CN117946074A - Compounds having PARP1 inhibitory activity and uses thereof - Google Patents

Compounds having PARP1 inhibitory activity and uses thereof Download PDF

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CN117946074A
CN117946074A CN202211290170.9A CN202211290170A CN117946074A CN 117946074 A CN117946074 A CN 117946074A CN 202211290170 A CN202211290170 A CN 202211290170A CN 117946074 A CN117946074 A CN 117946074A
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cancer
hydrogen
alkyl
compound
halogen
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高善云
候英杰
李晶晶
张朝波
许艳晓
屠汪洋
于冰
张毅翔
李乐平
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Shanghai Haihe Pharmaceutical Co Ltd
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Priority to PCT/CN2023/099434 priority patent/WO2024082654A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

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Abstract

The present invention relates to compounds having poly ADP-ribose polymerase inhibitory activity, pharmaceutical compositions and uses 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

Compounds having PARP1 inhibitory activity and uses 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 al25.18,2006) 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、X2 are each independently selected from N and C (H);
X 3 is selected from N and CR 10, wherein R 10 is hydrogen or halogen; preferably, R 10 is hydrogen or fluorine;
R 1 is hydrogen, C 1-C6 alkoxy, C 1-C6 alkyl which is unsubstituted or substituted by halogen; preferably hydrogen, C 1-C4 alkoxy, C 1-C4 alkyl which is unsubstituted or substituted by halogen; preferably hydrogen, C 1-C2 alkoxy, C 1-C4 alkyl or C 1-C4 fluoroalkyl; preferably hydrogen, methoxy, methyl, ethyl, 1-fluoroethyl, difluoromethyl, trifluoromethyl;
R 2 is hydrogen, halogen or C 1-C6 alkyl; preferably hydrogen, halogen or C 1-C4 alkyl; more preferably hydrogen or fluorine;
Each R 3、R4、R4'、R5、R6、R7 is independently hydrogen or C 1-C6 alkyl; preferably hydrogen or C 1-C4 alkyl;
R 8 is selected from hydrogen, halogen, unsubstituted or halogen substituted C 1-C6 alkyl; preferably, R 8 is selected from hydrogen, halogen, unsubstituted or halogen substituted C 1-C4 alkyl; more preferably, R 8 is selected from hydrogen, halogen, C 1-C4 alkyl and C 1-C4 fluoroalkyl;
R 9 is hydrogen, C 3-C6 cycloalkyl or C 1-C6 alkyl; preferably hydrogen or C 1-C4 alkyl;
m is 1 or 2; preferably 1;
The conditions are as follows:
When X 1 is N and X 2 and X 3 are C (H), then R 3、R4、R4'、R5、R6、R7 is not hydrogen at the same time;
When R 2 is H, then R 3、R4、R4'、R5、R6、R7 is not simultaneously hydrogen.
Preferably, provided that R 3、R4、R4'、R5、R6、R7 is not hydrogen at the same time.
Preferably, in the structure of formula (I)Part is selected from the following structures:
Preferably, the compound represented by (I) has a structure represented by the formulas (I-1) to (I-8) or a racemate thereof:
more preferably, the compound of formula (I) is selected from the following specific compounds:
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, 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" means a solvent addition form comprising a stoichiometric or non-stoichiometric amount of solvent. 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 an individual, 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. The subject is particularly intended to mean, 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 each example, the experimental instrument was described (for example 1 H NMR was recorded by Varian Mercury-300 or Varian Mercury-400 nuclear magnetic resonance apparatus, 13 C NMR was recorded by Varian Mercury-400 or Varian Mercury-500 or Varian Mercury-600 nuclear magnetic resonance apparatus, chemical shift was 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, and reversed phase preparative HPLC separation was performed using silica gel of 200-300 mesh).
