PARP / CK2 alpha double-target kinase inhibitor small molecule as well as pharmaceutical composition and application of PARP / CK2 alpha double-target kinase inhibitor small molecule

Abstract

The invention discloses a novel PARP / CK2 double-target kinase inhibitor small molecule as well as a pharmaceutical composition and application thereof, the small molecule takes benzofuran pyrimidone as a parent drug effect ring and a benzene ring as a connector in drug design, and is coupled with a 1-amino-2-methylisothiourea group, so that the novel PARP / CK2 double-target kinase inhibitor is obtained. The compound has good inhibitory activity on exogenous PARP enzyme, exogenous CK2 alpha enzyme and proliferation activity of various cancer cells, and can be used for preparing antitumor drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a small molecule PARP / CK2α dual-target kinase inhibitor and its pharmaceutical composition and application, and in particular to a novel small molecule dual-target kinase inhibitor with benzofuran pyrimidinone and benzothiophene pyrimidinone as the core skeleton structure. Background Art

[0002] The occurrence and development of cancer is a complex biological process involving multiple factors and multiple stages, which is related to the imbalance of multi-dimensional regulatory networks such as genetics, epigenetics, environmental exposure, and lifestyle. Although traditional cancer treatment methods have achieved certain curative effects, there are generally clinical problems such as high residual rate of tumor cells, high risk of metastasis and recurrence, insufficient target selectivity and cytotoxicity. In recent years, with the rapid development of tumor molecular biology, cell signal transduction pathways, and gene sequence analysis technologies, the emergence of small molecule targeted inhibitors has provided a new strategy for individualized precision medicine. By selectively regulating specific molecular targets, it significantly reduces the treatment-related side effects while prolonging the survival of patients. However, previous research and development of targeted drugs mainly focused on highly selective compounds targeting a single target. Subsequent studies found that this single-target inhibition strategy is prone to trigger the activation of compensatory signaling pathways in cancer cells, ultimately leading to drug resistance. To break through the limitations of single-target inhibitors, multi-target combination drug regimens are often used in clinical practice, but problems such as adverse interactions between different drugs, unpredictable pharmacokinetic characteristics, and patient compliance still need to be solved urgently. In contrast, developing multi-target inhibitors with independent intellectual property rights to achieve the coordinated regulation of multiple key molecular targets through a single chemical entity has become an important direction in current anti-cancer drug research and development.

[0003] PolyADP-ribose polymerase (PARP), as the core regulatory hub of the DNA damage repair pathway, dynamically maintains genomic integrity by catalyzing ADP-ribosylation reactions. This family contains 17 members, among which PARP-1, as the most representative subtype, its N-terminal zinc finger domain can specifically recognize DNA single-strand break sites, and the C-terminal catalytic domain consumes NAD +Generate poly(ADP-ribose) chains and recruit DNA repair proteins to the damage site. Recent studies have shown that PARP-1 can also play a key role in tumor microenvironment adaptation through multi-dimensional mechanisms such as regulating chromatin remodeling, cell cycle checkpoints, and inflammatory responses. PARP inhibitors developed based on the principle of "synthetic lethality" (such as olaparib, niraparib, etc.) have been successfully applied to the clinical treatment of solid tumors such as BRCA-mutated ovarian cancer and breast cancer, marking a major breakthrough in precision oncology. However, due to its highly dependent mechanism of action on homologous recombination repair defects, currently approved PARP inhibitors are only effective for tumors carrying germline mutations in BRCA1 / 2, and the gradually emerging drug resistance problems (such as secondary mutations in PARP1 / 2 genes, compensatory activation of DNA damage repair pathways, etc.) during long-term clinical medication significantly limit its clinical application scope. It is worth noting that recent studies have found that PARP inhibitors also show certain efficacy in treating tumors carrying ATM / ATR gene mutations, but the response rate of such patients still needs to be improved. Therefore, there is an urgent need to develop new strategies to break through the application bottleneck of existing inhibitors, including designing multi-target synergistic inhibitors, exploring combination drug regimens, and developing delivery systems targeting cancer stem cells, etc.

