CN104003940B - 2,4-difluoro-5-( phthalazone-1-methyl)-benzoyl piperazine compound and application thereof - Google Patents

Abstract

The invention relates to a 2,4-difluoro-5-(phthalazone-1-methyl)-benzoyl piperazine compound and application of the 2,4-difluoro-5-(phthalazone-1-methyl)-benzoyl piperazine compound. Specifically, the invention discloses a compound of the structure shown in the formula I, wherein the definition of the compound of the structure shown in the formula I can be seen in the specification. The compound has the excellent PARP activity inhibitory effect and the excellent antitumor effect.

Description

2,4-difluoro-5-(phthalazinone-1-methyl)-benzoylpiperazine compounds and their use

technical field

The invention belongs to the field of medicinal chemistry and pharmacotherapeutics. Specifically, the present invention relates to 2,4-difluoro-5-(phthalazinone-1-methyl)-benzoylpiperazine compounds and their preparation methods and uses. The compounds can be used as polyadenosine diphosphate-ribose polymerase (PARP) inhibitor antineoplastic drugs.

Background technique

Malignant tumors are a major threat to human health, and cancer treatment is always a major problem in today's world. Radiation therapy and traditional chemotherapy are still the main means of treatment.

However, during the treatment process, tumor cells can activate their own DNA damage repair mechanism to repair, and then develop resistance in drug therapy and radiotherapy. Therefore, in recent years, DNA repair pathways have become the focus of tumor research. Through further research, it is found that DNA repair enzymes are overexpressed in tumor cells, so blocking DNA repair pathways is an important new approach for tumor treatment. Polyadenosine diphosphate-ribose polymerase (PARP) plays an important role in DNA repair in tumor proliferation and growth, and has become an enzyme closely related to tumorigenesis. Currently, 18 proteins have been isolated from the PARP family, among which PARP-1 is the most abundant protein. PARP-1 plays a key role in physiological processes such as DNA repair, gene transcription and expression, cell apoptosis, and chromosome stability. PARP-1 is abundantly expressed in the nucleus of actively proliferating cells. Research results show that the expression of PARP-1 in cancer cells is significantly higher than that in normal cells, such as malignant lymphoma, liver cancer, colorectal cancer, leukemia, especially breast cancer and ovarian cancer. Therefore, research on PARP and PARP inhibitors has become a hotspot in cancer treatment.

Contents of the invention

The purpose of the present invention is to provide an effective PARP inhibitor.

Another object of the present invention is to provide an effective anticancer compound.

In the first aspect of the present invention, there is provided a compound with the structure shown in formula I or a pharmaceutically acceptable salt thereof,

In the formula, R ₁ is a substituted or unsubstituted aromatic ring group, a substituted or unsubstituted 5-6 membered heteroaryl ring group, a substituted or unsubstituted benzo 5-6 membered heteroaryl ring group, a substituted Or unsubstituted 5-6 membered heterocyclic group, substituted or unsubstituted benzo 5-6 membered heterocyclic group, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkane -sulfonyl,

Wherein, R ₂ is C3-C6 cycloalkyl, C1-C6 alkyl, substituted or unsubstituted aromatic ring group, substituted or unsubstituted 5-6 membered heteroaryl ring group, substituted or unsubstituted benzene And 5-6 membered heteroaromatic ring group, substituted or unsubstituted 5-6 membered heterocyclic group, substituted or unsubstituted benzo 5-6 membered heterocyclic group;

R ₃ is a substituted or unsubstituted aromatic ring group, a substituted or unsubstituted 5-6 membered heteroaryl ring group, a substituted or unsubstituted benzo 5-6 membered heteroaryl ring group, a substituted or unsubstituted 5-6 membered heterocyclic group, substituted or unsubstituted benzo 5-6 membered heterocyclic group, C3-C6 cycloalkyl;

Wherein, the heteroaromatic ring group or heterocyclic group contains 1 to 3 heteroatoms selected from N, O, and S; the substituted refers to being substituted by a substituent selected from the following group: hydroxyl, halogen, C1 -C3 alkyl, halogenated C1-C3 alkyl, C1-C3 alkoxy, halogenated C1-C3 alkoxy, cyano, tert-butylcarbamoyl, -O-(CH ₂ ) _n -O -; Wherein, n is an integer between 1-3.

In another preferred embodiment, the aromatic ring group is selected from the group consisting of phenyl, naphthyl; and/or

The 5-6 membered heteroaromatic ring group is selected from the group consisting of pyridyl, pyrimidyl, thiazolyl, isothiazolyl, furyl, thienyl, pyrrolyl; and/or

The benzo 5-6 membered heteroaryl ring group is selected from the group consisting of benzopyridyl, benzopyrimidinyl, benzothiazolyl, benzisothiazolyl, benzopyrrolyl, benzofuranyl, benzo Thienyl; and/or

The 5- to 6-membered heterocyclic group is selected from the group consisting of morpholinyl, tetrahydrofuryl, tetrahydrothiazolyl, dioxane, dioxane; and/or

The benzo 5-6 membered heterocyclic group is selected from the group consisting of benzodioxane, benzodioxane; and/or

The C1-C6 alkyl group is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl; and/or

The C3-C6 cycloalkyl group is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

In another preferred embodiment, the compound or a pharmaceutically acceptable salt thereof is selected from the following group:

N-cyclopropylformyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine ;

N-acetyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine;

N-benzoyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine;

N-butyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine;

N-ethyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine;

N-phenyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine;

N-cyclopropylmethyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine ;

N-(tetrahydrofuran-2formyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(morpholine-4-formyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzene Formyl}piperazine;

N-(2-pyridyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl} Piperazine;

N-(pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl} Piperazine hydrochloride;

N-(pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }piperazine hydrochloride;

N-(Methanesulfonyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Zinc;

N-(hydroxyethyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Zinc;

N-methyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine;

N-propyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine;

N-(5-bromo-pyrimidin-2-yl]-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Fluorobenzoyl}piperazine;

N-(4,6-Dimethyl-pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2 ,4-Difluorobenzoyl}piperazine;

N-(4-trifluoromethyl-pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine;

N-(4-fluorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(2-fluorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(4-methylphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine;

N-(4-bromophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(3-chlorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(5-chloro-2-methylphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Difluorobenzoyl}piperazine;

N-(4-chlorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(4-cyanophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine;

N-(4-methoxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzene Formyl}piperazine;

N-(2-methoxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzene Formyl}piperazine;

N-(5-chloro-piperidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Difluorobenzoyl}piperazine;

N-(3-chloro-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Fluorobenzoyl}piperazine;

N-Benzyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine;

N-Benzyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine sulfate ;

N-(pyridine-3-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine;

N-(pyridine-3-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine sulfate;

N-(5-methyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2,4-di Fluorobenzoyl}piperazine;

N-(5-methyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Difluorobenzoyl}piperazine hydrochloride;

N-(4-methyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Difluorobenzoyl}piperazine;

N-(5-iodo-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Fluorobenzoyl}piperazine;

N-(6-ethoxy-piperidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine;

N-(6-methoxy-piperidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine;

N-(4-Hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(4-Hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }piperazine lithium salt;

N-(3-tert-butylcarbamoyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2 ,4-Difluorobenzoyl}piperazine;

N-(4-methylbenzoyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzene Formyl}piperazine;

N-(2-Hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(2-Hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }piperazine lithium salt;

N-(1,4-benzodioxane-2-formyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]- 2,4-Difluorobenzoyl}piperazine;

N-(3,4-methylenedioxybenzyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine;

N-(3,4-methylenedioxybenzyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine sulfate;

N-(thiazol-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine;

N-(1,2-Benzisothiazol-3-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine;

N-(thiazole-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine;

N-(thiazole-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine sulfate;

N-(5-methyl-pyridine-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4 -Difluorobenzoyl}piperazine;

N-(5-methyl-pyridine-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4 -Difluorobenzoyl}piperazine hydrochloride.

In the second aspect of the present invention, a pharmaceutical composition is provided, which comprises the compound described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

In another preferred example, the pharmaceutical composition further includes other antitumor drugs.

In another preferred example, the other anti-tumor drugs are commercially available anti-tumor drugs, selected from the following group (including but not limited to): cisplatin, carboplatin, temozolomide, dacarbazine, doxorubicin ifosfamide, methotrexate, busulfan, and thiotepa.

In the third aspect of the present invention, there is provided the compound described in the first aspect of the present invention or its pharmaceutically acceptable salt or the pharmaceutical composition described in the second aspect of the present invention in the preparation of polyadenosine diphosphate-ribose polymerase (PARP ) applications in inhibitors.

In another preferred embodiment, the poly ADP-ribose polymerase (PARP) is poly ADP-ribose polymerase-1 (PARP-1).

In the fourth aspect of the present invention, there is provided the application of the compound according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to the second aspect of the present invention in the preparation of antitumor drugs.

In another preferred example, the anti-tumor drug is a PARP inhibitor anti-tumor drug.

In another preferred example, the tumor is a BRCA gene-related tumor; and/or the tumor is selected from the group consisting of breast cancer, ovarian cancer, pancreatic cancer, and prostate cancer.

In another preferred embodiment, the BRCA gene is BRCA1 or BRCA2.

In the fifth aspect of the present invention, there is provided a method for preparing the compound described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof, comprising the steps of: in an inert solvent, in the presence of a base and a condensing agent, compound VIII React with NR ₁ -piperazine to obtain a compound of formula I;

In another preferred example, the inert solvent is selected from the group consisting of DME, DMF, CH ₂ Cl ₂ , THF, and Et ₂ O.

In another preferred embodiment, the base is selected from the group consisting of N,N-diisopropylethylamine, Et ₃ N, and DMAP.

In another preferred example, the condensing agent is selected from the group consisting of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

In the fifth aspect of the present invention, a treatment method is provided, comprising the step of: administering a safe and effective amount of the compound described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof to a subject in need of treatment.

In another preferred example, the safe and effective dose is 0.5-100 mg/kg; preferably, 1-50 mg/kg.

In another preferred example, the administration is oral administration.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Fig. 1 is the time-pharmaceutical effect diagram of the experimental therapeutic effect of the compound of the present invention on the subcutaneous transplanted tumor of human breast cancer MDA-MB-436 nude mice.

Fig. 2 is a graph showing the influence of the compound of the present invention on the body weight of the mice during the experimental treatment of human breast cancer MDA-MB-436 subcutaneously transplanted tumors in nude mice.

detailed description

After extensive and in-depth research, the inventors have discovered a brand new 2,4-difluoro-5-(phthalazinone-1-methyl)-formylpiperazine compound, which has been shown in the PARP inhibition experiment. , BRCA gene-deficient tumor cell proliferation inhibition experiment and tumor-inhibitory activity test in mice transplanted tumors showed that the compound of the present invention exhibited excellent anti-tumor effect targeting PARP, and provided a basis for further design and development of new PARP inhibitor anti-tumor drugs in the future The structural foundation was laid. On this basis, the inventors have completed the present invention.

the term

The term "aryl ring group" refers to an aromatic group, preferably an aromatic ring group with 5-12 members, for example, selected from the following group (but not limited to): phenyl, naphthyl.

The term "5-6 membered heteroaromatic ring group" refers to a 5- to 6-membered aromatic group having one or more heteroatoms selected from nitrogen, oxygen or sulfur, which may be selected from the following group (but not limited to): pyridine Base, pyrimidinyl, thiazolyl, isothiazolyl, furyl, thienyl, pyrrolyl.

The term "benzo 5-6 membered heteroaromatic ring group" refers to a group in which a 5- to 6-membered aromatic group having one or more heteroatoms selected from nitrogen, oxygen or sulfur is fused with a benzene ring, optionally From the following group (but not limited to): benzopyridyl, benzopyrimidinyl, benzothiazolyl, benzisothiazolyl, benzopyrrolyl, benzofuranyl, benzothienyl.

The term "5-6 membered heterocyclic group" refers to a 5- to 6-membered cyclic group having one or more heteroatoms selected from nitrogen, oxygen or sulfur, which may be selected from the following group (but not limited to): Morpholinyl, tetrahydrofuranyl, tetrahydrothiazolyl, dioxanyl, dioxopentacyclyl.

The term "benzo 5-6 membered heterocyclic group" refers to a 5- to 6-membered cyclic group having one or more heteroatoms selected from nitrogen, oxygen or sulfur, which is fused with a benzene ring. selected from the group consisting of (but not limited to): benzodioxanyl, benzodioxanyl.

The term "C1-C6 alkyl" refers to a linear or branched alkyl group having 1 to 6 carbon atoms, which may be selected from the following group (but not limited to): methyl, ethyl, n-propyl, isopropyl Base, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl.

The term "C1-C6 alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, which may be selected from the following group (but not limited to): methoxy, ethoxy, n-propyl Oxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, n-pentoxy, isopentyloxy, neopentyloxy, n-hexyloxy.

The term "C3-C6 cycloalkyl" refers to a cyclic group with 3 to 6 carbon atoms, which can be selected from the following group (but not limited to): cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

The term "halogen" refers to fluorine, chlorine, bromine, iodine. The term "halo" refers to fluoro, chloro, bromo, iodo.

Each group in the present invention may be unsubstituted or substituted, and the substituted means being substituted by a substituent selected from the following group: hydroxyl, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, tert-butylcarbamoyl, -O-(CH ₂ ) _n -O-; wherein, n is an integer between 1-3. The substituents may be substituted at various positions of each group.

