A combination drug for treating triple negative breast cancer

Abstract

The present application relates to a kind of drugs for treating triple negative breast cancer and combination drug, belong to pharmaceutical field.The present application first finds out, targeting androgen receptor based on the protein degradation agent of ligand of E3 ligase of degree amine class can effectively prevent and / or treat triple negative breast cancer (including androgen receptor positive triple negative breast cancer), has good prospect in the preparation of drug for preventing and / or treating triple negative breast cancer.The present application first finds out, based on the protein degradation agent of ligand of E3 ligase of degree amine class androgen receptor is combined with targeted drug (including PI3K alpha inhibitor, PARP inhibitor), immunotherapy drug or chemotherapy drug can effectively prevent and / or treat triple negative breast cancer, has good prospect in the preparation of drug for preventing and / or treating triple negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and more specifically, this invention relates to a combination drug for treating triple-negative breast cancer. Background Technology

[0002] Breast cancer has become the most common malignant tumor in women worldwide, with the highest incidence and mortality rate among female cancers, seriously threatening and harming women's physical and mental health. Different types of breast cancer have significantly different biological characteristics and clinical manifestations. Currently, immunohistochemistry is mainly used clinically to classify breast cancer into four molecular subtypes based on the detection results of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2): luminal A (ER-positive or PR-positive, HER-2-negative, low Ki67 expression), luminal B (ER-positive or PR-positive, HER-2-positive), HER-2 overexpression type (ER and PR-negative, HER-2-positive, Ki67 mostly highly expressed), and basal-like type (ER, PR, and HER-2 all negative). Triple-negative breast cancer (TNBC), as the most malignant subtype of breast cancer, is characterized by negative ER, PR, and HER-2, and is characterized by high recurrence rate, high early metastasis rate, strong invasiveness, and poor prognosis.

[0003] Currently, chemotherapy is one of the main treatments for breast cancer. Cyclophosphamide, doxorubicin, and 5-fluorouracil (5-FU) have long been recognized as first-line chemotherapy drugs for advanced breast cancer. With the advent of drugs such as paclitaxel, more and more clinicians believe that paclitaxel and cisplatin can also be used as first-line chemotherapy regimens. Nevertheless, the therapeutic efficacy of these chemotherapy drugs still needs further improvement.

[0004] Androgen receptors (ARs) belong to the nuclear receptor family and are ligand-dependent transcription factors. Aberrant regulation of the AR signaling pathway plays a crucial role in the development and progression of prostate cancer; studies have shown that castration-resistant prostate cancer (CRPC) still depends on AR activity. Protein degradation-targeting chimeras (PROTACs), as small molecules capable of inducing target protein degradation, have attracted widespread attention. PROTACs, as bifunctional molecules, consist of a small molecule compound that binds to the target protein (POI), with a linker group introduced at an appropriate position, and then linked to a small molecule compound that binds to ubiquitin proteases. The resulting small probe can simultaneously bind to both the target protein and the ubiquitin protease, thereby promoting the ubiquitination of the target protein. The multiubiquitinated protein can then be recognized and degraded by the proteasome. Using the PROTACs strategy, protein degradation-targeting chimeras (AR PROTACs) capable of targeting and recognizing / binding androgen receptors were prepared. These can regulate androgen receptor levels through the intracellular ubiquitin-proteasome degradation system, induce androgen receptor degradation, and thus achieve therapeutic effects on androgen receptor-regulated diseases such as prostate cancer.

[0005] However, there are currently no reports of using androgen receptor protein degradation-targeting chimeras to treat triple-negative breast cancer. Summary of the Invention

[0006] One object of the present invention is to provide the use of a dynamamine-based E3 ligase ligand-targeting androgen receptor degrader in the preparation of a medicament for the prevention and / or treatment of triple-negative breast cancer.

[0007] Another object of the present invention is to provide the use of a dynoamine E3 ligase-based degrader targeting androgen receptors in combination with a targeted drug (including PI3Kα inhibitors, PARP inhibitors), an immunotherapy drug, or a chemotherapeutic drug in the preparation of a medicament for the prevention and / or treatment of triple-negative breast cancer.

[0008] Specifically, this invention provides the use of a degrading agent targeting androgen receptors based on amine E3 ligase ligands in the preparation of a medicament for the prevention and / or treatment of triple-negative breast cancer.

