WO2018192445A1 - Polycyclic compound having parp inhibition activity, and uses thereof - Google Patents

Abstract

Disclosed in the present invention are a polycyclic compound serving as a PARP inhibitor and uses thereof. The polycyclic compound in the present invention is represented in formula (I). The polycyclic compound in the present invention has PARP inhibition activity, provides a new commercial choice for PARP inhibitors, and can be used for treating and/or preventing tumors, strokes, myocardial ischemia, inflammation or diabetes.

Description

Polycyclic compound as PARP inhibitory activity and use thereof

The present application claims priority on Chinese Patent Application No. CN201710252002.3, filed on Apr. 17, 2017. This application cites the entire text of the above-mentioned Chinese patent application.

Technical field

The present invention belongs to the field of biomedicine and relates to a series of polycyclic compounds as PARP inhibitory activity, and in particular to a polycyclic compound of the formula I as a PARP inhibitory activity or a pharmaceutically acceptable salt thereof and use thereof.

Background technique

As a target for antitumor drugs, poly(ADP-ribose) polymerase (PARP) inhibitors have been actively explored for many years. Currently, AstraZeneca's first PARP inhibitor, Olaparib, has been proven to take advantage of defects in the DNA repair pathway to kill cancer cells first. This mode of action confers olaparib the potential to treat a wide range of tumor types with DNA repair defects. Olaparib has been used clinically to treat tumors with BRCA mutations, such as ovarian and ovarian cancer. Clovis Oncology also announced that the US FDA has accelerated the approval of its new drug Rubraca (rucaparib) as a monotherapy for the treatment of advanced ovarian cancer carrying harmful BRCA mutations (growth and/or somatic), which have been accepted by these women. More or more chemotherapy treatments. A series of compounds, whether as single-administration or as a combination therapy, are in clinical research, such as veliparib from Abbvie, niraparib from Tesaro, and talazoparib (BMN-673) from BioMarin.

Therefore, the development of a safer and more efficient new PARP inhibitor drug for cancer treatment has great social and economic benefits, and is also a research hotspot of major pharmaceutical companies. It is of great significance for the treatment of clinically relevant diseases to find new compounds with PARP inhibitory activity, improve the drug resistance and drug-forming properties of anti-tumor drugs, and improve their biological activity and bioavailability. Therefore, it is necessary to continue to develop novel compounds having PARP inhibitory activity.

Summary of the invention

The technical problem to be solved by the present invention is to provide a novel polycyclic compound, a pharmaceutical composition thereof, a preparation method and use thereof. The polycyclic compounds of the present invention have PARP inhibitory activity and provide a new commercial option for PARP inhibitors for the treatment and/or prevention of tumors, stroke, myocardial ischemia, inflammation or diabetes.

The present invention provides a polycyclic compound of the formula I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof,

Wherein the R ₁ is

The R ₃ and R _{4 are} independently

Said R _{5 is} independently H or C ₁ -C ₄ alkyl (such as methyl or isopropyl);

Said R ₂ is H, halogen (for example fluorine, chlorine or bromine), CN, OH, NH ₂ , C ₁ -C ₄ alkyl (for example methyl or isopropyl) or C ₁ -C ₄ alkoxy ( For example methoxy).

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

The R ₁ is

The R ₃ and R _{4 are} independently

The R _{5 is} independently H or C ₁ -C ₄ alkyl;

The R ₂ is H, halogen, CN, OH, NH ₂ , C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy; preferably, R ₂ is H, F, Cl, Br, methyl, Methoxy or isopropyl.

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

The R ₁ is

The R ₃ and R _{4 are} independently

Said R _{5 is} independently H or C ₁ -C ₄ alkyl (such as methyl or isopropyl);

The R ₂ is H, halogen (e.g., fluorine, chlorine or bromine) or CN.

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

The R ₁ is

The R ₃ and R _{4 are} independently

The R _{5 is} independently C ₁ -C ₄ alkyl (eg methyl or isopropyl);

The R ₂ is a halogen (e.g., fluorine, chlorine or bromine) or CN.

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

The R ₁ is

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

The R ₃ and R _{4 are} independently

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

The R _{5 is} independently a C ₁ -C ₄ alkyl group (e.g., methyl or isopropyl).

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

The R ₂ is H, halogen (e.g., fluorine, chlorine or bromine) or CN.

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

The R ₂ is a halogen (e.g., fluorine, chlorine or bromine) or CN.

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

The R ₂ is a halogen (e.g., fluorine, chlorine or bromine).

In a certain embodiment, the definition of each group in the compound I can be as follows (unannotated definitions are as described above):

R ₂ is H, F, Cl, Br, methyl, methoxy or isopropyl.

In the present invention, the solvate refers to a steadily present compound formed by combining a compound molecule with a solvent molecule, wherein the molar ratio of the compound molecule to the solvent molecule may be a common ratio of solvate, generally including 1:1, 1 : 2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. In the present invention, the solvent compound is preferably a hydrate such as a monohydrate, a dihydrate or the like.

Those skilled in the art will appreciate that, in accordance with the conventions used in the art, in the structural formula of the present application,

Used to delineate a chemical bond that is a point at which a moiety or substituent is attached to a core structure or a skeletal structure.

Thus, throughout the specification, one skilled in the art can select groups of R ₁ , R ₂ , R ₃ , R ₄ and substituents thereof in the compounds of Formula I to provide the present invention. A stable compound of formula I, or a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof, as described in the Examples.

In the present invention, the polycyclic compound as shown in Formula I may be any of the following compounds:

In the present invention, the stereoisomers include enantiomers, diastereomers, and mixtures of enantiomers or diastereomers.

The polycyclic compounds of the formula I according to the invention can be prepared according to conventional chemical synthesis methods in the art, the steps and conditions of which can be referred to the steps and conditions of similar reactions in the art.

The reaction solvent used in each reaction step of the present invention is not particularly limited, and any solvent which can dissolve the starting materials to some extent and does not inhibit the reaction is included in the present invention. In addition, many variations, equivalents, or equivalents to the solvents, solvent combinations, and solvent combinations described herein are considered to be within the scope of the invention.

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the polycyclic compound of formula I and a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient can be an excipient.

The present invention also provides the polycyclic compound of the formula I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof, or the use of the pharmaceutical composition in the preparation of a PARP inhibitor .

The PARP inhibitor can be used in vivo; it can also be used in vitro, mainly for experimental purposes, for example, providing alignment as a standard or control, or preparing a kit according to a conventional method in the art, and inhibiting PARP Provide fast detection.

The PARP can be PARP1.

The present invention also provides the polycyclic compound of the formula I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof, or the pharmaceutical composition for the treatment and/or prevention of PARP Use in drugs that rely on or PARP-mediated diseases or symptoms.

The PARP can be PARP1.

The present invention also provides the polycyclic compound of the formula I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof, or the pharmaceutical composition for the preparation of a tumor, a stroke, a myocardial Use in drugs for ischemia, inflammation or diabetes.

The tumor may be cancer or one or more of breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, leukemia, colon cancer, glioblastoma and lymphoma.

Unless otherwise specified, all technical and scientific terms used herein have the standard meaning of the claimed subject matter. In the event that there are multiple definitions for a term, the definitions herein prevail. When referring to URLs or other identifiers or addresses, it should be understood that such identifiers may change, specific information on the Internet may change, but equivalent information may be found by searching the Internet. The reference certifies that such information is available and publicly available.

It is to be understood that the above general description and the following detailed description are merely illustrative and not restrictive. The singular forms "a" or "an" Moreover, the term "comprising" is an open limitation and is not closed.

Unless otherwise indicated, the invention employs conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, or pharmacological detection, and the various steps and conditions can be referenced to routine procedures and conditions routine in the art. Unless otherwise indicated, the invention employs standard nomenclature and standard laboratory procedures and techniques for analytical chemistry, organic synthetic chemistry, and medical chemistry. In some cases, standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and drug delivery, and treatment of patients.