Abbreviations
Synthesis of key intermediates
Intermediate 1a:7- (bromomethyl) -8-fluoro-3-methylquinoxalin-2 (1H) -one
Step 1: synthesis of ethyl (4-bromo-3-fluoro-2-nitrophenyl) alaninate
To a dry 100mL single-necked flask was added compound 1a-1 (2.38 g,10 mmol), alanine ethyl ester hydrochloride (1.53 g,10 mmol), DIEA (3.87 g,30 mmol), DMF (50 mL) in this order at room temperature, and stirred overnight at room temperature. The reaction mixture was washed with water (100 mL), extracted three times with EA (100 mL), and the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, suction filtered, concentrated, and purified by silica gel column (PE: ea=4:1) to give product 1a-2 (1.5 g, orange solid), yield ：45％.1H-NMR(600MHz,CDCl3):7.42-7.38(m,1H),7.19-7.18(m,1H),6.35-6.32(m,1H),4.19-4.09(m,3H),1.95-1.92(m,1H),1.51-1.48(m,2H),1.24-1.18(m,3H).
Step 2: synthesis of 7-bromo-8-fluoro-3-methyl-3, 4-dihydroquinoxalin-2 (1H) -one
To a dry 100mL single-necked flask, 1a-2 (3 g,8.9 mmol), ammonium chloride (3.8 g,71.6 mmol), methanol (50 mL) and water (5 mL) were sequentially added at room temperature. Zinc powder (4.6 g,71.6 mmol) was added in portions under ice-bath conditions and the reaction stirred at room temperature for 2 hours. The reaction mixture was filtered, the filtrate was added with water (50 mL), extracted three times with ethyl acetate (50 mL), and the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, suction filtered, and the filtrate was concentrated. Methanol (15 mL), ethyl acetate (15 mL) and 4M hydrochloric acid gas (dioxane solution) (1 mL) were added to the residue, and stirred at room temperature for 2 hours, and the reaction was concentrated to give crude product 1a-3 (2.5 g, brown solid), yield: 45%. LCMS (ESI) m/z 257.1[ M-H ] -; rt=1.48 min (3.00 min).
Step 3: synthesis of 7-bromo-8-fluoro-3-methylquinoxalin-2 (1H) -one
1A-3 (2.5 g,9.7 mmol) and DCM (160 mL) were successively added to a dry 250mL single-neck flask at room temperature, 2, 3-dichloro-5, 6-dicyanobenzoquinone (2.6 g,11.6 mmol) was added in portions, and the reaction was stirred at room temperature for 2 hours. The reaction solution was concentrated, and a saturated sodium carbonate solution (50 mL) was added dropwise to the residue, followed by filtration, washing with water and drying. The solid was slurried with ethyl acetate and filtered to give product 1a-4 (1.6 g, light brown solid), yield: 64%. 1H-NMR(600MHz,DMSO-d6 ) 12.61 (s 1H), 7.53-7.47 (m, 2H), 2.40 (s, 3H).
Step 4: synthesis of 8-fluoro-7- (hydroxymethyl) -3-methylquinoxalin-2 (1H) -one
1A-4 (1.6G, 6.25 mmol), (tributyltin) methanol (2.4G, 7.5 mmol), x-phos Pd G2 (490 mg,0.625 mmol), dioxane (40 mL), heating overnight at 80℃under nitrogen, concentrating the reaction solution, purifying with silica gel column (PE: EA=1:1) to give product 1a-5 (1G, white solid), yield ：77％.1H-NMR(400MHz,DMSO-d6):12.42(s 1H),7.52-7.50(m,1H),7.34-7.30(m,1H),5.40(t,J＝5.6Hz,1H),4.63(d,J＝5.2Hz,2H),2.41(s,3H).