[0004] Casein kinase Ⅱ (CK2), as an important member of the serine / threonine kinase family, its heterotetrameric structure composed of α / α' catalytic subunits and β regulatory subunits endows it with unique substrate specificity. This kinase can phosphorylate more than 300 nuclear and cytoplasmic proteins and is widely involved in core biological processes such as cell proliferation (such as regulation of transcription factors like c-Myc, p53, etc.), apoptosis (such as phosphorylation of Bcl-2 family proteins), metabolic reprogramming (such as regulation of HIF-1α stability), and DNA damage repair (such as activation of ATM / ATR signaling pathways). It is worth emphasizing that CK2α plays a bidirectional regulatory role in DNA repair pathways such as non-homologous end joining (NHEJ) and homologous recombination (HR) by phosphorylating key factors such as X-ray repair cross-complementing protein 1 / 4 (XRCC1 / 4), heterochromatin protein 1-β (HP1β), DNA damage checkpoint mediator 1 (MDC1), and histone H4. Clinicopathological analysis shows that CK2α is abnormally highly expressed in most malignant tumor tissues, and its kinase activity is significantly positively correlated with tumor grade, metastatic potential, and poor patient prognosis. However, due to its unique structural characteristics and catalytic mechanism, there is currently no selective CK2α inhibitor approved for marketing. Summary of the Invention

[0005] Object of the Invention: The first object of the present invention is to provide a novel small molecule PARP / CK2α dual-target kinase inhibitor, the second object is to provide a pharmaceutical composition composed of the small molecule kinase inhibitor, and the third object is to provide a pharmaceutical use of the small molecule kinase inhibitor and its pharmaceutical composition.

[0006] Technical Solution: The small molecule PARP / CK2α dual-target kinase inhibitor of the present invention is selected from any one of the following compounds:

[0007]

[0008] The designed compound of the present invention uses CK2α as a co-therapeutic target, interferes with the compensatory activation of the DNA damage repair pathway by inhibiting its activity, thereby enhancing the anti-tumor efficacy of PARP-1 inhibitors, chemotherapeutic drugs, etc.; down-regulates cancer cell stemness by inhibiting its activity, thereby effectively reversing the acquired drug resistance and the like generated during the long-term use of targeted therapy, chemotherapy, radiotherapy, etc.

[0009] The specific synergistic mechanism is that PARP inhibitors capture PARP and inhibit its activity, resulting in the accumulation of unrepaired DNA single-strand breaks and further causing DNA double-strand damage. In normal cells and cancer cells with homologous recombination repair ability, DNA double-strand damage can be repaired by activating the homologous recombination pathway without generating cytotoxicity; however, in cancer cells with defective homologous recombination repair, DNA double-strand damage cannot be repaired by homologous recombination or is forced to rely on the error-prone non-homologous end joining pathway for repair, ultimately leading to cell death. The CK2α inhibitor inhibits DNA damage repair through multiple pathways and can effectively reduce cancer cell stemness. Based on the above mechanism of action, the present invention summarizes the structure-activity relationship of existing PARP inhibitors, uses benzofuran pyrimidinone as the parent nucleus, and couples the active fragment 1-amino-2-methylisothiourea group through a benzene ring as a linker (Linker). Experiments show that the designed compound of the present invention can simultaneously inhibit the activities of CK2α and PARP, thereby further enhancing the anti-tumor effect by inhibiting the activity of CK2α on the basis of maintaining the original anti-tumor effect of PARP inhibitors, expanding the scope of drug application, and overcoming the limitations of existing PARP inhibitors. This patent also systematically studies the application of this type of compound in the preparation of anti-tumor drugs.

[0010] The pharmaceutically acceptable salt of the small molecule PARP / CK2α dual-target kinase inhibitor of the present invention is a salt formed by the compound and any one of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic 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 or ferulic acid.

