 68 mg, yield 89%. 1 ¹H NMR (400 MHz, DMSO) δ 7.66-7.54 (m, 2H), 7.07 (d, J = 8.6 Hz, 2H), 6.94 (s, 2H), 6.38 (d, J = 7.8 Hz, 2H), 5.72 (t, J = 5.3 Hz, 1H), 3.36-3.28 (m, 2H), 2.71 (t, J = 6.4 Hz, 2H), 2.54 (d, J = 5.8 Hz, 4H), 2.32 (s, 6H), 1.71 (q, J = 3.4 Hz, 4H). 13 ¹³C NMR (101 MHz, DMSO) δ 161.92, 157.56, 154.64, 152.04, 146.41, 137.99, 135.44, 128.96, 128.93, 119.68, 119.02, 116.58, 101.73, 100.17, 83.56, 54.26, 53.92, 41.79, 23.59, 20.81. HRMS (ESI): m / z calcd for Chemical Formula: C 27 H 27 ClN₃O₂ [M-H]⁻, 460.1797; found, 460.1797.

[0096] Compound 16: 113 mg, yield 90%. 1 H NMR (400MHz, DMSO) δ7.61(d,J=8.3Hz,2H),7.07(d,J=8.3Hz,2H),6.93(s,2H),6.39(d,J=18.3Hz,2H),5.73(q,J=4.9Hz, 1H), 3.30 (q, J = 6.0Hz, 2H), 2.59 (t, J = 6.4Hz, 2H), 2.44 (s, 4H), 2.31 (s, 6H), 1.52 (q, J = 5.6Hz, 4H), 1.40 (q, J = 5.7Hz, 2H). 13 C NMR (101MHz, DMSO) δ161.82,157.53,154.67,152.08,146.45,137.96,135.47,128.95,128.92,11 9.63,119.02,116.51,101.84,100.34,83.67,56.51,54.06,25.92,24.33,20.79.HRMS(ESI):m / z calcd for Chemical Formula:C 28 H 29 ClN3O2[MH]-,474.1953; found,474.1952.

[0097] Example 6: Preparation of Compound 7

[0098]

[0099] Synthesis of Compound 7

[0100] Similar to compound C1, replacing 4-cyclohexyloxyphenylboronic acid with D2 yields compound 7 (113 mg, 50% yield). The NMR and mass spectrometry results for compound 7 are as follows: 1 H NMR (400MHz, DMSO) δ7.78-7.68(m,2H),7.09-7.00(m,2H),6.86(dd,J=10.9,1.3Hz,1H),6.84-6.78(m, 2H),6.69(s,2H),6.16(t,J=5.2Hz,1H),3.39(d,J=6.1Hz,2H),2.53(s,2H),2.26(s,6H),2.22(s,6H). 13C NMR(101MHz,DMSO)δ163.12,158.46,156.42,152.29,147.44,139.99,133.45,129.28,126.03 ,118.89,117.27,113.97,105.28,100.92,82.87,57.58,45.54,40.84,21.38.HRMS(ESI):m / z calcdfor Chemical Formula:C 25 H 27 FN3O[M+H] + ,404.2138; found,404.2140.

[0101] Example 7: Preparation of Compound 9

[0102]

[0103] (1) Synthesis of intermediate E1

[0104] To a solution of adamantane (690 mg, 4.53 mmol) and pyridine (394 mg, 4.99 mmol) in DCM (30 mL) at 0 °C, diphenylphosphine chloride (1000 mg, 4.53 mmol) was added dropwise. The mixture was stirred at room temperature for 1 hour. The evaporation was evaporated under reduced pressure, and the residue was purified by rapid chromatography (gradient: 0–5% EtOAc in petroleum) to give a colorless oil (1.22 g, 80% yield).

[0105] (2) Synthesis of intermediate D5

[0106] DIAD (601 mg, 2.97 mmol) was added dropwise to a DCM (30 mL) solution of E1 (1 g, 2.97 mmol). After about 5 minutes, the mixture of 4-hydroxyphenylboronic acid pinacol ester (719 mg, 3.27 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The evaporation was carried out under reduced pressure, and the residue was purified by rapid chromatography (gradient: 0-5% EtOAc in petroleum) to give a colorless oil (73 mg, 7% yield).