Step 5: synthesis of 7- (bromomethyl) -8-fluoro-3-methylquinoxalin-2 (1H) -one
To a dry 250mL one-necked flask, 1a-5 (800 mg,3.8 mmol) and methylene chloride (100 mL) were successively added at room temperature, and phosphorus tribromide (3.1 g,11.5 mmol) was added dropwise under ice-bath conditions. Stirring at room temperature for 2 hours, concentrating the reaction at low temperature, adding water (20 mL) to the residue, adjusting ph=8 with saturated aqueous sodium carbonate, filtering, washing the filter cake with water, and drying to give product 1a (600 mg, green solid), yield: 60%. LCMS (ESI) m/z 269.1[ M-H ] -; rt=1.37 min (3.00 min).
Intermediate 2a: synthesis of 7- (bromomethyl) -3-ethyl-8-fluoroquinoxalin-2 (1H) -one
Synthetic method reference is made to the synthesis of intermediate 1a, except that methyl 2-aminobutyrate hydrochloride is used as starting material instead of ethyl alaninate hydrochloride in step 1. LCMS (ESI) m/z 283[ M-H ] -; rt=1.56 min (3.00 min).
Intermediate 3a:7- (1-bromoethyl) -8-fluoro-3-methylquinoxalin-2 (1H) -one
Step 1: synthesis of 7-acetyl-8-fluoro-3-methylquinoxalin-2 (1H) -one
1A-4 (500 mg,1.95 mmol), tributyl (1-ethoxyvinyl) stannane (848 mg,2.34 mmol) and bis-triphenylphosphine palladium dichloride (69 mg,0.10 mmol) were added to a single-port flask containing 10mL dioxane, reacted at 95℃for 16 hours, diluted hydrochloric acid (2N) was added, reacted at 50℃for 0.5 hours, cooled to room temperature, and then saturated aqueous potassium fluoride was added, and reacted for 0.5 hours. The reaction solution was directly filtered, and after drying the filter cake, PE was used: ea=1: beating 1 gives product 3a-1 (290 mg, yellow solid). Yield: 67.6%, LCMS (ESI) m/z 221.1[ M+H ] +; rt=0.792 min (2.50 min).
Step 2: synthesis of 8-fluoro-7- (1-hydroxyethyl) -3-methylquinoxalin-2 (1H) -one
To a 100mL single-necked flask containing 10mL of methanol was added 3a-1 (0.15 g,0.68 mmol), and sodium borohydride (39 mg,1.02 mmol) was slowly added under ice bath, and the reaction was carried out at room temperature for 1 hour. The reaction solution was quenched with saturated ammonium chloride solution, extracted three times with dichloromethane, the organic phase was washed twice with water, saturated brine once, dried over anhydrous sodium sulfate, and concentrated to give crude product 3a-2 (100 mg, yellow solid) by column chromatography (PE: ea=1:1). Yield: 66.1%, LCMS (ESI) m/z 223.1[ M+H ] +; rt=1.12 min (2.50 min).
Step 3: synthesis of 7- (1-bromoethyl) -8-fluoro-3-methylquinoxalin-2 (1H) -one
To a 100mL single-necked flask containing 6mL of methylene chloride was added 3a-2 (100 mg,0.45 mmol) triphenylphosphine (354 mg,1.35 mmol) and carbon tetrabromide (299 mg,0.90 mmol), and the mixture was reacted at room temperature for 2 hours. The reaction was directly concentrated to dryness by column chromatography (PE: ea=2:1) to give product 3a (70 mg, yellow solid). Yield: 54.7%, purity: 50%, LCMS (ESI) m/z 283.1[ M-H ] -; rt=1.234 min (2.50 min).
Intermediate 4a:7- (1-bromoethyl) -8-fluoro-3-ethylquinoxalin-2 (1H) -one
Synthetic method reference is made to the synthesis of intermediate 3a, except thatReplace/>As starting material, and,/>Is obtained by using methyl 2-aminobutyrate hydrochloride instead of ethyl alaninate hydrochloride as a starting material in the step 1 of preparing the intermediate 1 a. LCMS (ESI) m/z 296.9[ M-H ] -; rt=1.574 (3.00 min).