Poly ADP-ribose polymerase (PARP)

PARP can participate in the polyadenosine diphosphate ribosylation process of eukaryotic proteins, and PARP can regulate many physiological processes in the body through the modification of various target proteins, such as single-strand DNA repair and cell cycle progression. After the DNA of the body is damaged by stimuli such as ionizing radiation and chemical drugs, PARP-1 can recognize the damage through the zinc finger structure of its DNA binding region, and then bind to the DNA gap and form a homodimer. 10-500 times more active. Activated PARP-1 uses its catalytic region to use nicotinamide adenine dinucleotide (NAD ⁺ ) as a substrate, decomposes it into nicotinamide and ADP ribose, and uses the latter to polymerize ADP on itself and other target proteins ribosylation. The poly-ADP-ribosylated histone is based on the strong negative charge of this chain and forms a strong repulsive force between DNA and relaxes the chromosome to facilitate the access of various DNA repair enzymes, such as: XRCC1, LIG-IIIα.

In tumor cells, when PARP activity is inhibited, single-strand DNA damage repair will go wrong, and with the increase of error-prone DNA damage, tumor cells will die, which serves the purpose of treating tumors. Most current PARP-1 inhibitors target at the C-terminus. PARP-1 inhibitors are similar in structure to the substrate NAD ⁺ , and compete for the binding of NAD ⁺ to the catalytic active site of PARP, thereby inhibiting PARP.

Combining PARP inhibitors with chemotherapy or radiotherapy can increase the sensitivity of tumor cells to chemotherapy or radiotherapy. In addition, tumor cells with BRCA gene or other key protein gene deletions often show abnormal sensitivity to PARP inhibitors. BRCA1/BRCA2 are two tumor suppressor genes that can inhibit the occurrence of malignant tumors, and play an important role in the homologous recombination repair pathway of DNA double-strand damage. Individuals or families with BRCA1 or BRCA2 gene defects have a high susceptibility to breast cancer, usually at a young age, and are prone to cancer on both breasts, and often have ovarian cancer at the same time. For such BRCA1/BRCA2 mutant tumors, PARP inhibitors can be used to simultaneously inhibit DNA repair and produce a synergistic lethal effect. Therefore, PARP inhibitors can be used alone or in combination with other drugs to treat BRCA gene-deficient tumors.

Preparation

The preparation method of the compound of formula (I) of the present invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compound of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in the specification or known in the art. Such a combination can be easily performed by those skilled in the art to which the present invention belongs.

A preferred preparation process is as follows:

Dissolve intermediate VIII in an inert solvent (such as N,N-dimethylacetamide), and sequentially add a base (such as N,N-diisopropylethylamine) and a condensing agent (such as benzotriazepine azole-N,N,N',N'-tetramethyluronium hexafluorophosphate), after stirring, add piperazine substituted by R ₁ , and react for a period of time at a certain temperature (such as room temperature or 20-30°C) ( Such as 10-30 hours or 24 hours), the compound of formula I is obtained. In the formula, R ₁ has the same meaning as above.

Specifically include the following steps:

Dissolve intermediate VIII in N,N-dimethylacetamide, add N,N-diisopropylethylamine, benzotriazole-N,N,N',N'-tetra Methylurea hexafluorophosphate, after stirring evenly, add piperazine substituted by R, react at room temperature for ₂₄ hours, stop the reaction, add water to the system, extract with dichloromethane, take the organic layer, evaporate the solvent under reduced pressure, The residue was subjected to column chromatography to obtain the target compound I.

The preparation method of the intermediate VIII may comprise the following steps:

Specifically, each step is as follows:

1) Add 2,4-difluorotoluene to the trifluoroacetic acid solution at 0-5°C, then add N-iodosuccinimide to the system in batches, react at room temperature for 10-20 hours, and stop the reaction , the solvent was removed under reduced pressure, the residue was added to aqueous sodium thiosulfate solution, extracted with ethyl acetate, the organic layer was taken, the solvent was removed under reduced pressure, and the residue was subjected to column chromatography to obtain intermediate II.

2) Dissolve intermediate II in N,N-dimethylformamide, add cuprous cyanide to the system, reflux at 150-180°C for 2-5 hours, cool to room temperature, and add 10% Ammonia, extracted with ethyl acetate, the organic layer was taken, the solvent was removed under reduced pressure, and the residue was subjected to column chromatography to obtain intermediate III.

3) Dissolve intermediate III and acetic anhydride in glacial acetic acid, keep the temperature at 0-5°C, then slowly add concentrated sulfuric acid to the system dropwise, after the dropwise addition, add chromium trioxide in batches to the reaction within 2 hours system, and keep the temperature below 5°C, then react for another 2 to 5 hours. After the reaction, pour the reaction solution into ice water, add excess sodium thiosulfate, extract with ethyl acetate and saturated sodium bicarbonate, take the organic layer, wash with saturated brine, dry, suction filter, concentrate, and the residue Intermediate IV was obtained by column chromatography.

4) The intermediate IV was dissolved in ethanol, and an equal amount of water was added as a solvent, and concentrated sulfuric acid was slowly added dropwise to the system. After the dropwise addition, the reaction was refluxed at 100° C. for 2 hours. The reaction was stopped, the system was distilled under reduced pressure, the residue was added with water, extracted with ethyl acetate, the organic layer was taken, the solvent was removed under reduced pressure, and the residue was subjected to column chromatography to obtain intermediate V.

5) Add the metal sodium wire into the methanol solution. After it is completely dissolved, add dimethyl phosphite to the system and keep the temperature at about 0°C. Then, within 20 minutes, the methanol solution of o-carboxybenzaldehyde was added to the system in batches, and the temperature was kept below 5°C. After the addition was complete, the reaction was carried out at 20°C for 2 hours. Methanesulfonic acid was then added dropwise to the solution over 35 minutes to stop the reaction. The system was distilled under reduced pressure, the residue was added with water, extracted with dichloromethane, the organic layer was taken, dried, filtered with suction, and the residue was subjected to column chromatography to obtain intermediate VI.

6) Add intermediates V and VI to the anhydrous tetrahydrofuran solution. After dissolving, slowly add triethylamine dropwise to the system and keep the temperature below 15°C, then react at room temperature for 5 hours, and distill the system under reduced pressure. Add water to the residue, continue the reaction for 30 minutes, stop the reaction, filter with suction, and wash the filter cake with water, n-hexane, and ether in sequence to obtain intermediate VII.

7) Add the intermediate VII to an appropriate amount of water to make an aqueous suspension, and add 13 mol/L sodium hydroxide solution to it. Then react at a temperature of 90° C. for 1 hour, add hydrazine hydrate to the system after cooling to 70° C., and react at 70° C. for 20 hours. After cooling to room temperature, adjust the reaction solution to pH=4 with dilute hydrochloric acid, and continue the reaction for 10 minutes. After suction filtration, the filter cake was washed with water and ether successively, and dried to obtain intermediate VIII.

active ingredient

As used herein, the term "compound of the present invention" refers to a compound represented by formula (I). The term also includes pharmaceutically acceptable salts of compounds of formula (I).

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or a base which is suitable for use as a medicine. Pharmaceutically acceptable salts include inorganic salts and organic salts. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid. Suitable bases for the formation of salts include, but are not limited to, alkali metal hydroxides (eg, lithium hydroxide, sodium hydroxide, potassium hydroxide, and the like).

Pharmaceutical compositions and methods of administration

Since the compound of the present invention has excellent inhibitory activity against polyadenosine diphosphate-ribose polymerase (PARP), the compound of the present invention and its pharmaceutically acceptable salt, as well as the pharmaceutical composition containing the compound of the present invention as the main active ingredient can be used For the treatment, prevention and alleviation of diseases mediated by polyadenosine diphosphate-ribose polymerase. According to the prior art, the compounds of the present invention are useful in the treatment of the following diseases: cancer and the like.

The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof within a safe and effective amount range and a pharmaceutically acceptable excipient or carrier. Wherein, "safe and effective dose" refers to: the amount of the compound is sufficient to obviously improve the condition without causing severe side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-200 mg of the compound of the present invention per dose.

"Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low enough toxicity. "Compatibility" herein means that the components of the composition can be blended with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as spit ), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.

The administration method of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but not limited to): oral administration, intratumoral administration, parenteral administration (intravenous, intramuscular) and the like.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents and, from such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner. Examples of usable embedding components are polymeric substances and waxy substances. The active compounds can also be in microencapsulated form, if desired, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

Besides such inert diluents, the compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage when administered, for a person with a body weight of 60kg, the daily The dosage is usually 1-2000 mg, preferably 20-500 mg. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

The advantages of the present invention mainly include: the present invention provides a compound with the structure shown in formula I, which has excellent inhibitory activity on polyadenosine diphosphate-ribose polymerase (PARP) and excellent antitumor activity.

The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Example 1 Preparation of 1,5-difluoro-2-iodo-4-toluene (intermediate II)

At 0°C, 4 ml of 2,4-difluorotoluene was put into the flask, 10 ml of trifluoroacetic acid was added, and then N-iodosuccinimide was added to the system in batches, totaling 9.1 g. After the feeding was completed, it was turned to room temperature to react overnight. After the reaction, distill under reduced pressure to remove the solvent, add an appropriate amount of saturated aqueous sodium thiosulfate solution to the residue, extract twice with ethyl acetate (50mL×2), take the organic layer, and wash with saturated brine (100mL×2) , dried over anhydrous magnesium sulfate, filtered, and distilled under reduced pressure, and the residue was separated by column chromatography to obtain 6.5 g of a colorless liquid (intermediate II), with a yield of 73%. ¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.57 (t, J=7.6 Hz, 1H), 6.81 (dd, J=9.3, 7.9 Hz, 1H), 2.24 (t, J=1.5 Hz, 3H).

Example 2 Preparation of (Intermediate III) of 2,4-difluoro-5-methylbenzonitrile

Put 5 grams of intermediate II into the flask, add 70 milliliters of N,N-dimethylformamide to dissolve, then add 2.7 grams of cuprous cyanide to the system, and reflux at 160°C for 3 hours. After the reaction, cool to room temperature, pour the reaction solution into 400 ml of 10% ammonia water, extract with ethyl acetate, take the organic layer, wash with saturated brine, dry over anhydrous magnesium sulfate, filter, distill under reduced pressure, and separate by column chromatography , 1.9 g of a colorless solid (Intermediate III) was obtained with a yield of 62%. ¹ H NMR (400MHz, CDCl ₃ ) δ: 7.49 (t, J=7.5Hz, 1H), 6.95 (t, J=9.0Hz, 1H), 2.30 (dd, J=2.1, 1.0Hz, 3H).

Example 3 Preparation of 2,4-difluoro-5-cyanobenzaldehyde diacetate (intermediate IV)

Put 3.4 grams of intermediate III into the flask, add 35 milliliters of acetic anhydride and 35 milliliters of glacial acetic acid, and keep the temperature at 0°C, then slowly add 5.24 milliliters of concentrated sulfuric acid to the system dropwise. After the addition, 6.23 grams of Chromium trioxide is added to the reaction system in batches within 2 hours, and the temperature is kept below 5°C, then the system is reacted for another 2 hours, the reaction is stopped, the reaction solution is poured into ice water, and an appropriate amount of saturated sodium thiosulfate solution is added, Extract twice with ethyl acetate, take the organic layer, then extract twice with saturated sodium bicarbonate, combine the organic layers, wash with saturated brine, dry over anhydrous magnesium sulfate, filter, distill under reduced pressure, and separate by column chromatography , to obtain 1.79 g of light yellow granular solid (Intermediate IV), the yield is 30%. ¹ H NMR (400MHz, CDCl3) δ: 7.93-7.80 (m, 2H), 7.06 (t, J=9.0Hz, 1H), 2.18 (s, 5H).

Example 4 Preparation of 2,4-difluoro-5-cyanobenzaldehyde (intermediate V)

Dissolve 3 grams of intermediate IV in 27 milliliters of ethanol and 27 milliliters of water, then slowly add 2.4 milliliters of concentrated sulfuric acid dropwise to the system. After the addition is complete, reflux at 100°C for 2 hours. Stop the reaction, distill under reduced pressure, remove the solvent, add an appropriate amount of water to the residue, extract twice with ethyl acetate solution, combine the organic layers, dry over anhydrous magnesium sulfate, filter, distill under reduced pressure, and separate by column chromatography to obtain 1.51 g White solid (Intermediate V), yield 81%. ¹ H NMR (400MHz, CDCl ₃ ) δ: 10.29 (s, 1H), 8.27 (t, J=7.2Hz, 1H), 7.16 (t, J=9.0Hz, 1H).

Example 5 Preparation of (3-oxo-1,3-dihydroisobenzofuran-1-yl) dimethyl phosphate (intermediate VI)

Put 24ml of anhydrous methanol into the three-necked flask, and install a drying tube and a rubber stopper. At 0°C, quickly add 0.78 g of metallic sodium wire into the three-necked flask. Inject 2.91 ml of dimethyl phosphite into the system and keep the temperature at around 0°C. Dissolve 4 grams of o-carboxybenzaldehyde in 4 milliliters of methanol, and then use a needle tube to add to the system in batches within 20 minutes, keeping the temperature below 5°C. After the addition, the temperature was raised to 20°C, and the reaction was carried out at this temperature for 2 hours. Finally methanesulfonic acid was added to the solution over 35 minutes to stop the reaction. Distilled under reduced pressure, removed solvent, added appropriate amount of water to the residue, extracted with dichloromethane, took the organic layer, dried over anhydrous magnesium sulfate, filtered, distilled under reduced pressure, and separated by column chromatography to obtain 3.36 grams of white solid (intermediate VI), yield 52%. ¹ H NMR (400MHz, CDCl ₃ ) δ: 8.03-7.93 (m, 1H), 7.84-7.72 (m, 2H), 7.69-7.58 (m, 1H), 5.75 (d, J=10.9Hz, 1H), 3.96 (d, J=10.8Hz, 3H), 3.62 (d, J=10.6Hz, 3H).

