CN118005637A - Fused tricyclic PARP1 inhibitor, preparation method and application thereof - Google Patents

Abstract

The invention relates to a fused tricyclic PARP1 inhibitor, a preparation method and application thereof. In particular to a compound for inhibiting the activity of poly ADP-ribose polymerase, a pharmaceutical composition and application thereof. The invention relates in particular to compounds of formula (I) and also to pharmaceutical compositions comprising these compounds and to the use of these compounds for the preparation of a medicament for the prophylaxis and/or treatment of diseases, in particular diseases which are ameliorated by the inhibition of PARP 1.

Description

Fused tricyclic PARP1 inhibitor, preparation method and application thereof

Technical Field

The invention relates to a compound with the activity of inhibiting poly ADP-ribose polymerase 1 (PARP 1) and application thereof, in particular to a quinolone compound, a pharmaceutical composition containing the compound and application of the compound in medicines for improving diseases, especially tumor diseases, by inhibiting PARP 1.

Background

Poly ADP-ribose polymerase (PARPs) is an emerging enzyme family with the ability to catalyze the transfer of ADP-ribose to target proteins (poly ADP-ribosylation), at least 18 PARP family members being encoded by different genes sharing homology in conserved catalytic domains (Morales et al, CRITICAL REVIEWS ^TM in Eukaryotic Gene Expression 24.1,2014). PARP1 is known as poly (ADP-ribose) polymerase 1, i.e., poly ADP ribose polymerase 1, and PARP1 is a abundant nucleoprotein (Murai et al CANCER RESEARCH 72.21,2012). PARP1 is capable of catalyzing the transfer of ADP ribose residues from nad+ to a target substrate protein or nucleic acid, creating a poly (ADP-ribose) chain that adds to a downstream target protein, a post-translational modification called PAR glycosylation (PARylation). PARPs play an important role in several cellular processes including cell proliferation and cell death (Murai et al CANCER RESEARCH 72.21,2012). The primary function of PARP is to participate in DNA damage repair, DNA Single Strand Breaks (SSBs), the most common type of damage, can be converted to potentially disruptive and deadly DNA Double Strand Breaks (DSBs), and PARP1 binds to damaged DNA at single strand DNA breaks (SSBs) and other DNA lesions, which event causes a series of allosteric changes in the structure of PARP1, thereby activating its catalytic function (Lord et al, science 355.6330,2017).

BRCA1 and BRCA2 proteins are critical for repair of double-stranded DNA breaks (DSBs), a process known as Homologous Recombination Repair (HRR), a form of DNA repair that utilizes homologous DNA sequences to direct repair at DSBs (Lord et al, science 355.6330,2017). HRR is typically a "conservative" mechanism because it restores the original DNA sequence at the site of DNA damage. When cells develop HRR defects, whether driven by defects in BRCA1, BRCA2, or other pathway components, non-conserved forms of DNA repair predominate, such as non-homologous end joining (NHEJ).

PARP inhibitors exert anticancer effects by blocking DNA damage repair in highly mutated cancer cells, thereby causing "toxic damage" to kill cells deleted for Homologous Recombination Repair (HRR). There are multiple signaling pathways for repair DNA within healthy cells, so inhibiting only PARP is not too toxic to it; however, for some tumor cells, because specific genetic mutations such as BRCA disrupt other DNA repair pathways, the DNA repair pathway will depend on PARP-1 and thus will be particularly sensitive to PARP inhibitors, which is why ovarian and breast cancer patients carrying BRCA mutations are more likely to benefit from PARP inhibitors. The PARP2 content is lower and only accounts for 5% -10% of the total PARP activity. PARP1 knockdown significantly reduced PARP activity (ye lamos et al, the EMBO journ 25.18,2006) compared to PARP2 knockdown (< 10%), PARP1 knockdown blocked The inhibitory activity of olaparib (Olaparib) on PARP and also abrogated The cytostatic effect of olaparib (Olaparib) (Murai et al CANCER RESEARCH, 2012). These data indicate that the key to PARP is PARP1, which determines PARPi efficacy. There are corresponding literature reports that mice survive requiring intact PARP2 in bone marrow; the deletion of PARP2 results in a reduction in RBC, WBC and BM cell numbers (farres et al, blood, the Journal of THE AMERICAN Society of Hematology 122.1,2013); PARP2 knockdown reduced The number of T cells and RBCs compared to PARP1 knockdown, whereas PARP1 knockdown did not significantly affect The number of T cells (yf lamos et al, blood, the EMBO journ al 25.18,2006) and RBCs (farres et al CELL DEATH & Differentiation 22.7,2015). Thus, inhibition of PARP1 is the major source of drug efficacy and inhibition of PARP2 is the major source of toxicity. The development of highly selective PARP1/2 inhibitors may significantly reduce the toxicity of PARP2 without significantly reducing the efficacy.

In view of the foregoing, there is a strong need in the art to develop highly potent and safe inhibitors of PARP, particularly inhibitors with high selectivity for PARP 1.

Disclosure of Invention

The invention relates to a compound shown in a general formula (I), or a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt, a crystal form, a solvate, a hydrate or a prodrug thereof,

Wherein,

X ¹ is independently selected from the group consisting of-N-, -NR ¹⁴-、-CR⁷ -, O, and S;

X ² is independently selected from the group consisting of-N- -NR ¹⁵ -and-CR ⁸ -, O and S;

X ³ is independently selected from the group consisting of-N- -NR ¹⁶ -and-CR ⁹ -, O and S;

X ⁴、X⁸ are each independently selected from the group consisting of-N-and-C-;

Is a single bond or a double bond; and X ¹、X²、X³、X⁴、X⁸ together form a five membered heteroaryl group;

r ⁷、R⁸、R⁹ is independently selected from hydrogen, halogen, unsubstituted or substituted C ₃-C₆ cycloalkyl or unsubstituted or substituted C ₁-C₆ alkyl; r ⁷、R⁸、R⁹ is preferably hydrogen, halogen or C ₁-C₄ alkyl; r ⁷、R⁸、R⁹ is more preferably hydrogen, F or methyl;

R ¹⁴、R¹⁵、R¹⁶ is independently selected from hydrogen, unsubstituted or substituted C ₃-C₆ cycloalkyl or unsubstituted or substituted C ₁-C₆ alkyl; r ¹⁴、R¹⁵、R¹⁶ is preferably selected from hydrogen or C ₁-C₃ alkyl; more R ¹⁴、R¹⁵、R¹⁶ is preferably selected from methyl;

X ⁵、X⁶ is each independently selected from the group consisting of-N-and-CR ¹⁰-;R¹⁰ is selected from the group consisting of hydrogen, halogen, cyano, unsubstituted or substituted C ₁-C₆ alkoxy, unsubstituted or substituted C ₁-C₆ alkyl; r ¹⁰ is preferably hydrogen, halogen, cyano, C ₁-C₄ alkyl; r ¹⁰ is more preferably hydrogen or fluorine, chlorine or methyl;

X ⁷ is-N-and-CR ¹⁷-;R¹⁷ is selected from hydrogen, halogen, cyano, unsubstituted or substituted C ₁-C₆ alkoxy, unsubstituted or substituted C ₁-C₆ alkyl; r ¹⁷ is preferably hydrogen, halogen, cyano, C ₁-C₄ alkyl; r ¹⁷ is more preferably hydrogen or fluorine, chlorine or methyl;

R ¹、R^1'、R²、R³、R⁴、R⁵ is independently selected from hydrogen, unsubstituted or substituted C ₁-C₆ alkyl; or R ⁴、R⁵ together with the carbon atom to which they are attached form a C ₃-C₆ cycloalkyl group;

s and n are each independently selected from 0, 1 or 2;

Y is selected from N or CH;

r ⁶ is selected from:

Each independently R ¹¹ is selected from halogen, cyano, C ₁-C₃ alkoxy, carbonyl, -CONHR ¹³, amino, preferably selected from halogen, -CONHR ¹³ and cyano;

m is 0,1,2 or 3;

R ¹² is selected from hydrogen, halogen, cyano, unsubstituted or substituted C ₁-C₄ alkyl;