Intermediate 5a: (3-ethyl-8-fluoro-2-oxo-1, 2-dihydro-1, 6-naphthyridin-7-yl) methylsulfonic acid methyl ester
Step 1: synthesis of (4-amino-6-chloro-5-fluoropyridin-3-yl) methanol
5A-1 (5.0 g,22.9 mmol), anhydrous toluene (70 mL) and diisopropylaluminum hydride (91.5 mmol,91.5 mL) were added dropwise at-78deg.C under nitrogen at room temperature to react for 16 hours. Methanol (3.66 mL), 15% sodium hydroxide solution (3.66 mL) and methanol (3.66 mL) were added dropwise in this order under an ice bath. The reaction was concentrated and purified by column chromatography (PE: ea=1:1) to give 5a-2 (2.45 g, white solid), yield: 61.2%, LCMS (ESI): m/z 177.1[ M+H ] +; rt=0.707 min (2.50 min).
Step 2: synthesis of 4-amino-6-chloro-5-fluoronicotinaldehyde
In a one-necked flask with 40mL of DCM, compound 5a-2 (2.45 g,13.9 mmol) and manganese dioxide (12 g,139 mmol) were added at room temperature. Stirred at room temperature and reacted for 16 hours. The reaction solution was concentrated by filtration to give Compound 5a-3 (2.2 g, gray solid), yield ：90.8％,LCMS(ESI):m/z 175.1[M+H]+;RT＝1.210min(2.50min).1H-NMR(400MHz,DMSO-d6)：δ9.95(d,J＝1.6Hz,1H),8.37(s,1H),7.97(s,2H).
Step 3: synthesis of 7-chloro-3-ethyl-8-fluoro-1, 6-naphthyridin-2 (1H) -one
In a dry 250mL three-necked flask, compound 5a-3 (3.0 g,17.2 mmol), 4-dimethylaminopyridine (207 mg,1.7 mmol) and DIEA (15.5 g,15.5 mmol) were added in an ice-bath, n-butyryl chloride (12.8 g,120 mmol) was slowly added dropwise, and the mixture was stirred at room temperature and allowed to react overnight. The reaction was concentrated under reduced pressure, and the residue was purified by column chromatography (PE: ea=10:1) to give compound 5a-4 (1.3 g, yellow solid), yield: 33.4%.1H-NMR (400 MHz, DMSO-d 6): delta 12.47 (s, 1H), 8.52 (s, 1H), 7.89 (s, 1H), 2.54-2.49 (m, 2H), 1.19-1.16 (m, 3H).
Step 4: synthesis of methyl 3-ethyl-8-fluoro-2-oxo-1, 2-dihydro-1, 6-naphthyridine-7-carboxylate
5A-4 (370 mg,1.63 mmol), methanol (10 mL), triethylamine (0.68 mL,4.90 mmol) and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (237 mg,0.33 mmol) were sequentially added to a dry 50mL single neck flask at room temperature. Carbon monoxide was replaced 3 times and reacted at 80℃for 24 hours. The reaction solution was concentrated under reduced pressure. The residue was purified by chromatography (PE: ea=2:1) to give 5a-5 (190 mg, yellow solid), yield ：46.51％,LCMS(ESI):m/z 251.0[M+H]+;RT＝1.114min(2.50min).1H-NMR(400MHz,DMSO-d6)：δ12.52(s,1H),8.72(s,1H),7.92(s,1H),3.91(s,3H),2.58-2.54(m,2H),1.19(t,J＝7.6Hz,3H).
Step 5: synthesis of 3-ethyl-8-fluoro-7- (hydroxymethyl) -1, 6-naphthyridin-2 (1H) -one
5A-5 (190 mg,0.76 mmol), tetrahydrofuran (5 mL) and lithium borohydride-tetrahydrofuran (2.0M, 0.76mmol,1.52 mmol) were added sequentially at 0deg.C to a dry 25mL single-neck flask. Stirred at room temperature and reacted for 2 hours. Quench with saturated water (20 mL), extract EA (30 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 using prep plate (pure ethyl acetate) to give 5a-6 (60 mg, off-white solid) in yield ：35.56％,LCMS(ESI):m/z 223.1[M+H]+;RT＝0.875min(2.50min).1H-NMR(400MHz,DMSO-d6)：δ12.23(s,1H),8.61(s,1H),7.87(s,1H),5.34(t,J＝6.0Hz,1H),4.63-4.61(m,2H),2.55-2.51(m,2H),1.18(t,J＝7.6Hz,3H).
