Piperazinone-containing phthalazinone compound as well as preparation method, pharmaceutical composition and application thereof

Abstract

The invention belongs to the field of medicinal chemistry, and discloses a new-structure phthalazinone compound containing piperazinone as a PARP1 / 2 / 7 inhibitor, and a preparation method, a pharmaceutical composition and application thereof. Specifically, the invention relates to phthalazinone compounds with novel structures as shown in a formula I, pharmaceutically acceptable salts of the phthalazinone compounds, a preparation method of the phthalazinone compounds, a composition containing one or more of the compounds, and application of the compounds in inhibition of PARP1, PARP2 and PARP7 (PARP1 / 2 / 7), treatment of diseases related to PARP1 / 2 / 7 and preparation of drugs for prevention and / or treatment of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and relates to phthalazinone compounds containing piperazinone as PARP1 / 2 / 7 inhibitors, their preparation methods, pharmaceutical compositions containing the compounds, and the compounds as drugs, especially as anti-tumor drugs, in combination with anti-tumor drugs as anti-tumor drug sensitizers, or in combination with anti-tumor drugs as immunomodulators. Background Art

[0002] PARPs (Poly ADP-ribose polymerase) are a class of protein nucleases that modify substrate proteins by ADP-ribosylation [Kirby, I.T. et al. Curr. Top. Microbiol. Immunol. 2019, 420, 211–231]. ADP-ribosylation is a post-translational modification that refers to the catalytic transfer of ADP-ribose from NAD + (nicotinamide adenine dinucleotide) to target proteins. ADP-ribosylation can change the functions, activities, and stabilities of target proteins, thereby affecting behaviors such as DNA repair and transcription and cellular metabolic processes [Rodriguez, K.M. et al. Elife 2021, 10.e60480.].

[0003] The PARP family contains 17 subtypes. According to the degree of ADP-ribosylation, they can be divided into two categories. One is polyADP-ribose polymerase (polyPARP), including four members, PARP1, PARP2, PARP5a, and PARP5b, which can catalyze the synthesis of polyADP-ribose (PAR) chains on their target substrates. The other is monoADP-ribose polymerase (monoPARP), including twelve members, PARP3, PARP4, PARP6, PARP7, PARP8, PARP9, PARP10, PARP11, PARP12, PARP14, PARP15, PARP16, PARP17. This type of PARP only transfers one ADP-ribose to the substrate protein); in addition, no ADP-ribosyltransferase activity has been found in PARP13, [Fehr, A.R. et al. Genes Dev. 2020, 34, 341–359].

[0004] Among the PARP family, PARP-2 and PARP1 have the highest homology and play a key role in DNA damage repair. Inhibiting the activities of PARP1 / 2 can effectively block the repair of damaged DNA, thereby enhancing the anti-tumor effects of chemotherapeutic drugs [Curtin, N.J. et al. Nat. Rev. Drug Discov. 2020, 19, 711–736]. Since 2014, six PARP1 / 2 inhibitors, namely olaparib (AZD2281), rucaparib (AG014699), niraparib (MK4827), talazoparib (BMN673), pamiparib (BGB290), and fluzoparib (SHR3162), have been successively marketed, demonstrating that PARP1 / 2 is an effective anti-tumor target [Chen, Y. et al. Biomed. Pharmacother. 2018, 99, 552–560; Boussios, S. et al. Drugs R D 2020, 20, 55–73; Wang, H. et al. J. Med. Chem. 2020, 63, 15541–15563; Wang, L. et al. Cancer Sci. 2019, 110, 1064–1075; Balasubramaniam, S. et al. Clin. Cancer Res. 2017, 23, 7165–7170; Ison, G. et al. Clin. Cancer Res. 2018, 24, 4066–4071; Hoy, S.M. Drugs 2018, 78, 1939–1946].

[0005] PARP7 belongs to monoPARP, is highly expressed in various tumor tissues, plays a key role in cell proliferation, tumor formation, especially in immune regulation, and is considered a negative regulator of nucleic acid sensing in tumor cells. In tumor models, when the expression of PARP7 increases, the IFN-β signaling pathway is inhibited, which helps tumor cells evade the recognition of the cellular immune system [Borden, E.C. Nat. Rev. Drug Discov. 2019, 18, 219–234]. In addition, PARP7 can also inhibit T cell-mediated anti-tumor immune responses [Rasmussen, M. et al. Cells 2021, 10, 623]. Therefore, inhibiting PARP7 can promote the nucleic acid signaling of IFN-I in a TBK1-dependent manner, stimulate innate or adaptive anti-tumor immune responses, and ultimately lead to the death of tumor cells [Gozgit, J.M. et al. Cancer Cell 2021, 39, 1214–1226].

[0006] RBN-2397, as a PARP7 selective inhibitor, has entered Phase II clinical trials for the treatment of solid tumors [https: / / data.pharmacodia.com]. In addition, PARP inhibitors - Thioparib and YCH1899, exhibit significant inhibitory activity against multiple PARP subtypes, with inhibitory activity against PARP1 and PARP2 reaching the nanomolar level and having certain inhibitory activity against PARP7. Both of these compounds have significant in vivo antitumor activity [Wang, L.M. et al. EMBO Mol. Med. 2023, 15, e16235; Sun, Y.T. et al. J. Med. Chem. 2023, 66, 17, 12284 - 12303]. Based on the above research results, it is speculated that triple-target inhibition of PARP1, PARP2, and PARP7 is a novel and promising antitumor drug. This patent designed and synthesized phthalazinone compounds containing piperazinone with a new structure, which have significant inhibitory activity against PARP1, PARP2, and PARP7. These compounds can not only inhibit the DNA damage repair process of tumor cells but also induce an immune response, inhibit the immune escape of tumor cells, and achieve the purpose of inhibiting tumor growth through multiple mechanisms, which is a more effective means of treating tumors. The purpose of this invention is to provide a completely new material basis for the treatment of diseases related to PARP1 / 2 / 7, especially for the treatment of tumors. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide phthalazinone derivatives containing piperazinone shown in Formula I, their preparation methods, pharmaceutical compositions, and their uses in preparing PARP1 / 2 / 7 inhibitors and their potential drugs, as well as their uses in preparing antitumor drugs, antitumor drug sensitizers, or tumor immunotherapy.

[0008] To solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0009] The first aspect of the technical solution of the present invention is to provide phthalazinone derivatives containing piperazinone shown in General Formula I or physiologically acceptable salts:

[0010]

[0011] In Formula I,

[0012] R1, R2, R3, and R4 are independently selected from H, F, Cl, Br;

[0013] R5 is selected from the following atoms or groups or structural fragments:

[0014] (1) Hydrogen, substituted or unsubstituted C1, C2, C3, C4, C5, C6, C7, C8 straight-chain or branched-chain alkyl groups, where the substituents are selected from F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylmethylene, cyclobutyl, oxetanyl, cyclopentyl, cyclohexyl, and where the Rc1 is independently selected from H, C1, C2, C3, C4 straight-chain or branched-chain alkyl groups;

[0015] (2) Substituted or unsubstituted C3, C4, C5, C6, C7 cycloalkyl groups, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered oxacycloalkyl groups, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered azacycloalkyl groups, where the substituents are selected from methyl, ethyl, propyl, isopropyl, F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylmethylene, cyclobutyl, and where the Rc1 is independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclopropylmethylene, cyclobutyl; the oxacycloalkyl and azacycloalkyl groups may contain one heteroatom or multiple heteroatoms simultaneously.

[0016] The second aspect of the technical solution of the present invention is to provide phthalazinone derivatives containing piperazinone or physiologically acceptable salts as shown in general formula IA:

[0017]

[0018] In formula IA,

[0019] R2 is independently selected from H, F, Cl, Br;

[0020] R5 is selected from the following atoms or groups or structural fragments:

[0021] (1) Hydrogen, substituted or unsubstituted C1, C2, C3, C4, C5, C6, C7, C8 straight-chain or branched-chain alkyl groups, where the substituents are selected from F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylmethylene, cyclobutyl, oxetanyl, cyclopentyl, cyclohexyl, and where the Rc1 is independently selected from H, C1, C2, C3, C4 straight-chain or branched-chain alkyl groups;

[0022] (2) Substituted or unsubstituted C3, C4, C5, C6, C7 cycloalkyl groups, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered oxacycloalkyl groups, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered azacycloalkyl groups, where the substituents are selected from methyl, ethyl, propyl, isopropyl, F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylmethylene, cyclobutyl, and where the Rc1 is independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclopropylmethylene, cyclobutyl; the oxacycloalkyl and azacycloalkyl groups may contain one heteroatom or multiple heteroatoms simultaneously.