[0009] The present invention also provides the use of a degrading agent based on amine E3 ligase ligand targeting androgen receptors in combination with a targeted drug in the preparation of a drug for the prevention and / or treatment of triple-negative breast cancer.

[0010] Furthermore, the targeted drug is a PI3K inhibitor.

[0011] Furthermore, the PI3K inhibitor is a PI3Kα inhibitor.

[0012] Further, the PI3K inhibitor is the following compound, or its optical isomer, tautomer, salt, prodrug, hydrate, or solvate: Alpelisib, GDC-0077, TAK-117, AZD-8186, IPI-549, Idelalisib, Buparlisib, Pilaralisib, Copanlisib, PX-866, Paxalisib, Duvelisib, Umbralisib, Taselisib, Perifosine, Buparlisib, Dactolisib, CUDC-907, and Voxtalisib.

[0013] Furthermore, the targeted drug is a PARP inhibitor.

[0014] Furthermore, the PARP inhibitor is olaparib, rucaparib, tapolaparib, niraparib, pamiparib, or fluzoparib.

[0015] Furthermore, the targeted drug is Alpelisib, and the molar ratio of the androgen receptor degrader to Alpelisib is 1:27-3:1;

[0016] Alternatively, the targeted drug is Duvelisib, and the molar ratio of the androgen receptor degrader to Duvelisib is 1:30-1:10;

[0017] Alternatively, the targeted drug is Idelalisib, and the molar ratio of the androgen receptor degrader to Idelalisib is 1:270-1:90.

[0018] The present invention also provides the use of a degrading agent targeting androgen receptor based on amine E3 ligase ligand in combination with an immunotherapy drug in the preparation of a drug for the prevention and / or treatment of triple-negative breast cancer.

[0019] The present invention also provides the use of a degrading agent targeting androgen receptor based on amine E3 ligase ligand in combination with a chemotherapeutic drug in the preparation of a drug for the prevention and / or treatment of triple-negative breast cancer.

[0020] Furthermore, the chemotherapy drug is Paclitaxel or 5-Fluorouracil.

[0021] Furthermore, the chemotherapy drug is Paclitaxel, and the molar ratio of the androgen receptor degrader to Paclitaxel is 20:1-5000:1;

[0022] Alternatively, the chemotherapy drug is 5-Fluorouracil, and the molar ratio of the androgen receptor degrader to 5-Fluorouracil is 1.85:1 to 5.54:1.

[0023] Furthermore, the degrading agent targeting the androgen receptor is a PROTAC bifunctional chimeric molecule of formula (I), or its optical isomer, its tautomer, or its pharmaceutically acceptable salt:

[0024]

[0025] ARB is the ligand for the androgen receptor, L is the linker, and U is the ligand for the E3 ligase.

[0026] Furthermore, the U-type CRBN-based E3 ligase ligand has the following structure:

[0027]

[0028] Wherein, E represents unsubstituted and substituted benzene rings, five-membered aromatic heterocycles, six-membered aromatic heterocycles, benzo[a]aromatic heterocycles, and benzene-unsaturated heterocycles;

[0029] E X It is free of halogens, oxygen, sulfur, nitrogen, carbonyl, and -(CH2)-.

[0030] E Y None, H, carbonyl group.

[0031] Furthermore, the U is a CRBN-type E3 ligase ligand.

[0032] Furthermore, the structure of U is selected from:

[0033]

[0034]

[0035] Furthermore, the PROTAC bifunctional chimeric molecule is selected from ARV-110, ARV-766, HP518, AC0176, GT20029, ASN-1780, ARD-2128, and ARD-2585.

[0036] Furthermore, the structure of the PROTAC bifunctional chimeric molecule is shown in formula (II):

[0037]

[0038] Where X is selected from F, Cl, Br, Me, CF3;

[0039] Y is selected from CH or N;

[0040] W is selected from O, S, NMe;

[0041] Ring A is selected from R 1 R 2 R 3 and / or R 4 The following groups are substituted: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane;

[0042] R 1 R 2 R 3 R 4 R 5 Each is independently selected from hydrogen and Me;

[0043] Ring B is selected from 5-membered aromatic heterocycles, 6-membered aromatic rings, and 6-membered aromatic heterocycles;

[0044] G 1 G 2 G 3 Selected independently from CR 6 , N; where R 6 Selected from H, halogen, hydroxyl;

[0045] n1, n2, n3, n4, n5, and n6 are each independently selected from 1 or 2;

[0046] M is selected from CR 7 R 8 , where R 7 R 8 Each is independently selected from H and Me;

[0047] Z is selected from H, F, Cl, Me.

