CN116143700A - Phthalazinone compound and its preparation method and application - Google Patents

Abstract

The phthalazinone compound and its preparation method and application belong to the technical field of medicine. The purpose of the present invention is to provide a PARP inhibitor with better activity and a PARP/HDACs dual-target inhibitor represented by general formula I. Studies have found that most of the compounds have significant inhibitory activity on PARP, and some compounds have strong inhibitory activity on PARP and HDACs. The pharmacological activity screening shows that the compounds provided by the present invention have good antitumor activity. Human tumor cells have a significant inhibitory effect and can be used in the preparation of drugs for the treatment of diseases caused by the abnormal expression of PARP and/or HDAC, especially in the preparation of drugs for the treatment and/or prevention of cancer, neurodegenerative diseases and inflammation .

Description

Phthaloxizone compounds and preparation methods and applications thereof

Technical Field

The present invention belongs to the field of medical technology, and specifically relates to a method for preparing a phthalazinone compound or a pharmaceutically acceptable salt thereof, and application of a pharmaceutical composition containing the compound.

Background Art

Poly ADP-ribose polymerase (PARP) is a ribozyme that has the ability to catalyze the transfer of ADP-ribose to target proteins and participates in many cellular processes including gene regulation, chromatin remodeling, DNA repair and apoptosis. PARP was first reported in 1963 and has been for more than 50 years. Its important role has received widespread attention from scholars, especially in recent years, great progress has been made in the study of the relationship between PARP and tumorigenesis and the use of PARP inhibition to improve the therapeutic effect of tumors. It is currently known that the PARP family contains at least 18 molecular subtypes, among which PARP-1 was discovered the earliest, is the most abundant in the PARP family, and plays about 90% of the functions in the PARP family, so it has been studied most extensively and thoroughly.

PARP-1 mainly repairs single-strand damaged DNA through the base-excision repair (BER) pathway. BRAC1/2 genes play a role in DNA damage repair through the homologous recombination (HR) pathway. When the BRCA gene mutates, the homologous recombination repair pathway is defective. At this time, when the function of PARP-1 is inhibited, base excision repair is blocked, leading to the formation of double-strand breaks (DSBs) of DNA. In cells with homologous recombination defects, they can only turn to the non-homologous end joining repair pathway, causing chromosome instability, cell cycle arrest and cell apoptosis, that is, synthetic lethality (SL). Based on the theory of synthetic lethality, PARP-1 inhibitors can have a synergistic lethal effect with tumor cells with homologous recombination repair defects, but will not kill normal cells. PARP-1 inhibitors can not only be used alone to treat homologous recombination-deficient tumors, but also make tumor cells more sensitive to cytotoxic drugs (chemoradiotherapy drugs, platinum, alkylating agents and topoisomerase inhibitors, etc.) by inhibiting PARP-1-mediated DNA damage repair.

Histone deacetylase (HDAC) is a class of proteases that are ubiquitous in eukaryotic cells. Currently, the research on HDACs inhibitors for cancer is mainly focused on Zn2 ⁺ -dependent proteases. Studies have shown that overexpression of HDACs is closely related to cancer. It disrupts the balance of histone acetylation levels, leading to an imbalance in gene expression, thereby affecting cell proliferation and cell cycle regulation, and then leading to cell carcinogenesis. HDACs inhibitors are hot targets in the field of tumor treatment in recent years. They can inhibit tumor cell differentiation and thus inhibit the growth of various tumor cells, and have the characteristics of high tolerance and low cytotoxicity.

A large number of studies have shown that simultaneous inhibition of HDACs and PARP can enhance the killing effect on tumor cells. In various tumor cells treated with PARPi and HDACi, synergistic inhibition of tumor cells was observed both in vivo and in vitro, indicating that the combined use of the two inhibitors can be used to improve the anti-tumor effect. Therefore, designing and synthesizing new PARP/HDACs dual inhibitors for the treatment of tumors is a potential strategy.

Summary of the invention

The primary purpose of the present invention is to provide a phthalazinone compound represented by general formula I or a pharmaceutically acceptable salt thereof.

in:

X ² , X ³ , and X ⁴ are each independently CR ³ ;

R ³ is selected from hydrogen, fluorine;

n为1或2；Ring A is selected from

n is 1 or 2;

R ² is selected from (C ₁ -C ₈ ) alkyl, and the (C ₁ -C ₈ ) alkyl may be optionally substituted by 1 to 3 R ⁴ ;

所述的苯基、苯基(C₂-C₄)烯基、吡啶基可任选被

或1-3个R⁷取代；R ⁴ is selected from trifluoromethyl, difluoromethyl, amino, hydroxy, phenyl, pyridyl, (C ₁ -C ₄ ) alkoxy, phenyl (C ₂ -C ₄ ) alkenyl,

The phenyl, phenyl(C ₂ -C ₄ )alkenyl, pyridyl may be optionally

or 1-3 R ⁷ substitutions;

R ⁵ and R ⁶ are each independently selected from hydrogen, hydroxy, (C ₁ -C ₄ )alkyl, hydroxy(C ₁ -C ₄ )alkyl, (C ₃ -C ₆ )cycloalkyl; the (C ₁ -C ₄ )alkyl may be optionally substituted by 1 to 3 R ¹¹ ;

or R ⁵ and R ⁶ together with the nitrogen atom to which they are attached form a 5-6 membered nitrogen-containing heterocyclic ring, wherein the nitrogen-containing heterocyclic ring contains 1-3 heteroatoms selected from N, O and S, and the nitrogen-containing heterocyclic ring may optionally include 1-2 carbon-carbon double bonds or triple bonds, and the nitrogen-containing heterocyclic ring may be optionally substituted by 1-3 identical or different R ¹⁰ , and the carbon atoms on the ring may be oxo-substituted;

R ¹¹ is selected from hydroxy, (C ₁ -C ₆ )alkoxy, (C ₁ -C ₆ )alkylamino;

R ⁷ is selected from hydrogen, halogen, trifluoromethyl, (C ₁ -C ₄ )alkyl, amino, hydroxyl, cyano;

R ¹⁰ is selected from hydrogen, (C ₁ -C ₄ )alkyl, hydroxy(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, hydroxy, amino, (C ₁ -C ₄ )acyl.

The present invention preferably comprises a phthalazinone compound represented by general formula I or a pharmaceutically acceptable salt thereof, wherein:

^R2 is selected from:

The phthalazinone compound or its pharmaceutically acceptable salt represented by the general formula I of the present invention is preferably any one of the following compounds:

The phthalazinone compounds of general formula I in the present invention can form pharmaceutically acceptable salts with acids. The pharmaceutically acceptable salts include salts formed with inorganic acids, organic acids, and alkali metal ions. The inorganic acid is selected from: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid; the organic acid is selected from: fumaric acid, succinic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid; the alkali metal ion is selected from: potassium ion, sodium ion, lithium ion.

代表取代基连接处。In the present invention, "halogen" refers to fluorine, chlorine, bromine or iodine; "alkyl" refers to a straight chain or branched alkyl; "cycloalkyl" refers to a substituted or unsubstituted cycloalkyl; "aryl" refers to a phenyl or naphthyl group with no substituent or with a substituent; "heteroaryl" refers to a monocyclic or polycyclic ring system containing one or more heteroatoms selected from N, O, S, and the ring system is aromatic, such as imidazolyl, pyridyl, pyrazolyl, (1,2,3)- and (1,2,4)-triazolyl, furanyl, thienyl;

Represents the place where substituents are attached.

The present invention can contain the phthalazinone compound of the above formula I or its pharmaceutically acceptable salt as an active ingredient, and mix it with a pharmaceutically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier or excipient includes a diluent, adhesive, wetting agent, disintegrant, lubricant, and glidant known in the art. Diluents include starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, and calcium hydrogen phosphate; wetting agents include water, ethanol, isopropanol, etc.; adhesives include starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, and polyethylene glycol; disintegrants include dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, and sodium dodecyl sulfate; lubricants and glidants include talc, silicon dioxide, and polyethylene glycol.

The pharmaceutical composition of the present invention can be prepared into pharmaceutically acceptable dosage forms, including injections, tablets, and capsules.

The phthalazinone compound or a pharmaceutically acceptable salt thereof of the present invention is used in combination with other active ingredients to achieve a better therapeutic effect.

The present invention also provides the use of the phthalazinone compound of general formula I or its pharmaceutically acceptable salt in the preparation of a drug for preventing and/or treating cancers, neurodegenerative diseases and inflammations related to PARP enzymes and HDAC enzymes. The cancer is any one of breast cancer, pancreatic cancer, ovarian cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, cervical cancer, testicular cancer, melanoma, nasopharyngeal cancer, oral cancer, malignant glioblastoma, bladder cancer, prostate cancer, esophageal cancer, brain tumor, reproductive system tumor, respiratory system tumor, lymphatic system tumor, digestive system tumor and skin tumor.

The present invention also provides the following synthetic routes (Routes 1-2), all of which are prepared by the methods described in these synthetic routes, by methods well known to those skilled in the art of organic chemistry, or are commercially available. All of the final compounds of the present invention are prepared by the methods described in these synthetic routes or by methods similar thereto, which are well known to those skilled in the art of organic chemistry. R ² , X ² , X ³ , and X ⁴ are corresponding groups at corresponding positions of the compounds shown in Formula I, and n=1 or 2.

Route 1:

Substituted or unsubstituted o-formylbenzoic acid is used as a starting material to react with dimethyl phosphite to obtain a phosphorus ylide intermediate 1; intermediate 1 is subjected to a Wittig-Horner reaction to obtain intermediate 2; intermediate 2 is subjected to cyanide hydrolysis and cyclization reaction to obtain intermediate 3; intermediate 3 is subjected to condensation and Boc protection removal reaction to obtain intermediate 4; intermediate 4 is subjected to carbon disulfide addition to obtain intermediate 5; intermediate 5 and a haloamine (amide or ester) are subjected to a nucleophilic substitution reaction to obtain the target compound of formula II.