R ¹³ is hydrogen, unsubstituted or substituted C ₁-C₆ alkyl, unsubstituted or substituted C ₃-C₈ cycloalkyl, unsubstituted or substituted C ₁-C₆ alkoxy, unsubstituted or substituted 3-8 membered heterocycloalkyl; preferably, R ¹³ is hydrogen, unsubstituted or halogen substituted C ₁-C₄ alkyl, C ₃-C₆ cycloalkyl, C ₁-C₆ alkoxy; the heterocycloalkyl group refers to a heterocycloalkyl group containing 1 to 3 heteroatoms selected from N, O, S;

R¹、R^1'、R²、R³、R⁴、R⁵、R⁷、R⁸、R⁹、R¹⁰、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷ Wherein said substitution is by one or more selected from the group consisting of C ₁-C₄ alkyl, halogen, hydroxy, cyano, amino, carboxy, C ₃-C₆ cycloalkyl;

The conditions are as follows:

The conditions are as follows:

When X ⁵ and X ⁸ are both-N-, at least one of X ¹、X²、X³、X⁴ is-N-;

When X ⁴ and X ⁵ are both-N-, X ³ is also-N-;

When X ³ is oxygen and X ¹、X²、X⁴、X⁸ is-C-, X ⁷ is-CR ¹⁷ -, R ¹、R^1'、R¹⁷ is not both hydrogen;

The preferred conditions are:

When X ⁸ is-N-, at least one of X ¹、X²、X³ is-N-;

When X ⁴ and X ⁵ are both-N-, X ³ is also-N-;

When X ³ is oxygen and X ¹、X²、X⁴、X⁸ is-C-, X ⁷ is-CR ¹⁷ -, R ¹、R^1'、R¹⁷ is not both hydrogen.

Preferably, in formula (I)The following structure is preferred:

More preferably selected from the following structures:

Wherein,

In the general formula (I)The following structure is preferred:

More preferably, in formula (I) The following structure is preferred:

Wherein R ²、R³、R⁴、R⁵、R¹²、R¹³ is as defined above.

More preferably, the compound of formula (I) is selected from the following specific compounds:

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in another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate, or prodrug thereof, and a pharmaceutically acceptable carrier.

In certain embodiments of the pharmaceutical composition, the pharmaceutical composition is formulated for intravenous administration, intramuscular administration, oral administration, rectal administration, inhalation administration, nasal administration, topical administration, ocular administration, or otic administration. In other embodiments of the pharmaceutical composition, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, solution, emulsion, ointment, eye or ear drop. In other embodiments of the pharmaceutical composition, it further comprises one or more additional therapeutic agents.

In another aspect, the present invention provides the use of a compound of formula (I), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, form, solvate, hydrate or prodrug thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for the prevention, treatment or amelioration of a disease by inhibition of PARP 1.

In another aspect, the present invention provides a method of preventing, treating or ameliorating a disease by inhibiting PARP1, the method comprising administering to a subject in need of such treatment an effective amount of a compound of formula (I), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystalline form, solvate, hydrate or prodrug thereof, or a pharmaceutical composition as described.

In some embodiments of the invention, the disease includes, but is not limited to, cancer.

In some embodiments of the invention, the cancer includes, but is not limited to, a malignancy, such as any one of ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, biliary tract cancer, osteosarcoma, cervical cancer, head and neck tumor, germ cell and embryo cancer, esophageal cancer, glioblastoma, ewing's sarcoma, pancreatic cancer, melanoma, cholangiocarcinoma, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, and hematological cancer.

In some embodiments of the invention, the genome of the cancer is of the type in which homologous recombination repair is absent.

In some embodiments of the invention, the cancer repair the deleted pathway by homologous recombination depending on DNA double strand damage.

In some embodiments of the invention, the cancer comprises one or more cancer cells that lack the ability to repair DNA double strand breaks by homologous recombination relative to normal cells.

In some embodiments of the invention, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2, or have a type of BRCA1 or BRCA2 mutation.

Description of the terms

In the present invention, unless explicitly stated otherwise, terms used in the present invention have the meanings defined below. Terms not explicitly defined herein have the general meaning commonly understood by those skilled in the art.

As used herein, the term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.

The terms "optional," "optional," or "optionally" as used herein mean that the subsequently described substitution pattern, event, or circumstance may or may not occur, and that the description includes instances where said substitution pattern occurs and instances where said substitution pattern does not occur. For example, "optionally substituted alkyl" includes "unsubstituted alkyl" and "substituted alkyl" as defined herein. It will be appreciated by those skilled in the art that for any group containing one or more substituents, the group does not include any sterically impractical, chemically incorrect, synthetically infeasible and/or inherently unstable substitution patterns.

As used herein, the term "pharmaceutically acceptable salt" refers to salts that retain the biological effects and properties of the compounds of the present invention, and which are not biologically or otherwise undesirable. Non-limiting examples of such salts include non-toxic, inorganic or organic base or acid addition salts of the compounds of the present invention. In many cases, the compounds of the present invention are capable of forming acid and/or base salts due to the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts may be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts may be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts may be derived include, for example, primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, and the like, such as, inter alia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound (basic or acidic moiety) by conventional chemical methods. Generally, the salts can be prepared as follows: the free acid form of the compound is reacted with a stoichiometric amount of a suitable base (e.g., na, ca, mg or K hydroxide, carbonate, bicarbonate, etc.) or the free base form of the compound is reacted with a stoichiometric amount of a suitable acid. Such reactions are generally carried out in water or an organic solvent or a mixed solvent of both. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred when feasible. Other suitable salts can be found in Remington's pharmaceutical sciences (Remington's Pharmaceutical Sciences), 20 th edition, mack publishing company (Mack Publishing Company), easton, pa., (1985), incorporated herein by reference.

As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic, absorption delaying agents, salts, preservatives, pharmaceuticals, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, the like, and combinations thereof, as known to those of ordinary skill in the art (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, mack publishing Company (MACK PRINTING Company), 1990, pp.1289-1329, incorporated herein by reference). Unless any conventional carrier is not compatible with the active ingredient, it is contemplated that it may be used in therapeutic or pharmaceutical compositions.

As used herein, the term "solvate" is meant to encompass stoichiometric or non-stoichiometric solvent addition forms. If the solvent is water, the solvate formed is a hydrate, and when the solvent is ethanol, the solvate formed is an ethanolate. Hydrates are formed by one or more molecules of water with a molecule of the substance, wherein the water retains its molecular state of H ₂ O, and such a combination is capable of forming one or more hydrates, such as hemihydrate, monohydrate, and dihydrate.

As used herein, "prodrug" refers to a chemically modified active or inactive compound that upon administration to a subject, becomes a compound of the invention through physiological actions in the body (e.g., hydrolysis, anabolism, etc.). Adaptations and techniques for making and using prodrugs are well known to those skilled in the art.

The term "therapeutically effective amount" of a compound of the invention refers to an amount of a compound of the invention that can elicit a biological or medical response in an individual or ameliorate symptoms, slow or delay the progression of a disease, or prevent a disease, etc.

As used herein, the term "individual" refers to an animal. Preferably, the animal is a mammal. Individual also refers to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like. In a preferred embodiment, the individual is a human.

As used herein, the term "inhibition" refers to the alleviation or inhibition of a particular patient, symptom or condition or disease, or a significant reduction in biological activity or process baseline activity.

As used herein, the term "treating" any disease or disorder in one embodiment refers to ameliorating the disease or disorder (i.e., preventing or slowing the progression of the disease or at least one clinical symptom thereof). In another embodiment, "treating" refers to improving at least one physical parameter that may not be perceived by the patient. In another embodiment, "treating" refers to modulating a disease or condition physically (e.g., stabilizing a perceived symptom) or physiologically (e.g., stabilizing a parameter of the body) or both.

Advantageous effects

The compound has the main advantages of high selectivity to PARP1, fewer side effects than olaharib (AZD-2281) and high clinical application value.

Detailed Description

The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified. The starting materials may generally be obtained from commercial sources or readily prepared using methods known to those skilled in the art.

In various embodiments, the experimental apparatus or materials used are as follows:

¹ H NMR was recorded by Varian Mercury-300 or Varian Mercury-400 nuclear magnetic resonance, ¹³ C NMR was recorded by Varian Mercury-400 or Varian Mercury-500 or Varian Mercury-600 nuclear magnetic resonance, chemical shift expressed as delta (ppm); mass spectra were recorded by Finnigan/MAT-95 (EI) and FINNIGAN LCQ/DECA and Micromass Ultra Q-TOF (ESI) type mass spectrometers; silica gel for reversed phase preparative HPLC separation was 200-300 mesh.