Term definition and description

The term "pharmaceutically acceptable" as used in the present invention is directed to those compounds, materials, compositions and/or dosage forms which are within the scope of sound medical judgment and are suitable for use with humans and animals. Tissue contact use without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to a salt of a compound of the invention prepared from a compound having a particular substituent found in the present invention and a relatively non-toxic acid or base. When a relatively acidic functional group is contained in the compound of the present invention, a base addition salt can be obtained by contacting a neutral amount of such a compound with a sufficient amount of a base in a neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When a relatively basic functional group is contained in the compound of the present invention, an acid addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of an acid in a neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogencarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and an organic acid salt, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; and salts of amino acids (such as arginine, etc.) And salts of organic acids such as glucuronic acid (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the invention contain both basic and acidic functional groups which can be converted to any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the neutral form of the compound. The parent form of the compound differs from the form of its various salts by certain physical properties, such as differences in solubility in polar solvents.

The "pharmaceutically acceptable salt" as used in the present invention is a derivative of the compound of the present invention, wherein the parent compound is modified by salt formation with an acid or with a base. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid groups such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example, salts formed from non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic acids and organic acids selected from the group consisting of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, Benzenesulfonic acid, benzoic acid, hydrogencarbonate, carbonic acid, citric acid, edetic acid, ethane disulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, Hydrobromic acid, hydrochloric acid, hydroiodide, hydroxynaphthalene, isethionate, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, dihydroxy Naphthenic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannin, tartaric acid and p-toluenesulfonic acid.

The "pharmaceutically acceptable salts" of the present invention can be synthesized from the parent compound containing an acid group or a base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in water or an organic solvent or a mixture of the two via a free acid or base form with a stoichiometric amount of a suitable base or acid. Generally, a nonaqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.

In addition to the form of the salt, the compounds provided herein also exist in the form of prodrugs. Prodrugs of the compounds described herein are readily chemically altered under physiological conditions to convert to the compounds of the invention. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound of formula I) is a prodrug within the scope and spirit of the invention. For example, a compound containing a carboxyl group can form a physiologically hydrolyzable ester which is prepared by hydrolysis in vivo to give the compound of formula I itself. The prodrug is preferably administered orally because hydrolysis occurs in many cases primarily under the influence of digestive enzymes. Parenteral administration can be used when the ester itself is active or hydrolysis occurs in the blood. Furthermore, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an in vivo setting.

Certain compounds of the invention may exist in unsolvated or solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are included within the scope of the invention. Certain compounds of the invention may exist in polycrystalline or amorphous form.

The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, radiolabeled compounds can be used, such as tritium ⁽³ H), iodine -125 ⁽¹²⁵ I) or ^C-14 (14 C). Alterations of all isotopic compositions of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.

In some embodiments, the compounds described herein exist as stereoisomers in which asymmetric or chiral centers are present. Stereoisomers are named according to the substituent configuration around the chiral carbon atom.

Or (S). Terms used in this article

And (S) is the configuration defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem, (1976), 45: 13-30, the contents of which are incorporated herein by reference. Embodiments described herein specifically include various stereoisomers and mixtures thereof. Stereoisomers include enantiomers, diastereomers, and mixtures of enantiomers or diastereomers. In some embodiments, each stereoisomer of a compound is prepared synthetically from a commercial starting material containing an asymmetric or chiral center, or by preparing a racemic mixture, followed by resolution. The method of resolution is, for example: (1) combining a mixture of enantiomers with a chiral auxiliary, and releasing a mixture of diastereomers by recrystallization or chromatography to release an optically pure product from the auxiliary; or (2) Direct separation of the mixture of optical enantiomers on a chiral column.

The term "excipient" generally refers to the carrier, diluent and/or vehicle required to formulate an effective pharmaceutical composition.

The term "effective amount" or "therapeutically effective amount" with respect to a pharmaceutical or pharmacologically active agent refers to a sufficient amount of a drug or agent that is non-toxic but that achieves the desired effect. For oral dosage forms in the present invention, an "effective amount" of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.

The term "active ingredient", "therapeutic agent", "active substance" or "active agent" refers to a chemical entity that is effective in treating a target disorder, disease or condition.

The term "alkoxy" refers to an alkyl group, as defined herein, appended to the remainder of the molecule through an oxygen atom. Examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tert-butoxy.

The term "alkyl" refers to a straight or branched chain hydrocarbon radical containing from 1 to 4 carbon atoms. Examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl. Herein, "C ₁ -C ₄ alkyl" means a straight or branched hydrocarbon group having 1 to 4 carbon atoms.

The term "bond" or "single bond" refers to a chemical bond between two atoms or between two moieties.

The term "ring" refers to any covalently closed structure. The ring includes, for example, a carbocyclic ring (for example, an aryl group and a cycloalkyl group), a heterocyclic ring (for example, a heteroaryl group and a heterocycloalkyl group), an aromatic group (for example, an aryl group and a heteroaryl group), and a non-aromatic group (for example, Cycloalkyl and heterocycloalkyl).

The term "ring system" refers to two or more rings in which two or more rings are fused. The term "fused" refers to a structure in which two or more rings share one or more bonds.

The term "halogen" means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

The term "comprising" is an open-ended expression that includes the subject matter of the invention, but does not exclude other aspects.

In the present invention, unless otherwise specified, a group which does not indicate a substitution means an unsubstituted, for example, "C ₁ -C ₄ alkoxy" means an unsubstituted C ₁ -C ₄ alkoxy group.

Provided herein is a therapeutic agent comprising a packaging material, a compound and a label provided herein within the packaging material, wherein the compound is effective for modulating the activity of PARP, or for treating, preventing or ameliorating PARP dependence Or one or more symptoms of a PARP-mediated disease or condition, and wherein the label indicates the compound or composition, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable N-oxide, pharmaceutically active metabolism A pharmaceutically acceptable prodrug or pharmaceutically acceptable solvate is used to treat, prevent or ameliorate one or more symptoms of a PARP-dependent or PARP-mediated disease or condition. Any combination of the various variables of the above is also within the scope of the description herein.

The PARP can be PARP1.

In one embodiment, disclosed herein is a pharmaceutical composition comprising a compound or stereoisomer of Formula I, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate, a pharmaceutically acceptable prodrug. In some embodiments, the pharmaceutical composition further contains a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises a second pharmaceutically active ingredient.

The compound of the formula I of the present invention has good PARP inhibitory activity, and the compound of the present invention can be effectively used as a PARP inhibitor for treating one or more diseases related to PARP activity, and is prepared for having The PARP inhibitory activity drug has good clinical application and medical use.

The PARP can be PARP1.

Tests for the in vitro metabolic stability of the compounds of formula I according to the invention show that the compounds of the invention exhibit good metabolic stability and provide an important basis for further preclinical studies.

The PARP inhibitors provided by the present invention can be used to treat a wide range of diseases including tumors, stroke, myocardial ischemia, inflammation and diabetes. Tumors that can be treated by PARP inhibitors include, but are not limited to, breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, and leukemia.

The PARP can be PARP1.

Some embodiments provide the use of a compound of Formula I, or a therapeutically acceptable salt thereof, in the manufacture of a medicament for PARP inhibitory activity in an individual considered to be in need of treatment.

The PARP can be PARP1.

Some embodiments provide the use of a compound of Formula I or a therapeutically acceptable salt thereof for the manufacture of a medicament for inhibiting tumor growth in an individual considered to be in need of treatment.

Some embodiments provide the use of a compound of Formula I, or a therapeutically acceptable salt thereof, in the manufacture of a medicament for treating cancer in an individual considered to be in need of treatment.

Some embodiments provide the use of a compound of Formula I or a therapeutically acceptable salt thereof for the manufacture of a medicament for the treatment of leukemia, colon cancer, glioblastoma, lymphoma in an individual considered to be in need of treatment.

