A peptidomimetic compound and its use in preparing 2019-nCoV main protease inhibitor

Abstract

The present invention belongs to the technical field of drug development, and relates to a new peptidomimetic compound, a pharmaceutically acceptable salt thereof, a stereoisomer thereof or an isotope label thereof, and further discloses its use for preparing a 2019-nCoV main protease inhibitor. The peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels of the present invention are based on the known 2019-nCoV main protease inhibitor PF-07321332 structure, and a new structure compound is obtained by introducing a halogen or aliphatic substituent. Compared with the applied inhibitor PF-07321332, the compound of the present invention has a good improvement in the inhibitory activity and pharmacokinetic properties of the 2019 new coronavirus main protease, and has a good application prospect in the treatment of new coronavirus pneumonia (COVID-19) caused by 2019 new coronavirus infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug research and development, and relates to a new peptidomimetic compound, a pharmaceutically acceptable salt thereof, a stereoisomer or an isotope label thereof, and further discloses its use in preparing a 2019-nCoV main protease inhibitor. Background Art

[0002] The 2019 novel coronavirus (2019-nCoV, also known as SARS-CoV-2) is a single-stranded, positive-sense RNA virus belonging to the betacoronavirus subgenus of the Sarcovirus subgenus. It is a zoonotic virus that can also be transmitted between people. The 2019 novel coronavirus is mainly transmitted through respiratory droplets and contact. When a person is infected with the coronavirus, it often leads to novel coronavirus infection. Common symptoms include respiratory symptoms, fever, cough, shortness of breath and difficulty breathing. Some infected patients will progress to severe illness, causing severe acute respiratory syndrome, renal failure, and even death. In view of this, how to curb the spread of the virus and better treat novel coronavirus infection has become an urgent common issue for all mankind.

[0003] It is reported that the 2019 novel coronavirus mutates rapidly, and at least 6 mutant strains have been reported. Omicron, reported in November 2021, has replaced Delta as the main prevalent variant. Relatively speaking, the Omicron variant has stronger immune escape ability (https: / / www.nature.com / articles / s41392-021-00852-5) and transmission ability (Schmidt, F. et al. Nature https: / / doi.org / 10.1038 / s41586-021-04005-0(2021)). This is mainly because Omicron has 32 mutations on its spike protein (SARS-CoV-2 variants of concern and variants under investigation in England, Technical briefing 29), which reduces or even ineffective the protective efficacy of vaccines and neutralizing antibodies.

[0004] Studies have shown that the main protease of the 2019 novel coronavirus (also known as 3C-like protease) is a relatively conservative protease, which is responsible for catalyzing the hydrolysis of the virus's polyproteins (pp1a and pp1ab) and producing multiple functional non-structural proteins to form a replication-transcription complex to complete the subsequent replication of genetic material and the synthesis of structural proteins. Therefore, by inhibiting the function of the 2019 novel coronavirus main protease, the virus replication process can be interfered with to achieve an antiviral effect; and since there is no protease with similar structural functions in the human body (Jin, Z. et al., 2020; Rathnayake, AD et al., 2020), it is an ideal antiviral target. In view of this, the field expects to develop new and highly effective drugs that can inhibit the 2019-nCoV main protease, providing new ideas for the prevention and treatment of the 2019 novel coronavirus. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a new peptidomimetic compound, a pharmaceutically acceptable salt thereof, a stereoisomer or an isotope label thereof, and apply the same to the treatment of novel coronavirus pneumonia caused by 2019 novel coronavirus infection;

[0006] The second technical problem to be solved by the present invention is to provide the use of the above-mentioned peptidomimetic compound for preparing the main protease inhibitor of the 2019 novel coronavirus.

[0007] In order to solve the above technical problems, the present invention provides a peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope-labeled substances, wherein the compound has a structure as shown in the following formula (I):

[0008]

[0009] in,

[0010] The R 1 and R 2 are independently selected from hydrogen, halogen, C 1-4 Alkyl, aryl or 4-6 membered heterocyclic ring, the above groups are unsubstituted or optionally substituted by 0-5 F atoms; said R 1 and R 2 Independently or selectively bonded to each other to form a 3-6 membered ring structure substituted by 0-5 F atoms;

[0011] The R 3 and R 4 are independently selected from hydrogen, halogen, C 1-4 Alkyl, COR 5, aryl or 4-6 membered heterocyclic ring, the above groups are unsubstituted or optionally substituted by 0-5 F atoms; and said R 3 and R 4 Not at the same time as hydrogen.

[0012] The peptidomimetic compounds of the present invention can be administered as prodrugs of the compounds of formula (I). According to the understanding of those skilled in the art, certain derivatives of the compounds of formula (I) which may have little or no pharmacological activity themselves will be converted into compounds of formula (I) having the desired activity (for example) by hydrolysis, especially by esterase or peptidase-promoted hydrolysis, when administered into or onto the body, and such derivatives are called "prodrugs". Those skilled in the art can obtain the corresponding prodrugs by modifying appropriate functional groups present in the compounds of formula (I) (H. Bundgaard, Design of Prodrugs, Elsevier, 1985).

[0013] The peptidomimetic compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. Unless otherwise specified, all stereoisomeric forms of the compounds of the present invention and mixtures thereof, including racemic mixtures, are part of the present invention.

[0014] All pharmaceutically acceptable isotope-labeled compounds of formula (I) of the peptidomimetic compounds of the present invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Substitution by heavier isotopes such as deuterium may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements, and may therefore be preferred in certain circumstances.