[0107] (3) Synthesis of intermediate C10

[0108] Similar to C1, replacing 4-cyclohexyloxyphenylboronic acid with D5 yields C10 (24 mg, 30% yield).

[0109] (4) Synthesis of compound 9

[0110] Similar to compound 2, replacing C1 with C10 yields compound 9 (13 mg, 80% yield). The NMR and mass spectrometry results for compound 9 are as follows: 1 H NMR (400MHz, DMSO) δ10.80 (s, 1H), 7.58-7.48 (m, 2H), 7.06 (d, J = 8.2Hz, 2H), 6.42 (d, J = 5.8Hz, 2H), 5.9 6-5.76(m,1H),2.98(s,2H),2.58(s,2H),2.15(s,6H),1.84(d,J=3.0Hz,6H),1.58(s,3H),1.24(s,6H). 13 C NMR(101MHz,DMSO)δ163.23,154.79,150.52,146.88,135.36,127.93,125.19,119.48,1 12.03,102.07,100.17,78.02,63.14,47.71,42.83,40.64,36.04,30.70.HRMS(ESI):m / z calcd for Chemical Formula:C 27 H 34 N3O2[M+H] + ,432.2651; found,432.2648.

[0111] Example 8: Preparation of compounds 10 and 13

[0112]

[0113] (1) Synthesis of intermediates C11 and C12

[0114] Similar to C1, 4-cyclohexyloxyphenylboronic acid was replaced with 4-phenoxyphenylboronic acid and then suzuki coupled with B1 and B2 respectively to obtain white solid C11 (102 mg, 52% yield) and white solid C12 (115 mg, 55% yield).

[0115] (2) Synthesis of compounds 10 and 13

[0116] Similar to compound 2, replacing C1 with C11 yields white solid 10 (72 mg, 90% yield), and replacing it with C12 yields white solid 13 (69 mg, 85% yield). The NMR and mass spectrometry results for compounds 10 and 13 are as follows:

[0117] Compound 10: 11H NMR (400 MHz, DMSO) δ 7.62 (d, J=8.2 Hz, 2H), 7.42 (t, J=7.7 Hz, 2H), 7.18 (t, J=7.5 Hz, 1H), 7.07 (d, J=8.2 Hz, 4H), 6.39 (d, J=19.7 Hz, 2H), 5.57 (t, J=4.8 Hz, 1H), 3.28 (q, J=5.7 Hz, 2H), 2.53 (d, J=6.6 Hz, 2H), 2.22 (s, 6H). 13 13C NMR (101 MHz, DMSO) δ 162.11, 157.62, 156.71, 151.95, 146.49, 135.42, 130.60, 128.94, 124.29, 119.50, 119.08, 116.68, 101.88, 100.05, 83.52, 57.57, 45.40, 40.55. HRMS (ESI): m / z calcd for ChemicalFormula:C 23 H 22 N3O2[M-H] - , 372.1717; found, 372.1715.

[0118] Compound 13: 1 1H NMR (400 MHz, DMSO) δ 7.65-7.57 (m, 2H), 7.42 (t, J=7.7 Hz, 2H), 7.18 (t, J=7.4 Hz, 1H), 7.07 (d, J=8.1 Hz, 4H), 6.37 (d, J=12.5 Hz, 2H), 5.64 (t, J=5.0 Hz, 1H), 3.23 (q, J=5.9 Hz, 2H), 2.65 (t, J=6.2 Hz, 2H), 2.53 (d, J=7.2 Hz, 4H), 0.99 (t, J=7.1 Hz, 6H). 13 13C NMR (101 MHz, DMSO) δ 161.86, 157.62, 156.72, 152.14, 146.49, 135.39, 130.61, 128.92, 124.30, 119.49, 119.09, 116.55, 101.77, 100.28, 83.56, 51.03, 46.79, 40.69, 12.45. HRMS (ESI): m / z calcd for Chemical Formula:C 25 H 26 N3O2[M-H] - , 400.2030; found, 400.2029.

[0119] Example 9: Preparation of Compound 11

[0120]

[0121] (1) Synthesis of intermediate F1

[0122] Similar to compound D1, replacing the starting materials m-tolylboronic acid and 4-hydroxyphenylboronic acid pinacol ester with 3,5-dimethyl-4-nitrophenol and 4-bromophenylboronic acid yields F1, a colorless oil (135 mg, 7% yield).