The above preferred conditions can be arbitrarily combined without departing from the ordinary knowledge in the art, that is, preferred embodiments of the present invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progressive effect of the present invention is that the polycyclic compound of the present invention has PARP inhibitory activity, and has good water solubility and good stability, and provides a new commercial option for PARP inhibitors, which can be used for treating and/or preventing tumors, Stroke, myocardial ischemia, inflammation or diabetes.

detailed description

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in the art or in accordance with the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

An embodiment of the invention provides a compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer or prodrug thereof, for the preparation of a compound of formula iridium or a pharmaceutically acceptable compound thereof Methods and intermediates of salts, hydrates, solvates, stereoisomers or prodrugs, pharmaceutical compositions, and the use of the compounds and pharmaceutical compositions of the invention in the manufacture of a medicament.

Example 1 Preparation of a compound of formula I-7

In the present example, a compound of the formula I-7 was synthesized using a compound of the formula 700 and a compound of the formula 710 as a starting compound.

Specifically, a compound of the formula 700 (300 mg, 1.0 mmol), a mixture of the compound of formula 710 (700 mg, 10 mmol) in methanol (10 ml) was heated to 60 ° C and stirred for 1 hour, then Sodium cyanoborohydride (1257 mg, 20 mmol) was added, and the mixture was stirred at 60 ° C for 12 hours, and concentrated under reduced pressure to remove methanol. The residue was purified by column chromatography to afford compound ESI-MS (m) /z): 356(M+1) ⁺ .

Example 2 Preparation of a compound of formula I-43

In the present example, the compound of the formula I-43 was synthesized using the compound of the formula 720 and the compound of the formula 730 as a starting compound.

Specifically, a compound of the formula 720 (315 mg, 1.0 mmol), a mixture of the compound of formula 730 (840 mg, 10 mmol) in methanol (15 ml) was heated to 55 ° C and stirred for 1 hour, then Sodium cyanoborohydride (1257 mg, 20 mmol) was added, and the mixture was stirred at 55 ° C for 15 hours, and concentrated under reduced pressure to remove methanol. The residue was purified by column chromatography to yield compound ESI-MS (m) /z): 384(M+1) ⁺ .

Example 3 Preparation of a compound of formula I-35

synthetic route:

Synthesis steps:

Step 1: Preparation of the compound of formula I-35-2

Ethyl cyanoacetate (8.8 mL, 82.9 mmol), ammonium acetate (1.7 g, 22.6 mmol) and acetic acid (3.8 mL) were added to a solution containing formula I-35-1 in a round bottom flask under a nitrogen atmosphere. A solution of the compound (15 g) in anhydrous toluene (150 mL) was obtained. The mixture was heated to 120 ° C for 48 hours. Evaporated and extracted with dichloromethane (20 mL x 3). The organic layers were combined, dried over Na ₂ SO _4, filtered and evaporated. Purification by chromatography (petroleum ether (PE): ethyl acetate (PA) = 10:1 (volume ratio)) afforded the compound of formula I-35-2 as a yellow oil (15.6 g, yield 71 %). _{^{1 H NMR (400MHz, CDCl 3}} ) δ4.31-4.28 (m, 2H), 3.60 (t, J = 5.6Hz, 2H), 3.54 (t, J = 5.2Hz, 2H), 3.12 (t, J = 5.6 Hz, 2H), 2.77 (t, J = 5.6 Hz, 2H), 1.48 (s, 9H), 1.37-1.35 (m, 3H).

Step 2: Preparation of the compound of formula I-35-3

Under a nitrogen atmosphere, 3M methylmagnesium bromide (53.1 mL, 159.3 mmol) was added dropwise to a mixture of CuI (15.1 g, 79.7 mmol) in dry THF (100 mL) at -60 °C - -70 °C. After stirring at 0 ° C for 1 h and cooling to -60 ° C to -70 ° C, a solution of the compound of formula I-35-2 (15.6 g, 53.1 mmol) in THF (50 mL) was added and the mixture was stirred at 20 ° C overnight. The reaction was quenched with aqueous saturated ₄ Cl NH. Filtered and the aqueous layer was extracted with EtOAc EtOAc. The combined organic layer was dried with Na ₂ SO _4, filtered and evaporated to give the crude compound of Formula I-35-3 as shown (9.9 g of), as a yellow oil, which was used without further purification in the next reaction . _{^{1 H NMR (400MHz, CDCl 3}} ) δ4.27-4.25 (m, 2H), 3.73 (s, 2H), 3.15-3.12 (m, 2H), 1.63-1.60 (m, 4H), 1.45 (s, 9H ), 1.34-1.31 (m, 3H), 1.22 (s, 3H).

Step 3: Preparation of the compound of formula I-35-4

To a solution of the compound of formula I-35-3 (9.9 g, 32 mmol) in DMSO (50 ml) and water (0.5 ml), lithium chloride (1.8 g, 44.8 mmol). The reaction was carried out at ° C for 2.5 hours. The reaction was cooled to room temperature and water was added. The mixture was extracted with ethyl acetate (50mL × 3), dried over Na ₂ SO _4, filtered and evaporated. The residue was purified by EtOAc EtOAc EtOAc (EtOAc:EtOAc _{^{1 H NMR (400MHz, CDCl 3}} ) δ3.61-3.58 (m, 2H), 3.23 (s, 2H), 2.30 (s, 2H), 1.45 (s, 13H), 1.16 (s, 3H).

Step 4: Preparation of the compound of formula I-35-5

2M LDA (diisopropylamino group) was added dropwise to a mixture of the compound of formula I-35-4 (7.1 g, 29.8 mmol) in THF (100 mL) at -60 ° C to -70 ° C under a nitrogen atmosphere. Lithium) (22 mL, 44.7 mmol). After stirring at -60 ° C to -70 ° C for 1 hour, ethyl formate (4.4 g, 59.6 mmol) was added dropwise until completion, then slowly warmed to room temperature and stirred for further 4 hours. Upon completion, the reaction was quenched with aqueous ₄ Cl NH. The (50mL × 3) the aqueous layer was extracted with ethyl acetate, dried over Na ₂ SO _4, filtered and evaporated to give a residue, purified by column chromatography (petroleum ether: ethyl acetate = 10: 1 (volume ratio)) was purified The product of the compound of formula I-35-5 was obtained as white solid (3.0 g, yield 38%).

Step 5: Preparation of the compound of formula I-35-12

At 0 deg.] C, was added dropwise NaNO ₂ (2.8g of 2,6-dibromo-4- (compound shown in Formula I-35-11) fluoroaniline (10g, 38mmol) in concentrated HCl (50mL) in a mixture, 41 mmol) of an aqueous solution. After stirring at 0 ℃ 40 minutes, the reaction mixture dropwise at 0 ℃ was added dropwise to the _{SnCl 2 · 2H 2 O (12g} , 57mmol) in concentrated HCl solution (100mL). The resulting mixture was slowly warmed to 20 ° C and stirred for 12 hours. The solid was collected by EtOAc (EtOAc)EtOAc. ^{1 H NMR (400MHz, DMSO-} d 6) δ9.87 (brs, 3H), 7.81 (d, J = 8.0Hz, 2H), 7.09 (s, 1H).

Step 6: Preparation of the compound of formula I-35-6

A mixture of the compound of formula I-35-5 (3 g, 11.2 mmol), KOAc (1.6 g, 16.8 mmol) and the compound of formula I-35-12 (4.3 g, 13.4 mmol) in ethanol (25 ml) The reaction was stirred at 60 ° C for 5 hours. After completion of the reaction, NaHCO ₃ (1.8 g, 22.4 mmol) was added to the mixture while stirring at 80 ° C for further 15 hours. After cooling to room temperature, the resulting mixture was evaporated. The (20mL × 3) the aqueous layer was extracted with EtOAc, dried over Na ₂ SO _4, filtered and evaporated. The residue was purified by column chromatography (EtOAc: EtOAc = EtOAc) _{^{1 H NMR (400MHz, CDCl 3}} ) δ7.46 (s, 1H), 7.44 (d, J = 5.4Hz, 2H), 3.63 (s, 2H), 3.28 (s, 4H), 2.05 (s, 2H) , 1.46 (s, 11H), 1.30 (s, 3H).

