Small molecule compound for targeted degradation of Pol theta and application thereof

Abstract

The invention discloses a small molecular compound for targeted degradation of Pol theta, and a salt, a pharmaceutical composition and an application thereof. The structure of the small molecule compound is shown as a formula I, the small molecule compound can effectively degrade Pol theta protein and selectively kill BRCA1 / 2 mutant tumor cells, a new candidate drug is provided for treatment of BRCA1 / 2 mutant tumors, and a new thought is provided for development of targeted Pol theta tumor drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a PROTAC small molecule compound and its salt, pharmaceutical composition and application, and in particular to a PROTAC small molecule compound and its salt, pharmaceutical composition and application for targeting the degradation of Polθ. Background Art

[0002] DNA polymerase θ (Polθ) is a member of the DNA polymerase A family and is encoded by the POLQ gene in mammalian cells. Polθ is a key enzyme in the microhomology end joining (MMEJ) pathway of DNA double-strand break (DSB) repair. As an alternative pathway for DNA double-strand break repair, the MMEJ pathway, in which Polθ participates, is inactive in cells with a normally functioning homologous recombination (HR) repair pathway. However, in BRCA1 / 2 mutant tumor cells, due to loss of BRCA1 / 2 function and the loss of the HR repair pathway, the MMEJ pathway plays a key role in DNA double-strand break repair. The survival of BRCA1 / 2 mutant tumor cells is highly dependent on MMEJ-mediated DNA repair. Therefore, inhibiting Polθ in homologous recombination repair-deficient (HRD) tumor cells can induce a "synthetic lethality" effect.

[0003] Polθ is a "synthetic lethality" target for BRCA1 / 2 mutant tumors, and the development of its inhibitors has been relatively slow. Currently, many inhibitors targeting Polθ polymerase and helicase activity are in the clinical or preclinical research stage, and no related drugs have yet been marketed. Summary of the Invention

[0004] Objectives of the invention: The first objective of the present invention is to provide a small molecule compound targeting Polθ polymerase based on the PROTAC strategy, the second objective is to provide a pharmaceutically acceptable salt of the small molecule compound, the third objective is to provide a pharmaceutical composition containing the small molecule compound or its salt, and the fourth objective is to provide a pharmaceutical application of the small molecule compound or its salt and the pharmaceutical composition.

[0005] Technical solution: The small molecule compound targeted for degradation of Polθ of the present invention has a structure of formula I,

[0006]

[0007] Wherein, R1 is selected from halogen; R2 and R3 are selected from C 1-6 Alkyl, halogenated C 1-6 alkyl;

[0008] R4 is selected from any one of the following structures:

[0009]

[0010] Linker is selected from any of the following structures:

[0011] n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0012] R5 is selected from any one of the following structures:

[0013]

[0014] Protein targeting chimera (PROTAC) is a bifunctional molecule that can target target proteins for ubiquitination degradation. Structurally, PROTAC mainly consists of a linker that covalently links the target protein ligand and the E3 ligase ligand to form a ternary complex, which degrades the target protein through ubiquitination.

[0015] Unlike current polymerase and helicase inhibitors, the present invention utilizes the targeted protein degradation technology "PROTAC" to design small molecule drugs that degrade Polθ at the protein level. It can effectively degrade Polθ and selectively kill BRCA1 / 2 mutant tumor cells, providing a new candidate drug for the treatment of BRCA1 / 2 mutant tumors and a new idea for the development of tumor drugs targeting Polθ.

[0016] Preferably, in the structure, R1 is selected from fluorine, chlorine, and iodine; R2 and R3 are selected from C 1-4 Alkyl, halogenated C 1-4 alkyl;

[0017] Linker is selected from any of the following structures:

[0018] n is selected from 1, 2, and 3.

[0019] More preferably, in the structure, R2 and R3 are selected from methyl, ethyl, trifluoromethyl, and trifluoroethyl.

