CN112300074A - Indazole formamide compound and preparation method and application thereof - Google Patents
Indazole formamide compound and preparation method and application thereof Download PDFInfo
- Publication number
- CN112300074A CN112300074A CN201910709883.6A CN201910709883A CN112300074A CN 112300074 A CN112300074 A CN 112300074A CN 201910709883 A CN201910709883 A CN 201910709883A CN 112300074 A CN112300074 A CN 112300074A
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- compound
- reaction
- pharmaceutically acceptable
- solution
- indazole
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
- C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
- C07D401/10—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 carbon chain containing aromatic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Biomedical Technology (AREA)
- Neurology (AREA)
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Abstract
The invention belongs to the field of chemical medicines, and particularly relates to an indazole formamide compound and a preparation method and application thereof. The invention provides an indazole carboxamide compound or pharmaceutically acceptable salt thereof, wherein the indazole carboxamide compound is shown as a formula I: wherein X, Y is hydrogen or halogen, and the halogen is fluorine, chlorine, bromine or iodine; r1Is aryl, heteroaryl, 5-7 membered heterocycle containing a heteroatom; the heteroaryl, 5-7 membered heterocycle containing the heteroatom may be further substituted with one or more substituents including alkyl, alkanoyl, aroyl, heteroaroyl. The invention provides a novel indazole formamide compound which has a good inhibition effect on PARP1 enzyme and PARP2 enzyme.
Description
Technical Field
The invention belongs to the field of chemical medicines, and particularly relates to an indazole formamide compound and a preparation method and application thereof.
Background
PARP, a polyadenylic diphosphribose polymerase, is a ribozyme present in eukaryotic cells that is involved in DNA repair. (1) It is capable of inhibiting the repair process of single-stranded damage to DNA, but this single-stranded damage to DNA can be converted to double-stranded Damage (DSB) during DNA replication to form replication forks, while DSB can still be repaired by the Homologous Recombination (HR) pathway. The synthetic lethal effect of PARP inhibitors and homologous recombination repair defects on tumor cells can result if the tumor cells have homologous recombination repair defects (including BRCA1 and BRCA2 mutations) that render DSB lesions irreparable. (2) While BRCA1 and BRCA2 are only part of HR repair, other proteins such as EMSY and PTEN are equally important for the HR pathway, PARP inhibitors may produce single drug antitumor activity through synthetic lethality if these genes are mutated or expression silenced in the HR repair pathway.
PARP, classified by structure and function into 4 classes: DNA-dependent PARP: PARP-1, PARP-2 and PARP-3, all activated by DNA damage through DNA binding regions; ② terminal anchor kinases (tan-kyrase) -1 and-2: ankyrin, all with large fragments, promote protein-protein interactions; ③ PARP containing Cys-his (ccch) fragments: PARP-7, PARP-11, PARP-12 and PARP-13, all comprising CCCH zinc fingers that bind to RNA and comprising a Trp-Trp-Glu (WWE) fragment capable of binding to ADP ribopolymer [ poly (ADP-ribose), PAR ]; PARP of large structure: PARP-9, PARP-14 and PARP-15, all contain a suprastructure domain that promotes the binding of proteins to mono ADP ribose (mono), MAR and PAR.
Studies have shown that by inhibiting PARP, PARP can be blocked from participating in DNA damage repair, exacerbating DNA damage in tumor cells. In view of the above, the present invention provides an indazole carboxamide compound as a PARP inhibitor, which provides a new choice for tumor treatment.
Disclosure of Invention
In order to solve the technical problems, the invention provides an indazole formamide compound and a preparation method and application thereof.
In a first aspect of the present invention, there is provided an indazole carboxamide compound, or a pharmaceutically acceptable salt thereof, represented by formula I:
wherein X, Y is hydrogen or halogen, and the halogen is fluorine, chlorine, bromine or iodine;
R1is aryl, heteroaryl, 5-7 membered heterocycle containing a heteroatom;
the heteroaryl, 5-7 membered heterocycle containing the heteroatom may be further substituted with one or more substituents including alkyl, alkanoyl, aroyl, heteroaroyl.
In certain embodiments, the R is1Is heteroaryl or 5-7 membered heterocycle containing a heteroatom.
In some embodimentsIn the scheme, the R is1Is composed of
And the like.
In certain embodiments, the R is1Is composed of
And the like.
In certain embodiments, the indazole carboxamides are
In certain embodiments, the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt; preferably, the inorganic acid salt is a sulfate, hydrochloride, nitrate, phosphate, or hydrobromide salt.
In certain embodiments, the organic acid salt is any of acetate, formate, methanesulfonate, trifluoroacetate, maleate, tartrate, succinate, fumarate, citrate, benzenesulfonate, benzoate, lactate, malate, and amino acid salts; preferably, the amino acid salt is aspartate, glutamate, glycinate, alanate, valinate, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, caseinate, cysteine, methionine, asparagine, glutamine, or threonine.
In a second aspect of the present invention, there is provided a method for preparing the indazole carboxamide compound or the pharmaceutically acceptable salt thereof, which comprises the following synthetic routes:
wherein, X, Y, R1Is as defined in any of the embodiments of the present invention.
The third aspect of the invention provides an application of the indazole carboxamide compound or the pharmaceutically acceptable salt thereof in preparing medicines for treating diseases improved by PARP activity inhibition.
Preferably, the disease ameliorated by the inhibition of PARP activity is vascular disease, septic shock, ischemic injury, neurotoxicity, hemorrhagic shock, inflammatory disease or multiple sclerosis.
As a third aspect of the present invention, there is provided a use of the indazole carboxamide compound or the pharmaceutically acceptable salt thereof for preparing an adjuvant drug for tumor therapy.
As the application of the third aspect of the invention, the indazole carboxamide compound or the pharmaceutically acceptable salt thereof is applied to preparing medicines for tumor enhancement radiotherapy.
As a third aspect of the present invention, there is provided a use of the indazole carboxamide compound or the pharmaceutically acceptable salt thereof for the preparation of a medicament for cancer chemotherapy.
As a use of the third aspect of the invention, the use of an indazole carboxamide compound as described above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for personalized cancer therapy lacking Homologous Recombination (HR) dependent DNA Double Strand Break (DSB) repair.
Preferably, the cancer is a cancer containing cancer cells in which the ability of one or more DSBs to repair DNA by HR is reduced or lost relative to normal cells.
Preferably, the cancer is a cancer having a BRCA-1 or BRCA-2 deficiency, mutant phenotype.
Preferably, the cancer is breast, ovarian, pancreatic or prostate cancer.
In a fourth aspect, the present invention provides a pharmaceutical composition of the indazole carboxamide compound, which comprises: the indazole carboxamide compound or pharmaceutically acceptable salt thereof is used as an active ingredient and at least one pharmaceutically acceptable auxiliary material; preferably, the composition is administered orally, by injection, transdermally, nasally, mucosally, and by inhalation; more preferably, the composition is in a general dosage form, sustained release, controlled release, localized or immediate release dosage form.
Compared with the prior art, the invention has the following technical effects:
1. the invention provides a novel indazole formamide compound which has a good inhibition effect on PARP1 enzyme and PARP2 enzyme.
2. According to the preparation method of the indazole formamide compound, cheap and easily-obtained chemical products are used as starting raw materials, and the synthesis yield of each step is high, so that the production cost is low, and the indazole formamide compound is more suitable for industrial production.
Detailed Description
The indazole carboxamides of the present invention, their preparation and use are described in the following examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. It should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
The reaction was carried out under conventional reaction conditions, although the conditions in the examples are not specified. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1 preparation
Compound 2:
to CCl of Compound 1(120g, 615mmol, 1.00eq) at 15 deg.C4(2400mL) was added NBS (383g, 2.15mol, 3.50eq) and CCl of AIBN (20.2g, 123mmol, 0.20eq) was added dropwise4(200mL) of the solution. The reaction solution was stirred at 80 ℃ for 48 h. TLC (petroleum ether: ethyl acetate: 5:1, product: R)f0.3) detection showed the reaction was complete. The reaction was filtered with suction and the filter cake was washed three times with DCM (1000 mL. times.3). Adding water into the filtrate, extracting, back-extracting the water phase with DCM (1000mL), extracting the combined organic phase with saturated saline water, and drying the organic phase with anhydrous Na2SO4Filtering, and concentrating the filtrate under vacuum. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 2(230g, 839mmol, yield 68.2%) was obtained as a white solid.
1H NMR:(400MHz，CDCl3)δ：7.96-7.98(m,1H),7.74-7.76(m,1H),7.59-7.74(m,1H),4.46(s,2H),3.90-3.91(m,3H).
Compound 3:
to compound 2(100g, 365mmol, 1.00eq) in CH at 20 deg.C3NMO (N-methylmorpholine-N-oxide) (85.5g, 730mmol, 77.0mL, 2.00eq) was added in portions to a CN (650mL) solution over 30min, followed by 4A molecular sieves (100g, 365mmol, 1.00 eq). The reaction mixture was stirred at 20 ℃ for 16 h. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.4) detection showed the reaction was complete. Suction filtering the reaction liquid, using CH as filter cake3CN (200mLx 3) was washed three times. The filtrate was added to water (1000mL) and extracted. The aqueous phase was back-extracted twice with EtOAc (200mL x 2), the organic phase was extracted with saturated brine, then the organic phase was Na anhydrous2SO4Drying, filtering, and concentrating the filtrate under vacuum. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 3(65.0g, 311mmol, yield 85.2%) was obtained as a pale white solid.
