Compound containing PAPR1 degradation agent and BET inhibitor and application of compound in antitumor drugs

Abstract

The invention discloses application of a compound containing a PAPR1 degradation agent and a BET inhibitor in preparation of antitumor drugs, and particularly relates to the technical field of biological medicines. The PAPR1 degradation agent is a compound which has tumor selectivity and induces PARP1 degradation based on a CRBN ligand, and pharmaceutically acceptable salts and hydrates thereof. The anti-tumor medicine composition comprises a novel PAPR1 degradation agent and a BET (Brunauer, Emmett and Teller) inhibitor. According to the compound disclosed by the invention, the BET inhibitor JQ1 is used for inhibiting the function of BET protein so as to inhibit DNA repair and influence the DNA damage level, and a strategy for selectively damaging homologous recombination repair of cancer cells is constructed, so that related tumors of non-BRCA gene mutation are sensitive to PARP inhibition, and the clinical application range of PARP targets is effectively expanded; in view of the fact that acquired homologous recombination repair and protein mutation are two most common resistance mechanisms for PARP inhibition, the PARP1 protein is effectively degraded by utilizing PARP1-PROTAC, and the resistance to PARP inhibitor treatment can be overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a complex containing a PAPR1 degrader and a BET inhibitor and application thereof in anti-tumor drugs. Background Art

[0002] Poly(ADP-ribose) polymerase (PARP) is a DNA damage repair enzyme that can repair DNA single-strand breaks (SSBs) through the base excision repair pathway, which is essential for the body to maintain the stability of the genome. PARP inhibitors are small molecule targeted drugs based on the "synthetic lethality" theory. Their mechanism of action is to inhibit the repair of SSBs, promote the accumulation of DNA double-strand breaks (DSBs), and thus lead to cell death. For tumors with homologous recombination (HR) defects (such as tumors with BRCA1 / 2 mutations), BRCA-mutated tumors are sensitive to PARP inhibitors because their DSB damage repair pathway is missing. In non-BRCA-mutated tumors, the HR pathway for repairing DSBs is intact, so PARP inhibitors have almost no therapeutic effect on them, which leads to the fact that PARP inhibitors are mainly used to treat tumors with BRCA gene mutations, and there are no broad patients who benefit. In addition, cancer cells may develop resistance to inhibitors through mutations or other mechanisms. The above problems limit the clinical application of PARP inhibitors. Therefore, it is urgent to explore new strategies to prevent or reverse PARP inhibitor resistance or expand its indications.

[0003] The protein degradation targeting chimera (PROTAC) technology can degrade key proteins in the occurrence and development of tumors, thereby achieving functional regulation of target proteins. Compared with PARP inhibitors, PARP-PROTAC directly removes or depletes target proteins through degradation, which is beneficial to overcome drug resistance caused by mutation or overexpression of target proteins, thereby improving the therapeutic effect on tumors. Chinese patent CN201910881556.9 proposes a compound based on CRBN ligand-induced PARP-1 degradation, a preparation method and an application. In the invention, a connecting chain is used to connect the PARP1 small molecule inhibitor Olaparib and the cereblon protein ligand in the E3 ubiquitin ligase complex to obtain a PROTAC molecule that can selectively induce PARP1 degradation. However, the anti-tumor activity of the molecule has no obvious advantages over PARP inhibitors, and effective application of non-BRCA mutated tumors has not been achieved. On the other hand, since PARP1 protein is widely present in body cells, and PROTAC drugs generally lack tumor targeting, it is easy to cause off-target effects to bring toxic side effects to normal cells, which also hinders its further development. Therefore, developing new tumor-selective PROTACs to reduce degradation toxicity to normal cells is the focus of future PROTACs research.