[0020] Preferably, in the structure, R1 is selected from fluorine; R2 and R3 are selected from trifluoromethyl;

[0021] Linker is selected from any of the following structures:

[0022]

[0023] Preferably, the PROTAC small molecule compound is selected from any one of the following compounds:

[0024]

[0025] The pharmaceutically acceptable salt of the small molecule compound targeted for degradation of Polθ described in the present invention is a salt formed by the small molecule compound targeted for degradation of Polθ and any one of the following acids: hydrochloric acid, carbonic acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and ferulic acid.

[0026] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, Citric acid, tartaric acid, methanesulfonic acid and similar acids; organic acid salts also include salts of amino acids (such as arginine, etc.), glucuronic acid and other organic acids. Certain specific compounds of the present invention contain basic and acidic functional groups and can be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner and the parent compound is separated to regenerate the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

[0027] The "pharmaceutically acceptable salts" of the present invention can be synthesized from parent compounds containing acid or basic groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0028] The pharmaceutical composition of the present invention comprises the PROTAC small molecule compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0029] The pharmaceutically acceptable carrier can be an excipient widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, so that the active ingredient dissolves at a desired rate after administration to the subject, or promotes effective absorption of the active ingredient after administration of the composition to the subject. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0030] The pharmaceutical composition of the present invention can be prepared by any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, and lyophilizing.

[0031] The pharmaceutical compositions of the present invention can be administered in any form, including transmucosal, oral (solid and liquid formulations), inhalation, ophthalmic, rectal, topical, and parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention can also be in a controlled-release or sustained-release dosage form (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, hard capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or transmucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, and drops. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other formulation forms include, but are not limited to, eye drops, other ophthalmic formulations; aerosols, such as nasal sprays, inhalers; and suppositories and lozenges suitable for parenteral administration.

[0032] The small molecule compound targeted for degradation of Polθ or its pharmaceutically acceptable salt and pharmaceutical composition described in the present invention are used in the preparation of anti-tumor drugs.

[0033] Preferably, the drug is an anti-BRCA1 / 2 mutant tumor drug.

[0034] More preferably, the drug is a drug for ovarian cancer, colon cancer, or breast cancer with BRCA1 / 2 mutations.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0036] The small molecule compound designed in the present invention for the targeted degradation of Polθ recognizes and binds to the target protein by forming a ternary complex and performs ubiquitination, thereby effectively degrading the Polθ protein; and after targeting the Polθ protein, it aggravates the DNA damage of BRCA1-deficient cells and selectively kills BRCA1 / 2 mutant tumor cells, providing a new candidate drug for the treatment of BRCA1 / 2 mutant tumors and a new idea for the development of tumor drugs targeting Polθ. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The degradation effect of the compound of the present invention on Polθ protein;

[0038] Figure 2 The inhibitory effect of the compounds of the present invention on tumor cell proliferation;

[0039] Figure 3 This is the result of the effect of the compound of the present invention on the expression of γH2AX in tumor cells;

[0040] Figure 4 This is the result of the effect of the compounds of the present invention on inducing ubiquitination of Polθ protein;

[0041] Figure 5 This is the result of the effect of the compounds of the present invention on the expression level of γH2AX-positive micronuclei in tumor cells;

[0042] Figure 6 This is the result of the mechanism of action of the compounds of the present invention in targeting and degrading Polθ protein. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0044] The general method for synthesizing the compound is as follows:

[0045]

[0046] Example 1: Ethyl 4-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)but-2-yn-1-yl-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)carbamate (Compound PP5-12)

[0047]

[0048] (1) Preparation of tert-butyl (2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)carbamate (Intermediate 2)

[0049] 2-(2,6-Dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (0.1 g, 0.36 mmol), a mono-Boc diamino derivative (0.36 mmol) and DIPEA 0.1 mL were added to DMF 5 mL and reacted at 90°C for 4-5 h. 50 mL of water was added and the mixture was extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol: dichloromethane = 1:20) to obtain intermediate 2, 70 mg, with a yield of 70%.