Abbreviations

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Synthesis of key intermediates

Intermediate 1a (R) -6-fluoro-N-methyl-5- (2-methylpiperazin-1-yl) pyridine amide hydrochloride

Step 1: synthesis of methyl 5-bromo-6-fluoropyridine formate

To a 50mL one-necked flask was successively added compound 1a-1 (1 g,4.6 mmol), acetonitrile (30 mL), silver difluoride (1.76 g,13.9 mmol), and the mixture was stirred overnight. The reaction was filtered, the filtrate was concentrated and purified by silica gel column (PE: ea=5:1) to give 1a-2 (450 mg, white solid), yield: 42%. LCMS (ESI) m/z 233.9[ M+H ] ⁺; rt=1.51 min (3.00 min).

Step 2: synthesis of (R) -4- (2-fluoro-6- (methoxycarbonyl) pyridin-3-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester

To a 50mL one-necked flask was successively added 1a-2 (450 mg,1.9 mmol), (R) -4-Boc-2-methylpiperazine (577 mg,2.9 mmol), ruphos Pd G3 (1599 mg,0.19 mmol), cesium carbonate (1.5 g,4.7 mmol), dioxane (6 mL). Heating overnight at 80deg.C under nitrogen, concentrating the reaction solution, and purifying with silica gel column (PE: EA=2:1) to obtain product 1a-3 (300 mg, yellow solid), yield: 44%. LCMS (ESI) m/z 354.1[ M+H ] ⁺; rt=1.80 min (3.00 min).

Step 3: tert-butyl (R) -4- (2-fluoro-6- (methylcarbamoyl) pyridin-3-yl) -3-methylpiperazine-1-carboxylate

To a 50mL single-necked flask was added 1a-3 (100 mg,0.28 mmol) and a solution of methylamine in ethanol (2 mL), and the reaction mixture was stirred overnight. Concentration gave 1a-4 (80 mg, yellow solid), yield: 80%. LCMS (ESI) M/z297.1[ M-56+H ] ⁺; rt=1.70 min (3.00 min).

Step 4: synthesis of (R) -6-fluoro-N-methyl-5- (2-methylpiperazin-1-yl) pyridine carboxamide hydrochloride

Into a 20mL one-necked flask, 1a-4 (80 mg,0.23 mmol), EA (2 mL), a 4M dioxane solution of hydrochloric acid (2 mL) was added, and the mixture was stirred at room temperature for 2 hours. Concentration gave 1a (65 mg, yellow oil), yield: 100%. LCMS (ESI) m/z 253.2[ M+H ] ⁺; rt=0.99 min (3.00 min).

Synthesis of intermediate 2a N-methyl-5- (piperazin-1-yl) pyridine amide hydrochloride

Step 1: synthesis of tert-butyl 4- (2-fluoro-6- (methoxycarbonyl) pyridin-3-yl) piperazine-1-carboxylate

1A-2 (500 mg,2.1 mmol), 1-t-butoxycarbonylpiperazine (600 mg,3.2 mmol), ruphos Pd G (180 mg,0.21 mmol), cesium carbonate (1.7 g,5.2 mmol), dioxane (15 mL) were added to a 50mL flask, heated at 80 ℃ overnight under nitrogen, the reaction mixture concentrated, and purified by silica gel column (PE: ea=2:1) to give 2a-1 (640 mg, yellow solid), yield: 88%. LCMS (ESI) m/z 340.1[ M+H ] ⁺; rt=1.74 min (3.00 min).

Step 2: synthesis of 5- (4- (t-butoxycarbonyl) piperazin-1-yl) -6-fluoropicolinic acid

To a dry 50mL single-neck flask was added 2a-1 (320 mg,0.94 mmol), tetrahydrofuran (8 mL), lithium hydroxide monohydrate (200 mg,4.7 mmol) in water (8 mL) was added dropwise, stirred for 2 hours, the reaction solution was adjusted to ph=6 with 1M hydrochloric acid solution, and the reaction solution was concentrated through a reverse phase column (2% -40% acetonitrile in water) to give 2a-2 (300 mg, yellow solid), yield: 92%. LCMS (ESI) m/z 326.1[ M+H ] ⁺; rt=1.26 min (3.00 min).

Step 3: synthesis of tert-butyl 4- (2-fluoro-6- (methylcarbamoyl) pyridin-3-yl) piperazine-1-carboxylate

To a dry 50mL single vial was added compound 2a-2 (300 mg,0.92 mmol), 1-hydroxybenzotriazole (149 mg,1.1 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (211 mg,1.1 mmol), DIEA (237 mg,1.84 mmol), DMF (6 mL), methylamine hydrochloride (123 mg,1.84 mmol) and stirred overnight. The reaction solution was added with water (20 mL), extracted three times with EA (30 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered with suction, and concentrated in a reverse phase column (20% -70% acetonitrile in water) to give 2a-3 (250 mg, white solid), yield: 80%. LCMS (ESI) m/z 283.1[ M-100+H ] ⁺; rt=1.66 min (3.00 min).

Step 4: synthesis of 6-fluoro-N-methyl-5- (piperazin-1-yl) pyridine amide hydrochloride

To a dry 20mL single-necked flask was added 2a-3 (250 mg,0.74 mmol), EA (3 mL), 4M dioxane hydrochloride solution (1 mL) at room temperature, stirred at room temperature for 2 hours, and concentrated to give product 2a (200 mg, yellow solid), yield: 100%. LCMS (ESI) m/z 239.1[ M+H ] ⁺; rt=0.91 min (3.00 min).

¹H-NMR(600MHz,CD₃OD):7.98-7.96(m,1H),7.67-7.64(m,1H),3.50-3.48(m,4H),3.45-3.43(m,4H),2.93(d,J＝3.6Hz,3H).

Synthesis of intermediate 3a N-methyl-5- (piperazin-1-yl) pyridine carboxamide hydrochloride

Synthetic method referring to the synthesis of intermediate 1a, except that Boc-piperazine was used instead of (R) -4-Boc-2-methylpiperazine in step 2 in the preparation method of intermediate 1a, and step 2 was performed with compound 1a-1 instead of compound 1 a-2. LCMS (ESI) m/z 221.2[ M+H ] ⁺; rt=0.285 min (6.00 min).

Intermediate 4a:7- (bromomethyl) pyrrole [1,2-a ] quinoxalin-4 (5H) -one

Step 1: synthesis of methyl 1- (4- (methoxycarbonyl) -2-nitrophenyl) -1H-pyrrole-2-carboxylate

In a 100mL single vial containing 50mL DMF was added 4a-1 (4.0 g,20.1 mmol), 1H-pyrrole-2-carboxylic acid methyl ester (2.76 g,22.1 mmol) and cesium carbonate (7.80 g,24.1 mmol), reacted at 60℃for 3H, the reaction solution diluted with water, extracted twice with EA, washed with saturated sodium chloride solution, dried and concentrated and column chromatographed (PE: EA=5:1) to give 4a-2 (4.0 g, yellow solid). Yield: 65.5%, LCMS (ESI) m/z 305.1[ M+H ] ⁺; rt=1.603 min (2.50 min).

Step 2: synthesis of methyl 4-oxo-4, 5-dihydropyrrolo [1,2-a ] quinoxaline-7-carboxylate

In a 100mL single-necked flask containing 30mL of acetic acid, 4a-2 (2.0 g,6.58 mmol), iron powder (7.37 g,132 mmol) was added, the reaction was allowed to react at 110℃for 3 hours, EA was added to the reaction solution, the filtrate was directly filtered, concentrated to dryness, saturated sodium bicarbonate solution was added, the extract was extracted three times with EA, the organic phase was washed twice with water, saturated brine was washed once, dried over anhydrous sodium sulfate, and concentrated to give 4a-3 (550 mg, yellow solid). Yield: 34.6%, LCMS (ESI) m/z 243.2[ M+H ] ⁺; rt=0.50 min (2.00 min).