Example 6 Preparation of 2,4-difluoro-5-[(Z/E)-(3-oxo-2-isobenzofuranone-1-methine]benzonitrile (intermediate VII)

Add 1.52 g of intermediate V and 3.08 g of intermediate VI into 20 ml of anhydrous tetrahydrofuran. After dissolving, slowly add 1.57 ml of triethylamine dropwise to the system and keep the temperature below 15°C, then react at room temperature for 5 After 1 hour, the solvent was evaporated, and 25 milliliters of water was added to the residue. After the reaction was continued for 30 minutes, the reaction was stopped, suction filtered, and the filter cake was washed with water, n-hexane and ether successively to obtain 2.22 grams of light yellow solid (intermediate VII). is 86%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 8.65(t, J=7.6Hz, 2H), 8.02(dd, J=8.4, 2.0Hz, 3H), 7.90-7.77(m, 5H), 7.71-7.62(m ,5H),7.18(t,J=8.8Hz,1H),7.07(dd,J=9.9,8.6Hz,2H),6.62(s,1H),6.57(s,2H).

Example 7 Preparation of 5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoic acid (Intermediate VIII)

Add 14 milliliters of water to 2.2 grams of intermediate VII to make an aqueous suspension, add 3.09 milliliters of 13 mol/liter sodium hydroxide solution, heat up to 90 ° C for 1 hour, then cool to 70 ° C, add 6.27 Add a milliliter of hydrazine hydrate (98%) and react at 70°C for 20 hours, cool to room temperature, adjust the reaction solution to pH=4 with 8mol/L dilute hydrochloric acid, continue stirring for 10 minutes, filter with suction, and wash the filter cake with water and ether in sequence , and dried to obtain 1.84 g of a khaki solid (intermediate VIII). The yield is 75%. ¹ HNMR (400MHz, DMSO-d6) δ: 12.57(s, 1H), 8.28(d, J=7.7Hz, 1H), 7.95(d, J=5.2Hz, 2H), 7.87(dd, J=9.8, 4.2Hz, 1H), 7.57(d, J=8.8Hz, 1H), 7.07(t, J=10.0Hz, 1H), 4.29(s, 2H).

Example 8 N-Cyclopropylformyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl } The preparation of piperazine (I-1)

Dissolve 200 mg of intermediate VIII in 5 ml of N,N-dimethylacetamide, add 454 microliters of N,N-diisopropylethylamine, 360 mg of benzotriazole-N, N,N',N'-Tetramethyluronium hexafluorophosphate, after stirring evenly, add 107.27 mg of 1-cyclopropanoylpiperazine, react at room temperature for 24 hours, stop the reaction, add water to the system, and use dichloro Methane was extracted, and the organic layer was taken, followed by column chromatography to obtain 85.8 mg of (I-1) as a white solid, with a yield of 30%. Mp: 116-122°C (CH ₂ Cl ₂ ); purity: 91%. ¹ HNMR (300MHz, CDCl ₃ ) δ: 10.10 (s, 1H, NH), 8.52-8.44 (m, 1H, ArH), 7.80 (qd, J=8.4, 7.7, 3.2Hz, 2H, ArH), 7.33- 7.26(m,2H,ArH),6.92(td,J=9.4,2.8Hz,1H,ArH),4.28(s,2H,CH ₂ ),3.75(s,4H,CH ₂ ),3.60(s,2H ,CH ₂ ),3.31(s,2H,CH ₂ ),1.90-1.66(m,1H,CH),1.06-0.95(m,2H,CH ₂ ),0.80(s,2H,CH ₂ ); HR- MS (ESI): m/z calc. C ₂₄ H ₂₃ F ₂ N ₄ O ₃ [M+H] ⁺ , 453.17382; Actual 453.17327.

Example 9 N-acetyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Preparation of oxazine (I-2)

Except that 1-cyclopropanylpiperazine is replaced by 1-acetylpiperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 50 mg of white solid (I-2), and the yield is 19 %. Mp: 126-136°C (CH ₂ Cl ₂ ); purity: 100%. ¹ HNMR (300MHz, CDCl ₃ ) δ: 10.26 (s, 1H, NH), 8.47 (d, J = 7.4Hz, 1H, ArH), 7.80 (d, J = 8.1Hz, 3H, ArH), 7.37-7.27 (m,1H,ArH),6.92(t,J=9.5Hz,1H,ArH),4.28(s,2H,CH ₂ ),3.86-3.16(m,8H,CH ₂ ),2.11(d,J= 13.6Hz, 3H, CH ₃ ); HR-MS (EI): m/z theoretical for C ₂₂ H ₂₀ F ₂ N ₄ O ₃ , 426.1503; actual 426.1508.

Example 10 N-benzoyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl} Preparation of piperazine (I-3)

Except that 1-cyclopropanoylpiperazine is changed into 1-benzoylpiperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 56 mg of white solid (I-3), and the yield is 18%. Mp: 132-147°C (CH ₂ Cl ₂ ); purity: 100%. ¹ HNMR (400MHz, CDCl3) δ: 9.98 (s, 1H, NH), 8.49 (d, J = 7.5Hz, 1H, ArH), 7.95-7.74 (m, 3H, ArH), 7.51-7.31 (m, 6H ,ArH),6.94(s,1H,ArH),4.30(s,2H,CH ₂ ),3.95-3.35(m,8H,CH ₂ ); HR-MS(EI): m/z theoretical value C ₂₇ H ₂₂ F ₂ N ₄ O ₃ [M] ⁺ ,488.1660; actual value 488.1661.

Example 11 N-butyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Preparation of oxazine (I-4)

Except that 1-cyclopropamoylpiperazine is replaced by 1-butylpiperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 60 mg of white solid (I-4), and the yield is 28.7 %. Mp: 82-91°C (CH ₂ Cl ₂ ); purity: 100%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.66 (s, 1H, NH), 8.49 (d, J = 7.9Hz, 1H, ArH), 7.83 (dt, J = 11.5, 7.5Hz, 3H, ArH), 7.33 -7.25(m,1H,ArH),6.92(t,J=9.3Hz,1H,ArH),4.30(s,2H,CH ₂ ),3.78(s,2H,CH ₂ ),3.32(s,2H, CH ₂ ), 2.51(t, J=5.1Hz, 2H, CH ₂ ), 2.38(d, J=7.1Hz, 4H, CH ₂ ), 1.52-1.25(m, 4H, CH ₂ ) 0.93(t, J =7.5Hz, 3H, CH ₃ ); HR-MS (EI): m/z theoretical value C ₂₄ H ₂₆ F ₂ N ₄ O ₂ [M] ⁺ , 440.2024; actual value 440.2026.

Example 12 N-ethyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Preparation of oxazine (I-5)

Except that 1-cyclopropanylpiperazine is replaced with 1-ethylpiperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 40 mg of white solid (I-5), and the yield is 15 %. Mp: 93-102°C (CH ₂ Cl ₂ ); purity: 100%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.09(s,1H,NH),8.56-8.43(m,1H,ArH),7.94-7.75(m,3H,ArH),7.33-7.26(q,1H,ArH ), 6.93 (t, J=9.4Hz, 1H, ArH), 4.30 (s, 2H, CH ₂ ), 3.87 (s, 2H, CH ₂ ), 3.41 (s, 2H, CH ₂ ), 2.74-2.39 ( m, 6H, CH ₂ ), 1.17 (t, J=7.2Hz, 3H, CH ₃ ); HR-MS (EI): m/z theoretical value C ₂₂ H ₂₂ F ₂ N ₄ O ₂ [M] ⁺ , 412.1711; actual value 412.1709.

Example 13 N-phenyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Preparation of oxazine (I-6)

Except that 1-cyclopropamoylpiperazine is replaced by 1-phenylpiperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 49 mg of white solid (I-6), and the yield is 16.8 %. Mp: 108-116°C (CH ₂ Cl ₂ ); purity: 97%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 9.91(s,1H,NH),8.53-8.45(m,1H,ArH),7.90-7.78(m,3H,ArH),7.37-7.30(m,3H,ArH ), 6.95 (dd, J=10.6, 8.0Hz, 4H, ArH), 4.31 (s, 2H, CH ₂ ), 3.94 (s, 2H, CH ₂ ), 3.49 (s, 2H, CH ₂ ), 3.27 ( t,J=5.1Hz,2H,CH ₂ ), 3.13(s,2H,CH ₂ ); HR-MS(EI): m/z theoretical value C ₂₆ H ₂₂ F ₂ N ₄ O ₂ [M] ⁺ , 460.1711; actual value 460.1706.

Example 14 N-cyclopropylmethyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl } The preparation of piperazine (I-7)

Except that 1-cyclopropamoylpiperazine is replaced with 1-(cyclopropylmethyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 33.3 mg of white solid (I-7), The yield is 12%. Mp: 105-112°C (CH ₂ Cl ₂ ); purity: 100%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.35 (s, 1H, NH), 8.56-8.44 (m, 1H, ArH), 7.84 (ddd, J=16.0, 10.9, 6.2Hz, 3H, ArH), 7.32 ( s,1H,ArH),6.92(t,J=9.3Hz,1H,ArH),4.30(s,2H,CH ₂ ),3.85(s,2H,CH ₂ ),3.38(s,2H,CH ₂ ) ,2.66(s,2H,CH ₂ ),2.52(s,2H,CH ₂ ),2.35(d,J=5.5Hz,2H,CH ₂ ),0.90(s,1H,CH),0.57(d,J =7.2Hz, 2H, CH ₂ ), 0.15 (d, J = 4.3Hz, 2H, CH ₂ ); HR-MS (EI): m/z theoretical value C ₂₄ H ₂₄ F ₂ N ₄ O ₂ [M] ⁺ ,438.1867; actual value 438.1869.

Example 15 N-(tetrahydrofuran-2formyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Preparation of Benzoyl}piperazine (I-8)

Except that 1-cyclopropamoylpiperazine is replaced with 1-(2-tetrahydrofuroyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 62.7 mg of white solid (I-8) , and the yield was 20.5%. Mp: 112-118°C (CH ₂ Cl ₂ ); purity: 100%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.18(s, 1H, NH), 8.49(d, J=6.6Hz, 1H, ArH), 7.93–7.74(m, 3H, ArH), 7.33(d, J= 6.3Hz,1H,ArH),6.94(t,J＝9.1Hz,1H,ArH),4.69–4.50(m,1H,CH),4.30(s,2H,CH ₂ ),3.99–3.24(m,10H , CH ₂ ), 2.37 (s, 1H, CH ₂ ), 2.00 (dd, J=39.1, 13.8Hz, 3H, CH ₂ ); HR-MS (EI): m/z theoretical value C ₂₅ H ₂₄ F ₂ N ₄ O ₄ [M] ⁺ ,482.1766; actual value 482.1760.

Example 16 N-(morpholine-4-formyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Preparation of Difluorobenzoyl}piperazine (I-9)

Except that 1-cyclopropanoylpiperazine is changed into 1-(4-morpholineformyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 84.6 mg of white solid (I-9 ), the yield was 26.9%. Mp: 110-124°C (CH ₂ Cl ₂ ); purity: 99%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 9.82(s, 1H, NH), 8.49(d, J=7.9Hz, 1H, ArH), 7.90–7.77(m, 3H, ArH), 7.32(d, J= 7.8Hz, 1H, ArH), 6.94(t, J=9.4Hz, 1H, ArH), 4.30(s, 2H, CH ₂ ), 3.77(d, J=7.0Hz, 2H, CH ₂ ), 3.71(dd , J=8.3, 4.0Hz, 4H, CH ₂ ), 3.39–3.26 (m, 8H, CH ₂ ), 3.24 (s, 2H, CH ₂ ); HR-MS (EI): m/z theoretical value C ₂₅ H ₂₅ F ₂ N ₅ O ₄ [M] ⁺ , 497.1875; actual value 497.1870.

Example 17 N-(2-pyridyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzene Formyl}piperazine (I-10) and N-(pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl ]-2, the preparation of 4-difluorobenzyl}piperazine hydrochloride (I-10-HCl)

Except that 1-cyclopropamoylpiperazine is replaced with 1-(2-pyridyl)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 75 mg of white solid (I-10), The yield is 25.7%. Mp: 104-114°C (CH ₂ Cl ₂ ); purity: 97%. I-10: ¹ H NMR (400MHz, CDCl ₃ ) δ: 10.19 (s, 1H), 8.54–8.45 (m, 1H), 8.22 (d, J=3.6Hz, 1H), 7.91–7.76 (m, 3H) ,7.55(t,J=7.2Hz,1H),7.34(t,J=7.8Hz,1H),6.95(t,J=9.4Hz,1H),6.75–6.65(m,2H),4.31(s, 2H), 3.89(s, 2H), 3.61(d, J=20.7Hz, 4H), 3.44(s, 2H); HR-MS(ESI): m/z theoretical value C ₂₅ H ₂₂ F ₂ N ₅ O ₂ [M+H] ⁺ ,462.17416; actual value 462.17361.