Route 2:

Intermediate 3 is used as a raw material, and intermediate 6 is prepared through condensation and Boc removal reaction; intermediate 6 is subjected to carbon disulfide addition to prepare intermediate 7; intermediate 7 and haloamine (amide or ester) are subjected to nucleophilic substitution reaction to prepare the target compound of formula III.

Beneficial effects of the present invention:

The compounds provided by the present invention have strong single PARP inhibition effect and PARP/HDAC co-inhibition effect, and can be used to prepare drugs for treating diseases caused by abnormal expression of PARP and/or HDAC, especially for preparing drugs for treating and/or preventing cancer, neurodegenerative diseases and inflammation.

The purpose of the present invention is to develop PARP inhibitors and PARP/HDACs dual-target inhibitors with better activity. Through experiments, it can be seen that most compounds have significant inhibitory activity on PARP, and some compounds have strong inhibitory activity on both PARP and HDACs. Pharmacological activity screening shows that the compounds provided by the present invention have good anti-tumor activity and have significant inhibitory effects on a variety of human tumor cells, and have medicinal value for further development.

DETAILED DESCRIPTION

The examples are intended to illustrate rather than limit the scope of the present invention. The H NMR spectra of the compounds were measured using a Bruker AVANCE III 600 or Bruker ARX-600 NMR instrument, with tetramethylsilane as an internal standard; the mass spectra were measured using an Agilent 1100 quadrupole LC/MS instrument; all reagents used were analytically pure or chemically pure.

Example 1: Preparation of 2-(dimethylamino)ethyl-4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 1)

Step A: Preparation of (3-oxo-1,3-dihydro-isobenzofuran-1-yl)phosphonic acid dimethyl ester (Intermediate 1)

At 0°C, dimethyl phosphite (16.5 mL, 0.18 mol) was added dropwise to a solution of sodium methoxide (77.2 g, 0.19 mol) in methanol (150 mL). After the addition was complete, o-formylbenzoic acid (22.5 g, 0.15 mol) was added to the reaction mixture in batches within 20 min, and the temperature was kept below 5°C. The reaction solution was slowly heated to 20°C and reacted for 2 h. After the reaction was completed, methanesulfonic acid (13.6 mL, 0.21 mol) was added dropwise to the reaction solution within 30 min. After the addition was complete, the solvent was evaporated and the obtained white residue was dissolved in water (90 mL), extracted with dichloromethane three times (3×70 mL), the organic layers were combined, the organic layers were backwashed with water twice (2×20 mL), and dried with magnesium sulfate. Magnesium sulfate was removed by filtration, and the filtrate was evaporated to dryness and frozen to obtain 25.8 g of a crystalline white solid with a yield of 71.2%, which was used directly in the next step without purification. MS (ESI) m/z (%): 243.04 [M+H] ⁺ .

Step B: Preparation of 2-fluoro-5-(3-oxo-1,(3H)-dihydro-isobenzofuranylidenemethyl)benzonitrile (Intermediate 2a)

24.2g (0.1mol) of intermediate 1 and 2-fluoro-5-formylbenzonitrile (0.1mol) were dissolved in 200mL of tetrahydrofuran, and triethylamine (10mL, 0.1mol) was slowly added dropwise over 25min, keeping the temperature below 15°C. After the addition was complete, the reaction solution was slowly heated to 20°C and reacted for 1h. After the reaction was complete, most of the solvent was evaporated, and the reaction solution was poured into 150mL of water and continued to stir for 30min. The solid was collected by filtration, washed twice with water (2×20mL), twice with n-hexane (2×20mL), and twice with ether (2×20mL), and dried to obtain a white solid (E:Z 4:3), with a yield of 91.2%. The isomer was used directly in the next step without separation, MS (ESI) m/z (%): 264.35 [MH] ^- .

According to the operation method of step B, intermediates 2b-2d were prepared respectively.

3-Fluoro-5-(3-oxo-1,(3H)-dihydro-isobenzofuranylidenemethyl)benzonitrile (2b): white solid, yield 89.2%, MS (ESI) m/z (%): 264.09 [MH] ^- .

4-Fluoro-3-(3-oxo-1,(3H)-dihydro-isobenzofuranylidenemethyl)benzonitrile (2c): white solid, yield 90.5%, MS (ESI) m/z (%): 264.28 [MH] ^- .

3-(3-Oxo-1,(3H)-dihydro-isobenzofuranylidenemethyl)benzonitrile (2d): white solid, yield 93.1%, MS (ESI) m/z (%): 246.12 [MH] ^- .

Step C: Preparation of 2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (Intermediate 3a)

Under stirring, add NaOH aqueous solution (13N, 32mL) to a suspension of intermediate 2a (0.09mol) in 130mL of water, heat to 90℃, and react for 1h. Cool the reaction solution to 70℃, slowly add 80% hydrazine hydrate (65mL, 1.3mol), and react at 70℃ for 18h. After the reaction is completed, cool to room temperature and acidify to pH 4 with 8N hydrochloric acid solution. Continue stirring at room temperature for 10min, filter the reaction solution, wash the filter cake with water (2×40mL) and ether (2×30mL) in turn, and dry to obtain a light pink powder with a yield of 71%. ¹ H NMR (600MHz, DMSO-d ₆ ) δ13.28(bs,1H),12.61(s,1H),8.28(dd,J＝7.9,1.0Hz,1H),7.99(d,J＝8.0Hz,1H),7.91(td,J＝7.9,1.3Hz,1H),7.86–7.81(m,2H ),7.61–7.57(m,1H),7.25(dd,J＝10.7,8.5Hz,1H),4.37(s,2H).MS(ESI)m/z(%):297.24[MH] ^- .

According to the operation method of step C, intermediates 3b-3d were prepared respectively.

3-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (3b): white solid, yield 69.2%. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 13.30 (bs, 1H), 12.62 (s, 1H), 8.29–8.26 (m, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.94–7.90 (m, 1H), 7.87–7.83 (m, 1H), 7.75–7.73 (m, 1H), 7.54–7.48 (m, 2H), 4.42 (s, 2H). MS (ESI) m/z (%): 299.13 [M+H] ⁺ .

4-Fluoro-3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (3c): white solid, yield 70.1%. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 13.02 (s, 1H), 12.54 (s, 1H), 8.29 (d, J=7.6 Hz, 1H), 8.03 (d, J=8.0 Hz, 1H), 7.97 (t, J=7.3 Hz, 1H), 7.93-7.86 (m, 3H), 7.34 (t, J=9.0 Hz, 1H), 4.42 (s, 2H). MS (ESI) m/z (%): 297.24 [MH] ^- .

3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (3d): white solid, yield 75.5%. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 12.97 (bs, 1H), 12.62 (s, 1H), 8.29–8.26 (m, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.91–7.87 (m, 2H), 7.85–7.81 (m, 1H), 7.79 (d, J=7.8 Hz, 1H), 7.59 (d, J=7.7 Hz, 1H), 7.44 (t, J=7.7 Hz, 1H), 4.39 (s, 2H). MS (ESI) m/z (%): 279.30 [MH] ^- .

Step D: Preparation of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)-2H-phthalazin-1-one (Intermediate 4a)

Intermediate 3a (2.1 g, 7.0 mmol), N-Boc piperazine (1.6 g, 8.4 mmol), HBTU (3.2 g, 8.4 mmol), and triethylamine (1.4 g, 14.0 mmol) were added to DMF (20 mL) and reacted at room temperature for 2 h. After the reaction was completed, the reaction solution was poured into water and stirred for more than 1 h. The suspension was filtered and the filter cake was washed with cold DMF-H ₂ O (1:1, 2×5 mL), cold water (2×5 mL), cold isopropanol (2×5 mL) and cold ether (2×5 mL) in sequence, and dried to obtain 3.1 g of a white solid. HCl (6N, 15 mL) was added to an ethanol (7 mL) solution of the intermediate prepared above (3.1 g, 6.5 mmol) at room temperature, and stirred at room temperature for 3 h. The ethanol was evaporated, and the aqueous solution was adjusted to pH 10 with ammonia water, extracted with dichloromethane three times (3×10 mL), and the organic layers were combined, backwashed once with 10 mL of water, dried over anhydrous sodium sulfate, and concentrated to obtain 2.1 g of a white crystalline solid with a total yield of 85%. ¹ H NMR (600 MHz, DMSO-d ₆ )δ12.59(s,1H),8.28–8.24(m,1H),7.99–7.94(m,1H),7.92–7.86(m,1H),7.86–7.80(m,1H),7.43–7.38(m,1H),7.34–7.30(m,1H),7.24–7.19(m, 1H),4.32(s,2H),3.57–2.54(m,8H).MS(ESI)m/z(%):367.17[M+H] ⁺ .

According to the operation method of step D, intermediates 4b-4e and intermediate 6 were prepared respectively.

4-(3-Fluoro-5-(piperazine-1-carbonyl)benzyl)-2H-phthalazin-1-one (4b): white solid, yield 76.2%, MS (ESI) m/z (%): 367.74 [M+H] ⁺ .

4-(2-Fluoro-5-(piperazine-1-carbonyl)benzyl)-2H-phthalazin-1-one (4c): white solid, yield 72.5%, MS (ESI) m/z (%): 367.23 [M+H] ⁺ .

4-(3-(Piperazine-1-carbonyl)benzyl)-2H-phthalazin-1-one (4d): white solid, yield 82.5%, ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 12.61 (s, 1H), 8.31–8.21 (m, 1H), 7.99–7.92 (m, 1H), 7.91–7.79 (m, 2H), 7.43–7.34 (m, 2H), 7.32–7.26 (m, 1H), 7.24–7.16 (m, 1H), 4.35 (s, 2H), 3.57–3.04 (m, 5H), 2.78–2.54 (m, 4H). MS (ESI) m/z (%): 349.02 [M+H] ⁺ .