[0011] "Pharmaceutically acceptable salts" refer to salts of compounds prepared from compounds having specific substituents with relatively non-toxic acids or bases. When a compound contains a relatively acidic functional group, the base addition salt can be obtained by contacting the free form of such a compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When a compound contains a relatively basic functional group, the acid addition salt can be obtained by contacting the free form of such a compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, where the inorganic acids include, for example, 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, etc.; and organic acid salts, where the organic acids include, for example, 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 organic acids such as amino acids (such as arginine, etc.), glucuronic acid, etc. When certain specific compounds contain both basic and acidic functional groups, they can thus be converted into either base or acid addition salts. Preferably, the salt is contacted with a base or an acid in a conventional manner, and then the parent compound is separated, thereby regenerating 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.

[0012] "Pharmaceutically acceptable salts" can be synthesized from parent compounds containing acid radicals or basic groups by conventional chemical methods. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of both, these compounds in the form of free acids or bases are reacted with a stoichiometric amount of an appropriate base or acid. Generally, non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0013] The compounds described in the present invention also include their tautomers, prodrugs, solvates, isotopic compounds, crystals.

[0014] Preferably, the tautomers are isomers formed by the conjugation and interconversion of double bonds in unsaturated heterocycles, including carbon-carbon double bond interconversion, carbon-heteroatom, heteroatom-heteroatom double bond interconversion, such as the tautomers formed by the double bond interconversion in the imidazole ring system and pyrazole ring.

[0015] Preferably, the prodrugs are esters and amides prodrugs introduced by carboxyl, hydroxyl, amino groups, and more preferably C1-C4 alkyl esters, C1-C4 carboxylic acid esters, C1-C4 alkyl amides.

[0016] Preferably, the solvate is a small molecule bound state formed by the compound and solvent molecules, more preferably a hydrate or an alcoholate; the solvate can further form a salt with the corresponding acid to obtain a salt of the solvate.

[0017] Preferably, the isotope compound is a compound in which hydrogen in the compound is replaced by deuterium.

[0018] Preferably, the crystal form is a specific crystal structure formed during the crystallization process of the compound, including different crystal forms of the compound itself, and also including different crystal forms of its salts, solvates, and salts of solvates.

[0019] The synthesis route of the small molecule PARP / CK2α dual-target kinase inhibitor of the present invention is as follows:

[0020]

[0021] The pharmaceutical composition of the present invention comprises the small molecule PARP / CK2α dual-target kinase inhibitor of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0022] Preferably, the pharmaceutically acceptable carrier is selected from binders, suspending agents, emulsifying agents, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, wetting agents, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents or sweeteners.

[0023] Preferably, the dosage form of the pharmaceutical composition is selected from injections, mucosal agents, inhalants, eye drops, implantable preparations, subcutaneous preparations, powders, granules, hard capsules, capsule tablets, soft capsules, tablets, suspensions, emulsions, elixirs, solutions, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, serum preparations, freeze-dried powders for injection, suspensions for injection, emulsions for injection or suppositories.

[0024] "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 drug composition, and can also provide methods to enable the active ingredient to dissolve at a desired rate after the subject receives the administration, or to promote the effective absorption of the active ingredient after the subject receives the composition administration. The pharmaceutical excipients can be inert fillers or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipients can include one or more of the following excipients: binder, suspending agent, emulsifier, diluent, filler, granulating agent, adhesive, disintegrant, lubricant, anti-adhesive agent, glidant, wetting agent, gelling agent, absorption retardant, dissolution inhibitor, enhancer, adsorbent, buffer, chelating agent, preservative, coloring agent, flavoring agent and sweetening agent.

[0025] The pharmaceutical composition described in the present invention can be prepared by any method known to those skilled in the art according to the disclosed content. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.

[0026] The pharmaceutical composition described in the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be in a controlled-release or sustained-release dosage form (such as liposomes or microspheres). Examples of solid oral preparations include but are not limited to powders, capsules, caplets, soft gelatin capsules and tablets. Examples of liquid preparations for oral or mucosal administration include but are not limited to suspensions, emulsions, elixirs and solutions. Examples of topical preparations include but are not limited to emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Examples of preparations for parenteral administration include but are not limited to injection solutions, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, injection suspensions and injection emulsions. Examples of other suitable preparations of the pharmaceutical composition include but are not limited to eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0027] The small molecule of the PARP / CK2α dual-target kinase inhibitor described in the present invention or its pharmaceutically acceptable salt, and the pharmaceutical composition are used in the preparation of drugs for selective PARP-1 inhibitor and / or CK2α inhibitor.