[0050] 'H NMR (300MHz, CDCl3) δ8.75 (s, 1H), 7.60-7.49 (m, 1H), 7.15 (d, J=7.1Hz, 1H), 6.98 (d, J=8.5Hz, 1H), 6.55 (t, J=5.6Hz, 1H), 5.36 (s, 1H), 5.17 (s, 1H), 4.98 (q, J=5.3Hz, 1H), 4.17 (q, J=7.1Hz, 5H), 3.78 (t, J=5.2Hz, 2H), 3.73 (s, 4H), 3.71-3.64 (m, 2H), 3.63-3.47 (m, 2H), 3.52 (s, 3H), 3. 36 (d, J=6.1Hz, 3H), 2.99-2.74 (m, 3H), 2.22-2.12 (m, 1H), 2.10 (s, 7H), 1.31 (t, J=7.1Hz, 8H).

[0051] (2) Preparation of 4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Intermediate 3)

[0052] To a solution of intermediate 2 (0.1 g) in dichloromethane was added trifluoroacetic acid, and the mixture was stirred at room temperature overnight. The mixture was evaporated to dryness under reduced pressure to obtain the crude intermediate 3, which was used directly in the next step.

[0053] (3) Preparation of 4-((tert-butyldiphenylsilyloxy)but-2-yn-1-ol (Intermediate 5):

[0054] To a DMF solution of compound 4 (1 g, 11.6 mmol) and imidazole (0.32 g, 1 mmol) was slowly added tert-butyl(chloro)diphenylsilane (58 mmol), and the reaction was allowed to rise from 0°C to room temperature for 3-4 h. Water and ethyl acetate were added to the reaction solution, and the mixture was extracted three times. The organic phase was dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and then purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to afford 780 mg of intermediate 5 in a 78% yield.

[0055] 1H NMR (300MHz, CDCl3) δ7.75 (dq, J=6.3, 2.3Hz, 1H), 7.54-7.33 (m, 2H), 4.39 (t, J=1.7Hz, 0H), 4.23 (t, J=1.8Hz, 0H), 1.09 (s, 2H).

[0056] Preparation of (4) ((4-bromobut-2-yn-1-yl)oxy) (tert-butyl) diphenylsilane (Intermediate 6)

[0057] To a dichloromethane solution of intermediate 5 (400 mg, 1.23 mmol) and CBr4 (1.38 mmol) was added PPh3 (1.38 mmol) in batches over 30 min at 0°C, followed by triethylamine (1.38 mmol). The mixture was allowed to react at room temperature for 3-4 h. The reaction solution was extracted three times with water and dichloromethane. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) under reduced pressure to give intermediate 6, 380 mg, in a yield of 95%.

[0058] (5) Preparation of N-(4-((tert-butyldiphenylsilyloxy)but-2-yn-1-yl)-4-fluoroaniline (Intermediate 7)

[0059] To a DMF solution of potassium carbonate (3.09 mmol), potassium iodide (1.03 mmol), and intermediate 6 (400 mg, 1.03 mmol) was slowly added 4-fluoroaniline (1.03 mmol) and allowed to react at room temperature. After completion of the reaction, the reaction solution was extracted three times with water and ethyl acetate, and evaporated to dryness under reduced pressure and subjected to silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to obtain intermediate 7, 240 mg, with a yield of 60%.

[0060] 1H NMR (300MHz, CDCl3) δ7.80-7.63 (m, 1H), 7.42 (ddd, J=14.2, 7.7, 6.1Hz, 1H), 6.93 (t, J=8.7Hz , 0H), 6.58 (dd, J=9.0, 4.4Hz, 0H), 4.33 (t, J=1.9Hz, 1H), 3.86 (t, J=1.9Hz, 0H), 1.07 (s, 2H).