Step 7: Preparation of the compound of formula I-35-7

The compounds of formula I-35-6 (950mg, 1.8mmol) , CuI (103mg, 0.54mmol), N, N- dimethyl-ethane-1,2-diamine (95mg, 1.1mmol), and K ₃ PO mixture of ₄ (1.4g, 5.4mmol) in DMF (15mL), at 70 deg.] C under N ₂ atmosphere was stirred overnight. After cooling to room temperature, the resulting mixture was filtered through a pad of Celite. The filtrate was evaporated and the residue was purifiedjjjjjjlililililililililililili _{^{1 H NMR (400MHz, CDCl 3}} ) δ9.26 (s, 1H), 7.67-7.65 (m, 1H), 7.11-7.09 (m, 1H), 6.98 (s, 1H), 3.52 (s, 2H), 3.35 (s, 2H), 1.99 (s, 2H), 1.71-1.64 (m, 2H), 1.44 (s, 9H), 1.32 (s, 3H).

Step 8: Preparation of the compound of formula I-35-8

The compound of the formula I-35-7 (850 mg, 1.9 mmol), Zn(CN) ₂ (444 mg, 3.8 mmol), Pd ₂ (dba) ₃ (174 mg, 0.19 mmol), DPPF (1,1'-double (diphenylphosphino) ferrocene) (210mg, 0.38mmol) and Zn (247mg, mixture of 3.8 mmol), and the in DMF (15mL),, stirred for 5 hours under an atmosphere of N ₂ at 120 ℃. After cooling to room temperature, the mixture was filtered, and the filtrate was evaporated to give crystals crystals crystals crystals ( Yield 64%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.76 (s, 1H), 7.73 (s, 1H), 7.29-7.27 (m, 1H), 7.17 (d, J = 8.4 Hz, 1H), 3.53-3.52 ( m, 2H), 3.40-3.36 (m, 2H), 2.00 (s, 2H), 1.67-1.63 (m, 2H), 1.46 (s, 9H), 1.35 (s, 3H).

Step 9: Preparation of the compound of formula I-35-9

At 0 deg.] C, a compound (480mg, 1.2mmol) to formula I-35-8 and 1N NaOH (1.5mL) in DMSO (25mL) was added dropwise 30% H ₂ O ₂ (1mL). After completion of the dropwise addition, the reaction solution was warmed to 45 ° C and stirred for 2 hours, then diluted with water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO _4, filtered and evaporated. The residue was purified by column chromatography (EtOAc: EtOAc: EtOAc) _{^{1 H NMR (400MHz, CDCl 3}} ) δ11.21 (s, 1H), 8.85 (s, 1H), 7.90 (d, J = 9.6Hz, 1H), 7.61 (s, 1H), 7.19 (s, 1H) , 6.09 (s, 1H), 3.52 (s, 2H), 3.44 (s, 2H), 2.01 (s, 2H), 1.71-1.69 (m, 2H), 1.47 (s, 9H), 1.39 (s, 3H) ).

Step 10: Preparation of the compound of formula I-35-10

To a solution of the compound (1 mg, EtOAc, m. The mixture was evaporated to give the title compound m.

Step 11: Preparation of the compound of formula I-35

3-Chloropropionyl chloride (36 mg, 0.29 mmol) was added dropwise to a solution of the compound of formula I-35-10 (200 mg), triethylamine (TEA) (48 mg, 0.48 mmol) in DCM (3 mL) The mixture was stirred at 0 ° C for 1 hour. Quenched with water and washed with aqueous NaHCO _3. The organic layer was dried over Na ₂ SO _4, filtered and evaporated. The mixture was purified by column chromatography (yield: EtOAc = EtOAc = EtOAc) ^{1 H NMR (400MHz, DMSO-} d 6) δ12.10 (s, 1H), 10.56 (s, 1H), 8.12 (s, 1H), 7.60 (d, J = 10.8Hz, 1H), 7.46 (d, J=8.2Hz, 1H), 6.82 (dd, J=16.8Hz, 10.8Hz, 1H), 6.08(d, J=16.4Hz, 1H), 5.66(d, J=10.4Hz, 1H), 3.88-3.68 (m, 2H), 3.43 (s, 2H), 2.11-2.08 (m, 2H), 1.64-1.62 (m, 2H), 1.34 (s, 3H).

Example 4 Preparation of a compound of formula I-34

synthetic route:

Synthesis steps:

a mixture of the compound of formula I-35-10 (160 mg, 0.45 mmol), DBU (158 mg, 1.04 mmol) Stir at 70 ° C overnight. Washed with water (10mL × 3), dried and the organic layer was dried over Na ₂ SO _4. Filter and evaporate. The mixture was purified by EtOAc EtOAc EtOAc EtOAc (EtOAc) The compound of formula I-34 (7 mg, 4%) was obtained as white solid. ^{1 H NMR (400MHz, DMSO-} d 6) δ12.22 (s, 1H), 10.52 (s, 1H), 9.43-9.33 (br s, 1H), 8.13 (s, 1H), 7.89-7.82 (m, 1H), 7.62 (d, J = 11.2 Hz, 1H), 7.52 (d, J = 6.0 Hz, 1H), 5.85 - 5.75 (m, 1H), 5.56 - 5.31 (m, 2H), 4.05 - 3.85 (m , 1H), 2.83-2.67 (m, 2H), 2.33-1.88 (m, 3H), 1.44-1.41 (m, 2H), 1.31-1.27 (m, 6H).

Example 5 Preparation of a compound of formula I-46

synthetic route:

Synthesis steps:

First step: Preparation of the compound of formula I-46-2

2M NaHMDS (sodium bis(trimethylsilyl)amide) (13.4 mL) was added dropwise to a solution of the compound of formula I-46-1 (5 g, 20.5 mmol) in 50 mL of THF at -78 ° C and N ₂ . 26.8 mmol). The mixture was stirred at -78 ° C for 0.5 hours. A solution of MeI (3.8 g, 26.7 mmol) in THF (10 mL) was then added dropwise. The mixture was stirred at room temperature for 20 hours. The reaction solution was treated with saturated aqueous NH ₄ Cl (100 mL) and extracted with EA (100mL × 2). The combined organics were dried with Na ₂ SO _4, filtered and evaporated to give a yellow oil, is a compound of formula I-46-2 (4.7 g, yield 88%).

Step 2: Preparation of the compound of formula I-46-3

At 0 ℃, THF to a compound of formula I-46-2 (4.4g, 17mmol) in (100 mL) was added LiBH ₄ (565mg, 25mmol). The mixture was stirred at room temperature overnight. The mixture obtained after completion of the reaction was adjusted to pH 5-6 with 1N HCl and extracted with ethyl acetate (100 mL×2). The combined organics were dried with Na ₂ SO _4, filtered and evaporated to give a colorless oil, as a compound of formula I-46-3 (3.9 g of, 99% yield).

Step 3: Preparation of the compound of formula I-46-4

Triethylamine (3.4 g, 34 mmol) and the compound of formula I-46-3 (3.9 g, 17 mmol) were added to 50 mL of DCM. A solution of MsCl (2.9 g, 25.5 mmol) in DCM (20 mL). The mixture was stirred for 2 hours at 0 ℃, and treated with saturated aqueous NaHCO ₃ (50mL). The organic layer was dried over Na ₂ SO _4, filtered and evaporated. The residue was purified by EtOAc EtOAcjjjjjjjj ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.40 (s, 2H), 3.59-3.29 (m, 3H), 3.02 (s, 3H), 1.71-1.56 (m, 4H), 1.45 (s, 9H), 1.38-1.25 (m, 1H), 0.99 (s, 3H).