1H NMR:(400MHz，CDCl3)δ：9.98(s,1H),8.27-8.29(m,1H),8.17-8.19(m,1H),7.76-7.80(m,1H),3.95(s,3H).
Compound 4:
NaN3a solution of (24.7g, 380mmol, 1.06eq) in EtOAc (560mL) and DMSO (560mL) was stirred at 20 ℃ for 30 min. Compound 3(75g, 359mmol, 1.00eq) was then added to the mixture. The reaction mixture was heated to 40 ℃ and stirred at 40 ℃ for 3 h. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.8) detection showed the reaction was complete. The reaction was cooled to room temperature, poured into ice water (1000mL), and extracted twice with EtOAc (300 mL. times.2). The organic phase is back extracted by saturated salt solution, and the organic phase is dried by anhydrous dry Na2SO4The mixture was filtered, and the filtrate was concentrated to 100mL under vacuum. Used directly in the next step without purification. Compound 4(74.0g, crude) was obtained as a yellow solid.
Compound 2 a:
to a solution of compound 1a (150g, 1.06mol, 113mL, 1.00eq) in DMF (750mL) at 15 deg.C was added the compound N-tert-butoxycarbonylpiperazine (198g, 1.06mol, 1.00eq) and K2CO3(294g, 2.13mol, 2.00 eq). The reaction mixture was stirred at 50 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 5:1, product: R)fDetection ═ 0.2) indicated completion of the reaction. The reaction mixture was cooled to room temperature, poured into water (2.0L) and filtered with suction. The filter cake was dried under vacuum. Used directly in the next step without purification. Crude 326g of compound 2a was obtained as a yellow solid.
1H NMR:(400MHz，CDCl3)δ：8.12-8.14(m,2H),6.81-6.83(m,2H),3.59-3.61(m,4H),3.41-3.43(m,4H),1.49(s,9H).
Compound 3 a:
to a solution of compound 2a (108g, 351mmol, 1.00eq) in methanol (490mL) under nitrogen was added Pd/C (10.8g, 351mmol, 10% Pd by weight, 1.00 eq). The suspension was evacuated and charged with hydrogen and replaced three times. The mixture was stirred at 50 ℃ under hydrogen (50psi) for 12 h. TLC (petroleum ether: ethyl acetate: 2:1, product: R)f0.3) detection showed the reaction was complete. The reaction was filtered with suction and the filter cake was washed three times with EtOAc (1000 mL. times.3). Concentrating the organic phase under vacuum to obtainAnd (4) residue. Used directly in the next step without purification. Compound 3a (86.4g, 312mmol, yield 88.9%) was obtained as a red solid.
1H NMR:(400MHz，CDCl3)δ：6.80-6.83(m,2H),6.65-6.67(m,2H),3.56-3.58(m,4H),3.46(s,2H),2.96-2.98(m,4H),1.49(s,9H).
Compound 4 a:
to compound 3a (47.0g, 169mmol, 1eq) in toluene (1000mL) was added a solution of compound 4(37.0g, 179mmol, 1.06eq) in EtOAc. The reaction solution is concentrated to 15V at 45-50 ℃ under vacuum condition. TFA (96.6mg, 847umol, 62.7uL, 0.005eq) was then added and stirred at 25 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.5) detection showed the reaction was complete. Used directly in the next step without purification. Compound 4a (75.0g, crude) (toluene solution) was obtained as a yellow solid.
Compound 5 a:
a solution of copper triflate (1.17g, 3.22mmol, 0.02eq) in THF (500mL) was heated to 60 ℃. A solution of compound 4a (75.0g, 161mmol, 1.00eq) in toluene was then added dropwise at 60 ℃ and after 1h the addition was complete. The reaction mixture was stirred at 60 ℃ for 1 hr. TLC (petroleum ether: ethyl acetate: 2:1, product: R)f0.6) detection showed the reaction was complete. The reaction was concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 5a (50.0g, 110mmol, yield 68.4%, purity 96.2%) was obtained as a green solid.
1H NMR:(400MHz，CDCl3)δ：8.45(s,1H),8.12-8.14(m,1H),7.94-7.96(m,1H),7.86-7.88(m,2H),7.16-7.20(m,1H),7.02-7.04(m,2H),4.05(s,3H),3.61-3.64(m,4H),3.22-3.24(m,4H),1.51(s,9H).
Compound 6 a:
to compound 5a (37.0g, 84.8mmol, 1.00eq) in MeOH (250mL) at 20 deg.C was added NH3MeOH (10M, 186mL, 22.0 eq). The reaction solution was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 1, product: R)f0.3) Detection indicated completion of the reaction. The reaction mixture was concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 6a (28.0g, 64.5mmol, yield 76.1%, purity 97.1%) was obtained as an off-white solid.
1H NMR:(400MHz，CDCl3)δ：9.08(s,1H),8.45(s,1H),8.29-8.31(m,1H),7.89-7.91(m,1H),7.77-7.80(m,2H),7.24-7.28(m,1H),7.04-7.06(m,2H),6.01(m,1H),3.62-3.65(m,4H),3.24-3.26(m,4H),1.51(s,9H).
Compound 7 a:
to compound 6a (43.0g, 102mmol, 1.00eq) in DCM (300mL) was added HCl/EtOAc (4M, 255mL, 10.0eq) dropwise at 25 ℃. The reaction mixture was stirred at 25 ℃ for 3 h. TLC (petroleum ether: ethyl acetate: 0:1, product: R)f0) detection indicated completion of the reaction. The reaction was filtered with suction and the filter cake was washed with EtOAc (50 mL. times.2). The solid was dried under vacuum. Used directly in the next step without purification. Compound 7a (36.0g, 99.3mmol, 97.3% yield, 98.7% purity, HCl) was obtained as a gray solid.
1H NMR:(400MHz，DMSO)δ：9.53(s,2H),9.25(s,1H),8.58(s,1H),8.00-8.04(m,3H),7.87(s,1H),7.19-7.23(m,4H),3.49-3.52(m,4H),3.23(s,4H).
Compound 101:
to a solution of compound 7a (31.0g, 86.6mmol, 1.00eq, HCl) in DCM (210mL) at 0 deg.C was added TEA (17.5g, 173mmol, 24.1mL, 2.00eq) dropwise. The reaction was stirred at 0 ℃ for 30min, followed by addition of cyclopropylcarbonyl chloride (13.6g, 130mmol, 11.8mL, 1.50 eq). The reaction mixture was stirred at 25 ℃ for 12 h. TLC (dichloromethane: methanol ═ 5:1, product: Rf0.3) detection showed the reaction was complete. The reaction mixture was filtered with suction and the filter cake was washed three times with DCM (50mL × 3). The filtrate and washings were combined and concentrated under vacuum to give a residue. The residue was purified by preparative-HPLC (column: Agela Durashell 10u 250 x 50 mM; mobile phase: [ water (10mM NH)4HCO3)-ACN](ii) a B%: 25% -45%, 20 min). Compound 101(6.10g, 15.5mmol) was obtained in 17.9% yield and purity99.1% as a yellow solid.
1H NMR:(400MHz，DMSO)δ：9.19(s,1H),8.60(s,1H),8.03-8.05(m,1H),7.97-8.00(m,3H),7.87(s,1H),7.23-7.26(m,1H),7.15-7.17(m,2H),3.85(s,2H),3.64(s,2H),3.23-3.32(m,4H),2.02-2.05(m,4H),0.73-0.77(m,4H).
Example 2 preparation
Compound 2 a:
to a solution of compound 1a (150g, 1.06mol, 113mL, 1.00eq) in DMF (750mL) at 15 deg.C was added the compound N-tert-butoxycarbonylpiperazine (198g, 1.06mol, 1.00eq) and K2CO3(294g, 2.13mol, 2.00 eq). The reaction mixture was stirred at 50 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 5:1, product: R)fDetection ═ 0.2) indicated completion of the reaction. The reaction mixture was cooled to room temperature, poured into water (2.0L) and filtered with suction. The filter cake was dried under vacuum. Used directly in the next step without purification. Crude 326g of compound 2a was obtained as a yellow solid.
1H NMR:(400MHz，CDCl3)δ：8.12-8.14(m,2H),6.81-6.83(m,2H),3.59-3.61(m,4H),3.41-3.43(m,4H),1.49(s,9H).
Compound 3 a:
to a solution of compound 2a (108g, 351mmol, 1.00eq) in methanol (490mL) under nitrogen was added Pd/C (10.8g, 351mmol, 10% Pd by weight, 1.00 eq). The suspension was evacuated and charged with hydrogen and replaced three times. The mixture was stirred at 50 ℃ under hydrogen (50psi) for 12 h. TLC (petroleum ether: ethyl acetate: 2:1, product: R)f0.3) detection showed the reaction was complete. The reaction was filtered with suction and the filter cake was washed three times with EtOAc (1000 mL. times.3). The organic phase was concentrated under vacuum to give a residue. Used directly in the next step without purification. Compound 3a (86.4g, 312 mmol) is obtainedYield 88.9%) as a red solid.
1H NMR:(400MHz，CDCl3)δ：6.80-6.83(m,2H),6.65-6.67(m,2H),3.56-3.58(m,4H),3.46(s,2H),2.96-2.98(m,4H),1.49(s,9H).