[0004] In order to improve the universality (effectiveness) of cancer patients, the development of strategies to selectively damage HR in cancer cells and sensitize non-HR-deficient cancers to PARP-related agents may provide new clinical applications. Bromodomain and extraterminal (BET) proteins contribute to the proliferation and survival of cancer cells by recruiting transcriptional machinery or indirectly maintaining and promoting oncogene transcription by binding to enhancers. Studies have shown that BET inhibitors can completely block the HR repair pathway by inhibiting the expression of BRCA1 and RAD51 proteins, and the combination of PARP inhibitors and BRD4 inhibitors has the potential for synergistic enhancement. Currently, this drug combination inhibits tumor progression in ovarian cancer and breast cancer xenograft models. However, the inhibition of PARP1 and BRD4 inhibitors requires maintaining high drug concentrations, and some inhibitors can even bind to multiple targets, which is prone to off-target or toxic side effects. Therefore, the development of a new generation of combined drug modes of PARP degraders with good efficacy and high safety and post-BET inhibitors is worth exploring and has important significance and great commercial value. Summary of the invention

[0005] To this end, the present invention provides a complex containing a PARP1 degrader and a BET inhibitor and its use in anti-tumor drugs to solve the problems of prominent drug resistance of existing PARP inhibitors, poor anti-tumor activity, no tissue targeting and limited application range of existing targeted PARP1 degraders. The present invention provides a combination of a protein degradation targeted chimera (PROTAC) of poly (ADP-ribose) polymerase 1 (PARP1) and a bromodomain and exogenous (BET) inhibitor and its use in anti-tumor.

[0006] The present invention aims to provide a novel PARP1 protein degradation targeted chimera that has excellent anti-tumor effect and good tumor selectivity under catalytic dosage conditions. Another purpose of the present invention is to explore a new generation of combined drug modes of PARP degraders and BET inhibitors, and to explore the application of the combined drug combination in a variety of tumors to produce synergistic anti-tumor effects (especially non-BRCA mutation tumors).

[0007] The present invention first provides a novel PARP1 protein degradation targeted chimera that still has excellent anti-tumor effects and good tumor selectivity under catalytic dosage conditions, which can effectively degrade PARP1 and solve the problems of tumor cell resistance and degradation toxicity. Secondly, the present invention combines a PARP1 degrader with a BET inhibitor, and uses the BET inhibitor to induce homologous recombination repair deficiency in tumor cells, increase the accumulation of DNA damage in tumor cells, make non-BRCA gene mutation-related tumors sensitive to PARP inhibition, effectively inhibit the proliferation of tumor cells, and expand the clinical application range of PARP targets. The combined drug involved in the present invention has an excellent synergistic effect, which is conducive to reducing drug dosage, reducing side effects, and improving the biosafety of clinical applications.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] According to one aspect of the present invention, a compound containing a PAPR1 degrader and a BET inhibitor is provided for use in the preparation of an anti-tumor drug.

[0010] Furthermore, the PAPR1 degrader is a tumor-selective compound based on CRBN ligand-induced PARP1 degradation, and the structure is a compound shown in Formula I and its pharmaceutically acceptable salts and hydrates:

[0011]

[0012] Among them, the linker is n is an integer from 1 to 12; preferably, n is an integer from 5 to 12, and the preferred compounds have relatively good activity in inducing PARP1 degradation and anti-tumor activity; as an example, n is more preferably an integer from 9 to 11, and the preferred compounds have better activity inducing PARP1 degradation and anti-tumor activity.

[0013] The PARP-targeted degrader of the present invention is a PROTAC molecule that selectively degrades PARP1, has excellent activity in inhibiting tumor cell proliferation, and can be used to improve anti-tumor research.

[0014] As an example, the PROTAC targeting the degradation of PARP1 is compound LB23, and the structural formula is shown in Formula II:

[0015]

[0016] Furthermore, the PAPR1 degrader selects Olaparib as the target protein ligand and pomalidomide as the ligand for recruiting CRBNE3 ligase, and connects the two ligand molecules through a linker to form a ternary complex to make a heterobifunctional PROTACs molecule. This molecule can selectively induce PARP1 degradation in tumor cells and exhibits good anti-tumor effects, while having little effect on the PARP1 protein of non-tumor cells and is safe.