Step 4: Preparation of the compound of formula I-46-5

A mixture of the compound of formula I-46-4 (614 mg, 2 mmol), CsF (456 mg, 3 mmol) and TMSCN (trimethylsilyl cyanide) (300 mg, 3 mmol) in 5 mL DMSO at 140 ° C in a microwave reaction Stir in the machine for 40 minutes. The mixture was treated with EA (100 mL) and washed with brine (50 <RTIgt; The organic layer was dried over Na ₂ SO _4, filtered and evaporated. The residue was purified by EtOAc EtOAcjjjjjj:

Step 5: Preparation of the compound of formula I-46-6

To a solution of the compound of formula I-46-5 (2.8 g, 11.8 mmol) in 20 mL DCM was added TFA (trifluoroacetic acid) (10 mL). The mixture was stirred at room temperature overnight. The solution was evaporated to remove DCM and TFA. With saturated NaHCO ₃ aqueous residue was adjusted to pH 7-9 and extracted with DCM (50mL × 4). The combined organics were dried with Na ₂ SO _4, filtered and evaporated to give a yellow oil, is a compound represented by formula I-46-6 (1.3g, 81% yield).

Step 6: Preparation of the compound of formula I-46-7

A mixture of the compound of the formula I-46-6 (1.3 g, 9.4 mmol), EtOAc (EtOAc, EtOAc. NaBH ₃ CN (1.2 g, 18.8 mmol) was then added at 0 °C. The mixture was stirred at room temperature overnight, and treated with saturated aqueous NaHCO ₃ (20mL). The resulting suspension was evaporated to remove a portion of MeOH and extracted with ethyl acetate (50 <RTIgt; The combined organics were dried with Na ₂ SO _4, filtered and evaporated. The residue was purified by EtOAc EtOAcjjjjj: _{^{1 H NMR (400MHz, CDCl 3}} ) δ7.19 (d, J = 8.4Hz, 2H), 6.85 (d, J = 8.4Hz, 2H), 3.80 (s, 3H), 3.43-3.34 (m, 2H) , 2.70-2.66 (m, 2H), 2.35-2.31 (m, 2H), 2.05-1.90 (m, 2H), 1.59-1.55 (m, 4H), 1.05 (s, 3H).

Step 7: Preparation of the compound of formula I-46-8

40mL THF solution at -70 ℃ and N _2, to a compound of formula I-46-7 as shown in (1.5g, 5.8mmol) was added dropwise 2M LDA (8.7mL, 17.4mmol). The mixture was stirred at -70 ° C for 1 hour. A solution of ethyl formate (1.3 g, 17.4 mmol) in THF (10 mL) was then added. The resulting mixture was stirred at room temperature for 14 hours. The reaction was treated with saturated aqueous NH ₄ Cl (30mL) and extracted with ethyl acetate (40mL × 3). The combined organics were dried with Na ₂ SO _4, filtered and evaporated to give a yellow oil, is a compound represented by formula I-46-8 (2.3g, crude). ESI-MS (m/z): 286.9 [M+H] ⁺ .

Step 8: Preparation of the compound of formula I-46-9

A mixture of the compound of formula I-46-8 (2.3 g, 8 mmol) and compound of formula I-35-12 (3.8 g, 12 mmol) was stirred in EtOAc (15 mL) EtOAc. The mixture was evaporated to remove most of the AcOH. With saturated NaHCO ₃ aqueous residue was adjusted to pH 7-9, and extracted with EA (50mL × 3). The combined organics were dried with Na ₂ SO _4, filtered and evaporated. The residue was purified by silica gel column chromatography (EtOAc/EtOAc EtOAc (EtOAc) The yield in two steps is 96%). _{^{1 H NMR (400MHz, CDCl 3}} ) δ7.50-7.42 (m, 3H), 7.13 (s, 2H), 6.84 (d, J = 7.2Hz, 2H), 3.79 (s, 3H), 3.66-3.63 ( m,1H), 3.27-3.19 (m, 2H), 2.96-2.87 (m, 1H), 2.03-2.00 (m, 1H), 1.81-1.77 (m, 2H), 1.60-1.42 (m, 3H), 1.25 (s, 3H).

Step 9: Preparation of the compound of formula I-46-10

Compound of formula I-46-9 (3.1 g, 5.6 mmol), Pd ₂ (dba) ₃ (413 mg, 0.45 mmol), xantphos (4,5-bis(diphenylphosphino)-9,9-di A mixture of methyl oxazide (521 mg, 0.9 mmol) and Cs ₂ CO ₃ (3.6 g, 11.2 mmol) was stirred in 10 mL DMF at 130 ° C under N ₂ overnight. The mixture was diluted with EA (200 mL) and washed with brine (100 mL×5). The organic layer was dried over Na ₂ SO _4, filtered and evaporated. The residue was purified by EtOAc EtOAcjjjjjjjj ^{1 H NMR (400MHz, DMSO-} d6) δ11.57 (s, 1H), 7.63 (s, 1H), 7.29-7.21 (m, 4H), 6.86 (d, J = 8.4Hz, 2H), 3.71 (s , 3H), 3.48-3.40 (m, 2H), 2.73-2.60 (m, 1H), 2.32-2.15 (m, 2H), 1.85-1.77 (m, 1H), 1.56-1.46 (m, 4H), 1.30 (s, 3H).

Step 10: Preparation of the compound of formula I-46-11

The compound of the formula I-46-10 (1.7 g, 3.6 mmol), ZnCN ₂ (844 mg, 7.2 mmol), Pd ₂ (dba) ₃ (264 mg, 0.29 mmol), Zn (468 mg, 7.2 mmol) and DPPF ( the mixture 319mg, 0.58mmol) in 15mL DMF, at a 120 deg.] C, stirring overnight under N _2. The mixture was diluted with EA (200 mL) and filtered. The filtration was washed with brine (100 mL x 5). The organic layer was dried over Na ₂ SO _4, filtered and evaporated. The residue was purified to silica gel elution elution elution elution elution elution ^{1 H NMR (400MHz, DMSO-} d6) δ7.69 (s, 1H), 7.53-7.51 (m, 2H), 7.18 (d, J = 7.6Hz, 2H), 6.82 (d, J = 7.6Hz, 2H ), 3.67 (s, 3H), 3.52-3.45 (m, 2H), 2.73-2.63 (m, 1H), 2.27-2.06 (m, 2H), 1.86-1.73 (m, 1H), 1.52-1.35 (m , 4H), 1.26 (s, 3H).

Step 11: Preparation of the compound of formula I-46-12

At 0 ℃, the compound shown in Formula I-46-11 (922mg, 2.2mmol) and 1N NaOH (3.3mL, 3.3mmol) in DMSO (10mL) was added dropwise _{30% H 2 O 2 (5mL} ). After the completion of the dropwise addition, the reaction solution was stirred at 50 ° C for 3 hours. The mixture was diluted with EA (200 mL) and washed with brine (100 mL×4). The organic layer was dried over Na ₂ SO _4, filtered and evaporated to give a yellow solid as the compound of formula I-46-12 (831 mg, 86% yield). ^{1 H NMR (400MHz, DMSO-} d6) δ11.85 (s, 1H), 10.59 (s, 1H), 8.09 (s, 1H), 7.76 (s, 1H), 7.63-7.57 (m, 1H), 7.49 -7.44 (m, 1H), 7.22 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 3.71 (s, 3H), 3.48-3.38 (m, 2H), 2.82 2.61 (m, 1H), 2.32 - 2.11 (m, 2H), 1.94-1.78 (m, 1H), 1.65-1.39 (m, 4H), 1.33 (s, 3H).

Step 12: Preparation of the compound of formula I-46-13

As shown in Formula I-46-12, HCOOH (1mL), piperidine (1 mL) and 10% Pd / C (300mg) of the compound (270mg, 0.62mmol) in MeOH (5mL) and H ₂ O (5mL) of The mixture was stirred at 55 ° C for 2 hours in a microwave reactor under N ₂ . The mixture was filtered and washed with MeOH (10 mL). With saturated aqueous NaHCO ₃ and the filtrate was adjusted to pH 7-9. The resulting mixture was evaporated. The residue was purified to silica gel elution elution elution elution ¹ H NMR (400 MHz, CD ₃ OD) δ 7.74 (s, 1H), 7.67-7.65 (m, 1H), 7.36-7.34 (m, 1H), 3.28-3.18 (m, 2H), 2.95-2.90 ( m, 1H), 2.22-2.16 (m, 1H), 1.82-1.62 (m, 4H), 1.36 (s, 3H).