[0028] Preferably, the drug is an anti-tumor drug.

[0029] Further preferably, the drug is for treating multiple myeloma, lung cancer, melanoma, liver cancer, kidney cancer, leukemia, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, rectal cancer, colon cancer, ovarian cancer, testicular cancer, breast cancer, bladder cancer, gallbladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastric cancer, astrocytoma, neuroblastoma, glioma, schwannoma or mesothelioma.

[0030] Even more preferably, the drug is for treating BRCA1 / 2 mutant ovarian cancer or breast cancer.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0032] The present invention uses benzofuran pyrimidinone as the core, and obtains a class of novel compounds by coupling a benzene ring with 1-amino-2-methylisothiourea group. This class of compounds shows good inhibitory effects on exogenous PARP enzyme activity, exogenous CK2α enzyme activity and the proliferation of various cancer cells, and the inhibitory activity reaches the micromolar concentration level or even the nanomolar concentration level, having the potential to become anti-tumor drugs. Specific embodiments

[0033] The technical solution of the present invention will be further described below in conjunction with the embodiments.

[0034] All reagents are of analytical purity. The nuclear magnetic resonance spectra of the compounds were measured by a Bruker ARX-600 nuclear magnetic resonance spectrometer with TMS as the internal standard; the high-resolution mass spectrometry was measured by an Agilent 6224 TOF LC / MS instrument.

[0035] Example 1: Preparation of intermediate IM-3a

[0036] At room temperature, potassium carbonate (43.50 g, 315.0 mmol) and compound 2-bromoacetamide (IM-2a) (15.98 g, 115.0 mmol) were successively added to a DMF (200 mL) solution of compound 5-fluoro-2-hydroxybenzonitrile (IM-1a) (16.26 g, 105.0 mmol), and then the reaction was carried out at 80 °C for 4 hours. The reaction process was monitored by TLC (PE / EA = 3 / 1, R f = 0.3). After the raw material IM-1a was completely reacted, the reaction solution was cooled to room temperature, poured into 600 mL of water, extracted with 900 mL of ethyl acetate (300 mL × 3), the organic phases were combined, washed with 200 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and then separated and purified by silica gel column chromatography (PE / EA = 5 / 1) to obtain a white solid product IM-3a (14.77 g, yield: 67%). 1HNMR(600MHz, DMSO-d6): δ 7.93 (d, J = 2.7 Hz, 1H), 7.72 - 7.70 (m, 1H), 7.51 (s, 1H), 7.44 (s, 1H), 7.09 (d, J = 9.1 Hz, 1H), 4.70 (s, 2H) ppm.

[0037] Example 2: Preparation of Intermediate IM-3b

[0038] Referring to the synthetic method of IM-3a, using IM-1b as the raw material instead of IM-1a to participate in the reaction, a white solid compound IM-3b (yield: 47%) was obtained. 1 HNMR(600MHz, DMSO-d6): δ 7.72 - 7.68 (m, 1H), 7.55 (s, 1H), 7.46 (s, 1H), 7.08 (t, J = 8.6 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 4.73 (s, 2H) ppm.

[0039] Example 3: Preparation of Intermediate IM-4a

[0040] At room temperature, KOH (12.98 g, 230.0 mmol) was added to a solution of compound 2-(4-chloro-2-cyanophenoxy)acetamide (IM-3a, 15.10 g, 71.9 mmol) in EtOH (200 mL), and then the reaction was carried out at 75 °C for 1 hour. Monitored by TLC (PE / EA = 1 / 1, R f = 0.2), after the raw material IM-3a reacted completely, the reaction solution was cooled to room temperature and directly concentrated under reduced pressure. The product was separated and purified by silica gel column chromatography (PE / EA = 3 / 1) to obtain a white solid product IM-4a (11.17 g, yield: 74%). 1 HNMR(600MHz, DMSO-d6): δ 7.84 (d, J = 7.8 Hz, 1H), 7.44 - 7.40 (m, 1H), 7.24 - 7.21 (m, 3H), 6.01 (s, 2H) ppm.