Compound 4 a:
to compound 3a (47.0g, 169mmol, 1eq) in toluene (1000mL) was added a solution of compound 4(37.0g, 179mmol, 1.06eq) in EtOAc (preparation of compound 4 used the procedure of example 1). The reaction solution is concentrated to 15V at 45-50 ℃ under vacuum condition. TFA (96.6mg, 847umol, 62.7uL, 0.005eq) was then added and stirred at 25 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.5) detection showed the reaction was complete. Used directly in the next step without purification. Compound 4a (75.0g, crude) (toluene solution) was obtained as a yellow solid.
Compound 5 a:
a solution of copper triflate (1.17g, 3.22mmol, 0.02eq) in THF (500mL) was heated to 60 ℃. A solution of compound 4a (75.0g, 161mmol, 1.00eq) in toluene was then added dropwise at 60 ℃ and after 1h the addition was complete. The reaction mixture was stirred at 60 ℃ for 1 hr. TLC (petroleum ether: ethyl acetate: 2:1, product: R)f0.6) detection showed the reaction was complete. The reaction was concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 5a (50.0g, 110mmol, yield 68.4%, purity 96.2%) was obtained as a green solid.
1H NMR:(400MHz，CDCl3)δ：8.45(s,1H),8.12-8.14(m,1H),7.94-7.96(m,1H),7.86-7.88(m,2H),7.16-7.20(m,1H),7.02-7.04(m,2H),4.05(s,3H),3.61-3.64(m,4H),3.22-3.24(m,4H),1.51(s,9H).
Compound 6 a:
to compound 5a (37.0g, 84.8mmol, 1.00eq) in MeOH (250mL) at 20 deg.C was added NH3MeOH (10M, 186mL, 22.0 eq). The reaction solution was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 1, product: R)f0.3) Detection indicated completion of the reaction. Inverse directionThe mixture was concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 6a (28.0g, 64.5mmol, yield 76.1%, purity 97.1%) was obtained as an off-white solid.
1H NMR:(400MHz，CDCl3)δ：9.08(s,1H),8.45(s,1H),8.29-8.31(m,1H),7.89-7.91(m,1H),7.77-7.80(m,2H),7.24-7.28(m,1H),7.04-7.06(m,2H),6.01(m,1H),3.62-3.65(m,4H),3.24-3.26(m,4H),1.51(s,9H).
Compound 102:
to compound 6a (43.0g, 102mmol, 1.00eq) in DCM (300mL) was added HCl/EtOAc (4M, 255mL, 10.0eq) dropwise at 25 ℃. The reaction mixture was stirred at 25 ℃ for 3 h. TLC (petroleum ether: ethyl acetate: 0:1, product: R)f0) detection indicated completion of the reaction. The reaction was filtered with suction and the filter cake was washed with EtOAc (50 mL. times.2). The solid was dried under vacuum. Used directly in the next step without purification. Compound 102(36.0g, 99.3mmol, 97.3% yield, 98.7% purity, HCl) was obtained as a gray solid.
1H NMR:(400MHz，DMSO)δ：9.53(s,2H),9.25(s,1H),8.58(s,1H),8.00-8.04(m,3H),7.87(s,1H),7.19-7.23(m,4H),3.49-3.52(m,4H),3.23(s,4H).
Example 3 preparation
Compound 2 a:
to a solution of Compound 1a (150g, 1.06mol, 113mL, 1.00eq) in DMF (750mL) at 15 deg.C was added piperidin-4-ol (107.2g, 1.06mol, 1.00eq) and K2CO3(294g, 2.13mol, 2.00 eq). The reaction mixture was stirred at 50 ℃ for 12 h. TLC (petroleum ether: ethyl acetate 2: 12) detection indicated completion of the reaction. The reaction mixture was cooled to room temperature, poured into water (2.0L) and filtered with suction. Filter cake in vacuum stripDrying under the condition of the parts. Used directly in the next step without purification. Crude 201g of compound 2a was obtained as a yellow oil.
Compound 3 a:
to a solution of compound 2a (77.9g, 351mmol, 1.00eq) in methanol (490mL) under nitrogen was added Pd/C (10.8g, 351mmol, 10% Pd by weight, 1.00 eq). The suspension was evacuated and charged with hydrogen and replaced three times. The mixture was stirred at 50 ℃ under hydrogen (50psi) for 12 h. TLC (petroleum ether: ethyl acetate 1: 1) detection indicated completion of the reaction. The reaction was filtered with suction and the filter cake was washed three times with EtOAc (1000 mL. times.3). The organic phase was concentrated under vacuum to give a residue. Used directly in the next step without purification. Compound 3a (58.56g, 305mmol, yield 87%) was obtained as a red solid.
Compound 4 a:
to compound 3a (32.45g, 169mmol, 1eq) in toluene (1000mL) was added a solution of compound 4(37.0g, 179mmol, 1.06eq) in EtOAc (preparation of compound 4 used the procedure of example 1). The reaction solution is concentrated to 15V at 45-50 ℃ under vacuum condition. TFA (96.6mg, 847umol, 62.7uL, 0.005eq) was then added and stirred at 25 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate 1: 1) detection indicated completion of the reaction. Used directly in the next step without purification. Compound 4a (58.0g, crude) (toluene solution) was obtained as a yellow solid.
Compound 5 a:
a solution of copper triflate (1.17g, 3.22mmol, 0.02eq) in THF (500mL) was heated to 60 ℃. A solution of compound 4a (45.48g, 120mmol, 1.00eq) in toluene was then added dropwise at 60 ℃ and after 1h the addition was complete. The reaction mixture was stirred at 60 ℃ for 1 hr. TLC (petroleum ether: ethyl acetate 1: 1) detection indicated completion of the reaction. The reaction was concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 5a (26.1g, 74.4mmol, yield 62%) was obtained as a solid.
Compound 103:
to compound 5a (26.1g, 74.4mmol, 1.00eq) in MeOH (250mL) at 20 deg.C was added NH3MeOH (10M, 186mL, 22.0 eq). The reaction solution was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate 1: 1) detection indicated completion of the reaction. The reaction mixture was concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 103(17.5g, 52mmol, yield 70%, purity 97.1%) was obtained as an off-white solid.
1H NMR:(400MHz，CDCl3)δ：9.07(s,1H),8.44(s,1H),8.28-8.30(m,1H),7.88-7.90(m,1H),7.76-7.79(m,2H),7.23-7.27(m,1H),7.03-7.05(m,2H),6.00(m,1H),3.72-3.75(m,2H),3.61-3.64(m,4H),3.23-3.25(m,4H).
Example 4 preparation
Compound 2 d: to a solution of compound 1d (100g,629mmol,69.4mL,1.00eq) in DMF (500mL) at 15 deg.C was added compound 1(117g,629mmol,1.00eq) and K2CO3(174g,1.26mol,2.00 eq). The reaction mixture was stirred at 50 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 5:1, product: R)fDetection ═ 0.2) indicated completion of the reaction. The reaction mixture was cooled to room temperature, poured into water (1.50L) and filtered with suction. The filter cake was concentrated under vacuum. Used directly in the next step without purification. Compound 2d (197g,605mmol, 96.3% yield) was obtained as a yellow solid.
1H NMR:(400MHz CDCl3)δ：8.98-8.01(m,1H),7.91-7.94(m,1H),6.90-6.94(m,1H),3.60-3.63(m,4H),3.24-3.26(m,4H),1.49(s,9H).
Compound 3 d: to a solution of compound 2d (98.5g,303mmol,1.00eq) in EtOH (450mL) under nitrogen was added Pd/C (10g,303mmol, 10% purity, 1.00 eq). The suspension was degassed under vacuum and purged three times with hydrogen. The mixture was stirred at 50 ℃ under hydrogen (50psi) for 12 h. TLC (petroleum ether: ethyl acetate ═3:1, product Rf0.4) detection showed the reaction was complete. The reaction mixture was cooled to room temperature. The two reactions were combined. The reaction mixture was filtered with suction and the filter cake was washed three times with EtOH (1000mL x 3). The filtrate was concentrated under vacuum to give a residue. Used directly in the next step without purification. Compound 3d (175g,567mmol, 93.6% yield, 95.7% purity) was obtained as a pink solid.
1H NMR:(400MHz CDCl3)δ：6.76-6.81(m,1H),6.39-6.45(m,2H),3.57(s,6H),2.91(s,4H),1.48-1.49(m,9H).
Compound 4: NaN3A solution of (16.5g,253mmol,1.06eq) in EtOAc (350mL) and DMSO (350mL) was stirred at 25 ℃ for 30 min. Compound 3(50.0g,239mmol,1eq) was then added to the mixture. The reaction was stirred at 40 ℃ for 2 h. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.8) detection showed the reaction was complete. The reaction was cooled to room temperature, poured into ice water (800mL), and extracted three times with EtOAc (500 mL. times.3). Extracting the combined organic phase with saturated saline solution, and drying the organic phase with anhydrous Na2SO4The filtrate was concentrated under vacuum. Used directly in the next step without purification. Compound 4(49.0g, crude) was obtained as a yellow solid (EtOAc solution).
Compound 4 d: to a solution of compound 3d (66.0g,223mmol,1.00eq) in toluene (1000mL) was added a solution of compound 4(48.0g,237mmol,1.06eq) in EtOAc. The reaction solution is concentrated to 15V at 45-50 ℃ under vacuum condition. TFA (127mg,1.12mmol,82.7uL,0.005eq) was then added to the above solution and stirred at 20 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.5) detection showed the reaction was complete. Used directly in the next step without purification. Compound 4d (95.0g, crude) (toluene solution) was obtained as a yellow solid.