Step 6: synthesis of methyl (3-ethyl-8-fluoro-2-oxo-1, 2-dihydro-1, 6-naphthyridin-7-yl) methylsulfonate 5a-6 (40 mg,0.18 mmol), tetrahydrofuran (3 mL), triethylamine (0.08 mL,0.54 mmol) and methylsulfonyl chloride (0.02 mL,0.22 mmol) were added sequentially at room temperature in a dry 25mL single neck flask. Stirred at room temperature and reacted for 2 hours. Dilute with water (10 mL), extract EA (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 5a (48 mg, yellow solid), LCMS (ESI): m/z 301.1[ M+H ] +; rt=1.170 min (2.50 min).
Intermediate 6a (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 6a-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 6a-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 6a-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) and 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 6a-3 (300 mg, yellow solid), yield: 44%. LCMS (ESI) m/z 354.1[ M+H ] +; rt=1.80 min (3.00 min).
Step 3: synthesis of tert-butyl (R) -4- (2-fluoro-6- (methylcarbamoyl) pyridin-3-yl) -3-methylpiperazine-1-carboxylate
A50 mL single-necked flask was successively charged with a solution of Compound 6a-3 (100 mg,0.28 mmol) in methylamine ethanol (2 mL), and the reaction mixture was stirred overnight. The reaction was concentrated to give 6a-4 (80 mg, yellow solid), yield: 80%. LCMS (ESI) m/z 297.1[ 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
To a 20mL one-necked flask, compound 6a-4 (80 mg,0.23 mmol), EA (2 mL), 4M hydrochloric acid gas (dioxane solution) (2 mL) was successively added, and the mixture was stirred at room temperature for 2 hours. The reaction was concentrated to give 6a (65 mg, yellow oil), yield: 100%. LCMS (ESI) m/z 253.2[ M+H ] +; rt=0.99 min (3.00 min).
Synthesis of intermediate 7a, (R) -N-methyl-5- (2-methylpiperazin-1-yl) pyridine carboxamide hydrochloride
Synthesis method referring to the synthesis of intermediate 6a, except that step 1 was not performed, and synthesis was started from step 2 by directly using 6a-1 instead of 6a-2 in step 2 LCMS(ESI):m/z 235.1[M+H]+;RT＝0.82min(3.00min).1H-NMR(400MHz,DMSO-d6):12.42(s 1H),7.52-7.50(m,1H),7.34-7.30(m,1H),5.40(t,J＝5.6Hz,1H),4.63(d,J＝5.2Hz,2H),2.41(s,3H).
Synthesis of intermediate 8a N-methyl-5- (piperazin-1-yl) pyridine carboxamide hydrochloride
Synthetic method referring to the synthesis of intermediate 6a, the synthesis was started from step 2 (please confirm if it is correct) with the exception that step 1 was not performed, instead of step 2, 6a-2 was directly used, and 4-Boc piperazine was used instead of (R) -4-Boc-2-methylpiperazine. LCMS (ESI) m/z 221.2[ M+H ] +; rt=0.285 min (6.00 min).
Intermediate 9a (S) -N-methyl-5- (2-methylpiperazin-1-yl) pyridine amide hydrochloride
Synthetic method referring to the synthesis of intermediate 6a, the synthesis was started from step 2, except that instead of step 1, 6a-1 was directly used instead of 6a-2 in step 2, and (S) -4-Boc-2-methylpiperazine was used instead of (R) -4-Boc-2-methylpiperazine. LCMS (ESI) m/z 235.2[ M+H ] +; rt=0.307 min (3.00 min).