Step 13: Preparation of the compound of formula I-46

TEA (81 mg, 0.8 mmol) and the compound of formula I-46-13 (50 mg, 0.16 mmol) were added to 10 mL of DCM. To the mixture was added a solution of 3-chloropropanoyl chloride (24 mg, 0.19 mmol) in DCM (1 mL). The reaction was stirred at room temperature for 72 hours. The mixture was diluted with DCM (50 mL) and brine. The organic layer was dried over Na ₂ SO _4, filtered and evaporated. The residue was purified by silica gel column chromatography (EtOAc/EtOAc (EtOAc) ^{1 H NMR (400MHz, DMSO-} d6) δ12.15 (s, 1H), 10.52 (s, 1H), 8.09 (s, 1H), 7.80 (d, J = 28.0Hz, 1H), 7.60 (d, J = 10.8 Hz, 1H), 7.49 (s, 1H), 6.93-6.77 (m, 1H), 6.10-6.04 (m, 1H), 5.66-5.60 (m, 1H), 3.98-3.84 (m, 1H), 3.74-3.66 (m, 1H), 3.58-3.50 (m, 2H), 2.21-2.09 (m, 1H), 1.76-1.49 (m, 3H), 1.28 (s, 3H).

Example 6 Preparation of a compound of formula I-47

synthetic route:

Synthesis steps:

A mixture of the compound of formula I-46-13 (50 mg, 0.16 mmol), 3-chloro-1-butene (86 mg, 0.96 mmol) and DBU (73 mg, 0.48 mmol) in 1 mL DMF 16 hours. The mixture was diluted with EA (50 mL) and washed with brine (30 mL×5). The organic layer was dried over Na ₂ SO _4, filtered and evaporated. The residue was purified by silica gel column chromatography (EtOAc/EtOAc (EtOAc) _{^{1 H NMR (400MHz, CD 3}} OD) δ7.75 (s, 1H), 7.69-7.65 (m, 1H), 7.36-7.34 (m, 1H), 5.95-5.86 (m, 1H), 5.18-5.11 ( m, 2H), 3.17-3.09 (m, 1H), 3.04-2.87 (m, 1H), 2.80-2.65 (m, 1H), 2.49-2.36 (m, 2H), 2.01-1.93 (m, 1H), 1.70-1.54 (m, 3H), 1.38 (s, 3H), 1.20 (d, J = 6.4 Hz, 3H).

Formula I-1 to Formula I-6, Formula I-8 to Formula I-33, Formula I-36 to Formula I-42, and Formula I-44 to I-45 were synthesized in a similar manner to Example 1 or 2. The compounds shown (see the section for specific structural formulas) differ only in that the starting compounds are adapted to the type of substituents R ₁ and R ₂ of the product and will not be described again. Among them, the ESI-MS (m/z) data of the obtained final product are summarized in Table 1 below. The ESI-MS (m/z) data for the compounds of the formulae I-1 to I-47 are as follows:

Table 1

Effect Example 1 In vitro study

In the present example, the biological activities of the compounds of the above formula I-1 to I-45 (sometimes referred to herein as compounds 1 to 45, respectively) were investigated.

Cell PARylation analysis

HCC1937 cells were seeded in 96-well plates at 4 x 104 cells/well and cultured overnight in a 37 °C incubator. The cells were treated with the test compound for 30 minutes and then treated with 1 mM hydrogen peroxide for 10 minutes. Cells were washed twice with 200 UL pre-cooled PBS and fixed with 100 ul of pre-cooled methanol/acetone (7:3) for 30 minutes in an ice bath. After air drying, the cells were blocked with PBS-Tween-20 blocking solution (0.05%) in 5% skim milk powder for 30 minutes at room temperature. The cells and the anti-PAR10H antibody were incubated at a ratio of 1:100 in blocking solution for 1 hour at room temperature, then washed three times with PBS-Tween-20, and then fluorescein-5(6)-isosulfide containing goat anti-mouse was added. The cyanate ester (FITC)-conjugated secondary antibody and 1 μg/mL DAPI blocking solution were incubated for 1 hour at room temperature in the dark. After rinsing three times with PBS-Tween-20, the data was analyzed using a fluorescent microplate counter (Flexstation III, Molecular Device).

PARP enzyme assay (according to the HT Universal PARP1 Colorimetric Assay Kit).

Histones were packaged in 96-well plates and incubated overnight at 4 °C. After washing the plate 3 times with 200UL PBST solution, it was blocked with a blocking solution, incubated at room temperature for 30 minutes, and then washed 3 times with a PBST solution. The test compound was treated to be added to the well plate, and then 20 μl of diluted PARP1 (1 nM) or 20 μl of PARP2 (3 nM) solution was added to the reaction system for 1 or 2 hours. A mixture of 50 μl streptavidin-HRP (1:50) was added to the well plate and incubated for 30 minutes at room temperature, and washed three times with PBST buffer. 100 μl (HRP) (chemiluminescent substrate A and substrate B (1:1)) were added to the well plates. Immediately read on a microplate reader (Envision, PerkinElmer).

Antiproliferative test

MDA-MB-436 and MDA-MB-231 cells were seeded in 96-well plates at a density of 500 and 2000 cells per well, respectively, and cultured overnight. The medium was RPMI 1640 containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. After the test compound was added, it was treated for 8 days. Cell viability was measured by the CCK8 kit. Specific Methods 10UL CCK8 reagent was added to each well and incubated at 37 ° C in a 5% CO 2 incubator for 3 hours. After shaking for 10 minutes, the light absorption value (OD value) was measured with a Flexstation III (Molecular Device) 450 nm.

PARP-1 enzyme inhibition a compound of the present invention to provide IC ₅₀ in Table 2 below. Compound IC ₅₀ of between 1 and is designated as +++ 100nM; IC ₅₀ compound is between 101 to 1000nm denoted ++, IC ₅₀ greater than 1000nm compound is designated as +.

Table 2

Compound number	IC 50 (nM)	Compound number	IC 50 (nM)	Compound number	IC 50 (nM)
I-1	+++	I-16	+++	I-31	+++
I-2	+++	I-17	+++	I-32	+++
I-3	+++	I-18	+++	I-33	+++
I-4	+++	I-19	+++	I-34	+++
I-5	+++	I-20	+++	I-35	+++
I-6	+++	I-21	+++	I-36	+++
I-7	+++	I-22	+++	I-37	+++
I-8	+++	I-23	+++	I-38	+++
I-9	+++	I-24	+++	I-39	+++
I-10	+++	I-25	+++	I-40	+++
I-11	+++	I-26	+++	I-41	+++
I-12	+++	I-27	+++	I-42	+++
I-13	+++	I-28	+++	I-43	+++
I-14	+++	I-29	+++	I-44	+++
I-15	+++	I-30	+++	I-45	+++

The test results show that the compounds of the formulae I-1 to I-45 of the present invention all have good PARP kinase inhibitory activity, whereby the compound of the present invention can be used as a PARP inhibitor for treating one or one The above tumor diseases related to PARP activity are used for preparing tumor drug.

Effect Example 2 IC ₅₀ value test of the inhibitory activity of the compound of the present invention against PARP1 enzyme in vitro

In the present example, the biological activities of the compounds of the above formula I-1 to I-47 (sometimes referred to herein as compounds 1 to 47, respectively) were investigated.

(1) Experimental purpose: The inhibitory activity and the half-activity inhibitory concentration (IC ₅₀ ) of the compound represented by the formula I-1 to I-47 of the present invention against the PARP1 enzyme were measured.