[0015] The term "alkyl" as used herein refers to a n H (2n+1) Non-limiting examples of straight or branched monovalent hydrocarbon groups include methyl, ethyl, propyl, butyl, 2-methyl-propyl, 1,1-dimethylethyl, pentyl, and hexyl.

[0016] The term "cycloalkyl" as used herein refers to a radical of the formula -C n H (2n-1) The term "cyclopentyl" refers to a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group.

[0017] The term "alkoxy" as used herein refers to an alkyl substituent attached through an oxygen atom, non-limiting examples of which include methoxy, ethoxy, propoxy, and butoxy.

[0018] The term "alkoxycarbonyloxy" as used herein refers to an alkoxy group attached through a carbonyl (-CO-) group, non-limiting examples of which include methoxycarbonyl, ethoxycarbonyl, and propoxycarbonyl.

[0019] The term "alkylcarbonyloxy" used herein refers to an alkyl group attached through a carbonyloxy group (-C(=O)O-), and representative examples include methylcarbonyloxy, ethylcarbonyloxy and tert-butylcarbonyloxy.

[0020] The term "alkoxycarbonyloxy-alkoxy" used herein refers to an alkoxycarbonyloxy group connected via an alkoxy group, and the term halogen used herein refers to F, Cl, Br, and I.

[0021] The term "heterocycle" as used herein refers to alicyclic and aromatic heterocyclic compounds having 4 to 7 carbon atoms, wherein the cyclomethylene (-CH 2 -) is replaced by a group selected from -O-, -S- or -N-, wherein the valence requirement of -N- is satisfied by H or by as a point of attachment; aromatic heterocycle refers to a 5-membered or 6-membered heterocyclic ring system that conforms to Hückel's rule and contains 1-2 atoms selected from O, S and N atoms.

[0022] The aryl group used in the present application refers to a functional group or substituent derived from a simple aromatic ring (such as a benzene ring, a naphthalene ring, etc.) without a heteroatom.

[0023] Specifically, the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels, wherein R 5 Selected from C 1-3 Alkyl or cycloalkyl; preferably, the R 5 Selected from C 1-3 Alkyl or C 1-3 Cycloalkyl.

[0024] Specifically, in the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels, the 4-6 membered heterocycle is a heterocycle containing 1-2 heteroatoms selected from nitrogen, oxygen and sulfur.

[0025] Preferably, the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels:

[0026] The R 1 and R 2 are independently selected from hydrogen, fluorine or C substituted by 0-3 fluorine atoms 1-4 Alkyl, the R 1 and R 2Independently or selectively bonded to each other to form a 3-6 membered ring structure;

[0027] The R 3 and R 4 are independently selected from hydrogen, halogen, C 1-3 Alkyl or COR 5 , wherein the R 5 Selected from C 1-3 Alkyl or C 1-3 Cycloalkyl.

[0028] Preferably, the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels:

[0029] The R 1 , R 2 are independently selected from fluorine or C substituted by 0-3 fluorine atoms 1-4 alkyl;

[0030] The R 3 , R 4 are independently selected from hydrogen, fluorine or C 1-3 alkyl.

[0031] Preferably, the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels are selected from the following compounds (1)-(31):

[0032]

[0033]

[0034] Preferably, the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope-labeled substances are selected from the group consisting of compounds (1), (2), (3), (4), (5), (6), (7), (8), (11), (12), (13), (14), (22), (23), (24), (25), (26), (28), (29), (30) and (31);

[0035] Preferably, the compound is selected from compound (1), (2), (3), (5), (7), (12), (13), (14), (22), (23), (24), (25), (28), (29), (30), (31);

[0036] More preferably, the compound is selected from compound (1), (3), (12), (13), (14), (22), (23), (24), (25), (31);

[0037] Most preferably, the compound is selected from compound (31).

[0038] The present invention also discloses a method for preparing the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels, comprising the step of introducing aliphatic, heterocyclic or aromatic substituents of selected structures into the rings in the compound molecules shown in formula (I).

[0039] The present invention also discloses the use of the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels for preparing 2019-nCoV main protease inhibitors.

[0040] The present invention also discloses the use of the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels for preparing drugs for treating novel coronavirus pneumonia caused by 2019 novel coronavirus infection.

[0041] Specifically, the peptidomimetic compound and its derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope labels can be administered alone or in combination with pharmacokinetic enhancers or other anti-2019 novel coronavirus drugs.

[0042] The peptidomimetic compounds and their derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope-labeled substances of the present invention are based on the known structure of the 2019-nCoV main protease inhibitor PF-07321332, and new structural compounds are obtained by introducing halogen or aliphatic substituents. Compared with the applied inhibitor PF-07321332, the compounds of the present invention have improved inhibitory activity and pharmacokinetic properties on the 2019 novel coronavirus main protease; in particular, the compounds obtained by substitution based on the introduction of fluorine atoms have greatly improved inhibitory effects, greater exposure in animals, better absorbability and pharmacokinetic properties, and have good application prospects in the treatment of novel coronavirus pneumonia (COVID-19) caused by 2019 novel coronavirus infection.