Compound 5 d: cu (OTf)2A solution of (1.42g,3.93mmol,0.02eq) in THF (560mL) was heated to 60 ℃. A solution of compound 4d (95.0g,196mmol,1.00eq) in toluene was then added dropwise at 60 ℃ over 1 h. The reaction mixture was stirred at 60 ℃ for 2 h. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.1) detection displayThe reaction was complete. The reaction mixture was cooled to room temperature and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 5d (65.0g,136mmol, 69.6% yield, 95.6% purity) was obtained as a brown solid.
1H NMR:(400MHz CDCl3)δ：8.46(s,1H),8.13-8.15(m,1H),7.93-7.96(m,1H),7.75-7.79(m,1H),7.65-7.68(m,1H),7.17-7.21(m,1H),7.02-7.06(m,1H),4.05(s,3H),3.62-3.65(m,4H),3.09-3.12(m,4H),1.50(s,9H).
Compound 6 d: to a solution of methyl compound 5d (65.0g,143mmol,1.00eq) in MeOH (450mL) at 20 deg.C was added NH3MeOH (10M,315mL,22.0 eq). The reaction was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 2:1, product: R)fDetection ═ 0.2) indicated completion of the reaction. The reaction mixture was filtered with suction and the filter cake was washed three times with MeOH (50 mL. times. 3). The solid was concentrated under vacuum to give a residue. The residue was used directly in the next step without purification. Compound 6d (52.0g,117mmol, 81.7% yield, 98.8% purity) was obtained as a yellow solid.
1H NMR:(400MHz CDCl3)δ：8.97-8.98(m,1H),8.46(s,1H),8.29-8.31(m,1H),7.87-7.90(m,1H),7.65-7.68(m,1H),7.57-7.60(m,1H),7.24-7.28(m,1H),7.04-7.08(m,1H),6.16-6.17(m,1H),3.63-3.65(m,4H),3.10-3.13(m,4H),1.50(s,9H).
Compound 7 d: to a solution of compound 6d (52.0g,118mmol,1.00eq) in DCM (360mL) was added dropwise HCl (g)/EtOAc (4M,296mL,10.0eq) at 25 ℃. The reaction solution was stirred at 25 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 0:1, product: R)f0) detection indicated completion of the reaction. The reaction mixture was filtered with suction and the filter cake was washed twice with EtOAc (50mL × 2). The solid was concentrated under vacuum to give a residue. The residue was used directly in the next step without purification. Compound 7d (30.0g,79.6mmol, 67.3% yield, 99.7% purity, HCl) was obtained as a pink solid.
1H NMR:(400MHz DMSO)δ：9.31(s,1H),8.52(s,1H),7.94-8.16(m,4H),7.86(m,1H),7.24-7.33(m,2H),3.24-3.36(m,8H).
Compound 104: to a solution of compound 7d (30.0g,79.8mmol,1.00eq, HCl) in DCM (210mL) at 0 deg.C was added TEA (16.2g,159mmol,22.2mL,2.00eq) dropwise. The reaction mixture was stirred at 0 ℃ for 30min, then a solution of compound 5(12.5g,120mmol,10.9mL,1.50eq) in DCM (20mL) was added dropwise at 0 ℃. The reaction solution was stirred at 25 ℃ for 12 h. TLC (dichloromethane: methanol ═ 5:1, product: RfDetection ═ 0.2) indicated completion of the reaction. The reaction mixture was filtered with suction and the filter cake was washed three times with DCM (50mL X3). The combined organic phases were concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 104(8.40g,20.0mmol, 25.0% yield, 97.1% purity) was obtained as a brown solid.
1H NMR:(400MHz DMSO)δ：9.28(s,1H),8.53(s,1H),8.09-8.13(m,1H),8.04-8.06(m,1H),7.99-8.01(m,1H),7.93-7.95(m,1H),7.84-7.85(m,1H),7.23-7.28(m,2H),3.87(s,2H),3.66(s,2H),3.07-3.14(m,4H),0.72-0.78(m,4H).
Example 5 preparation
Compound 2: at 25 deg.C, NaN3(17.6g,271mmol,1.05eq) was dissolved in a mixed solution of EtOAc (185mL) and DMSO (185 mL). The reaction mixture was stirred at 25 ℃ for 30 min. To the above reaction mixture was added compound 1(54.0g,258mmol,1.00eq) at 25 ℃. The reaction was stirred at 40 ℃ for 4 h. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.6) detection showed the reaction was complete. The reaction mixture was cooled to room temperature, poured into water (250mL), and extracted twice with EtOAc (200 mL. times.2). Extracting the organic phase with saturated saline solution and anhydrous Na2SO4Drying, filtering, and concentrating the filtrate under vacuum. Used directly in the next step without purification. Methyl 2-azido-3-formyl-benzoic acid methyl ester (52.0g, crude) was obtained as a yellow solid (EtOAc)A solution).
Compound 3: to a solution of compound 2a (41.0g,238mmol,1.00eq) in toluene (300mL) at 25 deg.C was added compound 2(51.8g,252mmol,1.06eq) (EtOAc solution). The reaction was concentrated to 5V under vacuum at 45 ℃. Toluene (200mL) and TFA (136mg,1.19mmol,88.2uL,0.005eq) were then added to the reaction mixture at 25 ℃. The reaction mixture was stirred at 25 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate: 5:1, product: R)f0.5) detection reaction was complete. The solution was concentrated to 3V under vacuum. Used directly in the next step without purification. Compound 3(80.0g, crude) was obtained as a yellow solid (toluene solution).
Compound 4: cu (OTf)2A solution of (1.61g,4.45mmol,0.02eq) in THF (550mL) was heated to 60 ℃. Then compound 3(80.0g,223mmol,1.00eq) (toluene solution) was added at 60 ℃. The reaction mixture was stirred at 60 ℃ for 3 h. TLC (petroleum ether: ethyl acetate: 5:1, product: R)f0.3) detection showed the reaction was complete. The reaction was cooled to room temperature and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 4(55.0g,166mmol, 74.5% yield) was obtained as a brown solid.
1H NMR:(400MHz CDCl3)δ：8.52(s,1H),8.14-8.16(m,1H),7.95-7.97(m,1H),7.88-7.90(m,2H),7.66-7.68(m,2H),7.19-7.23(m,1H),4.06(s,3H).
Compound 5: to a solution of methyl compound 4(50.0g,151mmol,1.00eq) in MeOH (300mL) at 25 deg.C was added NH3MeOH (10M,332mL,22.0 eq). The reaction was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 2:1, product: Rf: 0.3) detection indicated completion of the reaction. The reaction was cooled to room temperature and concentrated under vacuum to give a residue. Used directly in the next step without purification. Compound 5(40.0g,126mmol, 83.8% yield) was obtained as an off-white solid.
1H NMR:(400MHz CDCl3)δ：8.96(s,1H),8.52(s,1H),8.32-8.34(m,1H),7.90-7.92(m,1H),7.79-7.82(m,2H),7.69-7.71(m,2H),7.27-7.31(m,2H),6.07(s,1H).
Compound 6: to a solution of compound 5(40.0g,126mmol,1.00eq) in DMF (280mL) at 25 deg.C was added BPD (35.3g,139mmol,1.10eq) and KOAc (37.3g,379mmol,3.00 eq). The reaction solution was bubbled with nitrogen at 25 ℃ for 30 min. Pd (dppf) Cl was then added at 25 ℃ under nitrogen2.CH2Cl2(5.17g,6.33mmol,0.05eq) and the solution was purged three times with nitrogen. The reaction mixture was stirred at 85 ℃ for 2 h. LCMS (ET20729-131-P1A1) detection showed the reaction was complete. The reaction mixture was cooled to room temperature, filtered with suction and the filter cake was washed three times with EtOAc (50mLx 3). The solution was poured into water and separated. The aqueous phase was extracted with EtOAc (50mL), the organic phase was extracted with saturated brine, and the organic phase was then extracted with anhydrous Na2SO4Drying, filtering, and concentrating the filtrate under vacuum condition to obtain residue. The residue was triturated with EtOAc (10mL) at 25 ℃ for 30 min. Compound 6(25.0g, crude) was obtained as a brown solid.
1H NMR:(400MHz DMSO)δ：9.40(s,1H),8.55(s,1H),8.19-8.22(m,2H),8.06-8.08(m,1H),8.01-8.03(m,1H),7.92-7.93(m,1H),7.88-7.91(m,2H),7.26-7.30(m,1H),1.33(s,12H).
Compound 105: to compound 6(25.0g,68.8mmol,1.00eq) and compound 6a (11.2g,68.8mmol,1.00eq) in DMF (170mL) and H at 25 deg.C2To O (25mL), K was added2CO3(19.0g,138mmol,2.00 eq). The reaction mixture was bubbled with nitrogen for 30min, then Pd (dppf) Cl was added at 25 ℃ under nitrogen2.CH2Cl2(3.93g,4.82mmol,0.07 eq). The reaction was stirred at 90 ℃ for 5 h. TLC (petroleum ether: ethyl acetate: 1, product: R)fDetection ═ 0.2) indicated completion of the reaction. The reaction was cooled to room temperature, filtered with suction, and the filter cake was washed five times with DCM (100mL × 5). The solution was poured into water (500mL), separated and the aqueous phase extracted with DCM (100 mL). Extracting the organic phase with saturated saline solution, and then extracting the organic phase with anhydrous Na2SO4Drying, filtering, and concentrating the filtrate under vacuum condition to obtain residue. The residue was subjected to pre-HPLC (column: Agela Durashell 10u 250 x 50 mM; mobile phase: [ water (10mM NH)4HCO3)-ACN]；B％:20％-40％,20min) purification. Compound 105(1.43g,4.25mmol, 6.17% yield, 94.6% purity) was obtained as an off-white solid.