[0123] (2) Synthesis of intermediate D6

[0124] To a solution of F1 (120 mg, 0.37 mmol) in 1,4-dioxane (5 mL), KOAc (110 mg, 1.12 mmol), Pin₂B₂ (113 mg, 0.45 mmol), and (dppf)₂PdCl₂ (27 mg, 0.037 mmol) were added. The mixture was stirred at 100 °C under argon for 16 hours. The evaporation was evaporated under reduced pressure, and the residue was purified by rapid chromatography (gradient: 0–5% EtOAc in petroleum) to give a colorless oil (83 mg, 60% yield).

[0125] (3) Synthesis of intermediate C13

[0126] Similar to C1, replacing 4-cyclohexyloxyphenylboronic acid with D6 and then performing Suzuki coupling with B2 resulted in C13, a white solid (83 mg, 60% yield).

[0127] (4) Synthesis of compound 11

[0128] Similar to 2, replacing C1 with C13 yields 11, a white solid (21 mg, 83% yield). The NMR and mass spectrometry results for compound 11 are as follows: 1 H NMR (400MHz, DMSO) δ7.56-7.51(m,2H),6.95-6.89(m,2H),6.63(s,2H),6.36(dd,J=16.2,1.4Hz,2H) ,5.66-5.56(m,1H),4.48(s,2H),3.33-3.29(m,2H),2.60(q,J=4.8Hz,2H),2.28(s,6H),2.10(s,6H). 13C NMR (101MHz, DMSO) δ161.83,160.01,151.85,146.75,145.35,141.72,133.65,129.37,128.63 ,127.27,122.64,120.16,117.09,101.67,100.02,83.27,57.37,45.17,18.35.HRMS(ESI):m / z calcd for ChemicalFormula:C 25 H 27 N4O2[MH] - ,415.2139;found,415.2140.

[0129] Example 10: Preparation of compounds 17-19

[0130]

[0131] (1) Synthesis of compound 17

[0132] To a solution of compound 16 (100 mg, 0.21 mmol) and 4-dimethylaminopyridine (2.57 mg, 0.021 mmol) in DCM (5 mL), 2,4-difluorobenzenesulfonyl chloride (89 mg, 0.42 mmol) was added. The mixture was stirred at room temperature for 12 hours. The evaporation was evaporated under reduced pressure, and the crude product was purified by thin-layer chromatography (PE / THF = 2) to give a white solid (54 mg, 40% yield). The NMR and mass spectrometry results of compound 17 are as follows: 1 H NMR (400MHz, DMSO) δ7.76(d,J=8.3Hz,1H),7.61(d,J=8.3Hz,2H),7.27(d,J =8.3Hz,1H),7.07(d,J=8.3Hz,2H),7.03(s,1H),6.93(s,2H),6.39(d,J=18 .3Hz,2H),5.73(q,J=4.9Hz,1H),3.30(q,J=6.0Hz,2H),2.59(t,J=6.4Hz,2 H),2.44(s,4H),2.31(s,6H),1.52(q,J=5.6Hz,4H),1.40(q,J=5.7Hz,2H). 13C NMR (101MHz, DMSO) δ164.91,161.82,160.62,157.53,154.67,152.08,146.45,137.96,135.47,131.31,128.95,128.92,126. 13,119.63,119.02,116.51,112.48,106.12,101.84,100.34,83.67,56.51,54.06,25.92,24.33,20.79.HRMS(ESI):m / zcalcd for Chemical Formula:C 34 H 32 ClF₂N₃O₄ S[M+H] + ,652.1848; found,652.1849.

[0133] (2) Synthesis of compounds 18 and 19

[0134] Similar to compound 17, replacing it with different benzenesulfonyl chlorides yields compounds 18 and 19. The NMR and mass spectrometry results for compounds 18 and 19 are as follows:

[0135] Compound 18: 52 mg, yield 38%. 1 H NMR (400MHz, DMSO) δ8.48(d,J=8.3Hz,2H),8.09(d,J=8.3Hz,2H),7.61(d,J=8.3Hz,2H),7.07(d,J=8.3Hz,2H),6.93(s,2H),6.39(d,J=18.3Hz, 2H),5.73(q,J=4.9Hz,1H),3.30(q,J=6.0Hz,2H),2.59(t,J=6.4Hz,2H) ,2.44(s,4H),2.31(s,6H),1.52(q,J=5.6Hz,4H),1.40(q,J=5.7Hz,2H). 13 C NMR (101MHz, DMSO) δ161.82,157.53,154.67,152.08,151.73,146.45,141.52,137.96,135.47,130.13,128.95,12 8.92,123.81,119.63,119.02,116.51,101.84,100.34,83.67,56.51,54.06,25.92,24.33,20.79.HRMS(ESI):m / z calcd for Chemical Formula:C 34 H 34ClN4O6S[M+H] + ,661.1887; found,661.1889.