[0023] The third aspect of the technical solution of the present invention is to provide a phthalazinone derivative containing piperazinone represented by the general formula IA-1 or a physiologically acceptable salt thereof:

[0024]

[0025] In formula IA-1,

[0026] R2 is independently selected from H, F, Cl, Br.

[0027] The fourth aspect of the technical solution of the present invention is to provide a phthalazinone derivative containing piperazinone represented by the general formula IB or a physiologically acceptable salt thereof:

[0028]

[0029] In formula IB,

[0030] R3 is independently selected from H, F, Cl, Br;

[0031] R5 is selected from the following atoms or groups or structural fragments:

[0032] (1) hydrogen, substituted or unsubstituted C1, C2, C3, C4, C5, C6, C7, C8 straight-chain or branched-chain alkyl, wherein the substituent is selected from F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylmethylene, cyclobutyl, oxetanyl, cyclopentyl, cyclohexyl, wherein the said Rc1 is independently selected from H, C1, C2, C3, C4 straight-chain or branched-chain alkyl;

[0033] (2) substituted or unsubstituted C3, C4, C5, C6, C7 cycloalkyl, substituted or unsubstituted 3, 4, 5, 6, 7, 8-membered oxacycloalkyl, substituted or unsubstituted 3, 4, 5, 6, 7, 8-membered azacycloalkyl, wherein the said substituent is selected from methyl, ethyl, propyl, isopropyl, F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylmethylene, cyclobutyl, wherein the said Rc1 is independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclopropylmethylene, cyclobutyl; the oxacycloalkyl and azacycloalkyl may contain 1 heteroatom or may contain multiple heteroatoms simultaneously.

[0034] The fifth aspect of the technical solution of the present invention is to provide a phthalazinone derivative containing piperazinone represented by the general formula IB-1 or a physiologically acceptable salt thereof:

[0035]

[0036] In formula IB-1,

[0037] R3 is independently selected from H, F, Cl, Br.

[0038] In the sixth aspect of the technical solution of the present invention, for the purpose of achieving the object of the present invention, the preferred compounds include but are not limited to:

[0039]

[0040] The seventh aspect of the technical solution of the present invention is to provide a preparation method of the compounds described in the first to sixth aspects. The technical solution adopted includes the following steps: Dimethyl phosphite undergoes an addition cyclization reaction with differently substituted o-formylbenzoic acid under alkaline conditions (such as sodium methoxide) to obtain intermediate 2. Intermediate 2 reacts with 2-fluoro-5-formylbenzonitrile under the catalysis of alkaline conditions (such as DBU) to obtain intermediate 3. Intermediate 3 reacts with hydrazine hydrate to obtain intermediate 4. Intermediate 4 undergoes a hydrolysis reaction under alkaline conditions (such as sodium hydroxide) to obtain intermediate 5; Piperazinone reacts with benzyloxycarbonyl chloride under alkaline conditions (such as sodium carbonate) to obtain intermediate 7. Intermediate 7 reacts with different haloalkanes or sulfonic acid esters substituted with different alkyl groups under alkaline conditions (such as NaH) to obtain intermediate 8. Intermediate 8 removes the protecting group under catalytic hydrogenation conditions to obtain intermediate 9; Intermediate 5 reacts with intermediate 9 under the catalysis of a condensing agent to obtain the compound shown in general formula I;

[0041]

[0042] Reagents and reaction conditions: (i) Sodium metal, methanol, dimethyl phosphite, methanesulfonic acid, 0 °C - room temperature; (ii) 3-cyano-4-fluorobenzaldehyde, DBU, room temperature; (iii) Hydrazine hydrate, ethanol, 60 °C; (iv) 2.5N aqueous sodium hydroxide solution, 90 °C, dilute hydrochloric acid; (v) Benzyloxycarbonyl chloride, sodium carbonate, ethyl acetate, water, ice bath - room temperature; (vi) Sodium hydride, bromoalkane or alkyl-substituted p-toluenesulfonate, N,N-dimethylformamide (DMF), room temperature or 60 °C; (vii) 10% Pd / C, hydrogen, room temperature, atmospheric pressure; (viii) 1-ethyl-3(3-dimethylpropylamine)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF), room temperature;

[0043] Wherein the definitions of R1, R2, R3, R4, and R5 are the same as those described in any one of the first to fifth aspects of the technical solution.

[0044] In addition, the starting materials and intermediates in the above reactions are readily available, and each step of the reaction can be easily synthesized according to the reported literature or by conventional methods in organic synthesis that are well-known to those skilled in the art. The compounds described by General Formula I may exist in the form of solvates or non-solvates, and different solvates may be obtained by crystallization using different solvents. The pharmaceutically acceptable salts described by General Formula I include salts of different acids, such as salts of the following inorganic acids or organic acids: hydrochloric acid, acetic acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, lycium acid, maleic acid, tartaric acid, fumaric acid, citric acid, lactic acid. The pharmaceutically acceptable salts described by General Formula I also include different alkali metal salts (lithium, sodium, potassium salts), alkaline earth metal salts (calcium, magnesium salts), and ammonium salts, and salts of organic bases that can provide physiologically acceptable cations, such as salts of methylamine, dimethylamine, trimethylamine, piperidine, morpholine, and tris(2-hydroxyethyl)amine. All of these salts within the scope of the present invention can be prepared by conventional methods.

[0045] The eighth aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which comprises the compound or its pharmaceutically acceptable salt described in the first aspect to the fifth aspect of the technical solution of the present invention and a pharmaceutically common carrier.

[0046] This composition comprises at least one compound of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition is selected from tablets, capsules, pills, injections, sustained-release preparations, controlled-release preparations, or various particulate drug delivery systems. The pharmaceutical composition can be prepared according to methods well-known in the art. It can be made into any dosage form suitable for human or animal use by combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants. The content of the compounds of the present invention in their pharmaceutical compositions is usually 0.1 - 95% by weight. The compounds of the present invention or the pharmaceutical compositions containing them can be administered in unit dosage forms, and the administration routes can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc. The dosage forms for administration can be liquid dosage forms, solid dosage forms, or semi-solid dosage forms. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w type, w / o type, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nose drops, lotions, and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, aerosols (powder aerosols), sprays, etc.; semi-solid dosage forms can be ointments, gels, pastes, etc.

[0047] The compounds of the present invention can be formulated into conventional preparations, as well as sustained-release preparations, controlled-release preparations, targeted preparations and various particulate drug delivery systems. These preparations are prepared by methods well known to those skilled in the art. The excipients used for manufacturing tablets, capsules and coatings are conventional auxiliaries, such as starch, gelatin, gum arabic, silica, polyethylene glycol, and the solvents used for liquid dosage forms such as water, ethanol, propylene glycol, vegetable oils such as corn oil, peanut oil, olive oil, etc. Other auxiliaries may also be present in the preparations containing the compounds of the present invention, such as surfactants, lubricants, disintegrants, preservatives, flavoring agents, pigments, etc.

[0048] In order to formulate the compounds of the present invention into tablets, various excipients well known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropanol, etc.; the binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecylsulfonate, etc.; the lubricants and glidants can be talc, silica, stearates, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0049] The tablets can be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets.

[0050] In order to formulate the dosage unit into capsules, the active ingredient, the compound of the present invention, can be mixed with a diluent and a glidant, and the mixture can be directly placed into hard capsules or soft capsules. Alternatively, the active ingredient, the compound of the present invention, can be first made into granules or pellets with a diluent, a binder and a disintegrant, and then placed into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants, glidants used for preparing the tablets of the compounds of the present invention can also be used for preparing the capsules of the compounds of the present invention.

[0051] To prepare the compound of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent, and appropriate solubilizers, cosolvents, pH regulators, and osmotic pressure regulators commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. When preparing a freeze-dried powder injection, mannitol, glucose, etc. can also be added as a bulking agent.

[0052] In addition, if necessary, coloring agents, preservatives, fragrances, flavoring agents or other additives can also be added to the pharmaceutical preparation.