[0048] Furthermore, the structure of the PROTAC bifunctional chimeric molecule is selected from:

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Furthermore, the triple-negative breast cancer is androgen receptor-positive triple-negative breast cancer.

[0075] This invention is the first to discover that protein degraders targeting androgen receptors can effectively prevent and / or treat triple-negative breast cancer (including androgen receptor-positive triple-negative breast cancer), showing promising potential in the preparation of drugs for the prevention and / or treatment of triple-negative breast cancer. This invention is also the first to discover that the combined use of androgen receptor protein degraders with targeted drugs (including PI3Kα inhibitors, PARP inhibitors), immunotherapeutic drugs, or chemotherapeutic drugs can effectively prevent and / or treat triple-negative breast cancer, showing promising potential in the preparation of drugs for the prevention and / or treatment of triple-negative breast cancer.

[0076] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0077] In this invention, "aromatic ring" refers to an all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group with a conjugated π-electron system, such as phenyl and naphthyl. The aromatic ring may be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of connection to the parent group must be on a carbon atom of a ring with a conjugated π-electron system.

[0078] "Heteroaromatic ring" refers to a heteroaromatic group containing one or more heteroatoms. Heteratoms include oxygen, sulfur, and nitrogen. Examples include furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, and tetrazolyl. The heteroaromatic ring can be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaromatic ring.

[0079] In this invention, the degrading agent targeting the androgen receptor refers to the androgen receptor degrading agent.

[0080] "Pharmaceutical acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0081] "Salt" is an acidic and / or basic salt formed by a compound or its stereoisomer with an inorganic and / or organic acid and / or base, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compound. Alternatively, they can be obtained by mixing the compound, or its stereoisomer, with an appropriate amount (e.g., equimolar amounts) of an acid or base. These salts may form a precipitate in solution and be collected by filtration, or be recovered after solvent evaporation, or be prepared by freeze-drying after reaction in an aqueous medium. The salts described in this invention can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compound.

[0082] "Solvate" refers to a solvate formed by the compound of the present invention and a solvent, wherein the solvent includes (but is not limited to): water, ethanol, methanol, isopropanol, propylene glycol, tetrahydrofuran, and dichloromethane.

[0083] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0084] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0085] Figure 1 The effect of compound 279 and Alpelisib in inhibiting the growth of MDA-MB-453 cells.

[0086] Figure 2 The effect of compound 279 and Alpelisib in inhibiting the growth of MDA-MB-453 cells.

[0087] Figure 3 The effect of compound 281 and Alpelisib in inhibiting the growth of MDA-MB-453 cells.

[0088] Figure 4 The effect of compound 281 and Alpelisib in inhibiting the growth of MDA-MB-453 cells.

[0089] Figure 5 The effect of compound 282 and Alpelisib in inhibiting the growth of MDA-MB-453 cells.

[0090] Figure 6 The effect of compound 282 and Alpelisib in inhibiting the growth of MDA-MB-453 cells.

[0091] Figure 7 The effect of combined use of ARV-110 and Alpelisib on inhibiting the growth of MDA-MB-453 cells.

[0092] Figure 8 The effect of combined use of ARV-110 and Alpelisib on inhibiting the growth of MDA-MB-453 cells.

[0093] Figure 9 The effect of combined use of ARV-766 and Alpelisib on inhibiting the growth of MDA-MB-453 cells.

[0094] Figure 10The effect of combined use of ARV-766 and Alpelisib on inhibiting the growth of MDA-MB-453 cells.

[0095] Figure 11 The effect of compound 279 and Duvelisib in inhibiting the growth of MDA-MB-453 cells.

[0096] Figure 12 The effect of compound 279 and Duvelisib in inhibiting the growth of MDA-MB-453 cells.

[0097] Figure 13 The effect of compound 281 and Duvelisib in inhibiting the growth of MDA-MB-453 cells.

[0098] Figure 14 The effect of compound 281 and Duvelisib in inhibiting the growth of MDA-MB-453 cells.

[0099] Figure 15 The effect of compound 282 and Duvelisib in inhibiting the growth of MDA-MB-453 cells.

[0100] Figure 16 The effect of compound 282 and Duvelisib in inhibiting the growth of MDA-MB-453 cells.