(2) Materials and instruments: Multi-plate reader SpectraMax M4 Microplate Reader (Molecular Devices), PARP1 Colorimetric Assay Kit (BPS, Cat#80580), PBS (Life Technologies, Cat#003000), Tween-20 (Sigma, Cat) #P9416-100ml), H2SO4 (Chinese medicine, Cat#10021618), and the compounds of the formulas I-1 to I-47 of the present invention.

(3) Experimental methods and steps:

3.1 PARP1 Colorimetric Assay

3.1.1 PARP1 The Colorimetric Assay Kit contains:

PARP1 5μg

5x histone mixture 1ml

10x assay mixture containing biotinylated substrate 300μl

10x PARP assay buffer 1ml

Blocking buffer 25ml

Activated DNA 500μl

Streptavidin-HRP 100μl

Colorimetric HRP substrate 10ml

96-well plate

3.1.2 reagent preparation:

1x PBS: Take a packet of PBS powder and add 1L of deionized water to dissolve completely;

Add Tween-20 to PBST: 1x PBS;

2M H2SO4: dilute H2SO4 to 2M with deionized water;

1x PARP assay buffer: Dilute 10x PARP assay buffer in 1:10 with deionized water to obtain 1x PARP assay buffer.

3.1.3 Compound dilution

The compound was dissolved in DMSO to a 10 mM stock solution and further diluted to 1 mM or 100 μM in DMSO for later use.

For the primary screening test, 1 mM and 100 μM of the compound stock solution was diluted to 100 μM and 10 μM with 1× PARP assay buffer, and 5 μl of the diluted compound (total volume 50 μl) was added to each well, such that the final concentration of the compound was 10 μM and 1 μM.

For the IC50 value test, the 100 μM compound stock solution was diluted to 10 μM with 1× PARP assay buffer, and then the compound was serially diluted 10-fold with 1× PARP assay buffer containing 10% DMSO to obtain a series of concentrations of the compound for use. 5 μl of the diluted compound (total volume 50 μl) was added to each well, such that the final concentration of the compound was 1 μM starting at a series of 10-fold dilutions.

3.1.4 Reaction steps

3.1.4.1 coating

1) Dilute 5x histone mixture in a ratio of 1:5 with 1x PBS to obtain a 1x coating solution;

2) 50 μl of diluted coating solution per well was added at 4 ° C overnight;

3) Discard the coating solution and wash 3 times with 200 μl PBST buffer per well;

4) Add 200 μl Blocking buffer to each well and incubate for 90 min at room temperature;

5) Discard the blocking buffer and wash it 3 times with PBST;

3.1.4.2 PARP1 reaction experiment

1) A reaction solution was prepared in a ratio of 2.5 μl of 10x PARP buffer + 2.5 μl of 10x PARP Assaymixture + 5 μl of Activated DNA + 15 μl of deionized water per well, and 25 μl of the reaction solution was added to each well (see Table 1).

2) Add 5 μl of the diluted compound to the sample test well. Add an equal volume of 1x PARP buffer containing 10% DMSO to the whole live control well and blank well (see Table 3).

Table 3 PARP1 reaction system

	Full live control hole	Sample test hole	Blank hole
10x PARP buffer	2.5μl	2.5μl	2.5μl
10x Assay mixture	2.5μl	2.5μl	2.5μl
Activated DNA	5μl	5μl	5μl
Deionized water	15μl	15μl	15μl
Dilute test compound	-	5μl	-
1x PARP buffer with 10% DMSO	5μl	-	5μl
1x PARP buffer	-	-	20μl
PARP1 (2.5ng/ul)	20ul	20μl	-
total capacity	50μl	50μl	50μl

3) Add 20 μl of 1x PARP buffer to the blank well.

4) Thaw PARP1 on ice, dilute to 2.5 ng/ul with 1x PARP buffer, add 20 μl of diluted PARP1 to all wells outside the blank well, mix and react at room temperature for 1 hr.

6) The reaction solution was discarded and washed 3 times with PBST.

3.1.4.3 Detection

1) Dilute Streptavidin-HRP in a ratio of 1:50 with Blocking buffer, add 50 μl of diluted Streptavidin-HRP to all wells, and incubate for 30 min at room temperature.

3) Discard HRP and wash 3 times with PBST.

4) Add 100 μl of colorimetric HRP substrate to each well and react at room temperature for 20 min.

5) Add 100 μl of 2M H2SO4 to each well. Read OD 450 nm on a microplate reader.

3.2 Calculating the inhibition rate

The inhibition rate is calculated using the following formula:

Inhibition rate = (ODsample-OD0%) / (OD100% - OD0%) × 100%

ODsample: the OD value of the sample test well;

OD0%: the OD value of the blank hole;

OD100%: OD value of the whole live control well.

Results and discussion

The PARP1 Colorimetric Assay kit was used to detect the inhibitory activity of the compound represented by Formulas I-1 to I-47 on the PARP1 enzyme. IC ₅₀ data PARP1 enzyme inhibiting compounds of the invention are provided in Table 4 below. IC ₅₀ of the compound between 1 to 50nM are denoted +++; Compound IC ₅₀ of between 101 to 1000nm is denoted ++, IC ₅₀ greater than 1000nm compound is designated as +.

Table 4

Compound number	IC 50 (nM)	Compound number	IC 50 (nM)	Compound number	IC 50 (nM)
I-1	+++	I-16	+++	I-31	+++
I-2	+++	I-17	+++	I-32	+++
I-3	+++	I-18	+++	I-33	+++
I-4	+++	I-19	+++	I-34	2.964
I-5	+++	I-20	+++	I-35	31.61
I-6	+++	I-21	+++	I-36	+++
I-7	+++	I-22	+++	I-37	+++
I-8	+++	I-23	+++	I-38	+++
I-9	+++	I-24	+++	I-39	+++
I-10	+++	I-25	+++	I-40	+++
I-11	+++	I-26	+++	I-41	+++
I-12	+++	I-27	+++	I-42	+++
I-13	+++	I-28	+++	I-43	+++
I-14	+++	I-29	+++	I-44	+++
I-15	+++	I-30	+++	I-45	+++

I-46

7.722

I-47

8.324

The experimental results show that the compounds of the formulae I-1 to I-47 of the present invention have an IC ₅₀ value of about 1 nM to 100 nM for the PARP1 enzyme, and both show strong inhibition. All of the present invention have good PARP kinase inhibitory activity, whereby the compounds of the present invention are useful as PARP inhibitors for the treatment of one or more tumor diseases associated with PARP activity for the preparation of tumors. drug.

Effect Example 3 Kinetic Solubility Test:

In the present example, the kinetic solubility of the compounds of the formulae I-1 to I-47 of the present invention was tested. Among them, the test of kinetic solubility is commonly used in high-throughput screening of drugs during the drug discovery phase. In kinetic analysis, a good solubility should help produce reliable data in vitro and in vivo. Since the kinetic solubility is pH dependent, the pH of the aqueous phase is always specified, usually measured at a pH of 7.4 (physiological pH of body fluids).

Test method: Weighed quantitative compound samples were dissolved in pure DMSO to a final concentration of 10 mM, and the test compound and the control compound (10 mM DMSO mother liquor, 10 μL per well) were added to a 96-well plate containing 490 μL of buffer per well. After 2 minutes of vortexing, the sample plates were incubated for 24 hours at room temperature (22 ± 2 °C) on a shaker. 200 μL of the sample was then transferred to a MultiScreen filter plate (polycarbonate membrane), filtered through a millipore vacuum manifold and the filtrate was collected. The concentration of the compound in the filtrate was determined by HPLC-UV. Three different concentrations of the UV standard solution and the solubility test sample were injected successively. Each sample was inserted twice, and the standard curve was taken to calculate the concentration and averaged.

The experimental results show that the compounds of the formulae I-1 to I-47 of the present invention all have good water solubility.

Effect Example 4 In vitro metabolic stability test:

In the present example, the in vitro metabolic stability of the compounds of the formulae I-1 to I-47 of the present invention was tested. Among them, the in vitro metabolic stability test assesses the clearance rate of a compound in one-phase metabolism and predicts its intrinsic clearance rate in hepatocytes and in vivo. We evaluated the metabolic stability of some of the compounds of the present invention in human and rat liver microsomes by in vitro metabolic stability experiments. The control sample is ABT888.