[0017] Furthermore, the BET inhibitor is a BRD4 inhibitor.

[0018] Furthermore, the BRD4 inhibitor includes JQ1.

[0019] According to another aspect of the present invention, an anti-tumor pharmaceutical composition is provided, comprising a PAPR1 degrader and a BET inhibitor.

[0020] The tumors include breast cancer, pancreatic cancer, colorectal cancer, and ovarian cancer, and particularly refer to the above tumors without homologous recombination repair defects.

[0021] Furthermore, the molar ratio of the PAPR1 degrader to the BET inhibitor is 2 to 6:1; as an example, 2:1 is preferred.

[0022] Furthermore, the drug also includes pharmaceutically acceptable common excipients.

[0023] The present invention has the following advantages:

[0024] The series of novel PARP degraders synthesized by the present invention are protein degradation targeted chimeras that can efficiently and selectively degrade PARP1 in tumor cells, with little effect on the PARP1 protein of normal cells. These compounds have excellent anti-tumor activity and biosafety, and are ideal PARP1 degraders, showing good application prospects in anti-tumor applications.

[0025] On the one hand, the combined drug composition of the present invention utilizes the BET inhibitor JQ1 to inhibit the function of BET protein to inhibit DNA repair and affect the level of DNA damage, thereby constructing a strategy for selectively destroying homologous recombination repair of cancer cells, making non-BRCA gene mutation-related tumors sensitive to PARP inhibition, and effectively expanding the clinical application range of PARP targets. On the other hand, given that acquired homologous recombination repair and protein mutation are the two most common resistance mechanisms to PARP inhibition, this method can also overcome resistance to PARP inhibitor treatment by utilizing PARP1-PROTAC to effectively degrade PARP1 protein. In addition, PARP1 degraders can still have good anti-tumor effects under catalytic dosage conditions and can act on target proteins in a cycle, which is beneficial to reducing drug dosage, reducing side effects, and improving the biosafety of clinical applications. In summary, combined drug use can activate the advantages of the two drugs respectively, improve the synergistic effect of drug anti-tumor activity, and has great application prospects in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0027] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0028] Figure 1 The NMR spectrum of the compound LB23 provided in Example 1 of the present invention;

[0029] Figure 2 This is a graph showing the degradation activity of the compound LB23 provided in Example 2 of the present invention on PARP1 in wild-type human breast cancer cells MDA-MB-231;

[0030] Figure 3 The IC values ​​of the anti-tumor proliferation activity of the compound LB23 provided in Example 3 of the present invention on various tumor cells are shown in Table 1. 50 Value statistics chart;

[0031] Figure 4 This is a graph showing the degradation activity of the compound LB23 provided in Example 3 of the present invention compared with another highly efficient PARP1 degrader on PARP1 in normal liver cells LO2;

[0032] Figure 5 This is a graph showing the inhibition of the proliferation activity of pancreatic cancer cells by the PARP1 degrader LB23 and the BET inhibitor JQ1 alone or in combination as provided in Example 4 of the present invention;

[0033] Figure 6 A diagram showing the mechanism of the synergistic anti-tumor activity of the PARP1 degrader LB23 and the BET inhibitor JQ1 provided in Example 5 of the present invention;

[0034] Figure 7 This is a diagram showing that the PARP1 degrader LB23 provided in Example 6 of the present invention is used in combination with the BET inhibitor JQ1 to synergistically promote cell cycle inhibition and apoptosis;

[0035] Figure 8 This is a curve chart showing changes in body weight over time in mouse transplanted tumor models in different treatment groups provided in Example 7 of the present invention;

[0036] Fig. 9 This is a curve chart showing the change in tumor volume over time in the mouse transplanted tumor model in different treatment groups provided in Example 7 of the present invention. DETAILED DESCRIPTION

[0037] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1

[0039] This example provides a PROTAC molecule that selectively degrades PARP1. The molecular compound is LB23, and the synthesis route is as follows:

[0040]

[0041] Specific preparation method:

[0042] Weigh 1-[5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoyl]piperazine L1 (120 mg, 0.328 mmol, 1 eq), HATU (150 mg, 0.39 mmol, 1.2 eq) and L1 and 11-(Boc-amino)undecanoic acid as reaction raw materials linker (1.2 eq) and dissolve in anhydrous DMF, add DIPEA (129 mg, 1 mmol, 3 eq) and stir the reaction at room temperature. The reaction is monitored by TLC. Post-treatment: After adding a large amount of water to quench the reaction, extract with EA 3 times, combine the organic layers, and backwash with saturated brine 2 times. The organic layer is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM / CH 3 OH system) to obtain intermediate 1 as a light yellow solid with a yield of 89%. 1 The structures of the compounds were confirmed by H NMR and high-resolution mass spectrometry.

[0043] 1 H NMR(400MHz,Chloroform-d)δ10.66(br,1H),8.50–8.43(m,1H),7.80–7.67(m,3H),7.39–7.30(m,2H),7.05(t,J=9.1Hz,1H),4.53(s,1H ),4.29(s,2H),3.85–3.02(m,10H),2.34–2.26(m,2H),1.60–1.56(m,2H),1.44(s,9H),1.33–1.26(m,14H).HRMS(DART-TOF)calculated for C 36 H 48 FN 5 NaO 5 + [M+Na] + m / z 672.3537, found 672.3535.

[0044]

[0045] Intermediate 1;

[0046] Synthesis of compound P2: Pomalidomide (500 mg, 1.83 mmol) was added to a 50 ml round-bottom flask, dissolved with tetrahydrofuran, and the reaction mixture was stirred in an ice-water bath, and chloroacetyl chloride (620 mg, 5.5 mmol) was slowly added dropwise. After the addition was completed, the reaction was moved into a 60 ° C oil bath and heated under reflux for 6 h. The reaction was monitored by TLC to complete. After removing THF by evaporation under reduced pressure, the mixture was extracted three times with ethyl acetate and water, the organic layers were combined, and dried over anhydrous sodium sulfate. After the organic layers were concentrated under reduced pressure, ice ether was added to precipitate a large amount of yellow precipitate, and the filter cake was collected by filtration. The filter cake layer was washed twice with ice water and ice ethanol respectively, and a light yellow solid powder was obtained after drying, which was compound P2, a total of 574 mg, with a yield of 89.8%. The product does not need to be purified and can be directly used in the next step.

[0047] 1 H NMR(400MHz,Chloroform-d)δ10.49(s,1H),8.83(d,J=8.5Hz,1H),8.02(s,1H),7.77(t,J=7.9Hz,1H),7.6 3(d,J=7.2Hz,1H),5.04–4.94(m,1H),4.24(s,2H),2.95–2.72(m,3H),2.22–2.14(m,1H).HRMS(DART-TOF)

[0048] calculated for C 15 H 12 C1N 3 KO 5 + [M+K] + m / z 388.0061, found 388.0060.

[0049]

[0050] Weigh the intermediate 1 (0.12 mmol) into a 25 mL round-bottom flask, dissolve it with about 10 mL of TFA / DCM (1:2), and stir the reaction at room temperature for 1 to 2 h. After the reaction is completed by TLC monitoring, evaporate the solvent under reduced pressure to remove the crude product, which can be directly used in the next step. Add K 2 CO 3(0.3mmol) and KI (0.05mmol) were dissolved in anhydrous acetonitrile. Compound P2 (0.1mmol) was added to the mixture under stirring, and the reaction was transferred to an 80°C oil bath for reflux reaction for 6-8h. The reaction was monitored by TLC. The reaction solution was concentrated under reduced pressure to remove excess solvent, water was added to quench the reaction, and EA was used for extraction 3 times to collect the organic layer. After drying over anhydrous sodium sulfate, it was filtered and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (DCM / CH 3 OH system = 20:1) to obtain the target compound LB23 as a yellow solid with a yield of 75.2%. The correct structure was confirmed by high-resolution mass spectrometry and nuclear magnetic resonance. The nuclear magnetic spectrum is shown in Figure 1 .