[0043] The peptidomimetic compounds and their derivatives, pharmaceutically acceptable salts, stereoisomers, tautomers or isotope-labeled substances of the present invention have a good inhibitory effect on the main protease of the 2019 novel coronavirus, can inhibit the replication of the virus by inhibiting the main protease of the 2019 novel coronavirus, and can be used to prepare drugs for treating novel coronavirus pneumonia caused by infection with the 2019 novel coronavirus. The inhibitors can be administered alone or in combination with pharmacokinetic enhancers and other anti-2019 novel coronavirus drugs, providing a new drug option for the treatment of novel coronavirus infection. DETAILED DESCRIPTION

[0044] In the following embodiments of the present invention, the common abbreviations used include: CDC1 3 is deuterated chloroform; CO 2 Et is ethyl carboxylate; DCM is dichloromethane; DIPEA is N,N-diisopropylethylamine; DMF is dimethylformamide; DMSO is dimethyl sulfoxide; EtOAc / EA is ethyl acetate; H or h or hr is hour; Na 2 SO 4 is sodium sulfate; NADH is nicotinamide adenine dinucleotide (reduced form); NAD + is nicotinamide adenine dinucleotide (oxidized form); PEP is phosphoenolpyruvate; RT or rt is room temperature; TFA is trifluoroacetic acid; THF is tetrahydrofuran; PE is petroleum ether; DMAP is 4-dimethylaminopyridine; TMSCN is trimethylsilyl cyanide; HATU is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIBAL is diisobutylaluminum hydride; DAST is diethylaminosulfur trifluoride.

[0045] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present application will be further described below in conjunction with specific embodiments.

[0046] Example 1

[0047] This example is used to prepare compound (31), whose name is (2S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, and its structural formula is shown below:

[0048]

[0049] The synthetic route of compound (31) described in this example is as follows, which specifically comprises the following steps:

[0050]

[0051] Step 1: ((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamic acid Tert-butyl ester

[0052] At room temperature, (S)-2-(tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoic acid methyl ester (0.50 g, 1.74 mmol) was dissolved in aqueous ammonia (10.0 mL), and the reaction solution was stirred at room temperature for 3.0 hours; the reaction solution was filtered, and the filter cake was dried to obtain the target compound as a white solid (0.35 g, 74.1%); the product detection data was: MS (ESI, pos.ion) m / z: 272.1 [M+H] + ;

[0053] Step 2: tert-Butyl ((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamate

[0054] At room temperature, tert-butyl ((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (0.350 g, 1.29 mmol) was dissolved in dichloromethane (4.0 mL), and trifluoroacetic anhydride (0.8 mL) was added. The reaction solution was stirred at room temperature for 5.0 hours. Saturated sodium chloride was added to the reaction solution to quench, and ethyl acetate (100 mL×2) was used for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA=1:1) to obtain the target compound as a white solid (0.13 g, 39.8%). The product detection data was as follows: MS (ESI, pos.ion) m / z: 254.1 [M+H] + ;

[0055] Step 3: (S)-2-Amino-3-((S)-2-oxopyrrolidin-3-yl)propionitrile

[0056] At room temperature, tert-butyl ((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamate (0.13 g, 0.51 mmol) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (2.0 mL) was added. The reaction solution was stirred at room temperature for 1.5 hours, and the reaction solution was concentrated under reduced pressure to obtain the target compound as a light yellow solid (70 mg, 89.7%); the product detection data is: MS (ESI, pos.ion) m / z: 154.0 [M+H] + ;

[0057] Step 4: (3R,7aS)-3-Phenyltetrahydro-3H,5H-pyrrolo[1,2-c]oxazol-5-one

[0058] At room temperature, (S)-5-(Hydroxymethyl)pyrrolidin-2-one (25.0 g, 217.2 mmol) was dissolved in toluene (130.0 mL) solution, and then benzaldehyde (27.6 g, 260.6 mmol) and p-toluenesulfonic acid (1.9 g, 10.9 mmol) were added, and the mixture was heated to reflux and stirred overnight; the reaction solution was cooled, water (200 mL) was added to the reaction solution, and ethyl acetate (400 mL×2) was used for extraction, the organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the resulting mixture was subjected to silica gel column chromatography (PE:EA=4:1) to obtain the target compound (19.4 g, 44.0%) as a yellow oil; the product detection data were: MS (ESI, pos.ion) m / z: 204.0 [M+H] + ;

[0059] Step 5: (3R,7aS)-6-Fluoro-3-phenyltetrahydro-3H,5H-pyrrolo[1,2-c]oxazol-5-one

[0060] At room temperature, diisopropylamine (7.5 g, 73.8 mmol) and THF (150.0 mL) were added to the reaction bottle. Under nitrogen protection, the temperature was lowered to -30 ° C, and n-butyl lithium (32.5 mL, 81.2 mmol) was added dropwise. The temperature was kept at -20 ° C and stirred for 20 minutes. The temperature was then lowered to -50 ° C, (3R, 7aS)-3-phenyltetrahydro-3H, 5H-pyrrolo[1,2-c]oxazol-5-one (10.0 g, 49.2 mmol) was added, and the reaction was carried out at low temperature for 30 minutes. N-fluorobisbenzenesulfonamide (31.0 g, 98.4 mmol) was dissolved in 80 mL The mixture was added dropwise to the reaction system at low temperature in THF, and the reaction was kept at low temperature for 20 minutes, and the temperature was naturally raised to room temperature; saturated ammonium chloride (200 mL) was added to the reaction solution to quench, and extracted with ethyl acetate (300 mL × 3), the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the obtained mixture was subjected to silica gel column chromatography (PE: EA = 3: 1) to obtain the target compound (5.2 g, 47.8%) as a yellow oil; the product detection data is: MS (ESI, pos.ion) m / z: 222.0 [M+H] + ;

[0061] Step 6: (3R,7aS)-6-Fluoro-3-phenyl-6-(phenylseleno)tetrahydro-3H,5H-pyrrolo[1,2-c]oxazole- 5-Keto