Step 3: synthesis of 7- (hydroxymethyl) pyrrole [1,2-a ] quinoxalin-4 (5H) -one

In a 50mL three-necked flask containing 10mL tetrahydrofuran, 4a-3 (300 mg,1.24 mmol) at room temperature was cooled to 0deg.C, then a tetrahydrofuran solution (3.10 mL,3.10mmol,1 mol/L) of lithium aluminum hydride was slowly added, the reaction was carried out at 0deg.C for 2 hours, the reaction solution was quenched with water, and a small amount of 15% sodium hydroxide solution was added, stirred at room temperature for 20 minutes, then anhydrous magnesium sulfate was added to dry, filtered, and the filtrate was concentrated to give 4a-4 (180 mg, yellow solid) in yield ：67.9％.LCMS(ESI):m/z215.1[M+H]⁺;RT＝0.33min(2.00min).¹H NMR(400MHz,DMSO-d₆):δ11.25(s,1H),8.16-8.15(m,1H),7.99(d,J＝8.4Hz,1H),7.29-7.28(m,1H),7.14-7.12(m,1H),7.02-7.01(m,1H),6.68-6.66(m,1H),5.32-5.30(m,1H),4.53(d,J＝4.0Hz,2H).

Step 4: synthesis of 7- (bromomethyl) pyrrole [1,2-a ] quinoxalin-4 (5H) -one

To a 100mL single-necked flask containing 8mL of methylene chloride was added 4a-4 (80 mg,0.37 mmol), triphenylphosphine (293 mg,1.12 mmol) and carbon tetrabromide (248 mg,0.75 mmol), and the mixture was reacted at room temperature for 2 hours. The reaction solution was directly concentrated to dryness to give crude 4a (80 mg, yellow solid). LCMS (ESI) m/z 277.0[ M+H ] ⁺; rt=1.429 min (2.50 min).

Intermediate 5a (6-fluoro-4-oxo-4, 5-dihydropyrrolo [1,2-a ] quinoxalin-7-yl) methylsulfonic acid methyl ester

Step 1: synthesis of 1-bromo-2, 4-difluoro-3-nitrobenzene

In a single flask containing 250mL of 5a-1 (25.0 g,157 mmol), 100mL of concentrated sulfuric acid, N-bromosuccinimide (33.6 g,188 mmol) was slowly added in an ice bath and reacted overnight at 80℃and the reaction solution was slowly poured into ice water for dilution, EA extraction twice, and column chromatography (PE) after drying and concentration gave the product 5a-2 (36 g, yellow oil). Yield: 96.6%, ¹H NMR(400MHz,DMSO-d₆): delta 8.19-8.14 (m, 1H), 7.56-7.51 (m, 1H).

Step 2: synthesis of methyl 1- (4-bromo-3-fluoro-2-nitrophenyl) -1H-pyrrole-2-carboxylate

In a 100mL single vial containing 15mL DMF was added 5a-2 (0.5 g,2.1 mmol), methyl 1H-pyrrole-2-carboxylate (244 mg,1.89 mmol) and cesium carbonate (1.37 g,4.2 mmol), reacted at room temperature for 5H, the reaction solution diluted with water, extracted twice with EA, dried and concentrated followed by column chromatography (PE: EA=5:1) to give 5a-3 (4.0 g, yellow solid). Yield: 65.5%, LCMS (ESI) m/z 345.0[ M+H ] ⁺; rt=1.783 min (2.50 min).

Step 3: synthesis of 7-bromo-6-fluoropyrrolo [1,2-a ] quinoxalin-4 (5H) -one

In a single flask containing 250mL of acetic acid was added 5a-3 (6.5 g,18.9 mmol), iron powder (21 g,37.9 mmol), reacted at 110℃for 3h, the reaction solution was directly filtered after adding EA, the filtrate was concentrated, saturated sodium bicarbonate solution was added, extracted three times with EA, and the organic phase was washed with water and dried, and concentrated to give 5a-4 (650 mg, yellow solid). Yield is good ：12.2％,¹H NMR(400MHz,DMSO-d₆):δ11.46(s,1H),8.22(m,1H),7.89(d,J＝8.0Hz,1H),7.53-7.48(m,1H),7.10(d,J＝3.2Hz,1H),6.74-6.73(m,1H).

Step 4: synthesis of 6-fluoro-7- (hydroxymethyl) pyrrole [1,2-a ] quinoxalin-4 (5H) -one

5A-4 (700 mg,2.49 mmol), chloro (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium (II) (197mg, 0.25 mmol) and tri-tert-butyltin methanol (960 mg,2.99 mmol) were added to a three-necked flask containing 15mL of anhydrous dioxane, reacted overnight at 100℃and the reaction mixture was quenched with potassium fluoride solution, filtered, and the filtrate was concentrated to give 5a-5 (430 mg, yellow solid) in yield ：74.4％.LCMS(ESI):m/z 233.1[M+H]⁺;RT＝0.877min(2.00min).¹H NMR(400MHz,DMSO-d₆):δ11.23(s,1H),8.18-8.17(m,1H),7.87(d,J＝8.4Hz,1H),7.28-7.24(m,1H),7.08-7.07(s,1H),6.72-6.70(m,1H),5.36-5.33(m,1H),4.59(d,J＝5.6Hz,2H).

Step 5: synthesis of methyl (6-fluoro-4-oxo-4, 5-dihydropyrrolo [1,2-a ] quinoxalin-7-yl) methylsulfonate

5A-5 (150 mg,0.65 mmol) and triethylamine (164 mg,1.63 mmol) were added to a 100mL three-necked flask containing 8mL tetrahydrofuran, and methanesulfonyl chloride (89 mg,0.78 mmol) was added while cooling on ice, and the reaction was carried out at room temperature for 3 hours. The reaction solution was directly concentrated to dryness to give 5a (120 mg, yellow solid). LCMS (ESI) m/z 309.1[ M-H ] ^-; rt=1.241 min (2.50 min).

Intermediate 6a 7- (1-bromoethyl) pyrrolo [1,2-a ] quinoxalin-4 (5H) -one

Step 1: synthesis of methyl 1- (4-bromo-2-nitrophenyl) -1H-pyrrole-2-carboxylate

6A-1 (500 mg,2.27 mmol), methyl 1H-pyrrole-2-carboxylate (341.26 mg,2.73 mmol) and cesium carbonate (1.48 g,4.55 mmol) were dissolved in DMF (10 mL) and the reaction solution stirred at 80℃for 16H. The reaction mixture was diluted with water (20 mL), extracted with EA (20 mL. Times.3), and concentrated by drying to give 6a-2 (383 mg,1.18mmol, yield: 51.9%) as a result of column chromatography (PE: EA=10:1). LCMS (ESI) m/z 325.0[ M ]; rt=1.341 min (2.50 min).

Step2: synthesis of 7-bromopyrrolo [1,2-a ] quinoxalin-4 (5H) -one

6A-2 (1.5 g,4.61 mmol) and iron powder (5.15 g,92.27 mmol) were dissolved in acetic acid (50 mL) and stirred at 110℃for 3 hours. Concentrating to obtain a dark brown solid, dissolving the solid in dichloromethane, washing the organic phase with supersaturated aqueous sodium bicarbonate (30 mL. Times.2) and saturated saline (30 mL. Times.2), drying over anhydrous sodium sulfate, and concentrating under vacuum to obtain 6a-3 (1.3 g,4.49mmol, yield: 100%, yellow solid) ).¹H NMR(400MHz,DMSO-d₆):δ11.35(s,1H),8.20(s,1H),8.03(d,J＝8.4Hz,1H),7.45(s,1H),7.37(d,J＝8.4Hz,1H),7.06(d,J＝3.6Hz,1H),6.71(d,J＝2.8Hz,1H).

Step 3: synthesis of 7-acetylpyrrolo [1,2-a ] quinoxalin-4 (5H) -one

6A-3 (500 mg,1.9 mmol), tributyl (1-ethoxyethylene) tin (1.37 g,3.8 mmol) and PdCl ₂(PhP₃ (200 mg,0.29 mmol) were dissolved in a solution of 1, 4-dioxane (25 mL) and reacted at 100℃for 16 hours. Cooled to room temperature, 1M aqueous hydrochloric acid (10 mL) was added to the reaction solution and stirred for 10 minutes. EA (10 mL. Times.3) was extracted, dried and concentrated followed by column chromatography (tetrahydrofuran: petroleum ether=1:1) to give crude 6a-4 (220 mg,0.97mmol, yield: 51%). LCMS (ESI) m/z 227[ M+H ] ⁺; rt=0.947 (2.50 min).