[0053] To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

[0054] The dosage of the pharmaceutical composition of the compound of the present invention can vary within a wide range depending on the nature and severity of the disease to be prevented or treated, the individual conditions of the patient or animal, the administration route and dosage form, etc. Generally speaking, the appropriate daily dosage range of the compound of the present invention is 0.1 - 1000 mg / Kg body weight, preferably 0.1 - 200 mg / Kg body weight. The above dosage can be administered in one dosage unit or divided into several dosage units, which depends on the doctor's clinical experience and the dosing regimen including the use of other treatment means.

[0055] The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When there is a synergistic effect between the compound of the present invention and other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0056] The ninth aspect of the technical solution of the present invention is to provide the application of the compound described in the first to sixth aspects of the present invention in the preparation of PARP1 / 2 / 7 inhibitors, in the preparation of drugs for preventing and / or treating diseases related to PARP1 / 2 / 7, in the preparation of anti-tumor drugs, and in the preparation of drugs for diseases related to tumors, wherein the tumors are selected from melanoma, gastric cancer, lung cancer, breast cancer, kidney cancer, liver cancer, oral epidermal cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, colon cancer, bladder cancer, glioma, leukemia, lymphoma.

[0057] Beneficial technical effects:

[0058] This patent design synthesizes phthalazinone compounds containing piperazinone with a new structure, which have significant inhibitory activities against PARP1, PARP2, and PARP7. These compounds can not only inhibit the DNA damage repair process of tumor cells, but also induce immune responses and inhibit the immune escape of tumor cells. They can achieve the purpose of inhibiting tumor growth and killing tumors through multiple mechanisms, and are more effective means for treating tumors. This invention provides brand-new multi-target phthalazinone compounds for the treatment of diseases related to PARP1 / 2 / 7, especially for the treatment of tumors. Brief Description of the Drawings:

[0059] This patent includes three drawings, which are respectively:

[0060] Figure 1 After treating THP-1 cells with Compound 1, immunoblot analysis of the protein levels of phosphorylated TBK1 (p-TBK1) and γH2AX;

[0061] Figure 2 Inhibitory effect of Compound 1 on tumors in the MC-38 syngeneic tumor mouse model;

[0062] Figure 3 In the MC-38 syngeneic tumor mouse model, CD8 + / CD3 + Percentage of T cells. Detailed Embodiments:

[0063] The following will further illustrate the invention in combination with embodiments, but does not limit the scope of the invention.

[0064] The structure of the compound is determined by nuclear magnetic resonance (NMR) or high-resolution mass spectrometry (HRMS). The NMR measurement is carried out using Varian mercury 300 or Varian mercury 400, and the measurement solvents are CDCl3, DMSO-d6, acetone-d6, CD3OD. The internal standard is TMS, and the chemical shift is given in ppm as the unit. The m.p. is the melting point given in °C, and the temperature is not corrected. Silica gel column chromatography generally uses silica gel with 200 - 300 mesh as the carrier.

[0065] Abbreviation List:

[0066] TLC: Thin layer chromatography;

[0067] DIEA: Diisopropylethylamine; DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene

[0068] DMF: N,N-Dimethylformamide; THF: Tetrahydrofuran; PE: Petroleum ether; EA: Ethyl acetate

[0069] DCM: Dichloromethane; MeOH: Methanol; EtOH: Ethanol;

[0070] EDC or EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0071] HOBt: 1-Hydroxybenzotriazole; NaH: Sodium hydride; NaOH: Sodium hydroxide;

[0072] Cbz-Cl: Benzyloxycarbonyl chloride; K2CO3: Potassium carbonate;

[0073] min: Minute; r.t.: Room temperature; h: Hour;

[0074] DMSO-d6: Dimethyl sulfoxide-d6; CDCl3: Chloroform-d;

[0075] Preparation of intermediates:

[0076] (1) Preparation of 1-Pentan-3'-ylpiperazin-2-one

[0077]

[0078] a) Preparation of N-Benzyloxycarbonyl-3-oxopiperazine

[0079]

[0080] Piperazin-2-one (1 g, 10 mmol) was added to ethyl acetate (40 mL) and water (20 mL). K2CO3 (6.9 g, 50 mmol) was added under stirring at room temperature. Benzyloxycarbonyl chloride (2.1 mL, 15 mmol) was added dropwise to the reaction flask. After the addition, the reaction was stirred at room temperature. The reaction was stopped the next day. The layers were separated. The organic layer was washed with saturated NaCl solution (20 mL × 2), dried over anhydrous magnesium sulfate, and purified by column chromatography (DCM:MeOH = 75:1) to obtain 1.4 g of a white solid with a yield of 59.8%.

[0081] 1 1H-NMR (400 MHz, CDCl3): δ (ppm) 7.32–7.40 (m, 5H), 6.94 (brs, 1H), 5.16 (s, 2H), 4.16 (s, 2H), 3.70 (t, J = 4.8 Hz, 2H), 3.40 (brs, 2H).

[0082] b) Benzyl 4-Pentyl-3'-yl-3-oxopiperazine-1-carboxylate

[0083]

[0084] N-Carbobenzyloxy-3-oxopiperazine (8 g, 10.25 mmol) was added to DMF (80 mL). Under argon protection, NaH (2.7 g, 68.33 mmol) was added at room temperature. The mixture was stirred at room temperature for 1 h, then 3-bromopentane (10.56 mL, 84.29 mmol) was added. The reaction was heated to 50 °C. After 3 days, the reaction was stopped. Water was added, and the mixture was extracted with 200 mL of ethyl acetate. The organic layer was washed with saturated NaCl solution (50 mL × 2), dried over anhydrous magnesium sulfate, and purified by column chromatography to obtain 4.6 g of the product with a yield of 44.3%.

[0085] 1 H-NMR (400 MHz, CDCl3): δ (ppm) 7.28–7.36 (m, 5H), 5.16 (s, 2H), 4.45–4.50 (m, 1H), 4.19 (s, 2H), 3.68 (t, J = 5.2 Hz, 2H), 3.16 (brs, 2H), 1.35–1.58 (m, 4H), 0.85 (t, J = 7.6 Hz, 6H).

[0086] c) 1-Pentan-3'-ylpiperazin-2-one

[0087]

[0088] Benzyl 4-pentan-3'-yl-3-oxopiperazine-1-carboxylate (4.5 g) was added to EtOH (60 mL), and 10% Pd / C (1.35 g) was added. The reaction was carried out under normal temperature and pressure for hydrogenation. After 4 h, the reaction was stopped. The mixture was filtered and concentrated to obtain 2.5 g of a colorless oil with a yield of 99.6%.

[0089] 1 H-NMR (400 MHz, CDCl3): δ (ppm) 4.44–4.51 (m, 1H), 3.58 (s, 2H), 3.09 (d, J = 6.0 Hz, 4H), 1.39–1.56 (m, 4H), 0.86 (t, J = 7.2 Hz, 6H).

[0090] (2) 2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid

[0091]

[0092] a) Dimethyl (3-oxo-1,3-dihydroisobenzofuran-1-yl) phosphate

[0093]

[0094] Take metallic Na (276 mg, 12 mmol) and add it to 10 mL of methanol under an ice bath. After the reaction of metallic sodium is complete, add dimethyl phosphite (0.97 mL, 10.6 mmol) dropwise to the reaction solution. After the addition is complete, continue stirring for 20 min under an ice bath, then add a 6 mL methanol solution of o - formylbenzoic acid (1.2 g, 8 mmol) dropwise to the reaction flask. After the addition is complete, raise the temperature to 20 °C for reaction. After 1 h, the raw materials disappear. Then add methanesulfonic acid (0.78 mL, 12 mmol) dropwise, stir for 20 min, add water, extract with DCM (30 mL × 2), combine the organic layers, wash with saturated NaCl solution (15 mL × 2), and recrystallize with DCM and PE to obtain 1.4 g of white solid, with a yield of 72.8%. 1 1H - NMR (400 MHz, CDCl3): δ (ppm) 7.96 (d, J = 8.0 Hz, 1H), 7.73–7.79 (m, 2H), 7.61 (t, J = 7.2 Hz, 1H), 5.73 (d, J = 11.2 Hz, 1H), 3.94 (d, J = 10.8 Hz, 3H), 3.60 (d, J = 10.4 Hz, 3H).