[0061] (6) Preparation of 2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-((tert-butyldiphenylsilyloxy)but-2-yn-1-yl)-N-(4-fluorophenyl)acetamide (Intermediate 8)

[0062] To a DMF solution of intermediate 7 (1 g, 2.4 mmol), 2,4-bis(trifluoromethyl)phenylacetic acid (4.7 mmol), HATU (4.7 mmol), and DMAP (0.72 mmol) was added DIPEA (4.7 mmol) and reacted at room temperature for 4 h. The reaction solution was extracted three times with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to obtain intermediate 8, 600 mg, with a yield of 60%.

[0063] 1H NMR (300MHz, CDCl3) δ7.87 (s, 1H), 7.78 (dd, J=8.2, 1.9Hz, 1H), 7.73-7.65 (m, 4H), 7.54 (d, J=8.1Hz, 1H), 7.49-7.34 (m, 7H ), 7.27 (dp, J=5.5, 3.4Hz, 2H), 7.19-7.07 (m, 2H), 4.50 (t, J=2.0Hz, 2H), 4.31 (t, J=1.9Hz, 2H), 3.60 (s, 2H), 1.07 (s, 9H).

[0064] (7) Preparation of 2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)-N-(4-hydroxybut-2-yn-1-yl)acetamide (Intermediate RP)

[0065] To a THF solution of Intermediate 8 (1.28 g, 1.9 mmol) was added TBAF (3.8 mmol) and acetic acid (4 ml) and the mixture was allowed to stand at room temperature overnight. The reaction mixture was neutralized with saturated sodium bicarbonate and extracted three times with water and ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. Silica gel column chromatography (petroleum ether:ethyl acetate = 4:1) was performed to obtain Intermediate 9, 896 mg, in a 70% yield.

[0066] 1 H NMR (300MHz, CDCl3) δ7.92 (s, 1H), 7.84 (d, J=8.1Hz, 1H), 7.59 (d, J=8.1Hz, 1H), 7.44-7.32 (m, 2H), 7.24 (dt, J=8.5, 4.3Hz, 2H), 4.57 (d, J=2.2Hz, 2H), 4.33-4.26 (m, 2H), 3.67 (s, 2H).

[0067] (8) Preparation of 4-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)but-2-yn-1-yl-4-nitrobenzoate (Intermediate 10)

[0068] To a dichloromethane solution of intermediate RP (50 mg, 0.115 mmol) were added triethylamine (0.23 mmol) and 4-nitrophenyl chloroformate (0.23 mmol), and the reaction was carried out at room temperature for 12 h. The reaction solution was extracted three times with water and dichloromethane. The organic phase was dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain intermediate 10, 30 mg, with a yield of 60%.

[0069] (9) Preparation of ethyl 4-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetylamino)but-2-yn-1-yl-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)carbamate (Compound PP5-12)

[0070] Intermediate 10 (50 mg, 0.083 mmol), intermediate 3 (0.083 mmol), and DIPEA (0.66 mmol) were dissolved in 400 μl of DMF and reacted at room temperature for 12 h. The mixture was then extracted three times with water and ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to afford compound PP5-12, 20 mg, in a 40% yield.

[0071] Using similar operations as in Example 1, the following compound was obtained:

[0072]

[0073]

[0074] Example 2: Evaluation of the Degradation Effect of Compounds on Polθ Protein

[0075] (1) Experimental materials

[0076] RIPA Lysis Buffer: 1 M Tris, pH 8.0, 0.5 mM EDTA, 5 M NaCl, NP-40, 10% Sodium deoxycholate, 0.1% SDS, 1× protease inhibitor cocktail.

[0077] Compound working solution: Add the test compound to 100% DMSO and dilute to the desired concentration.

