WO2020228478A1 - Anti-tumor diazobicyclic apoptosis protein inhibitor - Google Patents

Anti-tumor diazobicyclic apoptosis protein inhibitor Download PDF

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Publication number
WO2020228478A1
WO2020228478A1 PCT/CN2020/085124 CN2020085124W WO2020228478A1 WO 2020228478 A1 WO2020228478 A1 WO 2020228478A1 CN 2020085124 W CN2020085124 W CN 2020085124W WO 2020228478 A1 WO2020228478 A1 WO 2020228478A1
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compound
group
isomer
amino
reaction
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PCT/CN2020/085124
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French (fr)
Chinese (zh)
Inventor
宋志春
张崇光
何东伟
包金远
张孝清
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南京华威医药科技集团有限公司
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Publication of WO2020228478A1 publication Critical patent/WO2020228478A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the field belongs to the field of medicinal chemistry, and specifically relates to an inhibitor of apoptosis protein and a preparation method and application thereof.
  • Apoptosis or programmed cell death is a genetically and biochemically regulated mechanism, which plays an important role in the development and homeostasis of invertebrates and vertebrates.
  • Abnormal apoptosis that leads to premature cell death has been linked to a variety of developmental disorders.
  • Defects in apoptosis that result in a lack of cell death have been linked to cancer and chronic viral infections.
  • IAP protein effectively inhibits the apoptosis of cancer cells induced by many kinds of apoptosis stimuli (including chemotherapeutics, radiation and immunotherapy), and is a new target of the apoptosis pathway.
  • Patent applications for inventions involving IAP inhibitors include WO2011018474A1, WO2008016893A1, WO2014047024A1, CN101484151A, etc.
  • IAP inhibitors currently under development include LCL161, Birinapant, BV6, GDC-0152, AZD5582, AT406, etc.
  • AT-406 is an effective, oral Smac mimic, an antagonist of IAPs, which can inhibit XIAP.
  • the structural formulas of cIAP1 and cIAP2 proteins are as follows:
  • IAP inhibitors have a synergistic immune checkpoint inhibitor treatment
  • the role of cancer has very good application prospects, but there are still many major challenges in the development of this species.
  • the purpose of the present invention is to provide a new inhibitor of apoptosis protein or a pharmaceutically acceptable salt or isomer thereof, characterized in that the compound has the following general formula:
  • X represents a C or N atom, when X is a C atom, Y is a CONHR 3 and Z represents a H atom; when X is a N atom, Z is a CONHR 3 and Y does not exist;
  • the R 1 and R 2 are each independently selected from C 1-20 alkylamino groups, C 5-8 cycloalkylamino groups, C 1 ⁇ 20 alkyl group, Or ⁇ -amino acid residues,
  • the ⁇ -amino acid residue is a substituent formed by the deletion of the carboxyl group on the ⁇ -carbon in the ⁇ -amino acid;
  • the Ar group represents an aryl or heteroaryl group with 5-8 atoms,
  • the m and n are each independently selected from any integer of 1 to 3;
  • R 3 is selected from substituted or unsubstituted benzyl, and the substituent is optionally selected from phenyl, halogen, C 1-4 alkyl or p-fluorophenyl;
  • a compound or a pharmaceutically acceptable salt or isomer thereof the compound has the following general formula:
  • R 1 is selected from C 1-20 alkylamino groups, C 1 ⁇ 20 alkyl group, Or any of the ⁇ -amino acid residues,
  • R 2 is selected from C 1-20 alkylamino group, C 5-8 cycloalkylamino group, C 1-20 alkyl group, Or any of the ⁇ -amino acid residues,
  • the ⁇ -amino acid residue is a substituted residue formed by the deletion of the carboxyl group on the ⁇ -carbon in the ⁇ -amino acid
  • the Ar group represents an aryl group and a heteroaryl group with 5-8 atoms.
  • the m and n are each independently selected from any integer of 1 to 3,
  • R 3 is selected from substituted or unsubstituted benzyl, and the substituent is optionally selected from phenyl, halogen, C 1-4 alkyl or p-fluorophenyl;
  • the alkylamino group described in formula I or II is And its isomers
  • the ⁇ -amino acid residue is or
  • R 2 in formula I or II is selected from C 1-13 alkyl
  • the aryl group described in formula I or II is phenyl
  • heteroaryl group described in formula I or II is imidazolyl
  • the halogen is F.
  • the present invention also provides methods for preparing compounds of formula I and formula II and their salts or isomers, but are not limited to the methods described below. All raw materials are prepared according to the group characteristics of the target molecule in accordance with the general formula, and are prepared through the schemes in these routes and methods well known to those of ordinary skill in the organic chemistry field or directly purchased.
  • the compounds of the present invention can be synthesized by combining the following methods with synthetic methods known in the field of synthetic organic chemistry or related modification methods recognized by those skilled in the art.
  • the preparation route of the compound of the present invention is as follows:
  • Option 1 includes the following steps:
  • N-Boc pyrrolidine-2-carboxylic acid methyl ester as starting material, react with allyl bromide under the action of lithium bistrimethylsilylamide, etc. to produce compound 1.
  • the reaction temperature is selected from -80°C to -20°C
  • the reaction solvent is selected from inert aprotic solvents such as tetrahydrofuran and toluene;
  • the Boc protecting group of compound 1 is removed under acidic conditions to obtain compound 2.
  • the acid can be hydrochloric acid, p-toluenesulfonic acid, etc.
  • the reaction solvent can be methanol, ethanol, tetrahydrofuran, etc.;
  • P 1 and P 2 are protecting groups for amino groups, and you can choose tert-butoxycarbonyl (Boc), 9-fluorenylmethylenoxycarbonyl (Fmoc), Boxycarbonyl (Cbz), and allyloxycarbonyl (Alloc), triphenylmethyl (Trt) and any suitable protecting groups, P 1 is not equal to P 2 (in embodiments, P 1 and P 2 are orthogonal protecting groups).
  • the condensing agent can be selected from N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7- Nitrobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and other condensing agents;
  • the carbon-carbon double bond is oxidized to an aldehyde with one less carbon to obtain compound 4.
  • the reaction temperature is from -80°C to -20°C
  • the reaction solvent is tetrahydrofuran, dichloromethane, methanol
  • the reducing agent is selected Triphenylphosphine, sodium sulfite, triethylamine, etc.;
  • Compound 4 removes the protective group P 2 under acidic conditions (when P 2 is the Boc protecting group), palladium-carbon hydrogenation (when P 2 is the Cbz protecting group) or piperidine (when P 2 is the Fmoc protecting group), and further Reductive amination to obtain macrocyclic compound 5, the reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene, etc., and the reducing agent is sodium borohydride, sodium cyanoborohydride, etc.;
  • Compound 5 is in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and a carboxylic acid compound containing R 1 group undergoes condensation reaction to obtain compound 6, temperature Choose from 25°C to 120°C, choose N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, condensing agent 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluoro Phosphate etc.;
  • an inert organic solvent such as acetonitrile, toluene, chloro
  • Compound 6 is saponified under alkaline conditions, and then the pH value is controlled under acidic conditions to obtain compound 7.
  • the organic solvent is methanol, ethanol and other water-soluble solvents, and the alkali is inorganic alkalis such as lithium hydroxide and sodium hydroxide;
  • Compound 7 is in inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and an amino compound containing R 3 group is condensed to obtain compound 8, and the temperature is selected At 25°C to 120°C, the condensing agent is N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2 -(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate Salt etc.;
  • Compound 8 removes the protective group P 1 under acidic conditions (when P 1 is a Boc protecting group), palladium-carbon hydrogenation (when P 1 is a Cbz protecting group), or piperidine (when P 1 is a Fmoc protecting group), etc.
  • Amino compound 9 the reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene, etc.;
  • Compound 9 is in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and a carboxylic acid compound containing R 2 group undergoes a condensation reaction to obtain compound I.
  • an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide
  • N,N'-diisopropylcarbodiimide choose from 25°C to 120°C, choose N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, condensing agent 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluoro Phosphate etc.
  • Option 2 includes the following steps:
  • the ring is closed under the action of inorganic bases such as potassium carbonate and cesium carbonate to produce compound 11.
  • inorganic bases such as potassium carbonate and cesium carbonate
  • the reaction temperature is 25°C to 120°C
  • the reaction solvent is acetonitrile, tetrahydrofuran, and N,N-dimethylformaldehyde.
  • Inert aprotic solvents such as amides;
  • P 1 and P 2 are protecting groups for amino groups, and you can choose tert-butoxycarbonyl (Boc), 9-fluorenylmethylenoxycarbonyl (Fmoc), Boxycarbonyl (Cbz), and allyloxycarbonyl (Alloc), triphenylmethyl (Trt), etc., P 1 is not equal to P 2 (in embodiments, P 1 and P 2 are orthogonal protecting groups).
  • Condensing agent selected N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7-azobenzene Triazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, etc.;
  • the carbon-carbon double bond of compound 12 is oxidized to an aldehyde with one less carbon to obtain compound 13.
  • the reaction temperature is selected from -80°C to -20°C
  • the reaction solvent is tetrahydrofuran, dichloromethane, methanol
  • the reducing agent is selected Triphenylphosphine, sodium sulfite, triethylamine, etc.;
  • Compound 13 removes the protective group P 2 under acidic conditions (when P 2 is the Boc protecting group), palladium-carbon hydrogenation (when P 2 is the Cbz protecting group), or piperidine (when P 2 is the Fmoc protecting group), and further Reductive amination to obtain macrocyclic compound 14, the reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene, etc., and the reducing agent is sodium borohydride, sodium cyanoborohydride, etc.;
  • Compound 14 is in inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and a carboxylic acid compound containing R 1 group undergoes condensation reaction to obtain compound 15.
  • inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide
  • N,N'-diisopropylcarbodiimide choose from 25°C to 120°C, choose N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, condensing agent 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluoro Phosphate etc.;
  • Compound 15 is saponified under alkaline conditions, and then the pH value is controlled under acidic conditions to obtain compound 16.
  • the organic solvent is methanol, ethanol and other water-soluble solvents, and the alkali is inorganic alkalis such as lithium hydroxide and sodium hydroxide;
  • Compound 16 is in inert organic solvents such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and an amino compound containing R 3 group undergoes condensation reaction to obtain compound 17, and the temperature is selected
  • the condensing agent is N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2 -(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate Salt etc.;
  • Compound 17 removes the protective group P 1 under acidic conditions (when P 1 is a Boc protecting group), palladium-carbon hydrogenation (when P 1 is a Cbz protecting group), or piperidine (when P 1 is a Fmoc protecting group), etc.
  • Amino compound 18, the reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene, etc.;
  • Compound 18 is in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and a carboxylic acid compound containing R 2 group undergoes condensation reaction to obtain compound II, temperature Choose from 25°C to 120°C, choose N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, condensing agent 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluoro Phosphate etc.
  • an inert organic solvent such as acetonitrile, toluene, chloro
  • the P 1 and P 2 groups are the protecting groups of the amino group, and the R 1 , R 2 and R 3 groups are defined as described above in the specification.
  • the method for preparing compounds involves protecting and deprotecting various chemical groups, and those skilled in the art can easily select a suitable protecting group according to the needs of protection and deprotection.
  • the above-mentioned group features mentioned in the present invention or the features mentioned in the examples can be combined arbitrarily on the basis of compliance with the laws of pharmacy.
  • Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. .
  • the disclosed features are only general examples of the same or similar features.
  • Another aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of any compound selected from the compound of formula I'or formula I or formula II or a pharmaceutically acceptable salt or isomer thereof as an active ingredient , And one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • the pharmaceutical composition preferably contains a compound of formula I'or formula I or formula II or a pharmaceutically acceptable salt or isomer thereof in a weight ratio of 1% to 90% as an active ingredient, more preferably a weight ratio of 10 %-80% active ingredients.
  • C 1 ⁇ 20 alkyl group means a saturated aliphatic hydrocarbon group of 1 to 20 carbon atoms, including straight-chain and branched-chain groups (the numerical ranges mentioned in this application, for example, “1-20”, refers to The group, in this case an alkyl group, can contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms).
  • the alkyl group in the present invention includes "alkylene".
  • An alkyl group containing 1 to 6 carbon atoms is called a lower alkyl group. When a lower alkyl group has no substituent, it is called an unsubstituted lower alkyl group.
  • the alkyl group is a medium-sized alkyl group having 1-10 carbon atoms, such as methyl, ethyl, ethylene, propyl, propylene, 2-propyl, n-butyl, isopropyl Butyl, butylene, tert-butyl, pentyl, etc.
  • the alkyl group is a lower alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, butylene, isobutyl or tert-butyl.
  • Alkyl groups can be substituted or unsubstituted.
  • C 1-20 alkylamino group means an R-amino compound, and the definition of R is as described for the C 1-20 alkyl group.
  • C 5-8 cycloalkaneamino group means an amine compound formed by substituting one carbon on a C 5-8 cycloalkane with an amino group, for example Wait.
  • ⁇ -amino acid residue means a substitution residue formed after the carboxyl group on the ⁇ -carbon of an amino acid is deleted.
  • benzyl means "benzyl", with a simple structure of C 6 H 5 CH 2 -, or Bn (abbreviation for Benzyl).
  • aryl refers to an all-carbon monocyclic or fused polycyclic group of 5 to 12 carbon atoms with a fully conjugated ⁇ -electron system.
  • Non-limiting examples of aryl groups are phenyl, naphthyl and anthracenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring.
  • Aryl groups can be substituted or unsubstituted.
  • the substituent is preferably one or more, more preferably one, two or three, and even more preferably one or two, independently selected from lower alkyl, trihaloalkyl, halogen, and hydroxyl , Lower alkoxy, mercapto, (lower alkyl)thio, cyano, acyl, thioacyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thioamino Formyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido.
  • the aryl group is a 5-membered monocyclic aryl group or a 6-membered monocyclic aryl group.
  • heteroaryl means a monocyclic or condensed ring group of 5 to 12 ring atoms, containing one, two, three or four ring heteroatoms selected from N, O or S, and the remaining ring atoms are C, in addition has a fully conjugated ⁇ electron system.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, and the ring connected to the parent structure is a heteroaryl ring.
  • Heteroaryl groups can be substituted or unsubstituted. When substituted, the substituent is preferably one or more, more preferably one, two or three, and still more preferably one or two.
  • Non-limiting examples of unsubstituted heteroaromatic bases are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyrimidine, quinoline, isoquinoline, purine, tetrazole, triazine and carbazole; preferably, Heteroaryl groups are nitrogen-containing 5-membered monocyclic heteroaryl groups and nitrogen-containing 6-membered monocyclic heteroaryl groups.
  • isomer can be understood to cover the conventional compound “stereoisomerism” in the art, including enantiomers and diastereomers, where diastereomers include cis and trans isomers, for example, From its cis isomer, trans isomer or a mixture of cis and trans isomers.
  • halogen means fluorine, chlorine, bromine or iodine.
  • salts that retain the biological effectiveness and properties of the parent compound. Such salts include:
  • a salt with an acid obtained by the reaction of the free base of the parent compound with an inorganic acid or organic acid, such as (but not limited to) hydrochloric acid, hydrobromic acid, etc., and organic acid such as (but not limited to) acetic acid, Malic acid, fumaric acid, maleic acid, p-toluenesulfonic acid, tartaric acid, citric acid, lactic acid, succinic acid or malonic acid, etc.
  • organic acid such as (but not limited to) acetic acid, Malic acid, fumaric acid, maleic acid, p-toluenesulfonic acid, tartaric acid, citric acid, lactic acid, succinic acid or malonic acid, etc.
  • Such salts have safety, effectiveness and due biological activity when used in mammals.
  • “Pharmaceutical composition” refers to one or more of the compounds described herein or their pharmaceutically acceptable salts, isomers and prodrugs and other chemical components, such as pharmaceutically acceptable carriers and Mixture of excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
  • “Pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to organisms and does not interfere with the biological activity and properties of the administered compound.
  • Excipient refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound.
  • excipients include (not limited to) lactose, sucrose, microcrystalline cellulose, sorbitol, polyvinylpyrrolidone, cellulose, water, methylcellulose, and the like.
  • the pharmaceutical composition may also contain: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl benzoate, etc.; sweeteners and flavoring agents.
  • lubricants such as talc, magnesium stearate and mineral oil
  • wetting agents such as talc, magnesium stearate and mineral oil
  • emulsifiers and suspending agents such as methyl benzoate, etc.
  • preservatives such as methyl benzoate, etc.
  • sweeteners and flavoring agents such as talc, magnesium stearate and mineral oil.
  • composition of the present invention is formulated, dosed and administered in a manner consistent with good medical practice.
  • the factors to be considered in this situation include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the location where the agent is delivered, the method of administration, the dosing regimen, and what is known to the medical practitioner Other factors.
  • the present invention also provides the use of the compound of formula I'or formula I or formula II or a pharmaceutically acceptable salt or isomer thereof:
  • IAP protein for example, c-IAP1, c-IAP2, X-IAP or ML-IAP.
  • the compounds of the present invention can be used to treat all types of cancer drugs that fail to undergo apoptosis.
  • cancer types include neuroblastoma, bowel cancer such as rectal cancer, colon cancer, familial adenomatous polyp cancer and hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, Tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine body cancer, endometrial cancer, choriocarcinoma, pancreatic cancer , Prostate cancer, testicular cancer, breast cancer, urinary system cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, Ho
  • the positive control compound AT-406 of the compound of the present invention is an inhibitor of cIAP1 and cIAP2, and its inhibitory effect on XIAP is weaker than that of cIAP1 and cIAP2, while the compound of the present invention has stronger inhibitory activity on the three proteins of XIAP, cIAP1 and cIAP2. Therefore, the compound of the present invention has a stronger inhibitory effect on apoptosis proteins as a whole;
  • the compound of the present invention has a strong inhibitory effect on MDA-MB-231 breast cancer and PC-3 pancreatic cancer cells, while the positive control compound AT-406 only has a better effect on MDA-MB-231 breast cancer. Strong, the inhibitory effect on PC-3 pancreatic cancer cells is relatively weak;
  • the compound of the present invention has good IAP inhibitory activity, and can be developed into a cancer treatment drug used alone in the later stage, or can be combined with other targeted drugs to treat cancer;
  • the compound of the present invention has good binding affinity with XIAP, cIAP1, and cIAP2 proteins, and has a good inhibitory effect on cell growth in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines. Its medicinal value and broad market prospects.
  • the dichloromethane solution prepared in the first step was added dropwise to the yellow clear solution, and stirred at room temperature for 6 hours to obtain a reddish brown solution. TLC detected that the reaction was complete.
  • the solvent was evaporated under reduced pressure, the residue was dissolved in 400 mL ethyl acetate, washed with 1M HCl solution, saturated sodium bicarbonate solution, saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure.
  • Diazaoctane-10a-carboxylic acid 1.8g (3.3mmol), 2.6g PyBop (5.0mmol) in a 100mL single-mouth reaction flask, add 50mL DMF, take another 0.85g DIEA (6.6mmol) , Stir at room temperature for 30 minutes. Another 1.2 g (6.6 mmol) of benzhydrylamine was added to the system, heated to 90° C. and stirred for 12 hours.
  • Example 46 Preparation of 1-acetaldehyde-2-((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino)propionyl)-pyrazole-3-carboxylic acid ethyl ester
  • Triazaoctane-8-carboxylic acid 0.95g (2.3mmol), 1.8g PyBop (3.6mmol) in a 100mL single-necked reaction flask, add 50mL DMF, another 0.60g DIEA (4.6mmol), and stir at room temperature for 30 minutes. Another 0.84 g (4.6 mmol) of benzhydrylamine was added to the system, heated to 90° C. and stirred for 12 hours. TLC detected that the reaction was complete.
  • Example 65 Test of the binding affinity of the compound to XIAP, cIAP1, and cIAP2
  • a fluorescence polarization (FP)-based competitive assay method was used for the assay.
  • fluorescently labeled Smac mimics Smac-2F
  • the K d values of Smac-2F, cIAP1-BIR3, and cIAP2-BIR3 are determined by monitoring the total fluorescence polarization of a mixture composed of a solid concentration fluorescent probe and an increasing concentration of protein up to complete saturation.
  • the K i value of the compound is determined by a dose-dependent competitive binding experiment, in which a serial dilution of the compound competes with a fixed concentration of fluorescent probe to bind to a fixed concentration of protein (usually the Kd value measured above is 2-3 times) .
  • 5ul test compound in DMSO and 120ul pre-incubated protein/tracer in assay buffer solution (100mM potassium phosphate, pH 7.5; 100ug/ml bovine gamma globulin; 0.02% sodium azide, Invitrogen)
  • the final concentrations of protein and probe were 3nm and 1nm, 5nm and 1nm for cIAP1-BIR3 and cIAP2-BIR3, respectively.
  • a negative control containing only protein/probe complexes (equivalent to 0% inhibition) and a positive control containing only free probes (equivalent to 100% inhibition) were included in each assay plate.
  • the IC50 value was determined by nonlinear regression fitting of the competition curve.
  • the Ki value of the competitive inhibitor is calculated using the previously described derivation equation based on the measured IC50 value, the Kd value of the probe and different proteins, and the concentration of the protein and probe in the refining assay.
  • the FP-based assay for the XIAP-BIR2-BIR3 protein was performed using the same procedure.
  • a bivalent fluorescent-tagged peptide Smac mimic (Smac-1F) was used as a fluorescent probe, and the Kd value of XIAP-BIR2-BIR3 was measured similarly to the saturation experiment.
  • 0.01% Triton X-100 was added to the assay buffer to achieve the stable fluorescence and polarization values of the dimer fluorescent probe.
  • the final protein and probe concentrations used in the competition assay were 2nm and 1nm, respectively.
  • the test results of the compounds of the present invention are shown in the following table.
  • Example 67 Cell growth inhibition test in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines
  • the effects of the compounds of the invention on the growth of different cancer cell lines were tested.
  • the cells were seeded on a 96-well flat-bottom cell culture plate together with the test compound at a density of 3000 cells/well and the cells were cultured at 37°C in an atmosphere of 95% air and 5% CO 2 for 4 days.
  • Use WST-8 kit and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H- Tetrazolium monosodium salt was used to determine the cell growth inhibition rate after treatment with different concentrations of the compound.
  • WST-8 was added to each well at a final concentration of 10%, and then the plate was incubated at 37°C for 2-3 hours.
  • the absorbance of the sample was measured at 450 nm using a ULTRATecan reader (Molecular Device). By comparing the absorbance of untreated cells and the treated cells with the test compound, the test compound is calculated inhibitory concentration (IC 50) 50% of cell growth. The test results are shown in the table below.
  • the compound of the present invention has a good inhibitory effect on cell growth in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines.

Abstract

The present invention provides a novel apoptosis protein inhibitor or a pharmaceutically acceptable salt thereof, an isomer thereof, a preparation method therefor and a pharmaceutical composition, wherein the definition of each group is as shown in the description. The present invention also provides use of the compound, the pharmaceutically acceptable salt thereof and the isomer thereof in the preparation of a medicament for diseases related to IAP proteins. The compound of the present invention has a better binding affinity to XIAP, cIAP1 and cIAP2 proteins, has a better inhibiting effect on cell growth of MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines, and has larger pharmaceutical value and a broad market prospect.