Intermediate 10a (S) -N-methyl-5- (3-methylpiperazin-1-yl) picolinamide hydrochloride
Synthetic method referring to the synthesis of intermediate 6a, the synthesis was started from step 2, except that instead of step 1, 6a-1 was directly used instead of 6a-2 in step 2, and tert-butyl (S) -2-methylpiperazine-1-carboxylate was used instead of (R) -4-Boc-2-methylpiperazine. LCMS (ESI) m/z 235.2[ M+H ] +; rt=0.81 min (3.00 min).
Intermediate 11a (R) -N-methyl-5- (3-methylpiperazin-1-yl) picolinamide hydrochloride
Synthetic method referring to the synthesis of intermediate 6a, the synthesis was started from step 2, except that instead of step 1, 6a-1 was directly used instead of 6a-2 in step 2, and tert-butyl (R) -2-methylpiperazine-1-carboxylate was used instead of (R) -4-Boc-2-methylpiperazine.
Intermediate 12a: 6-fluoro-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
6A-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) and dioxane (15 mL) were sequentially added to a dry 50mL single-neck flask. Heating overnight at 80deg.C under nitrogen, concentrating the reaction solution, and purifying with silica gel column (PE: EA=2:1) to obtain 12a-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-necked flask, 12a-1 (320 mg,0.94 mmol), tetrahydrofuran (8 mL) and an aqueous solution (8 mL) of lithium hydroxide monohydrate (200 mg,4.7 mmol) were sequentially added, the reaction solution was 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% to 40% acetonitrile aqueous solution) to obtain 12a-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 one-necked flask, 12a-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) were successively added and stirred at room temperature overnight. The reaction solution was added with water (20 mL), EA extracted (30 ml×3), the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, suction filtered, and the filtrate was concentrated and passed through a reverse phase column (20% -70% acetonitrile aqueous solution) to give 12a-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
Into a dry 20mL one-necked flask, 12a-3 (250 mg,0.74 mmol) was successively added, ethyl acetate (3 mL), 4M hydrochloric acid gas (dioxane solution) (1 mL), stirring was carried out at room temperature for 2 hours, and the reaction solution was concentrated to obtain 12a (200 mg, yellow solid), yield ：100％.LCMS(ESI):m/z 239.1[M+H]+;RT＝0.91min(3.00min).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).
Intermediate 13a methyl (R) -6-fluoro-5- (3-methylpiperazin-1-yl) pyridine hydrochloride
Synthetic method reference was made to the synthesis of intermediate 12a, except that tert-butyl (R) -2-methylpiperazine-1-carboxylate was used instead of 1-tert-butoxycarbonylpiperazine .LCMS(ESI):m/z 253.1[M+H]+;RT＝0.342&0.576min(6.00min).1H-NMR(400MHz,DMSO-d6)：δ9.62(s,1H),9.47(s,1H),8.45(dd,J＝4.4,9.2Hz,1H),7.87(dd,J＝1.2,8.0Hz,1H),7.72-7.67(m,1H),3.63-3.59(m,2H),3.39-3.45(m,2H),3.24-3.13(m,2H),3.04-2.98(m,1H),2.78(d,J＝4.8Hz,3H),1.31(d,J＝6.4Hz,3H).
Intermediate 14a 7- (1-bromoethyl) -3-methylquinoxalin-2 (1H) -one
Synthetic method reference is made to the synthesis of intermediate 3a, except thatInstead ofAs a starting material, moreover, 14a-4 was obtained by using 4-bromo-1-fluoro-2-nitrobenzene as a starting material in place of 1a-1 in step 1 of preparing intermediate 1 a. LCMS (ESI) m/z 267.0[ M+H ] +; rt=1.327 min (2.50 min).