[0078] (2) Experimental steps

[0079] 1) One day before the experiment, A2780 and KGN cells were plated so that the cell density was 50% to 60% on the next day.

[0080] 2) Four compounds were added to each well to give final concentrations of 0 μM, 1 μM, 5 μM, 10 μM, and 20 μM, and the cells were treated for 24 h.

[0081] 3) Harvest cells: Aspirate the old culture medium, wash twice with PBS, and add quantitative RIPA lysis buffer (1M Tris, pH 8.0, 0.5mM EDTA, 5M NaCl, NP-40, 10% sodium deoxycholate, 0.1% SDS, 1x protease inhibitor cocktail). Scrape the cells with a cell scraper, sonicate on ice for 32 seconds, and let them rest on ice for 30 minutes. Centrifuge at 12,000 rpm for 15 minutes, remove the supernatant, and boil the sample for 6 minutes.

[0082] 4) A total of 20 μg of protein (40 μg for standard immunoblotting experiments) was loaded onto an SDS-PAGE gel and then transferred to a nitrocellulose membrane. The nitrocellulose membrane was then blocked with Tris-buffered saline (BioRad) containing 0.1% Tween-20 and 5% milk. The membrane was incubated with the primary antibody overnight at 4°C and with the secondary antibody for 1 hour at room temperature. The blot was developed using enhanced chemiluminescence and exposed to autoradiographic film.

[0083] (3) Experimental results

[0084] like Figure 1 As shown in the results, compounds PP5-12, PPQC, and PP3-0 can effectively degrade Polθ protein, and the degradation effect is concentration-dependent.

[0085] Example 3: Evaluation of the inhibitory effect of compounds on tumor cell proliferation

[0086] (1) Experimental methods

[0087] A2780 shBRCA1-1 / 2 cell line was constructed and BRCA1 knockout was verified.

[0088] Cells were seeded in 24-well or 6-well plates. At the end of the group-addition assay, the cell colonies were stained with methylene blue or crystal violet and counted.

[0089] (2) Experimental results

[0090] like Figure 2 As shown in Figure c, BRCA1 was knocked out; Figure 2 As shown in b, PP5-12 compound can significantly inhibit cell proliferation (**P<0.01).

[0091] Compound PP5-12 has a significant selective inhibitory effect on the proliferation of BRCA1 / 2 mutated tumor cells, and is particularly superior to the intermediate RP, indicating that the PROTAC strategy adopted in the present invention is very effective.

[0092] Example 4: Evaluation of the regulatory effect of compounds on γH2AX expression in tumor cells

[0093] (1) Experimental methods

[0094] Cells were seeded in 6-well plates and treated the next day with equal concentrations of ART558, RP, PP5-12, PP3-0, and PP4-0 for 24 hours. The cells were then harvested. A total of 20 μg of protein (40 μg for standard immunoblotting experiments) was loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes. The nitrocellulose membranes were then blocked with Tris-buffered saline (BioRad) containing 0.1% Tween-20 and 5% milk. The membranes were incubated with the primary antibody overnight at 4°C and with the secondary antibody for 1 hour at room temperature. The blots were developed using enhanced chemiluminescence and exposed to autoradiographic film.

[0095] (2) Experimental results

[0096] like Figure 3 As shown in the results, compound PP5-12 can significantly upregulate the expression level of γH2AX in BRCA1 / 2 mutant tumor cells (**P < 0.01), and can exert an anti-tumor effect; in particular, it is better than the intermediate RP and the Polθ inhibitor ART558, indicating that the compound designed by the PROTAC strategy adopted in the present invention is better than the Polθ inhibitor ART558 and the intermediate RP.