Description

一种抗肿瘤的重氮双环类细胞凋亡蛋白抑制剂An anti-tumor inhibitor of diazobicyclic apoptotic protein
本申请要求2019年5月16日向中国国家知识产权局提交的，专利申请号为201910408971.2，发明名称为“一种抗肿瘤的重氮双环类细胞凋亡蛋白抑制剂”在先申请的优先权。该申请的全文通过引用的方式结合于本申请中。This application claims the priority of the prior application filed with the State Intellectual Property Office of China on May 16, 2019. The patent application number is 201910408971.2, and the invention title is "an anti-tumor inhibitor of diazobicyclic apoptosis protein". The full text of this application is incorporated into this application by reference.
技术领域Technical field
本领域属于医药化学领域，具体涉及一种细胞凋亡蛋白抑制剂及其制备方法和用途。The field belongs to the field of medicinal chemistry, and specifically relates to an inhibitor of apoptosis protein and a preparation method and application thereof.
背景技术Background technique
细胞凋亡或程序性细胞死亡是在基因和生物化学方面受调节的机制，其在无脊椎动物及脊椎动物的发育和体内平衡方面发挥重要作用。已将导致过早细胞死亡的细胞凋亡异常与多种发育障碍关联起来。导致细胞死亡缺乏的细胞凋亡缺陷已与癌症和慢性病毒感染关联起来。Apoptosis or programmed cell death is a genetically and biochemically regulated mechanism, which plays an important role in the development and homeostasis of invertebrates and vertebrates. Abnormal apoptosis that leads to premature cell death has been linked to a variety of developmental disorders. Defects in apoptosis that result in a lack of cell death have been linked to cancer and chronic viral infections.
目前的癌症疗法，包括化疗剂、放射和免疫疗法，间接诱导癌细胞中的细胞凋亡。因此，癌细胞由于正常细胞凋亡机制中的缺陷而无法执行细胞凋亡程序通常与对化疗、放射或免疫疗法诱导的细胞凋亡的抵抗增加相关。癌症由于细胞凋亡缺陷而对目前疗法的此类原发性或获得性抗性是目前癌症疗法中的主要问题。一类细胞凋亡的中心负调节剂是细胞凋亡蛋白(IAP)的抑制剂。这一类别包括蛋白例如XIAP、cIAP1、cIAP2、ML-IAP、HIAP、KIAP、TSIAP、NAIP、生存素、livin、ILP-2、apollon和BRUCE。IAP蛋白有效抑制相当多种细胞凋亡刺激(包括化疗剂、放射和免疫疗法)诱导的癌细胞细胞凋亡，是细胞凋亡通路的新靶点。Current cancer therapies, including chemotherapeutics, radiation and immunotherapy, indirectly induce apoptosis in cancer cells. Therefore, the inability of cancer cells to perform the apoptosis program due to defects in the normal apoptosis mechanism is usually associated with increased resistance to apoptosis induced by chemotherapy, radiation or immunotherapy. Such primary or acquired resistance of cancer to current therapies due to defects in apoptosis is a major problem in current cancer therapies. One type of central negative regulator of apoptosis is the inhibitor of apoptosis protein (IAP). This category includes proteins such as XIAP, cIAP1, cIAP2, ML-IAP, HIAP, KIAP, TSIAP, NAIP, survivin, livin, ILP-2, apollon and BRUCE. IAP protein effectively inhibits the apoptosis of cancer cells induced by many kinds of apoptosis stimuli (including chemotherapeutics, radiation and immunotherapy), and is a new target of the apoptosis pathway.
涉及IAP抑制剂的发明专利申请有WO2011018474A1、WO2008016893A1、WO2014047024A1、CN101484151A等。Patent applications for inventions involving IAP inhibitors include WO2011018474A1, WO2008016893A1, WO2014047024A1, CN101484151A, etc.
目前开发阶段的IAP抑制剂有LCL161，Birinapant，BV6，GDC-0152，AZD5582，AT406等，其中AT-406是一种有效的，可口服的Smac模拟物，为IAPs的拮抗剂，能够抑制XIAP，cIAP1和cIAP2蛋白，其结构式如下所示：IAP inhibitors currently under development include LCL161, Birinapant, BV6, GDC-0152, AZD5582, AT406, etc. Among them, AT-406 is an effective, oral Smac mimic, an antagonist of IAPs, which can inhibit XIAP. The structural formulas of cIAP1 and cIAP2 proteins are as follows:
Figure PCTCN2020085124-appb-000001
Figure PCTCN2020085124-appb-000001
目前临床上单独用免疫检测点抑制剂对实体瘤作用较弱，IAP抑制剂与免疫检测点抑制剂进行联合治疗实体瘤可以取得较好的治疗效果，IAP抑制剂具有协同免疫检测点抑制剂治疗癌症的作用，具有非常好的应用前景，但该品种的开发仍旧存在较多重大挑战。为了满足目前临床上对细胞凋亡蛋白抑制剂的需要，达到更好的肿瘤疾病治疗效果，我们致力于一系列高效低毒的IAP抑制剂的药物 设计等研究开发，这对于医药领域具有重大的意义。At present, the clinical use of immune checkpoint inhibitors alone has a weak effect on solid tumors. The combination of IAP inhibitors and immune checkpoint inhibitors can achieve better therapeutic effects in solid tumors. IAP inhibitors have a synergistic immune checkpoint inhibitor treatment The role of cancer has very good application prospects, but there are still many major challenges in the development of this species. In order to meet the current clinical needs for apoptosis protein inhibitors and achieve better treatment effects for tumor diseases, we are committed to the research and development of a series of high-efficiency and low-toxic IAP inhibitors, which are of great importance to the medical field. significance.
发明内容Summary of the invention
本发明的目的在于提供一种新的细胞凋亡蛋白抑制剂或其药学可接受的盐、异构体，其特征在于所述化合物具有如下通式：The purpose of the present invention is to provide a new inhibitor of apoptosis protein or a pharmaceutically acceptable salt or isomer thereof, characterized in that the compound has the following general formula:
Figure PCTCN2020085124-appb-000002
Figure PCTCN2020085124-appb-000002
其中，X代表C或N原子，当X为C原子时,Y为CONHR 3，Z代表H原子；当X为N原子时，Z为CONHR 3，Y不存在； Wherein, X represents a C or N atom, when X is a C atom, Y is a CONHR 3 and Z represents a H atom; when X is a N atom, Z is a CONHR 3 and Y does not exist;
所述的R 1和R 2各自独立地任意选自C 1～20烷胺基、C 5～8环烷胺基、
Figure PCTCN2020085124-appb-000003
C 1～20烷基、
Figure PCTCN2020085124-appb-000004
Figure PCTCN2020085124-appb-000005
或者α-氨基酸残基， The R 1 and R 2 are each independently selected from C 1-20 alkylamino groups, C 5-8 cycloalkylamino groups,
Figure PCTCN2020085124-appb-000003
C 1～20 alkyl group,
Figure PCTCN2020085124-appb-000004
Figure PCTCN2020085124-appb-000005
Or α-amino acid residues,
其中所述的α-氨基酸残基为α-氨基酸中α-碳上的羧基缺失后所形成的取代基；所述的Ar基团代表原子个数为5～8的芳基或杂芳基，所述的m和n各自独立选自1～3任意整数；The α-amino acid residue is a substituent formed by the deletion of the carboxyl group on the α-carbon in the α-amino acid; the Ar group represents an aryl or heteroaryl group with 5-8 atoms, The m and n are each independently selected from any integer of 1 to 3;
R 3选自取代或非取代的苄基，所述的取代基任意选自苯基、卤素、C 1～4烷基或对氟苯基； R 3 is selected from substituted or unsubstituted benzyl, and the substituent is optionally selected from phenyl, halogen, C 1-4 alkyl or p-fluorophenyl;
进一步地，一种化合物或其药学可接受的盐或异构体，所述化合物具有如下通式：Further, a compound or a pharmaceutically acceptable salt or isomer thereof, the compound has the following general formula:
Figure PCTCN2020085124-appb-000006
或者
Figure PCTCN2020085124-appb-000007
Figure PCTCN2020085124-appb-000006
or
Figure PCTCN2020085124-appb-000007
其中，上述式I或II中，Wherein, in the above formula I or II,
所述的R 1选自C 1～20烷胺基、
Figure PCTCN2020085124-appb-000008
C 1～20烷基、
Figure PCTCN2020085124-appb-000009
Figure PCTCN2020085124-appb-000010
或者α-氨基酸残基中的任意一种， Said R 1 is selected from C 1-20 alkylamino groups,
Figure PCTCN2020085124-appb-000008
C 1～20 alkyl group,
Figure PCTCN2020085124-appb-000009
Figure PCTCN2020085124-appb-000010
Or any of the α-amino acid residues,
R 2选自C 1～20烷胺基、C 5～8环烷胺基、C 1～20烷基、
Figure PCTCN2020085124-appb-000011
Figure PCTCN2020085124-appb-000012
或者α-氨基酸残基中的任意一种， R 2 is selected from C 1-20 alkylamino group, C 5-8 cycloalkylamino group, C 1-20 alkyl group,
Figure PCTCN2020085124-appb-000011
Figure PCTCN2020085124-appb-000012
Or any of the α-amino acid residues,
其中所述的α-氨基酸残基为α-氨基酸中α-碳上的羧基缺失后所形成的取代残基，所述的Ar基团代表原子个数为5～8的芳基和杂芳基，所述的m和n各自独立选自1～3任意整数，The α-amino acid residue is a substituted residue formed by the deletion of the carboxyl group on the α-carbon in the α-amino acid, and the Ar group represents an aryl group and a heteroaryl group with 5-8 atoms. , The m and n are each independently selected from any integer of 1 to 3,
R 3选自取代或非取代的苄基，所述的取代基任意选自苯基、卤素、C 1～4烷基或对氟苯基； R 3 is selected from substituted or unsubstituted benzyl, and the substituent is optionally selected from phenyl, halogen, C 1-4 alkyl or p-fluorophenyl;
在一种方案中，式I或II中所述的烷胺基为
Figure PCTCN2020085124-appb-000013
及其异构体； In one approach, the alkylamino group described in formula I or II is
Figure PCTCN2020085124-appb-000013
And its isomers;
在一种方案中，式I或II中所述的C 5～6环烷胺基为
Figure PCTCN2020085124-appb-000014
In one scheme, the C 5-6 cycloalkylamino group described in formula I or II is
Figure PCTCN2020085124-appb-000014
在一种方案中，所述的α-氨基酸残基为
Figure PCTCN2020085124-appb-000015
或者
Figure PCTCN2020085124-appb-000016
In one scheme, the α-amino acid residue is
Figure PCTCN2020085124-appb-000015
or
Figure PCTCN2020085124-appb-000016
在一种方案中，式I或II中R 2选自C 1～13烷基； In one scheme, R 2 in formula I or II is selected from C 1-13 alkyl;
在一种方案中，式I或II中所述的芳基为苯基；In one approach, the aryl group described in formula I or II is phenyl;
在一种方案中，式I或II中所述的杂芳基为咪唑基；In one approach, the heteroaryl group described in formula I or II is imidazolyl;
优选地，R 3基团中，所述的卤素为F。 Preferably, in the R 3 group, the halogen is F.
本发明的化合物的举例性的、非限制性的具体实例如下所示：Illustrative, non-limiting specific examples of the compounds of the present invention are shown below:
Figure PCTCN2020085124-appb-000017
Figure PCTCN2020085124-appb-000017
Figure PCTCN2020085124-appb-000018
Figure PCTCN2020085124-appb-000018
Figure PCTCN2020085124-appb-000019
Figure PCTCN2020085124-appb-000019
Figure PCTCN2020085124-appb-000020
Figure PCTCN2020085124-appb-000020
Figure PCTCN2020085124-appb-000021
Figure PCTCN2020085124-appb-000021
本发明还提供式I、式II化合物及其盐或异构体的制备方法，但不仅限于以下描述的方法。所有的原料都是根据符合通式规律的目标分子的基团特征，并通过这些路线中的方案、有机化学领域普通技术人员熟知的方法制备或者直接购买的。可将用下述方法和合成有机化学领域中已知的合成方法或本领域技术人员意识到的有关改变方法结合在一起，合成本发明化合物。The present invention also provides methods for preparing compounds of formula I and formula II and their salts or isomers, but are not limited to the methods described below. All raw materials are prepared according to the group characteristics of the target molecule in accordance with the general formula, and are prepared through the schemes in these routes and methods well known to those of ordinary skill in the organic chemistry field or directly purchased. The compounds of the present invention can be synthesized by combining the following methods with synthetic methods known in the field of synthetic organic chemistry or related modification methods recognized by those skilled in the art.
在一种方案中，本发明的化合物的制备路线如下所示：In one scheme, the preparation route of the compound of the present invention is as follows:
Figure PCTCN2020085124-appb-000022
Figure PCTCN2020085124-appb-000022
方案1plan 1
方案1包括如下步骤:Option 1 includes the following steps:
以N-Boc吡咯烷-2-甲酸甲酯为起始原料，在双三甲基硅基胺基锂等作用下与烯丙基溴反应生成化合物1，反应温度选取-80℃至-20℃，反应溶剂选取四氢呋喃，甲苯等惰性非质子溶剂；Take N-Boc pyrrolidine-2-carboxylic acid methyl ester as starting material, react with allyl bromide under the action of lithium bistrimethylsilylamide, etc. to produce compound 1. The reaction temperature is selected from -80℃ to -20℃ , The reaction solvent is selected from inert aprotic solvents such as tetrahydrofuran and toluene;
化合物1在酸性条件下脱除Boc保护基，得到化合物2，酸可以选取盐酸，对甲苯磺酸等，反应溶剂选取甲醇、乙醇、四氢呋喃等；The Boc protecting group of compound 1 is removed under acidic conditions to obtain compound 2. The acid can be hydrochloric acid, p-toluenesulfonic acid, etc., and the reaction solvent can be methanol, ethanol, tetrahydrofuran, etc.;
化合物2在缩合剂的作用下，与
Figure PCTCN2020085124-appb-000023
反应生成化合物3，P 1、P 2为氨基的保护基，可以选择叔丁氧羰基(Boc)、9-芴基甲基烯氧羰基(Fmoc)、 卞氧羰基(Cbz)、烯丙氧羰基(Alloc)、三苯基甲基(Trt)等任意合适的保护基，P 1不等于P 2(在实施方案中，P 1和P 2是正交保护基)。缩合剂可以选自N,N'-二异丙基碳二亚胺、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯等缩合剂中的任意一种； Compound 2 under the action of condensing agent, and
Figure PCTCN2020085124-appb-000023
The reaction produces compound 3, P 1 and P 2 are protecting groups for amino groups, and you can choose tert-butoxycarbonyl (Boc), 9-fluorenylmethylenoxycarbonyl (Fmoc), Boxycarbonyl (Cbz), and allyloxycarbonyl (Alloc), triphenylmethyl (Trt) and any suitable protecting groups, P 1 is not equal to P 2 (in embodiments, P 1 and P 2 are orthogonal protecting groups). The condensing agent can be selected from N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7- Nitrobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and other condensing agents;
化合物3在臭氧条件下，碳-碳双键氧化成少一个碳的醛，得到化合物4，反应温度选取在-80℃至-20℃，反应溶剂选取四氢呋喃、二氯甲烷、甲醇，还原剂选用三苯基膦、亚硫酸钠、三乙胺等；Under ozone conditions, the carbon-carbon double bond is oxidized to an aldehyde with one less carbon to obtain compound 4. The reaction temperature is from -80℃ to -20℃, the reaction solvent is tetrahydrofuran, dichloromethane, methanol, and the reducing agent is selected Triphenylphosphine, sodium sulfite, triethylamine, etc.;
化合物4在酸性(P 2为Boc保护基时)或钯碳加氢(P 2为Cbz保护基时)或哌啶(P 2为Fmoc保护基时)等条件下脱除保护基P 2，进一步还原胺化，得到大环化合物5，反应溶剂选取四氢呋喃、二氯甲烷、甲醇、甲苯等，还原剂选用硼氢化钠、氰基硼氢化钠等； Compound 4 removes the protective group P 2 under acidic conditions (when P 2 is the Boc protecting group), palladium-carbon hydrogenation (when P 2 is the Cbz protecting group) or piperidine (when P 2 is the Fmoc protecting group), and further Reductive amination to obtain macrocyclic compound 5, the reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene, etc., and the reducing agent is sodium borohydride, sodium cyanoborohydride, etc.;
化合物5在惰性有机溶剂如乙腈、甲苯、氯仿、二氯甲烷、四氢呋喃、二甲亚砜中，在缩合剂作用下和包含R 1基团的羧酸类化合物进行缩合反应，得到化合物6，温度选取在25℃至120℃，缩合剂选取N,N'-二异丙基碳二亚胺、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、1H-苯并***-1-基氧三吡咯烷基六氟磷酸盐等； Compound 5 is in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and a carboxylic acid compound containing R 1 group undergoes condensation reaction to obtain compound 6, temperature Choose from 25℃ to 120℃, choose N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, condensing agent 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluoro Phosphate etc.;
化合物6在碱性条件下皂化，然后控制PH值在酸性条件下，得到化合物7，有机溶剂选取甲醇、乙醇等水溶性溶剂，碱选取无机碱氢氧化锂、氢氧化钠等；Compound 6 is saponified under alkaline conditions, and then the pH value is controlled under acidic conditions to obtain compound 7. The organic solvent is methanol, ethanol and other water-soluble solvents, and the alkali is inorganic alkalis such as lithium hydroxide and sodium hydroxide;
化合物7在惰性有机溶剂如乙腈、甲苯、氯仿、二氯甲烷、四氢呋喃、二甲亚砜中，在缩合剂作用下和包含R 3基团的氨基类化合物进行缩合反应，得到化合物8，温度选取在25℃至120℃，缩合剂选取N,N'-二异丙基碳二亚胺、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、1H-苯并***-1-基氧三吡咯烷基六氟磷酸盐等； Compound 7 is in inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and an amino compound containing R 3 group is condensed to obtain compound 8, and the temperature is selected At 25°C to 120°C, the condensing agent is N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2 -(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate Salt etc.;
化合物8在酸性(P 1为Boc保护基时)或钯碳加氢(P 1为Cbz保护基时)或哌啶(P 1为Fmoc保护基时)等条件下脱除保护基P 1，得到氨基化合物9，反应溶剂选取四氢呋喃、二氯甲烷、甲醇、甲苯等； Compound 8 removes the protective group P 1 under acidic conditions (when P 1 is a Boc protecting group), palladium-carbon hydrogenation (when P 1 is a Cbz protecting group), or piperidine (when P 1 is a Fmoc protecting group), etc. Amino compound 9, the reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene, etc.;
化合物9在惰性有机溶剂如乙腈、甲苯、氯仿、二氯甲烷、四氢呋喃、二甲亚砜中，在缩合剂作用下和包含R 2基团的羧酸类化合物进行缩合反应，得到化合物I，温度选取在25℃至120℃，缩合剂选取N,N'-二异丙基碳二亚胺、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、1H-苯并***-1-基氧三吡咯烷基六氟磷酸盐等。 Compound 9 is in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and a carboxylic acid compound containing R 2 group undergoes a condensation reaction to obtain compound I. Choose from 25℃ to 120℃, choose N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, condensing agent 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluoro Phosphate etc.
在另一种方案中，本发明的化合物的制备路线如下所示：In another scheme, the preparation route of the compound of the present invention is as follows:
Figure PCTCN2020085124-appb-000024
Figure PCTCN2020085124-appb-000024
方案2Scenario 2
方案2包括如下步骤:Option 2 includes the following steps:
以化合物10为起始原料，在碳酸钾、碳酸铯等无机碱的作用下关环生成化合物11，反应温度选取25℃至120℃，反应溶剂选取乙腈、四氢呋喃、N,N-二甲基甲酰胺等惰性非质子溶剂；Using compound 10 as the starting material, the ring is closed under the action of inorganic bases such as potassium carbonate and cesium carbonate to produce compound 11. The reaction temperature is 25°C to 120°C, and the reaction solvent is acetonitrile, tetrahydrofuran, and N,N-dimethylformaldehyde. Inert aprotic solvents such as amides;
化合物11在缩合剂的作用下，与
Figure PCTCN2020085124-appb-000025
反应生成化合物12，P 1，P 2为氨基的保护基，可以选择叔丁氧羰基(Boc)，9-芴基甲基烯氧羰基(Fmoc)，卞氧羰基(Cbz)，烯丙氧羰基(Alloc)，三苯基甲基(Trt)等，P 1不等于P 2(在实施方案中，P 1和P 2是正交保护基)。缩合剂选取N,N'-二异丙基碳二亚胺，苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯等； Compound 11 under the action of condensing agent, and
Figure PCTCN2020085124-appb-000025
The reaction produces compound 12, P 1 and P 2 are protecting groups for amino groups, and you can choose tert-butoxycarbonyl (Boc), 9-fluorenylmethylenoxycarbonyl (Fmoc), Boxycarbonyl (Cbz), and allyloxycarbonyl (Alloc), triphenylmethyl (Trt), etc., P 1 is not equal to P 2 (in embodiments, P 1 and P 2 are orthogonal protecting groups). Condensing agent selected N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7-azobenzene Triazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, etc.;
化合物12在臭氧条件下，碳-碳双键氧化成少一个碳的醛，得到化合物13，反应温度选取在-80℃至-20℃，反应溶剂选取四氢呋喃、二氯甲烷、甲醇，还原剂选用三苯基膦、亚硫酸钠、三乙胺等；Under ozone conditions, the carbon-carbon double bond of compound 12 is oxidized to an aldehyde with one less carbon to obtain compound 13. The reaction temperature is selected from -80°C to -20°C, the reaction solvent is tetrahydrofuran, dichloromethane, methanol, and the reducing agent is selected Triphenylphosphine, sodium sulfite, triethylamine, etc.;
化合物13在酸性(P 2为Boc保护基时)或钯碳加氢(P 2为Cbz保护基时)或哌啶(P 2为Fmoc保护基时)等条件下脱除保护基P 2，进一步还原胺化，得到 大环化合物14，反应溶剂选取四氢呋喃、二氯甲烷、甲醇、甲苯等，还原剂选用硼氢化钠、氰基硼氢化钠等； Compound 13 removes the protective group P 2 under acidic conditions (when P 2 is the Boc protecting group), palladium-carbon hydrogenation (when P 2 is the Cbz protecting group), or piperidine (when P 2 is the Fmoc protecting group), and further Reductive amination to obtain macrocyclic compound 14, the reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene, etc., and the reducing agent is sodium borohydride, sodium cyanoborohydride, etc.;
化合物14在惰性有机溶剂如乙腈、甲苯、氯仿、二氯甲烷、四氢呋喃、二甲亚砜中，在缩合剂作用下和包含R 1基团的羧酸类化合物进行缩合反应，得到化合物15，温度选取在25℃至120℃，缩合剂选取N,N'-二异丙基碳二亚胺、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、1H-苯并***-1-基氧三吡咯烷基六氟磷酸盐等； Compound 14 is in inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and a carboxylic acid compound containing R 1 group undergoes condensation reaction to obtain compound 15. Choose from 25℃ to 120℃, choose N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, condensing agent 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluoro Phosphate etc.;
化合物15在碱性条件下皂化，然后控制PH值在酸性条件下，得到化合物16，有机溶剂选取甲醇、乙醇等水溶性溶剂，碱选取无机碱氢氧化锂、氢氧化钠等；Compound 15 is saponified under alkaline conditions, and then the pH value is controlled under acidic conditions to obtain compound 16. The organic solvent is methanol, ethanol and other water-soluble solvents, and the alkali is inorganic alkalis such as lithium hydroxide and sodium hydroxide;
化合物16在惰性有机溶剂如乙腈、甲苯、氯仿、二氯甲烷、四氢呋喃、二甲亚砜中，在缩合剂作用下和包含R 3基团的氨基类化合物进行缩合反应，得到化合物17，温度选取在25℃至120℃，缩合剂选取N,N'-二异丙基碳二亚胺、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、1H-苯并***-1-基氧三吡咯烷基六氟磷酸盐等； Compound 16 is in inert organic solvents such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and an amino compound containing R 3 group undergoes condensation reaction to obtain compound 17, and the temperature is selected At 25°C to 120°C, the condensing agent is N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2 -(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate Salt etc.;
化合物17在酸性(P 1为Boc保护基时)或钯碳加氢(P 1为Cbz保护基时)或哌啶(P 1为Fmoc保护基时)等条件下脱除保护基P 1，得到氨基化合物18，反应溶剂选取四氢呋喃、二氯甲烷、甲醇、甲苯等； Compound 17 removes the protective group P 1 under acidic conditions (when P 1 is a Boc protecting group), palladium-carbon hydrogenation (when P 1 is a Cbz protecting group), or piperidine (when P 1 is a Fmoc protecting group), etc. Amino compound 18, the reaction solvent is tetrahydrofuran, dichloromethane, methanol, toluene, etc.;
化合物18在惰性有机溶剂如乙腈、甲苯、氯仿、二氯甲烷、四氢呋喃、二甲亚砜中，在缩合剂作用下和包含R 2基团的羧酸类化合物进行缩合反应，得到化合物II，温度选取在25℃至120℃，缩合剂选取N,N'-二异丙基碳二亚胺、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、1H-苯并***-1-基氧三吡咯烷基六氟磷酸盐等。 Compound 18 is in an inert organic solvent such as acetonitrile, toluene, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, under the action of a condensing agent, and a carboxylic acid compound containing R 2 group undergoes condensation reaction to obtain compound II, temperature Choose from 25℃ to 120℃, choose N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, condensing agent 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluoro Phosphate etc.