[0062] At room temperature, diisopropylamine (0.79 g, 7.79 mmol) and THF (20 mL) were added to the reaction bottle. Under nitrogen protection, the temperature was lowered to -30°C, and n-butyl lithium (3.4 mL, 8.57 mmol) was added dropwise. The temperature was kept at -30°C with stirring for 30 minutes, and then the temperature was lowered to -70°C, and (3R, 7aS)-6-fluoro-3-phenyltetrahydro-3H, 5H-pyrrolo[1,2-c]oxazol-5-one (1.15 g, 5.19 mmol) was added. The reaction was carried out at low temperature for 0.5 hour. Phenylselenobromide (1.35 g, 5.7 mmol) was dissolved in 2 mL of THF, and the mixture was added dropwise to the reaction system at low temperature. The reaction was kept at low temperature for 30 minutes, and the temperature was naturally raised to room temperature. Saturated ammonium chloride (40 mL) was added to the reaction solution to quench, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow oily crude product (1.25 g).

[0063] Step 7: (3R,7aS)-6-Fluoro-3-phenyl-1,7a-dihydro-3H,5H-pyrrolo[1,2-c]oxazol-5-one

[0064] (3R, 7aS)-6-fluoro-3-phenyl-6-(phenylseleno)tetrahydro-3H, 5H-pyrrolo[1,2-c]oxazol-5-one (1.25 g) and dichloromethane (10.0 mL) were added to the reaction bottle. Under an ice bath, 30% hydrogen peroxide (2.0 mL) and pyridine (1.0 mL) were added, and then the temperature was naturally raised to room temperature. The reaction was completed by spotting. Water (50 mL) was added to the reaction solution, and dichloromethane (50 mL×3) was used for extraction. The product was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: EA = 1: 1) to obtain the target compound (0.23 g, 20.0%) as a colorless oil. The product detection data was: MS (ESI, pos.ion) m / z: 220.0 [M+H] + ;

[0065] Step 8: (3R,6bS)-5a-Fluoro-6,6-dimethyl-3-phenyltetrahydro-3H-cyclopropyl[3,4]pyrrolo[1,2- c]Oxazol-5(1H)-one

[0066] Add isopropyltriphenylphosphonium bromide (1.3 g, 3.42 mmol) and THF (20 mL) to the reaction bottle, add KHMDS (4.5 mL, 4.56 mmol) dropwise under ice bath conditions, keep stirring at zero degree for 1.5 minutes, add (3R, 7aS)-6-fluoro-3-phenyl-1,7a-dihydro-3H,5H-pyrrolo[1,2-c]oxazol-5-one (0.50 g, 2.28 mmol), react at low temperature for 2.0 hours, and naturally warm to room temperature. Saturated sodium chloride was added to the reaction solution to quench, and ethyl acetate (80 mL × 3) was used for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: EA = 3: 1) to obtain the target compound (0.14 g, 23.5%) as a colorless oil; the product detection data were: MS (ESI, pos.ion) m / z: 262.1 [M+H] + ;

[0067] Step 9: ((2S)-3-Benzyl-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-yl)methanol

[0068] Under ice bath condition, lithium aluminum tetrahydride (582.3 mg, 1.53 mmol) was added to a solution of (3R,6bS)-5a-fluoro-6,6-dimethyl-3-phenyltetrahydro-3H-cyclopropyl[3,4]pyrrolo[1,2-c]oxazol-5(1H)-one (400.0 mg, 1.53 mmol) in tetrahydrofuran (10.0 mL), and the reaction solution was stirred at room temperature for 3.0 hours; water (50 mL) was added to quench the mixture, and the mixture was filtered. The filtrate was extracted twice with ethyl acetate (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=50:1) to obtain the target compound (165.0 mg, 43.3%) as a colorless oil; the product detection data were: MS (ESI, pos.ion) m / z: 250.1 [M+H] + ;

[0069] Step 10: ((2S)-5-Fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-yl)methanol

[0070] Palladium carbon (10%, 30 mg) was added to a solution of ((2S)-3-benzyl-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-yl)methanol (300 mg, 1.2 mmol) in methanol (5.0 mL) at room temperature, the air was replaced with hydrogen twice, and then stirred at room temperature under a hydrogen atmosphere overnight; after the reaction was completed, the mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the target compound (145.0 mg, yield 76.0%) as a colorless oil; the product detection data were: MS (ESI, pos.ion) m / z: 160.1 [M+H] + ;

[0071] Step 11: tert-(4S)-1-Fluoro-4-(hydroxymethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylic acid Butyl Ester

[0072] To a solution of ((2S)-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-yl)methanol (100.0 mg, 0.63 mmol) in THF (2.0 mL) was added Boc 2 O (205.6 mg, 0.94 mmol), the reaction solution was stirred at room temperature for 1.0 hour; water (60 mL) was added, extracted with ethyl acetate (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: EA = 1: 2) to obtain the target compound (128.0 mg, 78.4%) as a colorless oil; the product detection data is: MS (ESI, pos.ion) m / z: 260.1 [M+H] + ;

[0073] Step 12: (2S)-3-(tert-Butyloxycarbonyl)-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-yl Formic acid

[0074] Jones reagent (2M, 0.5 mL) was added to a solution of (4S)-1-fluoro-4-(hydroxymethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester (100.0 mg, 0.39 mmol) in acetone (1.0 mL) at room temperature. The reaction solution was stirred at room temperature for 2.0 hours, and extracted three times with 100 mL of water and 100 mL of ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The organic phases were concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA=1:2) to obtain the target compound (80.0 mg, yield 75.1%) as a colorless oil. The product detection data were: MS (ESI, pos.ion) m / z: 274.0 [M+H] + ;