[0095] b) 2 - fluoro - 5 - ((3 - oxoisobenzofuran - 1(3H) - methylene)methyl)benzonitrile

[0096]

[0097] Take dimethyl (3 - oxo - 1,3 - dihydroisobenzofuran - 1 - yl) phosphate (726 mg, 3 mmol) and 2 - fluoro - 5 - formylbenzonitrile (447 mg, 3 mmol), add 15 mL of THF, and dropwise add Et3N to the reaction solution. Control the temperature below 15 °C during the addition. After the addition is complete, raise the temperature to 20 °C for reaction. Stop the reaction after 2 h, concentrate, add 20 mL of water, stir for 30 min, then filter by suction. Wash the filter cake with water, n - hexane, and ether to obtain 755 mg of pale yellow solid, with a yield of 94.9%. There are Z - type and E - type in the product, and the next reaction is carried out without separation.

[0098] c) 2 - fluoro - 5 - ((4 - oxo - 3,4 - dihydrophthalazin - 1 - yl)methyl)benzonitrile

[0099]

[0100] The compound 2-fluoro-5-((5-fluoro-3-oxoisobenzofuran-1(3H)-ylidene)methyl)benzonitrile (676 mg, 2.55 mmol) was added to ethanol (12 mL), heated to about 60 °C, hydrazine hydrate (4 mL, 89.6 mmol) was added, and the reaction was continued at about 60 °C. After 5 h, the reaction was stopped, water was added, and the mixture was extracted with ethyl acetate (50 mL × 2). The organic layers were combined, washed with saturated NaCl solution (20 mL × 2), dried over anhydrous magnesium sulfate, concentrated, and purified by column chromatography (DCM:MeOH, MeOH = 2%) to obtain 557 mg of a white solid with a yield of 79.8%.

[0101] 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 12.57 (s, 1H), 8.27 (dd, J1 = 8.0 Hz, J2 = 0.8 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.95–7.89 (m, 2H), 7.84 (td, J1 = 7.6 Hz, J2 = 0.8 Hz, 1H), 7.72 (ddd, J1 = 7.6 Hz, J2 = 5.2 Hz, J3 = 2.4 Hz 1H), 7.47 (t, J = 9.2 Hz, 1H), 4.37 (s, 2H).

[0102] d) 2-Fluoro-5-((4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzoic acid

[0103]

[0104] The compound 2-fluoro-5-((3-oxoisobenzofuran-1(3H)-ylidene)methyl)benzonitrile (525 mg, 1.88 mmol) was added to 2.5 N aqueous NaOH solution (9.28 mL, 23.2 mmol), heated to 90 °C for reaction for 1 h, then the reaction was stopped and cooled to room temperature. The reaction solution was extracted with ether, and the aqueous layer was adjusted to pH 2 - 3 with dilute HCl solution, and a solid precipitated. The solid was filtered by suction, and the filter cake was washed with water to obtain 515 mg of a white solid with a yield of 91.8%.

[0105] 1 H-NMR (400 MHz, DMSO-d6): δ (ppm) 8.25 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.88 (t, J = 7.2 Hz, 1H), 7.78–7.84 (m, 2H), 7.55 (brs, 1H), 7.21 (t, J = 10.4 Hz, 1H).

[0106] (3) 2-Fluoro-5-((6-fluoro-4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzoic acid

[0107]

[0108] a) Dimethyl (5-fluoro-3-oxo-1,3-dihydroisobenzofuran-1-yl) phosphate

[0109]

[0110] Add metallic Na (350 mg, 15 mmol) to 15 mL of methanol under an ice bath. After the complete reaction of metallic sodium, add dimethyl phosphite (1.2 mL, 13 mmol) dropwise to the reaction solution. After the addition is complete, continue stirring for 30 min under an ice bath, then add a 10 mL methanol solution of compound 5-fluoro-2-formylbenzoic acid (1.68 g, 10 mmol) dropwise to the reaction flask. After the addition is complete, raise the temperature to 20 °C for reaction. After 1 h, the raw materials disappear. Add methanesulfonic acid (1 mL, 15 mmol) dropwise and stir the reaction overnight. The next day, extract with DCM (40 mL × 2), combine the organic layers, wash with saturated NaCl solution (20 mL × 2), dry over anhydrous magnesium sulfate, concentrate, and perform column chromatography (DCM:EA, EA = 10%), obtaining 1.85 g of a white solid with a yield of 71.2%.

[0111] 1 H-NMR (400 MHz, CDCl3): δ (ppm) 7.75 (dddt, J1 = 8.4 Hz, J2 = 4.4 Hz, J3 = 2.0 Hz, J4 = 1.2 Hz, 1H), 7.61 (dd, J1 = 6.8 Hz, J2 = 2.0 Hz, 1H), 7.46 (td, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H), 5.69 (dt, J1 = 10.8 Hz, J2 = 1.2 Hz, 1H), 3.94 (d, J = 10.8 Hz, 3H), 3.55 (d, J = 10.4 Hz, 3H).

[0112] b) 2-Fluoro-5-((5-fluoro-3-oxoisobenzofuran-1(3H)-ylidene)methyl)benzonitrile

[0113]

[0114] Take dimethyl (5-fluoro-3-oxo-1,3-dihydroisobenzofuran-1-yl) phosphate (1.5 g, 5.77 mmol) and 2-fluoro-5-formylbenzonitrile (860 mg, 5.77 mmol), add 15 mL of THF, and dropwise add DBU (0.85 mL, 5.77 mmol) to the reaction solution. After the addition is complete, stir the reaction at room temperature. Stop the reaction after 2 h, concentrate, add 20 mL of water, stir for 30 min, then filter by suction. Wash the filter cake with water, n-hexane, and ether to obtain 1.63 g of an off-white solid with a yield of 100%. There are Z-form and E-form in the product, and the next reaction is continued without separation.

[0115] c) 2-Fluoro-5-((6-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzonitrile

[0116]

[0117] Take 2-fluoro-5-((5-fluoro-3-oxoisobenzofuran-1(3H)-ylidene)methyl)benzonitrile (1.5 g, 5.3 mmol), add ethanol (30 mL), heat to about 60 °C, add hydrazine hydrate (8 mL, 132.5 mmol), continue to react at about 60 °C. Stop the reaction after 5 h, add water, extract with ethyl acetate (50 mL × 2), combine the organic layers, wash with saturated NaCl solution (20 mL × 2), dry over anhydrous magnesium sulfate, concentrate, and perform column chromatography (DCM:MeOH, MeOH = 1%) to obtain 1.05 g of a white solid with a yield of 66.8%.

[0118] 1 1H-NMR (400 MHz, DMSO-d6): δ (ppm) 12.68 (s, 1H), 8.10 (dd, J1 = 8.8 Hz, J2 = 5.2 Hz, 1H), 7.94 (dd, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H), 7.91 (dd, J1 = 9.6 Hz, J2 = 2.4 Hz, 1H), 7.81 (td, J1 = 8.8 Hz, J2 = 2.8 Hz, 1H), 7.71 (ddd, J1 = 8.8 Hz, J2 = 5.2 Hz, J3 = 2.0 Hz 1H), 7.47 (t, J = 8.8 Hz, 1H), 4.37 (s, 2H).

[0119] d) 2-Fluoro-5-((6-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid

[0120]

[0121] Compound 2-fluoro-5-((6-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzonitrile (940 mg, 3.16 mmol) was added to an aqueous solution of 2.5 N NaOH (18 mL, 47.4 mmol). The mixture was heated to 90 °C for reaction. After 1 h, the reaction was stopped and cooled to room temperature. The reaction solution was extracted with diethyl ether. The aqueous layer was adjusted to pH 2 - 3 with dilute HCl solution, and a solid precipitated. It was filtered by suction, and the filter cake was washed with water to obtain 990 mg of white solid, with a yield of 99.2%.

[0122] 1 1H-NMR (500 MHz, DMSO-d6): δ (ppm) 13.27 (s, 1H), 12.73 (s, 1H), 8.11 (dd, J1 = 9.5 Hz, J2 = 5.5 Hz, 1H), 7.94 (dd, J1 = 8.5 Hz, J2 = 3.0 Hz, 1H), 7.79–7.83 (m, 2H), 7.56–7.59 (m, 1H), 7.26 (t, J = 8.5 Hz, 1H), 4.37 (s, 2H).