[0136] Compound 19: 23 mg, yield 22%. 1 H NMR(400MHz,DMSO)δ9.05(s,1H),8.87(d,J=8.3Hz,1H),8.32(d,J=8.3Hz,1 H),7.61(d,J=8.3Hz,2H),7.07(d,J=8.3Hz,2H),6.93(s,2H),6.39(d,J=18 .3Hz,2H),5.73(q,J=4.9Hz,1H),3.30(q,J=6.0Hz,2H),2.59(t,J=6.4Hz,2 H),2.44(s,4H),2.31(s,6H),1.52(q,J=5.6Hz,4H),1.40(q,J=5.7Hz,2H). 13 C NMR (101MHz, DMSO) δ161.82,157.53,154.67,152.66,152.08,148.93,146.45,144.72,137.96,135.47,131.34,129.93,12 8.95,128.92,119.63,119.02,116.51,115.51,101.84,100.34,83.67,56.51,54.06,25.92,24.33,20.79.HRMS(ESI):m / z calcd for Chemical Formula:C 34 H 33 ClN5O8 S[M+H] + ,706.1738; found,706.1740.

[0137] Example 11: Detection of the ability of the above compound to dissociate Hsp70 / Bim dimer in vitro by fluorescence polarization analysis.

[0138] A 21-amino acid BimBH3 peptide (amino acids: 79-99: QEDIIRNIARHLAQVGDSMDR) was synthesized and labeled with 6-carboxyfluorescein succinimide (FAM) at the N-terminus as a fluorescent tag (FAM-Bim). The reaction system used in the competitive binding assay consisted of GST-Hsp70 protein (300 nM) and FAM-Bim peptide (20 nM) dissolved in reaction buffer (100 mM K3PO4, pH 7.5; 100 μg / mL bovine γ-albumin; 0.02% sodium azide). In a 96-well plate, 100 μL of the reaction system was added to each well, followed by 1 μL of different concentrations of the target compound dissolved in DMSO (0-10 mM), resulting in a final concentration of 0-100 μM. Two control groups were set up: one control group contained only Hsp70 and FAM-Bim in the reaction system (equivalent to 0% inhibition rate), and the other control group contained only the FAM-Bim peptide in the reaction system. After 1 hour of incubation in the dark, the 96-well plates were analyzed using a microplate reader. Fluorescence polarization (mP) was measured at an emission wavelength of 530 nm generated by excitation at 485 nm. IC50 50 The value was calculated using SPSS.

[0139] The ability of compounds 1-19 to dissociate the Hsp70 / Bim dimer was tested using the same experimental method described above, with known Hsp70 inhibitors MKT-077 and S1g-10 as controls. The results showed that compounds 1-19 of the present invention, except for compound 3, could effectively dissociate the Hsp70 / Bim dimer, and their competitive binding to the Hsp70 protein was IC50. 50 The values ​​are in the range of 60 nM to 10 μM. Among them, the compounds of the preferred technical solutions (2, 5, 6, 8, 11-16) have a competitive dissociation constant of less than 1.0 μM with Hsp70 protein, which belongs to relatively strong competitive dissociation compounds. This indicates that the compounds of the present invention do have a strong ability to competitively bind to Bim with Hsp70 protein. The specific results are shown in Table 1.

[0140] Table 1. Experimental results on the ability of the compounds of the present invention to dissociate Hsp70 / Bim dimers.

[0141]

[0142] Example 12: Detection of apoptosis induced by the above compounds in leukemia cell lines, breast cancer cell lines, and lung cancer cells using flow cytometry.