110 milligrams of I-10 were dissolved in 4 milliliters of dichloromethane, and HCl gas was introduced into the reaction for 5 minutes, the solvent was evaporated to dryness, 6 milliliters of methanol was added to the residue, and the solvent was evaporated to dryness to obtain 100 milligrams of white solid (I- 10-HCl), the yield was 84.3%. Mp: 174-176°C; purity: 95%. I-10-HCl: ¹ HNMR (400MHz, DMSO) δ: 12.55(s, 1H), 8.28(d, J=7.9Hz, 1H), 8.11-7.92(m, 4H), 7.89(t, J=7.4 Hz, 1H), 7.53-7.40(m, 2H), 7.37(d, J=9.2Hz, 1H), 7.00(t, J=6.6Hz, 1H), 4.38(s, 2H), 3.84(s, 2H ), 3.80(s,2H), 3.73(s,2H), 3.44(s,2H).

Example 18 N-(pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Benzoyl}piperazine (I-11) and N-(pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methanol Preparation of ]-2,4-difluorobenzoyl}piperazine hydrochloride (I-11-HCl)

Except that 1-cyclopropanylpiperazine was replaced with 1-(2-pyrimidinyl)piperazine, all other required raw materials, reagents and preparation methods were the same as in Example 8 to obtain 35 mg of white solid (I-11), The yield is 12%. Mp: 105-114°C (CH ₂ Cl ₂ ); purity: 97%. I-11: ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.17(s, 1H), 8.55-8.45(m, 1H), 8.39(d, J=4.7Hz, 2H), 7.92-7.75(m, 3H) ,7.34(t,J=7.7Hz,1H),6.96(t,J=9.4Hz,1H),6.63(t,J=4.6Hz,1H),4.31(s,2H),3.98(s,2H) , 3.87 (s, 4H), 3.41 (s, 2H); HR-MS (ESI): m/z theoretical for C ₂₄ H ₂₁ F ₂ N ₆ O ₂ [M+H] ⁺ ,463.16941 actual 463.16886.

110 milligrams of I-11 were dissolved in 4 milliliters of dichloromethane, and HCl gas was introduced into the reaction for 5 minutes, and the solvent was evaporated to dryness. 6 milliliters of methanol was added to the residue, and the solvent was evaporated to dryness to obtain 102 milligrams of white solid (I- 11-HCl), the yield was 85.9%. Mp: 156-157°C; purity: 96%. I-11-HCl: ¹ HNMR (400MHz, DMSO) δ: 12.55(s, 1H), 8.42(d, J=4.8Hz, 2H), 8.28(d, J=7.1Hz, 1H), 8.03(d, J＝7.9Hz, 1H), 8.00-7.93(m, 1H), 7.88(t, J＝7.5Hz, 1H), 7.49-7.33(m, 2H), 6.72(t, J＝4.8Hz, 1H), 4.37 (d, J=6.1Hz, 2H), 3.82 (d, J=5.0Hz, 2H), 3.70 (s, 4H), 3.31 (s, 2H).

Example 19 N-(methanesulfonyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Preparation of acyl}piperazine (I-12)

Except that 1-cyclopropanylpiperazine is changed into 1-methanesulfonylpiperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 45 mg of white solid (I-12), and the yield is 15.4%. Mp: 96-103°C (CH ₂ Cl ₂ ); purity: 99%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.10 (s, 1H, NH), 8.53-8.46 (m, 1H, ArH), 7.84 (ddt, J=12.6, 8.5, 6.5Hz, 3H, ArH), 7.33 ( d,J=7.8Hz,1H,ArH),6.95(t,J=9.4Hz,1H,ArH),4.30(s,2H,CH ₂ ),3.43(s,2H,CH ₂ ),3.32(s, 2H,CH ₂ ), 3.19(s,2H,CH ₂ ), 3.00(s,2H,CH ₂ ), 2.83(s,3H,CH ₃ ); HR-MS(ESI): m/z theoretical value C ₂₁ H ₂₁ F ₂ N ₄ O ₄ S[M+H] ⁺ ,463.12516 Actual value 463.12461.

Example 20 N-(hydroxyethyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Preparation of acyl}piperazine (I-13)

Except that 1-cyclopropanylpiperazine is changed into 1-hydroxyethylpiperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 106 mg of white solid (I-13), and the yield is 39.1%. Mp: 96-102°C (CH ₂ Cl ₂ ); purity: 100%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.40 (s, 1H, NH), 8.48 (d, J = 8.5Hz, 1H, ArH), 7.84 (dt, J = 11.4, 7.0Hz, 3H, ArH), 7.33 (t,J=7.7Hz,1H,ArH),6.94(t,J=9.3Hz,1H,ArH),4.30(s,2H,CH ₂ ),4.00(s,2H,CH ₂ ),3.84(s ,2H,CH ₂ ), 3.56(s,2H,CH ₂ ), 2.90(d,J=19.2Hz,6H,CH ₂ ); HR-MS(ESI): m/z theoretical value C ₂₂ H ₂₃ F ₂ N ₄ O ₃ [M+H] ⁺ , 429.17382; actual value 429.17327.

Example 21 N-methyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Preparation of oxazine (I-14)

Except that 1-cyclopropamoylpiperazine is replaced by 1-methylpiperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 106 mg of white solid (I-14), and the yield is 46 %. Mp: 94-101°C (CH ₂ Cl ₂ ); purity: 97%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.32(s, 1H, NH), 8.54-8.45(m, 1H, ArH), 7.90-7.75(m, 3H, ArH), 7.31(d, J=8.0Hz, 1H, ArH), 6.92 (t, J=9.3Hz, 1H, ArH), 4.30 (s, 2H, CH ₂ ), 3.83 (s, 2H, CH ₂ ), 3.36 (s, 2H, CH ₂ ), 2.54 (s, 2H, CH ₂ ), 2.39 (d, J=13.7Hz, 5H, CH ₂ , CH ₃ ); HR-MS (ESI): m/z theoretical value C ₂₁ H ₂₁ F ₂ N ₄ O ₂ [ M+H] ⁺ ,399.16326; actual value 399.16271.

Example 22 N-propyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Preparation of oxazine (I-15)

Except that 1-cyclopropanylpiperazine is replaced by 1-propylpiperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8, and 30 mg of white solid (I-15) is obtained, and the yield is 11 %. Mp: 85-90°C (CH ₂ Cl ₂ ); purity: 97%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.10 (s, 1H, NH), 8.49 (d, J = 7.2Hz, 1H, ArH), 7.84 (ddd, J = 18.5, 11.0, 6.1Hz, 3H, ArH) ,7.32(d,J=7.8Hz,1H,ArH),6.93(t,J=9.4Hz,1H,ArH),4.30(s,2H,CH ₂ ),3.94(s,2H,CH ₂ ),3.49 (s, 2H, CH ₂ ), 2.63 (d, J=70.4Hz, 6H, CH ₂ ), 1.70-1.85 (m, 2H, CH ₂ ), 0.96 (t, J=7.3Hz, 3H, CH ₃ ) ; HR-MS (ESI): m/z calc. C ₂₃ H ₂₄ F ₂ N ₄ O ₂ Na[M+Na] ⁺ , 449.17650; Actual 449.17595.

Example 21 N-(5-bromo-pyrimidin-2-yl]-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, Preparation of 4-Difluorobenzoyl}piperazine (Compound I-16)

Except that 1-cyclopropanylpiperazine is replaced with 1-[2-(5-bromo)pyrimidinyl]pyrimidine, all the other required raw materials, reagents and preparation methods are the same as in Example 8, and 58 mg of white solid (I -16), the yield was 17%. Mp: 239-240°C (EtOAc); purity: 98%. ¹ HNMR (500MHz, CDCl ₃ ) δ: 9.89 (s, 1H, NH), 8.54 (d, J = 7.6Hz, 1H, ArH), 8.43 (s, 2H, ArH), 7.90 (dt, J = 23.7, 6.9Hz, 3H, ArH), 7.39(t, J=7.6Hz, 1H, ArH), 7.01(t, J=9.3Hz, 1H, ArH), 4.36(s, 2H, CH ₂ ), 3.99(s, 2H,CH ₂ ), 3.88(s,4H,CH ₂ ), 3.45(s,2H,CH ₂ ); HR-MS(EI): m/z theoretical value C ₂₄ H ₁₉ BrF ₂ N ₆ O ₂ [M ] ⁺ ,540.0721; the actual value is 540.0723.

Example 22 N-(4,6-dimethyl-pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl Preparation of ]-2,4-difluorobenzoyl}piperazine (I-17)

Except that 1-cyclopropamoylpiperazine is replaced by 1-[2-(4,6-dimethyl)pyrimidinyl]piperazine, the rest of the required raw materials, reagents and preparation methods are the same as in Example 8 to obtain white Solid 26 mg (I-17), yield 8.3%. Mp: 237-238°C (EtOAc); purity: 99%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 9.83(s, 1H, NH), 8.47(d, J=7.4Hz, 1H, ArH), 7.89-7.74(m, 3H, ArH), 7.33-7.28(m, 1H, ArH), 6.92(t, J=9.4Hz, 1H, ArH), 6.38(s, 1H, ArH), 4.28(s, 2H, CH ₂ ), 3.91(d, J=61.7Hz, 6H, CH ₂ ), 3.41(s,2H,CH ₂ ), 2.38(s,6H,CH ₃ ); HR-MS(EI): m/z theoretical value C ₂₆ H ₂₄ F ₂ N ₆ O ₂ [M] ⁺ , 490.1929; actual value 490.1927.

Example 23 N-(4-trifluoromethyl-pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl] Preparation of -2,4-difluorobenzoyl}piperazine (I-18)

Except that 1-cyclopropamoylpiperazine is replaced by 1-[2-(4-trifluoromethyl)pyrimidinyl]piperazine, the rest of the required raw materials, reagents and preparation methods are the same as in Example 8 to obtain a white solid 65 mg (I-18), yield 19.2%. Mp: 120-122°C (CH ₂ Cl ₂ ); purity: 99%. ¹ HNMR (500MHz, CDCl ₃ ) δ: 9.81 (s, 1H, NH), 8.52 (s, 1H, ArH), 8.47 (d, J = 7.7Hz, 1H, ArH), 7.81 (dd, J = 24.5, 7.6Hz,3H,ArH),7.31(s,1H,ArH),6.93(t,J＝9.3Hz,1H,ArH),6.83(s,1H,ArH),4.29(s,2H,CH ₂ ), 3.97(s,2H,CH ₂ ), 3.84(s,4H,CH ₂ ), 3.38(s,2H,CH ₂ ); HR-MS(EI): m/z theoretical value C ₂₅ H ₁₉ F ₅ N ₆ O ₂ [M] ⁺ ,530.1490; actual value 530.1491.

Example 24 N-(4-fluorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Preparation of Benzoyl}piperazine (I-19)

Except that 1-cyclopropamoylpiperazine is replaced by 1-(4-fluorophenyl)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 160 mg of white solid (I-19) , and the yield was 52.9%. Mp: 188-190°C (EtOAc); purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.03(s, 1H, NH), 8.47(d, J=7.2Hz, 1H, ArH), 7.89-7.72(m, 3H, ArH), 7.32(t, J= 7.8Hz,1H,ArH),7.09-6.85(m,5H,ArH),4.28(s,2H,CH ₂ ),3.95(s,2H,CH ₂ ),3.50(s,2H,CH ₂ ),3.18 (s,2H,CH ₂ ), 3.04(s,2H,CH ₂ ); HR-MS(EI): m/z theoretical value C ₂₆ H ₂₁ F ₃ N ₄ O ₂ [M] ⁺ ,478.1617; actual value 478.1613.

Example 25 N-(2-fluorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Preparation of Benzoyl}piperazine (I-20)

Except that 1-cyclopropanylpiperazine is replaced with 1-(2-fluorophenyl)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 128 mg of white solid (I-20) , and the yield was 42.3%. Mp: 215-217°C (EtOAc); purity: 99%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.07(s, 1H, NH), 8.47(d, J=7.4Hz, 1H, ArH), 7.89-7.74(m, 3H, ArH), 7.32(t, J= 7.7Hz, 1H, ArH), 7.22(s, 1H, ArH), 7.09(dd, J=13.9, 5.5Hz, 3H, ArH), 6.93(t, J=9.3Hz, 1H, ArH), 4.29(s ,2H,CH ₂ ), 4.03(s,2H,CH ₂ ), 3.57(s,2H,CH ₂ ), 3.24(s,2H,CH ₂ ), 3.11(s,2H,CH ₂ ); HR-MS (EI): m/z theoretical value C ₂₆ H ₂₁ F ₃ N ₄ O ₂ [M] ⁺ , 478.1617; actual value 478.1619.

Example 26 N-(4-methylphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Preparation of Fluorobenzoyl}piperazine (I-21)

Except that 1-cyclopropanylpiperazine is changed into 1-(4-methylphenyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 92 mg of white solid (I-21 ), the yield is 30.7%. Mp: 223-224°C (EtOAc); purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.00(s, 1H, NH), 8.47(d, J=7.4Hz, 1H, ArH), 7.88-7.74(m, 3H, ArH), 7.33(t, J= 7.6Hz,1H,ArH),7.14(s,3H,ArH),6.93(t,J=9.3Hz,2H,ArH),4.28(s,2H,CH ₂ ),4.01(s,2H,CH ₂ ) , 3.54 (s, 2H, CH ₂ ), 3.20 (d, J=53.4Hz, 4H, CH ₂ ), 2.31 (s, 3H, CH ₃ ); HR-MS (EI): m/z theoretical value C ₂₇ H ₂₄ F ₂ N ₄ O ₂ [M] ⁺ , 474.1867; actual value 474.1861.