进一步地，在上述方案1和方案2中，P 1，P 2基团为氨基的保护基，R 1、R 2和R 3基团的定义如说明书上文所述。 Further, in the above scheme 1 and scheme 2, the P 1 and P 2 groups are the protecting groups of the amino group, and the R 1 , R 2 and R 3 groups are defined as described above in the specification.
式I‘化合物的制备可以参照方案1或者方案2的方法进行。The preparation of the compound of formula I'can refer to the method of Scheme 1 or Scheme 2.
在上述制备方法中，制备化合物的方法如涉及将各种化学基团保护和脱保护，本领域技术人员可容易地根据保护和脱保护的需要选择适宜的保护基团。本发明提到的上述基团特征，或实施例提到的特征可以在符合药学规律基础上任意组合，说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。除有特别说明，揭示的特征仅为相同或形似特征的一般性例子。In the above preparation methods, the method for preparing compounds involves protecting and deprotecting various chemical groups, and those skilled in the art can easily select a suitable protecting group according to the needs of protection and deprotection. The above-mentioned group features mentioned in the present invention or the features mentioned in the examples can be combined arbitrarily on the basis of compliance with the laws of pharmacy. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. . Unless otherwise specified, the disclosed features are only general examples of the same or similar features.
本发明另一方面提供了一种药物组合物，其中含有治疗有效量的任意选自式式I‘或者式I或者式II化合物或其药学上可接受的盐或异构体的化合物作为活性成分，以及一种或多种药学上可接受的载体、稀释剂和/或赋型剂。Another aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of any compound selected from the compound of formula I'or formula I or formula II or a pharmaceutically acceptable salt or isomer thereof as an active ingredient , And one or more pharmaceutically acceptable carriers, diluents and/or excipients.
该药物组合物优选含有重量比为1％-90％的式I‘或者式I或者式II化合物或其药学上可接受的盐或异构体的化合物作为活性成分，更优选含有重量比为10％-80％的活性成分。The pharmaceutical composition preferably contains a compound of formula I'or formula I or formula II or a pharmaceutically acceptable salt or isomer thereof in a weight ratio of 1% to 90% as an active ingredient, more preferably a weight ratio of 10 %-80% active ingredients.
除非另有说明，下列用在权利要求书和说明书中的术语有如下含义或特征：Unless otherwise stated, the following terms used in the claims and specification have the following meanings or features:
术语“C 1～20烷基”表示1-20个碳原子的饱和的脂烃基，包括直链和支链基团(本申请书中提到的数字范围，例如“1-20”，是指该基团，此时为烷基，可以含1个碳原子、2个碳原子、3个碳原子等，直至包括20个碳原子)。本发明中的烷基包含“亚烷基”。含1－6个碳原子的烷基称为低级烷基。当低级烷基没有取代基时，称其为未取代的低级烷基。更优选的是，烷基是有1-10个碳原子的中等大小的烷基，例如甲基、乙基、亚乙基、丙基、亚丙基、2-丙基、正丁基、异丁基、亚丁基、叔丁基、戊基等。最好是，烷基为有1-5个碳原子的低级烷基，例如甲基、乙基、丙基、2-丙基、正丁基、亚丁基、异丁基或叔丁基等。烷基可以是取代的或未取代的。 The term "C 1 ~ 20 alkyl group" means a saturated aliphatic hydrocarbon group of 1 to 20 carbon atoms, including straight-chain and branched-chain groups (the numerical ranges mentioned in this application, for example, "1-20", refers to The group, in this case an alkyl group, can contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms). The alkyl group in the present invention includes "alkylene". An alkyl group containing 1 to 6 carbon atoms is called a lower alkyl group. When a lower alkyl group has no substituent, it is called an unsubstituted lower alkyl group. More preferably, the alkyl group is a medium-sized alkyl group having 1-10 carbon atoms, such as methyl, ethyl, ethylene, propyl, propylene, 2-propyl, n-butyl, isopropyl Butyl, butylene, tert-butyl, pentyl, etc. Preferably, the alkyl group is a lower alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, butylene, isobutyl or tert-butyl. Alkyl groups can be substituted or unsubstituted.
术语“C 1～20烷胺基”表示R-氨基类化合物，R的定义如C 1～20烷基所述。 The term "C 1-20 alkylamino group" means an R-amino compound, and the definition of R is as described for the C 1-20 alkyl group.
术语“C 5～8环烷胺基”表示C 5～8环烷上的一个碳被氨基取代所形成的胺类化合物，例如
Figure PCTCN2020085124-appb-000026
等。 The term "C 5-8 cycloalkaneamino group" means an amine compound formed by substituting one carbon on a C 5-8 cycloalkane with an amino group, for example
Figure PCTCN2020085124-appb-000026
Wait.
术语“α-氨基酸残基”表示-氨基酸中α-碳上的羧基缺失后所形成的取代残基。The term "α-amino acid residue" means a substitution residue formed after the carboxyl group on the α-carbon of an amino acid is deleted.
术语“苄基”表示“苯甲基”，结构简式C 6H 5CH 2-，或用Bn(Benzyl的缩写)表示。 The term "benzyl" means "benzyl", with a simple structure of C 6 H 5 CH 2 -, or Bn (abbreviation for Benzyl).
术语“芳基”表示5至12个碳原子的全碳单环或稠合多环基团，具有完全共轭的π电子***。芳基的非限制性实例有苯基、萘基和蒽基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环。芳基可以是取代的或未取代的。当被取代时，取代基优选为一个或多个，更优选为一个、两个或三个，进而更优选为一个或两个，独立地选自由低级烷基、三卤烷基、卤素、羟基、低级烷氧基、巯基、(低级烷基)硫基、氰基、酰基、硫代酰基、O-氨基甲酰基、N-氨基甲酰基、O-硫代氨基甲酰基、N-硫代氨基甲酰基、C-酰氨基、N-酰氨基、硝基、N-磺酰氨基、S-磺酰氨基。优选地，芳基为5元单环芳基、6元单环芳基。The term "aryl" refers to an all-carbon monocyclic or fused polycyclic group of 5 to 12 carbon atoms with a fully conjugated π-electron system. Non-limiting examples of aryl groups are phenyl, naphthyl and anthracenyl. The aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring. Aryl groups can be substituted or unsubstituted. When substituted, the substituent is preferably one or more, more preferably one, two or three, and even more preferably one or two, independently selected from lower alkyl, trihaloalkyl, halogen, and hydroxyl , Lower alkoxy, mercapto, (lower alkyl)thio, cyano, acyl, thioacyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thioamino Formyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido. Preferably, the aryl group is a 5-membered monocyclic aryl group or a 6-membered monocyclic aryl group.
术语“杂芳基”表示5至12个环原子的单环或稠合环基团，含有一个、两个、三个或四个选自N、O或S的环杂原子，其余环原子是C，另外具有完全共轭的π电子***。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环。杂芳基可以是取代的或未取代的。当被取代时，取代基优选为一个或多个，更为优选为一个、两个或三个，进而更为优选一个或两个。未取代的杂芳基地非限制性实例有吡咯、呋喃、噻吩、咪唑、噁唑、噻唑、吡唑、嘧啶、喹啉、异喹啉、嘌呤、四唑、三嗪和咔唑；优选地，杂芳基为含氮5元单环杂芳基、含氮6元单环杂芳基。The term "heteroaryl" means a monocyclic or condensed ring group of 5 to 12 ring atoms, containing one, two, three or four ring heteroatoms selected from N, O or S, and the remaining ring atoms are C, in addition has a fully conjugated π electron system. The heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, and the ring connected to the parent structure is a heteroaryl ring. Heteroaryl groups can be substituted or unsubstituted. When substituted, the substituent is preferably one or more, more preferably one, two or three, and still more preferably one or two. Non-limiting examples of unsubstituted heteroaromatic bases are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyrimidine, quinoline, isoquinoline, purine, tetrazole, triazine and carbazole; preferably, Heteroaryl groups are nitrogen-containing 5-membered monocyclic heteroaryl groups and nitrogen-containing 6-membered monocyclic heteroaryl groups.
术语“异构体”可理解涵盖了本领域常规的化合物“立体异构”，包括对映异构体和非对映异构体，其中非对映异构又包括顺反异构，例如选自其顺式异构体、反式异构体或者顺反异构体的混合物。The term "isomer" can be understood to cover the conventional compound "stereoisomerism" in the art, including enantiomers and diastereomers, where diastereomers include cis and trans isomers, for example, From its cis isomer, trans isomer or a mixture of cis and trans isomers.
术语“卤素”表示氟、氯、溴或碘。The term "halogen" means fluorine, chlorine, bromine or iodine.
术语“药学上可接受的盐”表示保留母体化合物的生物有效性和性质的那些 盐。这类盐包括：The term "pharmaceutically acceptable salts" means those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:
(1)与酸成盐，通过母体化合物的游离碱与无机酸或有机酸的反应而得，无机酸例如(但不限于)盐酸、氢溴酸等，有机酸例如(但不限于)乙酸、苹果酸、富马酸、马来酸、对甲苯磺酸、酒石酸、柠檬酸、乳酸、琥珀酸或丙二酸等。这类盐用于哺乳动物体内具有安全性、有效性和应有的生物活性。(1) Forming a salt with an acid, obtained by the reaction of the free base of the parent compound with an inorganic acid or organic acid, such as (but not limited to) hydrochloric acid, hydrobromic acid, etc., and organic acid such as (but not limited to) acetic acid, Malic acid, fumaric acid, maleic acid, p-toluenesulfonic acid, tartaric acid, citric acid, lactic acid, succinic acid or malonic acid, etc. Such salts have safety, effectiveness and due biological activity when used in mammals.
“药用组合物”指的是在此描述的一种或多种化合物或者它们的药学上可接受的盐、异构体和前药等与其它的化学成分，例如药学上可接受的载体和赋形剂的混合物。药用组合物的目的是促进化合物对生物体的给药。"Pharmaceutical composition" refers to one or more of the compounds described herein or their pharmaceutically acceptable salts, isomers and prodrugs and other chemical components, such as pharmaceutically acceptable carriers and Mixture of excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
“药学上可接受的载体”指的是对有机体不引起明显的刺激性和不干扰所给予化合物的生物活性和性质的载体或稀释剂。"Pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to organisms and does not interfere with the biological activity and properties of the administered compound.
“赋形剂”指的是加入到药用组合物中以进一步便利于给予化合物的惰性物质。赋形剂的实例包括(不局限于)乳糖、蔗糖、微晶纤维素、山梨醇、聚乙烯吡咯烷酮、纤维素、水、和甲基纤维素等。"Excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound. Examples of excipients include (not limited to) lactose, sucrose, microcrystalline cellulose, sorbitol, polyvinylpyrrolidone, cellulose, water, methylcellulose, and the like.
药物组合物还可含有：润滑剂例如滑石粉、硬脂酸镁和矿物油等；湿润剂；乳化剂和悬浮剂；防腐剂例如苯甲酸甲酯等；甜味剂和矫味剂。The pharmaceutical composition may also contain: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl benzoate, etc.; sweeteners and flavoring agents.
本发明的组合物以符合良好医学实践的方式进行配制、定剂量(dosed)和给药。在该情形中供考虑的因素包括所治疗的特定病症、所治疗的特定哺乳动物、个体患者的临床状况、病症的原因、递送药剂的位置、给药方法、给药方案以及医学从业者已知的其他因素。The composition of the present invention is formulated, dosed and administered in a manner consistent with good medical practice. The factors to be considered in this situation include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the location where the agent is delivered, the method of administration, the dosing regimen, and what is known to the medical practitioner Other factors.
本发明还提供了所述的式I‘或者式I或者式II化合物或其药学上可接受的盐或异构体的用途：The present invention also provides the use of the compound of formula I'or formula I or formula II or a pharmaceutically acceptable salt or isomer thereof:
(1)用于在细胞中诱导细胞凋亡，或使细胞、特别是癌细胞对细胞凋亡信号敏感。(1) It is used to induce apoptosis in cells, or make cells, especially cancer cells, sensitive to apoptosis signals.
(2)用于在过表达IAP蛋白(例如，c-IAP1、c-IAP2、X-IAP或ML-IAP)的细胞中诱导细胞凋亡。(2) It is used to induce apoptosis in cells overexpressing IAP protein (for example, c-IAP1, c-IAP2, X-IAP or ML-IAP).
(3)制备与IAP蛋白相关疾病的药物；更广泛地，所述化合物可用于治疗癌症。(3) Preparation of drugs for diseases related to IAP protein; more broadly, the compounds can be used to treat cancer.
(4)进一步地，本发明的化合物可以用于治疗未能经历细胞凋亡的所有癌症类型药物。此类癌症类型的实例包括神经母细胞瘤、肠癌诸如直肠癌、结肠癌、家族性腺瘤性息肉癌及遗传性非息肉性结肠直肠癌、食道癌、唇癌、喉癌、下咽癌、舌癌、唾液腺癌、胃癌、腺癌、甲状腺髓样癌、***状甲状腺癌、肾癌、肾实质癌、卵巢癌、子***、子宫体癌、子宫内膜癌、绒毛膜癌、胰腺癌、***癌、睪丸癌、乳腺癌、泌尿***癌、黑素瘤、脑瘤诸如胶质母细胞瘤、星形细胞瘤、脑膜瘤、髓母细胞瘤及周围神经外胚层瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、伯基特淋巴瘤、急性淋巴性白血病(ALL)、慢性淋巴性白血病(CLL)、急性髓性白血病(AML)、慢性髓性白血病(CML)、成人T-细胞白血病淋巴瘤、肝细胞癌、胆囊癌、支气管癌、小细胞肺癌、非小细胞肺癌、多发性骨髓瘤、基底细胞瘤、畸胎瘤、视网膜母细胞瘤、脉络膜黑素瘤、***瘤、横纹肌肉瘤、颅咽 瘤(craniopharyngeoma)、骨肉瘤、软骨肉瘤、肌肉瘤、脂肪肉瘤、纤维肉瘤、尤因肉瘤及浆细胞瘤。可用于治疗实体瘤。还可用于治疗乳腺癌、胰腺癌或恶性黑素瘤等多种重大疾病的药物。(4) Further, the compounds of the present invention can be used to treat all types of cancer drugs that fail to undergo apoptosis. Examples of such cancer types include neuroblastoma, bowel cancer such as rectal cancer, colon cancer, familial adenomatous polyp cancer and hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, Tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine body cancer, endometrial cancer, choriocarcinoma, pancreatic cancer , Prostate cancer, testicular cancer, breast cancer, urinary system cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, Hodgkin's lymph Tumor, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T- Cell leukemia lymphoma, hepatocellular carcinoma, gallbladder cancer, bronchial cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, spermatogonia Tumor, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, sarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma and plasmacytoma. Can be used to treat solid tumors. It can also be used to treat many major diseases such as breast cancer, pancreatic cancer or malignant melanoma.
本发明化合物的阳性对照化合物AT-406为cIAP1、cIAP2抑制剂，对于XIAP的抑制作用比cIAP1、cIAP2弱，而本发明的的化合物对XIAP、cIAP1、cIAP2三种蛋白的抑制活性均较强，因此本发明化合物对细胞凋亡蛋白整体上具有更强的抑制效果；The positive control compound AT-406 of the compound of the present invention is an inhibitor of cIAP1 and cIAP2, and its inhibitory effect on XIAP is weaker than that of cIAP1 and cIAP2, while the compound of the present invention has stronger inhibitory activity on the three proteins of XIAP, cIAP1 and cIAP2. Therefore, the compound of the present invention has a stronger inhibitory effect on apoptosis proteins as a whole;
从细胞药理数据发现，本发明化合物对MDA-MB-231乳腺癌和PC-3胰腺癌细胞具有很强的抑制作用，而阳性对照化合物AT-406仅对MDA-MB-231乳腺癌的作用较强，对PC-3胰腺癌细胞的抑制作用相对弱一些；From cell pharmacological data, it is found that the compound of the present invention has a strong inhibitory effect on MDA-MB-231 breast cancer and PC-3 pancreatic cancer cells, while the positive control compound AT-406 only has a better effect on MDA-MB-231 breast cancer. Strong, the inhibitory effect on PC-3 pancreatic cancer cells is relatively weak;
本发明的化合物具有较好的IAP抑制活性，后期可以开发成单独用药的癌症治疗药物，也可以与其他靶向药物联合用药治疗癌症；The compound of the present invention has good IAP inhibitory activity, and can be developed into a cancer treatment drug used alone in the later stage, or can be combined with other targeted drugs to treat cancer;
综上所述，本发明化合物与XIAP、cIAP1、cIAP2蛋白的结合亲和力较好，对MDA-MB-231乳腺癌和PC-3胰腺癌细胞系中细胞生长具有较好的抑制作用，具有较大的药用价值和广阔的市场化前景。In summary, the compound of the present invention has good binding affinity with XIAP, cIAP1, and cIAP2 proteins, and has a good inhibitory effect on cell growth in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines. Its medicinal value and broad market prospects.
具体实施例Specific embodiment
以下具体实施例用来进一步说明本发明，但本发明绝非仅限于这些例子(除另有注明外，所有原料均有市售)。The following specific examples are used to further illustrate the present invention, but the present invention is by no means limited to these examples (unless otherwise noted, all raw materials are commercially available).
(本例中所有涉及手性化合物均由手性柱制备分离纯化)(All the chiral compounds involved in this example are prepared and purified by chiral column)
实施例1：吡咯烷-2-甲酸甲酯的制备Example 1: Preparation of methyl pyrrolidine-2-carboxylate
Figure PCTCN2020085124-appb-000027
Figure PCTCN2020085124-appb-000027
取脯氨酸40g(0.35mol)和250mL甲醇于500mL单口反应瓶中，冷却至0℃，另取二氯亚砜45g(0.38mol)于250mL恒压滴液漏斗中，慢慢滴加至上述溶液体系中，随滴加体系放热，控制内温＜5℃，约20分钟滴加完毕。升温至70℃，得澄清溶液，回流3小时。降至室温后，减压蒸除溶剂，得到淡黄色油状液体43g，粗品直接用于下一步反应。Take 40g (0.35mol) of proline and 250mL of methanol in a 500mL single-necked reaction flask, cool to 0℃, take another 45g (0.38mol) of thionyl chloride in a 250mL constant pressure dropping funnel, and slowly add dropwise to the above In the solution system, heat is released with the dropping system, and the internal temperature is controlled to be less than 5°C, and the dropping is completed in about 20 minutes. The temperature was raised to 70°C to obtain a clear solution, which was refluxed for 3 hours. After cooling to room temperature, the solvent was evaporated under reduced pressure to obtain 43 g of light yellow oily liquid, and the crude product was directly used in the next reaction.
实施例2：N-Boc吡咯烷-2-甲酸甲酯的制备Example 2: Preparation of Methyl N-Bocpyrrolidine-2-carboxylate
Figure PCTCN2020085124-appb-000028
Figure PCTCN2020085124-appb-000028
取43g(0.33mol)吡咯烷-2-甲酸甲酯和400mL二氯甲烷于2L单口反应瓶中，加入88g(0.87mol)三乙胺,得到白色乳浊液，室温下搅拌15分钟，冷却至0℃。Take 43g (0.33mol) of methyl pyrrolidine-2-formate and 400mL of dichloromethane in a 2L single-necked reaction flask, add 88g (0.87mol) of triethylamine to obtain a white emulsion, stir at room temperature for 15 minutes, and cool to 0°C.
另取Boc酸酐152g(0.69mol)溶于600mL二氯甲烷于恒压漏斗中，滴加入上述体系中，随滴加放出大量气泡，放热，控制滴加速度，维持体系内温＜10℃。滴加完毕，得白色悬浊液，室温搅拌12小时。过滤，除去白色沉淀，滤液减压 蒸出溶剂，得白色悬浊液，加入300mL柠檬酸溶液(0.5M)洗涤，***萃取(3*500mL),合并有机层，饱和盐水洗涤，取有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯:石油醚＝1:5)纯化，得到清亮无色油状液体75g，收率94％。In addition, 152 g (0.69 mol) of Boc anhydride was dissolved in 600 mL of dichloromethane in a constant pressure funnel, and added dropwise to the above system. A large number of bubbles were released with the dropwise addition, heat was released, and the dropping rate was controlled to maintain the internal temperature of the system <10°C. After the addition was completed, a white suspension was obtained, which was stirred at room temperature for 12 hours. The white precipitate was removed by filtration, and the filtrate was evaporated under reduced pressure to obtain a white suspension, which was washed with 300 mL of citric acid solution (0.5M), extracted with ether (3*500 mL), combined the organic layers, washed with saturated brine, and used It was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 75 g of a clear and colorless oily liquid with a yield of 94%.