[0101] Figure 17 The effect of combined use of ARV-110 and Duvelisib on inhibiting the growth of MDA-MB-453 cells.

[0102] Figure 18 The effect of combined use of ARV-110 and Duvelisib on inhibiting the growth of MDA-MB-453 cells.

[0103] Figure 19 The effect of compound 279 and Idelalisib in inhibiting the growth of MDA-MB-453 cells.

[0104] Figure 20 The effect of compound 279 and Idelalisib in inhibiting the growth of MDA-MB-453 cells.

[0105] Figure 21 The effect of compound 281 and Idelalisib in inhibiting the growth of MDA-MB-453 cells.

[0106] Figure 22The effect of compound 281 and Idelalisib in inhibiting the growth of MDA-MB-453 cells.

[0107] Figure 23 The effect of compound 282 and Idelalisib in inhibiting the growth of MDA-MB-453 cells.

[0108] Figure 24 The effect of compound 282 and Idelalisib in inhibiting the growth of MDA-MB-453 cells.

[0109] Figure 25 The effect of combined use of ARV-110 and Idelalisib on inhibiting the growth of MDA-MB-453 cells.

[0110] Figure 26 The effect of combined use of ARV-110 and Idelalisib on inhibiting the growth of MDA-MB-453 cells.

[0111] Figure 27 The effect of combined use of ARV-766 and Idelalisib on inhibiting the growth of MDA-MB-453 cells.

[0112] Figure 28 The effect of combined use of ARV-766 and Idelalisib on inhibiting the growth of MDA-MB-453 cells.

[0113] Figure 29 The effect of compound 279 and Paclitaxel in inhibiting the growth of MDA-MB-453 cells.

[0114] Figure 30 The effect of compound 279 and Paclitaxel in inhibiting the growth of MDA-MB-453 cells.

[0115] Figure 31 The effect of compound 281 and Paclitaxel in inhibiting the growth of MDA-MB-453 cells.

[0116] Figure 32 Compound 282 and Paclitaxel in combination inhibited the growth of MDA-MB-453 cells. Figure 33 The effect of combined use of ARV-110 and Paclitaxel on inhibiting the growth of MDA-MB-453 cells. Detailed Implementation

[0117] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0118] Example 1: Efficacy of androgen receptor protein degraders in the treatment of triple-negative breast cancer (in vitro experiment)

[0119] 1. Experimental Methods

[0120] Triple-negative breast cancer MDA-MB-453 cells in the logarithmic growth phase were harvested at a concentration of 4 × 10⁻⁶ cells / cells. 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates. The plates were incubated at 37°C, 5% CO2, and saturated humidity for 24 or 48 hours. The negative control wells were added with the same volume of RPMI 1640 cell culture medium containing 10% fetal bovine serum. After incubation for 24 or 48 hours, AR PROTACs compound 162, compound 177, compound 230, compound 259, compound 279, compound 281, compound 282, compound 325, compound 357, compound 400, ARV-110, ARV-766, and the AR inhibitor HC-1119 were added to the 96-well plates, respectively. The concentrations of compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766 were 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM, or 10 μM; the concentrations of HC-1119 were 0 μM, 1.23 μM, 3.70 μM, 11.11 μM, 33.33 μM, or 100 μM.

[0121] After incubating with the drug for 7-10 days, add 10 μL of CCK-8 to each well and continue incubation for 2 hours. Measure the absorbance (OD) of each well using a microplate reader at a wavelength of 450 nm. Calculate the survival rate using Prism software: relative survival rate = (OD value of drug-treated wells / OD value of control wells) × 100%. Alternatively, discard 100 μL of culture medium from each well and add 50 μL of CTG. Shake in the dark for 2 min, incubate for 10 min, and read the values ​​using a microplate reader in Luminescence mode. Calculate the survival rate using Prism software: relative survival rate = (RLU value of drug-treated wells / RLU value of control wells) × 100%.

[0122] 2. Experimental Results

[0123] (1) IC50 values ​​of compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766 on MDA-MB-453 cells 50Measurement

[0124] IC50 values ​​of compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766 on MDA-MB-453 cells 50 The results are shown in Table 1. The judgment criteria are as follows: when IC50 ≤ 2 μM, it is labeled as A; when IC50 > 2 μM, it is labeled as B. Table 1 shows that compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766 have varying degrees of inhibitory effects on the growth of MDA-MB-453 cells and can be used for the effective treatment of triple-negative breast cancer.