Step 4: synthesis of 7- (1-hydroxyethyl) pyrrolo [1,2-a ] quinoxalin-4 (5H) -one

6A-4 (220 mg,0.97 mmol) was dissolved in methanol (5 mL) and sodium borohydride (73.58 mg,1.94 mmol) was added slowly at 0deg.C. The reaction was carried out at room temperature for 2 hours. Saturated aqueous ammonium chloride (5 mL) was quenched, extracted with EA (10 ml×3), and concentrated by drying followed by column chromatography (dichloromethane: methanol=1:1) to give 6a-5 (200 mg,0.88mmol, yield: 90.3%, white solid) ).LCMS(ESI):m/z 229.1[M-H]^-;RT＝0.877min(2.50min).¹H NMR(400MHz,DMSO-d₆):δ11.23(s,1H),8.15(s,1H),7.98(d,J＝8.4Hz,1H),7.33(s,1H),7.16(d,J＝8.4Hz,1H),7.02(d,J＝3.6Hz,1H),6.67(t,J＝3.2Hz,1H),5.30(d,J＝4.0Hz,1H),4.78-4.75(m,1H),1.35(d,J＝6.4Hz,3H).

Step 5: synthesis of 7- (1-bromoethyl) pyrrolo [1,2-a ] quinoxalin-4 (5H) -one

6A-5 (200 mg,0.87 mmol) was dissolved in dichloromethane (5 mL) and phosphine tribromide (711.55 mg,2.63 mmol) was added slowly at 0deg.C. The reaction was carried out at room temperature for 2 hours. The reaction was quenched with aqueous (5 mL), extracted with ethyl acetate (10 mL. Times.3), and dried and concentrated to give crude product 6a (200 mg,0.69mmol, yield: 79.0%, white solid). LCMS (ESI) m/z 289.1[ M-H ] ^-; rt= 1.447 (2.50 min).

Intermediate 7a:

the synthesis method referred to intermediate 6a, except that 5a-4 prepared in step 3 of intermediate 5a was used in place of 6a-3 used in step 4 in intermediate 6 a. LCMS (ESI) m/z 310.0[ M+H ] ⁺; rt=1.202 min (2.50 min).

Intermediate 8a 7- (bromomethyl) pyrazolo [1,5-a ] quinoxalin-4 (5H) -one

Step 1: synthesis of N- (5-bromo-2-fluorophenyl) -1H-pyrazole-5-carboxamide

In a 100mL single vial containing 30mL DMF was added 8a-1 (3.0 g,15.8 mmol), 1H-pyrazole-5-carboxylic acid (1.95 g,17.4 mmol), DIEA (6.1 g,47.4 mmol) and HATU (9.0 g,23.7 mmol), reacted at room temperature for 16H, the reaction was diluted with water, the EA extracted twice, washed with saturated sodium chloride solution, dried and concentrated and then column chromatographed (PE: EA=5:1) to give 8a-2 (2.0 g, yellow solid). Yield: 44.8%, LCMS (ESI) m/z 286.0[ M+H ] ⁺; rt=1.03 min (2.00 min).

Step 2: synthesis of 7-bromopyrazol [1,5-a ] quinoxalin-4 (5H) -one

To a three-necked flask containing 30mL of N, N-dimethylacetamide was added 8a-2 (2.0 g,7.07 mmol), sodium hydride (560 mg,14.1 mmol) was added under ice bath, and the mixture was reacted at 145℃for 16 hours, and after the reaction mixture was quenched with a saturated ammonium chloride solution, a solid was separated out, and after filtration and drying, 8a-3 (1.5 g, yellow solid) was obtained. Yield is good ：80.7％,LCMS(ESI):m/z 264.0/266.0[M+H]⁺;RT＝0.93min(2.00min).¹H NMR(400MHz,DMSO-d₆):δ11.50(s,1H),8.03-7.97(m,2H),7.47-7.39(m,2H),7.10(s,1H).

Step 3: synthesis of 7- (hydroxymethyl) pyrazolo [1,5-a ] quinoxalin-4 (5H) -one

In a single vial containing 10mL dioxane was added 8a-3 (500 mg,1.90 mmol), tributyltin methanol (671 mg,2.09 mmol) and XPhos Pd G2 (75 mg,0.095 mmol), and reacted at 80℃for 16h under nitrogen protection, the reaction solution was directly concentrated to column chromatography (DCM: methanol=15:1) to give the product 8a-4 (190 mg, yellow solid), yield: 45.0%. LCMS (ESI) m/z 214.1[ M-H ] ^-; rt=0.984 min (2.50 min).

Step 4: synthesis of 7- (bromomethyl) pyrazolo [1,5-a ] quinoxalin-4 (5H) -one

In a 100mL single-necked flask containing 8mLDCM was added 8a-4 (80 mg,0.37 mmol), triphenylphosphine (293 mg,1.12 mmol) and carbon tetrabromide (248 mg,0.74 mmol), and the mixture was reacted at room temperature for 2 hours. The reaction solution was directly concentrated to dryness to give product 8a (80 mg, yellow solid). Crude, LCMS (ESI) m/z 278.0[ M+H ] ⁺; rt= 1.405min (2.50 min).

Intermediate 9a 7- (1-bromoethyl) pyrazolo [1,5-a ] quinoxalin-4 (5H) -one

The synthesis method referred to intermediate 6a, except that 8a-3 prepared in step 2 of intermediate 8a was used in place of 6a-3 used in step 4 in intermediate 6 a. LCMS (ESI) m/z 290.0[ M-H ] ^-; rt=1.300 min (2.50 min).

Synthesis of intermediate 10a (6-fluoro-4-oxo-4, 5-dihydropyrazolo [1,5-a ] quinoxalin-7-yl) methanesulfonic acid methyl ester

Step 1: synthesis of N- (2, 6-difluorophenyl) acetamide

In a one-necked flask, compound 10a-1 (2.00 g,15.49 mmol), DCM (20 mL), and acetic anhydride (1.53 mL,16.27 mmol) were sequentially added. Stirred at room temperature and reacted for 16 hours. The reaction solution was concentrated under reduced pressure. The residue was diluted with water (20 mL), the pH was adjusted to 8 with saturated aqueous sodium bicarbonate solution, and a white solid precipitated. Filtration and collection of the filter cake gave 10a-2 (2.60 g, white solid), yield ：98.07％,LCMS(ESI):m/z 172.2[M+H]⁺;RT＝1.036min(2.50min).1H-NMR(400MHz,DMSO-d6)：δ9.69(s,1H),7.37-7.30(m,1H),7.17-7.13(m,2H),2.08(s,3H).

Step 2: synthesis of N- (3-bromo-2, 6-difluorophenyl) acetamide

In a flask, compound 10a-2 (1.00 g,5.84 mmol), concentrated sulfuric acid (10 mL) and N-bromosuccinimide (1.04 g,5.84 mmol) were added sequentially at room temperature. Stirred at room temperature and reacted for 16 hours. The reaction mixture was poured into ice water (50 mL) and a white solid was precipitated. Filtration under reduced pressure collected the filter cake to give 10a-3 (1.26 g, white solid), yield: 86.24%, LCMS (ESI) m/z 291.0[ M+H+MeCN ] ⁺; rt=1.290 min (2.50 min).

Step 3: synthesis of 3-bromo-2, 6-difluoroaniline

To a dry one-necked flask, 10a-3 (1.00 g,4.00 mmol), ethanol (6 mL) and concentrated hydrochloric acid (3 mL) were sequentially added at room temperature. Heated to 70℃and reacted for 2 hours. The reaction solution was concentrated under reduced pressure. The residue was extracted with saturated aqueous sodium bicarbonate to adjust ph=8, ea (30 ml×2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 10a-4 (650 mg, yellow solid), yield ：78.14％,LCMS(ESI):m/z 207.9[M+H+MeCN]⁺;RT＝1.636min(2.50min).¹H-NMR(400MHz,DMSO-d6)：δ6.93-6.88(m,1H),6.81-6.76(m,1H),5.54(s,2H).