4-(3-(1,4-diazepane-1-carbonyl)-4-fluorobenzyl)-2H-phthalazin-1-one (4e): light yellow solid, yield 65.4%, ¹ H NMR (600 MHz, DMSO-d ₆ )δ12.61(s,1H),8.26(d,J＝7.7Hz,1H),7.99–7.94(m,1H),7.92–7.86(m,1H),7.85–7.81(m,1H),7.47–7.37(m,2H),7.27–7.18(m,1H),4.33(s,2H) ,3.84–3.55(m,2H),3.31–2.77(m,6H),1.93–1.62(m,2H).MS(ESI)m/z(%):381.03[M+H] ⁺ .

2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-(piperidin-4-yl)benzamide (Intermediate 6): Intermediate 3a and 4-amino-N-Boc-piperidine were used as raw materials to obtain Intermediate 6, a light yellow solid, with a yield of 66.8%. ¹ HNMR (600 MHz, DMSO-d ₆ )δ12.60(s,1H),8.49(bs,1H),8.28–8.24(m,1H),8.00–7.97(m,1H),7.92–7.88(m,1H),7.85–7.81(m,1H),7.52–7.48(m,1H),7.47–7.42(m,1H),7 .22–7.18(m,1H),4.33(s,2H),4.04–3.96(m,1H),3.26–1.67(m,8H).MS(ESI)m/z(%):381.35[M+H] ⁺ .

Step E: Preparation of sodium 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Intermediate 5a)

Intermediate 4a (0.37 g, 1 mmol), NaOH (0.04 g, 1 mmol), and carbon disulfide (0.1 g, 1 mmol) were dissolved in methanol (5 mL) and reacted at room temperature for 4 h. After the reaction was completed, 0.4 g of a light yellow solid was obtained by filtration. The yield was 88.0%. MS (ESI) m/z (%): 486.89 [M+Na] ⁺ .

According to the operation method of step E, intermediates 5b-5e and intermediate 7 were prepared respectively.

Sodium 4-(3-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (5b): light yellow solid, yield 84.5%, MS (ESI) m/z (%): 486.83 [M+Na] ⁺ .

Sodium 4-(4-fluoro-3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (5c): light yellow solid, yield 85.3%, MS (ESI) m/z (%): 486.83 [M+Na] ⁺ .

Sodium 4-(3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (5d): light yellow solid, yield 83.8%, MS (ESI) m/z (%): 468.87 [M+Na] ⁺ .

Sodium 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)1,4-diazepane-1-carbodisulfate (5e): light yellow solid, yield 75.8%, MS (ESI) m/z (%): 500.88 [M+Na] ⁺ .

Sodium 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Intermediate 7): Intermediate 7 was prepared using Intermediate 6 as a raw material. The yield was 82.8%, and the result was as a light yellow solid. MS (ESI) m/z (%): 500.88 [M+Na] ⁺ .

Step F: Preparation of 2-(dimethylamino)ethyl-4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 1)

Intermediate 5a (0.24 g, 0.5 mmol), 2-dimethylaminoethyl chloride hydrochloride (0.08 g, 0.5 mmol) and triethylamine (0.06 g, 0.6 mmol) were dissolved in methanol (5 mL) and reacted at 50 °C for 4 h. After the reaction was completed, the mixture was cooled to room temperature and filtered to obtain 0.23 g of a white solid with a yield of 90%. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.26 (d, J＝7.7 Hz, 1H), 7.96 (d, J＝7.9 Hz, 1H), 7.90 (t, J＝7.3 Hz, 1H), 7.84 (t, J＝7.4 Hz, 1H), 7.48–7.43 (m, 1H), 7.39 (d, J＝4.9 Hz, 1H), 7.25 (t, J＝8 .9Hz,1H),4.34(s,2H),4.32–3.80(m,4H),3.76–3.69(m,2H),3.41–3.37(m,2H),3.35–3.33(m,2H),2.55–2.52(m,2H),2.18(s,6H).MS(ESI)m/z(%) :514.39[M+H] ⁺ .

According to the preparation method of compound 1, intermediates 5 and 7 are used as raw materials, and various small molecular halogenated amines (amides, esters) are reacted through substitution reactions and the preparation method of step F to obtain compounds 2-46.

Example 2: Preparation of 2-(diethylamino)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 2)

Using intermediate 5a and 2-diethylaminoethyl chloride hydrochloride as raw materials, a white solid was obtained according to step F with a yield of 86.4%. ¹ H NMR (600MHz, CDCl ₃ -d ₆ ) δ10.88(s,1H),8.43–8.39(m,1H),7.73–7.68(m,2H),7.67–7.63(m,1H),7.32–7.25(m,2H),6.98(t,J＝8.8Hz,1H),4.23(s,2 H),4.23–3.74(m,6H),3.43–3.38(m,2H),3.38–3.30(m,2H),2.77–2.70(m,2H),2.64–2.53(m,4H),1.01(t,J＝6.8Hz,6H).MS(ESI)m/z:542.42[M+H] ⁺ .

Example 3: Preparation of 2-(pyrrolidin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 3)

Using intermediate 5a and N-(2-chloroethyl)pyrrolidine hydrochloride as raw materials, a white solid was obtained according to step F with a yield of 83.5%. ¹ H NMR (600 MHz, DMSO-d ₆ )δ12.60(s,1H),8.26(d,J＝7.3Hz,1H),7.96(d,J＝7.6Hz,1H),7.92–7.87(m,1H),7.86–7.80(m,1H),7.50–7.43(m,1H),7.42–7.35(m,1H),7.25(t,J＝8.6Hz,1H), 4.34(s,2H),4.34–3.80(m,4H),3.78–3.68(m,2H),3.49–3.42(m,2H),2.90–2.75(m,2H),2.69–2.60(m,2H),2.50(s,4H),1.73(s,4H).MS(ESI)m/z :540.25[M+H] ⁺ .

Example 4: Preparation of 2-(piperidin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 4)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.28–8.25 (m, 1H), 7.95 (d, J = 8.0Hz, 1H), 7.91–7.87 (m, 1H), 7.83(t,J＝7.5Hz,1H),7.46(ddd,J＝7.7,5.0,2.1Hz,1H),7.38(dd,J＝6.4,2.1Hz,1H),7.25(t,J＝9.0Hz ,1H),4.34(s,2H),4.34–3.78(m,4H),3.76–3.69(m,2H),3.45–3.35(m,2H),3.35–3.33(m,2H),2.56–2.51 (m,2H),2.38(s,4H),1.48(s,4H),1.37(s,2H).MS(ESI)m/z:554.46[M+H] ⁺ .

Example 5: Preparation of 2-(4-methylpiperazin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 5)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.26 (d, J = 7.5Hz, 1H), 7.95 (d, J = 8.0Hz, 1H), 7.91–7.88 (m, 1H),7.85–7.82(m,1H),7.46(ddd,J＝7.7,4.9,2.1Hz,1H),7.38(dd,J＝6.4,2.1Hz,1H),7.25(t,J＝9.0Hz ,1H),4.34(s,2H),4.34–3.79(m,4H),3.77–3.68(m,2H),3.40(t,J＝7.1Hz,2H),3.35–3.33(m,2H), 2.56(t,J＝7.1Hz,2H),2.49–2.23(m,8H),2.17(s,3H).MS(ESI)m/z:569.31[M+H] ⁺ .

Example 6: Preparation of 2-morpholinoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 6)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.26 (d, J = 7.4Hz, 1H), 7.96 (d, J = 7.3Hz, 1H), 7.90 (t, J = 6.6Hz,1H),7.84(t,J＝6.7Hz,1H),7.48–7.43(m,1H),7.41–7.35(m,1H),7.25(t,J＝8.5Hz,1 H),4.34(s,2H),4.34–3.80(m,4H),3.77–3.70(m,2H),3.56(s,4H),3.45–3.39(m,2H),3.35–3.33(m, 2H),2.60–2.55(m,2H),2.41(s,4H).MS(ESI)m/z:556.18[M+H] ⁺ .

Example 7: Preparation of 2-(dimethylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 7)

¹ H NMR (600 MHz, DMSO-d ₆ )δ12.60(s,1H),8.26(d,J＝7.7Hz,1H),7.96(d,J＝7.9Hz,1H),7.90(t,J＝7.3Hz,1H),7.83(t,J＝7.3Hz,1H),7.49–7.44(m,1H),7.43–7.38(m,1H),7.25 (t,J＝8.9Hz,1H),4.34(s,2H),4.30(s,2H),4.34–3.84(m,4H),3.79–3.69(m,2H),3.38–3.34(m,2H),3.08(s,3H),2.84(s,3H).MS(ESI)m/z:550.10[ M+Na] ⁺ .

Example 8: Preparation of 2-(diethylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 8)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.26 (d, J = 7.7Hz, 1H), 7.96 (d, J = 8.1Hz, 1H), 7.93–7.88 (m, 1H),7.86–7.81(m,1H),7.49–7.44(m,1H),7.43–7.38(m,1H),7.25(t,J＝9.0Hz,1H),4.34(s,2H),4.32 (s ,2H),4.34–3.85(m,4H),3.81–3.67(m,2H),3.42(q,J＝7.0Hz,2H),3.38–3.34(m,2H),3.28(q,J＝7.0 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3H), 1.01 (t, J = 7.0 Hz, 3H). MS (ESI) m/z: 578.14 [M + Na] ⁺ .

Example 9: Preparation of 2-(cyclopropylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 9)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),8.33–8.17(m,2H),8.00–7.94(m,1H),7.93–7.88(m,1H),7.86–7.80 (m,1H),7.50–7.44(m,1H),7.43–7.37(m,1H),7.29–7.21(m,1H),4.34(s,2H),4.34 –4.01(m,3H),3.96(s,2H),3.94–3.82(m,1H),3.80–3.68(m,2H),3.40–3.34(m,2H),2.64–2.58(m,1H) ,0.65–0.56(m,2H),0.46–0.33(m,2H).MS(ESI)m/z:562.14[M+Na] ⁺ .