[0123] (4) 2-Fluoro-5-((7-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid

[0124]

[0125] a) Dimethyl (6-fluoro-3-oxo-1,3-dihydroisobenzofuran-1-yl)phosphate

[0126]

[0127] Metallic sodium (205 mg, 8.93 mmol) was added to 10 mL of methanol under an ice bath. After the complete reaction of metallic sodium, dimethyl phosphite (0.71 mL, 7.74 mmol) was added dropwise to the reaction solution. After the addition, the mixture was stirred for another 30 min under an ice bath. Then, a 10 mL methanol solution of compound 4-fluoro-2-formylbenzoic acid (1 g, 5.95 mmol) was added dropwise to the reaction flask. After the addition, the temperature was raised to 20 °C for reaction. After 1 h, the raw materials disappeared. Methanesulfonic acid (0.6 mL, 8.93 mmol) was added dropwise and the reaction was stirred overnight. The next day, it was extracted with DCM (30 mL × 2), the organic layers were combined, washed with saturated NaCl solution (20 mL × 2), dried over anhydrous magnesium sulfate, concentrated, and purified by column chromatography (DCM:EA, EA = 10%) to obtain 0.50 g of white solid, with a yield of 31.8%.

[0128] 11H-NMR (400 MHz, CDCl3): δ (ppm) 7.95 (dd, J1 = 8.4 Hz, J2 = 4.8 Hz, 1H), 7.44 (dt, J1 = 8.4 Hz, J2 = 2.4 Hz, 1H), 7.30 (tt, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H), 5.67 (d, J1 = 11.6 Hz, 1H), 3.94 (d, J = 11.2 Hz, 3H), 3.68 (d, J = 10.8 Hz, 3H).

[0129] b) 2-Fluoro-5-((6-fluoro-3-oxoisobenzofuran-1(3H)-ylidene)methyl)benzonitrile

[0130]

[0131] Take dimethyl (6-fluoro-3-oxo-1,3-dihydroisobenzofuran-1-yl) phosphate (400 mg, 1.54 mmol) and 2-fluoro-5-formylbenzonitrile (229 mg, 1.54 mmol), add 8 mL of THF, and dropwise add DBU (0.23 mL, 1.54 mmol) to the reaction solution. After the addition is complete, stir the reaction at room temperature. Stop the reaction after 1.5 h, concentrate, add 20 mL of water, stir for 30 min, then filter by suction. Wash the filter cake with water, n-hexane, and ether to obtain 435 mg of an off-white solid with a yield of 100%. There are Z-form and E-form in the product, and they are not separated and continue to the next step of the reaction.

[0132] c) 2-Fluoro-5-((7-fluoro-4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzonitrile

[0133]

[0134] Take 2-fluoro-5-((6-fluoro-3-oxoisobenzofuran-1(3H)-ylidene)methyl)benzonitrile (400 g, 1.41 mmol), add 10 mL of ethanol, heat to about 60 °C, add hydrazine hydrate (2.1 mL, 35 mmol), continue to react at about 60 °C. Stop the reaction after 7 h, add water, extract with ethyl acetate (30 mL × 2), combine the organic layers, wash with saturated NaCl solution (15 mL × 2), dry over anhydrous magnesium sulfate, concentrate, and perform column chromatography (DCM:MeOH, MeOH = 3%) to obtain 210 mg of a light pink solid with a yield of 50.2%.

[0135] 1H-NMR (400 MHz, DMSO-d6): δ (ppm) 12.65 (s, 1H), 8.33 (dd, J1 = 8.8 Hz, J2 = 5.6 Hz, 1H), 7.92 (dd, J1 = 6.0 Hz, J2 = 2.0 Hz, 1H), 7.85 (dd, J1 = 10.0 Hz, J2 = 2.4 Hz, 1H), 7.68 - 7.76 (m, 2H), 7.48 (t, J = 8.8 Hz, 1H), 4.35 (s, 2H).

[0136] d) 2-Fluoro-5-((7-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid

[0137]

[0138] Take the compound 2-fluoro-5-((7-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzonitrile (200 mg, 0.67 mmol), add 2.5 N aqueous NaOH solution (4 mL, 10 mmol), heat the reaction to 90 °C, stop the reaction after 1 h, cool to room temperature, extract the reaction solution with ether, adjust the pH of the aqueous layer to 2 - 3 with dilute HCl solution, a solid precipitates, filter by suction, wash the filter cake with water, and obtain 200 mg of white solid with a yield of 94.7%.

[0139] 1 H-NMR (400 MHz, DMSO-d6): δ (ppm) 13.23 (s, 1H), 12.67 (s, 1H), 8.33 (dd, J1 = 8.8 Hz, J2 = 5.6 Hz, 1H), 7.85 (d, J = 2.8 Hz, 1H), 7.83 (t, J = 2.4 Hz, 1H), 7.70 (td, J1 = 8.8 Hz, J2 = 4.8 Hz, 1H), 7.60 (ddd, J1 = 8.8 Hz, J2 = 4.8 Hz, J3 = 2.4 Hz, 1H), 7.25 (dd, J1 = 10.8 Hz, J2 = 8.4 Hz, 1H), 4.34 (s, 2H).

[0140] (5) 2-Fluoro-5-((7-bromo-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid

[0141]

[0142] a) Dimethyl (6-bromo-3-oxo-1,3-dihydroisobenzofuran-1-yl) phosphate

[0143]

[0144] Take metallic Na (245 mg, 10.5 mmol) and add it to 15 mL of methanol under an ice bath. After the reaction of metallic sodium is complete, add dimethyl phosphite (0.84 mL, 9.1 mmol) dropwise to the reaction solution. After the addition is complete, continue stirring for 30 min under an ice bath. Then, add a 10 mL methanol solution of compound 4-bromo-2-formylbenzoic acid (1.6 g, 7 mmol) dropwise to the reaction flask. After the addition is complete, raise the temperature to 20 °C for reaction. After 1 h, the raw materials disappear. Then, add methanesulfonic acid (0.7 mL, 10.5 mmol) dropwise and stir the reaction overnight. The next day, extract with DCM (40 mL × 2), combine the organic layers, wash with saturated NaCl solution (30 mL × 2), dry over anhydrous magnesium sulfate, concentrate, and perform column chromatography (DCM:EA, EA = 10%), obtaining 1.6 g of a white solid with a yield of 61.36%.

[0145] 1 1H-NMR (400 MHz, CDCl3): δ (ppm) 7.92 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.74 (dt, J1 = 8.0 Hz, J2 = 2.8 Hz, 1H), 5.68 (d, J1 = 11.2 Hz, 1H), 3.94 (d, J = 10.8 Hz, 3H), 3.69 (d, J = 10.8 Hz, 3H).

[0146] b) 2-Fluoro-5-((6-bromo-3-oxoisobenzofuran-1(3H)-ylidene)methyl)benzonitrile

[0147]

[0148] Take dimethyl (6-bromo-3-oxo-1,3-dihydroisobenzofuran-1-yl) phosphate (700 mg, 2.19 mmol) and compound 2-fluoro-5-formylbenzonitrile (329 mg, 2.19 mmol), add 15 mL of THF, and add DBU (0.32 mL, 2.19 mmol) dropwise to the reaction solution. After the addition is complete, stir the reaction at room temperature. Stop the reaction after 2 h, concentrate, add 20 mL of water, stir for 30 min, then filter by suction. Wash the filter cake with water, n-hexane, and ether to obtain 750 mg of a pale yellow solid with a yield of 100%. There are Z-form and E-form in the product, and they are not separated and continue to the next step.

[0149] c) 2-Fluoro-5-((7-bromo-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzonitrile

[0150]

[0151] The compound 2-fluoro-5-((6-bromo-3-oxoisobenzofuran-1(3H)-ylidene)methyl)benzonitrile (685 g, 2 mmol) was added to ethanol (15 mL). After heating to about 60 °C, hydrazine hydrate (3 mL, 50 mmol) was added. The reaction was continued at about 60 °C. After 5 h, the reaction was stopped. The reaction mixture was cooled to room temperature, filtered by suction. The filter cake was washed with water and then with ethanol to obtain 590 mg of a white solid with a yield of 82.6%.