[0075] Step 13: (4S)-4-(((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamoyl)- 1-Fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester

[0076] At room temperature, (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propionitrile (86.3 mg, 0.56 mmol) and HATU (251.0 mg, 0.66 mmol) were added to a solution of (2S)-3-(tert-butoxycarbonyl)-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (140.0 mg, 0.51 mmol) in DMF (3.0 mL), and the mixture was stirred at room temperature for 1.0 hour. After the reaction was completed, 100 mL of water and 120 mL of ethyl acetate were added for extraction twice, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA=1:1) to obtain the target compound as a white solid (165.0 mg, yield 79.1%). The product detection data were as follows: MS (ESI, pos.ion) m / z: 409.2 [M+H] + ;

[0077] Step 14: (2S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-fluoro-6,6-dihydro- Methyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

[0078] To a solution of (4S)-tert-butyl 4-(((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamoyl)-1-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (140.0 mg, 0.34 mmol) in DCM (1.0 mL) was added trifluoroacetic acid (1.0 mL) at room temperature, and the mixture was stirred at room temperature for 3.0 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain the target compound as a white solid (100.0 mg, yield 95.4%). The product detection data were as follows: MS (ESI, pos.ion) m / z: 309.1 [M+H] + ;

[0079] Step 15: ((2S)-1-((4S)-4-(((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)amino (1-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutane-2-yl) tert-Butyl)carbamate

[0080] To a solution of (2S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (200.0 mg, 0.65 mmol) in DMF (3.0 mL) was added (S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoic acid (165.0 mg, 0.71 mmol) and HATU ( 370.5 mg, 0.98 mmol), stirred at room temperature for 1.0 hour; after the reaction was completed, 100 mL of water and 100 mL of ethyl acetate were added for extraction twice, the organic phases were combined, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (PE: EA = 1: 1) to obtain a white solid target compound (210.0 mg, yield 62.0%); the product detection data is: MS (ESI, pos.ion) m / z: 522.3 [M+H] + ;

[0081] Step 16: (2S)-3-((S)-2-amino-3,3-dimethylbutanoyl)-N-((S)-1-cyano-2-((S)-2-oxo 2-(4-(2-pyrrolidin-3-yl)ethyl)-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

[0082] To a solution of tert-butyl ((2S)-1-((4S)-4-(((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamoyl)-1-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (200.0 mg, 0.38 mmol) in DCM (1.0 mL) was added trifluoroacetic acid (1.0 mL) at room temperature, and the mixture was stirred at room temperature for 2.0 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain the target compound as a yellow solid (150.0 mg, yield 93.7%). The product detection data were as follows: MS (ESI, pos.ion) m / z: 422.1 [M+H] + ;

[0083] Step 17: (2S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- Formamide

[0084] To a solution of (2S)-3-((S)-2-amino-3,3-dimethylbutanoyl)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-fluoro-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (70.0 mg, 0.17 mmol) in DCM (2.0 mL) was added trifluoroacetic anhydride (69.8 mg, 0.33 mmol) and triethylamine. Amine (51.5 mg, 0.51 mmol), the reaction solution was stirred at room temperature for 2.0 hours, 60 mL of water and 60 mL of ethyl acetate were added for extraction three times, the organic phases were combined, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (PE: EA = 1: 3) to obtain a white solid target compound (45.0 mg, yield 51.2%); the product detection data is: MS (ESI, pos.ion) m / z: 518.2 [M+H] + ; 1 HNMR (500MHz, CD 3 OD): δ4.48-4.51 (m, 1H), 4.32-4.28 (m, 1H), 4.24 (s, 1H), 3.69-3.27 (m, 2H), 3.20-3.15 (m, 2H), 2.20-2.13 (m, 3H), 2.10-1.95 (m, 3H), 0.92 (s, 9H), 0.87 (s, 6H). It can be seen that the structure of the compound prepared in this example is correct.

[0085] Example 2

[0086] This example is used to prepare compound (9), whose name is 1-acetyl-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamide)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, and its structural formula is shown below:

[0087]

[0088] The synthetic route of compound (9) described in this example is shown below, which specifically comprises the following steps:

[0089]

[0090] Step 1: N-(tert-Butyl)-3-methylbut-2-en-1-amine

[0091] Under ice bath, tert-butylamine (10.0 g, 136.0 mmol) was dissolved in THF (100 mL) solution, and then 1-bromo-3-methylbut-2-ene (10.2 g, 68.4 mmol) and triethylamine (13.7 g, 136.0 mmol) were added in sequence, and stirred at room temperature for 5 hours; water (150 mL) was added to the reaction solution, and extracted with ethyl acetate (300 mL × 2), the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting mixture was chromatographed on a silica gel column (PE: EA = 1: 2) to obtain the target compound as a white solid (1.5 g, 15.4%); the product detection data is: MS (ESI, pos.ion) m / z: 142.0 [M+H] + ;