[0143] Apoptosis is a fundamental characteristic of cells, playing a crucial role in embryonic development, tissue repair, and homeostasis. In normal cells, phosphatidylserine (PS) is only distributed on the inner side of the cell membrane lipid bilayer. However, in the early stages of apoptosis, PS flips from the inner to the outer side of the lipid membrane. Annexin V, a Ca-dependent phospholipid-binding protein with a molecular weight of 35-36 kDa, binds specifically and with high affinity to PS that flips outward during apoptosis. Using FITC-labeled annexin V as a fluorescent probe, apoptosis can be detected using flow cytometry or fluorescence microscopy. Propidium iodide (PI) is a DNA dye that is excited by ultraviolet light at around 535 nm and emits red fluorescence at 615 nm. Therefore, cells carrying annexin V-FITC are apoptotic cells, and the number of cells stained with PI represents the total cell count. By counting the different cell numbers, the proportion of apoptotic cells can be calculated.

[0144] This experiment used the Annexin-V / propidium iodide staining by flow cytometry kit (Beyotime, Suzhou, China C1063) to detect the apoptosis of compounds 2, 5, 6, 8, 11-19 (a series of different concentrations) after 24 hours in human leukemia cell lines (K562, KCL22, BV173), human leukemia TKI-resistant cell lines (K562-R3), human breast cancer cell lines (MCF7, T47D), human breast cancer tamoxifen-resistant cell lines (MCF7 / TAM-R, T47D / TAM-R), human cervical cancer cell line HeLa, human liver cancer cell line HepG2, human lung cancer cell lines (H23, A549), near-normal somatic cell line U937, and human embryonic kidney cell line HEK293.

[0145] The main experimental steps are as follows:

[0146] 1. Cell collection: After treatment with the above compounds for 24 hours, the suspended cells were directly collected into 10 mL centrifuge tubes, with 5 × 10⁶ cells per sample. 6 / mL, centrifuge at 1000r / min for 5min, and discard the culture medium;

[0147] 2. Wash once with incubation buffer, then centrifuge at 500-1000 rpm for 5 min;

[0148] 3. Resuspend the cells in 100 μL of labeling solution (Annexin-V / PI) and incubate at room temperature in the dark for 10–15 min;

[0149] Centrifuge at 4,500–1,000 rpm for 5 minutes to pellet the cells, then wash once with incubation buffer.

[0150] 5. Add fluorescent (SA-FLOUS) solution and incubate at 4°C for 20 min, protected from light and with occasional shaking. Detect the apoptosis induced by the compound using flow cytometry. Use cells without the test compound as a control group.

[0151] Flow cytometry analysis: The excitation wavelength of the flow cytometer was 488 nm. A passband filter with a wavelength of 515 nm was used to detect FITC fluorescence, and another filter with a wavelength greater than 560 nm was used to detect PI.

[0152] Results Interpretation: On the scatter plot of a bivariate flow cytometer, the lower left quadrant shows viable cells (FITC- / PI-); the upper right quadrant shows non-viable cells, i.e., necrotic cells (FITC+ / PI+); and the lower right quadrant shows apoptotic cells (FITC+ / PI-). Based on these results, the percentage of apoptotic cells in the total cell count is calculated, and then expressed as logarithmic. 10 The compound concentration is plotted on the horizontal axis, and the percentage of apoptotic cells is plotted on the vertical axis to fit an apoptosis curve. The compound concentration at which 50% of cells undergo apoptosis is calculated using the curve, i.e., the half-maximal effective concentration (EC50) for apoptosis induction. 50 The specific results are shown in Table 2.

[0153]

[0154] The results showed that compounds 2, 5, 6, 8, 11-16, and 19 of the present invention could efficiently induce tumor cell apoptosis (EC) in leukemia cell lines (such as K562, BV173, KCL22, etc.), breast cancer cell lines (such as MFC7, T47D, etc.), and lung cancer cell lines (such as A549, H23, etc.). 50 The concentration <5 μM indicates that the compounds of this invention exhibit good selectivity in inhibiting and killing leukemia cells, breast cancer cells, and lung cancer cells. Furthermore, while compound 19, as a prodrug, does not exhibit strong Hsp70-Bim dissociation ability in vitro, it can be deprotected by glutathione highly expressed in tumor cells to yield compound 16. Compound 19 achieves a similar tumor cell apoptosis-inducing ability as compound 16, further improving the safety of this series of compounds.