Step 4: synthesis of N- (3-bromo-2, 6-difluorophenyl) -1H-pyrazole-5-carboxamide

1H-pyrazole-5-carboxylic acid (3.23 g,28.85 mmol), anhydrous dichloromethane (40 mL), oxalyl chloride (2.28 mL,26.92 mmol) and N, N-dimethylformamide (0.10 mL) were sequentially added to a dry 100mL three-necked flask at 0deg.C. After stirring at room temperature and 3 hours of reaction, the mixture was slowly added to a mixed solution of 10a-4 (2.00 g,9.62 mmol) in DCM (30 mL) and pyridine (15 mL) at 0deg.C. Stirred at room temperature and reacted for 16 hours. The reaction mixture was diluted with saturated aqueous ammonium chloride (100 mL) and extracted with methylene chloride (80 mL. Times.2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether: ethyl acetate=5:1) to give 10a-5 (1.80 g, off-white solid), yield ：61.97％,LCMS(ESI):m/z 303.9[M+H]⁺;RT＝1.287min(2.50min).1H-NMR(400MHz,DMSO-d6)：δ13.49(s,1H),10.01(s,1H),7.92(d,J＝1.6Hz,1H),7.75-7.69(m,1H),7.26-7.21(m,1H),6.77(t,J＝2.0Hz,1H).

Step 5: synthesis of 7-bromo-6-fluoropyrazolo [1,5-a ] quinoxalin-4 (5H) -one

Sodium hydride (60% wt,185mg,4.63 mmol) and N, N-dimethylacetamide (5 mL) and 10a-5 (700 mg,2.32 mmol) in sequence were added to a dry 50mL three-necked flask at 0deg.C. Heated to 145℃and reacted for 16 hours. Diluted with saturated aqueous ammonium chloride (50 mL) and precipitated, filtered, and the filtrate was extracted with ethyl acetate (15 mL. Times.2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was slurried with ethyl acetate (10 mL), filtered, and the filter cake collected to give crude product 10a-6 (180 mg, yellow solid) ),LCMS(ESI):m/z 282.0[M+H]⁺;RT＝1.470min(2.50min).¹H-NMR(400MHz,DMSO-d6)：δ12.08(s,1H),8.13(d,J＝2.0Hz,1H),7.89(dd,J1＝1.6Hz,J2＝9.6Hz,1H),7.59-7.56(m,1H),7.22(d,J＝2.0Hz,1H).

Step 6: synthesis of 6-fluoro-7- (hydroxymethyl) pyrazolo [1,5-a ] quinoxalin-4 (5H) -one

10A-6 (180 mg,0.64 mmol), 1, 4-dioxane (5 mL) and chloro (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium (50 mg,0.06 mmol) were sequentially added to a dry 25mL three-necked flask at room temperature. 3 times with nitrogen, tributyltin methanol (246 mg,0.77 mmol) was added. Heated to 80℃and reacted for 16 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was slurried with ethyl acetate (10 mL), filtered, and the cake collected to give compound 10a-7 (48 mg, yellow solid), yield: 32.25%, LCMS (ESI): m/z 234.1[ M+H ] +; rt=0.998 min (2.50 min).

Step 7: synthesis of methyl (6-fluoro-4-oxo-4, 5-dihydropyrazolo [1,5-a ] quinoxalin-7-yl) mesylate

To a dry 25mL single-necked flask, 10a-7 (48 mg,0.21 mmol), tetrahydrofuran (5 mL), triethylamine (0.09 mL,0.62 mmol) and methylsulfonyl chloride (0.02 mL,0.25 mmol) were added sequentially at room temperature. Stirred at room temperature and reacted for 1 hour. Concentrating under reduced pressure. Dilute with water (10 mL), extract with ethyl acetate (10 ml×2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to give 10a (58 mg, yellow solid), LCMS (ESI) m/z 312.0[ M+H ] ⁺; rt=1.223 min (2.50 min).

Synthesis of intermediate 11a 7- (bromomethyl) -6-fluoro-1-methyl-1, 5-dihydro-4H-pyrazolo [4,3-c ] quinolin-4-one

Step 1: synthesis of 3-bromo-2-fluoro-6-iodobenzoic acid

11A-1 (4.0 g,13.3 mmol) was added sequentially at room temperature to a three-necked flask containing 40mL of tetrahydrofuran, lithium diisopropylamide (7.3 mL,14.6 mmol) was added at 78℃and the reaction was allowed to proceed for 1 hour and then allowed to stand at room temperature overnight. The reaction solution was quenched with saturated ammonium chloride, diluted with water, adjusted to pH 8-9 with dilute sodium hydroxide solution, extracted twice with EA, the aqueous phase adjusted to pH acidic with dilute hydrochloric acid, extracted twice with EA, the organic phases combined and washed twice with water, once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 11a-2 (3.2 g, yellow solid). Yield: 69.8%, ¹H NMR(400MHz,CDCl₃): δ7.68 (d, j=8.4 hz, 1H), 7.57-7.53 (m, 1H).

Step 2: synthesis of (3-bromo-2-fluoro-6-iodophenyl) carbamic acid tert-butyl ester

In a three-necked flask containing 40mL of toluene, 11a-2 (3.0 g,8.7 mmol), diphenyl azide phosphate (2.87 g,10.4 mmol), triethylamine (1.05 g,10.4 mmol), tert-butanol (tert-butanol) were added after 1 hour of reaction at 120℃and the reaction was continued for 3 hours, and the reaction solution was directly concentrated to dryness (PE/EA=30/1) to give 11a-3 (2.9 mg, white solid). LCMS (ESI): m/z 413.8[ M-H ] ^-; rt=1.684 min (2.50 min).

Step 3: synthesis of tert-butyl (3-bromo-2-fluoro-6- (1-methyl-1H-pyrazol-5-yl) phenyl) carbamate

In a flask containing 15mL dioxane and 5mL water was added 11a-3 (1.4 g,3.37 mmol), 1-methyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-pyrazole (431 mg,3.04 mmol), potassium carbonate (1.40 g,10.1 mmol), 1-bis (diphenylphosphine) bis-iron palladium dichloride (catalytic amount). The reaction was stirred at 70℃for 3h. The reaction solution was cooled to room temperature, diluted with water, extracted twice with EA, the organic phases were combined, washed twice with brine, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (EA: pe=1:10). Yield 11a-4 (900 mg, yellow solid) was obtained: 72.3%; LCMS (ESI) m/z 370.0[ M+H ] ⁺; rt=1.444 min (2.50 min).

Step 4: synthesis of 3-bromo-2-fluoro-6- (1-methyl-1H-pyrazol-5-yl) aniline

In a 50mL single-necked flask containing 15mL of methanol hydrochloride was added 11a-4 (900 mg,2.44 mmol), after 3h of reaction at room temperature, the mixture was directly dried by spin-drying, diluted with water, pH was adjusted to alkaline with saturated sodium bicarbonate solution, extracted twice with ethyl acetate, the organic phases were combined and washed twice with water, saturated brine was washed once, dried over anhydrous sodium sulfate, and concentrated to give 11a-5 (580 mg, yellow solid) yield: 88.4%; LCMS (ESI) m/z 270.3/272.3[ M+H ] ⁺; rt=1.22 min (2.00 min).

Step 5: synthesis of 7-bromo-6-fluoro-1-methyl-1, 5-dihydro-4H-pyrazolo [4,3-c ] quinolin-4-one

In a 50mL single-necked flask containing 15mL DMF, 11a-5 (780 mg,2.9 mmol) and carbonyldiimidazole (1.41 g,8.70 mmol) were added, the reaction was carried out overnight at 170℃and the reaction mixture was diluted with water to precipitate a solid, which was filtered and dried to give 11a-6 (650 mg, brown solid) in yield ：76.0％.LCMS(ESI):m/z 296.2[M+H]⁺;RT＝0.85min(2.00min).¹H NMR(400MHz,DMSO-d₆):δ11.51(s,1H),8.15(s,1H),7.97(d,J＝8.8Hz,1H),7.57-7.53(m,1H),4.35(s,3H).

Step 6: synthesis of 6-fluoro-7- (hydroxymethyl) -1-methyl-1, 5-dihydro-4H-pyrazolo [4,3-c ] quinolin-4-one

To a single vial containing 10mL dioxane was added 11a-6 (200 mg,0.68 mmol), tributyltin methanol (239 mg,0.75 mmol) and chloro (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium (II) (27 mg,0.03 mmol), and the reaction mixture was directly concentrated at 80℃for 16 hours, slurried with EA, and filtered to give 11a-7 (140 mg, yellow solid). Yield: 83.8%, LCMS (ESI) m/z 248.2[ M+H ] ⁺; rt=0.895 min (2.50 min).