[0125] Table 1

[0126]

[0127] (2) IC50 of HC-1119 on MDA-MB-453 cells 50 Measurement

[0128] IC50 of HC-1119 on MDA-MB-453 cells 50 The test results are shown in Table 1. The judgment criteria are as follows: when IC50 ≤ 2 μM, it is labeled as A; when IC50 > 2 μM, it is labeled as B. Table 1 shows that HC-1119 has a poor inhibitory effect on the growth of MDA-MB-453 cells, indicating that the AR inhibitor HC-1119 is significantly less effective in treating triple-negative breast cancer than AR PROTACs compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766.

[0129] Example 2: Efficacy of combined use of androgen receptor protein degrader and PI3K inhibitor Alpelisib in the treatment of triple-negative breast cancer (in vitro experiment)

[0130] 1. Experimental Methods

[0131] Triple-negative breast cancer MDA-MB-453 cells in the logarithmic growth phase were harvested at a concentration of 4 × 10⁻⁶ cells / cells. 3Cells were seeded at a concentration of [number] cells / well in 96-well plates. The plates were incubated at 37°C, 5% CO2, and saturated humidity for 24 hours. The negative control wells were added with the same volume of RPMI 1640 cell culture medium containing 10% fetal bovine serum. After 24 hours of incubation, ARPROTACs compounds 279, 281, and 282, ARV-110, ARV-766, and the PI3K inhibitor Alpelisib were sequentially added to the 96-well plates.

[0132] The concentrations of compounds 279, 281, 282, ARV-110, and ARV-766 were 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM, or 10 μM; the concentrations of Alpelisib were 0 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM, or 10 μM.

[0133] After incubating with the drug for 7 days, add 10 μL of CCK-8 to each well and continue incubation for 2 hours. Measure the absorbance (OD) of each well using a microplate reader at a wavelength of 450 nm. Calculate the survival rate using Prism software: relative survival rate = (OD value of drug-treated wells / OD value of control wells) × 100%. Alternatively, discard 100 μL of culture medium from each well and add 50 μL of CTG. Shake in the dark for 2 min, incubate for 10 min, and read the values ​​using a microplate reader in Luminescence mode. Calculate the survival rate using Prism software: relative survival rate = (RLU value of drug-treated wells / RLU value of control wells) × 100%. Then, determine the drug combination effect using the Combination Index (CI). The determination criteria are as follows: when CI < 1, it indicates that the combined drug treatment has a synergistic effect.

[0134] 2. Experimental Results

[0135] (1) Results of combined use of compounds 279, 281, 282, ARV-110, ARV-766 and Alpelisib: Figures 1 to 10 As shown, when the molar ratio of compounds 279, 281, 282, ARV-110, ARV-766, and Alpelisib is 1:27 to 9:1, the combined use of these compounds has a synergistic effect in inhibiting the growth of MDA-MB-453 cells and can be used for the synergistic treatment of triple-negative breast cancer.

[0136] Example 3: Efficacy of combined use of androgen receptor protein degrader and PI3K inhibitor Duvelisib in the treatment of triple-negative breast cancer (in vitro experiment)

[0137] 1. Experimental Methods

[0138] Triple-negative breast cancer MDA-MB-453 cells in the logarithmic growth phase were harvested at a concentration of 4 × 10⁻⁶ cells / cells. 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates. The plates were incubated at 37°C, 5% CO2, and saturated humidity for 24 hours. The negative control wells were added with the same volume of RPMI 1640 cell culture medium containing 10% fetal bovine serum. After 24 hours of incubation, ARPROTACs compounds 279, 281, and 282, ARV-110, and the PI3K inhibitor Duvelisib were sequentially added to the 96-well plates.

[0139] The concentrations of compounds 279, 281, 282, and ARV-110 were: 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, and 0.370 μM, respectively.

[0140] 1.111 μM, 3.333 μM or 10 μM; Duvelisib concentrations are: 0 μM, 1.23 μM, 3.70 μM, 11.11 μM, 33.33 μM or 100 μM.