实施例3：N-Boc-2-丙烯基-吡咯烷-2-甲酸甲酯的制备Example 3: Preparation of N-Boc-2-propenyl-pyrrolidine-2-carboxylic acid methyl ester
Figure PCTCN2020085124-appb-000029
Figure PCTCN2020085124-appb-000029
取N-Boc吡咯烷-2-甲酸甲酯20g(87.3mmol)和200mL四氢呋喃于500mL三口瓶中，氮气保护，冷却至-78℃。Take 20 g (87.3 mmol) of N-Boc pyrrolidine-2-carboxylic acid methyl ester and 200 mL of tetrahydrofuran in a 500 mL three-necked flask under nitrogen protection and cool to -78°C.
另取双三甲基硅基胺基锂105mL(105mmol，1M)于恒压滴液漏斗，滴加入上述体系中，控制滴加速度，维持体系内温＜-78℃，得到黄褐色溶液。滴加完毕，于-78℃下搅拌1.5小时。In addition, 105 mL (105 mmol, 1M) of lithium bistrimethylsilylamide was added to the constant pressure dropping funnel and added dropwise to the above system to control the dropping rate and maintain the internal temperature of the system <-78°C to obtain a yellow-brown solution. After the addition is complete, stir at -78°C for 1.5 hours.
另取烯丙基溴15.9g(131mmol)溶于100mL四氢呋喃，滴加入上述体系，控制滴加速度，维持体系内温＜-78℃，滴加完毕，于-78℃下搅拌1小时。恢复至室温，搅拌1小时，得红褐色溶液。TLC检测反应完全，加入50mL水淬灭反应。乙酸乙酯萃取(3*200mL)合并有机层，饱和盐水洗涤，取有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯:石油醚＝1:10)纯化，得到无色油状液体18.7g，收率82％。In addition, 15.9 g (131 mmol) of allyl bromide was dissolved in 100 mL of tetrahydrofuran, and added dropwise to the above system, controlling the dropping rate, maintaining the internal temperature of the system <-78°C, after the addition, stirring at -78°C for 1 hour. Return to room temperature and stir for 1 hour to obtain a reddish brown solution. TLC detected that the reaction was complete, and 50 mL of water was added to quench the reaction. Ethyl acetate extraction (3*200 mL) and the combined organic layers were washed with saturated brine. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was subjected to column chromatography (eluent: ethyl acetate: petroleum ether = 1:10) Purification to obtain 18.7 g of colorless oily liquid with a yield of 82%.
实施例4：2-丙烯基-1-((3-苄氧羰基-氨基-2-叔丁氧羰基-氨基)丙酰基)吡咯烷-2-甲酸甲酯的制备Example 4: Preparation of 2-propenyl-1-((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino)propionyl)pyrrolidine-2-carboxylic acid methyl ester
Figure PCTCN2020085124-appb-000030
Figure PCTCN2020085124-appb-000030
取18g(66.9mmol)N-Boc-2-丙烯基-吡咯烷-2-甲酸甲酯和50mL无水乙醇于500mL单口反应瓶中，加入100mL盐酸乙醇，室温搅拌2小时，TLC检测，反应完全。减压蒸出溶剂及过量氯化氢，得到无色油状液体14g，溶于50mL二氯甲烷。Take 18g (66.9mmol) of N-Boc-2-propenyl-pyrrolidine-2-carboxylic acid methyl ester and 50mL of absolute ethanol in a 500mL single-necked reaction flask, add 100mL of hydrochloric acid ethanol, stir at room temperature for 2 hours, TLC detection, the reaction is complete . The solvent and excess hydrogen chloride were evaporated under reduced pressure to obtain 14 g of a colorless oily liquid, which was dissolved in 50 mL of dichloromethane.
另取(S)-2-(Boc-氨基)-3-(Cbz-氨基)丙酸27g(80.3mmol)和200mL二氯甲烷于500mL单口反应瓶中，加入38g HATU(100.35mmol)，17gDIEA(133.8mmol)，室温搅拌1小时，得黄色清液。Separately take 27g (80.3mmol) of (S)-2-(Boc-amino)-3-(Cbz-amino)propionic acid and 200mL of dichloromethane in a 500mL single-neck reaction flask, add 38g HATU (100.35mmol), 17gDIEA( 133.8mmol), stirred at room temperature for 1 hour to obtain a yellow clear liquid.
将第一步制得的二氯甲烷溶液滴加入上述黄色清液中，室温搅拌6小时，得到红褐色溶液，TLC检测反应完全。减压蒸出溶剂，残余物用400mL乙酸乙酯溶解，用1M HCl溶液洗涤，饱和碳酸氢钠溶液洗涤，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯:石油醚＝1:2)纯化，得到白色固体21.2g，收率65％。The dichloromethane solution prepared in the first step was added dropwise to the yellow clear solution, and stirred at room temperature for 6 hours to obtain a reddish brown solution. TLC detected that the reaction was complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 400 mL ethyl acetate, washed with 1M HCl solution, saturated sodium bicarbonate solution, saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:2) to obtain 21.2 g of white solid with a yield of 65%.
实施例5：2-乙醛基-1-((3-苄氧羰基-氨基-2-叔丁氧羰基-氨基)丙酰基)吡咯烷-2-甲酸甲酯的制备Example 5: Preparation of 2-acetaldehyde-1-((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino)propionyl)pyrrolidine-2-carboxylic acid methyl ester
Figure PCTCN2020085124-appb-000031
Figure PCTCN2020085124-appb-000031
取2-丙烯基-1-((3-苄氧羰基-氨基-2-叔丁氧羰基-氨基)丙酰基)吡咯烷-2-甲酸甲酯20g(40.9mmol)和200mL二氯甲烷于500mL三口反应瓶中，冷却至-78℃，通入臭氧约30分钟，至体系变为蓝色，于-78℃下搅拌30分钟，停止通入臭氧。通入氮气鼓泡除去多余臭氧至蓝色消失，得到无色溶液，加入100mL三乙胺，恢复至室温下，搅拌2小时，TLC检测反应完全。加入1M柠檬酸洗涤，饱和氯化钠水溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯:石油醚＝1:2)纯化，得到白色固体16.8g，收率84％。Take 20g (40.9mmol) of methyl 2-propenyl-1-((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino)propionyl)pyrrolidine-2-carboxylate and 200mL dichloromethane in 500mL In a three-necked reaction flask, cool to -78°C, and pass ozone for about 30 minutes until the system turns blue. Stir at -78°C for 30 minutes, stop passing ozone. Nitrogen was bubbled through to remove excess ozone until the blue color disappeared, and a colorless solution was obtained. 100 mL of triethylamine was added, returned to room temperature, and stirred for 2 hours. TLC detected that the reaction was complete. Wash with 1M citric acid, wash with saturated sodium chloride aqueous solution, dry the organic layer with sodium sulfate, filter, distill off the solvent under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:2) , To obtain 16.8 g of white solid with a yield of 84%.
实施例6：(5R,10aR)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯的制备Example 6: (5R,10aR)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrrolo[1,2-a][1,5]diazaoctane-10a- Preparation of methyl formate
Figure PCTCN2020085124-appb-000032
Figure PCTCN2020085124-appb-000032
取15g(30.5mmol)2-乙醛基-1-((3-N-苄氧羰基-2-N-叔丁氧羰基)丙酰基)吡咯烷-2-甲酸甲酯和260mL甲醇于500mL单口反应瓶中，溶解得无色清液，加入1.5g(10％)Pd/C，氢气气氛下，室温搅拌12小时，TLC检测反应完全。过滤除去 Pd/C，减压蒸出溶剂，加入200mL四氢呋喃溶解，加入氰基硼氢化钠2.9g(45.8mmol)，室温搅拌，TLC检测反应完全。加入20mL水淬灭反应，过滤，减压蒸出溶剂，残余物用200mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝10:1)纯化，得到白色固体7.8g，收率75％。Take 15g (30.5mmol) 2-acetaldehyde-1-((3-N-benzyloxycarbonyl-2-N-tert-butoxycarbonyl)propionyl)pyrrolidine-2-carboxylic acid methyl ester and 260mL methanol in 500mL single mouth In the reaction flask, it was dissolved into a colorless clear liquid, 1.5 g (10%) Pd/C was added, and the mixture was stirred at room temperature for 12 hours under a hydrogen atmosphere. TLC detected that the reaction was complete. The Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, 200 mL of tetrahydrofuran was added to dissolve, 2.9 g (45.8 mmol) of sodium cyanoborohydride was added, and the mixture was stirred at room temperature. TLC detected that the reaction was complete. The reaction was quenched by adding 20 mL of water, filtered, and the solvent was evaporated under reduced pressure. The residue was dissolved in 200 mL of ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. After purification (eluent: dichloromethane:methanol=10:1), 7.8 g of white solid was obtained with a yield of 75%.
实施例7：(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯的制备Example 7: (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrrole Preparation of and [1,2-a][1,5]diazaoctane-10a-methyl carboxylate
Figure PCTCN2020085124-appb-000033
Figure PCTCN2020085124-appb-000033
取N-Cbz-N-甲基-D-丙氨酸1.5g(6.5mmol)和60mL二氯甲烷于250mL单口反应瓶中，加入3.4g HATU(8.9mmol)，1.5gDIEA(11.8mmol)，室温搅拌1小时，得黄色清液。Take 1.5g (6.5mmol) of N-Cbz-N-methyl-D-alanine and 60mL of dichloromethane in a 250mL single-mouth reaction flask, add 3.4g HATU (8.9mmol), 1.5gDIEA (11.8mmol), room temperature Stir for 1 hour to obtain a yellow clear liquid.
取(5R,10aR)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯2g(5.9mmol)溶于20mL二氯甲烷，滴加入上述体系中，室温搅拌6小时，TLC检测反应完全。减压蒸出溶剂，残余物用200mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到褐色油状物2.1g，收率64％。Take (5R,10aR)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrrolo[1,2-a][1,5]diazaoctane-10a-carboxylic acid methyl ester 2 g (5.9 mmol) was dissolved in 20 mL of dichloromethane, added dropwise to the above system, stirred at room temperature for 6 hours, TLC detected that the reaction was complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 200 mL ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent was dichloromethane : Methanol=20:1) Purification to obtain 2.1 g of brown oil with a yield of 64%.
实施例8：(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-羧酸的制备Example 8: (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrrole Preparation of bi[1,2-a][1,5]diazaoctane-10a-carboxylic acid
Figure PCTCN2020085124-appb-000034
Figure PCTCN2020085124-appb-000034
取2g(3.6mmol)(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯和60mL甲醇于250mL单口反应瓶中，加入氢氧化钾0.4g(7.2mmol)，60mL水，加热至50℃，搅拌3小时，TLC检测反应完全。加入60mL水，减压蒸出甲醇，剩余水溶液用乙酸乙酯(2*100mL)萃取，弃去有机层，水溶液用浓盐酸调节pH至1-2，乙酸乙酯(3*100mL)萃取，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，得1.9g油状物，直接用于下一步反应。Take 2g (3.6mmol) (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-de Hydropyrrolo[1,2-a][1,5]diazepine-10a-methyl carboxylate and 60mL methanol in a 250mL single-necked reaction flask, add 0.4g potassium hydroxide (7.2mmol), 60mL water, Heat to 50°C and stir for 3 hours. TLC detects that the reaction is complete. 60mL of water was added, methanol was evaporated under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2*100mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, and extracted with ethyl acetate (3*100mL), organic The layer was dried with sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain 1.9 g of oil, which was directly used in the next reaction.
实施例9：(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺的制备Example 9: (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrrole Preparation of and [1,2-a][1,5]diazaoctane-10a-(N-diphenylmethyl)-carboxamide
Figure PCTCN2020085124-appb-000035
Figure PCTCN2020085124-appb-000035
取(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-羧酸1.8g(3.3mmol)，2.6g PyBop(5.0mmol)于100mL单口反应瓶中，加入50mLDMF，另取0.85g DIEA(6.6mmol),室温搅拌30分钟。另取二苯甲胺1.2g(6.6mmol)加入体系中，加热至90℃，搅拌12小时，TLC检测反应完全。反应液倒入300mL水中，乙酸乙酯(3*100mL)萃取，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到淡黄色固体1.7g，收率74％。Take (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrrolo[1 ,2-a][1,5]Diazaoctane-10a-carboxylic acid 1.8g (3.3mmol), 2.6g PyBop (5.0mmol) in a 100mL single-mouth reaction flask, add 50mL DMF, and take another 0.85g DIEA( 6.6mmol), stirred at room temperature for 30 minutes. Another 1.2 g (6.6 mmol) of benzhydrylamine was added to the system, heated to 90° C., and stirred for 12 hours. TLC detected that the reaction was complete. The reaction solution was poured into 300 mL of water, extracted with ethyl acetate (3*100 mL), washed with saturated sodium chloride solution, the organic layer was dried with sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent is two Chloromethane: methanol = 15:1) was purified to obtain 1.7 g of light yellow solid with a yield of 74%.
实施例10：(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺的制备Example 10: (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-amino-6-one-dehydropyrrolo[1,2-a] [1,5] Preparation of Diazaoctane-10a-(N-Diphenylmethyl)-Carboxamide
Figure PCTCN2020085124-appb-000036
Figure PCTCN2020085124-appb-000036
取0.5g(0.7mmol)(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺和10mL无水乙醇于100mL单口反应瓶中，加入20mL盐酸乙醇，室温搅拌2小时，TLC检测，反应完全。减压蒸出溶剂及过量氯化氢，得到无色油状液体0.5g，直接用于下一步反应。Take 0.5g (0.7mmol) (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one- Dehydropyrrolo[1,2-a][1,5]diazepine-10a-(N-diphenylmethyl)-carboxamide and 10mL absolute ethanol in a 100mL single-neck reaction flask, add 20mL hydrochloric acid Ethanol, stirring at room temperature for 2 hours, TLC detection, the reaction is complete. The solvent and excess hydrogen chloride were distilled off under reduced pressure to obtain 0.5 g of a colorless oily liquid, which was directly used in the next reaction.
实施例11：(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺的制备Example 11: (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrolo[ Preparation of 1,2-a][1,5]diazaoctane-10a-(N-diphenylmethyl)-carboxamide
Figure PCTCN2020085124-appb-000037
Figure PCTCN2020085124-appb-000037
取异戊酸82mg(0.8mmol)和10mL二氯甲烷于100mL单口反应瓶中，加入0.42g HATU(1.1mmol)，0.18gDIEA(1.4mmol)，室温搅拌30分钟，得淡黄色清液。Take 82 mg (0.8 mmol) of isovaleric acid and 10 mL of dichloromethane in a 100 mL single-necked reaction flask, add 0.42 g HATU (1.1 mmol), 0.18 g DIEA (1.4 mmol), and stir at room temperature for 30 minutes to obtain a light yellow clear liquid.
取(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺0.5g溶于10mL二氯甲烷，加入到上述体系中，得黄绿色溶液，室温搅拌3小时，TLC检测，反应完全。减压蒸出溶剂，残余物用100mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到淡黄色固体370mg，收率76％。Take (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-amino-6-one-dehydropyrrolo[1,2-a][1, 5] Diazaoctane-10a-(N-diphenylmethyl)-carboxamide 0.5g dissolved in 10mL dichloromethane, added to the above system to obtain a yellow-green solution, stirred at room temperature for 3 hours, TLC detection, reaction complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 100 mL ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent was dichloromethane : Methanol=20:1) Purification to obtain 370 mg of pale yellow solid with a yield of 76%.
实施例12：(5R,10aR)-3-((R)-2-甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺)(I-11)的制备Example 12: (5R,10aR)-3-((R)-2-methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrolo[1,2-a ][1,5]Diazaoctane-10a-(N-diphenylmethyl)-carboxamide)(I-11)
Figure PCTCN2020085124-appb-000038
Figure PCTCN2020085124-appb-000038
取(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺200mg(0.29mmol)和50mL甲醇于100mL单口反应瓶中，溶解得无色清液，加入20mg(10％)Pd/C，氢气气氛下，室温搅拌12小时，TLC检测反应完全。过滤除去Pd/C，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到淡黄色固体138mg，收率85％。Take (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrolo[1,2 -a][1,5] Diazaoctane-10a-(N-Benzhydryl)-carboxamide 200mg (0.29mmol) and 50mL methanol in a 100mL single-necked reaction flask, dissolved into a colorless clear liquid, add 20mg (10%) Pd/C, stirred at room temperature for 12 hours under hydrogen atmosphere, TLC detected that the reaction was complete. The Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane:methanol=15:1) to obtain 138 mg of pale yellow solid with a yield of 85%.
1H NMR(400MHz,d 6-DMSO):δ＝8.79-8.82(m,1H),7.90-8.10(m,1H),7.22-7.36(m,8H),7.13-7.15(m,2H),6.03-6.06(m,1H),4.75-4.90(m,1H),3.92-4.00(m,1H),3.65-3.80(m,1H),3.52(s, 1H),3.40-3.43(m,2H),2.90-3.20(m,3H),2.10-2.30(m,5H),1.60-2.00(m,5H),1.24(s,2H),1.05-1.15(m,3H),0.84-0.89(m,6H)，[M+H] +:m/z＝562.3 1 H NMR (400MHz, d 6 -DMSO): δ=8.79-8.82 (m, 1H), 7.90-8.10 (m, 1H), 7.22-7.36 (m, 8H), 7.13-7.15 (m, 2H), 6.03-6.06(m,1H),4.75-4.90(m,1H),3.92-4.00(m,1H),3.65-3.80(m,1H),3.52(s, 1H),3.40-3.43(m,2H) ), 2.90-3.20 (m, 3H), 2.10-2.30 (m, 5H), 1.60-2.00 (m, 5H), 1.24 (s, 2H), 1.05-1.15 (m, 3H), 0.84-0.89 (m ,6H), [M+H] + :m/z=562.3
实施例13：(5R,10aR)-3-苄氧羰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯的制备Example 13: (5R,10aR)-3-benzyloxycarbonyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrrolo[1,2-a][1,5]diazepine Preparation of Heterooctyl-10a-Methyl Formate
Figure PCTCN2020085124-appb-000039
Figure PCTCN2020085124-appb-000039
取(5R,10aR)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯5g(14.7mmol)、碳酸氢钠3.1g(36.8mmol)和30mL四氢呋喃于100mL单口反应瓶中，加入30mL水，冷却至5℃，另取3.0g(17.6mmol)Cbz-Cl溶于15mL四氢呋喃，滴加入其中，滴加完毕，室温搅拌6小时，TLC检测反应完全。过滤，滤液减压蒸出四氢呋喃，乙酸乙酯(3*100mL)萃取，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到白色固体6.4g，收率92％。Take (5R,10aR)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrrolo[1,2-a][1,5]diazaoctane-10a-carboxylic acid methyl ester 5g (14.7mmol), 3.1g (36.8mmol) of sodium bicarbonate and 30mL of tetrahydrofuran were put into a 100mL single-necked reaction flask, 30mL of water was added, cooled to 5°C, another 3.0g (17.6mmol) of Cbz-Cl was dissolved in 15mL of tetrahydrofuran, Add dropwise to it, after the dropwise addition is complete, stirring at room temperature for 6 hours, TLC detects that the reaction is complete. After filtration, the filtrate was evaporated under reduced pressure to remove tetrahydrofuran, extracted with ethyl acetate (3*100 mL), washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatography (eluent (Dichloromethane:methanol=20:1) to obtain 6.4 g of white solid with a yield of 92%.
实施例14：(5R,10aR)-3-苄氧羰基-5-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯的制备Example 14: (5R,10aR)-3-benzyloxycarbonyl-5-amino-6-one-dehydropyrrolo[1,2-a][1,5]diazaoctane-10a-formic acid methyl Preparation of ester
Figure PCTCN2020085124-appb-000040
Figure PCTCN2020085124-appb-000040
取6g(12.6mmol)(5R,10aR)-3-苄氧羰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯和30mL无水乙醇于100mL单口反应瓶中，加入30mL盐酸乙醇，室温搅拌2小时，TLC检测，反应完全。减压蒸出溶剂及过量氯化氢，得到无色油状液体5.2g，直接用于下一步反应。Take 6g (12.6mmol) (5R,10aR)-3-benzyloxycarbonyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrrolo[1,2-a][1,5] Diazaoctane-10a-methyl formate and 30 mL of absolute ethanol were put into a 100 mL single-necked reaction flask, 30 mL of hydrochloric acid ethanol was added, and the mixture was stirred at room temperature for 2 hours. TLC detected that the reaction was complete. The solvent and excess hydrogen chloride were distilled off under reduced pressure to obtain 5.2 g of a colorless oily liquid, which was directly used in the next reaction.
实施例15：(5R,10aR)-3-苄氧羰基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯的制备Example 15: (5R,10aR)-3-benzyloxycarbonyl-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrrolo[1 Preparation of ,2-a][1,5]diazaoctane-10a-methyl carboxylate
Figure PCTCN2020085124-appb-000041
Figure PCTCN2020085124-appb-000041
取N-Boc-N-甲基-D-丙氨酸2.8g(13.9mmol)和60mL二氯甲烷于250mL单口反应瓶中，加入7.2g HATU(18.9mmol)，3.3gDIEA(25.2mmol)，室温搅拌1小时，得黄色清液。Take 2.8g (13.9mmol) of N-Boc-N-methyl-D-alanine and 60mL of dichloromethane in a 250mL single-mouth reaction flask, add 7.2g HATU (18.9mmol), 3.3g DIEA (25.2mmol), room temperature Stir for 1 hour to obtain a yellow clear liquid.
取(5R,10aR)-3-苄氧羰基-5-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯5.2g溶于20mL二氯甲烷，滴加入上述体系中，室温搅拌6小时，TLC检测反应完全。减压蒸出溶剂，残余物用300mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到红褐色油状物5.1g，收率72％。Take (5R,10aR)-3-benzyloxycarbonyl-5-amino-6-one-dehydropyrrolo[1,2-a][1,5]diazaoctane-10a-carboxylic acid methyl ester 5.2g Dissolved in 20 mL of dichloromethane, added dropwise to the above system, stirred at room temperature for 6 hours, TLC detected that the reaction was complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 300 mL ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent is dichloromethane : Methanol=20:1) Purify to obtain 5.1 g of reddish brown oil with a yield of 72%.