Example 10: Preparation of 2-(cyclobutylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 10)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.42 (d, J = 7.6Hz, 1H), 8.28–8.24 (m, 1H), 7.96 (d, J = 8.0Hz, 1H),7.92–7.88(m,1H),7.85–7.81(m,1H),7.46(ddd,J＝7.8,4.9,2.1Hz,1H),7.40(dd,J＝6.4,2.0Hz,1H) ,7.25(t,J=9. 0Hz,1H),4.34(s,2H),4.34–4.03(m,3H),3.98(s,2H),3.95–3.80(m,1H),3.78–3.70(m,2H),3.38–3.33( m,2H),2.16–2.11(m,2H),1.92–1.85(m,2H),1.66–1.57(m,2H).MS(ESI)m/z:576.22[M+Na] ⁺ .

Example 11: Preparation of 2-(cyclopentylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 11)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),8.31–8.22(m,1H),8.18–8.09(m,1H),8.01–7.94(m,1H),7.93–7.87 (m,1H),7.86–7.77(m,1H),7.51–7.43(m,1H),7.43–7.33(m,1H),7.31–7.18(m,1H),4.34(s,2H),4.34 –3.82(m,7H),3.80–3.68(m,2H),3.40–3.35(m,2H),1.82–1.34(m,8H).MS(ESI)m/z:590.17[M+Na] ⁺ .

Example 12: Preparation of 2-(cyclohexylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 12)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),8.30–8.23(m,1H),8.07–8.01(m,1H),7.99–7.78(m,3H),7.49–7.37 (m,2H),7.31–7.20(m,1H),4.34(s,2H),4.34–4.06(m,3H),3.99(s,2H),3.92–3.85(m,1H),3.78–3.70 (m,2H),3.54–3.48(m,1H),3.35(m,2H),1.74–1.51(m,5H),1.24–1.07(m,5H).MS(ESI)m/z:604.19[ M+Na] ⁺ .

Example 13: Preparation of 2-oxo-2-(pyrrolidin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 13)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.26 (d, J = 7.7Hz, 1H), 7.96 (d, J = 8.0Hz, 1H), 7.90 (t, J = 7.4Hz,1H),7.84(t,J＝7.5Hz,1H),7.48–7.43(m,1H),7.42–7.38(m,1H),7.25(t,J＝9.0Hz,1H),4.34( s,2H),4.33–4.24(m,1H),4.2 2(s,2H),4.19–3.99(m,2H),3.99–3.82(m,1H),3.79–3.68(m,2H),3.56(t,J＝6.8Hz,2H),3.38–3.35( m,2H),3.29(t,J＝6.9Hz,2H),1.91(p,J＝6.8Hz,2H),1.78(p,J＝6.8Hz,2H).MS(ESI)m/z:576.22 [M+Na] ⁺ .

Example 14: Preparation of 2-oxo-2-(piperidin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 14)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.26 (d, J = 7.6Hz, 1H), 7.96 (d, J = 7.8Hz, 1H), 7.92–7.88 (m, 1H),7.83(t,J＝7.2Hz,1H),7.48–7.43(m,1H),7.42–7.37(m,1H),7.25(t,J＝8.7Hz,1H),4.34(s,2H ),4.32(s ,2H),4.23–4.00(m,3H),3.96–3.87(m,1H),3.79–3.70(m,2H),3.52–3.46(m,2H),3.45–3.40(m,2H),3.39 –3.35(m,2H),1.63–1.52(m,4H),1.46–1.39(m,2H).MS(ESI)m/z:590.11[M+Na] ⁺ .

Example 15: Preparation of 2-morpholino-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 15)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.26 (d, J = 7.8Hz, 1H), 7.96 (d, J = 8.0Hz, 1H), 7.90 (t, J = 7.4Hz,1H),7.84(t,J＝7.5Hz,1H),7.48–7.44(m,1H),7.42–7.38(m,1H),7.25(t,J＝8.9Hz,1H),4.34( s,2 H),4.33(s,2H),4.30–3.97(m,3H),3.96–3.81(m,1H),3.78–3.71(m,2H),3.65–3.60(m,2H),3.58–3.53( m,4H),3.46–3.42(m,2H),3.38–3.35(m,2H).MS(ESI)m/z:592.16[M+Na] ⁺ .

Example 16: Preparation of 2-thiomorpholino-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 16)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.26 (d, J = 7.6Hz, 1H), 7.96 (d, J = 7.8Hz, 1H), 7.90 (t, J = 6.7Hz,1H),7.84(t,J＝7.2Hz,1H),7.48–7.44(m,1H),7.42–7.38(m,1H),7.25(t,J＝8.9H z,1H),4.34(s,5H),4.19–4.03(m,2H),3.98–3.87(m,1H),3.83–3.79(m,2H),3.77–3.70(m,4H),2.74– 2.70(m,2H),2.56–2.53(m,2H).MS(ESI)m/z:608.16[M+Na] ⁺ .

Example 17: Preparation of 2-(4-methylpiperazin-1-yl)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 17)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.26 (d, J = 7.7Hz, 1H), 7.96 (d, J = 7.8Hz, 1H), 7.90 (t, J = 7.2Hz,1H),7.83(t,J＝7.2Hz,1H),7.48–7.44(m,1H),7.40(d,J＝4.9Hz,1H),7.25(t,J＝8.9Hz,1H) ,4.34(s,2H),4 .32(s,2H),4.31–3.84(m,4H),3.78–3.70(m,2H),3.56–3.51(m,2H),3.47–3.42(m,2H),3.38–3.34(m, 2H),2.38–2.33(m,2H),2.27–2.23(m,2H),2.18(s,3H).MS(ESI)m/z:605.28[M+Na] ⁺ .

Example 18: Preparation of 2-(piperidin-1-yl)ethyl 4-(3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 18)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61 (s, 1H), 8.26 (d, J = 7.7Hz, 1H), 7.96 (d, J = 8.0Hz, 1H), 7.89 (t, J = 7.4Hz,1H),7.84(d,J＝7.5Hz,1H),7.46–7.37(m,3H),7.29(d,J＝7.3Hz,1H),4.36(s,2H) ,4.36–3.81(m,4H),3.78–3.58(m,2H),3.48–3.36(m,4H),2.59–2.52(m,2H),2.49–2.30(m,4H),1.53–1.44( m,4H),1.41–1.34(m,2H).MS(ESI)m/z:536.21[M+H] ⁺ .

Example 19: Preparation of 2-(4-methylpiperazin-1-yl)ethyl 4-(3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 19)

¹ H NMR (600 MHz, DMSO-d ₆ )δ12.61(s,1H),8.26(d,J＝7.9Hz,1H),7.95(d,J＝8.0Hz,1H),7.89(t,J＝7.6Hz,1H),7.83(t,J＝7.5Hz,1H),7.43(d,J＝7.8Hz,1H),7.41–7.37(m,2H),7. 31–7.27(m,1H),4.36(s,2H),4.36–3.77(m,4H),3.75–3.55(m,2H),3.45–3.35(m,4H),2.59–2.53(m,2H),2.49–2.21(m,8H),2.16(s,3H).MS(ESI)m /z:551.26[M+H] ⁺ .

Example 20: Preparation of 2-(cyclopentylamino)-2-oxoethyl 4-(3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 20)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61(s,1H),8.33–8.21(m,1H),8.19–8.09(m,1H),8.02–7.77(m,3H),7.50–7.34 (m,3H),7.34–7.25(m,1H),4.36(s,2H),4.33–3.82(m,7H),3.81–3.59(m,2H),3.55–3.37(m,2H),1.83 –1.57(m,4H),1.53–1.34(m,4H).MS(ESI)m/z:572.12[M+Na] ⁺ .

Example 21: Preparation of 2-(cyclohexylamino)-2-oxoethyl 4-(3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 21)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61(s,1H),8.32–8.22(m,1H),8.09–8.01(m,1H),8.00–7.93(m,1H),7.92–7.86 (m,1H),7.86–7.79(m,1H),7.47–7.36(m,3H),7.34–7.26(m,1H),4.36(s,2H) ,4.36–3.84(m,7H),3.79–3.62(m,2H),3.56–3.40(m,3H),1.76–1.63(m,4H),1.57–1.50(m,1H),1.29–1.25( m,1H),1.22–1.08(m,4H).MS(ESI)m/z:587.17[M+Na] ⁺ .

Example 22: Preparation of 2-(piperidin-1-yl)ethyl 4-(3-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 22)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61 (s, 1H), 8.27 (d, J = 7.8Hz, 1H), 7.97 (d, J = 8.0Hz, 1H), 7.91 (t, J = 7.4Hz,1H),7.84(t,J＝7.5Hz,1H),7.31(d,J＝9.6Hz,1H),7.22(s,1H),7.16(d,J＝8.4Hz,1H),4.3 9(s,2H),4.36–3.78(m,4H),3.76–3.60(m,2H),3.48–3.34(m,4H),2.61–2.51(m,2H),2.49–2.28(m,4H ),1.56–1.43(m,4H),1.43–1.32(m,2H).MS(ESI)m/z:554.20[M+H] ⁺ .

Example 23: Preparation of 2-(4-methylpiperazin-1-yl)ethyl 4-(3-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 23)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.65 (s, 1H), 8.29 (d, J = 7.7Hz, 1H), 7.98 (d, J = 8.0Hz, 1H), 7.93–7.89 (m, 1H),7.87–7.83(m,1H),7.34–7.31(m,1H),7.25(s,1H),7.20–7.17(m,1H),4.40(s,2H),4.37– 4.15(m,2H),4.09–3.84(m,2H),3.78–3.65(m,2H),3.45–3.42(m,2H),3.42–3.38(m,2H),2.59(t,J＝7.1 Hz,2H),2.57–2.37(m,8H),2.26(s,3H).MS(ESI)m/z:569.24[M+H] ⁺ .