1H NMR:(400MHz DMSO)δ：9.39(s,1H),8.59(s,1H),8.57(s,1H),8.27-8.29(m,2H),8.20-8.22(m,2H),8.07-8.07(m,1H),8.03-8.05(m,1H),7.91(s,1H),7.26-7.30(m,1H),3.96(s,3H).
Example 6 preparation
Compound 2: at 25 deg.C, NaN3(17.6g,271mmol,1.05eq) was dissolved in a mixed solution of EtOAc (185mL) and DMSO (185 mL). The reaction mixture was stirred at 25 ℃ for 30 min. To the above reaction mixture was added compound 1(54.0g,258mmol,1.00eq) at 25 ℃. The reaction was stirred at 40 ℃ for 4 h. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.6) detection showed the reaction was complete. The reaction mixture was cooled to room temperature, poured into water (250mL), and extracted twice with EtOAc (200 mL. times.2). Extracting the organic phase with saturated saline solution and anhydrous Na2SO4Drying, filtering, and concentrating the filtrate under vacuum. Used directly in the next step without purification. Methyl 2-azido-3-formyl-benzoic acid methyl ester (52.0g, crude) was obtained as a yellow solid (EtOAc solution).
Compound 3: to a solution of compound 2a (41.0g,238mmol,1.00eq) in toluene (300mL) at 25 deg.C was added compound 2(51.8g,252mmol,1.06eq) (EtOAc solution). The reaction was concentrated to 5V under vacuum at 45 ℃. Toluene (200mL) and TFA (136mg,1.19mmol,88.2uL,0.005eq) were then added to the reaction mixture at 25 ℃. The reaction mixture was stirred at 25 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate: 5:1, product: R)f0.5) detection reaction was complete. The solution was concentrated to 3V under vacuum. Used directly in the next step without purification. Compound 3(80.0g, crude) was obtained as yellowA colored solid (toluene solution).
Compound 4: cu (OTf)2A solution of (1.61g,4.45mmol,0.02eq) in THF (550mL) was heated to 60 ℃. Then compound 3(80.0g,223mmol,1.00eq) (toluene solution) was added at 60 ℃. The reaction mixture was stirred at 60 ℃ for 3 h. TLC (petroleum ether: ethyl acetate: 5:1, product: R)f0.3) detection showed the reaction was complete. The reaction was cooled to room temperature and concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 4(55.0g,166mmol, 74.5% yield) was obtained as a brown solid.
1H NMR:(400MHz CDCl3)δ：8.52(s,1H),8.14-8.16(m,1H),7.95-7.97(m,1H),7.88-7.90(m,2H),7.66-7.68(m,2H),7.19-7.23(m,1H),4.06(s,3H).
Compound 5: to a solution of methyl compound 4(50.0g,151mmol,1.00eq) in MeOH (300mL) at 25 deg.C was added NH3MeOH (10M,332mL,22.0 eq). The reaction was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 2:1, product: Rf: 0.3) detection indicated completion of the reaction. The reaction was cooled to room temperature and concentrated under vacuum to give a residue. Used directly in the next step without purification. Compound 5(40.0g,126mmol, 83.8% yield) was obtained as an off-white solid.
1H NMR:(400MHz CDCl3)δ：8.96(s,1H),8.52(s,1H),8.32-8.34(m,1H),7.90-7.92(m,1H),7.79-7.82(m,2H),7.69-7.71(m,2H),7.27-7.31(m,2H),6.07(s,1H).
Compound 6: to a solution of compound 5(40.0g,126mmol,1.00eq) in DMF (280mL) at 25 deg.C was added BPD (35.3g,139mmol,1.10eq) and KOAc (37.3g,379mmol,3.00 eq). The reaction solution was bubbled with nitrogen at 25 ℃ for 30 min. Pd (dppf) Cl was then added at 25 ℃ under nitrogen2.CH2Cl2(5.17g,6.33mmol,0.05eq) and the solution was purged three times with nitrogen. The reaction mixture was stirred at 85 ℃ for 2 h. LCMS (ET20729-131-P1A1) detection showed the reaction was complete. The reaction mixture was cooled to room temperature, filtered with suction and the filter cake was washed three times with EtOAc (50mLx 3). The solution was poured into water and separated. The aqueous phase was extracted with EtOAc (50mL) and the organic phase was extractedExtracting with saturated salt solution, and extracting organic phase with anhydrous Na2SO4Drying, filtering, and concentrating the filtrate under vacuum condition to obtain residue. The residue was triturated with EtOAc (10mL) at 25 ℃ for 30 min. Compound 6(25.0g, crude) was obtained as a brown solid.
1H NMR:(400MHz DMSO)δ：9.40(s,1H),8.55(s,1H),8.19-8.22(m,2H),8.06-8.08(m,1H),8.01-8.03(m,1H),7.92-7.93(m,1H),7.88-7.91(m,2H),7.26-7.30(m,1H),1.33(s,12H).
Compound 106: to compound 6(25.0g,68.8mmol,1.00eq) and compound 6a (11.2g,68.8mmol,1.00eq) in DMF (170mL) and H at 25 deg.C2To O (25mL), K was added2CO3(19.0g,138mmol,2.00 eq). The reaction mixture was bubbled with nitrogen for 30min, then Pd (dppf) Cl was added at 25 ℃ under nitrogen2.CH2Cl2(3.93g,4.82mmol,0.07 eq). The reaction was stirred at 90 ℃ for 5 h. TLC (petroleum ether: ethyl acetate: 1, product: R)fDetection ═ 0.2) indicated completion of the reaction. The reaction was cooled to room temperature, filtered with suction, and the filter cake was washed five times with DCM (100mL × 5). The solution was poured into water (500mL), separated and the aqueous phase extracted with DCM (100 mL). Extracting the organic phase with saturated saline solution, and then extracting the organic phase with anhydrous Na2SO4Drying, filtering, and concentrating the filtrate under vacuum condition to obtain residue. The residue was subjected to pre-HPLC (column: Agela Durashell 10u 250 x 50 mM; mobile phase: [ water (10mM NH)4HCO3)-ACN](ii) a 20 percent to 40 percent of B percent, 20 min). Compound 106(1.09g,3.43mmol, 5% yield, 95.2% purity) was obtained as an off-white solid.
1H NMR:(400MHz DMSO)δ：9.38(s,1H),8.58(s,1H),8.56(s,1H),8.26-8.28(m,2H),8.19-8.21(m,2H),8.06-8.07(m,1H),8.02-8.04(m,1H),7.90(s,1H),7.25-7.29(m,1H),3.95(s,3H).
Example 7 preparation
Compound 2 a:
to a solution of compound 1a (150g, 1.06mol, 113mL, 1.00eq) in DMF (750mL) at 15 deg.C was added the compound N-tert-butoxycarbonylpiperazine (198g, 1.06mol, 1.00eq) and K2CO3(294g, 2.13mol, 2.00 eq). The reaction mixture was stirred at 50 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 5:1, product: R)fDetection ═ 0.2) indicated completion of the reaction. The reaction mixture was cooled to room temperature, poured into water (2.0L) and filtered with suction. The filter cake was dried under vacuum. Used directly in the next step without purification. Crude 326g of compound 2a was obtained as a yellow solid.
Compound 3 a:
to a solution of compound 2a (108g, 351mmol, 1.00eq) in methanol (490mL) under nitrogen was added Pd/C (10.8g, 351mmol, 10% Pd by weight, 1.00 eq). The suspension was evacuated and charged with hydrogen and replaced three times. The mixture was stirred at 50 ℃ under hydrogen (50psi) for 12 h. TLC (petroleum ether: ethyl acetate: 2:1, product: R)f0.3) detection showed the reaction was complete. The reaction was filtered with suction and the filter cake was washed three times with EtOAc (1000 mL. times.3). The organic phase was concentrated under vacuum to give a residue. Used directly in the next step without purification. Compound 3a (86.4g, 312mmol, yield 88.9%) was obtained as a red solid.
Compound 4 a:
to compound 3a (47.0g, 169mmol, 1eq) in toluene (1000mL) was added a solution of compound 4(37.0g, 179mmol, 1.06eq) in EtOAc. The reaction solution is concentrated to 15V at 45-50 ℃ under vacuum condition. TFA (96.6mg, 847umol, 62.7uL, 0.005eq) was then added and stirred at 25 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.5) detection showed the reaction was complete. Used directly in the next step without purification. Compound 4a (75.0g, crude) (toluene solution) was obtained as a yellow solid.
Compound 5 a:
copper triflate (1.17g, 3.22mmol, 0.02eq) in TThe HF (500mL) solution was heated to 60 ℃. A solution of compound 4a (75.0g, 161mmol, 1.00eq) in toluene was then added dropwise at 60 ℃ and after 1h the addition was complete. The reaction mixture was stirred at 60 ℃ for 1 hr. TLC (petroleum ether: ethyl acetate: 2:1, product: R)f0.6) detection showed the reaction was complete. The reaction was concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 5a (50.0g, 110mmol, yield 68.4%, purity 96.2%) was obtained as a green solid.