实施例16：(5R,10aR)-3-苄氧羰基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-羧酸的制备Example 16: (5R,10aR)-3-benzyloxycarbonyl-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrrolo[1 Preparation of ,2-a][1,5]diazaoctane-10a-carboxylic acid
Figure PCTCN2020085124-appb-000042
Figure PCTCN2020085124-appb-000042
取2g(3.6mmol)(5R,10aR)-3-苄氧羰基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯和60mL甲醇于250mL单口反应瓶中，加入氢氧化钾0.4g(7.2mmol)，60mL水，加热至50℃，搅拌3小时，TLC检测反应完全。加入60mL水，减压蒸出甲醇，剩余水溶液用乙酸乙酯(2*100mL)萃取，弃去有机层，水溶液用浓盐酸调节pH至1-2，乙酸乙酯(3*100mL)萃取，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，得1.9g油状物，直接用于下一步反应。Take 2g (3.6mmol) (5R, 10aR)-3-benzyloxycarbonyl-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrrolo [1,2-a][1,5] Diazaoctane-10a-methyl formate and 60mL methanol in a 250mL single-necked reaction flask, add 0.4g potassium hydroxide (7.2mmol), 60mL water, and heat to 50 After stirring for 3 hours at ℃, the reaction was complete as detected by TLC. 60mL of water was added, methanol was evaporated under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2*100mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, and extracted with ethyl acetate (3*100mL), organic The layer was dried with sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain 1.9 g of oil, which was directly used in the next reaction.
实施例17：(5R,10aR)-3-苄氧羰基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺的制备Example 17: (5R,10aR)-3-benzyloxycarbonyl-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrrolo[1 Preparation of ,2-a][1,5]diazaoctane-10a-(N-diphenylmethyl)-carboxamide
Figure PCTCN2020085124-appb-000043
Figure PCTCN2020085124-appb-000043
取(5R,10aR)-3-苄氧羰基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-羧酸1.8g(3.3mmol)，2.6g PyBop(5.0mmol)于100mL单口反应瓶中，加入50mLDMF，另取0.85g DIEA(6.6mmol),室温搅拌30分钟。另取二苯甲胺1.2g(6.6mmol)加入体系中，加热至90℃，搅拌12小时， TLC检测反应完全。反应液倒入300mL水中，乙酸乙酯(3*100mL)萃取，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到淡黄色固体1.6g，收率72％。Take (5R,10aR)-3-benzyloxycarbonyl-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrrolo[1,2- a] [1,5] Diazaoctane-10a-carboxylic acid 1.8g (3.3mmol), 2.6g PyBop (5.0mmol) in a 100mL single-mouth reaction flask, add 50mL DMF, take another 0.85g DIEA (6.6mmol) , Stir at room temperature for 30 minutes. Another 1.2 g (6.6 mmol) of benzhydrylamine was added to the system, heated to 90° C. and stirred for 12 hours. TLC detected that the reaction was complete. The reaction solution was poured into 300mL water, extracted with ethyl acetate (3*100mL), washed with saturated sodium chloride solution, the organic layer was dried with sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent is two Chloromethyl: methanol = 15:1) was purified to obtain 1.6 g of light yellow solid with a yield of 72%.
实施例18：(5R,10aR)-3-氨基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺的制备Example 18: (5R,10aR)-3-amino-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrrolo[1,2 -a] [1,5] Diazaoctane-10a-(N-diphenylmethyl)-formamide preparation
Figure PCTCN2020085124-appb-000044
Figure PCTCN2020085124-appb-000044
取(5R,10aR)-3-苄氧羰基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺0.5g(0.70mmol)和30mL甲醇于100mL单口反应瓶中，溶解得无色清液，加入50mg(10％)Pd/C，氢气气氛下，室温搅拌12小时，TLC检测反应完全。过滤除去Pd/C，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到淡黄色固体344mg，收率85％。Take (5R,10aR)-3-benzyloxycarbonyl-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrrolo[1,2- a] [1,5] Diazaoctane-10a-(N-diphenylmethyl)-carboxamide 0.5g (0.70mmol) and 30mL methanol are dissolved in a 100mL single-necked reaction flask to obtain a colorless clear liquid, add 50mg (10%) Pd/C, stirred at room temperature for 12 hours under hydrogen atmosphere, the reaction was complete as detected by TLC. The Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane:methanol=15:1) to obtain 344 mg of pale yellow solid with a yield of 85%.
实施例19：(5R,10aR)-3-(异戊酰基)-氨基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺的制备Example 19: (5R,10aR)-3-(isovaleryl)-amino-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one-dehydrogenation Preparation of pyrrolo[1,2-a][1,5]diazepine-10a-(N-diphenylmethyl)-carboxamide
Figure PCTCN2020085124-appb-000045
Figure PCTCN2020085124-appb-000045
取异戊酸72mg(0.7mmol)和10mL二氯甲烷于100mL单口反应瓶中，加入342mg HATU(0.9mmol)，154mgDIEA(1.2mmol)，室温搅拌30分钟，得淡黄色清液。Take 72 mg (0.7 mmol) of isovaleric acid and 10 mL of dichloromethane in a 100 mL single-necked reaction flask, add 342 mg HATU (0.9 mmol), 154 mg DIEA (1.2 mmol), and stir at room temperature for 30 minutes to obtain a light yellow clear liquid.
取(5R,10aR)-3-氨基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺344mg(0.6mmol)溶于10mL二氯甲烷，加入到上述体系中，得黄绿色溶液，室温搅拌3小时，TLC检测，反应完全。减压蒸出溶剂，残余物用100mL乙酸乙酯溶解，饱和氯化钠溶 液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到白色固体277mg，收率70％。Take (5R,10aR)-3-amino-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrrolo[1,2-a] [1,5] Diazepine-10a-(N-diphenylmethyl)-carboxamide 344 mg (0.6 mmol) was dissolved in 10 mL of dichloromethane, added to the above system to obtain a yellow-green solution, stirred at room temperature 3 Hours, TLC detection, the reaction is complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 100 mL ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent was dichloromethane : Methanol=20:1) Purification to obtain 277 mg of white solid with a yield of 70%.
实施例20：(5R,10aR)-3-(异戊酰基)-氨基-5-((R)-2-甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺)(I-12)的制备Example 20: (5R,10aR)-3-(isovaleryl)-amino-5-((R)-2-methylamino)-propionyl-amino-6-one-dehydropyrrolo[1,2 -a] [1,5] Diazaoctane-10a-(N-Diphenylmethyl)-Carboxamide) (I-12) Preparation
Figure PCTCN2020085124-appb-000046
Figure PCTCN2020085124-appb-000046
取277mg(0.42mmol)(5R,10aR)-3-(异戊酰基)-氨基-5-((R)-2-(叔丁氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-二苯甲基)-甲酰胺和10mL无水乙醇于100mL单口反应瓶中，加入10mL盐酸乙醇，室温搅拌2小时，TLC检测，反应完全。减压蒸出溶剂及过量氯化氢，得到无色油状液体，加入100mL乙酸乙酯，用饱和碳酸氢钠溶液调节pH至7-8，分液，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到白色固体160mg，收率68％。Take 277mg (0.42mmol) (5R,10aR)-3-(isovaleryl)-amino-5-((R)-2-(tert-butoxycarbonyl)methylamino)-propionyl-amino-6-one- Dehydropyrrolo[1,2-a][1,5]diazepine-10a-(N-diphenylmethyl)-carboxamide and 10mL absolute ethanol in a 100mL single-neck reaction flask, add 10mL hydrochloric acid Ethanol, stirring at room temperature for 2 hours, TLC detection, the reaction is complete. The solvent and excess hydrogen chloride were distilled off under reduced pressure to obtain a colorless oily liquid. Add 100 mL ethyl acetate, adjust the pH to 7-8 with saturated sodium bicarbonate solution, separate the layers, dry the organic layer over sodium sulfate, filter, and distill off under reduced pressure Solvent, the residue was purified by column chromatography (eluent: dichloromethane: methanol = 15:1) to obtain 160 mg of white solid with a yield of 68%.
1H NMR(400MHz,d 6-DMSO):δ＝8.82-8.79(m,1H),8.10-7.90(m,1H),7.36-7.22(m,8H),7.15-7.13(m,2H),6.06-6.03(m,1H),4.90-4.75(m,1H),4.00-3.92(m,1H),3.80-3.65(m,1H),3.52(s,1H),3.43-3.40(m,2H),3.20-2.90(m,3H),2.30-2.10(m,5H),2.00-1.60(m,5H),1.24(s,2H),1.15-1.05(m,3H),0.89-0.84(m,6H)，[M+H] +:m/z＝562.3 1 H NMR (400MHz, d 6 -DMSO): δ = 8.82-8.79 (m, 1H), 8.10-7.90 (m, 1H), 7.36-7.22 (m, 8H), 7.15-7.13 (m, 2H), 6.06-6.03 (m, 1H), 4.90-4.75 (m, 1H), 4.00-3.92 (m, 1H), 3.80-3.65 (m, 1H), 3.52 (s, 1H), 3.43-3.40 (m, 2H) ), 3.20-2.90 (m, 3H), 2.30-2.10 (m, 5H), 2.00-1.60 (m, 5H), 1.24 (s, 2H), 1.15-1.05 (m, 3H), 0.89-0.84 (m ,6H), [M+H] + :m/z=562.3
实施例21：(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯的制备Example 21: (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-amino-6-one-dehydropyrrolo[1,2-a] [1,5] Preparation of Methyl Diazacyclo-10a-carboxylate
Figure PCTCN2020085124-appb-000047
Figure PCTCN2020085124-appb-000047
取6g(10.7mmol)(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯和30mL无水乙醇于100mL单口反应瓶中，加入30mL盐酸乙醇，室温搅拌2小时，TLC检测，反应完全。减压蒸出溶剂及过量氯化氢，得到无色油状液体5.2g，直接用于下一步反应。Take 6g (10.7mmol) (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-de Hydropyrrolo[1,2-a][1,5]diazepine-10a-methyl formate and 30mL of absolute ethanol are added to a 100mL single-neck reaction flask, add 30mL of hydrochloric acid ethanol, stir at room temperature for 2 hours, TLC detection , The reaction is complete. The solvent and excess hydrogen chloride were distilled off under reduced pressure to obtain 5.2 g of a colorless oily liquid, which was directly used in the next reaction.
实施例22：(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯的制备Example 22: (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrolo[ Preparation of 1,2-a][1,5]diazepine-10a-methyl carboxylate
Figure PCTCN2020085124-appb-000048
Figure PCTCN2020085124-appb-000048
取异戊酸1.2g(11.8mmol)和20mL二氯甲烷于100mL单口反应瓶中，加入6.1g HATU(16.1mmol)，2.8gDIEA(21.4mmol)，室温搅拌30分钟，得黄色清液。Take 1.2 g (11.8 mmol) of isovaleric acid and 20 mL of dichloromethane in a 100 mL single-necked reaction flask, add 6.1 g HATU (16.1 mmol), 2.8 g DIEA (21.4 mmol), and stir at room temperature for 30 minutes to obtain a yellow clear liquid.
取(5R,10aR)3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯5.2g溶于20mL二氯甲烷，加入到上述体系中，得黄绿色溶液，室温搅拌3小时，TLC检测，反应完全。减压蒸出溶剂，残余物用100mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到白色固体4.7g，收率81％。Take (5R,10aR)3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-amino-6-one-dehydropyrrolo[1,2-a][1,5 ] 5.2 g of diazepine-10a-methyl formate was dissolved in 20 mL of dichloromethane, and added to the above system to obtain a yellow-green solution, which was stirred at room temperature for 3 hours, detected by TLC, and the reaction was complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 100 mL ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent was dichloromethane : Methanol=20:1) Purify to obtain 4.7 g of white solid with a yield of 81%.
实施例23：(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-羧酸的制备Example 23: (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrolo[ Preparation of 1,2-a][1,5]diazaoctane-10a-carboxylic acid
Figure PCTCN2020085124-appb-000049
Figure PCTCN2020085124-appb-000049
取470mg(0.86mmol)(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-甲酸甲酯和20mL甲醇于100mL单口反应瓶中，加入氢氧化钾150mg(2.59mmol)，20mL水，加热至50℃，搅拌3小时，TLC检测反应完全。加入20mL水，减压蒸出甲醇，剩余水溶液用乙酸乙酯(2*50mL)萃取，弃去有机层，水溶液用浓盐酸调节pH至1-2，乙酸乙酯(3*60mL)萃取，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，得460mg油状物，直接用于下一步反应。Take 470mg (0.86mmol) (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrole And [1,2-a][1,5]diazepine-10a-methyl formate and 20mL methanol in a 100mL single-necked reaction flask, add 150mg potassium hydroxide (2.59mmol), 20mL water, and heat to 50 After stirring for 3 hours at ℃, the reaction was complete as detected by TLC. 20mL of water was added, methanol was evaporated under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2*50mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, and extracted with ethyl acetate (3*60mL), organic The layer was dried over sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain 460 mg of oil, which was directly used in the next reaction.
实施例24：(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-2-苯基-苄基)-甲酰胺的制备Example 24: (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrolo[ Preparation of 1,2-a][1,5]diazepine-10a-(N-2-phenyl-benzyl)-carboxamide
Figure PCTCN2020085124-appb-000050
Figure PCTCN2020085124-appb-000050
取(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-羧酸460mg，670mg PyBop(1.29mmol)于100mL单口反应瓶中，加入20mLDMF，另取221mg DIEA(1.72mmol),室温搅拌30分钟。另取2-苯基卞胺173mg(0.95mmol)加入体系中，加热至90℃，搅拌12小时，TLC检测反应完全。反应液倒入300mL水中，乙酸乙酯(3*100mL)萃取，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到白色固体358mg，收率60％。Take (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrolo[1,2 -a] [1,5] Diazaoctane-10a-carboxylic acid 460mg, 670mg PyBop (1.29mmol) in a 100mL single-neck reaction flask, add 20mL DMF, another 221mg DIEA (1.72mmol), and stir at room temperature for 30 minutes. Another 173 mg (0.95 mmol) of 2-phenylbenzamide was added to the system, heated to 90° C. and stirred for 12 hours. TLC detected that the reaction was complete. The reaction solution was poured into 300 mL of water, extracted with ethyl acetate (3*100 mL), washed with saturated sodium chloride solution, the organic layer was dried with sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent is two Chloromethane: methanol = 15:1) was purified to obtain 358 mg of white solid with a yield of 60%.
实施例25：(5R,10aR)-3-((R)-2-甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-2-苯基-苄基)-甲酰胺)(I-02)的制备Example 25: (5R,10aR)-3-((R)-2-methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrolo[1,2-a ] [1,5] Diazaoctane-10a-(N-2-phenyl-benzyl)-carboxamide) (I-02)
Figure PCTCN2020085124-appb-000051
Figure PCTCN2020085124-appb-000051
取(5R,10aR)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡咯并[1,2-a][1,5]二氮杂辛环-10a-(N-2-苯基-苄基)-甲酰胺358g(0.52mmol)和20mL甲醇于100mL单口反应瓶中，溶解得无色清液，加入36mg(10％)Pd/C，氢气气氛下，室温搅拌12小时，TLC检测反应完全。过滤除去Pd/C，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到淡黄色固体256mg，收率88％。Take (5R,10aR)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrrolo[1,2 -a][1,5]diazepine-10a-(N-2-phenyl-benzyl)-carboxamide 358g (0.52mmol) and 20mL methanol in a 100mL single-necked reaction flask, dissolved into a colorless clear After adding 36mg (10%) Pd/C, stirring at room temperature for 12 hours under hydrogen atmosphere, TLC detected that the reaction was complete. The Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane:methanol=15:1) to obtain 256 mg of pale yellow solid, with a yield of 88%.
1H NMR(400MHz,d 6-DMSO):δ＝8.43-8.42(m,1H),7.97-7.95(m,1H),7.44-7.41(m,2H),7.38-7.29(m,6H),7.19-7.18(m,1H),4.80-4.78(m,1H),4.60-4.54(m,1H),3.96-3.91(m,2H),2.30-2.02(m,6H),2.09-1.74(m,6H),1.72-1.62(m,1H),1.05-1.03(d,2H),0.98-0.96(m,3H),0.83-0.78(m,6H)，[M+H] +:m/z＝562.3 1 H NMR (400MHz, d 6 -DMSO): δ=8.43-8.42(m,1H), 7.97-7.95(m,1H),7.44-7.41(m,2H), 7.38-7.29(m,6H), 7.19-7.18(m,1H), 4.80-4.78(m,1H), 4.60-4.54(m,1H), 3.96-3.91(m,2H), 2.30-2.02(m,6H), 2.09-1.74(m ,6H),1.72-1.62(m,1H),1.05-1.03(d,2H),0.98-0.96(m,3H),0.83-0.78(m,6H),[M+H] + :m/z =562.3
实施例26Example 26
Figure PCTCN2020085124-appb-000052
Figure PCTCN2020085124-appb-000052
1H NMR(400MHz,d 6-DMSO):δ＝8.82-8.79(m,1H),8.10-7.90(m,1H),7.36-7.22(m,8H),7.15-7.13(m,2H),6.06-6.03(m,1H),4.90-4.75(m,1H),4.00-3.92(m,1H),3.80-3.65(m,1H),3.52(s,1H),3.43-3.40(m,2H),3.20-2.90(m,3H),2.30-2.10(m,5H),2.00-1.60(m,5H),1.24(s,2H),1.15-1.05(m,3H),0.89-0.84(m,6H)，[M+H] +:m/z＝562.3 1 H NMR (400MHz, d 6 -DMSO): δ = 8.82-8.79 (m, 1H), 8.10-7.90 (m, 1H), 7.36-7.22 (m, 8H), 7.15-7.13 (m, 2H), 6.06-6.03 (m, 1H), 4.90-4.75 (m, 1H), 4.00-3.92 (m, 1H), 3.80-3.65 (m, 1H), 3.52 (s, 1H), 3.43-3.40 (m, 2H) ), 3.20-2.90 (m, 3H), 2.30-2.10 (m, 5H), 2.00-1.60 (m, 5H), 1.24 (s, 2H), 1.15-1.05 (m, 3H), 0.89-0.84 (m ,6H), [M+H] + :m/z=562.3
实施例27Example 27
Figure PCTCN2020085124-appb-000053
Figure PCTCN2020085124-appb-000053
H-NMR(400MHz,d 6-DMSO):δ＝8.83-8.81(d,1H),7.88-7.86(d,1H),7.37-7.24(m,8H),7.16-7.10(m,2H),6.06-6.04(d,1H),4.99-4.95(m,1H),3.95-3.92(m,2H),3.73-3.70(m,1H),3.59-3.40(m,3H),2.42-2.21(m,6H),2.05-1.92(m,3H),1.79-1.73(m,2H),1.34-1.18(m,6H),0.89-0.85(m,6H).[M+H] +:m/z＝562.3 H-NMR (400MHz, d 6 -DMSO): δ=8.83-8.81 (d, 1H), 7.88-7.86 (d, 1H), 7.37-7.24 (m, 8H), 7.16-7.10 (m, 2H), 6.06-6.04 (d, 1H), 4.99-4.95 (m, 1H), 3.95-3.92 (m, 2H), 3.73-3.70 (m, 1H), 3.59-3.40 (m, 3H), 2.42-2.21 (m ,6H),2.05-1.92(m,3H),1.79-1.73(m,2H),1.34-1.18(m,6H),0.89-0.85(m,6H).[M+H] + :m/z =562.3
实施例28Example 28
Figure PCTCN2020085124-appb-000054
Figure PCTCN2020085124-appb-000054
1H NMR(400MHz,d 6-DMSO):δ＝8.57(m,1H),8.03-7.95(m,1H),7.31-7.27(m,2H), 7.22-7.21(m,3H),4.93-4.79(m,1H),4.65-4.52(m,1H),4.05-3.93(m,10H),2.89(s,2H),2.73(s,2H),2.07(s,2H),2.02-1.98(s,12H),1.19-1.15(s,3H),1.00-0.98(m,2H),0.90-0.88(m,6H)，[M+H] +:m/z＝486.3 1 H NMR (400MHz, d 6 -DMSO): δ = 8.57 (m, 1H), 8.03-7.95 (m, 1H), 7.31-7.27 (m, 2H), 7.22-7.21 (m, 3H), 4.93 4.79(m, 1H), 4.65-4.52(m, 1H), 4.05-3.93(m, 10H), 2.89(s, 2H), 2.73(s, 2H), 2.07(s, 2H), 2.02-1.98( s,12H),1.19-1.15(s,3H),1.00-0.98(m,2H),0.90-0.88(m,6H), [M+H] + :m/z=486.3
实施例29Example 29
Figure PCTCN2020085124-appb-000055
Figure PCTCN2020085124-appb-000055