Example 24: Preparation of 2-(cyclopentylamino)-2-oxoethyl 4-(3-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 24)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61 (s, 1H), 8.27 (d, J = 7.8Hz, 1H), 8.14 (d, J = 6.9Hz, 1H), 7.98 (d, J = 8.0Hz,1H),7.91(t,J＝7.4Hz,1H),7.84(t,J＝7.4Hz,1H),7.31(d,J＝9.5Hz,1H),7.23(s,1H),7.17 (d,J＝8.3Hz,1H),4. 39(s,2H),4.34–4.05(m,3H),4.02–3.87(m,4H),3.76–3.63(m,2H),3.48–3.37(m,2H),1.82–1.73(m,2H ),1.67–1.58(m,2H),1.53–1.44(m,2H),1.42–1.34(m,2H).MS(ESI)m/z:590.05[M+Na] ⁺ .

Example 25: Preparation of 2-(cyclohexylamino)-2-oxoethyl 4-(3-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 25)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61(s,1H),8.33–8.21(m,1H),8.09–7.81(m,4H),7.37–7.13(m,3H),4.39(s ,2H),4.33–3.87(m,6H),3.78–3.61(m,2H),3.55–3.38(m,3H),1.75–1.61(m,4H),1.58–1.50(m,1H),1.30 –1.25(m,1H),1.22–1.05(m,4H).MS(ESI)m/z:604.19[M+Na] ⁺ .

Example 26: Preparation of 2-(piperidin-1-yl)ethyl 4-(4-fluoro-3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 26)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.55 (s, 1H), 8.28 (d, J = 7.8Hz, 1H), 8.00 (d, J = 8.0Hz, 1H), 7.95 (t, J = 7.5Hz,1H),7.87(t,J＝7.5Hz,1H),7.45–7.37(m,2H),7.31(t,J＝9.0Hz,1H),4.39(s,2H) ,4.34–3.76(m,4H),3.74–3.56(m,2H),3.49–3.34(m,4H),2.62–2.51(m,2H),2.49–2.28(m,4H),1.55–1.43( m,4H),1.42–1.33(m,2H).MS(ESI)m/z:554.65[M+H] ⁺ .

Example 27: Preparation of 2-(4-methylpiperazin-1-yl)ethyl 4-(4-fluoro-3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 27)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.55(s,1H),8.34–8.23(m,1H),8.04–7.92(m,2H),7.91–7.83(m,1H),7.46–7.36 (m,2H),7.35–7.26(m,1H),4.39(s,2H),4.32–3.80(m,4H),3.74–3.58(m,2H),3.45–3.36(m,4H),2.59 –2.55(m,2H),2.48–2.24(m,8H),2.17(s,3H).MS(ESI)m/z:570.27[M+H] ⁺ .

Example 28: Preparation of 2-(cyclopentylamino)-2-oxoethyl 4-(4-fluoro-3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbon disulfate (Compound 28)

¹ H NMR (600 MHz, DMSO-d ₆ )δ12.55(s,1H),8.35–8.22(m,1H),8.19–8.09(m,1H),8.04–7.92(m,2H),7.91–7.83(m,1H),7.49–7.37(m,2H),7.36–7.26(m,1H),4.39(s,2H),4 .32–3.86(m,7H),3.76–3.57(m,2H),3.56–3.39(m,2H),1.83–1.71(m,2H),1.67–1.57(m,2H),1.53–1.45(m,2H),1.42–1.33(m,2H).MS(ESI)m/z:5 89.98[M+Na] ⁺ .

Example 29: Preparation of 2-(cyclohexylamino)-2-oxoethyl 4-(4-fluoro-3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 29)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.55(s,1H),8.36–8.21(m,1H),8.09–7.92(m,3H),7.92–7.81(m,1H),7.51–7.24 (m,3H),4.39(s,2H),4.31–3.88(m,6H),3.76–3.60(m,2H),3.55–3.39(m,3H),1.77–1.62(m,4H),1.57 –1.49(m,1H),1.31–1.26(m,1H),1.22–1.07(m,4H).MS(ESI)m/z:604.13[M+Na] ⁺ .

Example 30: Preparation of 2-(piperidin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-1,4-diazepane-1-carbodisulfate (Compound 30)

MS (ESI) m/z: 568.92 [M+H] ⁺ .

Example 31: Preparation of 2-(4-methylpiperazin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-1,4-diazepane-1-carbodisulfate (Compound 31)

MS (ESI) m/z: 583.13 [M+H] ⁺ .

Example 32: Preparation of 2-(cyclopentylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-1,4-diazepane-1-carbodisulfate (Compound 32)

MS (ESI) m/z: 604.51 [M+Na] ⁺ .

Example 33: Preparation of 2-(cyclohexylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-1,4-diazepane-1-carbodisulfate (Compound 33)

MS (ESI) m/z: 618.27 [M+Na] ⁺ .

Example 34: Preparation of 2-(dimethylamino)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 34)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),8.35–8.21(m,2H),8.01–7.94(m,1H),7.92–7.79(m,2H),7.57–7.40 (m,2H),7.25–7.15(m,1H),5.20–5.07(m,1H),4.48–4.38(m,1H),4.33(s, 2H),4.21–4.10(m,1H),3.60–3.44(m,2H),3.37–3.26(m,2H),2.51–2.46(m,2H),2.17(s,6H),2.00–1.86( m,2H),1.57–1.43(m,2H).MS(ESI)m/z:528.02[M+Na] ⁺ .

Example 35: Preparation of 2-(diethylamino)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 35)

¹ H NMR (600 MHz, DMSO-d ₆ )δ12.60(s,1H),8.39–8.19(m,2H),8.03–7.79(m,3H),7.60–7.38(m,2H),7.28–7.13(m,1H),5.24–5.02(m,1H),4.50–4.38(m,1H),4.33(s,2H),4 .23–4.07(m,1H),3.59–3.45(m,2H),3.36–3.28(m,2H),2.78–2.51(m,6H),2.01–1.88(m,2H),1.57–1.43(m,2H),1.00(s,6H).MS(ESI)m/z:556.38[ M+H] ⁺ .

Example 36: Preparation of 2-(pyrrolidin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 36)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),8.35–8.22(m,2H),8.01–7.94(m,1H),7.93–7.80(m,2H),7.57–7.49 (m,1H),7.48–7.41(m,1H),7.24–7.16(m,1H),5.22–5.02(m,1H),4.47–4.37(m,1H),4 .33(s,2H),4.20–4.11(m,1H),3.58–3.49(m,2H),3.48–3.41(m,2H),3.01–2.56(m,6H),1.99–1.90(m, 2H),1.82–1.69(m,4H),1.55–1.46(m,2H).MS(ESI)m/z:554.46[M+H] ⁺ .

Example 37: Preparation of 2-(piperidin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidin-1-carbon disulfate (Compound 37)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),8.38–8.19(m,2H),8.03–7.79(m,3H),7.61–7.38(m,2H),7.28–7.13 (m,1H),5.23–5.01(m,1H),4.52–4.38(m,1H),4.33(s,2H),4.22–4.08(m,1H),3.58–3.39(m,4H),2.65 –2.50(m,6H),2.00–1.88(m,2H),1.60–1.36(m,8H).MS(ESI)m/z:568.35[M+H] ⁺ .

Example 38: Preparation of 2-morpholinoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 38)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.59 (s, 1H), 8.30 (d, J = 7.7Hz, 1H), 8.26 (d, J = 7.7Hz, 1H), 7.97 (d, J = 8.0Hz,1H),7.91–7.88(m,1H),7.85–7.81(m,1H),7.53(dd,J＝6.6,2.0Hz,1H),7.44(ddd,J＝7.3,4.5,2.1Hz ,1H),7.22–7.18(m,1H),5.17–5.10(m,1 H),4.45–4.39(m,1H),4.33(s,2H),4.18–4.13(m,1H),3.58–3.54(m,4H),3.54–3.45(m,2H),3.39(t, J＝7.0Hz,2H),2.56(t,J＝7.2Hz,2H),2.45–2.37(m,4H),1.98–1.90(m,2H),1.53–1.47(m,2H).MS(ESI )m/z:570.20[M+H] ⁺ .

Example 39: Preparation of 2-(diethylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 39)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.34 (d, J = 7.7Hz, 1H), 8.29–8.23 (m, 1H), 7.98 (d, J = 8.0Hz, 1H),7.92–7.87(m,1H),7.83(t,J＝7.5Hz,1H),7.56–7.50(m,1H),7.44(ddd,J＝7.6,4.7,2.3Hz,1H),7.23 –7.17(m,1H),5.15–5.02(m,1H),4.52–4.38( m,1H),4.33(s,2H),4.29(s,2H),4.21–4.11(m,1H),3.64–3.46(m,2H),3.42(q,J＝7.1Hz,2H),3.27 (q,J＝7.0Hz,2H),2.02–1.89(m,2H),1.57–1.47(m,2H),1.20(t,J＝7.1Hz,3H),1.01(t,J＝7.1Hz, 3H).MS(ESI)m/z:592.22[M+Na] ⁺ .

Example 40: Preparation of 2-oxo-2-(pyrrolidin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 40)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.59 (s, 1H), 8.33 (d, J = 7.4Hz, 1H), 8.26 (d, J = 7.7Hz, 1H), 7.98 (d, J = 8.0Hz,1H),7.89(t,J＝7.5Hz,1H),7.83(t,J＝7.5Hz,1H),7.55–7.51(m,1H),7.47–7.41(m,1H),7.20( t,J＝9.2Hz,1H),5.14–5.01 (m,1H),4.50–4.40(m,1H),4.33(s,2H),4.19(s,2H),4.18–4.13(m,1H),3.61–3.47(m,4H),3.29(t ,J＝6.8Hz,2H),1.99–1.87(m,4H),1.78(p,J＝6.8Hz,2H),1.56–1.48(m,2H).MS(ESI)m/z:590.11[M +Na] ⁺ .