Example 27 N-(4-bromophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Preparation of Benzoyl}piperazine (I-22)

Except that 1-cyclopropanylpiperazine is replaced by 1-(4-bromophenyl)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 164 mg of white solid (I-22) , and the yield was 48.1%. Mp: 243°C (EtOAc); Purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.16(s, 1H, NH), 8.46(d, J=7.5Hz, 1H, ArH), 7.89-7.75(m, 3H, ArH), 7.43(d, J= 8.6Hz, 2H, ArH), 7.34(t, J=7.7Hz, 1H, ArH), 7.03(s, 2H, ArH), 6.93(t, J=9.3Hz, 1H, ArH), 4.29(s, 2H ,CH ₂ ), 4.04(s,2H,CH ₂ ), 3.60(s,2H,CH ₂ ), 3.29(s,2H,CH ₂ ), 3.16(s,2H,CH ₂ ); HR-MS (EI ): m/z theoretical value C ₂₆ H ₂₁ BrF ₂ N ₄ O ₂ [M] ⁺ , 538.0816; actual value 538.0818.

Example 28 N-(3-chlorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Preparation of Benzoyl}piperazine (I-23)

Except that 1-cyclopropanylpiperazine is replaced by 1-(3-chlorophenyl)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 190 mg of white solid (I-23) , and the yield was 60.7%. Mp: 199-200°C (EtOAc); purity: 99%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 9.98(s, 1H, NH), 8.47(d, J=7.4Hz, 1H, ArH), 7.88-7.75(m, 3H, ArH), 7.32(t, J= 7.9Hz, 1H, ArH), 7.20(t, J=8.3Hz, 1H, ArH), 6.92(dd, J=17.5, 7.9Hz, 3H, ArH), 6.82(d, J=8.0Hz, 1H, ArH ),4.29(s,2H,CH ₂ ),3.92(s,2H,CH ₂ ),3.47(s,2H,CH ₂ ),3.26(s,2H,CH ₂ ),3.12(s,2H,CH ₂ ); HR-MS (EI): m/z theoretical value C ₂₆ H ₂₁ ClF ₂ N ₄ O ₂ [M] ⁺ , 494.1321; actual value 494.1320.

Example 29 N-(5-chloro-2-methylphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2 , Preparation of 4-difluorobenzoyl}piperazine (I-24)

Except that 1-cyclopropamoylpiperazine is replaced by 1-(5-chloro-2-methylphenyl)piperazine, the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 160 mg of white solid (I-24), the yield was 49.7%. Mp: 244-245°C (EtOAc); purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.16(s, 1H, NH), 8.47(d, J=7.3Hz, 1H, ArH), 7.89-7.74(m, 3H, ArH), 7.32(t, J= 7.8Hz,1H,ArH),7.11(d,J=8.1Hz,1H,ArH),7.05-6.87(m,3H,ArH),4.29(s,2H,CH ₂ ),3.95(s,2H,CH ₂ ), 3.50(s, 2H, CH ₂ ), 2.97(t, J=4.6Hz, 2H, CH ₂ ), 2.87(s, 2H, CH ₂ ), 2.29(s, 3H, CH ₃ ); HR- MS(EI): m/z calc. C ₂₇ H ₂₃ ClF ₂ N ₄ O ₂ [M] ⁺ , 508.1478; Actual 508.1483.

Example 30 N-(4-chlorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Preparation of Benzoyl}piperazine (I-25)

Except that 1-cyclopropanylpiperazine is replaced with 1-(4-chlorophenyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 130 mg of white solid (I-25) , and the yield was 41.5%. Mp: 237°C (EtOAc); Purity: 99%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.04(s, 1H, NH), 8.46(d, J=7.6Hz, 1H, ArH), 7.88-7.75(m, 3H, ArH), 7.04(s, 2H, ArH), 7.38-7.27 (m, 3H, ArH), 6.93 (t, J=9.3Hz, 1H, ArH), 4.28 (s, 2H, CH ₂ ), 4.02 (s, 2H, CH ₂ ), 3.57 ( s,2H,CH ₂ ), 3.27(s,2H,CH ₂ ), 3.13(s,2H,CH ₂ ); HR-MS(EI): m/z theoretical value C ₂₆ H ₂₁ ClF ₂ N ₄ O ₂ [M] ⁺ ,494.1321; actual value 494.1319.

Example 31 N-(4-cyanophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Preparation of Fluorobenzoyl}piperazine (I-26)

Except that 1-cyclopropanylpiperazine is changed into 1-(4-cyanophenyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 150 mg of white solid (I-26 ), the yield was 48.9%. Mp: 236-237°C (EtOAc); purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.44(s, 1H, NH), 8.47(d, J=7.5Hz, 1H, ArH), 7.92-7.73(m, 3H, ArH), 7.52(d, J= 8.8Hz, 2H, ArH), 7.34(t, J=7.7Hz, 1H, ArH), 6.99-6.83(m, 3H, ArH), 4.30(s, 2H, CH ₂ ), 3.92(s, 2H, CH ₂ ), 3.49(s,2H,CH ₂ ), 3.41(s,2H,CH ₂ ), 3.28(s,2H,CH ₂ ); HR-MS(EI): m/z theoretical value C ₂₇ H ₂₁ F ₂ N ₅ O ₂ [M] ⁺ ,485.1663; actual value 485.1664.

Example 32 N-(4-methoxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Preparation of Difluorobenzoyl}piperazine (I-27)

Except that 1-cyclopropanylpiperazine is replaced with 1-(4-methoxyphenyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 59 mg of white solid (I- 27), the yield was 18.9%. Mp: 190-192°C (EtOAc); purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 9.88 (s, 1H, NH), 8.47 (d, J = 7.8Hz, 1H, ArH), 7.81 (dt, J = 8.1, 7.0Hz, 4H, ArH), 7.33 (t,J=7.6Hz,1H,ArH),6.99–6.81(m,4H,ArH),4.28(s,2H,CH ₂ ),3.97(s,2H,CH ₂ ),3.79(s,3H, CH ₃ ), 3.50(s,2H,CH ₂ ), 3.16(s,4H,CH ₂ ); HR-MS(EI): m/z theoretical value C ₂₇ H ₂₄ F ₂ N ₄ O ₃ [M] ⁺ ,490.1816; actual value 490.1815.

Example 33 N-(2-methoxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Preparation of Difluorobenzoyl}piperazine (I-28)

Except that 1-cyclopropanylpiperazine is changed into 1-(2-methoxyphenyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 70.6 mg of white solid (I- 28), the yield was 22.7%. Mp: 184-185°C (EtOAc); purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.02(s, 1H, NH), 8.47(d, J=7.6Hz, 1H, ArH), 7.87-7.75(m, 3H, ArH), 7.32(t, J= 7.7Hz,1H,ArH),7.07(s,1H,ArH),7.00-6.80(m,4H,ArH),4.28(s,2H,CH ₂ ),3.96(s,2H,CH ₂ ),3.87( s,3H,CH ₃ ), 3.51(s,2H,CH ₂ ), 3.08(d,J=49.0Hz,4H,CH ₂ ); HR-MS(EI): m/z theoretical value C ₂₇ H ₂₄ F ₂ N ₄ O ₃ [M] ⁺ ,490.1816; actual value 490.1818.

Example 34 N-(5-chloro-piperidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2 , Preparation of 4-difluorobenzoyl}piperazine (I-29)

Except that 1-cyclopropamoylpiperazine is replaced with 1-(5-chloro-2-piperidinyl) piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 36.7 mg of white solid ( I-29), the yield was 11.7%. Mp: 230-231°C (EtOAc); purity: 96%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 9.93(s, 1H, NH), 8.47(d, J=7.7Hz, 1H, ArH), 8.13(d, J=2.2Hz, 1H, ArH), 7.82(dt ,J=11.1,6.6Hz,3H,ArH),7.48(d,J=9.1Hz,1H,ArH),7.31(t,J=7.7Hz,1H,ArH),6.93(t,J=9.4Hz, 1H,ArH),6.61(d,J=9.1Hz,1H,ArH),4.28(s,2H,CH ₂ ),3.86(s,2H,CH ₂ ),3.56(d,J=22.6Hz,4H, CH ₂ ), 3.41 (s, 2H, CH ₂ ); HR-MS (EI): m/z calc. C ₂₅ H ₂₀ ClF ₂ N ₅ O ₂ [M] ⁺ , 495.1274; Actual 495.1270.

Example 35 N-(3-chloro-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, Preparation of 4-difluorobenzoyl}piperazine (I-30)

Except that 1-cyclopropanylpiperazine is changed into 1-(3-chloro-2-pyridine) piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 66.7 mg of white solid (I- 30), the yield was 21.3%. Mp: 249°C (EtOAc); Purity: 95%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.17 (s, 1H, NH), 8.47 (d, J = 7.6Hz, 1H, ArH), 8.21 (d, J = 4.6Hz, 1H, ArH), 7.89-7.74 (m,3H,ArH),7.63(d,J=7.7Hz,1H,ArH),7.34(t,J=7.7Hz,1H,ArH),6.96-6.87(m,2H,ArH),4.29(s , 2H, CH ₂ ), 3.97-3.87 (m, 2H, CH ₂ ), 3.49-3.36 (m, 4H, CH ₂ ), 3.32 (d, J=4.1Hz, 2H, CH ₂ ); HR-MS ( EI): m/z theoretical value C ₂₅ H ₂₀ ClF ₂ N ₅ O ₂ [M] ⁺ , 495.1274; actual value 495.1281.

Example 36 N-Benzyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Oxazine (I-31) and N-benzyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzene Preparation of formyl}piperazine sulfate (I-31-H ₂ SO ₄ )

Except that 1-cyclopropamoylpiperazine is replaced by 1-benzylpiperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 110 mg of white solid (I-31), and the yield is 36.7 %. Mp: 92-94°C (CH ₂ Cl ₂ ); purity: 96%. I-31: ¹ HNMR (400MHz, CDCl ₃ ) δ: 9.97 (s, 1H, NH), 8.46 (d, J = 7.3Hz, 1H, ArH), 7.80 (dt, J = 18.4, 6.9Hz, 3H, ArH), 7.30(d, J=14.7Hz, 5H, ArH), 7.24(s, 1H, ArH), 6.89(t, J=9.3Hz, 1H, ArH), 4.26(s, 2H, CH ₂ ), 3.76(s,2H,CH ₂ ), 3.54(s,2H,CH ₂ ), 3.29(s,2H,CH ₂ ), 2.51(s,2H,CH ₂ ), 2.36(s,2H,CH ₂ ); HR-MS (EI): m/z theoretical value C ₂₇ H ₂₄ F ₂ N ₄ O ₂ [M] ⁺ , 474.1867; actual value 474.1863.

Dissolve 112 mg of I-31 in 2 ml of ethanol, add 13 microliters of concentrated sulfuric acid, react at room temperature for 20 minutes, evaporate the solvent to dryness, add 6 ml of ethyl acetate to the residue, perform supernatant treatment, suction filter, and place on the filter membrane The solid was dissolved in methanol again, and the solvent was evaporated to dryness to obtain 79 mg of white solid (I-31-H ₂ SO ₄ ), with a yield of 58.4%. Mp: 161-165°C; purity: 92%. I-31-H ₂ SO ₄ : ¹ HNMR (400MHz, DMSO) δ: 12.55(s, 1H), 9.83(s, 1H), 8.29(d, J=7.8Hz, 1H), 7.94(ddd, J= 31.0, 14.3, 7.6Hz, 3H), 7.58-7.30(m, 7H), 4.54(s, 2H), 4.36(s, 4H), 3.30-2.93(m, 6H).

Example 37 N-(pyridine-3-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Fluorobenzoyl}piperazine (I-32) and N-(pyridine-3-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl )Methyl]-2,4-difluorobenzoyl}piperazine sulfate (I-32-H ₂ SO ₄ ) preparation

Except that 1-cyclopropamoylpiperazine is replaced with 1-[(3-pyridyl)methyl]piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 75 mg of white solid (I- 32), the yield is 24.9%. Mp: 106-108°C (CH ₂ Cl ₂ ); purity: 99%. I-32: ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.13 (s, 1H, NH), 8.56 (s, 2H, ArH), 8.46 (d, J=7.4Hz, 1H, ArH), 7.81 (dt, J=18.0,6.8Hz,4H,ArH),7.36-7.24(m,2H,ArH),6.90(t,J=9.4Hz,1H,ArH),4.27(s,2H,CH ₂ ),3.82(s ,2H,CH ₂ ), 3.63(s,2H,CH ₂ ), 3.35(s,2H,CH ₂ ), 2.59(s,2H,CH ₂ ), 2.44(s,2H,CH ₂ ); HR-MS (EI): m/z theoretical value C ₂₆ H ₂₃ F ₂ N ₅ O ₂ [M] ⁺ , 475.1820; actual value 475.1821.

Dissolve 110 mg of I-32 in 2 ml of ethanol, add 13 microliters of concentrated sulfuric acid, react at room temperature for 20 minutes, evaporate the solvent to dryness, add 6 ml of ethyl acetate to the residue, perform supernatant treatment, filter with suction, and place on the filter membrane The solid was dissolved in methanol again, and the solvent was evaporated to dryness to obtain 55 mg of white solid (I-32-H ₂ SO ₄ ), with a yield of 41.4%. Mp: 180～185°C (CH ₂ Cl ₂ ); purity: 94%. I-32-H ₂ SO ₄ : ¹ HNMR(400MHz,DMSO)δ:12.55(s,1H),9.97(s,1H),8.72(s,2H),8.29(d,J＝7.7Hz,1H) ,7.94(ddd,J=30.9,14.1,7.5Hz,4H),7.63(s,1H),7.52-7.33(m,2H),4.65-4.15(m,2H),4.36(s,4H),3.60 -2.90(m,6H).