Compound 6 a:
to compound 5a (37.0g, 84.8mmol, 1.00eq) in MeOH (250mL) at 20 deg.C was added NH3MeOH (10M, 186mL, 22.0 eq). The reaction solution was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 1, product: R)f0.3) Detection indicated completion of the reaction. The reaction mixture was concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 6a (28.0g, 64.5mmol, yield 76.1%, purity 97.1%) was obtained as an off-white solid.
Compound 7 a:
to compound 6a (43.0g, 102mmol, 1.00eq) in DCM (300mL) was added HCl/EtOAc (4M, 255mL, 10.0eq) dropwise at 25 ℃. The reaction mixture was stirred at 25 ℃ for 3 h. TLC (petroleum ether: ethyl acetate: 0:1, product: R)f0) detection indicated completion of the reaction. The reaction was filtered with suction and the filter cake was washed with EtOAc (50 mL. times.2). The solid was dried under vacuum. Used directly in the next step without purification. Compound 7a (36.0g, 99.3mmol, 97.3% yield, 98.7% purity, HCl) was obtained as a gray solid.
Compound 8 a:
to a solution of compound 7a (31.0g, 86.6mmol, 1.00eq, HCl) in DCM (210mL) at 0 deg.C was added TEA (17.5g, 173mmol, 24.1mL, 2.00eq) dropwise. The reaction mixture was stirred at 0 ℃ for 30min, then tert-butyl (bromomethyl) (methyl) carbamate (29.12g, 130mmol, 1.50eq) was added. The reaction mixture was stirred at 25 ℃ for 12 h. TLC (dichloromethane: methanol ═ 5: 1) detection indicated completion of the reaction. Reaction mixtureSuction was applied and the filter cake was washed three times with DCM (50mL x 3). The filtrate and washings were combined and concentrated under vacuum to give a residue. The residue was purified by preparative-HPLC (column: Agela Durashell 10u 250 x 50 mM; mobile phase: [ water (10mM NH)4HCO3)-ACN](ii) a B%: 25% -45%, 20 min). Compound 8a (8.05g, 17.32mmol) was obtained in 20% yield as a yellow solid.
Compound 107:
to compound 8a (8.05g, 17.32mmol, 1.00eq) in DCM (300mL) was added HCl/EtOAc (4M, 255mL, 10.0eq) dropwise at 25 ℃. The reaction mixture was stirred at 25 ℃ for 3 h. TLC (petroleum ether: ethyl acetate ═ 0: 1) detection indicated completion of the reaction. The reaction was filtered with suction and the filter cake was washed with EtOAc (50 mL. times.2). The solid was dried under vacuum to give compound 107(5.7g, 15.8mmol, 91% yield, 98.7% purity, HCl) as a white solid.
1H NMR:(400MHz，DMSO)δ：9.17(s,1H),8.58(s,1H),8.01-8.03(m,1H),7.95-7.97(m,3H),7.85(s,1H),7.21-7.24(m,1H),7.13-7.15(m,2H),3.83(s,2H),3.62(s,2H),3.60(s,2H),3.21-3.30(m,4H),3.15(s,3H),2.07(s,1H),2.01-2.03(m,4H).
Example 8 preparation
Compound 2 a:
to a solution of compound 1a (150g, 1.06mol, 113mL, 1.00eq) in DMF (750mL) at 15 deg.C was added the compound N-tert-butoxycarbonylpiperazine (198g, 1.06mol, 1.00eq) and K2CO3(294g, 2.13mol, 2.00 eq). The reaction mixture was stirred at 50 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 5:1, product: R)fDetection ═ 0.2) indicated completion of the reaction. The reaction mixture was cooled to room temperature, poured into water (2.0L) and filtered with suction. The filter cake was dried under vacuum. Used directly in the next step without purification. To obtainCrude compound 2a, 326g, was a yellow solid.
Compound 3 a:
to a solution of compound 2a (108g, 351mmol, 1.00eq) in methanol (490mL) under nitrogen was added Pd/C (10.8g, 351mmol, 10% Pd by weight, 1.00 eq). The suspension was evacuated and charged with hydrogen and replaced three times. The mixture was stirred at 50 ℃ under hydrogen (50psi) for 12 h. TLC (petroleum ether: ethyl acetate: 2:1, product: R)f0.3) detection showed the reaction was complete. The reaction was filtered with suction and the filter cake was washed three times with EtOAc (1000 mL. times.3). The organic phase was concentrated under vacuum to give a residue. Used directly in the next step without purification. Compound 3a (86.4g, 312mmol, yield 88.9%) was obtained as a red solid.
Compound 4 a:
to compound 3a (47.0g, 169mmol, 1eq) in toluene (1000mL) was added a solution of compound 4(37.0g, 179mmol, 1.06eq) in EtOAc. The reaction solution is concentrated to 15V at 45-50 ℃ under vacuum condition. TFA (96.6mg, 847umol, 62.7uL, 0.005eq) was then added and stirred at 25 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate: 3:1, product: R)f0.5) detection showed the reaction was complete. Used directly in the next step without purification. Compound 4a (75.0g, crude) (toluene solution) was obtained as a yellow solid.
Compound 5 a:
a solution of copper triflate (1.17g, 3.22mmol, 0.02eq) in THF (500mL) was heated to 60 ℃. A solution of compound 4a (75.0g, 161mmol, 1.00eq) in toluene was then added dropwise at 60 ℃ and after 1h the addition was complete. The reaction mixture was stirred at 60 ℃ for 1 hr. TLC (petroleum ether: ethyl acetate: 2:1, product: R)f0.6) detection showed the reaction was complete. The reaction was concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 5a (50.0g, 110mmol, yield 68.4%, purity 96.2%) was obtained as a green solid.
Compound 6 a:
to compound 5a (37.0g, 84.8mmol, 1.00eq) in MeO at 20 deg.CH (250mL) addition of NH3MeOH (10M, 186mL, 22.0 eq). The reaction solution was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate: 1, product: R)f0.3) Detection indicated completion of the reaction. The reaction mixture was concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 6a (28.0g, 64.5mmol, yield 76.1%, purity 97.1%) was obtained as an off-white solid.
Compound 7 a:
to compound 6a (43.0g, 102mmol, 1.00eq) in DCM (300mL) was added HCl/EtOAc (4M, 255mL, 10.0eq) dropwise at 25 ℃. The reaction mixture was stirred at 25 ℃ for 3 h. TLC (petroleum ether: ethyl acetate: 0:1, product: R)f0) detection indicated completion of the reaction. The reaction was filtered with suction and the filter cake was washed with EtOAc (50 mL. times.2). The solid was dried under vacuum. Used directly in the next step without purification. Compound 7a (36.0g, 99.3mmol, 97.3% yield, 98.7% purity, HCl) was obtained as a gray solid.
Compound 108:
to a solution of compound 7a (31.0g, 86.6mmol, 1.00eq, HCl) in DCM (210mL) at 0 deg.C was added TEA (17.5g, 173mmol, 24.1mL, 2.00eq) dropwise. The reaction was stirred at 0 ℃ for 30min, then cyclobutylformyl chloride (15.3g, 130mmol, 11.8mL, 1.50eq) was added. The reaction mixture was stirred at 25 ℃ for 12 h. TLC (dichloromethane: methanol ═ 5:1, product: Rf0.3) detection showed the reaction was complete. The reaction mixture was filtered with suction and the filter cake was washed three times with DCM (50mL × 3). The filtrate and washings were combined and concentrated under vacuum to give a residue. The residue was purified by preparative-HPLC (column: Agela Durashell 10u 250 x 50 mM; mobile phase: [ water (10mM NH)4HCO3)-ACN](ii) a B%: 25% -45%, 20 min). Compound 108(5.58g, 13.86mmol) was obtained in 16% yield and 99.5% purity as a yellow solid.
1H NMR:(400MHz，DMSO)δ：9.19(s,1H),8.60(s,1H),8.03-8.05(m,1H),7.97-8.00(m,3H),7.87(s,1H),7.23-7.26(m,1H),7.15-7.17(m,2H),3.34-3.36(m,4H),3.30-3.35(m,4H),2.87-2.89(m,1H),2.02-2.05(m,6H).
Example 9 preparation
Compound 2 a:
to compound 1a (44.28g, 169mmol, 1eq) in toluene (1000mL) was added a solution of compound 4(37.0g, 179mmol, 1.06eq) in EtOAc (compound 4 prepared as in example 1). The reaction solution is concentrated to 15V at 45-50 ℃ under vacuum condition. TFA (96.6mg, 847umol, 62.7uL, 0.005eq) was then added and stirred at 25 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate 3: 1) detection indicated completion of the reaction. Used directly in the next step without purification. Compound 2a (72.0g, crude) (toluene solution) was obtained as a yellow solid.
Compound 3 a:
a solution of copper triflate (1.17g, 3.2mmol, 0.02eq) in THF (500mL) was heated to 60 ℃. A solution of compound 2a (75.0g, 160mmol, 1.00eq) in toluene was then added dropwise at 60 ℃ and after 1h the addition was complete. The reaction mixture was stirred at 60 ℃ for 1 hr. TLC (petroleum ether: ethyl acetate ═ 2: 1) detection indicated completion of the reaction. The reaction was concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 3a (43.78g, 104mmol, yield 65%) was obtained as a solid.