[0041] Example 4: Preparation of Intermediate IM-4b

[0042] Referring to the synthetic method of IM-4a, using IM-3b as the raw material instead of IM-3a to participate in the reaction, a white solid compound IM-4b (yield: 73%) was obtained. 1 HNMR(600MHz, DMSO-d6): δ 7.49 - 7.42 (m, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.06 - 7.03 (m, 1H), 5.78 (s, 2H) ppm.

[0043] Example 5: Preparation of Intermediate IM-6a

[0044] At room temperature, to a solution of compound 3-amino-5-chlorobenzofuran-2-carboxamide (IM-4a, 0.94 g, 4.5 mmol) in CH3CN (15 mL) were successively added compound terephthalaldehyde (IM-5, 1.50 g, 11.2 mmol) and I2 (1.26 g, 4.9 mmol), and then the reaction mixture was stirred at room temperature for 24 hours. Monitored by TLC (DCM / MeOH = 15 / 1, R f = 0.4), after the raw material IM-4a was completely reacted, the reaction was quenched with saturated aqueous sodium thiosulfate solution, the precipitated solid was filtered, and the filter cake was separated and purified by silica gel column chromatography (DCM / MeOH = 180 / 1) to obtain white solid compound IM-6a (0.82 g, yield: 56%). 1 1H NMR (600 MHz, DMSO-d6): δ 12.83 (s, 1H), 10.15 (s, 1H), 8.69 (d, J = 7.9 Hz, 1H), 8.31 (t, J = 7.7 Hz, 1H), 8.14 - 8.11 (m, 2H), 7.88 (d, J = 8.3 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H) ppm.

[0045] Example 6: Preparation of Intermediate IM-6b

[0046] Referring to the synthesis method of IM-6a, using IM-4b instead of IM-4a and terephthalaldehyde (IM-5) as raw materials to participate in the reaction, white solid compound IM-6b (yield: 66%) was obtained. 1 1H NMR (600 MHz, DMSO-d6): δ 13.42 (s, 1H), 10.12 (s, 1H), 8.34 (d, J = 8.1 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H), 7.74 - 7.71 (m, 2H), 7.36 - 7.33 (m, 1H) ppm.

[0047] Example 7: Preparation of Compound NT-01

[0048] At room temperature, to a solution of intermediate compound IM-6a (0.36 g, 1.1 mmol) in MeOH (10 mL) was added S-methylisothiosemicarbazide hydrochloride (IM-7, 0.25 g, 1.1 mmol), then two drops of acetic acid were added dropwise to the reaction solution, and then the reaction was carried out at 50 °C for 10 hours. Monitored by TLC (DCM / MeOH = 10 / 1, R fWhen (= 0), after the raw material IM-6a reacted completely, it was cooled and filtered. The filter cake was washed successively with ethanol, hot MeOH:DCM (v:v = 1:1), and cold ethanol (5 mL each), and the solid was collected and dried to obtain a pale yellow solid product NT-03 (0.10 g, yield: 74%). 1 H NMR (600 MHz, DMSO-d6): δ 13.16 (s, 1H), 8.34 (d, J = 10.3 Hz, 1H), 8.21 (d, J = 8.1 Hz, 2H), 8.15 (d, J = 9.6 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.14 (s, 1H), 2.39 (d, J = 21.6 Hz, 3H) ppm. 13 C NMR (150 MHz, DMSO-d6): δ 163.27, 154.69, 154.18, 153.44, 150.38, 142.79, 139.10, 138.46, 132.50, 129.80, 128.83, 128.10, 127.54, 123.99, 120.77, 114.84, 12.09 ppm. HR-MS (m / z) (ESI): calcd for C 19 H 14 ClN5O2S [M+H] + : 412.0635; found: 412.0625.