Intermediate 15a 7- (1-bromoethyl) -3-ethylquinoxalin-2 (1H) -one
Synthetic methods reference is made to the synthesis of intermediate 3a, except for the use/>Replace/>As a starting material, furthermore, 15a-4 was obtained by substituting 4-bromo-1-fluoro-2-nitrobenzene for 1a-1 and methyl 2-aminobutyrate hydrochloride for alanine ethyl ester hydrochloride in step 1 for the preparation of intermediate 1 a. LCMS (ESI) m/z 296.9[ M-H ] -; rt=1.574 min (2.50 min).
Synthesis of Compound 1
To a dry 25mL single-necked flask was added, in order, compound 2a (50 mg,0.175 mmol), 6a (61 mg,0.210 mmol), DIEA (68 mg,0.525 mmol) and acetonitrile (3 mL) at room temperature. Heated at 80℃for 2 hours. The reaction solution was purified by reverse phase preparation column and dried to give Compound 1 (25 mg,0.0548 mmol) as a white solid in yield ：31％.LCMS(ESI):m/z 457.2[M+H]+;RT＝1.62min(3.00min).1H-NMR(600MHz,DMSO-d6):12.44(s,1H),8.41-8.39(m,1H),7.84-7.82(m,1H),7.60-7.55(m,2H),7.32-7.30(m,1H),3.85-3.84(m,1H),3.67(s,2H),3.28-3.26(m,1H),3.04-3.02(m,1H),2.84-2.75(m,6H),2.55-2.51(m,1H),2.38-2.35(m,1H),1.28(m,3H),1.03(d,J＝6.0Hz,3H).
Compound 2-Compound 10
According to the procedure described for Compound 1, intermediate 2a and 6a were replaced with the intermediates in the following tables, respectively, to synthesize Compound 2-Compound 10, the structural formulae of the respective example compounds being shown in the preceding tables
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Synthesis of Compound 12
5A (27 mg,0.09 mmol), acetonitrile (3 mL), 8a (35 mg,0.13 mmol) and DIEA (0.07 mL,0.45 mmol) were added sequentially at room temperature to a dry 25mL single-neck flask. Heated to 75℃and reacted for 3 hours. The reaction solution was concentrated under reduced pressure, and the residue was prepared by prep-HPLC (0.1% formic acid) to give 12 (11.06 mg, white solid), yield ：28.98％,LCMS(ESI):m/z 425.05[M+H]+;RT＝3.134min(6.00min).1H-NMR(400MHz,DMSO-d6)：δ12.25(s,1H),8.62(s,1H),8.40-8.37(m,1H),8.24(d,J＝2.4Hz,1H),7.87(s,1H),7.81(d,J＝8.8Hz,1H),7.37-7.34(m,1H),3.76(d,J＝2.4Hz,2H),3.30-3.26(m,4H),2.77(d,J＝4.8Hz,3H),2.63-2.61(m,4H),2.55-2.51(m,2H),1.18(t,J＝7.6Hz,3H).
Compound 15 was synthesized according to the procedure described for compound 12, substituting intermediate 8a with 12a
Stereoisomers of these compounds may be obtained by conventional chiral resolution methods.
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 ScientificTM,cat.37535);Streptavidin(HRP)(Abcam,cat.ab7403); Peroxidase Chemiluminescent Substrate Kit(Seracare,cat.5430-0040);20xPBS(CST,cat.9808S);20xPBST(CST,cat.9809S);AZD2281(Selleck,cat.S1060)
The experimental method comprises the following steps:
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) 1 Xassay buffer a 2.5-fold final concentration of PARP1 or PARP2 solution was prepared. 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) The reaction was initiated by adding 15. Mu.L of substrate solution to each well at 1.67 times the final concentration using a1 Xassay buffer. 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++ HillSlope ((logIC 50 0-X))
TABLE 1 IC50 for the inhibition of PARP1/2 enzymes by the compounds of the present disclosure
As can also be seen from the results of table 1, the compounds of the present disclosure have very significant selective inhibitory effects on PARP1 compared to the compounds of the prior art.