[0097] Example 5: Evaluation of the induction effect of compounds on Polθ protein ubiquitination

[0098] (1) Experimental methods

[0099] For immunoprecipitation, 293T cells were left untreated (DMSO) or treated with the Polθ PROTAC degrader PP5-12 for 24 h and incubated with 20 μM MG132 for 4 to 6 hours. The cells were then lysed with EBC lysis buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 1 mM N-ethylmaleimide, 2 mM Na3VO4, 20 mM NaF, 1 mM PMSF, and 1× protease inhibitor cocktail). The cell lysate was sonicated and clarified by centrifugation at 12,000 g for 15 minutes at 4°C. The supernatant (1 mg) was pre-incubated with beads for pre-washing and then incubated with 1 μG of the corresponding antibody and beads overnight at 4°C. The beads were washed three times with 1 ml of Triton X-100 lysis buffer. Immune complexes were eluted from the beads by boiling at 95°C for 10 minutes and analyzed by immunoblotting. For ubiquitination analysis, 293T cells were transfected with HA-Ub plasmid using Lipofectamine 2000 for 24 hours and then left untreated (DMSO) or treated with EBC buffer as described above. Standard immunoprecipitation procedures were then performed.

[0100] (2) Experimental results

[0101] like Figure 4 As shown in the results, compound PP5-12 can significantly induce the ubiquitination of Polθ protein, thereby exerting protein degradation, and the induction effect is concentration-dependent.

[0102] Case Study 6: Evaluation of the Effects of Compounds on the Expression Levels of γH2AX-Positive Micronuclei in Tumor Cells

[0103] (1) Experimental methods

[0104] Cell plating: Add cell slides to 12-well plates one day in advance and plate A2780 cells and A2780 shBRCA1 cells to a cell density of 50%-60%.

[0105] RP, ART558, and PP5-12 (20 μM) were added and treated for 24 h.

[0106] The old culture medium of the 12-well plate was discarded, and 500 μl of ice-cold PBS was added to each well and washed twice, each wash for 5 minutes.

[0107] Fixation: Add 200-500 μl 4% PFA to each well and fix at room temperature for 15 minutes.

[0108] Discard PFA and wash with ice-cold PBS three times, 3 min each time

[0109] Permeabilization: Add 200-500 μl of 0.3% Triton X-100 to each well and permeabilize for 12 minutes at room temperature.

[0110] Discard TritonX-100 and wash once with ice-cold PBS for 3 min each time.

[0111] 500 μl of blocking solution (diluted with 10% goat serum) was added to each well, and the 12-well plate was placed in a 37° C. incubator for 1 hour.

[0112] Discard the blocking solution, take out the cell slide with cell forceps, place it in a humidified box (with the cell side facing up), drop the primary antibody (diluted 1:200 with 5% goat serum) on the cell slide, and incubate at 4°C overnight.

[0113] The next day, the cell slides were removed, placed back into the 12-well plate, and washed four times with PBST, each time for 5 min.

[0114] Then take out the cell slide, put it into a humidified box (with the cell side facing up), add secondary antibody (diluted with 5% goat serum at 1:500-1:2000), and protect from light for 1 hour.

[0115] Then take out the slide, put it back into the 12-well plate, and wash it 4 times with PBST, 5 minutes each time.

[0116] Take a clean glass slide, add a drop (10 μl) of fluorescence quencher (containing DIPA) on the slide, take out the cell slide, turn the cell side down, and protect from light throughout the process.

[0117] Seal the slides with nail polish to prevent them from drying out.

[0118] Observe the cell slides using a microscope.

[0119] (2) Experimental results

[0120] like Figure 5 As shown in the results, compound PP5-12 can significantly increase the expression of γH2AX in BRCA1-deficient cells, and can significantly increase the number of γH2AX-positive micronuclei in BRCA1-mutated or -deficient cells compared with ART558 and intermediate RP (**P < 0.01), indicating that the compounds designed in the present invention can effectively aggravate DNA damage in BRCA1-deficient cells after targeting Polθ.

[0121] Case Study 7: Evaluation of the Mechanism of Compounds Targeting Degradation of Polθ Protein

[0122] (1) Experimental methods

[0123] The cells were plated to a cell density of 70%-80%.