[0155] Example 13: Compound-induced apoptosis depends on the presence of Hsp70 / Bim dimer

[0156] The apoptosis-inducing effect of the compounds of this invention on leukemia cells, breast cancer cells, and lung cancer cells was verified through interfering RNA experiments, which showed that it depended on Hsp70 and Bim. The pGenesil plasmid was used to express the interfering RNA. The interfering sequences were 5'-GGG TTTCATCCAGGATCGA-3' targeting Hsp70 and 5'-CAGGACACAGAGGAGGTTT-3' targeting Bim. Transfection of the interfering plasmid was performed using Lipofectamine 2000. Human leukemia cells K562, human breast cancer cells MCF7, and human lung cancer cells A549 were cultured using standard methods to a density of 102. 5 After transfecting cells with 2 μg of Hsp70 and Bim interference plasmids (per mL), cells were collected by centrifugation after 24 hours. Cells were lysed in 2% CHAPS lysis buffer at 4°C for 10 minutes. 20 μL of each sample was taken for SDS-PAGE electrophoresis, transferred to a membrane, and incubated with Hsp70 and Bim antibodies, respectively. The bands were then exposed using ECL chromogenic reagent. Relative protein expression levels were quantified by optical density measurement of the immunoblotting bands. Using the expression levels of Hsp70 and Bim in wild-type K562 / MCF7 / A549 cells as a reference, cell lines showing a significant decrease in Hsp70 and Bim protein expression after transfection with the Hsp70 and Bim interference plasmids compared to wild-type cells were selected, demonstrating that this method effectively interferes with the expression of Hsp70 and Bim proteins.

[0157] Human leukemia cells K562, human breast cancer cells MCF7, and human lung cancer cells A549 were cultured using standard methods, and the density reached 10-1. 5 After transfecting each well with 2 μg of Hsp70 and Bim interference plasmids for 24 hours, 0-20 μM of compounds 2, 5, 6, 8, and 11-19 were added and incubated for 24 hours. Apoptosis was detected by flow cytometry, and the half-maximal effective concentration (EC50) for apoptosis induction was calculated. 50 The operation method of the flow cytometer is as described in Example 12, and the experimental results are shown in Table 3.

[0158] Experimental results showed that in human leukemia cells K562, human breast cancer cells MCF7, and human lung cancer cells A549, where Hsp70 and Bim were deficient, the apoptosis-inducing ability of compounds 2, 5, 6, 8, and 11-19 was inhibited compared to the wild-type experimental group. These results indicate that the apoptosis-inducing properties of the compounds of this invention depend on both Hsp70 and Bim proteins, meaning that the compounds' apoptosis-inducing effect depends on the presence of the Hsp70 / Bim dimer.

[0159]

[0160] Example 14: Safety performance of the compounds of the present invention

[0161] The safety performance of the compounds of this invention was examined through an acute toxicity test in mice. This experiment uses the most representative compound 16 as an example to illustrate the experimental procedure.

[0162] At 9:00 AM, six male Balb / C mice (weighing 28-29g) were randomly divided into three groups. Their body weight was measured, and the average weight was calculated. The mice were fasted at 9:00 PM that evening, but water was allowed. Compound 16 was prepared and divided into three dosage groups: Group A (final concentration 3mg / kg), Group B (final concentration 5mg / kg), and control group C (50% DMSO (solvent)). The drugs were administered intraperitoneally according to body weight to the final experimental concentration. Group C received the same volume of DMSO solution. The drug administration conditions for each group are shown in Table 4.

[0163] Table 4. Body weight and drug dosage of mice in each group during acute toxicity test in mice.

[0164]

[0165] The injection of the drug was completed at 9:30 a.m. on the second day, and the weight of each group of mice was measured. Then, the weight of each group of mice was measured and the condition of the mice was observed at 9:30 a.m. every day. The experiment was carried out for 7 days.

[0166] The experimental results showed that mice in groups A, B, and C all exhibited lethargy after drug administration, but resumed normal eating approximately one hour later. Throughout the experiment, all six mice in groups A, B, and C were in good condition, with healthy fur, bright red eyes, no adverse reactions, no deaths, normal food and water intake, and significant weight gain. Daily weight changes in the mice are shown below. Figure 1 .

[0167] After weighing, the mice were euthanized by dislocation, dissected, and their liver tissue was collected for further experiments.