Example 38 N-(5-methyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2, 4-Difluorobenzoyl}piperazine (I-33) and N-(5-methyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo Preparation of -phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine hydrochloride (I-33-HCl)

Except that 1-cyclopropanylpiperazine is replaced with 1-(5-methyl-2-pyridyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 35 mg of white solid ( I-33), the yield was 11.6%. Mp: 175-176°C (EtOAc); purity: 99%. I-33: ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.25(s, 1H), 8.47(d, J=7.5Hz, 1H), 8.03(s, 1H), 7.89-7.73(m, 3H), 7.46 -7.28(m,2H),6.92(t,J=9.4Hz,1H),6.62(d,J=8.3Hz,1H),4.29(s,2H),3.87(s,2H),3.53(d, J=15.8Hz, 4H), 3.42(s,2H), 2.22(s,3H); HR-MS(EI): m/z theoretical value C ₂₆ H ₂₃ F ₂ N ₅ O ₂ [M] ⁺ ,475.1820 ; Actual value 475.1819.

Dissolve 110 mg of I-33 in 4 ml of dichloromethane, feed HCl gas, ventilate and react for 5 minutes, evaporate the solvent to dryness, add 6 ml of methanol to the residue, and evaporate the solvent to obtain 109 mg of white solid (I- 33-HCl), the yield was 92.0%. Mp: 183-185°C; purity: 98%. I-33-HCl: ¹ HNMR (500MHz, DMSO) δ: 12.53(s, 1H), 8.28(d, J=7.8Hz, 1H), 8.06-7.81(m, 5H), 7.52-7.25(m, 3H ), 4.37(s,2H), 3.87-3.60(m,6H), 3.41(s,2H), 2.23(s,3H).

Example 39 N-(4-methyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2 , Preparation of 4-difluorobenzoyl}piperazine (I-34)

Except that 1-cyclopropamoylpiperazine is changed into 1-(4-methyl-2-pyridyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 50 mg of white solid ( I-34), the yield was 16.6%. Mp: 179～180°C (EtOAc); Purity: 99%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.10 (s, 1H, NH), 8.47 (d, J=7.9Hz, 1H, ArH), 8.07( s,1H,ArH),7.84(dd,J=16.9,9.3Hz,3H,ArH),7.34-7.28(m,1H,ArH),6.93(t,J=9.2Hz,1H,ArH),6.57( d,J=18.4Hz,2H,ArH),4.28(s,2H,CH ₂ ),3.88(s,2H,CH ₂ ),3.63(s,4H,CH ₂ ),3.44(s,2H,CH ₂ ), 2.32 (s, 3H, CH ₃ ); HR-MS (EI): m/z theoretical value C ₂₆ H ₂₃ F ₂ N ₅ O ₂ [M] ⁺ , 475.1820; actual value 475.1819.

Example 40 N-(5-iodo-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, Preparation of 4-difluorobenzoyl}piperazine (I-35)

Except that 1-cyclopropanylpiperazine is changed into 1-(5-iodo-2-pyridyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8, and 50 mg of white solid (I -35), the yield was 13.5%. Mp: 100-103°C (CH ₂ Cl ₂ ); purity: 99%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.46 (s, 1H, NH), 8.46 (d, J = 7.5Hz, 1H, ArH), 8.32 (d, J = 1.7Hz, 1H, ArH), 7.98-7.75 (m,4H,ArH),7.74-7.67(m,1H,ArH),7.32(t,J=7.7Hz,1H,ArH),6.93(t,J=9.3Hz,1H,ArH),4.29(s ,2H,CH ₂ ), 3.85(s,2H,CH ₂ ), 3.56(d,J=23.1Hz,4H,CH ₂ ), 3.40(s,2H,CH ₂ ); HR-MS(EI):m /z theoretical value C ₂₅ H ₂₀ F _2I N ₅ O ₂ [M] ⁺ , 587.0630; actual value 587.0629.

Example 41 N-(6-ethoxy-piperidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl] Preparation of -2,4-difluorobenzoyl}piperazine (I-36)

Except that 1-cyclopropamoylpiperazine is replaced by 1-(6-ethoxy-2-piperidinyl)piperazine, the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain a white solid 70.9 mg (I-36), the yield was 22.2%. Mp: 186°C (EtOAc); Purity: 99%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.11(s, 1H, NH), 8.47(d, J=7.6Hz, 1H, ArH), 7.88-7.74(m, 3H, ArH), 7.42(t, J= 8.0Hz, 1H, ArH), 7.32(t, J=7.7Hz, 1H, ArH), 6.92(t, J=9.3Hz, 1H, ArH), 6.15(dd, J=22.1, 8.0Hz, 2H, ArH ), 4.26 (dd, J=13.5, 6.3Hz, 4H, CH ₂ ), 3.86 (s, 2H, CH ₂ ), 3.58 (s, 2H, CH ₂ ), 3.50 (s, 2H, CH ₂ ), 3.41 (s, 2H, CH ₂ ), 1.36 (t, J=7.0Hz, 3H, CH ₃ ); HR-MS(EI): m/z theoretical value C ₂₇ H ₂₅ F ₂ N ₅ O ₃ [M] ⁺ ,505.1925; actual value 505.1923.

Example 42 N-(6-methoxy-piperidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl] Preparation of -2,4-difluorobenzoyl}piperazine (I-37)

Except that 1-cyclopropamoylpiperazine is replaced by 1-(6-methoxy-2-piperidinyl)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8, and a white solid 80.6 mg (I-37), the yield was 25.9%. Mp: 160-161°C (EtOAc); purity: 100%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.05(s, 1H, NH), 8.47(d, J=7.7Hz, 1H, ArH), 7.88-7.74(m, 3H, ArH), 7.43(t, J= 7.9Hz, 1H, ArH), 7.32(t, J=7.7Hz, 1H, ArH), 6.92(t, J=9.3Hz, 1H, ArH), 6.16(dd, J=17.9, 7.9Hz, 2H, ArH ),4.28(s,2H,CH ₂ ),3.85(s,5H,CH ₂ ,CH ₃ ),3.59(s,2H,CH ₂ ),3.51(s,2H,CH ₂ ),3.42(s,2H , CH ₂ ); HR-MS (EI): m/z theoretical for C ₂₆ H ₂₃ F ₂ N ₅ O ₃ [M] ⁺ , 491.1769; actual 491.1767.

Example 43 N-(4-hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Benzoyl}piperazine (I-38)N-(4-hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl Preparation of Lithium Salt of ]-2,4-difluorobenzoyl}piperazine (I-38-Li)

Except that 1-cyclopropamoylpiperazine is replaced by 1-(4-hydroxyphenyl)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 120 mg of white solid (I-38) , and the yield was 39.8%. Mp: 249-251°C (EtOAc); purity: 96%. I-38: ¹ HNMR (400MHz, DMSO) δ: 12.53 (s, 1H, NH), 8.90 (s, 1H, ArOH), 8.28 (d, J＝7.8Hz, 1H, ArH), 8.02 (d, J =7.6Hz,1H,ArH),7.99-7.93(m,1H,ArH),7.90-7.85(m,1H,ArH),7.44-7.35(m,2H,ArH),6.79(d,J=8.9Hz ,2H,ArH),6.66(d,J=8.8Hz,2H,ArH),4.37(s,2H,CH ₂ ),3.73(s,2H,CH ₂ ),3.33-3.27(s,2H,CH ₂ ), 2.98(s,2H,CH ₂ ), 2.84(s,2H,CH ₂ ); HR-MS(EI):m/z theoretical value C ₂₆ H ₂₂ F ₂ N ₄ O ₃ [M] ⁺ ,476.1660 ; Actual value 476.1658.

Dissolve 100 mg of I-38 in a mixed solution of 15 ml of dichloromethane and 10 ml of methanol, add 9.4 mg of lithium hydroxide monohydrate, react at room temperature for 3 hours, evaporate the solvent to dryness, add 6 ml of ether to the residue, and After treatment, suction filtration and drying, 81.2 mg of (I-38-Li) was obtained as a white solid, with a yield of 80.2%. Mp: 328-330°C; purity: 94%. I-38-Li: ¹ HNMR (400MHz, DMSO) δ: 8.22 (d, J = 7.6Hz, 1H), 7.79 (dd, J = 29.0, 9.3Hz, 3H), 7.43-7.23 (m, 2H), 6.66(d,J=8.5Hz,2H),6.50(d,J=8.3Hz,2H),4.33(s,2H),3.69(s,2H),3.27(s,2H),2.89(s,2H ), 2.73(s,2H).

Example 44 N-(3-tert-butylcarbamoyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl Preparation of ]-2,4-difluorobenzoyl}piperazine (I-39)

Except that 1-cyclopropylcarbamoylpiperazine is replaced with 1-(3-tert-butylcarbamoyl-pyridin-2-yl)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain white Solid 120 mg (I-39), yield 22.9%. Mp: 120～122°C (CH ₂ Cl ₂ ); purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.08 (s, 1H, NH), 8.47 (d, J = 7.9Hz, 1H, ArH), 8.36 (dd, J = 4.8, 1.8Hz, 1H, ArH), 8.23 (dd, J=7.6,1.8Hz,1H,ArH),8.14(s,1H,ArH),7.88-7.76(m,3H,ArH),7.35(t,J=7.7Hz,1H,ArH),7.10 (dd,J=7.6,4.8Hz,1H,ArH),6.93(t,J=9.3Hz,1H,ArH),4.29(s,2H,CH ₂ ),3.92(s,2H,CH ₂ ),3.47 (s,2H,CH ₂ ), 3.27(s,2H,CH ₂ ), 3.21(s,2H,CH ₂ ), 1.46(s,9H,CH ₃ ); HR-MS(EI): m/z theoretical Value C ₃₀ H ₃₀ F ₂ N ₆ O ₃ [M] ⁺ ,560.2347; actual value 560.2337.

Example 45 N-(4-methylbenzoyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Preparation of Difluorobenzoyl}piperazine (I-40)

Except that 1-cyclopropamoylpiperazine is changed into 1-(4-methylbenzoyl)piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 97 mg of white solid (I- 40), the yield was 30.5%. Mp: 133-135°C (CH ₂ Cl ₂ ); purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 9.96 (s, 1H, NH), 8.47 (d, J = 8.1Hz, 1H, ArH), 7.87–7.73 (m, 3H, ArH), 7.29 (dd, J = 7.5,5.5Hz,3H,ArH),7.22(d,J＝7.7Hz,2H,ArH),6.91(s,1H,ArH),4.28(s,2H,CH ₂ ),3.87-3.45(m,6H ,CH ₂ ), 3.32(s,2H,CH ₂ ), 2.38(s,3H,CH ₃ ); HR-MS(EI): m/z theoretical value C ₂₈ H ₂₄ F ₂ N ₄ O ₃ [M] ⁺ ,502.1816; actual value 502.1817.

Example 46 N-(2-Hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Benzoyl}piperazine (I-41) and N-(2-hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methanol Preparation of lithium]-2,4-difluorobenzoyl}piperazine (I-41-Li)

Except that 1-cyclopropamoylpiperazine is replaced by 1-(2-hydroxyphenyl)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 122 mg of white solid (I-41) , and the yield was 40.5%. Mp: 252～253°C (CH ₂ Cl ₂ ); purity: 96%. I-41: ¹ HNMR (400MHz, DMSO) δ: 12.53 (s, 1H, NH), 9.01 (s, 1H, ArOH), 8.28 (d, J = 7.6Hz, 1H, ArH), 7.94 (ddd, J =34.6,19.4,7.6Hz,3H,ArH),7.41(dd,J=16.8,8.8Hz,2H,ArH),6.91-6.69(m,4H,ArH),4.37(s,2H,CH ₂ ), 3.76(s,2H,CH ₂ ), 3.38-3.34(s,2H,CH ₂ ), 2.94(s,2H,CH ₂ ), 2.81(s,2H,CH ₂ ); HR-MS(EI):m /z theoretical value C ₂₆ H ₂₂ F ₂ N ₄ O ₃ [M] ⁺ , 476.1660; actual value 476.1660.

Dissolve 100 mg of I-44 in a mixed solution of 15 ml of dichloromethane and 10 ml of methanol, add 9.4 mg of lithium hydroxide monohydrate, react at room temperature for 3 hours, evaporate the solvent to dryness, add 6 ml of ether to the residue, and After treatment, suction filtration and drying, 75 mg of (I-41-Li) was obtained as a white solid with a yield of 74.1%. Mp: 330-335°C; purity: 96%. I-41-Li: ¹ HNMR (400MHz, DMSO) δ: 8.24 (d, J = 7.6Hz, 1H), 7.84 (ddd, J = 29.0, 17.2, 7.4Hz, 3H), 7.44-7.24 (m, 2H ),6.71-6.53(m,2H),6.43(d,J＝7.4Hz,1H),6.18(s,1H),4.34(s,2H),3.72(s,2H),3.30(s,2H) ,2.92(s,2H),2.76(s,2H).