[0141] After incubating with the drug for 7-10 days, add 10 μL of CCK-8 to each well and continue incubation for 2 hours. Measure the absorbance (OD) of each well using a microplate reader at a wavelength of 450 nm. Calculate the survival rate using Prism software: relative survival rate = (OD value of drug-treated wells / OD value of control wells) × 100%. Alternatively, discard 100 μL of culture medium from each well and add 50 μL of CTG. Shake in the dark for 2 min, incubate for 10 min, and read the values ​​using a microplate reader in Luminescence mode. Calculate the survival rate using Prism software: relative survival rate = (RLU value of drug-treated wells / RLU value of control wells) × 100%. Then, determine the drug combination effect using the Combination Index (CI). The determination criteria are as follows: when CI < 1, it indicates that the combined drug treatment has a synergistic effect.

[0142] 2. Experimental Results

[0143] The results of combined use of compounds 279, 281, 282, ARV-110, and Duvelisib are as follows: Figures 11 to 18As shown, when the molar ratio of compounds 279, 281, 282, ARV-110, and Duvelisib is 1:30 to 1:1.111, the combined use of these compounds has a synergistic effect in inhibiting the growth of MDA-MB-453 cells and can be used for synergistic treatment of triple-negative breast cancer.

[0144] Example 4: Efficacy of combined use of androgen receptor protein degrader and PI3K inhibitor Idelalisib in the treatment of triple-negative breast cancer (in vitro experiment)

[0145] 1. Experimental Methods

[0146] Triple-negative breast cancer MDA-MB-453 cells in the logarithmic growth phase were harvested at a concentration of 4 × 10⁻⁶ cells / cells. 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates. The plates were incubated at 37°C, 5% CO2, and saturated humidity for 24 hours. The negative control wells were added with the same volume of RPMI 1640 cell culture medium containing 10% fetal bovine serum. After 24 hours of incubation, ARPROTACs compounds 279, 281, and 282, ARV-110, ARV-766, and the PI3K inhibitor Idelalisib were sequentially added to the 96-well plates.

[0147] The concentrations of compounds 279, 281, 282, ARV-110, and ARV-766 were 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM, or 10 μM; the concentrations of Idelalisib were 0 μM, 1.23 μM, 3.70 μM, 11.11 μM, 33.33 μM, or 100 μM.

[0148] After incubating with the drug for 7-10 days, add 10 μL of CCK-8 to each well and continue incubation for 2 hours. Measure the absorbance (OD) of each well using a microplate reader at a wavelength of 450 nm. Calculate the survival rate using Prism software: relative survival rate = (OD value of drug-treated wells / OD value of control wells) × 100%. Alternatively, discard 100 μL of culture medium from each well and add 50 μL of CTG. Shake in the dark for 2 min, incubate for 10 min, and read the values ​​using a microplate reader in Luminescence mode. Calculate the survival rate using Prism software: relative survival rate = (RLU value of drug-treated wells / RLU value of control wells) × 100%. Then, determine the drug combination effect using the Combination Index (CI). The determination criteria are as follows: when CI < 1, it indicates that the combined drug treatment has a synergistic effect.

[0149] 2. Experimental Results

[0150] The results of combined use of compounds 279, 281, 282, ARV-110, ARV-766, and Idelalisib are as follows: Figures 19 to 28 As shown, when the molar ratio of compounds 279, 281, 282, ARV-110, ARV-766, and Idelalisib is 1:270 to 1:3.333, the combined use of these compounds exhibits a synergistic effect in inhibiting the growth of MDA-MB-453 cells, and may be used for the synergistic treatment of triple-negative breast cancer.

[0151] Example 5: Efficacy of combined use of androgen receptor protein degrader and chemotherapy drug Paclitaxel in the treatment of triple-negative breast cancer (in vitro experiment)

[0152] 1. Experimental Methods

[0153] Triple-negative breast cancer MDA-MB-453 cells in the logarithmic growth phase were harvested at a concentration of 4 × 10⁻⁶ cells / cells. 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates. The plates were incubated at 37°C, 5% CO2, and saturated humidity for 24 hours. The negative control wells were added with the same volume of RPMI 1640 cell culture medium containing 10% fetal bovine serum. After 24 hours of incubation, ARPROTACs compounds 279, 281, and 282, ARV-110, and the chemotherapy drug Paclitaxel were sequentially added to the 96-well plates.

[0154] The concentrations of compounds 279, 281, 282, and ARV-110 were: 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, and 0.370 μM, respectively.

[0155] 1.111 μM, 3.333 μM or 10 μM; Paclitaxel concentrations are: 0 nM, 0.025 nM, 0.074 nM, 0.222 nM, 0.667 nM or 2 nM.