Reference to the specific operation steps of this experimental method (Tang Minghai, Wang Hairong, Wang Chunyan, Ye Yuyu. In vitro metabolism of antitumor compound E7 in different species of liver microsomal enzymes[J]. Chinese Journal of Traditional Chinese Medicine, 2016, No.9, 1739 - Method of assay described in page 1743).

The experimental results show that the compounds of the formulas I-1 to I-47 of the present invention all show good metabolic stability and provide an important basis for further preclinical research.

Effect Example 5 Metabolic stability study

(1) Study on metabolic stability of compounds in plasma

experimental method:

1. Experimental materials and test systems

1) Plasma

species	Description	Anticoagulant	Store
Human	Human, male, mixed	EDTA	-80 ° C
Rat	S.D. rat, male, mixed	EDTA	-80 ° C
Dog	Beagle, male, mixed	EDTA	-80 ° C

2) Buffer solution: phosphate buffer (100 mM KH ₂ PO ₄ -K ₂ HPO ₄ ), made by Shanghai Medicil Biomedical Laboratory.

3) Internal standard solution: acetonitrile solution containing 200 ng/mL internal standard (tolbutamide).

2. The test article is a compound of the formula I-1 to I-47 described in the present invention, and a control sample ABT888 thereof.

3. Metabolic stability test: The compound of the present invention was mixed with plasma and incubated at 37 ° C for 0, 5, 15, 30, 60 and 120 minutes, respectively. At the indicated incubation time point, 4 times the volume of the internal standard solution was added to stop the reaction. Finally, the sample was centrifuged and the supernatant was taken for detection by LC-MS/MS.

(b) Study on the stability of compounds in rat, beagle and human liver microsomes:

experimental method:

1. Experimental materials and test systems

1) Liver microsomes

species	Description	Store	Liver weight (g liver/kg)
Human	Human, male, mixed	-80 ° C	26
Rat	S.D. rat, male, mixed	-80 ° C	40
Dog	Beagle, male, mixed	-80 ° C	32

2) NADPH: purchased from Roche, the number is 10621706001

3) Buffer solution: phosphate buffer (100 mM KH ₂ PO ₄ -K ₂ HPO ₄ ), made by Shanghai Medicil Biomedical Laboratory.

4) Internal standard solution: acetonitrile solution containing 200ng/mL internal standard (tolbutamide)

5) Positive control: Midazolam

2. The test product is a compound represented by the formula I-1 to I-47 according to the present invention, and a control sample ABT888 thereof

3. Metabolic stability experiment

Mix the compound with liver microsomes (final concentration 0.5 mg/mL) and reducing coenzyme NADPH (final concentration 2 mM) and incubate at 37 °C for 0, 5, 15, 30 and 45 minutes respectively. To the specified incubation time At the time of the reaction, the reaction was terminated by adding 3 volumes of the internal standard solution. Finally, the sample was centrifuged and the supernatant was taken for detection by LC-MS/MS.

4. Data analysis:

By mass spectrometry, the concentration of the compound in each sample was expressed by the peak area ratio (the ratio of the peak area of the compound to the internal standard peak area), and then the percentage of the compound remaining at each incubation time point was calculated with reference to the compound concentration of 0 minutes (%Remaining). ), the ln logarithm of the remaining percentage is linearly fitted to the incubation time, and the half-life and the intrinsic clearance are calculated according to the following formula.

%Remaining=PAR _{appointed time} /PAR _0-min x 100(PAR:peak area ratio)

The elimination rate constant(k)=(-gradient)

Half life(t _1/2 )(minutes)=0.693/k

Intrinsic clearance(CL _int )(mL/min/kg)=(0.693/t _1/2 )x(mL incubation/mg microsomal protein)x(mg microsome/g liver)x(gr liver/kg body weight)=( 0.693/t _1/2 )/0.5 (mg microsomal protein/mL incubation) x 45 (mg microsome/g liver) x (gr liver/kg body weight)

5. Acceptance criteria for control compounds: Metazolam's metabolic half-life should be <30 min

The experimental results show that the compounds I-34, I-35, I-46 and I-47 of the present invention show good metabolic stability in plasma and liver microsomes, and provide further preclinical studies. Important reference.

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" and the like means a specific feature described in connection with the embodiment or example, A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described, it is understood that the foregoing embodiments are illustrative and not restrictive Variations, modifications, alterations and variations of the above-described embodiments are possible within the scope of the invention.

Claims (15)

1. a polycyclic compound of formula I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof;

   

   Wherein the R ₁ is

   

   

   The R ₃ and R _{4 are} independently

   

   The R _{5 is} independently H or C ₁ -C ₄ alkyl;

   The R ₂ is H, halogen, CN, OH, NH ₂ , C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy.
2. The polycyclic compound I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof, according to claim 1, wherein when said R ₅ is C ₁ -C ₄ In the case of an alkyl group, the C ₁ -C ₄ alkyl group is a methyl group or an isopropyl group;

   And/or, when said R ₂ is halogen, said halogen is fluorine, chlorine or bromine;

   And/or, when the R ₂ is a C ₁ -C ₄ alkyl group, the C ₁ -C ₄ alkyl group is a methyl group or an isopropyl group;

   And/or, when said R ₂ is a C ₁ -C ₄ alkoxy group, said C ₁ -C ₄ alkoxy group is a methoxy group.
3. The polycyclic compound I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof, according to claim ₁ , wherein R ₁ is

   

   And/or, said R ₃ and R _{4 are} independently

   

   And/or, said R _{5 is} independently H or C ₁ -C ₄ alkyl;

   And/or, R ₂ is H, halogen or CN.
4. The polycyclic compound I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof according to claim 3, wherein said R _{5 is} independently C ₁ - C ₄ alkyl;

   And/or, R ₂ is halogen or CN.
5. The polycyclic compound I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof according to claim 4, wherein the R ₂ is a halogen.
6. The polycyclic compound I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof, according to claim ₁ , wherein R ₁ is

   

   The R ₃ and R _{4 are} independently

   

   The R _{5 is} independently H or C ₁ -C ₄ alkyl;

   The R ₂ is H, halogen, CN, OH, NH ₂ , C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy.
7. The polycyclic compound I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof according to claim 6, wherein R ₂ is H, F, Cl, Br, A Base, methoxy or isopropyl.
8. The solvate of the polycyclic compound I according to any one of claims 1 to 7, wherein in the solvate, the molar ratio of the compound of the formula I to the solvent molecule is 1:1, 1:2. , 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.
9. The polycyclic compound I, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof according to claim 1, wherein the polycyclic compound I is any one of the following compounds :

   

   

   
10. A pharmaceutical composition comprising the polycyclic compound I according to any one of claims 1 to 9, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof.
11. The polycyclic compound I according to any one of claims 1 to 9, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof or the pharmaceutical composition according to claim 10 in the preparation Use in medicines for inhibiting PARP.
12. The use according to claim 11, wherein the medicament is for inhibiting PARP in vivo or in vitro.
13. The polycyclic compound I according to any one of claims 1 to 9, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof or the pharmaceutical composition according to claim 10 in the preparation Use in medicine for the treatment and/or prevention of a PARP dependent or PARP mediated disease or condition.
14. The polycyclic compound I according to any one of claims 1 to 9, a pharmaceutically acceptable salt, solvate, metabolite, stereoisomer or prodrug thereof or the pharmaceutical composition according to claim 10 in the preparation Use in medicine for the treatment and/or prevention of tumors, stroke, myocardial ischemia, inflammation or diabetes.
15. A kit for inhibiting PARP, comprising the polycyclic compound I according to any one of claims 1 to 9, a pharmaceutically acceptable salt, solvate, metabolite thereof, stereoisomerism thereof A prodrug or prodrug or a pharmaceutical composition according to claim 10.