[0152] 1 1H-NMR (400 MHz, DMSO-d6): δ (ppm) 12.66 (s, 1H), 8.22 (d, J = 2.0 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H), 8.02 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H), 7.90 (dd, J1 = 6.4 Hz, J2 = 2.4 Hz, 1H), 7.73 (ddd, J1 = 8.8 Hz, J2 = 6.4 Hz, J3 = 2.4 Hz 1H), 7.48 (t, J = 8.8 Hz, 1H), 4.38 (s, 2H).

[0153] d) 2-Fluoro-5-((7-bromo-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid

[0154]

[0155] The compound 2-fluoro-5-((7-bromo-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzonitrile (550 mg, 1.54 mmol) was added to 2.5 N aqueous NaOH solution (9.2 mL, 23.10 mmol). The mixture was heated to 90 °C for reaction. After 40 min, the reaction solution was not clear. Ethanol (3 mL) was added and the reaction solution became basically clear. The reaction was continued at 90 °C. After 1 h, the reaction was stopped. The reaction mixture was concentrated, water was added, and the reaction solution was extracted with diethyl ether. The aqueous layer was adjusted to pH 2 - 3 with dilute HCl solution, and a solid precipitated. The solid was filtered by suction, and the filter cake was washed with water to obtain 500 mg of a brick-red solid with a yield of 86.35%.

[0156] 1 1H-NMR (500 MHz, DMSO-d6): δ (ppm) 13.13 (s, 1H), 12.70 (s, 1H), 8.22 (s, 1H), 8.17 (s, 1H), 8.01 (s, 1H), 7.82 (s, 1H), 7.58 (s, 1H), 7.25 (s, 1H), 4.37 (s, 2H).

[0157] Example 1

[0158] Synthesis of 4-(4-Fluoro-3-(3-oxo-4-(pentan-3'-yl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (Compound 1)

[0159]

[0160] Take 2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (745 mg, 2.5 mmol), add DMF (25 mL), EDC (960 mg, 5 mmol), HOBt (675 mg, 1.0 mmol), DIEA (1.3 mL, 7.5 mmol) and 1-(pentan-3'-yl)piperazin-2-one (492 mg, 2.90 mmol), stir and react at room temperature, stop the reaction the next day, add water, extract with EA (40 mL × 2), combine the organic layers, wash with saturated NaCl solution (30 mL × 2), dry over anhydrous magnesium sulfate, concentrate, column chromatography (DCM:MeOH, MeOH = 5%), and obtain 870 mg of white solid, with a yield of 77.3%.

[0161] 1 1H-NMR (400 MHz, CDCl3): δ (ppm) 10.80 (s, 0.4H), 10.67 (s, 0.6H), 8.46 (d, J = 6.8 Hz, 1H), 7.69–7.81 (m, 3H), 7.32–7.39 (m, 2H), 7.04 (t, J = 9.2 Hz, 1H), 4.42–4.51 (m, 2H), 4.28 (s, 2H), 4.04 (s, 1H), 3.96 (s, 1H), 3.52 (s, 1H), 3.24 (t, J = 5.2 Hz, 1H), 3.11 (s, 1H), 1.37–1.59 (m, 4H), 0.82–0.86 (m, 6H); HRMS (ESI) m / z: calcd for C 25 H 28 N4O3F [M + H] + , 451.2140; found, 451.2137.

[0162] Example 2

[0163] Synthesis of 7-Fluoro-4-(4-fluoro-3-(3-oxo-4-(pentan-3'-yl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (Compound 2)

[0164]

[0165] Take the compound 2-fluoro-5-((6-fluoro-4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzoic acid (158 mg, 0.5 mmol), add DMF (8 mL), EDC (192 mg, 1.0 mmol), HOBt (135 mg, 1.0 mmol), DIEA (0.28 mL, 1.5 mmol) and 1-pentan-3'-ylpiperazin-2-one (128 mg, 0.75 mmol). Stir the reaction at room temperature, stop the reaction the next day, add water, extract with EA (40 mL × 2), combine the organic layers, wash with saturated NaCl solution (30 mL × 2), dry over anhydrous magnesium sulfate, concentrate, and perform column chromatography (DCM:MeOH, MeOH = 3%) to obtain 190 mg of a white solid with a yield of 81.2%.

[0166] 1 H-NMR (500 MHz, CDCl3): δ (ppm) 10.60–10.75 (m, 1H), 8.10 (brs, 1H), 7.75 (brs, 1H), 7.49 (brs, 1H), 7.32–7.38 (m, 2H), 7.06 (brs, 1H), 4.43–4.50 (m, 1.5H), 4.28 (s, 2H), 3.96–4.06 (m, 2.5H), 3.48–3.53 (m, 1.5H), 3.25 (s, 1H), 3.14 (brs, 0.5H), 1.44–1.53 (m, 4H), 0.86 (brs, 6H); HRMS (ESI) m / z: calcd for C 25 H 27 N4O3F2 [M+H] + , 469.2046; found, 469.2053.

[0167] Example 3

[0168] Synthesis of 6-fluoro-4-(4-fluoro-3-(3-oxo-4-(pentan-3'-yl)-piperazine-1-carbonyl)-benzyl)phthalazin-1(2H)-one (Compound 3)

[0169]

[0170] Take the compound 2-fluoro-5-((7-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (158 mg, 0.5 mmol), add DMF (8 mL), EDC (192 mg, 1.0 mmol), HOBt (135 mg, 1.0 mmol), DIEA (0.28 mL, 1.5 mmol) and 1-pentan-3'-ylpiperazin-2-one (128 mg, 0.75 mmol), stir the reaction at room temperature, stop the reaction the next day, add water, extract with EA (40 mL × 2), combine the organic layers, wash with saturated NaCl solution (30 mL × 2), dry over anhydrous magnesium sulfate, concentrate, and perform column chromatography (DCM:MeOH, MeOH = 3%), to obtain 190 mg of a white solid with a yield of 81.2%.

[0171] 1 H-NMR (400 MHz, DMSO-d6): δ (ppm) 12.67 (s, 0.6H), 12.65 (s, 0.4H), 8.33 (dd, J1 = 8.4 Hz, J2 = 5.2 Hz, 1H), 7.80 (dt, J1 = 9.6 Hz, J2 = 2.8 Hz, 1H), 7.71 (tt, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H), 7.46–7.50 (m, 1H), 7.35–7.42 (m, 1H), 7.27 (t, J = 8.8 Hz, 1H), 4.32 (s, 2H), 4.19 (s, 2H), 3.80–3.84 (m, 2H), 3.41 (t, J = 5.2 Hz, 1H), 3.20 (t, J = 5.6 Hz, 1H), 3.05 (t, J = 5.2 Hz, 1H), 1.34–1.47 (m, 4H), 0.72–0.77 (m, 6H); HRMS (ESI) m / z: calcd for C 25 H 278 N4O3FBr[M+H] + , 529.1245; found, 529.1248.

[0172] Example 4

[0173] Synthesis of 6-bromo-4-(4-fluoro-3-(3-oxo-4-(pentan-3'-yl)-piperazine-1-carbonyl)-benzyl)phthalazin-1(2H)-one (Compound 4)

[0174]

[0175] Take the compound 2-fluoro-5-((7-bromo-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (230 mg, 0.61 mmol), add DMF (8 mL), EDC (235 mg, 1.22 mmol), HOBt (165 mg, 1.22 mmol), DIEA (0.32 mL, 1.83 mmol) and 1-pentan-3'-ylpiperazin-2-one (156 mg, 0.92 mmol), stir the reaction at room temperature, stop the reaction the next day, add water, extract with ethyl acetate (40 mL × 2), combine the organic layers, wash with saturated NaCl solution (30 mL × 2), dry over anhydrous magnesium sulfate, concentrate, and perform column chromatography (DCM:MeOH, MeOH = 3%) to obtain 270 mg of a white solid with a yield of 83.6%.

[0176] 1 1H-NMR (500 MHz, CDCl3): δ (ppm) 10.49 - 10.57 (m, 1H), 8.31–8.33 (m, 1H), 7.84–7.87 (m, 2H), 7.31–7.40 (m, 2H), 7.04–7.10 (m, 1H), 4.43–4.52 (m, 1.5H), 4.23–4.28 (m, 2.5H), 4.08 (s, 1H), 3.98 (brs, 1H), 3.59 (brs, 0.5H), 3.25 (t, J = 4.8 Hz, 1H), 3.11 - 3.13 (m, 1.5H), 1.36–1.57 (m, 4H), 0.83–0.88 (m, 6H); HRMS (ESI) m / z: calcd for C 25 H 28 N4O3F [M + H] + , 469.2046; found, 469.2035.