[0051] 1 H NMR (600 MHz, DMSO-d 6 )δ12.63(s,1H),11.18(s,1H),8.77(dd,J=11.4,8.2Hz,1H),8.27(dd,J=7.9,1.4Hz,1H),7.97(d,J=8.1H z,1H),7.91–7.82(m,3H),7.67–7.59(m,1H),7.45(dt,J=6.0,2.8Hz,1H),7.40–7.35(m,1H),7.25(td,J=9 .0,2.7Hz,1H),5.20–5.14(m,1H),4.34(s,2H),3.67–3.40(m,8H),3.23–3.08(m,3H),2.96–2.87(m,1H),2 .66–2.53(m,4H),2.28(dt,J=39.0,7.6Hz,2H),2.14–2.05(m,1H),1.54–1.42(m,4H),1.32–1.22(m,12H). 13 C NMR (151 MHz, DMSO-d 6)δ173.21,172.04,170.26,168.61,168.46,168.08,167.23,164.65,159.86,157.22,145.77 ,137.50,135.05,134.44,133.69,132.26,131.93,129.54,129.43,128.33,127.20,126.53,1 25.02,118.41,115.71,60.24,56.03,50.44,49.38,46.86(2C),45.84,42.17,36.89,32.72,3 1.90,29.94.4,29.91,29.47,29.03,26.69,25.07,23.19,21.24.HRMS(DART-TOF)calculated for C 46 H 52 FN 8 O 8 + [M+H] + m / z863.3892, found 863.3887.

[0052]

[0053] Example 2

[0054] This example provides the inhibitory activity of compound LB23 on tumor cell proliferation:

[0055] The MTT method was used to determine the inhibitory activity of the compounds on tumor cells. The cell lines used included: Hela (human cervical cancer cells), H2228, H3122 (human non-small cell lung cancer cells), SW620 (human colon cancer cells) and SKOV3 (human ovarian cancer cells, etc.).

[0056] The specific steps are:

[0057] Inoculate cells: Prepare a single cell suspension with RPMI1640 complete medium containing 10% fetal bovine serum, and inoculate 3000-5000 cells per well in a 96-well plate with a volume of 80 μL per well. Culture cells: Place the 96-well plate in a CO 2 Incubator, 37°C, 5% CO 2Culture under the conditions for 24 hours. Dosing: After the tumor cells adhere to the wall, the DMSO solution is diluted with the culture medium in a gradient manner, and the dosing concentration is set to 40, 13.3, 4.44, 1.48, 0.493, 0.164, 0.0548, 0.0182 μM, and 3 replicate wells are set for each concentration. Continue to culture for 5 to 7 days after dosing. Color: After the culture is completed, add 20 μL of MTT solution (5 mg / mL prepared with PBS) to each well. Continue incubation for 4 hours, terminate the culture, and carefully aspirate the culture supernatant in the well. For suspended cells, centrifuge and then aspirate the culture supernatant in the well. Add 150 μL DMSO to each well and shake for 10 minutes to fully dissolve the crystals. Colorimetry: Select 570 nm as the measurement wavelength, and measure the light absorption value of each well on the enzyme-linked immunosorbent monitor. According to the formula: cell proliferation inhibition rate = [1-(A sample well-A blank well) / (A control well-A blank well)], the cell proliferation inhibition activity IC of the test compound was calculated using GraphPadPrism 8. 50 The unit is μM), and the results are as follows Figure 2 The data results showed that the cell activity of LB23 was better than that of the positive drug Olaparib, especially in H3122 and MV-4-11 cells, showing a significant increase in activity.