[0092] Step 2: N-(tert-butyl)-N-(3-methylbutyl-2-en-1-yl)-3-oxobutanamide

[0093] At room temperature, N-(tert-butyl)-3-methylbut-2-en-1-amine (1.0 g, 7.0 mmol) was dissolved in toluene (10.0 mL) solution, and then tert-butyl 3-oxobutyrate (1.6 g, 10.5 mmol) and DMAP (0.17 g, 1.4 mmol) were added in sequence, and heated under reflux overnight; the reaction solution was cooled, and water (200 mL) and ethyl acetate (100 mL×3) were added to the reaction solution for extraction, and the organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting mixture was subjected to silica gel column chromatography (PE:EA=5:1) to obtain the target compound as a white solid (0.60 g, 38.0%); the product detection data were: MS (ESI, pos.ion) m / z: 226.0 [M+H] + ;

[0094] Step 3: 2-Bromo-N-(tert-butyl)-N-(3-methylbutyl-2-en-1-yl)-3-oxobutanamide

[0095] At room temperature, NBS (0.62 g, 3.5 mmol) was added to a solution of N-(tert-butyl)-N-(3-methylbutyl-2-en-1-yl)-3-oxobutanamide (0.60 g, 2.60 mmol) in ethyl acetate (10.0 mL), and the mixture was stirred at room temperature for 3.0 hours. Water was slowly added to quench the mixture, and the mixture was extracted with ethyl acetate (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA=5:1) to obtain the target compound as a white solid (0.31 g, 39.2%). The product detection data were: MS (ESI, pos.ion) m / z: 304.0 [M+H] + ;

[0096] Step 4: 3-Acetyl-4-(2-bromopropan-2-yl)-1-(tert-butyl)pyrrolidin-2-one

[0097] At -78°C, ytterbium trifluoromethanesulfonate (1.2 g, 1.97 mmol) and triethylboron solution (1.97 mL, 1.0 M) were added to a solution of 2-bromo-N-(tert-butyl)-N-(3-methylbutyl-2-en-1-yl)-3-oxobutanamide (0.30 g, 0.99 mmol) in tetrahydrofuran (5.0 mL) under an oxygen atmosphere, and the mixture was stirred at -78°C for 2 hours; the mixture was then slowly warmed to room temperature, water (100 mL) was added to the reaction solution to quench, the mixture was extracted with ethyl acetate (200 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA=3:1) to obtain the target compound (0.15 g, 50.0%) as a colorless oil; the product detection data were: MS (ESI, pos.ion) m / z: 304.0 [M+H] + ;

[0098] Step 5: 1-Acetyl-3-(tert-butyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-one

[0099] Under ice bath, sodium hydride (40.0 mg, 0.99 mmol) was added to a solution of 3-acetyl-4-(2-bromopropan-2-yl)-1-(tert-butyl)pyrrolidin-2-one (0.10 g, 0.33 mmol) in tetrahydrofuran (3.0 mL), and the reaction solution was stirred at room temperature for 0.5 hour; water (30 mL) was added to the reaction solution to quench, and the mixture was extracted with ethyl acetate (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA=5:1) to obtain the target compound (60.0 mg, 81.5%) as a colorless oil; the product detection data were: MS (ESI, pos.ion) m / z: 224.1 [M+H] + ;

[0100] Step 6: 1-Acetyl-3-(tert-butyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonitrile

[0101] At room temperature, zirconocene hydrochloride (228.5 mg, 0.90 mmol) was added to a solution of 1-acetyl-3-(tert-butyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-one (100 mg, 0.45 mmol) in tetrahydrofuran (5.0 mL), and the reaction solution was stirred at room temperature for 2 hours; then TMSCN (89.1 mg, 0.90 mmol) was added, and the mixture was stirred at room temperature for 5.0 hours. Water (30 mL) and ethyl acetate (100 mL×2) were added for extraction, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA=1:1) to obtain the target compound (45.0 mg, 56.1%) as a colorless oil; the product detection data were: MS (ESI, pos.ion) m / z: 235.1 [M+H] + ;

[0102] Step 7: 1-Acetyl-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonitrile

[0103] Trifluoroacetic acid (2.0 mL) was added to a solution of 1-acetyl-3-(tert-butyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonitrile (70.0 mg, 0.30 mmol) in DCM (1.0 mL) at room temperature. The mixture was stirred at room temperature for 5.0 hours under nitrogen protection. The reaction solution was concentrated under reduced pressure to obtain a crude product (45.0 mg, 84.0%), which was directly used in the next step without further purification. The product detection data was as follows: MS (ESI, pos.ion) m / z: 179.0 [M+H] + ;

[0104] Step 8: 1-Acetyl-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester

[0105] Under ice bath conditions, concentrated hydrochloric acid (2.0 mL) was added to a solution of 1-acetyl-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonitrile (110.0 mg, 0.62 mmol) in methanol (5.0 mL), and the reaction solution was heated under reflux with stirring for 5.0 hours; the reaction solution was cooled, water (50 mL) was added, and the pH was adjusted to neutral with 5% NaOH, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=10:1) to obtain the target compound (57.0 mg, 43.5%) as a colorless oil; the product detection data were: MS (ESI, pos.ion) m / z: 212.0 [M+H] + ;

[0106] Step 9: 1-acetyl-3-((S)-2-(tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-6,6-di Methyl-3-azabicyclo[3.1.0]hexane-2-carboxylate

[0107] At room temperature, (S)-2-((tert-butyloxycarbonyl)amino)-3,3-dimethylbutanoic acid (84.2 mg, 0.36 mmol) and HATU (250.8 mg, 0.66 mmol) were added to a solution of 1-acetyl-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester (70.0 mg, 0.33 mmol) in DMF (2.0 mL), and the mixture was stirred at room temperature for 2.0 hours. After the reaction was completed, 30 mL of water and 100 mL of ethyl acetate were added for extraction twice, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA=2:1) ​​to obtain the target compound as a white solid (110.0 mg, yield 78.6%). The product detection data were: MS (ESI, pos.ion) m / z: 425.2 [M+H] + ;