[0177] Pharmacological experiments:

[0178] I. Determination of PARP1 / 2 enzyme inhibitory activity

[0179] 1. Materials and reagents

[0180] 1.1. Protein

[0181] catPARP1 (PARP1 catalytic domain structure) or catPARP2.

[0182] 1.2. Test buffer

[0183] 1×PBS + 100 nM BSA: 135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, 100 nM BSA.

[0184] 1.3. Probe

[0185] ZJ-II-604, with a molecular weight of 870.25 Da.

[0186]

[0187] 2. Test method

[0188] Fluorescence polarization method, excitation wavelength 485 nm, emission wavelength 525 nm. The assay buffer for the test system is 1×PBS + 100 nM BSA (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, 100 nM BSA). Test in a black 96-well plate.

[0189] 2.1. Determination of the binding K d value of probe ZJ-II-604 to catPARP-1 / 2

[0190] First, proteins PARP1 and PARP2 with a concentration of 0.5 mM were diluted with the assay buffer. The concentrations (μM) of catPARP1 protein were: 2, 0.666, 0.2, 0.0666, 0.02, 0.00666, 0.002, 0.000666, 0.0002, 0.0000666, 0.00002, 0; the concentrations (μM) of catPARP2 protein were: 1.0, 0.4, 0.2, 0.1, 0.04, 0.02, 0.01, 0.004, 0.002, 0.001, 0; Preparation of the probe molecule solution: The probe molecule was first prepared into a 100 mM solution with DMSO and then diluted to a concentration of 100 μM with the buffer solution. 2.5 μL of the probe molecule at this concentration was added to 10 mL of the assay buffer for activity evaluation; 100 μL of the diluted PARP-1 or PARP-2 protein at different concentrations and the diluted probe were respectively added to the cell culture plate; Incubate overnight at 4°C in the dark, and fit formula 1 to obtain the probe dissociation constant K d .

[0191]

[0192] Y = fluorescence anisotropy value (mA); x = concentration of PARP1 / 2 (μM); A0 = Y min ; A max = Y max - Y min ; Cl = probe concentration (μM); K d = probe equilibrium dissociation constant (μM);

[0193] After measurement and calculation, the K value of ZJ-II-604 binding to PARP1 is 0.676 nM, with a R value of 99.89%. The K value of ZJ-II-604 binding to PARP2 is 0.6569 nM, with a R value of 99.73%. d value is 0.676 nM, with a R 2 value of 99.89%. The K value of binding to PARP2 is d 0.6569 nM, with a R 2 value of 99.73%.

[0194] 2.2. Compound K d (K i ) value determination

[0195] Preparation of compound solution (Solution A): Dilute the compound with a concentration of 100 μM (DMSO solution) to gradient concentrations (μM): 1000, 100, 10, 1, 0.1, 0.01, 0.001 using the assay buffer. Preparation of Solution B: First, dissolve the probe molecule in DMSO to prepare a 100 mM solution, and then dilute it to a concentration of 100 μM with the buffer solution. Take 2.5 μL of the probe with a concentration of 100 μM and 4 μL of PARP1 or PARP2 with a concentration of (0.5 mM) and add them to 10 mL of the assay buffer solution to obtain Solution B. Take 100 μL of the diluted compound with different concentrations (Solution A) and the solution of protein and probe 100 μL (Solution B) and add them to the cell culture plate. Incubate overnight at 4°C in the dark, and fit the formula 2 to obtain the compound inhibition constant K, that is, the dissociation constant K i , that is, the dissociation constant K d .

[0196]

[0197] Y = fluorescence anisotropy value (mA); x = Log value of compound concentration (Log μM); A0 = Y min ; A max = Y max -Y min ; Cl = probe concentration (μM); K d = probe equilibrium dissociation constant (μM); K i = compound equilibrium dissociation constant (μM)

[0198] The experimental results are shown in Table 1

[0199] Table 1. K values of compounds inhibiting PARP1 and PARP2 d

[0200]

[0201] II. Determination of PARP7 enzyme inhibitory activity

[0202] 1. Materials and reagents

[0203] 1.1. Protein

[0204] PARP7, purchased from BPS Bioscience.

[0205] 1.2. Test compounds

[0206] Compounds 1 - 4. The compounds were prepared at concentrations of 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM in assay buffer (50 mM Tris, 2 mM MgCl2, pH 8.0).

[0207] 2. Assay method

[0208] Coat a 96 - well plate with 50 μg / mL histone at 4°C overnight, and wash the plate 3 times with PBS containing 0.1% Triton X - 100. Set up blank control wells, enzyme - added control wells, and wells for test compounds. Add Biotin - NAD + (5 pmol) 30 μL, purification buffer (0.1 M NaCl, 50 mM Tris, 2 mM MgCl2, pH 8.0) 10 μL, DNA (1 μg / mL) 5 μL to the blank control wells; add Biotin - NAD + (5 pmol) 30 μL , PARP7 (32 nM) 10 μL, DNA (1 μg / mL) 5 μL to the enzyme - added control wells; add Biotin - NAD + (5 pmol) 30 μL, diluted test compound solution 5 μL, PARP7 (32 nM) 10 μL, DNA (1 μg / mL) 5 μL to the wells for test compounds. Then make up the volume of each well to 50 μL and react at room temperature for 1 h. Wash the plate 3 times with PBS containing 0.1% Triton X - 100. Then add HRP - labeled streptavidin antibody (1:500), incubate at room temperature for 30 min, and wash the plate 3 times with PBS containing 0.1% Triton X - 100. Then add 50 μL of TMB reaction solution, react in the dark at room temperature for 15 min, and then terminate the reaction with 50 μL of 2N sulfuric acid. Measure the absorbance at 450 nm. Calculate the inhibition rate of the test compound on PARP7.

[0209] The experimental results are shown in Table 2

[0210] Table 2. IC of compounds inhibiting PARP7 50

[0211]

[0212] III. Cell experiments:

[0213] Experimental method:

[0214] After treating THP-1 cells with Compound 1 at concentrations of 0.1 μM, 1 μM, and 10 μM for 24 hours, the cells were collected and lysed for Western blot analysis.

[0215] Experimental results:

[0216] The results showed that Compound 1 dose-dependently activated phosphorylated STAT1 and phosphorylated TBK1, suggesting that Compound 1 could restore T cell immune tolerance by inhibiting PARP7. The results are shown in Figure 1 . Compound 1 significantly increased the level of γH2AX protein, indicating that Compound 1 inhibited the DNA damage repair of tumor cells by inhibiting the activities of PARP1 / 2.

[0217] IV. In vivo pharmacodynamic experiments:

[0218] (I) Inhibitory effect of Compound 1 on human breast cancer MDA-MB-436 subcutaneous xenografts

[0219] Experimental method:

[0220] 1. Procedures

[0221] Under sterile conditions, 1×10 7 human breast cancer MDA-MB-436 cells were inoculated into the right axilla of female Balb / c-nu nude mice (6 - 8 weeks old). When the tumor volume grew to 1000 mm 3 , the tumor tissue was aseptically dissected, cut into tumor pieces of 2 mm × 2 mm in size, and evenly inoculated subcutaneously in the axillary and dorsal regions of the nude mice. When the tumor grew to 100 - 300 mm 3 , the animals were randomly grouped and drug administration was started (day 0).

[0222] AZD5305 and Compound 1 were administered orally every day. The body weight was measured twice a week, and the length and width of the tumor were measured with a vernier caliper. After 14 days of drug administration, the nude mice were sacrificed by cervical dislocation, the tumor tissue was dissected, and weighed. Finally, the tumor inhibition rate was calculated to evaluate the intensity of the anti-tumor effect.

[0223] 2. Grouping

[0224] Blank control group, positive drug AZD5305 group (5 mg / kg), Compound 1 (1 mg / kg) group, Compound 1 (2 mg / kg) group, and Compound 1 (5 mg / kg) group.

[0225] 3. Drug preparation

[0226] AZD5305 and Compound 1 were suspended in 0.5% sodium carboxymethylcellulose solution.