Step 7: synthesis of 7- (bromomethyl) -6-fluoro-1-methyl-1, 5-dihydro-4H-pyrazolo [4,3-c ] quinolin-4-one

To a 100mL single-necked flask containing 8mL of methylene chloride were added 11a-7 (70 mg,0.28 mmol), triphenylphosphine (222 mg,0.85 mmol) and carbon tetrabromide (188 mg,0.57 mmol), and the mixture was reacted at room temperature for 2 hours. The reaction solution was directly concentrated to dryness to give 11a (50 mg, yellow solid). Crude, LCMS (ESI) m/z 310.3[ M+H ] ⁺; rt=0.64 min (2.00 min).

Intermediate 12a N-methyl-5- (pyrrolidin-3-yloxy) picolinamide hydrochloride

Step 1: synthesis of methyl 5- ((1- (tert-butoxycarbonyl) pyrrolidin-3-yl) oxy) picolinate

Sodium hydride (641 mg,16 mmol) was added to a dry 250mL three-necked flask, nitrogen was substituted 3 times, tetrahydrofuran (40 mL) was added under an ice bath, 12a-1 (2 g,10.7 mmol), 12a-2 (1.66 g,10.7 mmol), and the reaction was carried out at room temperature for one hour. The reaction mixture was extracted with ethyl acetate (30 mL. Times.3), and the organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether: ethyl acetate=3:1) to give 12a-3 (1 g, yellow oil), yield: 29%, LCMS (ESI) m/z 323.2[ M+H ] ⁺; rt=1.59 min (2.50 min)

Step 2:3- ((6- (methylcarbamoyl) pyridin-3-yl) oxy) pyrrolidine-1-carboxylic acid tert-butyl ester

A dry 100mL single-necked flask was charged with 12a-3 (400 mg,1.24 mmol) and an ethanol solution of methylamine (30%, 6 mL). Stirred at room temperature and reacted for 16 hours. The reaction solution was concentrated under reduced pressure. The residue was diluted with water (10 mL) and extracted with ethyl acetate (10 mL. Times.3). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 12a-4 (380 mg, yellow oil)

LCMS(ESI):m/z 266[M+H]⁺;RT＝1.560min(2.50min).¹H-NMR(400MHz,DMSO-d₆)：δ8.57(d,J＝4Hz,1H),8.28(d,J＝2.4Hz,1H),7.98(d,J＝8.8Hz,1H),7.58-7.56(m,1H),5.18(s,1H),4.88(d,J＝3.2Hz,1H),3.44-3.40(m,3H),3.11(s,1H),2.79(d,J＝4.4Hz,3H),1.40(d,J＝5.6Hz,9H),1.19-1.56(m,1H).

Step 3: synthesis of N-methyl-5- (pyrrolidin-3-yloxy) pyridine amide hydrochloride

In a dry 100mL single-necked flask, 12a-4 (380 mg,1.18 mmol), methylene chloride (6 mL) and dioxane solution of hydrochloric acid (4.0M, 6 mL) were added under ice-bath. Stirred at room temperature and reacted for 16 hours. The reaction mixture was concentrated under reduced pressure to give 12a (300 mg, crude yellow solid), LCMS (ESI) m/z 220[ M+H ] ⁺; rt=0.603 min (2.5 min).

Synthesis of intermediate 13a 7- (1-bromoethyl) -6-fluoropyrazolo [1,5-a ] quinoxalin-4 (5H) -one

Synthetic method referring to intermediate 6a, except that 10a-6 prepared in step 5 of intermediate 10a was used in place of 6a-3 used in step 4 in intermediate 6 a. LCMS (ESI) m/z 310.0[ M-H ] ^-; rt=1.548 min (2.50 min).

Synthesis of Compound 1

In a 25mL single port flask containing 5mL of acetonitrile, 4a (80 mg,0.29 mmol), 3a (74 mg,0.29 mmol) and DIEA (187 mg,1.45 mmol) were added and reacted at 70℃for 2 hours to prepare compound 1 (31.68 mg, white solid) by Pre-HPLC (formic acid), yield ：26.2％.LCMS(ESI):m/z 417.1[M+H]⁺;RT＝3.144min(6.00min).¹H NMR(400MHz,DMSO-d₆):δ11.23(s,1H),8.39(d,J＝5.2Hz,1H),8.27(d,J＝2.8Hz,1H),8.17-8.16(m,1H),8.02(d,J＝8.4Hz,1H),7.83(d,J＝8.8Hz,1H),7.41-7.38(m,1H),7.31(s,1H),7.18(d,J＝8.0Hz,1H),7.03-7.02(m,1H),6.69-6.67(m,1H),3.58(s,2H),3.41-3.31(m,4H),2.78(d,J＝5.2Hz,3H),2.57-2.50(m,4H).

Compound 2-Compound 13

According to the procedure described for compound 1, intermediates 4a and 3a were each replaced with an intermediate in the following table, compound 2-compound 13 was synthesized, the structural formula of each example compound being as shown in the preceding table

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The invention is further described in detail below in connection with specific examples and with reference to the data. It should be understood that these examples are for the purpose of illustrating the invention only and are not intended to limit the scope of the invention in any way as regards the particular combination of the invention, the method of preparation and the function and effect thereof. The beneficial effects of the pharmaceutical combinations of the invention can also be determined by other test models known to those skilled in the relevant art.

Biological examples

Experimental example 1: evaluation of PARP1/2 inhibitory Activity of Compounds

In assays using Histone as a substrate, PARP1/2 inhibitory activity of the compounds of the present disclosure was tested. Experimental objective the compounds of the present application were tested for their IC50 values for inhibition of PARP1/2 enzyme activity according to established experimental methods. AZD-2281 (Olaparib) was used as a positive control compound.

Experimental reagent:

recombinant human PARP1 protein (Abcam, cat. Ab27963); recombinant human PARP2 protein (BPS, cat.80502); recombinant histones H1(Active Motif,cat.81126);NAD+,Biotin-Labeled(BPS,cat.80610);SuperBlock(TBS)Blocking Buffer(Thermo Scientific^TM,cat.37535);Streptavidin(HRP)(Abcam,cat.ab7403);LumiGLO Peroxidase Chemiluminescent Substrate Kit(Seracare,cat.5430-0040);20xPBS(CST,cat.9808S);20xPBST(CST,cat.9809S);AZD2281(Selleck,cat.S1060)

Experiment method one: PARP1 inhibitory Activity

1. Compound preparation compound was diluted with DMSO in 384 well plates to 1000-fold final concentration for use.

2. Coating a microplate:

1) Histone was diluted with PBS, 25uL Histone mixture was added to each well and incubated for 2h.

2) Each well was washed 5 times with PBST solution. The solution was removed on a clean paper towel.

3) 75UL of Blocking buffer was added to each well and incubated for 1h at room temperature.

4) Each well was washed 5 times with PBST solution. The solution was removed on a clean paper towel.

3. Ribosylation reaction:

1) 25nL of spare 1000-fold final concentration of compound was transferred to 384 reaction plates, and 25nL of 100% DMSO was added to Min control wells and Max control wells, respectively.

2) PARP1 solution was prepared at 2.5 times final concentration in 1x Assay buffer.

3) Adding 10uL of enzyme solution to each of the compound wells and the Max control wells; 10uL of 1 Xassay buffer was added to Min control wells.

4) Centrifugation at 1000rpm for 60 seconds and incubation at room temperature for 15 minutes.

5) 1.67 Times of final concentration of substrate solution was prepared by using 1 Xassay buffer, 500. Mu.M NAD+ was added to the substrate solution, and 15. Mu.L of substrate solution was added to each well to initiate the reaction.

6) Centrifuge at 1000rpm for 60 seconds and incubate at room temperature for 2h.

7) Each well was washed 5 times with PBST solution. The solution was removed on a clean paper towel.

4. And (3) detection: 1) A Strepitavidin-HRP solution was prepared, 25. Mu.L was added to each well, centrifuged at 1000rpm for 60 seconds, and incubated at room temperature for 30 minutes. 2) Each well was washed 5 times with PBST solution. The solution was removed on a clean paper towel. 3) 50 mu L ELISA Chemiluminescent Substrate were added per well. 4) Centrifuge at 1000rpm for 60 seconds, read with EnSight minutes later.