[0058] Example 3

[0059] This example provides a test of the degradation activity and toxicity of compound LB23 on PARP1 protein:

[0060] Based on the good activity of compound LB23 in inducing selective degradation of PARP1, this example selected it to study its protein degradation mechanism. This example selected MDA-MB-231 cells (human triple-negative breast cancer cell line) for immunoblotting experiments. The results are as follows Figure 3 shown.

[0061] In this example, the degradation activity of the target PROTACs molecule LB23 was studied at different concentrations. LB23 can show concentration-dependent degradation of PARP1 protein in MDA-MB-231 cells at a concentration of 0.06 to 1 μM. Its degradation activity of PARP1 protein DC 50 The maximum degradation efficiency is 53nM and Dmax>90%.

[0062] In fact, PARP and E3 ligases are widely expressed in tumors and normal tissues. If the target protein is not tumor-specific, PROTACs molecules may cause target toxicity. However, so far, there are few studies on the target toxicity of PROTACs. Therefore, in terms of considering the safety of PROTACs drugs, it is critical to develop PROTACs with tumor specificity / selectivity. Since SK-575 is the most effective PARP1 degrader reported so far, the degradation concentration of PARP1 in various human tumor cells is DC 50 This reaches the nanomolar or even picomolar level, which prompts this example to focus on the degradation toxicity of SK-575 on PARP1 in normal cells.

[0063] In this example, the toxicity of PARP1 degradation agent SK-575 as a control and LB23 inducing PARP1 degradation in L-O2 cell line (normal human liver cells) was studied. Experimental data show (eg Figure 4 ), SK-575 has a strong degradation toxicity to PARP1 in L-O2 cells, and its degradation toxicity DC 50 It is 2.89nM, while the PARP1 protein expression level in L-O2 cells treated with LB23 has no obvious change, and its degradation toxicity to L-O2 cells is measured to be 3.2μM, which is much lower than the degradation toxicity of SK-575. Further analysis of the degradation activity of SK-575 in MDA-MB-231 cells shows that the degradation activity of SK-575 in tumor cells is 0.23nM, and the degradation activity of LB23 in MDA-MB-231 cells is 53nM. It can be seen that the selectivity of LB23 for tumor cells is 60 times that of normal cells (3.2μM: 0.053μM), and the selectivity of LB23 for tumor cells is nearly 5 times higher than that of SK-575 (2.89nM: 0.23nM). Therefore, the PARP1-PROTAC LB23 in this example has less degradation toxicity to normal cells and has better biosafety.

[0064] Example 4

[0065] This example provides PARP1 degrader LB23 and BET small molecule inhibitor JQ1 to synergistically inhibit pancreatic cancer cell proliferation:

[0066] Pancreatic cancer is a highly lethal digestive tract tumor, and its treatment faces challenges such as being difficult to cure and prone to drug resistance. Currently, PARP inhibitors have been approved by the FDA for the treatment of pancreatic cancer with BRCA mutations, but they have disadvantages such as limited scope of application and easy to develop drug resistance. According to the above examples, the PARP1 degrader LB23 exhibits excellent PARP1 degradation activity and safety for normal cells, and JQ1 is one of the earliest available BET (BRD4) inhibitors, and JQ1 has been widely used in many studies. Therefore, this example chooses to use the BET inhibitor JQ1 to construct HR defects, and pioneeringly combines PROTAC compounds that target the degradation of PARP with BET small molecule inhibitors to explore their synergistic anti-tumor effects (especially non-BRCA mutation tumors). First, the CCK-8 method was used to detect the proliferation inhibitory activity of LB23 and JQ1 on two pancreatic cancer cells PANC-1 and BXPC-3 when used alone or in combination. According to Figure 5 The experimental results showed that JQ1 alone had good proliferation inhibitory activity against pancreatic cancer compared with LB23 alone, but more effective activity was observed in the combination administration group of JQ1 and LB23 than either single drug, indicating that the drug combination involved in the present invention has a favorable synergistic inhibitory effect on the proliferation of PANC-1 and BxPC-3 cells.