[0108] Step 10: 1-Acetyl-3-((S)-2-amino-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo [3.1.0] Methyl hexane-2-carboxylate

[0109] At room temperature, trifluoroacetic acid (1.0 mL) was added to a solution of 1-acetyl-3-((S)-2-(tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester (50.0 mg, 0.12 mmol) in DCM (1.0 mL), and the reaction solution was stirred at room temperature for 2.0 hours; the reaction solution was concentrated under reduced pressure to obtain the target compound as a light yellow solid (40.0 mg, yield 100.0%); the product detection data were: MS (ESI, pos.ion) m / z: 325.2 [M+H] + ;

[0110] Step 11: 1-Acetyl-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6- Methyl dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate

[0111] Trifluoroacetic anhydride (64.8 mg, 0.30 mmol) was added to a solution of 1-acetyl-3-((S)-2-amino-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester (50.0 mg, 0.15 mmol) in DCM (1.0 mL) at room temperature. The reaction solution was stirred at room temperature for 1.0 hour, and extracted three times with 40 mL of water and 200 mL of ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA=3:1) to obtain the target compound as a white solid (37.0 mg, yield 58.7%). The product detection data are: MS (ESI, pos.ion) m / z: 421.1 [M+H] + ;

[0112] Step 12: 1-Acetyl-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6- Methyl dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate

[0113] Lithium hydroxide (47.6 mg, 1.2 mmol) was added to a solution of 1-acetyl-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester (50.0 mg, 0.12 mmol) in methanol (2.0 mL) and water solution (2.0 mL) at room temperature. The reaction solution was stirred at room temperature for 5.0 hours, 40 mL of water was added, the pH was adjusted to neutral with 5% HCl, and the mixture was extracted twice with 100 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA=1:2) to obtain the target compound as a white solid (36.0 mg, yield 74.1%). The product detection data are as follows: MS (ESI, pos.ion) m / z: 407.1 [M+H] + ;

[0114] Step 13: 1-Acetyl-N-((1S)-1-cyano-2-(2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid amine

[0115] To a solution of methyl 1-acetyl-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (50.0 mg, 0.12 mmol) in DMF (2.0 mL) was added (2S)-2-amino-3-(2-oxopyrrolidin-3-yl)propionitrile (20.7 mg, 0.12 mmol) and HATU ( 93.4 mg, 0.25 mmol), stirred at room temperature for 1.0 hour; after the reaction was completed, 60 mL of water and 100 mL of ethyl acetate were added for extraction twice, the organic phases were combined, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (PE: EA = 1: 2) to obtain a white solid target compound (27.0 mg, yield 40.5%); the product detection data is: MS (ESI, pos.ion) m / z: 542.2 [M+H] + ; 1 HNMR (500 MHz, DMSO-d 6):δ9.20(s,1H),9.01(s,1H),7.1(s,1H)4.99-4.90(m,1H),4.42(s,1H),4.20(s,1H),3.93-3.90(m,1H),3.70-3.64(m,1H),3.18-3.02(m,2H),2.41-2.36(m,1H),2.17-2.08(m,2H),1.79-1.68(m,3H),1.58-1.51(m,1H),1.25-1.23(m,1H),0.92(s,10H),0.87(s,6H). It can be seen that the structure of the compound prepared in this embodiment is correct.

[0116] Example 3

[0117] This example is used to prepare compound (12), whose name is N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-1-(1,1-difluoroethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamide)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, and its structural formula is shown below:

[0118]

[0119] The specific preparation method of compound (12) described in this example comprises the following steps:

[0120] Step 1: 3-(tert-butyl)-1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-one

[0121] Under ice bath conditions, 1-acetyl-3-(tert-butyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-one (0.5 g, 2.2 mmol) was dissolved in DAST (5.0 mL) solution, and then a catalytic amount of ethanol was added, and the mixture was sealed and heated under reflux overnight; the reaction solution was cooled, and water (50 mL) and ethyl acetate (100 mL×2) were added to the reaction solution for extraction, the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the resulting mixture was subjected to silica gel column chromatography (PE:EA=5:1) to obtain a white solid target compound (0.26 g, 48.9%); the product detection data are: MS (ESI, pos.ion) m / z: 246.1 [M+H] + ;

[0122] Step 2: 3-(tert-butyl)-1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-ol

[0123] At room temperature, 3-(tert-butyl)-1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-one (100.0 mg, 0.45 mmol) was dissolved in THF (2.0 mL solution, and DIBAL (2M, 3.0 mL) was added and stirred at room temperature for 3.0 hours; water (100 mL) and ethyl acetate (100 mL×2) were added to the reaction solution for extraction, the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography (PE:EA=1:1) to obtain the target compound as a white solid (75.0 mg, 67.2%); the product detection data were: MS (ESI, pos.ion) m / z: 248.1 [M+H] + ;

[0124] Step 3: 3-(tert-butyl)-1-(1,1-difluoroethyl)-2-methoxy-6,6-dimethyl-3-azabicyclo [3.1.0] Hexane

[0125] At room temperature, p-toluenesulfonic acid (5 mg) and methanol (40 mg, 0.26 mmol) were added to a toluene (2.0 mL) solution of 3-(tert-butyl)-1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-ol (30 mg, 0.13 mmol), and the reaction solution was heated at 70°C for 5 hours. The reaction solution was cooled, slowly quenched with water, extracted with ethyl acetate (50 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA=1:1) to obtain the target compound as a white solid (20 mg, 64.0%); the product detection data are: MS (ESI, pos.ion) m / z: 262.1 [M+H] + ;