1H NMR(400MHz,d 6-DMSO):δ＝8.43-8.42(m,1H),7.97-7.95(m,1H),7.44-7.41(m,2H),7.38-7.29(m,6H),7.19-7.18(m,1H),4.80-4.78(m,1H),4.60-4.54(m,1H),3.96-3.91(m,2H),2.30-2.02(m,6H),2.09-1.74(m,6H),1.72-1.62(m,1H),1.05-1.03(d,2H),0.98-0.96(m,3H),0.83-0.78(m,6H)，[M+H] +:m/z＝562.3 1 H NMR (400MHz, d 6 -DMSO): δ=8.43-8.42(m,1H), 7.97-7.95(m,1H),7.44-7.41(m,2H), 7.38-7.29(m,6H), 7.19-7.18(m,1H), 4.80-4.78(m,1H), 4.60-4.54(m,1H), 3.96-3.91(m,2H), 2.30-2.02(m,6H), 2.09-1.74(m ,6H),1.72-1.62(m,1H),1.05-1.03(d,2H),0.98-0.96(m,3H),0.83-0.78(m,6H),[M+H] + :m/z =562.3
实施例30Example 30
Figure PCTCN2020085124-appb-000056
Figure PCTCN2020085124-appb-000056
H-NMR(400MHz,d 6-DMSO):δ＝8.77-8.75(d,1H),7.94-7.92(d,1H),7.37-7.34(m,2H),7.19-7.15(m,4H),7.11-7.06(m,2H),6.08-6.06(d,1H),4.94-4.92(m,1H),3.99-3.92(m,2H), 3.72-3.52(m,1H),3.48-3.41(m,3H),2.50-2.41(m,1H),2.18-2.13(m,6H),1.95-1.92(m,4H),1.25(s,2H),1.05-0.98(m,3H),0.87-0.84(m,6H).[M+H] +:m/z＝598.4 H-NMR (400MHz, d 6 -DMSO): δ = 8.77-8.75 (d, 1H), 7.94-7.92 (d, 1H), 7.37-7.34 (m, 2H), 7.19-7.15 (m, 4H), 7.11-7.06 (m, 2H), 6.08-6.06 (d, 1H), 4.94-4.92 (m, 1H), 3.99-3.92 (m, 2H), 3.72-3.52 (m, 1H), 3.48-3.41 (m ,3H), 2.50-2.41(m,1H), 2.18-2.13(m,6H),1.95-1.92(m,4H),1.25(s,2H),1.05-0.98(m,3H),0.87-0.84 (m,6H).[M+H] + :m/z=598.4
实施例31Example 31
Figure PCTCN2020085124-appb-000057
Figure PCTCN2020085124-appb-000057
H-NMR(400MHz,d 6-DMSO):δ＝8.80-8.78(d,1H),7.90-7.88(d,1H),7.34-7.32(m,4H),7.27-7.23(m,4H),7.11-7.09(m,2H),6.03-6.01(d,1H),4.92-5.05(m,1H),3.98-3.92(m,1H),3.86-3.68(m,1H),3.52-3.40(m,1H),3.38-3.10(m,8H),2.42-2.21(m,2H),2.15-2.02(m,2H),1.79-1.73(m,2H),1.42-1.38(m,1H),1.34-1.18(m,28H),1.12-0.95(m,1H),0.89-0.85(m,3H).[M+H] +:m/z＝716.6 H-NMR (400MHz, d 6 -DMSO): δ = 8.80-8.78 (d, 1H), 7.90-7.88 (d, 1H), 7.34-7.32 (m, 4H), 7.27-7.23 (m, 4H), 7.11-7.09 (m, 2H), 6.03-6.01 (d, 1H), 4.92-5.05 (m, 1H), 3.98-3.92 (m, 1H), 3.86-3.68 (m, 1H), 3.52-3.40 (m ,1H),3.38-3.10(m,8H),2.42-2.21(m,2H),2.15-2.02(m,2H),1.79-1.73(m,2H),1.42-1.38(m,1H),1.34 -1.18(m,28H),1.12-0.95(m,1H),0.89-0.85(m,3H).[M+H] + :m/z=716.6
实施例32Example 32
Figure PCTCN2020085124-appb-000058
Figure PCTCN2020085124-appb-000058
H-NMR(400MHz,d 6-DMSO):δ＝8.82-8.80(d,1H),7.98-7.95(d,1H),7.33-7.30(m,4H),7.29-7.21(m,9H),7.18-7.13(m,1H),6.08-6.06(d,1H),4.99-4.96(m,1H),3.98-3.95(m,1H),3.79-3.75(m,1H),3.26-3.24(m,2H),3.21-3.12(m,2H),2.89-2.80(s,1H),2.79-2.70(m,3H),2.60-2.51(m,1H),2.32-2.26(m,2H),2.19-2.15(s,3H),1.86-1.68(m,2H),1.36-1.30(m,1H),1.26-1.24(m,1H),1.07-0.98(m,4H).[M+H] +:m/z＝610.4 H-NMR (400MHz, d 6 -DMSO): δ = 8.82-8.80 (d, 1H), 7.98-7.95 (d, 1H), 7.33-7.30 (m, 4H), 7.29-7.21 (m, 9H), 7.18-7.13(m,1H), 6.08-6.06(d,1H), 4.99-4.96(m,1H), 3.98-3.95(m,1H), 3.79-3.75(m,1H), 3.26-3.24(m ,2H),3.21-3.12(m,2H),2.89-2.80(s,1H),2.79-2.70(m,3H),2.60-2.51(m,1H),2.32-2.26(m,2H),2.19 -2.15(s,3H),1.86-1.68(m,2H),1.36-1.30(m,1H),1.26-1.24(m,1H),1.07-0.98(m,4H).[M+H] + :m/z＝610.4
实施例33Example 33
Figure PCTCN2020085124-appb-000059
Figure PCTCN2020085124-appb-000059
1H-NMR(400MHz,d 6-DMSO):δ＝8.83-8.81(d,1H),8.17-8.15(m,1H),7.58-6.85(m,13H),6.07-6.05(d,1H),4.94-4.93(d,1H),4.16-4.14(m,2H),4.08-4.01(m,2H),3.95-3.92(d,3H),2.77-2.75(m,2H),2.39- 1.854(m,10H),1.07-1.02(m,3H).[M+H]+:m/z＝600.2 1 H-NMR (400MHz, d 6 -DMSO): δ=8.83-8.81(d,1H), 8.17-8.15(m,1H), 7.58-6.85(m,13H), 6.07-6.05(d,1H) , 4.94-4.93(d, 1H), 4.16-4.14(m, 2H), 4.08-4.01(m, 2H), 3.95-3.92(d, 3H), 2.77-2.75(m, 2H), 2.39- 1.854( m,10H),1.07-1.02(m,3H).[M+H]+:m/z=600.2
实施例34Example 34
Figure PCTCN2020085124-appb-000060
Figure PCTCN2020085124-appb-000060
H-NMR(400MHz,d 6-DMSO):δ＝8.89-8.86(d,1H),8.84-8.80(d,1H),7.38-7.18(m,8H),7.14-7.10(m,2H),6.06-6.04(d,1H),5.00-4.95(m,1H),4.10-3.75(m,2H),3.08-3.00(s,3H),2.52-2.10(m,6H),2.09-2.07(m,3H),1.11-1.38(m,3H),0.98-0.92(m,9H).[M+H] +:m/z＝562.3 H-NMR (400MHz, d 6 -DMSO): δ = 8.89-8.86 (d, 1H), 8.84-8.80 (d, 1H), 7.38-7.18 (m, 8H), 7.14-7.10 (m, 2H), 6.06-6.04(d,1H),5.00-4.95(m,1H),4.10-3.75(m,2H),3.08-3.00(s,3H),2.52-2.10(m,6H),2.09-2.07(m ,3H),1.11-1.38(m,3H),0.98-0.92(m,9H).[M+H] + :m/z=562.3
实施例35Example 35
Figure PCTCN2020085124-appb-000061
Figure PCTCN2020085124-appb-000061
H-NMR(400MHz,d 6-DMSO):δ＝8.78-8.76(d,1H),8.03-8.02(d,1H),7.37-7.24(m,8H),7.16-7.10(m,2H),6.06-6.04(d,1H),4.99-4.95(m,1H),3.95-3.92(m,1H),3.73-3.70(m,3H),3.59-3.40(m,2H),2.42-2.21(m,6H),2.05-1.92(m,2H),1.79-1.73(m,2H),1.34-1.18(m,3H),0.89-0.85(m,3H).[M+H] +:m/z＝590.4 H-NMR (400MHz, d 6 -DMSO): δ=8.78-8.76(d,1H), 8.03-8.02(d,1H), 7.37-7.24(m,8H), 7.16-7.10(m,2H), 6.06-6.04 (d, 1H), 4.99-4.95 (m, 1H), 3.95-3.92 (m, 1H), 3.73-3.70 (m, 3H), 3.59-3.40 (m, 2H), 2.42-2.21 (m ,6H),2.05-1.92(m,2H),1.79-1.73(m,2H),1.34-1.18(m,3H),0.89-0.85(m,3H).[M+H] + :m/z =590.4
实施例36Example 36
Figure PCTCN2020085124-appb-000062
Figure PCTCN2020085124-appb-000062
1H-NMR(400MHz,d6-DMSO):δ＝8.31-8.29(m,2H),8.02-8.00(m,1H),7.92-7.88(m,1H),7.29-7.18(m,10H),4.79-4.93(m,4H),4.52-4.65(m,1H),3.93-4.05(m,10H),2.89(s,2H),2.73(s,2H),2.07(s,2H),1.98-2.02(s,12H),1.15-1.19(s,3H),0.98-1.00(m,2H),0.88-0.90(m,6H)，[M+H]+:m/z＝500.4 1 H-NMR (400MHz, d6-DMSO): δ = 8.31-8.29 (m, 2H), 8.02-8.00 (m, 1H), 7.92-7.88 (m, 1H), 7.29-7.18 (m, 10H), 4.79-4.93(m,4H),4.52-4.65(m,1H),3.93-4.05(m,10H),2.89(s,2H),2.73(s,2H),2.07(s,2H),1.98- 2.02(s,12H),1.15-1.19(s,3H),0.98-1.00(m,2H),0.88-0.90(m,6H),[M+H]+:m/z=500.4
实施例37Example 37
Figure PCTCN2020085124-appb-000063
Figure PCTCN2020085124-appb-000063
1HNMR(400MHz,d 6-DMSO):δ＝8.70-9.00(m,1H),8.20-8.50(m,1H),7.18-7.40(m,5H),4.80-5.05(m,2H),3.80-4.20(m,2H),3.40-3.70(m,2H),2.98-3.18(m,1H),2.60-2.80(m,1H),2.30-2.40(m,1H),1.70-2.20(m,10H),1.30-1.60(m,7H),1.10-1.30(s,6H),0.85(s,6H)，[M+H] +:m/z＝500.4 1 HNMR (400MHz, d 6 -DMSO): δ = 8.70-9.00 (m, 1H), 8.20-8.50 (m, 1H), 7.18-7.40 (m, 5H), 4.80-5.05 (m, 2H), 3.80 -4.20(m,2H),3.40-3.70(m,2H),2.98-3.18(m,1H),2.60-2.80(m,1H),2.30-2.40(m,1H),1.70-2.20(m, 10H),1.30-1.60(m,7H),1.10-1.30(s,6H),0.85(s,6H),[M+H] + :m/z=500.4
实施例38Example 38
Figure PCTCN2020085124-appb-000064
Figure PCTCN2020085124-appb-000064
1H NMR(400MHz,d 6-DMSO):δ＝9.58(s,1H),8.95(s,1H),8.47(s,1H),8.10-8.02(m,1H),7.78-7.68(m,2H),7.50(s,1H),7.44(s,1H),7.36-7.22(m,10H),7.15-7.13(m,2H),6.42-6.38(s,1H),6.10-6.01(s,1H),4.75-4.90(s,1H),4.11-3.92(m,8H),3.82-3.75(s,1H),3.72-3.58(s,1H),3.15-2.92(m,1H),2.42-2.38(m,1H),2.10-2.00(m,6H),1.96-1.89(s,2H),1.78-1.62(s,1H),1.60-1.53(s,1H),1.51-1.48(s,1H),1.32-1.18(m,6H),1.15-1.05(m,3H),0.86-0.75(m,2H)，[M+H] +:m/z＝868.2 1 H NMR (400MHz, d 6 -DMSO): δ = 9.58 (s, 1H), 8.95 (s, 1H), 8.47 (s, 1H), 8.10-8.02 (m, 1H), 7.78-7.68 (m, 2H),7.50(s,1H),7.44(s,1H),7.36-7.22(m,10H),7.15-7.13(m,2H),6.42-6.38(s,1H),6.10-6.01(s, 1H),4.75-4.90(s,1H),4.11-3.92(m,8H),3.82-3.75(s,1H),3.72-3.58(s,1H),3.15-2.92(m,1H),2.42- 2.38 (m, 1H), 2.10-2.00 (m, 6H), 1.96-1.89 (s, 2H), 1.78-1.62 (s, 1H), 1.60-1.53 (s, 1H), 1.51-1.48 (s, 1H) ),1.32-1.18(m,6H),1.15-1.05(m,3H),0.86-0.75(m,2H),[M+H] + :m/z=868.2
实施例39：4-对甲苯磺酰氧基丁酸乙酯的制备Example 39: Preparation of ethyl 4-p-toluenesulfonyloxybutyrate
Figure PCTCN2020085124-appb-000065
Figure PCTCN2020085124-appb-000065
取4-羟基丁酸乙酯50g(0.38mol)和300mL二氯甲烷于1L单口反应瓶中，加入三乙胺76.8g(0.76mol)，冷却至5℃，另取对甲苯磺酰氯86.6g(0.46mol)溶于200mL二氯甲烷，滴加入上述体系中，滴加完毕，室温搅拌6小时，TLC检测，反应完全。加入300mL水洗涤，稀盐酸(1M)洗涤,饱和盐水洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯：石油醚＝1:20)纯化，得到无色油状液体100g，收率92％。Take 50g (0.38mol) of ethyl 4-hydroxybutyrate and 300mL of dichloromethane in a 1L single-necked reaction flask, add 76.8g (0.76mol) of triethylamine, cool to 5°C, and take 86.6g of p-toluenesulfonyl chloride ( 0.46 mol) was dissolved in 200 mL of dichloromethane and added dropwise to the above system. After the dropwise addition was completed, the mixture was stirred at room temperature for 6 hours, and TLC detected that the reaction was complete. Add 300mL of water to wash, wash with dilute hydrochloric acid (1M), wash with saturated brine, dry the organic layer with sodium sulfate, filter, distill off the solvent under reduced pressure, and column chromatography of the residue (eluent: ethyl acetate: petroleum ether = 1: 20) Purification to obtain 100 g of a colorless oily liquid with a yield of 92%.
实施例40：2-(叔丁氧羰基)肼基丁酸乙酯的制备Example 40: Preparation of ethyl 2-(tert-butoxycarbonyl)hydrazinobutyrate
Figure PCTCN2020085124-appb-000066
Figure PCTCN2020085124-appb-000066
取4-对甲苯磺酰氧基丁酸乙酯50g(0.17mol),叔丁氧羰基肼67g(0.51mol)和600mLDMF于1L单口反应瓶中，加热至90℃搅拌6小时，TLC检测，反应完全。加入2L水，乙酸乙酯萃取(3*300mL),饱和盐水洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯：石油醚＝1:10)纯化，得到无色油状液体25g，收率60％。Take 50g (0.17mol) of ethyl 4-p-toluenesulfonyloxybutyrate, 67g (0.51mol) of tert-butoxycarbonylhydrazine and 600mL of DMF in a 1L single-necked reaction flask, heat to 90℃ and stir for 6 hours, TLC detection, reaction complete. Add 2L water, extract with ethyl acetate (3*300mL), wash with saturated brine, dry the organic layer with sodium sulfate, filter, evaporate the solvent under reduced pressure, and column chromatography of the residue (eluent: ethyl acetate: petroleum ether = 1:10) Purification to obtain 25 g of colorless oily liquid with a yield of 60%.
实施例41：1-烯丙基-2-(叔丁氧羰基)肼基丁酸乙酯的制备Example 41: Preparation of ethyl 1-allyl-2-(tert-butoxycarbonyl)hydrazinobutyrate
Figure PCTCN2020085124-appb-000067
Figure PCTCN2020085124-appb-000067
取2-(叔丁氧羰基)肼基丁酸乙酯25g(0.10mol)和300mLDMF于500mL单口反应瓶中，加入碳酸钾27.6g(0.20mol),室温搅拌，另取烯丙基溴14.5g(0.12mol)加入其中，加热至90℃搅拌12小时，TLC检测，反应完全。加入1L水，乙酸乙酯萃取(3*300mL),饱和盐水洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯：石油醚＝1:15)纯化，得到无色油状液体21.5g，收率75％。Take 25g (0.10mol) of ethyl 2-(tert-butoxycarbonyl)hydrazinobutyrate and 300mL of DMF into a 500mL single-necked reaction flask, add 27.6g (0.20mol) of potassium carbonate, stir at room temperature, and take 14.5g of allyl bromide (0.12mol) was added to it, heated to 90°C and stirred for 12 hours, TLC detected, the reaction was complete. Add 1L water, extract with ethyl acetate (3*300mL), wash with saturated brine, dry the organic layer with sodium sulfate, filter, distill off the solvent under reduced pressure, and column chromatography of the residue (eluent: ethyl acetate: petroleum ether = 1:15) Purify to obtain 21.5 g of colorless oily liquid with a yield of 75%.
实施例42：1-烯丙基-2-(叔丁氧羰基)肼基-2-溴-丁酸乙酯的制备Example 42: Preparation of 1-allyl-2-(tert-butoxycarbonyl)hydrazino-2-bromo-butyric acid ethyl ester
Figure PCTCN2020085124-appb-000068
Figure PCTCN2020085124-appb-000068
取1-烯丙基-2-(叔丁氧羰基)肼基丁酸乙酯20g(70mmol)和200mL预干燥四氢呋喃于500mL三口反应瓶中，氮气保护，冷却至-60℃，取二异丙基氨基锂140mL(1M in THF,0.14mol),滴加入其中，维持-60℃下搅拌2小时，降温至-78℃，另取四溴化碳46g(0.14mol)溶于100mL四氢呋喃，滴加入上述体系中，维持-78℃下搅拌2小时,TLC检测，反应完全。加入20mL饱和氯化铵溶液淬灭反应，用稀盐酸(1M)洗涤，饱和盐水洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯：石油醚＝1:15)纯化，得到无色油状液体20.7g，收率81％。Take 20g (70mmol) of ethyl 1-allyl-2-(tert-butoxycarbonyl)hydrazinobutyrate and 200mL of pre-dried tetrahydrofuran in a 500mL three-necked reaction flask, under nitrogen protection, cool to -60°C, take diisopropyl 140mL (1M in THF, 0.14mol) of lithium amide, add dropwise to it, keep stirring at -60°C for 2 hours, cool to -78°C, take 46g (0.14mol) of carbon tetrabromide dissolved in 100mL tetrahydrofuran, and add dropwise In the above system, the temperature was maintained at -78°C and stirred for 2 hours. TLC detected that the reaction was complete. The reaction was quenched by adding 20 mL of saturated ammonium chloride solution, washed with dilute hydrochloric acid (1M), washed with saturated brine, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent is ethyl acetate Ester: petroleum ether = 1:15) purified to obtain 20.7 g of a colorless oily liquid with a yield of 81%.
实施例43：1-烯丙基-肼基-2-溴-丁酸乙酯的制备Example 43: Preparation of 1-allyl-hydrazino-2-bromo-butyric acid ethyl ester
Figure PCTCN2020085124-appb-000069
Figure PCTCN2020085124-appb-000069
取20g(54.8mmol)1-烯丙基-2-(叔丁氧羰基)肼基-2-溴-丁酸乙酯和100mL无水乙醇于500mL单口反应瓶中，加入100mL盐酸乙醇，室温搅拌2小时，TLC检测，反应完全。减压蒸出溶剂及过量氯化氢，得到无色油状液体16.2g，直接用于下一步反应。Take 20g (54.8mmol) 1-allyl-2-(tert-butoxycarbonyl)hydrazino-2-bromo-butyric acid ethyl ester and 100mL absolute ethanol in a 500mL single-neck reaction flask, add 100mL hydrochloric acid ethanol, and stir at room temperature After 2 hours, TLC detection showed that the reaction was complete. The solvent and excess hydrogen chloride were distilled off under reduced pressure to obtain 16.2 g of a colorless oily liquid, which was directly used in the next reaction.
实施例44：1-烯丙基-吡唑-3-甲酸乙酯的制备Example 44: Preparation of 1-allyl-pyrazole-3-carboxylic acid ethyl ester
Figure PCTCN2020085124-appb-000070
Figure PCTCN2020085124-appb-000070
取1-烯丙基-肼基-2-溴-丁酸乙酯16.2g和200mLDMF于500mL单口反应瓶中，加入碳酸钾18.9g(0.14mol)，加热至90℃搅拌12小时，TLC检测，反应完全。加入600mL水，乙酸乙酯萃取(3*300mL),饱和盐水洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯：石油醚＝1:10)纯化，得到白色固体6.3g，收率63％。Take 16.2g of 1-allyl-hydrazino-2-bromo-butyric acid ethyl ester and 200mL of DMF into a 500mL single-necked reaction flask, add 18.9g (0.14mol) of potassium carbonate, heat to 90℃ and stir for 12 hours, TLC detection, The reaction is complete. Add 600mL water, extract with ethyl acetate (3*300mL), wash with saturated brine, dry the organic layer with sodium sulfate, filter, distill off the solvent under reduced pressure, and column chromatography of the residue (eluent: ethyl acetate: petroleum ether = 1:10) Purification to obtain 6.3 g of white solid with a yield of 63%.
实施例45：1-烯丙基-2-((3-苄氧羰基-氨基-2-叔丁氧羰基-氨基)丙酰基)-吡唑-3-甲酸乙酯的制备Example 45: Preparation of 1-allyl-2-((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino)propionyl)-pyrazole-3-carboxylic acid ethyl ester
Figure PCTCN2020085124-appb-000071
Figure PCTCN2020085124-appb-000071
取(S)-2-(Boc-氨基)-3-(Cbz-氨基)丙酸13.9g(41mmol)和200mL二氯甲烷于500mL单口反应瓶中，加入20.8g HATU(54.7mmol)，8.8gDIEA(68.4mmol)，室温搅拌1小时，得黄色清液。Take 13.9g (41mmol) of (S)-2-(Boc-amino)-3-(Cbz-amino)propionic acid and 200mL of dichloromethane in a 500mL single-mouth reaction flask, add 20.8g HATU (54.7mmol), 8.8g DIEA (68.4mmol), stirred at room temperature for 1 hour to obtain a yellow clear liquid.
另取1-烯丙基-吡唑-3-甲酸乙酯6.3g(34.2mmol)溶于100mL二氯甲烷，滴加入上述黄色清液中，室温搅拌6小时，得到红褐色溶液，TLC检测反应完全。减压蒸出溶剂，残余物用400mL乙酸乙酯溶解，用1MHCl溶液洗涤，饱和碳酸氢钠溶液洗涤，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯:石油醚＝1:2)纯化，得到白色固体14.6g，收率85％。In addition, 6.3 g (34.2 mmol) of ethyl 1-allyl-pyrazole-3-carboxylate was dissolved in 100 mL of dichloromethane, and added dropwise to the above-mentioned yellow clear liquid, and stirred at room temperature for 6 hours to obtain a reddish-brown solution. The reaction was detected by TLC complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 400 mL ethyl acetate, washed with 1M HCl solution, saturated sodium bicarbonate solution, saturated sodium chloride solution, the organic layer was dried with sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:2) to obtain 14.6 g of white solid with a yield of 85%.
实施例46：1-乙醛基-2-((3-苄氧羰基-氨基-2-叔丁氧羰基-氨基)丙酰基)-吡唑-3-甲酸乙酯的制备Example 46: Preparation of 1-acetaldehyde-2-((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino)propionyl)-pyrazole-3-carboxylic acid ethyl ester
Figure PCTCN2020085124-appb-000072
Figure PCTCN2020085124-appb-000072
取1-烯丙基-2-((3-苄氧羰基-氨基-2-叔丁氧羰基-氨基)丙酰基)-吡唑-3-甲酸乙酯14.6g(29mmol)和200mL二氯甲烷于500mL三口反应瓶中，冷却至-78℃，通入臭氧约30分钟，至体系变为蓝色，于-78℃下搅拌30分钟，停止通入臭氧。通入氮气鼓泡除去多余臭氧至蓝色消失，得到无色溶液，加入100mL三乙胺，恢复至室温下，搅拌2小时，TLC检测反应完全。加入1M柠檬酸洗涤，饱和氯化钠水溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为乙酸乙酯:石油醚＝1:2)纯化，得到白色固体11.5g，收率78％。Take 1-allyl-2-((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino)propionyl)-pyrazole-3-carboxylic acid ethyl ester 14.6g (29mmol) and 200mL dichloromethane In a 500mL three-necked reaction flask, cool to -78°C, and pass ozone in for about 30 minutes until the system turns blue. Stir at -78°C for 30 minutes and stop passing ozone. Nitrogen was bubbled through to remove excess ozone until the blue color disappeared, and a colorless solution was obtained. 100 mL of triethylamine was added, returned to room temperature, and stirred for 2 hours. TLC detected that the reaction was complete. Wash with 1M citric acid, wash with saturated sodium chloride aqueous solution, dry the organic layer with sodium sulfate, filter, distill off the solvent under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:2) , To obtain 11.5 g of white solid with a yield of 78%.