Example 41: Preparation of 2-oxo-2-(piperidin-1-yl)ethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 41)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.33 (d, J = 7.5Hz, 1H), 8.26 (d, J = 7.8Hz, 1H), 7.98 (d, J = 8.0Hz,1H),7.89(t,J＝7.4Hz,1H),7.83(t,J＝7.5Hz,1H),7.55–7.51(m,1H),7.46–7.42(m,1H),7.20( t,J＝9.2Hz,1H),5.12–5.05(m, 1H),4.49–4.41(m,1H),4.33(s,2H),4.29(s,2H),4.20–4.13(m,1H),3.58(s,2H),3.50–3.47(m,2H) ,3.45–3.39(m,2H),2.00–1.90(m,2H),1.62–1.48(m,6H),1.45–1.40(m,2H).MS(ESI)m/z:604.19[M+Na ] ⁺ .

Example 42: Preparation of 2-morpholino-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 42)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.59 (s, 1H), 8.34 (d, J = 7.6Hz, 1H), 8.26 (d, J = 7.7Hz, 1H), 7.98 (d, J = 8.0Hz,1H),7.89(t,J＝7.3Hz,1H),7.83(t,J＝7.5Hz,1H),7.55–7.51(m,1H),7.46–7.42(m,1H),7.20( t,J＝9.3Hz,1H),5.12–5 .05(m,1H),4.48–4.41(m,1H),4.33(s,2H),4.30(s,2H),4.19–4.14(m,1H),3.64–3.60(m,2H),3.60 –3.51(m,6H),3.46–3.43(m,2H),1.98–1.89(m,2H),1.56–1.48(m,2H).MS(ESI)m/z:606.11[M+Na] ⁺ .

Example 43: Preparation of 2-(cyclopropylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 43)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.59 (s, 1H), 8.33 (d, J = 7.6Hz, 1H), 8.26 (d, J = 7.8Hz, 1H), 8.23–8.20 (m, 1H),7.98(d,J＝8.0Hz,1H),7.91–7.88(m,1H),7.83(t,J＝7.2Hz,1H),7.53(dd,J＝6.6,1.9Hz,1H), 7.44(ddd,J＝7.3,4.6,2.2Hz,1H),7.22–7.18(m,1H), 5.11–5.05(m,1H),4.44–4.38(m,1H),4.33(s,2H),4.18–4.13(m,1H),3.94(s,2H),3.58–3.48(m,2H), 2.63–2.59(m,1H),1.98–1.91(m,2H),1.52(q,J＝9.9Hz,2H),0.63–0.58(m,2H),0.43–0.37(m,2H).MS( ESI)m/z:576.16[M+Na] ⁺ .

Example 44: Preparation of 2-(cyclobutylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 44)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.40 (d, J = 7.2Hz, 1H), 8.33 (d, J = 7.4Hz, 1H), 8.26 (d, J = 7.7Hz,1H),7.98(d,J＝7.9Hz,1H),7.89(t,J＝7.4Hz,1H),7.83(t,J＝7.4Hz,1H),7.59–7.48(m,1H) ,7.47–7.40(m,1H),7.20(t,J ＝9.2Hz,1H),5.18–4.99(m,1H),4.46–4.37(m,1H),4.33(s,2H),4.22–4.09(m,2H),3.96(s,2H),3.64– 3.43(m,2H),2.20–2.07(m,2H),2.00–1.83(m,4H),1.66–1.47(m,4H).MS(ESI)m/z:590.11[M+Na] ⁺ .

Example 45: Preparation of 2-(cyclopentylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 45)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.33 (d, J = 7.5Hz, 1H), 8.26 (d, J = 7.8Hz, 1H), 8.11 (d, J = 7.0Hz,1H),7.98(d,J＝8.0Hz,1H),7.89(t,J＝7.5Hz,1H),7.83(t,J＝7.5Hz,1H),7.57–7.49(m,1H) ,7.47–7.40(m,1H),7.20(t,J＝9.2Hz,1H),5.14–5.03(m,1 H),4.47–4.37(m,1H),4.33(s,2H),4.19–4.11(m,1H),4.03–3.97(m,1H),3.96(s,2H),3.61–3.46(m, 2H),2.01–1.88(m,2H),1.83–1.70(m,2H),1.68–1.58(m,2H),1.57–1.43(m,4H),1.43–1.32(m,2H).MS( ESI)m/z:604.13[M+Na] ⁺ .

Example 46: Preparation of 2-(cyclohexylamino)-2-oxoethyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 46)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 8.33 (d, J = 7.5Hz, 1H), 8.26 (d, J = 7.8Hz, 1H), 8.02 (d, J = 7.7Hz,1H),7.98(d,J＝8.0Hz,1H),7.89(t,J＝7.5Hz,1H),7.83(t,J＝7.5Hz,1H),7.55–7.51(m,1H) ,7.46–7.42(m,1H),7.20(t,J＝9.2Hz,1H),5.15– 5.02(m,1H),4.48–4.37(m,1H),4.33(s,2H),4.19–4.12(m,1H),3.97(s,2H),3.60–3.47(m,3H),2.00– 1.89(m,2H),1.74–1.63(m,4H),1.56–1.49(m,3H),1.27–1.21(m,2H),1.19–1.10(m,3H).MS(ESI)m/z :618.15[M+Na] ⁺ .

Example 47: Preparation of 4-(hydroxyamino)-4-oxobutyl-4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 47)

Intermediate 5a (0.24 g, 0.5 mmol), ethyl 4-bromobutyrate (0.12 g, 0.6 mmol) and triethylamine (0.06 g, 0.6 mmol) were dissolved in methanol (5 mL) and reacted at 50 ° C for 4 h. After the reaction was completed, the mixture was cooled to room temperature and concentrated in vacuo. 4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-thiocarbonyl)butyric acid ethyl ester was obtained by column chromatography as a white solid (0.25 g, yield 90%).

The intermediate 4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-thiocarbonyl)butanoic acid ethyl ester (0.25 g, 0.45 mmol), DBU (0.04 g, 0.23 mmol) and hydroxylamine aqueous solution (0.27 g, 8.2 mmol) were dissolved in methanol (5 mL) and reacted at 40° C. for 3 h. After the reaction was completed, the mixture was cooled to room temperature, diluted with water (5 mL), adjusted to pH 6 with 2N HCl, and extracted with DCM:iPrOH (3:1) (3×5 mL). The organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo, and column chromatography was performed to obtain 0.15 g of a light red solid with a yield of 62%.

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61 (s, 1H), 10.42 (s, 1H), 8.72 (s, 1H), 8.27 (d, J = 6.7Hz, 1H), 7.98–7.93 ( m,1H),7.93–7.87(m,1H),7.86–7.81(m,1H),7.49–7.43(m,1H),7.42–7.37(m,1H),7.2 5(t,J＝7.5Hz,1H),4.34(s,3H),4.18–4.01(m,2H),3.93–3.81(m,1H),3.77–3.69(m,2H),3.40–3.34( m,2H),3.29–3.21(m,2H),2.13–2.02(m,2H),1.90–1.82(m,2H); ¹³ CNMR (151MHz, DMSO-d ₆ ) δ196.29,168.71,164.66,159.87,157.76,156.13,145.27,135.27,133.96,132.41,132.36,132.04,129.55,129.52,128 .38,126.56,125.94,123.84,123.72,116.55, 116.41, 45.82, 41.40, 36.94, 36.18, 31.77, 24.92. MS (ESI) m/z (%): 566.17 [M+Na] ⁺ .

According to the preparation method of Example 47, the compounds of Examples 48-63 were prepared from Intermediates 5 and 7 and various small molecule halogenated esters.

Example 48: Preparation of 5-(hydroxyamino)-5-oxopentyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 48)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61(s,1H),10.36(s,1H),8.69(s,1H),8.32–8.23(m,1H),8.02–7.77(m,3H ),7.52–7.35(m,2H),7.30–7.20(m,1H),4.34(s,2H),4.34––3.81(m,4H),3.79–3.63(m,2H),3.40–3.34( m,2H),3.30–3.20(m,2H),2.07–1.89(m,2H),1.72–1.47(m,4H).MS(ESI)m/z:580.19[M+Na] ⁺ .

Example 49: Preparation of 6-(hydroxyamino)-6-oxohexyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 49)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61 (s, 1H), 10.35 (s, 1H), 8.68 (s, 1H), 8.27 (d, J = 7.7Hz, 1H), 7.96 (d, J＝7.9Hz,1H),7.90(t,J＝7.4Hz,1H),7.84(t,J＝7.4Hz,1H),7.48–7.43(m,1H),7.41–7.37(m,1H), 7.25(t,J＝8.9Hz,1H),4.3 4(s,2H),4.34–3.80(m,4H),3.78–3.68(m,2H),3.35–3.32(m,2H),3.24(t,J＝7.3Hz,2H),1.95(t, J＝7.2Hz,2H),1.62(p,J＝7.3Hz,2H),1.55–1.47(m,2H),1.39–1.30(m,2H).MS(ESI)m/z:594.08[M+ Na] ⁺ .

Example 50: Preparation of 7-(hydroxyamino)-7-oxoheptyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 50)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),10.33(s,1H),8.66(s,1H),8.35–8.20(m,1H),8.00–7.79(m,3H ),7.51–7.33(m,2H),7.31–7.19(m,1H),4.34(s,2H),4.34–3.80(m,4H),3.80–3.64(m,2H),3.39–3.33(m ,2H),3.27–3.18(m,2H),2.03–1.83(m,2H),1.68–1.21(m,8H).MS(ESI)m/z:608.29[M+Na] ⁺ .