[0156] After incubating with the drug for 7 days, add 10 μL of CCK-8 to each well and continue incubation for 2 hours. Measure the absorbance (OD) of each well using a microplate reader at a wavelength of 450 nm. Calculate the survival rate using Prism software: relative survival rate = (OD value of drug-treated wells / OD value of control wells) × 100%. Alternatively, discard 100 μL of culture medium from each well and add 50 μL of CTG. Shake in the dark for 2 min, incubate for 10 min, and read the values ​​using a microplate reader in Luminescence mode. Calculate the survival rate using Prism software: relative survival rate = (RLU value of drug-treated wells / RLU value of control wells) × 100%. Then, determine the drug combination effect using the Combination Index (CI). The determination criteria are as follows: when CI < 1, it indicates that the combined drug treatment has a synergistic effect.

[0157] 2. Experimental Results

[0158] The results of combined use of compounds 279, 281, 282, ARV-110, and Paclitaxel are as follows: Figures 19 to 33 As shown, when the molar ratio of compounds 279, 281, 282, ARV-110, and Paclitaxel is 20:1 to 5000:1, the combined use of these compounds has a synergistic effect in inhibiting the growth of MDA-MB-453 cells and can be used for synergistic treatment of triple-negative breast cancer.

[0159] In summary, this invention is the first to discover that protein degraders targeting androgen receptors can effectively prevent and / or treat triple-negative breast cancer (including androgen receptor-positive triple-negative breast cancer), showing promising potential in the preparation of drugs for the prevention and / or treatment of triple-negative breast cancer. This invention is also the first to discover that the combined use of androgen receptor protein degraders with targeted drugs (including PI3Kα inhibitors and PARP inhibitors), immunotherapeutic drugs, or chemotherapeutic drugs can effectively prevent and / or treat triple-negative breast cancer, showing promising potential in the preparation of drugs for the prevention and / or treatment of triple-negative breast cancer.

Claims

1. The use of a degrader targeting androgen receptor based on a dynoamine E3 ligase ligand in combination with a targeted drug in the preparation of a drug for treating triple-negative breast cancer, characterized in that: The targeted drug is a PI3K inhibitor; The degrader targeting androgen receptors is a PROTAC bifunctional chimeric molecule, which is selected from ARV-110 or compounds with the following structures: ； The PI3K inhibitor is the following compound or its salt: Alpelisib, Duvelisib, Idelalisib; The triple-negative breast cancer mentioned above is androgen receptor-positive triple-negative breast cancer. When the targeted drug is Alpelisib, the degrading agent targeting the androgen receptor is compound 279 or compound 281, and the molar ratio of the degrading agent targeting the androgen receptor to Alpelisib is 3:

1. When the targeted drug is Duvelisib, the degrading agent targeting the androgen receptor is compound 279 or compound 281, the molar ratio of compound 279 to Duvelisib is 1:10, and the molar ratio of compound 281 to Duvelisib is 10:11.

11. When the targeted drug is Idelalisib, the degrading agent targeting the androgen receptor is compound 281, compound 282, or ARV-110, the molar ratio of compound 281 to Idelalisib is 10:33.33, the molar ratio of compound 282 to Idelalisib is 3.333:33.33 or 10:33.33, and the molar ratio of ARV-110 to Idelalisib is 3.333:33.33 or 10:33.

33.

2. The use according to claim 1, characterized in that: When the targeted drug is Alpelisib, the degrading agent targeting the androgen receptor is compound 279, and the molar ratio of the degrading agent targeting the androgen receptor to Alpelisib is 3:

1.

3. The use of a degrading agent targeting androgen receptors based on amine E3 ligase ligands in combination with chemotherapeutic drugs in the preparation of drugs for the prevention and / or treatment of triple-negative breast cancer, characterized in that: The chemotherapy drug is Paclitaxel; The degrader targeting androgen receptors is a PROTAC bifunctional chimeric molecule, which is selected from compounds with the following structures: ； The triple-negative breast cancer mentioned above is androgen receptor-positive triple-negative breast cancer. The molar ratio of the androgen receptor degrader to Paclitaxel is 20:1-5000:

1.

4. The use according to claim 3, characterized in that: The degrading agent targeting the androgen receptor is compound 279, and the molar ratio of compound 279 to Paclitaxel is 0.041 μM: 2 nM.

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