实施例47：(5S,8S)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-甲酸甲酯的制备Example 47: (5S,8S)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[1,2-a][1,2,5]triazaoctane Preparation of -8-methyl formate
Figure PCTCN2020085124-appb-000073
Figure PCTCN2020085124-appb-000073
取11.5g(22.7mmol)1-乙醛基-2-((3-苄氧羰基-氨基-2-叔丁氧羰基-氨基)丙酰基)-吡唑-3-甲酸乙酯和200mL甲醇于500mL单口反应瓶中，溶解得无色清液， 加入1.2g(10％)Pd/C，氢气气氛下，室温搅拌12小时，TLC检测反应完全。过滤除去Pd/C，减压蒸出溶剂，加入200mL四氢呋喃溶解，加入氰基硼氢化钠2.1g(34.1mmol)，室温搅拌，TLC检测反应完全。加入20mL水淬灭反应，过滤，减压蒸出溶剂，残余物用200mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝10:1)纯化，得到白色固体6.2g，收率80％。Take 11.5g (22.7mmol) 1-acetaldehyde-2-((3-benzyloxycarbonyl-amino-2-tert-butoxycarbonyl-amino)propionyl)-pyrazole-3-carboxylic acid ethyl ester and 200mL methanol in Dissolve in a 500 mL single-mouth reaction flask to obtain a colorless clear solution, add 1.2 g (10%) Pd/C, and stir at room temperature for 12 hours under a hydrogen atmosphere. TLC detects that the reaction is complete. The Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, 200 mL of tetrahydrofuran was added to dissolve, and 2.1 g (34.1 mmol) of sodium cyanoborohydride was added, stirred at room temperature, and TLC detected that the reaction was complete. The reaction was quenched by adding 20 mL of water, filtered, and the solvent was evaporated under reduced pressure. The residue was dissolved in 200 mL of ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. After purification (eluent: dichloromethane: methanol = 10:1), 6.2 g of white solid was obtained with a yield of 80%.
实施例48：(5S,8S)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-甲酸甲酯的制备Example 48: (5S,8S)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyridine Preparation of azolo[1,2-a][1,2,5]triazaoctane-8-methyl carboxylate
Figure PCTCN2020085124-appb-000074
Figure PCTCN2020085124-appb-000074
取N-Cbz-N-甲基-D-丙氨酸2.6g(10.9mmol)和80mL二氯甲烷于250mL单口反应瓶中，加入5.2g HATU(13.6mmol)，2.4gDIEA(18.1mmol)，室温搅拌1小时，得黄色清液。Take 2.6g (10.9mmol) of N-Cbz-N-methyl-D-alanine and 80mL of dichloromethane in a 250mL single-mouth reaction flask, add 5.2g HATU (13.6mmol), 2.4gDIEA (18.1mmol), room temperature Stir for 1 hour to obtain a yellow clear liquid.
取(5S,8S)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-甲酸甲酯3.1g(9.1mmol)溶于50mL二氯甲烷，滴加入上述体系中，室温搅拌6小时，TLC检测反应完全。减压蒸出溶剂，残余物用300mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到褐色油状物3.7g，收率72％。Take (5S,8S)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[1,2-a][1,2,5]triazaoctane-8- 3.1 g (9.1 mmol) of methyl formate was dissolved in 50 mL of dichloromethane, added dropwise to the above system, stirred at room temperature for 6 hours, TLC detected that the reaction was complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 300 mL ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent is dichloromethane : Methanol=20:1) Purification to obtain 3.7 g of brown oil with a yield of 72%.
实施例49：(5S,8S)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-羧酸的制备Example 49: (5S,8S)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyridine Preparation of azolo[1,2-a][1,2,5]triazaoctane-8-carboxylic acid
Figure PCTCN2020085124-appb-000075
Figure PCTCN2020085124-appb-000075
取1g(1.8mmol)(5S,8S)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-甲酸甲酯和30mL甲醇于250mL单口反应瓶中，加入氢氧化钾0.3g(5.4mmol)，30mL水，加热至50℃，搅拌3小时，TLC检测反应完全。加入60mL水，减压蒸出甲醇，剩余水溶液用乙酸乙酯(2*100mL)萃取，弃去有机层，水溶液用浓盐酸调节pH至1-2，乙酸乙酯(3*100mL)萃取，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，得0.98g油状物，直接用于下一步反应。Take 1g (1.8mmol) (5S, 8S)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-de Hydropyrazolo[1,2-a][1,2,5]triazaoctane-8-methyl carboxylate and 30mL methanol in a 250mL single-neck reaction flask, add 0.3g (5.4mmol) of potassium hydroxide, 30mL of water, heated to 50°C, stirred for 3 hours, TLC detected that the reaction was complete. 60mL of water was added, methanol was evaporated under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2*100mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, and extracted with ethyl acetate (3*100mL), organic The layer was dried with sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain 0.98 g of oil, which was directly used in the next reaction.
实施例50：(5S,8S)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺的制备Example 50: (5S,8S)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyridine Preparation of azolo[1,2-a][1,2,5]triazaoctane-8-(N-diphenylmethyl)-carboxamide
Figure PCTCN2020085124-appb-000076
Figure PCTCN2020085124-appb-000076
取(5S,8S)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-羧酸0.98g(1.8mmol)，1.4g PyBop(2.7mmol)于100mL单口反应瓶中，加入50mLDMF，另取0.46g DIEA(3.6mmol),室温搅拌30分钟。另取二苯甲胺0.66g(3.6mmol)加入体系中，加热至90℃，搅拌12小时，TLC检测反应完全。反应液倒入300mL水中，乙酸乙酯(3*100mL)萃取，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到淡黄色固体0.77g，收率60％。Take (5S,8S)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[ 1,2-a][1,2,5]Triazaoctane-8-carboxylic acid 0.98g (1.8mmol), 1.4g PyBop (2.7mmol) in a 100mL single-mouth reaction flask, add 50mL DMF, and another 0.46 g DIEA (3.6mmol), stir at room temperature for 30 minutes. Another 0.66 g (3.6 mmol) of benzhydrylamine was added to the system, heated to 90° C., and stirred for 12 hours. TLC detected that the reaction was complete. The reaction solution was poured into 300mL water, extracted with ethyl acetate (3*100mL), washed with saturated sodium chloride solution, the organic layer was dried with sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent is two Chloromethane: methanol = 15:1) was purified to obtain 0.77 g of light yellow solid with a yield of 60%.
实施例51：(5S,8S)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺的制备Example 51: (5S,8S)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-amino-6-one-dehydropyrazolo[1,2-a ][1,2,5]Triazaoctane-8-(N-diphenylmethyl)-carboxamide
Figure PCTCN2020085124-appb-000077
Figure PCTCN2020085124-appb-000077
取0.5g(0.7mmol)(5S,8S)3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺和10mL无水乙醇于100mL单口反应瓶中，加入20mL盐酸乙醇，室温搅拌2小时，TLC检测，反应完全。减压蒸出溶剂及过量氯化氢，得到无色油状液体0.45g，直接用于下一步反应。Take 0.5g (0.7mmol) (5S, 8S) 3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(tert-butyloxycarbonyl)-amino-6-one-de Hydropyrazolo[1,2-a][1,2,5]triazaoctane-8-(N-diphenylmethyl)-formamide and 10mL of absolute ethanol are added to a 100mL single-mouth reaction flask 20 mL of hydrochloric acid ethanol, stirred at room temperature for 2 hours, TLC detection, the reaction was complete. The solvent and excess hydrogen chloride were distilled off under reduced pressure to obtain 0.45 g of a colorless oily liquid, which was directly used in the next reaction.
实施例52：(5S,8S)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺的制备Example 52: (5S,8S)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrazolo Preparation of [1,2-a][1,2,5]Triazaoctane-8-(N-Diphenylmethyl)-carboxamide
Figure PCTCN2020085124-appb-000078
Figure PCTCN2020085124-appb-000078
取异戊酸82mg(0.8mmol)和10mL二氯甲烷于100mL单口反应瓶中，加入0.42g HATU(1.1mmol)，0.18gDIEA(1.4mmol)，室温搅拌30分钟，得淡黄色清液。Take 82 mg (0.8 mmol) of isovaleric acid and 10 mL of dichloromethane in a 100 mL single-necked reaction flask, add 0.42 g HATU (1.1 mmol), 0.18 g DIEA (1.4 mmol), and stir at room temperature for 30 minutes to obtain a light yellow clear liquid.
取(5S,8S)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺0.45g溶于10mL二氯甲烷，加入到上述体系中，得黄绿色溶液，室温搅拌3小时，TLC检测，反应完全。减压蒸出溶剂，残余物用100mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到白色固体345mg，收率71％。Take (5S,8S)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-amino-6-one-dehydropyrazolo[1,2-a][1 ,2,5]Triazaoctane-8-(N-diphenylmethyl)-carboxamide 0.45g was dissolved in 10mL of dichloromethane, added to the above system to obtain a yellow-green solution, stirred at room temperature for 3 hours, TLC The reaction is complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 100 mL ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent was dichloromethane : Methanol=20:1) Purification to obtain 345 mg of white solid with a yield of 71%.
实施例53：(5S,8S)-3-((R)-2-甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺(I-13)的制备Example 53: (5S,8S)-3-((R)-2-methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrazolo[1,2- a][1,2,5]Triazaoctane-8-(N-diphenylmethyl)-carboxamide (I-13)
Figure PCTCN2020085124-appb-000079
Figure PCTCN2020085124-appb-000079
取(5S,8S)-3-((R)-2-(苄氧羰基)甲氨基)-丙酰基-5-(异戊酰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺200mg(0.29mmol)和50mL甲醇于100mL单口反应瓶中，溶解得无色清液，加入20mg(10％)Pd/C，氢气气氛下，室温搅拌12小时，TLC检测反应完全。过滤除去Pd/C，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到白色固体138mg，收率85％。Take (5S,8S)-3-((R)-2-(benzyloxycarbonyl)methylamino)-propionyl-5-(isovaleryl)-amino-6-one-dehydropyrazolo[1, 2-a][1,2,5]Triazaoctane-8-(N-diphenylmethyl)-carboxamide 200mg (0.29mmol) and 50mL methanol in a 100mL single-necked reaction flask, dissolved into colorless clear After adding 20mg (10%) Pd/C, stirring at room temperature for 12 hours under a hydrogen atmosphere, TLC detected that the reaction was complete. Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane:methanol=15:1) to obtain 138 mg of white solid with a yield of 85%.
1H NMR(400MHz,d 6-DMSO):δ＝8.03-8.00(m,1H),7.49-7.32(m,2H),7.30-7.22(m,4H),7.19-7.10(m,4H),6.06-6.03(m,1H),5.05-4.99(m,1H),4.45-4.39(m,1H),3.80-3.62(m,1H),3.52(s,1H),3.43-3.40(m,2H),3.20-2.90(m,3H),2.78-2.47(m,2H),2.30-2.10(m,5H),2.00-1.60(m,5H),1.18-1.05(m,3H),0.89-0.82(m,6H)，[M+H] +:m/z＝563.3 1 H NMR (400MHz, d 6 -DMSO): δ=8.03-8.00 (m, 1H), 7.49-7.32 (m, 2H), 7.30-7.22 (m, 4H), 7.19-7.10 (m, 4H), 6.06-6.03 (m, 1H), 5.05-4.99 (m, 1H), 4.45-4.39 (m, 1H), 3.80-3.62 (m, 1H), 3.52 (s, 1H), 3.43-3.40 (m, 2H) ), 3.20-2.90 (m, 3H), 2.78-2.47 (m, 2H), 2.30-2.10 (m, 5H), 2.00-1.60 (m, 5H), 1.18-1.05 (m, 3H), 0.89-0.82 (m,6H), [M+H] + :m/z=563.3
实施例54：(5S,8S)-3-(异戊酰基)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-甲酸甲酯的制备Example 54: (5S,8S)-3-(isovaleryl)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[1,2-a][1,2 ,5] Preparation of methyl triazaoctane-8-carboxylate
Figure PCTCN2020085124-appb-000080
Figure PCTCN2020085124-appb-000080
取异戊酸1.1g(11mmol)和30mL二氯甲烷于100mL单口反应瓶中，加入5.7g HATU(15mmol)，2.6gDIEA(20mmol)，室温搅拌30分钟，得淡黄色清液。Take 1.1 g (11 mmol) of isovaleric acid and 30 mL of dichloromethane in a 100 mL single-neck reaction flask, add 5.7 g HATU (15 mmol), 2.6 g DIEA (20 mmol), and stir at room temperature for 30 minutes to obtain a light yellow clear liquid.
取(5S,8S)5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-甲酸甲酯3.4g(10mmol)溶于30mL二氯甲烷，加入到上述体系中，得黄绿色溶液，室温搅拌3小时，TLC检测，反应完全。减压蒸出溶剂，残余物用100mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到白色固体2.8g，收率66％。Take (5S,8S)5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[1,2-a][1,2,5]triazaoctane-8-carboxylic acid 3.4 g (10 mmol) of methyl ester was dissolved in 30 mL of dichloromethane, and added to the above system to obtain a yellow-green solution. The mixture was stirred at room temperature for 3 hours, detected by TLC, and the reaction was complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 100 mL ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent was dichloromethane : Methanol=20:1) Purify to obtain 2.8 g of white solid with a yield of 66%.
实施例55：(5S,8S)-3-(异戊酰基)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-羧酸的制备Example 55: (5S,8S)-3-(isovaleryl)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[1,2-a][1,2 ,5] Preparation of triazaoctane-8-carboxylic acid
Figure PCTCN2020085124-appb-000081
Figure PCTCN2020085124-appb-000081
取1g(2.3mmol)(5S,8S)-3-(异戊酰基)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-甲酸甲酯和30mL甲醇于100mL单口反应瓶中，加入氢氧化钾0.3g(5.9mmol)，30mL水，加热至50℃，搅拌3小时，TLC检测反应完全。加入30mL水，减压蒸出甲醇，剩余水溶液用乙酸乙酯(2*100mL)萃取，弃去有机层，水溶液用浓盐酸调节pH至1-2，乙酸乙酯(3*100mL)萃取，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，得0.95g油状物，直接用于下一步反应。Take 1g (2.3mmol) (5S,8S)-3-(isovaleryl)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[1,2-a][1 ,2,5]Triazaoctane-8-methyl formate and 30mL methanol in a 100mL single-necked reaction flask, add 0.3g (5.9mmol) of potassium hydroxide, 30mL of water, heat to 50℃, stir for 3 hours, TLC The detection reaction is complete. 30mL of water was added, methanol was evaporated under reduced pressure, the remaining aqueous solution was extracted with ethyl acetate (2*100mL), the organic layer was discarded, the aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid, and extracted with ethyl acetate (3*100mL). The layer was dried with sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain 0.95 g of oil, which was directly used in the next reaction.
实施例56：(5S,8S)-3-(异戊酰基)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺的制备Example 56: (5S,8S)-3-(isovaleryl)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[1,2-a][1,2 ,5] Preparation of triazaoctane-8-(N-diphenylmethyl)-carboxamide
Figure PCTCN2020085124-appb-000082
Figure PCTCN2020085124-appb-000082
取(5S,8S)-3-(异戊酰基)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-羧酸0.95g(2.3mmol)，1.8g PyBop(3.6mmol)于100mL单口反应瓶中，加入50mLDMF，另取0.60g DIEA(4.6mmol),室温搅拌30分钟。另取二苯甲胺0.84g(4.6mmol)加入体系中，加热至90℃，搅拌12小时，TLC检测反应完全。反应液倒入300mL水中，乙酸乙酯(3*100mL)萃取，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到淡黄色固体0.70g，收率53％。Take (5S,8S)-3-(isovaleryl)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[1,2-a][1,2,5] Triazaoctane-8-carboxylic acid 0.95g (2.3mmol), 1.8g PyBop (3.6mmol) in a 100mL single-necked reaction flask, add 50mL DMF, another 0.60g DIEA (4.6mmol), and stir at room temperature for 30 minutes. Another 0.84 g (4.6 mmol) of benzhydrylamine was added to the system, heated to 90° C. and stirred for 12 hours. TLC detected that the reaction was complete. The reaction solution was poured into 300mL water, extracted with ethyl acetate (3*100mL), washed with saturated sodium chloride solution, the organic layer was dried with sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent is two Chloromethane: methanol = 15:1) was purified to obtain 0.70 g of light yellow solid with a yield of 53%.
实施例57：(5S,8S)-3-(异戊酰基)-5-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺的制备Example 57: (5S,8S)-3-(isovaleryl)-5-amino-6-one-dehydropyrazolo[1,2-a][1,2,5]triazaoctane Preparation of -8-(N-diphenylmethyl)-formamide
Figure PCTCN2020085124-appb-000083
Figure PCTCN2020085124-appb-000083
取200mg(0.35mmol)(5S,8S)-3-(异戊酰基)-5-(叔丁基氧羰基)-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺和10mL无水乙醇于100mL单口反应瓶中，加入20mL盐酸乙醇，室温搅拌2小时，TLC检测，反应完全。减压蒸出溶剂及过量氯化氢，得到无色油状液体0.17g，直接用于下一步反应。Take 200mg (0.35mmol) (5S,8S)-3-(isovaleryl)-5-(tert-butyloxycarbonyl)-amino-6-one-dehydropyrazolo[1,2-a][1 ,2,,5]Triazaoctane-8-(N-diphenylmethyl)-carboxamide and 10mL of absolute ethanol were added to a 100mL single-necked reaction flask, 20mL of hydrochloric acid ethanol was added, stirred at room temperature for 2 hours, TLC detection, The reaction is complete. The solvent and excess hydrogen chloride were distilled off under reduced pressure to obtain 0.17 g of a colorless oily liquid, which was directly used in the next reaction.
实施例58：(5S,8S)-3-(异戊酰基)-5-((S)-2-(苄氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺的制备Example 58: (5S,8S)-3-(isovaleryl)-5-((S)-2-(benzyloxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrazolo Preparation of [1,2-a][1,2,5]Triazaoctane-8-(N-Diphenylmethyl)-carboxamide
Figure PCTCN2020085124-appb-000084
Figure PCTCN2020085124-appb-000084
取N-Cbz-N-甲基-D-丙氨酸99mg(0.42mmol)和20mL二氯甲烷于100mL单口反应瓶中，加入0.24g HATU(0.63mmol)，0.12gDIEA(0.88mmol)，室温搅拌1小时，得黄色清液。Take 99mg (0.42mmol) of N-Cbz-N-methyl-D-alanine and 20mL of dichloromethane in a 100mL single-mouth reaction flask, add 0.24g HATU (0.63mmol), 0.12g DIEA (0.88mmol), stir at room temperature In 1 hour, a yellow clear liquid was obtained.
取(5S,8S)-3-(异戊酰基)-5-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺0.17g溶于20mL二氯甲烷，滴加入上述体系中，室温搅拌6小时，TLC检测反应完全。减压蒸出溶剂，残余物用100mL乙酸乙酯溶解，饱和氯化钠溶液洗涤，有机层用硫酸钠干燥，过滤，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝20:1)纯化，得到褐色油状物160mg，收率66％。Take (5S,8S)-3-(isovaleryl)-5-amino-6-one-dehydropyrazolo[1,2-a][1,2,5]triazaoctane-8- 0.17 g of (N-diphenylmethyl)-formamide was dissolved in 20 mL of dichloromethane, added dropwise to the above system, and stirred at room temperature for 6 hours. TLC detected that the reaction was complete. The solvent was evaporated under reduced pressure, the residue was dissolved in 100 mL ethyl acetate, washed with saturated sodium chloride solution, the organic layer was dried over sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was column chromatographed (eluent was dichloromethane : Methanol=20:1) Purification to obtain 160 mg of brown oil with a yield of 66%.
实施例59：(5S,8S)-3-(异戊酰基)-5-((S)-2-甲氨基)-丙酰基-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺(I-14)的制备Example 59: (5S,8S)-3-(isovaleryl)-5-((S)-2-methylamino)-propionyl-amino-6-one-dehydropyrazolo[1,2- a][1,2,5]Triazaoctane-8-(N-diphenylmethyl)-carboxamide (I-14)
Figure PCTCN2020085124-appb-000085
Figure PCTCN2020085124-appb-000085
取(5S,8S)-3-(异戊酰基)-5-((S)-2-(苄氧羰基)甲氨基)-丙酰基-氨基-6-酮-脱氢吡唑并[1,2-a][1,2,5]三氮杂辛环-8-(N-二苯甲基)-甲酰胺160mg(0.23mmol)和50mL甲醇于100mL单口反应瓶中，溶解得无色清液，加入16mg(10％)Pd/C，氢气气氛下，室温搅拌12小时，TLC检测反应完全。过滤除去Pd/C，减压蒸出溶剂，残余物柱层析(洗脱剂为二氯甲烷:甲醇＝15:1)纯化，得到白色固体106mg，收率82％。Take (5S,8S)-3-(isovaleryl)-5-((S)-2-(benzyloxycarbonyl)methylamino)-propionyl-amino-6-one-dehydropyrazolo[1, 2-a][1,2,5]Triazaoctane-8-(N-diphenylmethyl)-carboxamide 160mg (0.23mmol) and 50mL methanol in a 100mL single-necked reaction flask, dissolve to a colorless clear After adding 16mg (10%) Pd/C, stirring at room temperature for 12 hours under hydrogen atmosphere, TLC detected that the reaction was complete. Pd/C was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane:methanol=15:1) to obtain 106 mg of white solid with a yield of 82%.