Example 51: Preparation of 4-(hydroxycarbamoyl)benzyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 51)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),11.20(s,1H),9.04(s,1H),8.31–8.20(m,1H),7.99–7.79(m,3H ),7.74–7.64(m,2H),7.52–7.41(m,3H),7.41–7.36(m,1H),7.29–7.21(m,1H),4.61(s,2H),4.34(s,2H ),4.34–3.80(m,4H),3.78–3.66(m,2H),3.38–3.34(m,2H).MS(ESI)m/z:614.05[M+Na] ⁺ .

Example 52: Preparation of (E)-4-(3-hydroxyamino)-3-oxoprop-1-en-1-ylbenzyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 52)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),10.79(s,1H),9.06(s,1H),8.32–8.22(m,1H),7.98–7.80(m,3H ),7.55–7.36(m,7H),7.29–7.21(m,1H),6.53–6.38(m,1H),4.58(s,2H),4.34(s,2H),4.34–3.82(m,4H ),3.79–3.67(m,2H),3.45–3.35(m,2H).MS(ESI)m/z:640.23[M+Na] ⁺ .

Example 53: Preparation of 6-(hydroxyamino)-6-oxohexyl 4-(3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 53)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 8.27 (d, J = 7.5Hz, 1H), 7.96 (d, J＝7.5Hz,1H),7.91–7.86(m,1H),7.86–7.80(m,1H),7.45–7.37(m,3H),7.33–7.26(m,1H),4.36(s,2H) ,4.3 6–3.84(m,4H),3.76–3.62(m,2H),3.51–3.37(m,2H),3.24(t,J＝7.0Hz,2H),1.95(t,J＝7.0Hz,2H) ,1.68–1.57(m,2H),1.55–1.46(m,2H),1.38–1.29(m,2H).MS(ESI)m/z:576.03[M+Na] ⁺ .

Example 54: Preparation of 7-(hydroxyamino)-7-oxoheptyl 4-(3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 54)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61 (s, 1H), 10.33 (s, 1H), 8.65 (s, 1H), 8.26 (d, J = 7.7Hz, 1H), 7.96 (d, J＝7.9Hz,1H),7.89(t,J＝7.3Hz,1H),7.83(t,J＝7.4Hz,1H),7.45–7.35(m,3H),7.29(d,J＝7.3Hz, 1H),4.36(s,2H),4.36–3. 80(m,4H),3.78–3.58(m,2H),3.47–3.35(m,2H),3.23(t,J＝7.1Hz,2H),1.93(t,J＝7.1Hz,2H),1.67 –1.57(m,2H),1.51–1.45(m,2H),1.38–1.32(m,2H),1.28–1.25(m,2H).MS(ESI)m/z:589.98[M+Na] ⁺ .

Example 55: Preparation of 6-(hydroxyamino)-6-oxohexyl 4-(3-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 55)

¹ H NMR (600 MHz, DMSO-d ₆ )δ12.61(s,1H),10.35(s,1H),8.68(s,1H),8.35–8.20(m,1H),8.03–7.82(m,3H),7.36–7.13(m,3H),4.39(s,2H),4.39–3.83(m,4H),3.76–3.62( m,2H),3.48–3.37(m,2H),3.29–3.19(m,2H),2.03–1.89(m,2H),1.68–1.58(m,2H),1.56–1.47(m,2H),1.38–1.30(m,2H).MS(ESI)m/z:594.02[M+Na] ⁺ .

Example 56: Preparation of 7-(hydroxyamino)-7-oxoheptyl 4-(3-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 56)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.61 (s, 1H), 10.34 (s, 1H), 8.66 (s, 1H), 8.27 (d, J = 7.6Hz, 1H), 7.98 (d, J＝7.7Hz,1H),7.91(t,J＝7.1Hz,1H),7.85(t,J＝7.2Hz,1H),7.31(d,J＝9.1Hz,1H),7.23(s,1H) ,7.17(d,J＝8.3Hz,1H),4.39(s,2H),4.35–4.1 3(m,2H),4.07–3.83(m,2H),3.76–3.62(m,2H),3.46–3.36(m,2H),3.24(t,J＝7.1Hz,2H),1.94(t, J＝7.1Hz,2H),1.66–1.58(m,2H),1.51–1.45(m,2H),1.39–1.32(m,2H),1.28–1.25(m,2H).MS(ESI)m/ z:608.23[M+Na] ⁺ .

Example 57: Preparation of 6-(hydroxyamino)-6-oxohexyl 4-(4-fluoro-3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 57)

¹ H NMR (600 MHz, DMSO-d ₆ )δ12.55(s,1H),10.34(s,1H),8.67(s,1H),8.37–8.20(m,1H),8.07–7.79(m,3H),7.50–7.24(m,3H),4.39(s,2H),4.31–3.84(m,4H),3.75–3.57( m,2H),3.53–3.37(m,2H),3.28–3.20(m,2H),2.05–1.84(m,2H),1.69–1.57(m,2H),1.56–1.46(m,2H),1.40–1.28(m,2H).MS(ESI)m/z:594.27[M+Na] ⁺ .

Example 58: Preparation of 7-(hydroxyamino)-7-oxoheptyl 4-(4-fluoro-3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazine-1-carbodisulfate (Compound 58)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.55 (s, 1H), 10.34 (s, 1H), 8.66 (s, 1H), 8.28 (d, J = 7.3Hz, 1H), 8.04–7.93 ( m,2H),7.91–7.83(m,1H),7.46–7.36(m,2H),7.31(t,J＝8.1Hz,1H),4.39(s,2H),4.34–3.81(m,4H) , 3.76–3.57(m,2H),3.52–3.36(m,2H),3.28–3.19(m,2H),2.00–1.88(m,2H),1.67–1.57(m,2H),1.52–1.44(m ,2H),1.39–1.32(m,2H),1.29–1.24(m,2H).MS(ESI)m/z:608.16[M+Na] ⁺ .

Example 59: Preparation of 6-(hydroxyamino)-6-oxohexyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-1,4-diazepane-1-carbodisulfate (Compound 59)

MS (ESI) m/z: 607.97 [M+Na] ⁺ .

Example 60: Preparation of 7-(hydroxyamino)-7-oxoheptyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-1,4-diazepane-1-carbodisulfate (Compound 60)

MS (ESI) m/z: 622.05 [M+Na] ⁺ .

Example 61: Preparation of 6-(hydroxyamino)-6-oxohexyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 61)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 10.34 (s, 1H), 8.67 (s, 1H), 8.35–8.22 (m, 2H), 7.98 (d, J＝7.7 Hz,1H),7.89(t,J＝7.2Hz,1H),7.83(t,J＝7.2Hz,1H),7.58–7.49(m,1H),7.49–7.40(m,1H),7.20(t ,J＝9.1Hz,1H),5.22–5.0 6(m,1H),4.49–4.37(m,1H),4.33(s,2H),4.20–4.11(m,1H),3.59–3.44(m,2H),3.26–3.17(m,2H), 2.03–1.85(m,4H),1.65–1.58(m,2H),1.55–1.46(m,4H),1.37–1.30(m,2H).MS(ESI)m/z:608.03[M+Na] ⁺ .

Example 62: Preparation of 7-(hydroxyamino)-7-oxoheptyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbon disulfate (Compound 62)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60 (s, 1H), 10.34 (s, 1H), 8.66 (s, 1H), 8.31 (d, J = 7.4Hz, 1H), 8.26 (d, J＝7.7Hz,1H),7.98(d,J＝7.8Hz,1H),7.89(t,J＝7.3Hz,1H),7.83(t,J＝7.3Hz,1H),7.56–7.51(m, 1H),7.47–7.41(m,1H),7.20(t,J＝9.2Hz,1H),5.21– 5.07(m,1H),4.48–4.38(m,1H),4.33(s,2H),4.21–4.09(m,1H),3.56–3.44(m,2H),3.25–3.16(m,2H), 1.98–1.88(m,4H),1.65–1.57(m,2H),1.52–1.44(m,4H),1.38–1.32(m,2H),1.28–1.24(m,2H).MS(ESI)m /z:622.05[M+Na] ⁺ .

Example 63: Preparation of 4-(hydroxycarbamoyl)benzyl 4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamido)piperidine-1-carbodisulfate (Compound 63)

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.60(s,1H),11.20(s,1H),9.03(s,1H),8.34–8.24(m,2H),8.00–7.94(m,1H ),7.92–7.87(m,1H),7.85–7.79(m,1H),7.69(d,J＝7.5Hz,2H),7.55–7.51(m,1H),7.49–7.38(m,3H), 7. 23–7.17(m,1H),5.17–5.06(m,1H),4.59(s,2H),4.44–4.36(m,1H),4.33(s,2H),4.20–4.13(m,1H), 3.60–3.48(m,2H),1.99–1.90(m,2H),1.56–1.46(m,2H).MS(ESI)m/z:628.07[M+Na] ⁺ .