5. Data analysis, inhibition rate was calculated using the following formula: inhibition% = (maximum signal-compound signal)/(maximum signal-minimum signal) ×100 where "minimum signal" is negative control Kong Junzhi and "maximum signal" is positive control Kong Junzhi.

Fitting dose-response curve: the log of concentration was used as the X-axis and the percent inhibition was Y-axis, using the analytical software GraphPadPrism5 log (inhibitor) vs. response-variable slope fit curve, to give the compounds of the present disclosure IC50 values for inhibition of enzyme activity. The fitting formula is: y=bottom+ (top-bottom)/(1+10 > ((logIC 50 0-X) × HillSlope)).

And the experimental method II comprises the following steps: PARP2 inhibitory Activity

PARP2 inhibition Activity assay the same assay as assay one, except that recombinant human PARP2 solution was used instead of the PARP1 solution used in "step 2" 3. Ribosylation reaction ".

TABLE 1 IC50 for the inhibition of PARP1/2 enzymes by the compounds of the present disclosure

From the results in table 1, it can be seen that the compounds of the present invention have high selectivity for PARP1, and may reduce toxicity produced by PARP2 without significantly reducing the efficacy. The inventors have also unexpectedly found that the compounds of the present disclosure have good physical and chemical stability, good bioavailability (e.g., low clearance) and good drug formation. Therefore, the compound has fewer side effects than olaharib (AZD-2281) and has high clinical application value.

Claims (10)

1. A compound shown in a general formula (I), or stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, crystal forms, solvates, hydrates or prodrugs thereof,

Wherein,

X ¹ is independently selected from the group consisting of-N-, -NR ¹⁴-、-CR⁷ -, O, and S;

X ² is independently selected from the group consisting of-N- -NR ¹⁵ -and-CR ⁸ -, O and S;

X ³ is independently selected from the group consisting of-N- -NR ¹⁶ -and-CR ⁹ -, O and S;

X ⁴、X⁸ are each independently selected from the group consisting of-N-and-C-;

Is a single bond or a double bond; and X ¹、X²、X³、X⁴、X⁸ together form a five membered heteroaryl group;

r ⁷、R⁸、R⁹ is independently selected from hydrogen, halogen, unsubstituted or substituted C ₃-C₆ cycloalkyl or unsubstituted or substituted C ₁-C₆ alkyl; r ⁷、R⁸、R⁹ is preferably hydrogen, halogen or C ₁-C₄ alkyl; r ⁷、R⁸、R⁹ is more preferably hydrogen, F or methyl;

R ¹⁴、R¹⁵、R¹⁶ is independently selected from hydrogen, unsubstituted or substituted C ₃-C₆ cycloalkyl or unsubstituted or substituted C ₁-C₆ alkyl; r ¹⁴、R¹⁵、R¹⁶ is preferably selected from hydrogen or C ₁-C₃ alkyl; more R ¹⁴、R¹⁵、R¹⁶ is preferably selected from methyl;

X ⁵、X⁶ is each independently selected from the group consisting of-N-and-CR ¹⁰-;R¹⁰ is selected from the group consisting of hydrogen, halogen, cyano, unsubstituted or substituted C ₁-C₆ alkoxy, unsubstituted or substituted C ₁-C₆ alkyl; r ¹⁰ is preferably hydrogen, halogen, cyano, C ₁-C₄ alkyl; r ¹⁰ is more preferably hydrogen or fluorine, chlorine or methyl;

X ⁷ is-N-and-CR ¹⁷-;R¹⁷ is selected from hydrogen, halogen, cyano, unsubstituted or substituted C ₁-C₆ alkoxy, unsubstituted or substituted C ₁-C₆ alkyl; r ¹⁷ is preferably hydrogen, halogen, cyano, C ₁-C₄ alkyl; r ¹⁷ is more preferably hydrogen or fluorine, chlorine or methyl;

R ¹、R^1'、R²、R³、R⁴、R⁵ is independently selected from hydrogen, unsubstituted or substituted C ₁-C₆ alkyl; or R ⁴、R⁵ together with the carbon atom to which they are attached form a C ₃-C₆ cycloalkyl group;

s and n are each independently selected from 0, 1 or 2;

Y is selected from N or CH;

r ⁶ is selected from:

Each independently R ¹¹ is selected from halogen, cyano, C ₁-C₃ alkoxy, carbonyl, -CONHR ¹³, amino, preferably selected from halogen, -CONHR ¹³ and cyano;

m is 0,1,2 or 3;

R ¹² is selected from hydrogen, cyano, halogen, unsubstituted or substituted C ₁-C₄ alkyl;

R ¹³ is hydrogen, unsubstituted or substituted C ₁-C₆ alkyl, unsubstituted or substituted C ₃-C₈ cycloalkyl, unsubstituted or substituted C ₁-C₆ alkoxy, unsubstituted or substituted 3-8 membered heterocycloalkyl; preferably, R ¹³ is hydrogen, unsubstituted or halogen substituted C ₁-C₄ alkyl, C ₃-C₆ cycloalkyl, C ₁-C₆ alkoxy; the heterocycloalkyl group refers to a heterocycloalkyl group containing 1 to 3 heteroatoms selected from N, O, S;

R¹、R^1'、R²、R³、R⁴、R⁵、R⁷、R⁸、R⁹、R¹⁰、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷ Wherein said substitution is by one or more selected from the group consisting of C ₁-C₄ alkyl, halogen, hydroxy, cyano, amino, carboxy, C ₃-C₆ cycloalkyl;

The conditions are as follows:

When X ⁵ and X ⁸ are both-N-, at least one of X ¹、X²、X³、X⁴ is-N-;

When X ⁴ and X ⁵ are both-N-, X ³ is also-N-;

When X ³ is oxygen and X ¹、X²、X⁴、X⁸ is-C-, X ⁷ is-CR ¹⁷ -, R ¹、R^1'、R¹⁷ is not both hydrogen;

The preferred conditions are:

When X ⁸ is-N-, at least one of X ¹、X²、X³ is-N-;

When X ⁴ and X ⁵ are both-N-, X ³ is also-N-;

When X ³ is oxygen and X ¹、X²、X⁴、X⁸ is-C-, X ⁷ is-CR ¹⁷ -, R ¹、R^1'、R¹⁷ is not both hydrogen.

2. The compound of claim 1, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, form, solvate, hydrate or prodrug thereof, wherein,

In the general formula (I)Selected from the following structures:

Wherein each substituent is as defined in claim 1.

3. The compound of claim 1, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, form, solvate, hydrate or prodrug thereof, wherein,

In the general formula (I)Selected from the following structures:

Wherein each substituent is as defined in claim 1.

4. The compound of claim 1, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, form, solvate, hydrate or prodrug thereof, wherein,

In the general formula (I)Selected from the following structures:

Wherein each substituent is as defined in claim 1.

5. The compound of claim 1, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, form, solvate, hydrate or prodrug thereof, wherein,

In the general formula (I)Selected from the following structures:

Wherein each substituent is as defined in claim 1.

6. The compound according to any one of claim 1 to 5, or a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt, a crystal form, a solvate, a hydrate, or a prodrug thereof, wherein,

The compound of formula (I) is selected from the following specific compounds:

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7. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystalline form, solvate, hydrate or prodrug thereof, as claimed in any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

8. Use of a compound of formula (I) according to any one of claims 1-6, or a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt, a crystal form, a solvate, a hydrate or a prodrug thereof, or a pharmaceutical composition according to claim 7, for the manufacture of a medicament for the prevention, treatment or amelioration of a disease by inhibition of PARP 1.

9. The use according to claim 8, wherein

The disease is cancer.

10. The use according to claim 9, wherein,

Including, but not limited to, malignant tumors, such as any of ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, biliary tract cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell and embryo cancer, esophageal cancer, glioblastoma, ewing sarcoma, pancreatic cancer, melanoma, cholangiocarcinoma, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoid cancer, and hematological cancer,

Preferably, the genome of the cancer is of the type in which homologous recombination repair is absent,

Preferably, the cancer relies on DNA double-stranded lesions to repair the deleted pathway by homologous recombination,

Preferably, the cancer comprises one or more cancer cells that lack the ability to repair a DNA double strand break by homologous recombination relative to normal cells,

Preferably, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2, or have a type of BRCA1 or BRCA2 mutation.