1H NMR(400MHz,d 6-DMSO):δ＝8.05-8.02(m,1H),7.47-7.29(m,2H),7.31-7.12(m,4H),7.18-7.08(m,4H),6.06-6.03(m,1H),5.00-4.93(m,1H),4.42-4.39(m,1H),3.92-3.73(m,1H),3.54(s,1H),3.41-3.39(m,2H),3.22-2.88(m,3H),2.70-2.33(m,2H),2.21-2.12(m,5H),2.05-1.78(m,5H),1.28-1.15(m,3H),0.92-0.85(m,6H)，[M+H] +:m/z＝563.3 1 H NMR (400MHz, d 6 -DMSO): δ=8.05-8.02 (m, 1H), 7.47-7.29 (m, 2H), 7.31-7.12 (m, 4H), 7.18-7.08 (m, 4H), 6.06-6.03(m,1H),5.00-4.93(m,1H),4.42-4.39(m,1H),3.92-3.73(m,1H),3.54(s,1H),3.41-3.39(m,2H) ), 3.22-2.88 (m, 3H), 2.70-2.33 (m, 2H), 2.21-2.12 (m, 5H), 2.05-1.78 (m, 5H), 1.28-1.15 (m, 3H), 0.92-0.85 (m,6H), [M+H] + :m/z=563.3
实施例60：Example 60:
Figure PCTCN2020085124-appb-000086
Figure PCTCN2020085124-appb-000086
1H NMR(400MHz,d 6-DMSO):δ＝8.03-8.00(m,1H),7.49-7.32(m,2H),7.30-7.22(m,4H),7.19-7.10(m,4H),6.06-6.03(m,1H),5.05-4.99(m,1H),4.45-4.39(m,1H),3.80-3.62(m,1H),3.52(s,1H),3.43-3.40(m,2H),3.20-2.90(m,3H),2.78-2.47(m,2H),2.30-2.10(m,5H),2.00-1.60(m,5H),1.18-1.05(m,3H),0.89-0.82(m,6H)，[M+H] +:m/z＝563.3 1 H NMR (400MHz, d 6 -DMSO): δ=8.03-8.00 (m, 1H), 7.49-7.32 (m, 2H), 7.30-7.22 (m, 4H), 7.19-7.10 (m, 4H), 6.06-6.03 (m, 1H), 5.05-4.99 (m, 1H), 4.45-4.39 (m, 1H), 3.80-3.62 (m, 1H), 3.52 (s, 1H), 3.43-3.40 (m, 2H) ), 3.20-2.90 (m, 3H), 2.78-2.47 (m, 2H), 2.30-2.10 (m, 5H), 2.00-1.60 (m, 5H), 1.18-1.05 (m, 3H), 0.89-0.82 (m,6H), [M+H] + :m/z=563.3
实施例61：Example 61:
Figure PCTCN2020085124-appb-000087
Figure PCTCN2020085124-appb-000087
1H NMR(400MHz,d 6-DMSO):δ＝8.05-8.02(m,1H),7.47-7.29(m,2H),7.31-7.12(m,4H),7.18-7.08(m,4H),6.06-6.03(m,1H),5.00-4.93(m,1H),4.42-4.39(m,1H),3.92-3.73(m,1H),3.54(s,1H),3.41-3.39(m,2H),3.22-2.88(m,3H),2.70-2.33(m,2H),2.21-2.12(m,5H),2.05-1.78(m,5H),1.28-1.15(m,3H),0.92-0.85(m,6H)，[M+H] +:m/z＝563.3 1 H NMR (400MHz, d 6 -DMSO): δ=8.05-8.02 (m, 1H), 7.47-7.29 (m, 2H), 7.31-7.12 (m, 4H), 7.18-7.08 (m, 4H), 6.06-6.03(m,1H),5.00-4.93(m,1H),4.42-4.39(m,1H),3.92-3.73(m,1H),3.54(s,1H),3.41-3.39(m,2H) ), 3.22-2.88 (m, 3H), 2.70-2.33 (m, 2H), 2.21-2.12 (m, 5H), 2.05-1.78 (m, 5H), 1.28-1.15 (m, 3H), 0.92-0.85 (m,6H), [M+H] + :m/z=563.3
实施例62：Example 62:
Figure PCTCN2020085124-appb-000088
Figure PCTCN2020085124-appb-000088
1HNMR(400MHz,d 6-DMSO):δ＝8.04-8.01(m,1H),7.52-7.36(m,1H),7.27-7.21(m,2H),7.16-7.05(m,2H),6.05-6.01(m,1H),5.04-4.97(m,1H),4.52-4.43(m,1H),4.00-3.92(m,3H),3.84-3.67(m,1H),3.58(s,1H),3.45-3.41(m,2H),3.27-2.93(m,3H),2.74-2.53(m,2H),2.35-2.18(m,5H),2.12-1.68(m,5H),1.21-1.09(m,3H),0.93-0.88(m,6H)，[M+H] +:m/z＝563.3 1 HNMR (400MHz, d 6 -DMSO): δ=8.04-8.01 (m, 1H), 7.52-7.36 (m, 1H), 7.27-7.21 (m, 2H), 7.16-7.05 (m, 2H), 6.05 -6.01(m,1H),5.04-4.97(m,1H),4.52-4.43(m,1H),4.00-3.92(m,3H),3.84-3.67(m,1H),3.58(s,1H) ,3.45-3.41(m,2H),3.27-2.93(m,3H),2.74-2.53(m,2H),2.35-2.18(m,5H),2.12-1.68(m,5H),1.21-1.09( m,3H),0.93-0.88(m,6H),[M+H] + :m/z=563.3
实施例63：Example 63:
Figure PCTCN2020085124-appb-000089
Figure PCTCN2020085124-appb-000089
1H-NMR(400MHz,d 6-DMSO):δ＝8.12-8.05(m,1H),7.41-7.39(m,2H),7.32-7.21(m,6H),7.13-7.06(m,1H),6.11-6.07(m,1H),5.07-4.97(m,1H),4.46-4.37(m,1H),3.81-3.64(m,1H),3.55(s,1H),3.46-3.41(m,2H),3.25-2.96(m,3H),2.73-2.49(m,2H),2.33-2.17(m,5H),2.06-1.68(m,5H),1.12-1.09(m,3H),0.92-0.88(m,6H)，[M+H] +:m/z＝563.6 1 H-NMR (400MHz, d 6 -DMSO): δ=8.12-8.05 (m, 1H), 7.41-7.39 (m, 2H), 7.32-7.21 (m, 6H), 7.13-7.06 (m, 1H) ,6.11-6.07(m,1H),5.07-4.97(m,1H),4.46-4.37(m,1H),3.81-3.64(m,1H),3.55(s,1H),3.46-3.41(m, 2H), 3.25-2.96 (m, 3H), 2.73-2.49 (m, 2H), 2.33-2.17 (m, 5H), 2.06-1.68 (m, 5H), 1.12-1.09 (m, 3H), 0.92- 0.88(m,6H), [M+H] + :m/z=563.6
实施例64：Example 64:
Figure PCTCN2020085124-appb-000090
Figure PCTCN2020085124-appb-000090
1H NMR(400MHz,d 6-DMSO):δ＝8.08-8.02(m,1H),7.44-7.38(m,1H),7.32-7.26(m,2H),7.19-7.15(m,4H),7.11-7.06(m,2H),6.06-6.03(m,1H),5.06-4.97(m,1H),4.44-4.35(m,1H),3.82-3.61(m,1H),3.58(s,1H),3.47-3.42(m,2H),3.23-2.96(m,3H),2.73-2.41(m,2H),2.38-2.12(m,5H),2.07-1.66(m,5H),1.11-1.08(m,3H),0.95-0.86(m,6H)，[M+H] +:m/z＝599.4 1 H NMR (400MHz, d 6 -DMSO): δ=8.08-8.02 (m, 1H), 7.44-7.38 (m, 1H), 7.32-7.26 (m, 2H), 7.19-7.15 (m, 4H), 7.11-7.06(m,2H),6.06-6.03(m,1H),5.06-4.97(m,1H),4.44-4.35(m,1H),3.82-3.61(m,1H),3.58(s,1H) ), 3.47-3.42 (m, 2H), 3.23-2.96 (m, 3H), 2.73-2.41 (m, 2H), 2.38-2.12 (m, 5H), 2.07-1.66 (m, 5H), 1.11-1.08 (m,3H),0.95-0.86(m,6H),[M+H] + :m/z=599.4
实施例65：化合物与XIAP、cIAP1、cIAP2的结合亲和力测试Example 65: Test of the binding affinity of the compound to XIAP, cIAP1, and cIAP2
为了试验本发明化合物与XIAP、cIAP1、cIAP2蛋白的结合亲和力，使用了基于荧光偏振(FP)的竞争性测定方法进行测定，对于cIAP1-BIR3、cIAP2-BIR3测定法，荧光标记的Smac模拟物(Smac-2F)用作荧光探针。Smac-2F与cIAP1-BIR3、cIAP2-BIR3的K d值通过监测用固体浓度的荧光探针和高达完全饱和的渐增浓度的蛋白构成的混合物的总荧光偏振来测定。使用Infinite M-1000酶标仪(Tecan U.S.,Research Triangle Park,NC)在Microfluor 2 96-孔黑色圆底板(Fisher Scientific)中测量荧光偏振值。在96-孔中，添加1nM Smac-2F和渐增浓度的蛋白至测定缓冲液(100mM磷酸钾，PH＝7.5；100ug/ml牛γ球蛋白；0.02％叠氮化钠，Invitrogen)中，最终体积为125ul。在振荡器上将所述板混合15分钟并在室温孵育1-3小时。在485nm激发波长和530nm发射波长，以毫偏振单位(millipolarization unit)测量偏振值。然后使用GraphPad Prism 5.0软件通过拟合S形剂量依赖性FP增加作为蛋白浓度的函数来计算平衡解离常数(K d) In order to test the binding affinity of the compounds of the present invention to XIAP, cIAP1, and cIAP2 proteins, a fluorescence polarization (FP)-based competitive assay method was used for the assay. For the cIAP1-BIR3 and cIAP2-BIR3 assay methods, fluorescently labeled Smac mimics ( Smac-2F) is used as a fluorescent probe. The K d values of Smac-2F, cIAP1-BIR3, and cIAP2-BIR3 are determined by monitoring the total fluorescence polarization of a mixture composed of a solid concentration fluorescent probe and an increasing concentration of protein up to complete saturation. The fluorescence polarization value was measured in a Microfluor 2 96-well black round bottom plate (Fisher Scientific) using an Infinite M-1000 microplate reader (Tecan US, Research Triangle Park, NC). In 96-wells, add 1nM Smac-2F and increasing concentrations of protein to the assay buffer (100mM potassium phosphate, pH=7.5; 100ug/ml bovine gamma globulin; 0.02% sodium azide, Invitrogen), and finally The volume is 125ul. The plates were mixed on a shaker for 15 minutes and incubated at room temperature for 1-3 hours. The polarization value was measured in millipolarization unit at 485nm excitation wavelength and 530nm emission wavelength. Then use GraphPad Prism 5.0 software to calculate the equilibrium dissociation constant (K d ) by fitting the S-shaped dose-dependent FP increase as a function of protein concentration
化合物的K i值通过符合剂量依赖性的竞争性结合实验来测定，其中化合物的系列稀释液与固定浓度的荧光探针竞争结合固定浓度的蛋白(通常为上述测定的Kd值得2-3倍)。将5ul的在DMSO中的测试化合物与120ul在测定缓冲溶液(100mM磷酸钾，PH＝7.5；100ug/ml牛γ球蛋白；0.02％叠氮化钠，Invitrogen)中的预孵育蛋白/示踪剂复合物混合物添加至测定板，并且在室温孵育1-3小时。蛋白和探针的终浓度对于cIAP1-BIR3和cIAP2-BIR3测定分别为3nm和1nm、5nm和1nm。仅含有蛋白/探针复合物的阴性对照(相当于0％抑制)和仅含有游离探针的阳性对照(相当于100％抑制)包含在每个测定板中。如上所述测量EP值。通过竞争曲线的非线性回归拟合测定IC50值。竞争性抑制剂的Ki值使用先前描述的推导方程，基于所测量的IC50值、探针与不同蛋白的Kd值和精制性测定中蛋白和探针的浓度来计算。 The K i value of the compound is determined by a dose-dependent competitive binding experiment, in which a serial dilution of the compound competes with a fixed concentration of fluorescent probe to bind to a fixed concentration of protein (usually the Kd value measured above is 2-3 times) . 5ul test compound in DMSO and 120ul pre-incubated protein/tracer in assay buffer solution (100mM potassium phosphate, pH=7.5; 100ug/ml bovine gamma globulin; 0.02% sodium azide, Invitrogen) The complex mixture is added to the assay plate and incubated at room temperature for 1-3 hours. The final concentrations of protein and probe were 3nm and 1nm, 5nm and 1nm for cIAP1-BIR3 and cIAP2-BIR3, respectively. A negative control containing only protein/probe complexes (equivalent to 0% inhibition) and a positive control containing only free probes (equivalent to 100% inhibition) were included in each assay plate. Measure the EP value as described above. The IC50 value was determined by nonlinear regression fitting of the competition curve. The Ki value of the competitive inhibitor is calculated using the previously described derivation equation based on the measured IC50 value, the Kd value of the probe and different proteins, and the concentration of the protein and probe in the refining assay.
对于XIAP-BIR2-BIR3蛋白的基于FP的测定使用相同的程序进行。在该测定中，二价荧光标签的肽类Smac模拟物(Smac-1F)用作荧光探针，其与XIAP-BIR2-BIR3的Kd值通过饱和实验类似的测定。将0.01％Triton X-100添加至测定缓冲液中以实现二聚体荧光探针的稳定性的荧光和偏振值。竞争性测定中利用的最终蛋白和探针浓度分别为2nm和1nm。本发明中化合物测试结果如下表所示。The FP-based assay for the XIAP-BIR2-BIR3 protein was performed using the same procedure. In this measurement, a bivalent fluorescent-tagged peptide Smac mimic (Smac-1F) was used as a fluorescent probe, and the Kd value of XIAP-BIR2-BIR3 was measured similarly to the saturation experiment. 0.01% Triton X-100 was added to the assay buffer to achieve the stable fluorescence and polarization values of the dimer fluorescent probe. The final protein and probe concentrations used in the competition assay were 2nm and 1nm, respectively. The test results of the compounds of the present invention are shown in the following table.
Figure PCTCN2020085124-appb-000091
Figure PCTCN2020085124-appb-000091
Figure PCTCN2020085124-appb-000092
Figure PCTCN2020085124-appb-000092
实验结果发现本发明化合物与XIAP、cIAP1、cIAP2蛋白的结合亲和力较好，具有较好的IAP抑制活性。The experimental results found that the compound of the present invention has good binding affinity with XIAP, cIAP1, and cIAP2 proteins, and has good IAP inhibitory activity.
实施例67：MDA-MB-231乳腺癌和PC-3胰腺癌细胞系中细胞生长抑制测试Example 67: Cell growth inhibition test in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines
测试了本发明化合物对不同癌细胞系的生长的影响。将细胞以3000细胞/孔的密度与试验化合物一起接种在96-孔平底细胞培养板上并将细胞在37℃下在95％空气和5％CO 2的气氛中培养4天。使用WST-8试剂盒和2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺苯基)-2H-四唑鎓一钠盐，测定在用不同浓度化合物处理之后的细胞生长抑制率。以10％的最终浓度将WST-8添加到 每一孔中，然后在37℃下培养所述板2-3小时。使用ULTRATecan读数器(MolecularDevice)在450nm下测量样品的吸光度。通过比较未处理的细胞和用试验化合物处理过的细胞中的吸光度，计算试验化合物抑制细胞生长50％的浓度(IC 50)。测试结果如下表所示。 The effects of the compounds of the invention on the growth of different cancer cell lines were tested. The cells were seeded on a 96-well flat-bottom cell culture plate together with the test compound at a density of 3000 cells/well and the cells were cultured at 37°C in an atmosphere of 95% air and 5% CO 2 for 4 days. Use WST-8 kit and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H- Tetrazolium monosodium salt was used to determine the cell growth inhibition rate after treatment with different concentrations of the compound. WST-8 was added to each well at a final concentration of 10%, and then the plate was incubated at 37°C for 2-3 hours. The absorbance of the sample was measured at 450 nm using a ULTRATecan reader (Molecular Device). By comparing the absorbance of untreated cells and the treated cells with the test compound, the test compound is calculated inhibitory concentration (IC 50) 50% of cell growth. The test results are shown in the table below.
Figure PCTCN2020085124-appb-000093
Figure PCTCN2020085124-appb-000093
Figure PCTCN2020085124-appb-000094
Figure PCTCN2020085124-appb-000094
本发明化合物对MDA-MB-231乳腺癌和PC-3胰腺癌细胞系中细胞生长具有较好的抑制作用。The compound of the present invention has a good inhibitory effect on cell growth in MDA-MB-231 breast cancer and PC-3 pancreatic cancer cell lines.

Claims (12)

  1. 一种化合物或其药学可接受的盐、异构体，其特征在于所述化合物具有如下通式：A compound or a pharmaceutically acceptable salt or isomer thereof, characterized in that the compound has the following general formula:
    Figure PCTCN2020085124-appb-100001
    Figure PCTCN2020085124-appb-100001
    其中，X代表C或N原子，当X为C原子时,Y为CONHR 3，Z代表H原子；当X为N原子时，Z为CONHR 3，Y不存在； Wherein, X represents a C or N atom, when X is a C atom, Y is a CONHR 3 and Z represents a H atom; when X is a N atom, Z is a CONHR 3 and Y does not exist;
    所述的R 1和R 2各自独立地任意选自C 1～20烷胺基、C 5～8环烷胺基、
    Figure PCTCN2020085124-appb-100002
    C 1～20烷基、
    Figure PCTCN2020085124-appb-100003
    Figure PCTCN2020085124-appb-100004
    或者α-氨基酸残基，     The R 1 and R 2 are each independently selected from C 1-20 alkylamino groups, C 5-8 cycloalkylamino groups,
    Figure PCTCN2020085124-appb-100002
    C 1～20 alkyl group,
    Figure PCTCN2020085124-appb-100003
    Figure PCTCN2020085124-appb-100004
    Or α-amino acid residues,
    其中所述的α-氨基酸残基为α-氨基酸中α-碳上的羧基缺失后所形成的取代基，所述的Ar基团代表原子个数为5～8的芳基或杂芳基，所述的m和n各自独立选自1～3任意整数；The α-amino acid residue is a substituent formed by the deletion of the carboxyl group on the α-carbon of the α-amino acid, and the Ar group represents an aryl or heteroaryl group with 5-8 atoms. The m and n are each independently selected from any integer of 1 to 3;
    R 3选自取代或非取代的苄基，所述的取代基任意选自苯基、卤素、C 1～4烷基或对氟苯基。 R 3 is selected from substituted or unsubstituted benzyl, and the substituent is optionally selected from phenyl, halogen, C 1-4 alkyl or p-fluorophenyl.
  2. 一种化合物或其药学可接受的盐或异构体，其特征在于所述化合物具有如下通式：A compound or a pharmaceutically acceptable salt or isomer thereof, characterized in that the compound has the following general formula:
    Figure PCTCN2020085124-appb-100005
    Figure PCTCN2020085124-appb-100005
    其中，上述式I或II中，Wherein, in the above formula I or II,
    所述的R 1选自C 1～20烷胺基、
    Figure PCTCN2020085124-appb-100006
    C 1～20烷基、
    Figure PCTCN2020085124-appb-100007
    Figure PCTCN2020085124-appb-100008
    或者α-氨基酸残基中的任意一种，     Said R 1 is selected from C 1-20 alkylamino groups,
    Figure PCTCN2020085124-appb-100006
    C 1～20 alkyl group,
    Figure PCTCN2020085124-appb-100007
    Figure PCTCN2020085124-appb-100008
    Or any of the α-amino acid residues,
    R 2选自C 1～20烷胺基、C 5～8环烷胺基、C 1～20烷基、
    Figure PCTCN2020085124-appb-100009
    Figure PCTCN2020085124-appb-100010
    或者α-氨基酸残基中的任意一种，     R 2 is selected from C 1-20 alkylamino group, C 5-8 cycloalkylamino group, C 1-20 alkyl group,
    Figure PCTCN2020085124-appb-100009
    Figure PCTCN2020085124-appb-100010
    Or any of the α-amino acid residues,
    其中所述的α-氨基酸残基为α-氨基酸中α-碳上的羧基缺失后所形成的取代残基，所述的Ar基团代表原子个数为5～8的芳基和杂芳基，所述的m和n各自独立选自1～3任意整数，The α-amino acid residue is a substituted residue formed by the deletion of the carboxyl group on the α-carbon in the α-amino acid, and the Ar group represents an aryl group and a heteroaryl group with 5-8 atoms. , The m and n are each independently selected from any integer of 1 to 3,
    R 3选自取代或非取代的苄基，所述的取代基任意选自苯基、卤素、C 1～4烷基或对氟苯基。 R 3 is selected from substituted or unsubstituted benzyl, and the substituent is optionally selected from phenyl, halogen, C 1-4 alkyl or p-fluorophenyl.
  3. 如权利要求2所述的化合物及其盐或异构体，其特征在于式I或II中所述的烷胺基为
    Figure PCTCN2020085124-appb-100011
    及其异构体。     The compound and salt or isomer thereof according to claim 2, wherein the alkylamino group in formula I or II is
    Figure PCTCN2020085124-appb-100011
    And its isomers.
  4. 如权利要求2所述的化合物及其盐或异构体，其特征在于式I或II中所述的 C 5～6环烷胺基为
    Figure PCTCN2020085124-appb-100012
    The compound and salt or isomer thereof according to claim 2, wherein the C 5-6 cycloalkylamino group in formula I or II is
    Figure PCTCN2020085124-appb-100012
  5. 如权利要求2所述的化合物及其盐或异构体，其特征在于所述的α-氨基酸残基为
    Figure PCTCN2020085124-appb-100013
    或者
    Figure PCTCN2020085124-appb-100014
    The compound and its salt or isomer according to claim 2, wherein the α-amino acid residue is
    Figure PCTCN2020085124-appb-100013
    or
    Figure PCTCN2020085124-appb-100014
  6. 如权利要求2所述的化合物及其盐或异构体，其特征在于式I或II中R 2选自C 1～13烷基。 The compound according to claim 2 and its salt or isomer, characterized in that R 2 in formula I or II is selected from C 1-13 alkyl.
  7. 如权利要求2所述的化合物及其盐或异构体，其特征在于式I或II中所述的芳基为苯基。The compound and its salt or isomer according to claim 2, characterized in that the aryl group in formula I or II is phenyl.
  8. 如权利要求2所述的化合物及其盐或异构体，其特征在于式I或II中所述的杂芳基为咪唑基。The compound and its salt or isomer according to claim 2, characterized in that the heteroaryl group in formula I or II is imidazolyl.
  9. 一种化合物，其特征在于其结构选自A compound characterized in that its structure is selected from
    Figure PCTCN2020085124-appb-100015
    Figure PCTCN2020085124-appb-100015
    Figure PCTCN2020085124-appb-100016
    Figure PCTCN2020085124-appb-100016
    Figure PCTCN2020085124-appb-100017
    Figure PCTCN2020085124-appb-100017
    Figure PCTCN2020085124-appb-100018
    Figure PCTCN2020085124-appb-100018
    Figure PCTCN2020085124-appb-100019
    Figure PCTCN2020085124-appb-100019
  10. 一种药物组合物，其特征在于含有治疗有效量的权利要求1至9中任意一项所述的化合物或其药学上可接受的盐或异构体作为活性成分，以及一种或多种药学上可接受的载体、稀释剂和/或赋型剂。A pharmaceutical composition characterized by containing a therapeutically effective amount of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt or isomer thereof as an active ingredient, and one or more pharmacological agents Acceptable carriers, diluents and/or excipients.
  11. 含有权利要求1至9中任意一项所述的化合物或其药学上可接受的盐或异构体在制备与IAP蛋白相关疾病的药物中的用途。The use of the compound described in any one of claims 1 to 9 or a pharmaceutically acceptable salt or isomer thereof in the preparation of a medicine for diseases related to IAP protein.
  12. 含有权利要求1至9中任意一项所述的化合物或其药学上可接受的盐或异构体在制备治疗未能经历细胞凋亡的所有癌症类型的药物中的用途。Use of a compound containing the compound described in any one of claims 1 to 9 or a pharmaceutically acceptable salt or isomer thereof in the preparation of a medicament for treating all types of cancers that have not undergone apoptosis.
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