[0049] Example 8: Preparation of Compound NT-02

[0050] Using intermediate IM-6b and IM-7 as raw materials and referring to the synthesis method of NT-01, a pale yellow solid compound NT-02 (yield: 63%) was obtained. 1 H NMR (600 MHz, DMSO-d6): δ 13.20 (s, 1H), 8.33 (s, 1H), 8.18 (d, J = 8.1 Hz, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.71 (s, 2H), 7.32 (d, J = 8.3 Hz, 1H), 7.13 (s, 1H), 2.39 (d, J = 20.4 Hz, 3H) ppm. 1313C NMR(150MHz,DMSO-d6):δ163.30,157.35(d,J=8.2Hz),157.18,155.50,154.21,153.12,150.38,141.87,138.37,137.90,130.96(d,J=7.6Hz),128.06,127.56,111.53(d,J=19.9Hz),110.32(d,J=18.2Hz),109.51,99.50,12.07ppm.HR-MS(m / z)(ESI):calcd for C 19 H 14 FN5O2S[M+H] + :396.0930;found:396.0925.

[0051] Example 9: Evaluation of the Enzyme Inhibitory Activity of Compounds

[0052] (1) Experimental Method

[0053] For the detection of the inhibitory activity of all compounds of the present invention against exogenous enzyme activity, commercially available PARP-1 and CK2α enzyme-linked immunosorbent assay kits provided by Shanghai Fusheng Industrial Co., Ltd. were used, and the operation was carried out strictly in accordance with the kit instructions. The experimental design included a blank group, a test group, and a positive control group. After adding the target protein dilution to each well, 1.0 μmol / L of the test compound solution was added to the test group and the positive control group respectively. The positive control drugs were Olaparib (for PARP-1) and CX-4945 (for CK2α). After sealing the plate, it was incubated in an incubator at 37 °C for 2 h. After washing 5 times with the washing solution, except for the blank group, 50 μL of the enzyme-labeled reagent was added to each well, and the plate was sealed again and incubated at 37 °C for another 30 min. Subsequently, 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B were added in sequence, shaken gently and evenly, and incubated at 37 °C in the dark for 10 min. Finally, 50 μL of the termination solution was added to each well. Zero was adjusted with the blank well, and the absorbance (OD value) of each well was measured in sequence at a wavelength of 490 nm using an enzyme-labeled instrument. Each compound was subjected to three parallel independent experiments, and the results were averaged to calculate the enzyme activity inhibition rate and IC 50 value, and the specific data are shown in Table 1.

[0054] (2) Experimental Results

[0055] Table 1 Inhibitory Effects of Target Compounds on Exogenous PARP-1 Enzyme and CK2α Enzyme

[0056]

[0057] Using human PARP ELISA and CK2α ELISA enzyme activity detection kits, the inhibitory effects of all target compounds on PARP and CK2α enzyme activities were determined, with Olaparib and CX-4945 as positive controls. The experimental results are shown in Table 1. Olaparib has a potent inhibitory effect on PARP, and the IC 50 value reached 7.80 nM. CX-4549 has a potent inhibitory effect on CK2α, and the IC 50 value reached 4.91 nM. The target compounds designed and synthesized with benzofuran pyrimidinone as the core in the present invention can effectively inhibit the enzyme activities of both PARP and CK2α at the same time. Among them, the IC 50 values of NT-01 for PARP and CK2α enzymes are 34.21 nM and 14.72 nM respectively; among them, the IC 50 values of NT-02 for PARP and CK2α enzymes are 19.57 nM and 11.64 nM respectively, indicating that NT-01 and NT-02 are effective dual-target kinase inhibitors.