Study on the biological activity of the compounds of the present invention

Compound inhibition of PARP-1 activity test

The assay used to measure PARP-1 kinase activity is based on an enzyme-linked immunosorbent assay (ELISA). The specific procedure is:

Prepare histone coated 384-well plate, add 25 μL histone solution to each well and incubate overnight at 4°C. Prepare PBST buffer, blocking buffer and assay buffer, wash histone coated 384-well plate 3 times with PBST buffer. Block with 50 μL blocking buffer for 1h at room temperature. Wash plate 3 times with PBST buffer. Prepare 1000x compound in source plate. Transfer 1 μL compound from source plate to 96-well middle plate, shake and centrifuge at 1000 rpm for 1min. Transfer 5 μL DMSO/compound to each well. Incubate PARP-1 and DNA mixture at 25°C for 10min. Add 10 μL PARP-1 & DNA, incubate with compound at room temperature for 10min and add 10 μL DNA to the minimal control of the assay plate. Add 10 μL 2.5x NAD ⁺ to each well and incubate at 25°C for 60min. Wash plate 3 times with PBST buffer. Add 20 μL of anti-poly/mono ADP ribose rabbit monoclonal antibody. Incubate at room temperature for 1.5 h and wash the plate 3 times with PBST buffer. Add 20 μL of diluent (1:2000 in blocking buffer) anti-rabbit IgG, HRP antibody. Incubate at room temperature for 1 h and then wash the plate 3 times with PBST buffer. Add 25 μL of a mixture of Femto-ECL substrate A and Femto-ECL substrate B (1:1). Read chemiluminescence immediately on Envision. The IC ₅₀ data for the example compounds against PARP-1 kinase are calculated in Table 1.

Table 1

Compound inhibition of HDAC-1 activity test

The assay used to measure HDAC-1 kinase activity is based on an enzyme-linked immunosorbent assay (ELISA). The specific procedure is:

Prepare 1x assay buffer (modified Tris buffer). Transfer the compound to the assay plate in 100% DMSO by Echo550. Prepare the enzyme solution in 1x assay buffer. Add trypsin and Ac peptide substrate to 1x assay buffer to make substrate solution. Transfer 15 μL of enzyme solution to the assay plate. For low control, transfer 15 μL of 1x assay buffer. Add 10 μL of substrate solution to each well to start the reaction. Read the plate on Paradigm with 355nm excitation and 460nm emission kinetics. The inhibition data of the example compounds on HDAC-1 kinase are shown in Table 2.

Table 2

In vitro anti-tumor cell activity

The phthalazinone compounds of the above formula I according to the present invention were screened for their in vitro inhibitory activity on human breast cancer cells MDA-MB-436, human breast cancer cells MCF-7 and human breast cancer cells MDA-MB-231.

After the cells are revived and passaged 2-3 times to stabilize, use trypsin solution (0.25%) to digest them from the bottom of the culture bottle. Pour the cell digestion solution into a centrifuge tube, and then add culture medium to stop digestion. Centrifuge the centrifuge tube at 800r/min for 10 minutes, discard the supernatant and add 5mL culture medium, blow and mix the cells, aspirate 10μL of cell suspension and add it to the cell counting plate for counting, and adjust the cell concentration to 104/well. In the 96-well plate, except for A1 well which is a blank well without cells, add 100μL of cell suspension to the rest. Place the 96-well plate in an incubator and culture for 24h.

Dissolve the test sample in 50 μL of dimethyl sulfoxide, then add an appropriate amount of culture medium to dissolve the sample into a 2 mg/mL drug solution, and then dilute the sample to 20, 4, 0.8, 0.16, and 0.032 μg/mL in a 24-well plate.

Each concentration was added to 3 wells, of which the growth of cells in the two rows and two columns around was greatly affected by the environment, and only the blank cell wells were used. The 96-well plate was placed in an incubator and cultured for 96 hours.

The drug-containing culture medium in the 96-well plate was discarded, and the cells were rinsed twice with phosphate buffer solution (PBS). 100 μL of MTT (0.5 mg/mL) was added to each well and placed in the incubator for 4 hours, then the MTT solution was discarded and 100 μL of dimethyl sulfoxide was added. The surviving cells and the MTT reaction product A were fully dissolved by shaking on a magnetic oscillator, and the results were measured in an ELISA instrument. The IC ₅₀ value of the drug can be obtained by the Bliss method.

The results of the compounds' inhibitory activity against human breast cancer cells MDA-MB-436, human breast cancer cells MCF-7 and human breast cancer cells MDA-MB-231 are shown in Table 3.

Table 3

It can be clearly seen from the above test results that the compound represented by the general formula I to be protected by the present invention has a high level of inhibitory activity against PARP and/or HDAC, as well as good in vitro anti-tumor cell proliferation activity.

Although the present invention has been described in terms of specific embodiments, modifications and equivalents will be apparent to those skilled in the art and are intended to be encompassed within the scope of the present invention.

Claims (9)

1. A phthalazinone compound or a pharmaceutically acceptable salt thereof, characterized in that the phthalazinone compound has the structural formula:

in: X ² , X ³ , and X ⁴ are each independently CR ³ ; R ³ is selected from hydrogen, fluorine; Ring A is selected from

n is 1 or 2; R ² is selected from (C ₁ -C ₈ ) alkyl, and the (C ₁ -C ₈ ) alkyl may be optionally substituted by 1 to 3 R ⁴ ; R ⁴ is selected from trifluoromethyl, difluoromethyl, amino, hydroxy, phenyl, pyridyl, (C ₁ -C ₄ ) alkoxy, phenyl (C ₂ -C ₄ ) alkenyl,

The phenyl, phenyl(C ₂ -C ₄ )alkenyl, pyridyl may be optionally

or 1-3 R ⁷ substitutions; R ⁵ and R ⁶ are each independently selected from hydrogen, hydroxy, (C ₁ -C ₄ )alkyl, hydroxy(C ₁ -C ₄ )alkyl, (C ₃ -C ₆ )cycloalkyl; the (C ₁ -C ₄ )alkyl may be optionally substituted by 1 to 3 R ¹¹ ; or R ⁵ and R ⁶ together with the nitrogen atom to which they are attached form a 5-6 membered nitrogen-containing heterocyclic ring, wherein the nitrogen-containing heterocyclic ring contains 1-3 heteroatoms selected from N, O and S, and the nitrogen-containing heterocyclic ring may optionally include 1-2 carbon-carbon double bonds or triple bonds, and the nitrogen-containing heterocyclic ring may be optionally substituted by 1-3 identical or different R ¹⁰ , and the carbon atoms on the ring may be oxo-substituted; R ¹¹ is selected from hydroxy, (C ₁ -C ₆ )alkoxy, (C ₁ -C ₆ )alkylamino; R ⁷ is selected from hydrogen, halogen, trifluoromethyl, (C ₁ -C ₄ )alkyl, amino, hydroxyl, cyano; R ¹⁰ is selected from hydrogen, (C ₁ -C ₄ )alkyl, hydroxy(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, hydroxy, amino, (C ₁ -C ₄ )acyl. 2. The phthalazinone compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, in the general formula I, R ² is selected from:

3. The phthalazinone compound or pharmaceutically acceptable salt thereof according to claim 2, characterized in that the phthalazinone compound represented by general formula I is any one of the following compounds:

4. The phthalazinone compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the pharmaceutically acceptable salt refers to a salt formed by the phthalazinone compound and an inorganic acid, an organic acid, or an alkali metal ion, wherein the inorganic acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid; the organic acid is selected from the group consisting of fumaric acid, succinic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, citric acid, oxalic acid, tartaric acid, and benzoic acid; and the alkali metal ion is selected from the group consisting of potassium ion, sodium ion, and lithium ion. 5. A pharmaceutical composition, characterized in that the phthalazinone compound or a pharmaceutically acceptable salt thereof according to claim 1 is used as an active ingredient and mixed with a pharmaceutically acceptable carrier or excipient to prepare the composition; the carrier or excipient is one or more of a diluent, a binder, a wetting agent, a disintegrant, a lubricant and a glidant; the diluent is one or more of starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol and calcium hydrogen phosphate; the wetting agent is one or more of water, ethanol and isopropanol; the binder is starch slurry, dextrin, sugar The invention relates to a pharmaceutical composition comprising: a pharmaceutical composition comprising: a pharmaceutically acceptable form of the pharmaceutical composition, wherein the pharmaceutical composition comprises one or more of: a pharmaceutically acceptable form of the pharmaceutical composition, wherein the pharmaceutical composition comprises one or more of: a pharmaceutically acceptable form of the pharmaceutical composition, and ... 6. Use of the phthalazinone compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the preparation of a medicament for preventing and/or treating cancer, neurodegenerative disease or inflammation associated with PARP enzyme and HDAC enzyme. 7. Use of the pharmaceutical composition according to claim 5 in the preparation of a drug for preventing and/or treating cancer, neurodegenerative disease or inflammation associated with PARP enzyme and HDAC enzyme. 8. The use according to claim 6 or 7, characterized in that the cancer is any one of breast cancer, pancreatic cancer, ovarian cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, cervical cancer, testicular cancer, melanoma, nasopharyngeal cancer, oral cancer, malignant glioblastoma, bladder cancer, prostate cancer, esophageal cancer, brain tumor, reproductive system tumor, respiratory system tumor, lymphatic system tumor, digestive system tumor and skin tumor. 9. A method for preparing the phthalazinone compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that it comprises the following synthetic route: The R ² , X ² , X ³ , and X ⁴ are corresponding groups at corresponding positions of the compound represented by the general formula I, and n=1 or 2; Route 1:

Substituted or unsubstituted o-formylbenzoic acid is used as a starting material to react with dimethyl phosphite to obtain a phosphorus ylide intermediate 1; intermediate 1 is subjected to a Wittig-Horner reaction to obtain intermediate 2; intermediate 2 is subjected to cyanide hydrolysis and cyclization reaction to obtain intermediate 3; intermediate 3 is subjected to condensation and Boc protection removal reaction to obtain intermediate 4; intermediate 4 is subjected to carbon disulfide addition to obtain intermediate 5; intermediate 5 is subjected to a nucleophilic substitution reaction with a halogenated amine, a halogenated amide or a halogenated ester to obtain a target compound of formula II; Route 2:

Intermediate 3 is used as a raw material, and intermediate 6 is prepared through condensation and Boc removal reaction; intermediate 6 is subjected to carbon disulfide addition to prepare intermediate 7; intermediate 7 is reacted with halogenated amine, halogenated amide or halogenated ester through nucleophilic substitution reaction to prepare the target compound of formula III.