[0067] Example 5

[0068] This example provides that JQ1 inhibits the expression of key proteins in the homologous recombination repair pathway in non-BRCA mutated pancreatic cancer:

[0069] Western Blot experiments were used to verify the mechanism of action of the BET small molecule inhibitor JQ1 on pancreatic cancer without BRCA mutations, that is, the expression of DNA damage marker γ-H2AX, DNA repair protein RAD51, and BRCA1 were detected respectively. Pancreatic cancer cells (PANC-1) treated with drug JQ1 were collected and total protein was extracted. Proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane (PVDF membrane), and identified with primary antibodies for γ-H2AX, RAD51, and BRCA1 and corresponding secondary antibodies. Protein expression was detected by ECL ultrasensitive immunoblotting detection reagent, and images were captured by chemiluminescence imaging system and analyzed by grayscale scanning.

[0070] Figure 6The experimental results showed that BRD4 was effectively inhibited by JQ1 in a dose-dependent manner and upregulated the expression of γ-H2AX, which confirmed that BRD4 is involved in the DNA damage response. In an in vitro model of pancreatic cancer without BRCA mutations, the combination of JQ1 and LB23 increased the level of the DNA damage marker γ-H2AX and reduced the expression of BRCA1 and RAD51 and was superior to the drug combination of JQ1 and OL (PARP1 inhibitor). BET inhibition was associated with reduced expression of BRCA1 and Rad51, thereby significantly increasing HR defects, which confirmed that downregulation of BRCA1 and RAD51 proteins contributes to the induction of homologous recombination deficiency. In summary, JQ1 inhibition of BET and LB23 degradation of PARP1 are beneficial to enhance DNA damage activity, possibly by blocking HR repair by weakening the expression of RAD51 and BRCA1.

[0071] Example 6

[0072] This example provides a combination of LB23 and JQ1 to synergistically promote cell cycle inhibition and apoptosis:

[0073] PANC-1 cells in the logarithmic growth phase were taken, the culture medium was discarded, and the cells were digested with trypsin and diluted with DMEM medium containing 10% fetal bovine serum to a concentration of 3×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate. 37°C, 5% CO 2 The prepared target compound was diluted to the administration concentration. 2 Incubate in the incubator for 24 hours. After trypsin digestion, transfer the cell suspension to a centrifuge tube and centrifuge for 5 minutes. Aspirate the supernatant, wash with pre-cooled PBS buffer, and centrifuge. Repeat the wash twice. Prepare the flow cytometry sample according to the instructions of the cell cycle and apoptosis detection kit. The results are shown in the figure. Figure 7 shown.

[0074] The cell cycle results showed that after 24 hours of incubation with OL or LB23, the cell cycle was blocked in the G2 / M phase in a dose-dependent manner, while JQ1 inhibited the G1 / S transition and significantly blocked the cell cycle progression in the G0 / G1 phase in a concentration-dependent manner. At the same time, compared with the OL (PARP1 inhibitor) treatment group, the percentage of PANC-1 cells in the G0 / G1 phase in the OL and JQ1 combined treatment group increased by 12.43%, while the G2 / M phase only increased slightly. This example found that not only did the cell cycle arrest of PANC-1 cells in the G0 / G1 phase increase from 43.66% in the LB23 group to 50.25% in the LB23 and JQ1 combination, but the LB23 and JQ1 combination significantly prolonged the G2 / M phase by 8.3%. Specifically, the combination of JQ1 and LB23 can simultaneously arrest the cell cycle in the G0 / G1 and G2 / M phases, confirming that the drug combination of PARP1 degrader and JQ1 has a better cycle arrest effect than the drug combination of PARP1 inhibitor and JQ1.

[0075] The results of cell apoptosis showed that there was no obvious apoptosis after the cells were treated with OL or LB23 alone, while the number of apoptotic cells increased significantly in the combination of OL and JQ1 or the combination of LB23 and JQ1 compared with the JQ1 treatment group. In terms of quantity, when treated with the combination of OL and JQ1, the apoptosis rate increased by 18.8% compared with the OL single-drug group, especially the combination of LB23 and JQ1, the proportion of apoptotic cells was significantly increased by 20.5% compared with the LB23 group.