Example 47 N-(1,4-benzodioxane-2-formyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methanol Preparation of ]-2,4-difluorobenzoyl}piperazine (I-42)

Except that 1-cyclopropamoylpiperazine is replaced by 1-[(1,4-benzodioxan-2-yl)carbonyl]piperazine, the rest of the required raw materials, reagents and preparation methods are the same as in Example 8 , 67 mg of (I-42) was obtained as a white solid with a yield of 19.4%. Mp: 129～131°C (CH ₂ Cl ₂ ); purity: 96%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.09 (s, 1H, NH), 8.47 (d, J = 7.0Hz, 1H, ArH), 7.81 (dt, J = 13.4, 6.8Hz, 3H, ArH), 7.39 -7.28(m,1H,ArH),7.04-6.70(m,5H,ArH),4.89-4.73(m,1H,CH),4.50(dd,J=11.9,2.3Hz,1H,CH ₂ ),4.36 (d, J=7.1Hz, 1H, CH ₂ ), 4.29 (s, 2H, CH ₂ ), 4.07-3.77 (m, 3H, CH ₂ ), 3.74-3.26 (m, 5H, CH ₂ ); HR- MS(EI): m/z calc. C ₂₉ H ₂₄ F ₂ N ₄ O ₅ [M] ⁺ , 546.1715; Actual 546.1714.

Example 48 N-(3,4-methylenedioxybenzyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl] -2,4-difluorobenzoyl}piperazine (I-43) and N-(3,4-methylenedioxybenzyl)-N'-{5-[(3,4-dihydro Preparation of -4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine sulfate (I-43-H ₂ SO ₄ )

Except that 1-cyclopropanoylpiperazine is replaced by 1-(3,4-methylenedioxybenzyl)piperazine, the rest of the required raw materials, reagents and preparation methods are the same as in Example 8 to obtain a white solid 70 mg (I-43), yield 21.3%. Mp: 201-202°C (EtOAc); purity: 96%. I-43: ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.18 (s, 1H, NH), 8.46 (d, J = 7.6Hz, 1H, ArH), 7.89-7.70 (m, 3H, ArH), 7.28 ( s,1H,ArH),7.26-7.26(m,1H,ArH),7.24(s,1H,ArH),6.89(t,J=9.2Hz,2H,ArH),6.75(s,2H,ArH), 5.95(s,2H,CH ₂ ),4.27(s,2H,CH ₂ ),3.79(s,2H,CH ₂ ),3.50(s,2H,CH ₂ ),3.33(s,2H,CH ₂ ), 2.47 (d, J = 58.8 Hz, 4H, CH ₂ ); HR-MS (ESI): m/z theoretical for C ₂₈ H ₂₅ F ₂ N ₄ O ₄ [M+H] ⁺ , 519.1844; actual 519.1843.

Dissolve 110 mg of I-43 in 2 ml of ethanol, add 12 microliters of concentrated sulfuric acid, react at room temperature for 20 minutes, evaporate the solvent to dryness, add 6 ml of ethyl acetate to the residue, perform supernatant treatment, suction filter, and place on the filter membrane The solid was dissolved in methanol again, and the solvent was evaporated to dryness to obtain 93 mg of white solid (I-43-H ₂ SO ₄ ), with a yield of 71.5%. Mp: 167-168°C (EtOAc); purity: 98%. I-43-H ₂ SO ₄ : ¹ HNMR (400MHz, DMSO) δ: 12.54(s, 1H), 9.71(s, 1H), 8.29(d, J=7.8Hz, 1H), 8.03-7.92(m, 2H), 7.88(t, J=7.5Hz, 1H), 7.51-7.36(m, 2H), 7.09-6.93(m, 3H), 6.08(s, 2H), 4.36(s, 2H), 4.26(s , 2H), 3.57(d, J=13.9Hz, 1H), 3.40(s, 1H), 3.34(s, 1H), 3.23(s, 1H), 3.16-2.83(m, 4H).

Example 49 N-(thiazol-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluoro Preparation of Benzoyl}piperazine (I-44)

Except that 1-cyclopropanylpiperazine is replaced with 1-(2-thiazole)piperazine, all other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 54.7 mg of white solid (I-44). The rate is 18.5%. Mp: 209-210°C (EtOAc); purity: 96%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.13 (s, 1H, NH), 8.52-8.43 (m, 1H, ArH), 7.82 (ddd, J=17.7, 10.5, 6.3Hz, 3H, ArH), 7.32 ( t, J=7.7Hz, 1Hv), 7.22(d, J=3.7Hz, 1H, ArH), 6.93(t, J=9.4Hz, 1H, ArH), 6.63(d, J=3.7Hz, 1H, ArH ), 4.28(s,2H,CH ₂ ), 3.90(s,2H,CH ₂ ), 3.54(d,J=17.9Hz,4H,CH ₂ ), 3.44(s,2H,CH ₂ ); HR-MS (EI): m/z theoretical value C ₂₃ H ₁₉ F ₂ N ₅ O ₂ S[M] ⁺ , 467.1228; actual value 467.1212.

Example 50 N-(1,2-Benzisothiazol-3-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl] Preparation of -2,4-difluorobenzoyl}piperazine (I-45)

Except that 1-cyclopropanylpiperazine is replaced by 1-[3-(1,2-benzisothiazole)]piperazine, the rest of the required raw materials, reagents and preparation methods are the same as in Example 8 to obtain a white solid 85.3 mg (I-45), yield 26.1%. Mp: 219-220°C (EtOAc); purity: 98%. ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.09(s, 1H, NH), 8.47(d, J=7.8Hz, 1H, ArH), 7.91-7.75(m, 5H, ArH), 7.53-7.45(m, 1H, ArH), 7.42-7.30 (m, 2H, ArH), 6.93 (t, J=9.3Hz, 1H, ArH), 4.29 (s, 2H, CH ₂ ), 3.99 (s, 2H, CH ₂ ), 3.64-3.57(m,2H,CH ₂ ), 3.57-3.45(m,4H,CH ₂ ); HR-MS(EI):m/z theoretical value C ₂₇ H ₂₁ F ₂ N ₅ O ₂ S[M] ⁺ ,517.1384; actual value 517.1385.

Example 51 N-(thiazole-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Fluorobenzoyl}piperazine (I-46) and N-(thiazole-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl )Methyl]-2,4-difluorobenzoyl}piperazine sulfate (I-46-H ₂ SO ₄ ) preparation

Except that 1-cyclopropanylpiperazine is changed into 1-[(2-thiazole) methyl]piperazine, all the other required raw materials, reagents and preparation methods are the same as in Example 8 to obtain 64.7 mg of white solid (I- 46), the yield is 21.2%. Mp: 109-110°C (CH ₂ Cl ₂ ); purity: 98%. I-46: ¹ HNMR (400MHz, CDCl ₃ ) δ: 10.16(s, 1H), 8.47(d, J=7.1Hz, 1H), 7.87-7.71(m, 4H), 7.37-7.27(m, 2H) ,6.89(t,J=9.3Hz,1H),4.27(s,2H),3.95(s,2H),3.82(s,2H),3.36(s,2H),2.63(d,J=50.9Hz, 4H); HR-MS (EI): m/z theoretical for C ₂₄ H ₂₁ F ₂ N ₅ O ₂ S[M] ⁺ , 481.1384; actual 481.1382.

Dissolve 110 mg of I-46 in 2 ml of ethanol, add 12 microliters of concentrated sulfuric acid, react at room temperature for 20 minutes, evaporate the solvent to dryness, add 6 ml of ethyl acetate to the residue, supernatant treatment, suction filtration, filter membrane The above solid was dissolved in methanol again, and the solvent was evaporated to dryness to obtain 120 mg of white solid (I-46-H ₂ SO ₄ ), with a yield of 90.6%. Mp: 178-180°C; purity: 96%. I-46-H ₂ SO ₄ : ¹ HNMR(500MHz,DMSO)δ:12.49(s,1H),8.27-8.21(m,1H),7.99-7.78(m,5H),7.47-7.29(m,2H ), 4.67 (s, 2H), 4.57-4.17 (m, 6H), 3.32-3.03 (m, 4H).

Example 52 N-(5-methyl-pyridine-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]- 2,4-Difluorobenzoyl}piperazine (I-47) and N-(5-methyl-pyridine-2-methyl)-N'-{5-[(3,4-dihydro-4 Preparation of -oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine hydrochloride (I-47-HCl)

Except that 1-cyclopropamoylpiperazine is replaced by 1-[2-(5-methyl)pyridylmethyl]piperazine, the rest of the required raw materials, reagents and preparation methods are the same as in Example 8 to obtain a white solid 54.7 mg (I-47), the yield was 18.5%. Mp: 195-196°C (CH ₂ Cl ₂ ); purity: 98%. I-47: ¹ HNMR (500MHz, CDCl ₃ ) δ: 10.21 (s, 1H, NH), 8.47 (dd, J=22.3, 15.0Hz, 2H, ArH), 7.89-7.74 (m, 3H, ArH), 7.50(d, J=6.9Hz, 1H, ArH), 7.30(d, J=8.3Hz, 2H, ArH), 6.90(t, J=9.3Hz, 1H, ArH), 4.28(s, 2H, CH ₂ ),3.80(s,2H,CH ₂ ),3.68(s,2H,CH ₂ ),3.32(s,2H,CH ₂ ),2.58(s,2H,CH ₂ ),2.45(s,2H,CH ₂ ), 2.34 (s, 3H, CH ₃ ); HR-MS (ESI): m/z theoretical for C ₂₇ H ₂₅ F ₂ N ₅ O ₂ Na[M+Na] ⁺ , 512.1874; actual 512.1904.

114 milligrams of I-47 were dissolved in 4 milliliters of dichloromethane, HCl gas was passed through, and the ventilated reaction was carried out for 5 minutes, the solvent was evaporated to dryness, 6 milliliters of methanol was added to the residue, and the solvent was evaporated to dryness to obtain 118 mg of white solid (I- 47-HCl), the yield was 96.3%. Mp: 174°C; Purity: 95%. I-47-HCl: ¹ HNMR (400MHz, DMSO) δ: 12.55(s, 1H), 8.56(s, 1H), 8.29(d, J=7.7Hz, 1H), 8.03-7.92(m, 2H), 7.86(dd, J=14.6,7.5Hz,2H),7.65(d,J=7.9Hz,1H),7.44(dd,J=12.1,7.6Hz,2H),4.45(s,2H),4.36(s ,2H), 3.92(s,2H), 3.56(s,2H), 3.30(s,2H), 3.16(d,J=6.6Hz,2H), 2.37(s,3H).

Example 53 Inhibition experiment of poly ADP-ribose polymerase-1 (PARP-1)

experimental method:

Enzyme-Linked Immunosorbent Assay (Enzyme-Linked Immunosorbent Assay, ELISA) (refer to the ELISA method described in the literature published by Decker P.; reference: Decker P. et al. An improved nonisotopic test to screen a large series of new inhibitor molecules of Poly (ADP-ribose) Polymerase activity for therapeutic applications. Clinical Cancer Research, 5: 1169-1172, 1999.). The principle is to coat the substrate histone on the adsorption 96-well plate, add PARP-1 recombinase, substrate NAD ⁺ , and activated DNA to make the PARP-1 recombinase enzymatically react, so that the histone produces the product PAR (poly ADP-ribose), and then add anti-PAR (anti-PAR) antibody to detect the intensity of the product PAR on the coated histone on the 96-well plate, thereby reflecting the activity of PARP recombinase.

The specific method is as follows:

1. Histones are recognized as important substrates of PARP-1. Coat it with PBS (10mM sodium phosphate buffer, 150mM NaCl, pH 7.2-7.4) without potassium ions to coat a 96-well microtiter plate, place it on a shaker at 37°C overnight; discard the liquid in the well. The plate was washed 5 times with 120 μL/well of PBS-T (PBS containing 0.1% Tween-20). Dry in an oven at 37°C.

2. Add NAD ⁺ (purchased from Sigma, final concentration 8μM/well), DNA (Synthetics, 100ng/well), PARP-1 (Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 10ng/well) (use PARP-1 enzyme reaction buffer solution, the buffer solution contains 50mM Tris (trishydroxymethylaminomethane), 2mM MgCl ₂ , pH8.0), and 10μL of a certain dilution concentration (final concentration starting from 10μM, 10-fold dilution of 6 gradients) is added to each well. For each compound, two replicates were set up for each concentration. The reaction system was 100 μL/well (supplemented with the aforementioned PARP-1 enzyme reaction buffer), and the shaker was reacted at 37°C for 1 h. Blank control wells (without enzyme) and positive control wells ( A positive inhibitor, AZD2281 (trade name Olaparib, purchased from LC Laboratories), and a negative control (without inhibitor) were added. Start the reaction, and place it on a shaker at 37°C for 1 hour.

3. Wash the plate three times with PBS-T, add the primary antibody anti-PAR MAb10H (purchased from Trevigen, 1:4000, diluted with PBS-T containing 5 μg/mL BSA), 100 μL/well, and incubate at 37 ° C for 1 h ;

4. Wash the plate three times with PBS-T, add peroxidase-labeled secondary antibody (anti-rabbit antibody) (purchased from Jackson ImmunoResearch, 1:2000, diluted with PBS-T containing 5 μg/mL BSA), 100 μL/ Well, react on a shaker at 37°C for 30 minutes;

5. Add 2 mg/mL of OPD (o-phenylenediamine hydrochloride) chromogenic solution 100 μL/well (diluted with 0.1M citric acid-sodium citrate buffer (pH=5.4) containing 0.03% H ₂ O ₂ ) , react in the dark at 25°C for 15 minutes (ultrasound is required for OPD dissolution, and the chromogenic solution must be prepared and used immediately).

6. Stop the reaction with 50 μL/well of 2M H ₂ SO ₄ , read with a Molecular Devices microplate reader (purchased from Molecular Instruments Corporation (MDC), USA, model SpectraMax 190 Microplate Reader (90V to 240V)), and measure the OD value at a wavelength of 490 nm. The _IC50 value was calculated according to the microplate reader's own software SofeMax Pro.