Compound 4 a:
to compound 3a (43.78g, 104mmol, 1.00eq) in MeOH (500mL) at 20 deg.C was added NH3MeOH (10M, 300 mL). The reaction solution was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate 1: 1) detection indicated completion of the reaction. The reaction mixture was concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 4a (32.9g, 81.1mmol, yield 78%) was obtained as an off-white solid.
Compound 5 a:
to compound 4a (32.9g, 81.1mmol, 1.00eq) in DCM (200mL) was added HCl/EtOAc (4M, 200mL) dropwise at 25 ℃. The reaction mixture was stirred at 25 ℃ for 3 h. TLC (petroleum ether: ethyl acetate ═ 0: 1) detection indicated completion of the reaction. The reaction was filtered with suction and the filter cake was washed with EtOAc (50 mL. times.2). The solid was dried under vacuum. Used directly in the next step without purification. Compound 5a (22.53g, 73.62mmol, 90% yield, HCl) was obtained as a grey solid.
Compound 109:
to a solution of compound 5a (22.53g, 73.62mmol, 1.00eq, HCl) in DCM (200mL) at 0 deg.C was added TEA (14.875g, 147.24mmol, 20.5mL, 2.00eq) dropwise. The reaction was stirred at 0 ℃ for 30min, followed by addition of cyclopropylcarbonyl chloride (11.6g, 110.43mmol, 1.50 eq). The reaction mixture was stirred at 25 ℃ for 12 h. TLC (dichloromethane: methanol ═ 5: 1) detection indicated completion of the reaction. The reaction mixture was filtered with suction and the filter cake was washed three times with DCM (50mL × 3). The filtrate and washings were combined and concentrated under vacuum to give a residue. The residue was purified by preparative-HPLC (column: Agela Durashell 10u 250 x 50 mM; mobile phase: [ water (10mM NH)4HCO3)-ACN](ii) a B%: 25% -45%, 20 min). Compound 109(4.13g, 11mmol) was obtained in 15% yield and 99.5% purity as a yellow solid.
1H NMR:(400MHz，DMSO)δ：9.18(s,1H),8.59(s,1H),8.02-8.04(m,1H),7.96-8.00(m,3H),7.86(s,1H),7.22-7.25(m,1H),7.14-7.16(m,2H),4.74(t,1H),3.42-3.46(m,2H),1.97-2.02(m,4H),0.72-0.76(m,4H).
Example 10 preparation
Compound 2 a:
to compound 1a (46.6g, 169mmol, 1eq) in toluene (1000mL) was added a solution of compound 4(37.0g, 179mmol, 1.06eq) in EtOAc (compound 4 prepared as in example 1). The reaction solution is concentrated to 15V at 45-50 ℃ under vacuum condition. TFA (96.6mg, 847umol, 62.7uL, 0.005eq) was then added and stirred at 25 ℃ for 2 h. The reaction solution is concentrated to 10V at 45-50 ℃ under vacuum condition. TLC (petroleum ether: ethyl acetate 3: 1) detection indicated completion of the reaction. Used directly in the next step without purification. Compound 2a (70.0g, crude) (toluene solution) was obtained as a yellow solid.
Compound 3 a:
a solution of copper triflate (1.17g, 3.02mmol, 0.02eq) in THF (500mL) was heated to 60 ℃. A solution of compound 2a (70.0g, 151mmol,1.00eq) in toluene was then added dropwise at 60 ℃ and after 1h the addition was complete. The reaction mixture was stirred at 60 ℃ for 1 hr. TLC (petroleum ether: ethyl acetate ═ 2: 1) detection indicated completion of the reaction. The reaction was concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 3a (39.4g, 90.6mmol, yield 60%) was obtained as a solid.
Compound 4 a:
to compound 3a (39.4g, 90.6mmol, 1.00eq) in MeOH (500mL) at 20 deg.C was added NH3MeOH (10M, 300 mL). The reaction solution was stirred at 100 ℃ for 12 h. TLC (petroleum ether: ethyl acetate 1: 1) detection indicated completion of the reaction. The reaction mixture was concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO)2And (3) purifying by using petroleum ether/ethyl acetate (100/1-0/1). Compound 4a (30.4g, 72.5mmol, yield 80%) was obtained as an off-white solid.
Compound 5 a:
to compound 4a (30.4g, 72.5mmol, 1.00eq) in DCM (200mL) was added HCl/EtOAc (4M, 200mL) dropwise at 25 ℃. The reaction mixture was stirred at 25 ℃ for 3 h. TLC (petroleum ether: ethyl acetate ═ 0: 1) detection indicated completion of the reaction. The reaction was filtered with suction and the filter cake was washed with EtOAc (50 mL. times.2). The solid was dried under vacuum. Used directly in the next step without purification. Compound 5a (21.3g, 66.7mmol, 92% yield, HCl) was obtained as a grey solid.
Compound 110:
to a solution of compound 5a (21.3g, 66.7mmol, 1.00eq, HCl) in DCM (200mL) at 0 deg.C was added TEA (13.5g, 133.4mmol, 18.6mL, 2.00eq) dropwise. The reaction was stirred at 0 ℃ for 30min, followed by addition of cyclopropylcarbonyl chloride (10.5g, 100mmol, 1.50 eq). The reaction mixture was stirred at 25 ℃ for 12 h. TLC (dichloromethane: methanol ═ 5: 1) detection indicated completion of the reaction. The reaction mixture was filtered with suction and the filter cake was washed three times with DCM (50mL × 3). The filtrate and washings were combined and concentrated under vacuum to give a residue. The residue was purified by preparative-HPLC (column: AgelaDurashell 10u 250 x 50 mM; mobile phase: [ water (10mM NH)4HCO3)-ACN](ii) a B%: 25% -45%, 20 min). Compound 110(3.1g, 8mmol) was obtained in 12% yield and 99.3% purity as a yellow solid.
1H NMR:(400MHz，DMSO)δ：9.17(s,1H),8.58(s,1H),8.01-8.03(m,1H),7.95-8.01(m,3H),7.85(s,1H),7.21-7.24(m,1H),7.13-7.15(m,2H),3.32-3.50(m,4H),2.75-2.78(m,1H),1.43-1.58(m,4H),0.71-0.75(m,4H).
Test example 1 evaluation of the inhibitory Effect of the Compound of the present invention on PARP1 enzyme
1.1 materials and instruments
1.2 Experimental procedures
1.2.1 Compound preparation and treatment
1.2.1.1 Compound DMSO stock preparation
All compounds were formulated in DMSO at the following concentrations:
| name of Compound | Molecular weight | Concentration (mM) |
| AZD2281 | 434.46 | 30 |
| Compound 101 | 389.45 | 30 |
| Compound 102 | 321.38 | 30 |
| Compound 103 | 336.39 | 30 |
| Compound 104 | 407.44 | 30 |
| Compound 105 | 318.33 | 30 |
| Compound 106 | 318.33 | 30 |
| Compound 107 | 364.44 | 30 |
| Compound 108 | 403.48 | 30 |
| Compound 109 | 374.44 | 30 |
| Compound 110 | 388.46 | 30 |
1.2.1.2 storage of Compounds
All compounds in dimethyl sulfoxide were stored in a desiccator at room temperature for a short period of time.
1.2.1.3 working stock preparation
All assays were serially diluted 3-fold from 10mM with DMSO at 10 concentration levels.
AZD2281 was serially diluted 3-fold from 0.05mM in DMSO at 10 concentration levels.
All compounds were diluted 10-fold with 1 × PARP buffer.
10 XPositive control (5. mu.M, AZD2281) and 10 Xnegative control (100% DMSO) were prepared
Preparation of 10 × negative control (100% DMSO)
The plate was shaken on a shaker for 5 minutes.
1.2.2 preparation of 1 × PARP buffer:
1 volume 10 XPARP buffer diluted with 9 volumes of distilled water
1.2.3 Compound screening:
a) 1 XHistone mixture with PBS
b) To each well 25. mu.l of diluted histone solution was added and incubated overnight at 4 ℃.
c) Each well was washed three times with 100. mu.l PBST buffer (1 XPBS, containing 0.05% Tween 20). The plate was tapped onto a clean paper towel to remove the liquid.
d) Each well was blocked by adding 100. mu.l of blocking buffer to each well. Incubate at room temperature for 90 minutes.
e) Plates were washed 3 times with 100 μ l PBST buffer per well. The plate was tapped onto a clean paper towel to remove the liquid.
f) Prepare 1X PARP assay mix and 1X activated DNA in 1X PARP buffer.
g) Add 12.5 μ Ι of solution (prepared in step f) to each well of the assay plate.
h) Transfer 2.5 μ l of compound dilution (prepared in 2.6.1.3) to each well of the assay plate;
i) formulation of PARP1 enzyme (1ng/ul) with 1 XPARP buffer
j) The reaction was started by adding 10. mu.L of parp1 enzyme (prepared in step i) to each well of the assay plate.
k) Shake for 30S at room temperature for 60 minutes.
l) the reaction mixture was discarded after 1 hour, the plate was washed three times with 100. mu.l PBST buffer, and the plate was tapped onto a clean paper towel as before.
m) Streptavidin-HRP was diluted 1:50 with blocking buffer. Mu.l of diluted Streptavidin-HRP was added to each well. Incubate at room temperature for 30 minutes.
n) washing three times with 100. mu.l PBST buffer per well. The plate was tapped onto a clean paper towel to remove the liquid.
o) before use, 25. mu.l of HRP chemiluminescent substrate A and 25. mu.l of HRP chemiluminescent substrate B were mixed on ice, 50. mu.l added per well.
p) immediately reading the plate with a microplate reader capable of reading the luminous photometric intensity.