[0076] Example 7

[0077] This example detects that the combination of LB23 and JQ1 has a tumor-killing effect in vivo at the animal level:

[0078] Balb / c nude female mice were selected and PANC-1 tumor cells were cultured to the logarithmic growth phase. After trypsin digestion, the cells were collected to prepare a cell suspension, added to the matrix gel, mixed well to make a cell suspension and placed on ice. Tumors (5×10 6 / ) to establish a xenograft tumor model. In vivo efficacy study: When the tumor volume grows to about 70-100mm 3The mice were randomly divided into a solvent control group, a combined drug group (LB23+JQ1) and a positive drug group administered alone. The drug dosages were as follows: vehicle group, OL (40 mg / kg), LB23 (40 mg / kg), JQ1 (20 mg / kg), OL+JQ1 (40+20 mg / kg) and LB23+JQ1 (40 mg / kg). All drugs were administered by intraperitoneal injection. The drug was administered once a day (two days of continuous administration and then two days of suspension), and the tumor size and body weight were tested every two days. The tumor volume was measured with a vernier caliper, and the test was continued for 21 days, and the inhibition rate of tumor growth was calculated. After the experiment, the mice were anesthetized with chloral hydrate and killed. The tumor tissues were taken, fixed with 4% paraformaldehyde and embedded in OCT and stored in a -80°C refrigerator. Western Blot and immunohistochemistry were used to analyze the expression of key proteins in the PARP-mediated signaling pathway. At the same time, the main organs (heart, liver, spleen, lung and kidney) of each group of mice were taken. The experimental results are shown in the following table. Figure 8 and Fig. 9 As shown, the combined drug group of the present invention can significantly inhibit the growth of tumors in vivo, and the tumor growth rate is significantly slower than that of the single drug treatment group. In addition, compared with the 80.7% tumor inhibition rate of the PARP inhibitor OL and JQ1 drug combination for pancreatic cancer, the combined drug group of the present invention has obvious advantages in the treatment of pancreatic cancer, and its tumor inhibition rate is as high as 98.5%. On the other hand, compared with the single drug treatment group, the combined drug group at the dosage used in the present invention did not have a significant effect on the weight of mice, and the weight of mice was not significantly different from that of the placebo group, which proved to a certain extent that the combined drug preparation of the present invention has good biosafety.

[0079] In summary, the combination therapy of PARP1 degrader and BET inhibitor has a more obvious tumor inhibitory effect than single therapy, and has fewer toxic side effects.

[0080] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Use of a complex containing a PAPR1 degrader and a BET inhibitor in the preparation of anti-tumor drugs.

2. The use according to claim 1, It is characterized in that The PAPR1 degrader is a tumor-selective compound based on CRBN ligand-induced PARP1 degradation, and the structure is a compound shown in Formula I and its pharmaceutically acceptable salts and hydrates: Among them, the linker is n is an integer from 1 to 12.

3. The use according to claim 1, It is characterized in that The PAPR1 degrader selects Olaparib as the target protein ligand and pomalidomide as the ligand for recruiting CRBN E3 ligase, and connects the two ligand molecules through a linker to form a ternary complex to prepare a heterobifunctional PROTACs molecule.

4. The use according to claim 1, It is characterized in that The BET inhibitor is a BRD4 inhibitor.

5. The use according to claim 4, It is characterized in that The BRD4 inhibitors include JQ1.

6. An anti-tumor drug composition, It is characterized in that Including PAPR1 degraders and BET inhibitors.

7. An anti-tumor pharmaceutical composition according to claim 6, It is characterized in that The molar ratio of the PAPR1 degrader to the BET inhibitor is 2 to 6:

1.

8. An anti-tumor pharmaceutical composition according to claim 6, It is characterized in that The medicine also includes pharmaceutically acceptable common excipients.

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