[0124] Transfection: Replace each cell group with serum-free and antibody-free DMEM. Simultaneously, add the plasmid and transfection reagent to each serum-free and antibody-free DMEM medium and incubate for 5 minutes. Mix the two and incubate for 15 minutes before adding the cells to the culture dish. After 4-6 hours, replace the medium with normal DMEM and continue culturing for 36-48 hours.

[0125] Harvest cells: discard the old culture medium, wash twice with PBS, add 1 ml of EBC Buffer to each 6 cm dish, scrape the cells with a cell scraper, sonicate, and lyse on ice for 30 minutes.

[0126] Centrifuge at 12,000 rpm for 15 minutes. Take 100 μl of the supernatant from each group as the input sample and cook it 5x at 100°C for 5 minutes. Add 30 μl of Myc-Beads (previously washed twice with PBS) to the remaining supernatant and incubate on a shaker at 4°C for at least 8 hours.

[0127] On the next day, the centrifuge tube was removed, and the tube was centrifuged at 2000 RFG for 2 min. The supernatant was discarded, and the tube was resuspended with PBS and centrifuged at 2000 RFG for 2 min. This process was repeated twice.

[0128] The supernatant was discarded and 2x loading was added to the precipitate (100°C, 5 min).

[0129] (2) Experimental results

[0130] like Figure 6 As shown, compound PP5-12 can enhance the interaction between the ubiquitin ligase CRBN and Polθ in a dose-dependent manner. Furthermore, when CRBN is knocked out, the ubiquitination effect induced by PP5-12 is inhibited. These results demonstrate that the compounds designed in this invention recognize and bind to target proteins by forming a ternary complex, leading to ubiquitination and degradation of the target proteins.

Claims

1. A small molecule compound targeting the degradation of Polθ, characterized in that: Having the structure of Formula I, Wherein, R1 is selected from halogen; R2 and R3 are selected from C 1-6 Alkyl, halogenated C 1-6 alkyl; R4 is selected from any one of the following structures: Linker is selected from any of the following structures: n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; R5 is selected from any one of the following structures:

2. The small molecule compound targeting degradation of Polθ according to claim 1, characterized in that In the structure, R1 is selected from fluorine, chlorine, and iodine; R2 and R3 are selected from bC 1-4 Alkyl, halogenated C 1-4 alkyl; Linker is selected from any of the following structures: n is selected from 1, 2, and 3.

3. The small molecule compound targeting degradation of Polθ according to claim 2, characterized in that In the structure, R2 and R3 are selected from methyl, ethyl, trifluoromethyl and trifluoroethyl.

4. The small molecule compound targeting degradation of Pol θ according to claim 1, characterized in that In the structure, R1 is selected from fluorine; R2 and R3 are selected from trifluoromethyl; Linker is selected from any of the following structures:

5. The small molecule compound targeting degradation of Pol θ according to claim 1, characterized in that It is selected from any one of the following compounds:

6. A pharmaceutically acceptable salt of the small molecule compound targeting Polθ degradation according to claim 1, characterized in that: A salt formed by the small molecule compound targeted for degradation of Pplθ and any one of the following acids: hydrochloric acid, carbonic acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and ferulic acid.

7. A pharmaceutical composition, characterized in that Comprising the small molecule compound targeted for degradation of Polθ according to claim 1 or the pharmaceutically acceptable salt according to claim 6 and a pharmaceutically acceptable carrier.

8. Use of the small molecule compound targeted for degradation of Polθ according to claim 1, the pharmaceutically acceptable salt according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of an anti-tumor drug.

9. The use according to claim 8, characterized in that The drug is a drug for treating BRCA1 / 2 mutant tumors.

10. The use according to claim 9, characterized in that The drug is a drug for ovarian cancer, colon cancer and breast cancer with BRCA1 / 2 mutation.

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