[0227] 4. Calculation method

[0228] Tumor volume (TV): V = 1 / 2 × a × b 2 , where a and b represent the length and width of the tumor, respectively.

[0229] Tumor inhibition rate: Inhibition(%) = (1 - T / C) × 100, where T is the TV or tumor weight of the treatment group, and C is the TV or tumor weight of the negative control group.

[0230] Experimental results:

[0231] Compound 1 has significant inhibitory activity against human breast cancer MDA-MB-436. The results are shown in Table 3.

[0232] Table 3. Inhibitory effect of Compound 1 on tumor growth in the MDA-MB-436 xenograft model

[0233]

[0234] (2) Inhibitory effect of Compound 1 on subcutaneous xenograft tumors of colon cancer MC38

[0235] Experimental method:

[0236] 1. Steps

[0237] Inoculate 1 × 10 7 mouse colon cancer MC38 cells. On the second day (day 1), the mice were randomly grouped and orally treated with olaparib (50 mg / kg, daily) or Compound 1 (10 mg / kg, daily) for 17 days. At the end of the experiment, tumor tissues were collected for flow cytometry analysis.

[0238] 2. Grouping

[0239] Blank control group, positive drug AZD2281 group (50 mg / kg), Compound 1 (10 mg / kg) group.

[0240] 3. Drug preparation

[0241] AZD2281 and Compound 1 were suspended in 0.5% sodium carboxymethylcellulose solution.

[0242] Experimental results:

[0243] The results showed that Compound 1 could significantly inhibit tumor growth and reverse the immune tolerance of the tumor microenvironment, manifested as a significant increase in infiltrating CD8 + T lymphocytes in the tumor tissue after administration of Compound 1. The results are shown in Figure 2 and Figure 3 .

Claims

1. A compound of the general formula I or a pharmaceutically acceptable salt thereof In formula I, R1, R2, R3 and R4 are independently selected from H, F, Cl, Br; R5 is selected from the following atoms or groups or structural fragments: (1) hydrogen, substituted or unsubstituted C1, C2, C3, C4, C5, C6, C7, C8 straight-chain or branched-chain alkyl, wherein the substituent is selected from F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylmethylene, cyclobutyl, oxetanyl, cyclopentyl, cyclohexyl, wherein said Rc1 is independently selected from H, C1, C2, C3, C4 straight-chain or branched-chain alkyl; (2) substituted or unsubstituted C3, C4, C5, C6, C7 cycloalkyl, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered oxacycloalkyl, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered azacycloalkyl, wherein the substituent is selected from methyl, ethyl, propyl, isopropyl, F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylmethylene, cyclobutyl, wherein said Rc1 is independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclopropylmethylene, cyclobutyl; the oxacycloalkyl and azacycloalkyl may contain 1 heteroatom or may contain multiple heteroatoms simultaneously.

2. The compound or its pharmaceutically acceptable salt according to claim 1, wherein The compound is as shown in general formula IA, In formula IA, R2 is independently selected from H, F, Cl, Br; R5 is selected from the following atoms, groups or structural fragments: (1) hydrogen, substituted or unsubstituted C1, C2, C3, C4, C5, C6, C7, C8 straight-chain or branched-chain alkyl groups, where the substituents are selected from F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylene, cyclobutyl, oxetanyl, cyclopentyl, cyclohexyl, and where the said Rc1 is independently selected from H, C1, C2, C3, C4 straight-chain or branched-chain alkyl groups; (2) substituted or unsubstituted C3, C4, C5, C6, C7 cycloalkyl groups, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered oxacycloalkyl groups, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered azacycloalkyl groups, where the said substituents are selected from methyl, ethyl, propyl, isopropyl, F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylene, cyclobutyl, and where the said Rc1 is independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclopropylene, cyclobutyl; the oxacycloalkyl and azacycloalkyl groups may contain 1 heteroatom or may contain multiple heteroatoms simultaneously.

3. The compound or its pharmaceutically acceptable salt according to claim 2, wherein The compound is as shown in general formula IA-1, In formula IA-1, R2 is independently selected from H, F, Cl, Br.

4. The compound or its pharmaceutically acceptable salt according to claim 1, wherein The compound is as shown in general formula IB In formula IB, R3 is independently selected from H, F, Cl, Br; R5 is selected from the following atoms, groups or structural fragments: (1) hydrogen, substituted or unsubstituted C1, C2, C3, C4, C5, C6, C7, C8 straight-chain or branched-chain alkyl groups, where the substituents are selected from F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylene, cyclobutyl, oxetanyl, cyclopentyl, cyclohexyl, and where the said Rc1 is independently selected from H, C1, C2, C3, C4 straight-chain or branched-chain alkyl groups; (2) substituted or unsubstituted C3, C4, C5, C6, C7 cycloalkyl groups, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered oxacycloalkyl groups, substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-membered azacycloalkyl groups, where the said substituents are selected from methyl, ethyl, propyl, isopropyl, F, Cl, Br, CN, ORc1, cyclopropyl, cyclopropylene, cyclobutyl, and where the said Rc1 is independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclopropylene, cyclobutyl; the oxacycloalkyl and azacycloalkyl groups may contain 1 heteroatom or may contain multiple heteroatoms simultaneously.

5. The compound or its pharmaceutically acceptable salt according to claim 4, characterized in that, The compound is as shown in general formula IB-1 In formula IB-1, R3 is independently selected from H, F, Cl, Br.

6. The compound or its pharmaceutically acceptable salt according to any one of claims 1-5, characterized in that, The said compound is selected from the following groups:

7. The compound or its pharmaceutically acceptable salt according to any one of claims 1-6, characterized in that, The pharmaceutical salts of the said compound are selected from salts formed by combination with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions or organic bases capable of providing physiologically acceptable cations, as well as ammonium salts.

8. The compound or its pharmaceutically acceptable salt according to claim 7, characterized in that, The inorganic acid described above is selected from hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; the organic acid is selected from acetic acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, lycium acid, maleic acid, tartaric acid, fumaric acid, citric acid or lactic acid; the alkali metal ion is selected from lithium ion, sodium ion, potassium ion; the alkaline earth metal ion is selected from calcium ion, magnesium ion; the organic base capable of providing a physiologically acceptable cation is selected from methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris(2-hydroxyethyl)amine.

9. The preparation method of the compound according to any one of claims 1-6, characterized in that, It includes the following steps: Dimethyl phosphite undergoes an addition ring-closing reaction with differently substituted o-formylbenzoic acid to obtain intermediate 2; intermediate 2 reacts with 2-fluoro-5-formylbenzonitrile to obtain intermediate 3; intermediate 3 reacts with hydrazine hydrate to obtain intermediate 4; intermediate 4 undergoes a hydrolysis reaction to obtain intermediate 5; piperazinone undergoes an acylation reaction with benzyloxycarbonyl chloride to obtain intermediate 7; intermediate 7 undergoes a substitution reaction with different bromoalkanes or differently alkyl-substituted p-sulfonic acid esters to obtain intermediate 8; intermediate 8 removes the protecting group to obtain intermediate 9; intermediate 5 and intermediate 9 undergo a condensation reaction to obtain the compound shown in general formula I; wherein the definitions of R1, R2, R3, R4, and R5 are as described in any one of claims 1-5.

10. A pharmaceutical composition, characterized in that, It contains an effective dose of the compound according to any one of claims 1-8 or its pharmaceutically acceptable salt and a pharmacodynamically acceptable carrier.

11. Use of the compound or its pharmaceutically acceptable salt according to any one of claims 1-8 in the preparation of a PARP-1 / 2 / 7 inhibitor.

12. Use of the compound or its pharmaceutically acceptable salt according to any one of claims 1-8 in the preparation of a drug for preventing and / or treating diseases related to PARP1 / 2 / 7.

13. Use of the compound or its pharmaceutically acceptable salt according to any one of claims 1-8 in the preparation of an anti-tumor drug, an anti-tumor drug sensitizer or a tumor immunotherapy drug.

14. According to the use of claim 13, characterized in that, The tumors described above are selected from melanoma, gastric cancer, lung cancer, breast cancer, triple-negative breast cancer, renal cancer, liver cancer, oral epidermal cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, colon cancer, bladder cancer, glioma, leukemia, lymphoma.

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