Claims (10)

1. A compound of formula (I), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, form, solvate, hydrate or prodrug thereof,
Wherein,
X 1、X2 are each independently selected from N and C (H);
X 3 is selected from N and CR 10, wherein R 10 is hydrogen or halogen;
R 1 is hydrogen, C 1-C6 alkoxy, C 1-C6 alkyl which is unsubstituted or substituted by halogen;
R 2 is hydrogen, halogen or C 1-C6 alkyl;
each R 3、R4、R4'、R5、R6、R7 is independently hydrogen or C 1-C6 alkyl;
R 8 is selected from hydrogen, halogen, unsubstituted or halogen substituted C 1-C6 alkyl;
r 9 is hydrogen, C 3-C6 cycloalkyl, C 1-C6 alkyl, unsubstituted or substituted by halogen;
m is 1 or 2;
The conditions are as follows:
When X 1 is N and X 2 and X 3 are C (H), then R 3、R4、R4'、R5、R6、R7 is not hydrogen at the same time; and
When R 2 is H, then R 3、R4、R4'、R5、R6、R7 is not simultaneously hydrogen.
2. The compound of claim 1, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, form, solvate, hydrate or prodrug thereof,
Wherein,
R 10 is hydrogen or fluorine;
r 1 is hydrogen, C 1-C4 alkoxy, C 1-C4 alkyl which is unsubstituted or substituted by halogen;
R 2 is hydrogen, halogen or C 1-C4 alkyl;
each R 3、R4、R4'、R5、R6、R7 is independently hydrogen or C 1-C4 alkyl;
r 8 is selected from hydrogen, halogen, unsubstituted or halogen substituted C 1-C4 alkyl;
R 9 is hydrogen, C 3-C6 cycloalkyl or C 1-C4 alkyl;
m is 1 or 2;
The conditions are as follows:
When X 1 is N and X 2 and X 3 are C (H), then R 3、R4、R4'、R5、R6、R7 is not hydrogen at the same time;
When R 2 is H, then R 3、R4、R4'、R5、R6、R7 is not simultaneously hydrogen.
3. The compound of claim 1, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, form, solvate, hydrate or prodrug thereof,
Wherein,
M is 1;
R 1 is hydrogen, C 1-C2 alkoxy, C 1-C4 alkyl or C 1-C4 fluoroalkyl;
r 2 is hydrogen or fluorine;
R 8 is selected from hydrogen, halogen, C 1-C4 alkyl, and C 1-C4 fluoroalkyl;
R 9 is hydrogen or C 1-C4 alkyl;
The conditions are as follows:
When X 1 is N and X 2 and X 3 are C (H), then R 3、R4、R4'、R5、R6、R7 is not hydrogen at the same time;
When R 2 is H, then R 3、R4、R4'、R5、R6、R7 is not simultaneously hydrogen;
the other substituents are 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 structure of formula (I)Part is selected from the following structures:
the other substituents are as defined in claim 1.
5. The compound of claim 1, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystalline form, solvate, hydrate, or prodrug thereof, wherein the compound of formula (I) has a structure represented by formulae (I-1) to (I-8) or a racemate thereof:
Each substituent is as defined in claim 1.
6. The compound of claim 1, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, form, solvate, hydrate or prodrug thereof, wherein the compound of formula (I) is selected from the following specific compounds:
7. a pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1-6, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate, or prodrug thereof, 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 or 8, 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 includes, but is not limited to, cancer,
Preferably, the genome of the cancer is of the type in which homologous recombination repair is absent,
Or preferably, the cancer relies on DNA double-strand damage to repair the deleted pathway by homologous recombination,
Or 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,
Or preferably, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2, or that have a type of BRCA1 or BRCA2 mutation.
10. The use of claim 9, wherein the cancer comprises 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 tumors, 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, lymphatic cancer, and blood cancer.
CN202211290170.9A 2022-10-20 2022-10-20 Compounds having PARP1 inhibitory activity and uses thereof Pending CN117946074A (en)

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