1.2.4 data analysis
1.2.4.1% inhibition was calculated as follows:
and (2) Lum: luminous intensity
1.2.4.2 calculating IC50 and plotting the effective dose curve of the compound
IC50 values were calculated using grahpad 5.0 software by applying a non-linear regression fit (dose response-variable slope) to the percent inhibition values and the log concentration of compound concentration.
Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*Hill Slope))
X:log of inhibitor concentration
Y:％Inhibition
1.2.5 reference
http://bpsbioscience.com/azd2281-olaparib-27003
2. Results
Inhibition of the PARP1 enzyme by the compounds of the present invention
The results show that all compounds of the present invention are highly potent inhibitors of PARP 1.
Test example 2 evaluation of inhibitory Effect of the Compound of the present invention on the enzymatic Activity of parp2
2.1 materials and instruments
2.2 Experimental procedures
2.2.1 Compound preparation and treatment
2.2.1.1 Compound DMSO stock preparation
All compounds were formulated in DMSO at the following concentrations:
| name of Compound | Molecular weight | Concentration (mM) |
| AZD2281 | 434.46 | 30 |
| Compound 101 | 389.45 | 30 |
| Compound 102 | 321.38 | 30 |
| Compound 103 | 336.39 | 30 |
| Compound 104 | 407.44 | 30 |
| Compound 105 | 318.33 | 30 |
| Compound 106 | 318.33 | 30 |
| Compound 107 | 364.44 | 30 |
| Compound 108 | 403.48 | 30 |
| Compound 109 | 374.44 | 30 |
| Compound 110 | 388.46 | 30 |
2.2.1.2 storage of Compounds
All compounds in dimethyl sulfoxide were stored in a desiccator at room temperature for a short period of time. (up to 3 months).
The remaining compound was stored for a long period of-20.
2.2.1.3 working stock preparation
All assays were serially diluted 3-fold from 10mM with DMSO at 10 concentration levels.
AZD2281 was serially diluted 3-fold from 0.01mM in DMSO at 10 concentration levels.
All compounds were diluted 10-fold with 1 XPPARP buffer
10 XPositive control (5. mu.M, AZD2281) and 10 Xnegative control (100% DMSO) were prepared
Preparation of 10 × negative control (100% DMSO)
The plate was shaken on a shaker for 5 minutes.
2.2.2 preparation of 1 × PARP buffer:
1 volume 10 XPARP buffer diluted with 9 volumes of distilled water
2.2.3 Compound screening:
a) 1 XHistone mixture with PBS
b) To each well 25. mu.l of diluted histone solution was added and incubated overnight at 4 ℃.
c) Each well was washed three times with 100. mu.l PBST buffer (1 XPBS, containing 0.05% Tween 20). The plate was tapped onto a clean paper towel to remove the liquid.
d) Each well was blocked by adding 100. mu.l of blocking buffer to each well. Incubate at room temperature for 90 minutes.
e) Plates were washed 3 times with 100 μ l PBST buffer per well. The plate was tapped onto a clean paper towel to remove the liquid.
f) Prepare 1X PARP assay mix and 1X activated DNA in 1X PARP buffer.
g) Add 12.5 μ Ι of solution (prepared in step f) to each well of the assay plate.
h) Transfer 2.5 μ l of compound dilution (prepared in 2.6.1.3) to each well of the assay plate;
i) formulation of PARP2 enzyme (1ng/ul) with 1 XPARP buffer
j) The reaction was started by adding 10. mu.L of parp2 enzyme (prepared in step i) to each well of the assay plate.
k) Shake for 30S at room temperature for 60 minutes.
l) the reaction mixture was discarded after 1 hour, the plate was washed three times with 100. mu.l PBST buffer, and the plate was tapped onto a clean paper towel as before.
m) Streptavidin-HRP was diluted 1:50 with blocking buffer. Mu.l of diluted Streptavidin-HRP was added to each well. Incubate at room temperature for 30 minutes.
n) washing three times with 100. mu.l PBST buffer per well. The plate was tapped onto a clean paper towel to remove the liquid.
o) before use, 25. mu.l of HRP chemiluminescent substrate A and 25. mu.l of HRP chemiluminescent substrate B were mixed on ice, 50. mu.l added per well.
p) immediately reading the plate with a microplate reader capable of reading the luminous photometric intensity.
2.2.4 data analysis
2.2.4.1% inhibition was calculated as follows:
and (2) Lum: luminous intensity
2.2.4.2 calculation of IC50 and plotting the effective dose curves of the compounds
IC50 values were calculated using grahpad 5.0 software by applying a non-linear regression fit (dose response-variable slope) to the percent inhibition values and the log concentration of compound concentration.
Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*Hill Slope))
X:log of inhibitor concentration
Y:％Inhibition
2.2.5 reference
http://bpsbioscience.com/azd2281-olaparib-27003
2.3. Results
Inhibition of the PARP2 enzyme by the compounds of the present invention
The results show that all compounds of the present invention are highly potent inhibitors of PARP 2.
Claims (18)
1. An indazole carboxamide compound or a pharmaceutically acceptable salt thereof, wherein said indazole carboxamide compound is represented by formula I:
wherein X, Y is hydrogen or halogen, and the halogen is fluorine, chlorine, bromine or iodine;
R1is aryl, heteroaryl, 5-7 membered heterocycle containing a heteroatom;
the heteroaryl, 5-7 membered heterocycle containing the heteroatom may be further substituted with one or more substituents including alkyl, alkanoyl, aroyl, heteroaroyl.
2. The indazolecarboxamide compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R is1Is heteroaryl or 5-7 membered heterocycle containing a heteroatom.
3. The indazolecarboxamide compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R is1Is composed of
4. The indazolecarboxamide compound or the pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein R is1Is composed of
5. The indazole carboxamide compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, whereinCharacterized in that the indazole carboxamide compound is
6. The indazolecarboxamide compound or the pharmaceutically acceptable salts thereof according to any one of claims 1 to 5, wherein the pharmaceutically acceptable salts are inorganic acid salts or organic acid salts; preferably, the inorganic acid salt is a sulfate, hydrochloride, nitrate, phosphate, or hydrobromide salt.
7. The indazole carboxamide compound according to claim 6, or a pharmaceutically acceptable salt thereof, wherein the organic acid salt is any one of acetate, formate, methanesulfonate, trifluoroacetate, maleate, tartrate, succinate, fumarate, citrate, benzenesulfonate, benzoate, lactate, malate, and amino acid salt; preferably, the amino acid salt is aspartate, glutamate, glycinate, alanate, valinate, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, caseinate, cysteine, methionine, asparagine, glutamine, or threonine.
8. A process for the preparation of the indazole carboxamides of any one of claims 1 to 7, or pharmaceutically acceptable salts thereof, which comprises the following synthetic route:
wherein, X, Y, R1Is as defined in claim 1.
9. Use of an indazole carboxamide compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of diseases which are ameliorated by the inhibition of PARP activity.
10. The use according to claim 9, wherein the disease ameliorated by the inhibition of PARP activity is vascular disease, septic shock, ischemic injury, neurotoxicity, hemorrhagic shock, inflammatory disease or multiple sclerosis.
11. Use of an indazolecarboxamide compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for the preparation of an adjuvant for the treatment of tumors.
12. Use of an indazole carboxamide compound as claimed in any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in tumor-intensive radiotherapy.
13. Use of an indazole carboxamide according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the chemotherapy of cancer.
14. Use of an indazolecarboxamide compound according to any one of claims 1-7, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for personalized cancer treatment lacking Homologous Recombination (HR) dependent DNA Double Strand Break (DSB) repair.
15. The use of claim 14, wherein the cancer is a cancer cell comprising one or more DSBs that have a reduced or lost ability to repair DNA by HR relative to normal cells.
16. The use according to claim 14, wherein said cancer is a cancer with a BRCA-1 or BRCA-2 deficient, mutant phenotype.
17. The use of claim 14, wherein the cancer is breast, ovarian, pancreatic or prostate cancer.
18. A pharmaceutical composition of the indazole carboxamide compound according to any one of claims 1 to 7, comprising: the indazole carboxamide compound or pharmaceutically acceptable salt thereof is used as an active ingredient and at least one pharmaceutically acceptable auxiliary material; preferably, the composition is administered orally, by injection, transdermally, nasally, mucosally, and by inhalation; more preferably, the composition is in a general dosage form, sustained release, controlled release, localized or immediate release dosage form.
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| CN101415686A (en) * | 2006-04-03 | 2009-04-22 | P.安杰莱蒂分子生物学研究所 | Amide substituted indazole and benzotriazole derivatives as poly(ADP-ribose)polymerase (PARP) inhibitors |
| CN103052633A (en) * | 2010-07-14 | 2013-04-17 | 浙江贝达药业有限公司 | Amids Substituted Indazole Derivativees As Ploy(adp-ribose)polymerase Inhibitors |
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| CN112457297B (en) * | 2020-11-24 | 2023-04-28 | 福建医科大学 | PARP protein degradation agent and preparation method and application thereof |
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