[0058] Example 10: Evaluation of in vitro cytotoxic activity of compounds

[0059] (1) Experimental method

[0060] The MTT method was used to test the cytotoxic activities of all compounds of the present invention. Cells in the logarithmic growth phase were counted and inoculated into a 96-well culture plate, with an inoculation volume of 8000 - 10000 cells per well. After culturing overnight until the cells adhered, the drug administration group and the control group were set. The test compounds were first prepared into stock solutions with DMSO and diluted into gradient concentrations with cell culture medium before use (the final concentration of DMSO ≤ 0.4%, the same below). Three replicates were set for each concentration. After adding the drugs, the cells were cultured for another 72 h. Then, 20 μL of MTT solution with a concentration of 5 mg / mL was added to each well and incubated at 37 °C for 4 h. The supernatant was carefully aspirated, and 150 μL of DMSO was added to each well to dissolve the crystals. The optical density (OD value) of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, the cell inhibition rate was calculated, and a concentration-inhibition rate curve was plotted to obtain the IC 50 value. The experimental results are shown in Table 2.

[0061] (2) Experimental results

[0062] Table 2 IC 50 values of target compounds against different cells

[0063]

[0064] In this invention, human ovarian cancer cell line SK-OV-3, human breast cancer cell line MCF-7, human prostate cancer cell line PC-3, human ovarian cancer cell line A2780 and human normal liver cell line L02 were selected to evaluate the inhibitory activity of the target compounds on the proliferation of cancer cells and normal cells. Olaparib and CX-4945 were used as positive controls. The results are shown in Table 2. Although Olaparib and CX-4945 have very high inhibitory activity against PARP and CK2α enzymes, their cytotoxicity is relatively weak. Among them, the IC 50 values of CX-4945 against the four tested cancer cells are all in the range of 13.87 - 17.01 μM. Olaparib has relatively strong cytotoxicity against MCF-7 and A2780 cells, and the IC 50 values are 8.76 and 8.03 μM respectively. Among the compounds designed in this invention, NT-01 has strong cytotoxicity against SKOV3 and A2780 cells, and the IC 50 values are 8.57 μM and 9.06 μM respectively; NT-02 has strong cytotoxicity against A2780 cells, and the IC 50 value is 7.63 μM.

Claims

1. A small molecule PARP / CK2α dual-target kinase inhibitor, characterized in that, Any one of the following compounds:

2. A pharmaceutically acceptable salt of the PARP / CK2α dual-target kinase inhibitor small molecule according to claim 1, characterized in that, A salt formed by the compound and any one of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic 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 or ferulic acid.

3. A method for preparing a small molecule PARP / CK2α dual-target kinase inhibitor as described in claim 1, characterized in that, The synthetic route is as follows:

4. A pharmaceutical composition, characterized in that, Comprising the PARP / CK2α dual-target kinase inhibitor small molecule according to claim 1 or the pharmaceutically acceptable salt according to claim 2 and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, wherein The pharmaceutically acceptable carrier is selected from binders, suspending agents, emulsifying agents, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, wetting agents, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents or sweetening agents.

6. The pharmaceutical composition according to claim 4, wherein Its preparation form is selected from injections, mucosal agents, inhalants, eye drops, implant preparations, subcutaneous preparations, powders, granules, hard capsules, capsule tablets, soft capsules, tablets, suspensions, emulsions, elixirs, solutions, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, serum preparations, freeze-dried powders for injection, suspensions for injection, emulsions for injection or suppositories.

7. Use of the PARP / CK2α dual-target kinase inhibitor small molecule according to claim 1, the pharmaceutically acceptable salt according to claim 2 or the pharmaceutical composition according to claim 4 in the preparation of a drug for selectively inhibiting PARP-1 and / or CK2α.

8. The application according to claim 7, characterized in that, The drug is an anti-tumor drug.

9. The application according to claim 8, wherein The drug for treating multiple myeloma, lung cancer, melanoma, liver cancer, kidney cancer, leukemia, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, rectal cancer, colon cancer, ovarian cancer, testicular cancer, breast cancer, bladder cancer, gallbladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastric cancer, astrocytoma, neuroblastoma, glioma, schwannoma or mesothelioma.

10. The application according to claim 9, wherein, The drug is a drug for treating BRCA1 / 2 mutant ovarian cancer or breast cancer.

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