Experimental results:

Table 1 shows the inhibitory activity data of the compounds of the present invention on PARP-1. The results show that all compounds of the present invention have strong inhibitory activity on PARP-1, among which there are 10 active compounds with half effective inhibitory concentration IC _{50 ≤ 10nM, and 14 active compounds with half effective inhibitory concentration 10nM<IC 50 ≤20nM} , there are 24 active compounds with half effective inhibitory concentration 20nM<IC ₅₀ ≤100nM. Compared with the positive control drug AZD2281, 28 compounds of the present invention have stronger PARP-1 inhibitory activity than AZD2281. This fully demonstrates that the compound of the present invention is a class of potent PARP-1 inhibitors.

Table 1 Inhibitory activity on PARP-1 (IC ₅₀ , nM)

Example 54 Anti-tumor cell proliferation inhibition experiment of VC-8 (BRCA2 ^-/- ) and V79 (BRCA2 ^+/+ ) in vitro

experimental method:

VC-8 (BRCA2 ^-/- ) and V79 (BRCA2 ^+/+ ) (derived from Leiden University, or conventionally cultured according to literature methods). Cells in the logarithmic growth phase were inoculated into 96-well culture plates (Corning, 3599) at 90 μL/well, and cultured in a 37°C, 5% _CO2 conditional incubator (Thermo, 3111) for 24 hours until the cells adhered. 10 μL of the compound to be tested at a certain dilution concentration was added to each well, and three replicates were set for each concentration. A corresponding concentration of DMSO solvent control (as a negative control) and a cell-free zero well (as a blank control) were set up. After adding the drug, the cells were cultured at 37°C and 5% _CO for 72 hours, and 10 μL/well of CellCounting Kit-8 (purchased from DojinDo, CK04) solution was added to continue culturing for 4 hours. MAX190) to measure the OD450nm value, and calculate the _IC50 value according to the software SofeMax Pro that comes with the microplate reader.

Experimental results:

Table 2 shows the inhibitory activity data of the compound of the present invention on BRCA2 gene mutant tumor cell VC-8 (BRCA2 ^−/− ) and wild type tumor cell V79 (BRCA2 ^+/+ ). As a result, it was found that 21 compounds of the present invention showed strong anti-tumor cell VC-8 (BRCA2 ^-/- ) proliferation inhibitory activity (IC ₅₀ <1000nM) and good selectivity to tumor cell V79 (BRCA2 ^+/+ ) (SI _V79/VC-8 >10). Compared with the positive control drug AZD2281, 8 compounds of the present invention are better than AZD2281 in inhibiting the proliferation inhibitory activity of tumor cell VC-8 (BRCA2 ^−/− ) and in the selectivity to tumor cell V79 (BRCA2 ^+/+ ). On the one hand, this proves that the compound of the present invention is a class of compounds with strong anti-tumor activity. On the other hand, it also shows that the compound of the present invention is more sensitive to BRCA2 gene mutant tumor cells, and indirectly confirms that its anti-tumor effect is to target PARP- 1.

Table 2 Anti-tumor cell proliferation activity data of VC-8 (BRCA2 ^-/- ) and V79 (BRCA2 ^+/+ ) (IC ₅₀ , nM)

Example 55 Anti-tumor cell MDA-MB-436 (BRCA1 ^-/- ) proliferation inhibition experiment in vitro

Experimental method: Same as Example 54, BRCA1 gene mutant tumor cell MDA-MB-436 (BRCA1 ^−/− ) (purchased from ATCC, USA).

Experimental results:

Table 3 shows the inhibitory activity data of the compound of the present invention on BRCA1 gene mutant tumor cell MDA-MB-436 (BRCA1 ^−/− ). As a result, it was found that 13 compounds of the present invention showed strong anti-proliferation inhibitory activity of tumor cell MDA-MB-436 (BRCA1 ^−/− ) (IC ₅₀ <1000 nM). Compared with the positive control drug AZD2281, 11 compounds of the present invention have better inhibitory activity on tumor cell MDA-MB-436 (BRCA1 ^-/- ) proliferation than AZD2281. On the one hand, this proves that the compound of the present invention is a class of compounds with strong anti-tumor activity, and on the other hand, it also shows that the compound of the present invention is more sensitive to BRCA1 gene mutant tumor cells.

Table 3. Anti-proliferation activity data of tumor cell MDA-MB-436(BRCA1 ^-/- ) (IC ₅₀ , nM)

Example 56 Therapeutic effect on human breast cancer MDA-MB-436 (BRCA1 ^-/- ) subcutaneously transplanted tumors in nude mice

experimental method:

Human breast cancer MDA-MB-436 cell line was purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

The cell line was used to subcutaneously inoculate the right axilla of nude mice, the inoculated amount of cells was 5×10 ⁶ /mouse, and the xenograft tumor was formed and then passed in nude mice for one generation before use. The tumor tissue in the vigorous growth stage was cut into about 1.5 mm ³ , and inoculated subcutaneously in the right armpit of nude mice under sterile conditions. The diameter of the subcutaneous transplanted tumor in nude mice was measured with a vernier caliper, and the animals were randomly divided into groups after the average volume of the tumor grew to 150-165 mm ³ . The test compounds were administered orally once a day in different doses for 21 consecutive days. Positive control drug AZD228130mg/kg was orally administered once a day for 21 consecutive days. During the whole experiment, the diameter of the transplanted tumor was measured twice a week, and the body weight of the mice was weighed at the same time. The formula for calculating tumor volume (TV) is: TV=1/2×a×b ² , where a and b represent length and width, respectively.

The relative tumor volume (RTV) was calculated according to the measurement results, and the calculation formula was: RTV=V _t /V ₀ , where V ₀ was the tumor volume measured during administration in separate cages (that is, d ₀ ), V _t is the tumor volume at each measurement.

The evaluation index of antitumor activity is the relative tumor proliferation rate T/C (%), and the calculation formula is as follows: T/C (%)=(T _RTV /C _RTV )×100%, T _RTV : treatment group RTV; C _RTV : Negative control group RTV.

Experimental results:

Compound I-10-HCl, I-11-HCl, I-33-HCl of the present invention, to human breast cancer MDA-MB-436 (BRCA1 ^-/- ) experimental therapeutic effect data of nude mouse subcutaneous tumor transplantation tumor are shown in table 4 and shown in Figure 1. It was found that, for the same dose group (30 mg/kg) of the positive control drug AZD2281, the in vivo anti-tumor effects of the three compounds of the present invention were all better than AZD2281 (T/C<43.67%). In the 10mg/kg dose group, the in vivo antitumor effects of the two compounds of the present invention (I-10-HCl and I-11-HCl) were significantly better than AZD2281 (T/C<43.67%), even when the dose was reduced to 1mg/kg kg, the in vivo anti-tumor effect of a compound of the present invention (I-11-HCl) is basically equivalent to that of AZD2281 (T/C=48.19%). This fully proves that the compounds of the present invention are a class of compounds with potent anti-tumor activity in vivo. Further, during the three in vivo anti-tumor experiments of the compounds of the present invention, the body weight of the mice did not appear any phenomenon of weight loss (see Figure 2), which also preliminarily confirmed that the compounds of the present invention have relatively low toxic and side effects.

Table 4. Experimental therapeutic effect on human breast cancer MDA-MB-436 subcutaneously transplanted tumor in nude mice

*p<0.05 **p<0.001

Possibility of industrial use

The preparation process of the compound of the present invention is simple, and the production cost is low. In the inhibition experiment of receptor polyadenosine diphosphate-ribose polymerase (PARP) closely related to tumor occurrence and development, tumor cells VC-8 (BRCA2 ^-/- ), V79 ( BRCA2 ^+/+ ) and MDA-MB-436 (BRCA1 ^-/- ) proliferation inhibition experiments, as well as human breast cancer MDA-MB-436 (BRCA1 ^-/- ) subcutaneous transplantation tumors in nude mice showed good results Therefore, it is not only expected to be developed as a PARP-targeted monotherapy drug for BRCA gene-deficient tumors, but also can be used in combination with existing anti-tumor chemotherapy drugs for anti-tumor therapy.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (14)

1. A compound with a structure as shown in formula I or a pharmaceutically acceptable salt thereof, In the formula, R ₁ is a substituted or unsubstituted aromatic ring group, a substituted or unsubstituted 5-6 membered heteroaryl ring group, an unsubstituted benzo 5-6 membered heteroaryl ring group, a substituted or unsubstituted 5-6 membered heterocyclic group, unsubstituted benzo 5-6-membered heterocyclic group, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkyl-sulfonyl, Among them, _R3 is an unsubstituted aromatic ring group, a substituted or unsubstituted 5-6 membered heteroaryl ring group, a substituted or unsubstituted benzo 5-6 membered heteroaryl ring group, a substituted or unsubstituted 5-6 membered heterocyclic group, substituted or unsubstituted benzo 5-6 membered heterocyclic group, C3-C6 cycloalkyl group; Wherein, the substituted refers to being substituted by a substituent selected from the following group: hydroxyl, halogen, C1-C3 alkyl, halogenated C1-C3 alkyl, C1-C3 alkoxy, cyano, tert-butyl Carbamoyl; Wherein, the aromatic ring group is selected from the group consisting of phenyl, naphthyl; and The 5-6 membered heteroaryl ring group is selected from the group consisting of pyridyl, pyrimidyl, thiazolyl, isothiazolyl, furyl, thienyl, pyrrolyl; and The benzo 5-6 membered heteroaromatic ring group is selected from the group consisting of benzopyridyl, benzopyrimidinyl, benzothiazolyl, benzisothiazolyl, benzopyrrolyl, benzofuranyl, benzo Thienyl; and The 5- to 6-membered heterocyclic group is selected from the group consisting of tetrahydrofuryl, tetrahydrothiazolyl, dioxane, and dioxopentacyclyl; and The benzo 5-6 membered heterocyclic group is selected from the group consisting of benzodioxane, benzodioxane; and The C1-C6 alkyl group is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl; and The C3-C6 cycloalkyl group is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the 5-6 membered heteroaryl ring group is selected from the group consisting of pyridyl, pyrimidyl, thiazolyl, isothiazole base. 3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the benzo 5-6 membered heteroaromatic ring group is selected from the group consisting of benzothiazolyl, benzisothiazole base. 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the benzo 5-6 membered heterocyclic group is benzodioxane. 5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the C3-C6 cycloalkyl group is cyclopropyl. 6. The compound according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of: N-butyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine; N-ethyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine; N-phenyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine; N-cyclopropylmethyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine ; N-(2-pyridyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl} Piperazine; N-(pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl} Piperazine hydrochloride; N-(pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine; N-(pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }piperazine hydrochloride; N-(Methanesulfonyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Zinc; N-(hydroxyethyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piper Zinc; N-propyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine; N-(5-bromo-pyrimidin-2-yl]-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Fluorobenzoyl}piperazine; N-(4,6-Dimethyl-pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2 ,4-Difluorobenzoyl}piperazine; N-(4-trifluoromethyl-pyrimidin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine; N-(4-fluorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine; N-(2-fluorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine; N-(4-methylphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine; N-(4-bromophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine; N-(3-chlorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine; N-(4-chlorophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine; N-(4-cyanophenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine; N-(4-methoxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzene Formyl}piperazine; N-(2-methoxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzene Formyl}piperazine; N-(3-chloro-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Fluorobenzoyl}piperazine; N-Benzyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine; N-Benzyl-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl}piperazine sulfate ; N-(pyridine-3-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine sulfate; N-(5-methyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2,4-di Fluorobenzoyl}piperazine; N-(5-methyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Difluorobenzoyl}piperazine hydrochloride; N-(4-methyl-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4- Difluorobenzoyl}piperazine; N-(5-iodo-pyridin-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-di Fluorobenzoyl}piperazine; N-(4-Hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine; N-(4-Hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }piperazine lithium salt; N-(2-Hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine; N-(2-Hydroxyphenyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }piperazine lithium salt; N-(3,4-methylenedioxybenzyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine; N-(3,4-methylenedioxybenzyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine sulfate; N-(thiazol-2-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzoyl }Piperazine; N-(1,2-Benzisothiazol-3-yl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2, 4-Difluorobenzoyl}piperazine; N-(thiazole-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine; N-(thiazole-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4-difluorobenzyl Acyl}piperazine sulfate; N-(5-methyl-pyridine-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4 -Difluorobenzoyl}piperazine; N-(5-methyl-pyridine-2-methyl)-N'-{5-[(3,4-dihydro-4-oxo-phthalazin-1-yl)methyl]-2,4 -Difluorobenzoyl}piperazine hydrochloride. 7. A pharmaceutical composition, characterized by comprising the compound according to any one of claims 1-6 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier. 8. The pharmaceutical composition according to claim 7, further comprising other antineoplastic drugs. 9. The compound or pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 6 or the pharmaceutical composition as claimed in claim 7 in the preparation of polyadenosine diphosphate-ribose polymerase, i.e. the application of PARP inhibitors . 10. Use of the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 7 in the preparation of antitumor drugs. 11. The application according to claim 10, wherein the antineoplastic drug is a PARP inhibitor antineoplastic drug. 12. The use according to claim 10 or 11, characterized in that the tumor is a BRCA gene-related tumor. 13. The use according to claim 10 or 11, wherein the tumor is selected from the group consisting of breast cancer, ovarian cancer, pancreatic cancer or prostate cancer. 14. The compound as claimed in claim 1 or the preparation method of pharmaceutically acceptable salt thereof, is characterized in that, comprises the step: in inert solvent, in the presence of base and condensing agent, compound VIII and NR ₁ - Piperazine is reacted, thereby obtains formula I compound; Wherein, R ₁ is as described in claim 1.