[0168] Anatomical observation: Six mice were dissected to observe the characteristics of their internal organs. No significant differences were found between the drug-treated groups (Groups A and B) and the control group (Group C). The kidneys and hearts were normal in color, bright red, and showed no toxicological signs. The livers were normal in color and shape, without deformities, and no toxicological symptoms were observed. As shown in Table 5, there was no significant difference in the average liver weight among the three groups, and liver weight was positively correlated with mouse body weight. Paraffin-embedded sections of liver tissue from Groups A and B showed no inflammatory reaction after HE staining.

[0169] Table 5. Liver and body weight of 6 mice dissected on day 7 after intraperitoneal injection.

[0170]

[0171] Acute toxicity test results in mice: A single injection of compound 16 at a dose of up to 5 mg / kg had no significant toxic side effects on mice. Dissection experiments further demonstrated that compound 16 had no toxic effects on the internal organs of mice; weight monitoring showed whether compound 16 had a significant effect on the weight of mice.

[0172] Compounds 14, 15, and 19 were all tested for acute toxicity in mice using the same method. The results showed that compounds 14, 15, and 19 had no toxicological effects on the internal organs of mice and had no significant effect on the weight of mice.

[0173] Example 15. The compound's inhibitory activity on the growth of leukemia cells, breast cancer cells, and lung cancer cells in a tumor-bearing mouse model.

[0174] Using a leukemia-bearing mouse model as an example, based on the results of acute toxicity tests, five groups of six-week-old nude mice (n=4 per group) were randomly selected. Human leukemia cell line K562 cells were subcutaneously transplanted into each group. One group served as a control, while the other groups were injected daily with 0.3 mg / kg of compounds 14-16 and 19, respectively. Specific experimental details are shown in Table 6.

[0175] Breast cancer and lung cancer tumor-bearing mouse models were constructed using the same method, and breast cancer cell line MCF7 cells and lung cancer cell line A549 cells were subcutaneously transplanted, respectively.

[0176] Table 6. Experimental findings on the in vivo inhibitory activity of compounds against leukemia in mice.

[0177]

[0178] Conclusion from mouse experiment: Figure 1 The experimental data showed that compound 16 of the present invention had no obvious toxic side effects on mice at doses below 5 mg / kg. When nude mice transplanted with human leukemia cells, breast cancer cells, and lung cancer cells were administered the drug at a dose of 0.3 mg / kg for 14 consecutive days, the tumor weight in the treated group was significantly smaller than that in the control group, and the tumor inhibition rate reached 50% (calculated by tumor weight). Figure 2 Moreover, their body weight did not change significantly compared to the control group.

[0179] Compounds 14-15 and 19 were used to determine their inhibitory activity against leukemia cells, breast cancer cells, and lung cancer cells in mouse models using the same method. The results showed that the tumor inhibition rates of compounds 14-15 and 19 were both above 50% (calculated based on tumor weight). Figure 2 The above results indicate that the compounds in this invention can effectively inhibit the growth of leukemia cells, breast cancer cells, and lung cancer cells, without significant toxic side effects, and can be effectively used for the prevention and treatment of leukemia, breast cancer, and lung cancer.

[0180] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of biphenyl compounds or their stereoisomers or pharmaceutically acceptable salts, characterized in that, The biphenyl compounds mentioned are selected from: 。 2. A pharmaceutical composition, characterized in that, It includes one or more of the biphenyl compounds of claim 1, their stereoisomers, or their pharmaceutically acceptable salts.

3. A single-dose pharmaceutical composition, characterized in that, It contains 1-1000 mg of one or more of the biphenyl compounds of claim 1, their stereoisomers, or their pharmaceutically acceptable salts.

4. A pharmaceutical formulation comprising the biphenyl compound of claim 1 or its stereoisomer or its pharmaceutically acceptable salt, the pharmaceutical composition of claim 2 or the single-dose pharmaceutical composition of claim 3, characterized in that, The pharmaceutical preparations include tablets, lozenges, drops, injectable preparations, granules, creams, ointments, suppositories, gels, or capsules.

5. The use of the biphenyl compound of claim 1 or its stereoisomer or pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 2 or the single-dose pharmaceutical composition of claim 3 in the preparation of a medicament for treating or preventing diseases that benefit from the regulation of Hsp70 protein / Bim protein dimer; The diseases mentioned are leukemia, breast cancer, or lung cancer.

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