[0126] Step 4: 3-(tert-butyl)-1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-yl Nitrile

[0127] At room temperature, TMSCN (0.19 g, 2.87 mmol) was added to a solution of 3-(tert-butyl)-1-(1,1-difluoroethyl)-2-methoxy-6,6-dimethyl-3-azabicyclo[3.1.0]hexane (0.15 g, 0.57 mmol) in tetrahydrofuran (3.0 mL), and the reaction solution was heated under reflux with stirring for 4 hours; the reaction solution was cooled, water (50 mL) was added to the reaction solution to quench, and the solution was extracted with ethyl acetate (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA=1:1) to obtain the target compound (0.11 g, 75.3%) as a colorless oil; the product detection data were: MS (ESI, pos.ion) m / z: 257.1 [M+H]+ ;

[0128] Step 5: 1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonitrile

[0129] At room temperature, 3-(tert-butyl)-1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonitrile (100.0 mg, 0.39 mmol) was dissolved in trifluoroacetic acid (2.0 mL) and stirred at room temperature for 5.0 hours under nitrogen protection; the reaction solution was concentrated under reduced pressure to obtain a crude product which was directly used in the next reaction (61.0 mg, 78.2%); the product detection data was: MS (ESI, pos.ion) m / z: 200.0 [M+H] + ;

[0130] Step 6: 1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester

[0131] Under ice bath conditions, concentrated hydrochloric acid (0.4 mL) was added to a solution of 1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonitrile (110.0 mg, 0.55 mmol) in methanol (3.0 mL), and the reaction solution was heated under reflux with stirring for 3.0 hours; the reaction solution was cooled, water (50 mL) was added, and the pH was adjusted to neutral with 5% NaOH solution, extracted with ethyl acetate (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM: MeOH = 30: 1) to obtain the target compound (49.0 mg, 38.2%) as a colorless oil; the product detection data are: MS (ESI, pos.ion) m / z: 234.1 [M+H] + ;

[0132] Step 7: 3-((S)-2-(tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-1-(1,1-difluoroethyl 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester

[0133] At room temperature, (S)-2-((tert-butyloxycarbonyl)amino)-3,3-dimethylbutanoic acid (99.1 mg, 0.43 mmol) and HATU (326.8 mg, 0.86 mmol) were added to a solution of 1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester (100 mg, 0.43 mmol) in DMF (3.0 mL), and the mixture was stirred at room temperature for 1.0 hour. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with ethyl acetate (60 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA=1:1) to obtain the target compound as a white solid (140.0 mg, yield 73.0%). The product detection data were as follows: MS (ESI, pos.ion) m / z: 447.2 [M+H]+ ;

[0134] Step 8: 3-((S)-2-amino-3,3-dimethylbutanoyl)-1-(1,1-difluoroethyl)-6,6-dimethyl-3- Methyl azabicyclo[3.1.0]hexane-2-carboxylate

[0135] Trifluoroacetic acid (1.0 mL) was added to a solution of methyl 3-(2-(tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (100.0 mg, 0.22 mmol) in DCM (1.0 mL) at room temperature, and the reaction solution was stirred at room temperature for 2.0 hours; the reaction solution was concentrated under reduced pressure to obtain the target compound as a light yellow solid (76.0 mg, yield 99.8%); the product detection data were: MS (ESI, pos.ion) m / z: 347.2 [M+H] + ;

[0136] Step 9: 1-(1,1-difluoroethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester

[0137] Trifluoroacetic anhydride (136.5 mg, 0.66 mmol) and triethylamine (66.6 mg, 0.66 mmol) were added to a solution of methyl 3-((S)-2-amino-3,3-dimethylbutanoyl)-1-(1,1-difluoroethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (75.0 mg, 0.22 mmol) in DCM (3.0 mL) at room temperature. The reaction solution was stirred at room temperature for 2.0 hours, and extracted twice with 100 mL of water and 200 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA=1:1) to obtain the target compound as a white solid (64.0 mg, yield 65.8%). The product detection data are as follows: MS (ESI, pos.ion) m / z: 443.1 [M+H] + ;

[0138] Step 10: 1-(1,1-difluoroethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester

[0139] At room temperature, lithium hydroxide (47.5 mg, 1.1 mmol) was added to a solution of 1-(1,1-difluoroethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester (50.0 mg, 0.11 mmol) in methanol (1.0 mL) and aqueous solution (1.0 mL). The reaction solution was stirred at room temperature for 2.0 hours, 40 mL of water was added, and the pH was adjusted to neutral with 5% HCl. The mixture was extracted three times with 50 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA=1:3) to obtain the target compound as a white solid (41.0 mg, yield 87.0%). The product detection data are as follows: MS (ESI, pos.ion) m / z: 429.1 [M+H] + ;

[0140] Step 11: N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-1-(1,1-difluoroethyl)- 3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane Alkane-2-carboxamide

[0141] To a solution of (1S,5R)-1-(1,1-difluoroethyl)-3-(3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (50.0 mg, 0.12 mmol) in DMF (3.0 mL) were added (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propionitrile (21.9 mg, 0.13 mmol) and HATU (66.1 mg, 0.17 mmol) at room temperature, and the mixture was stirred at room temperature for 3.0 hours. After the reaction was completed, 50 mL of water and 100 mL of ethyl acetate were added for extraction twice, the organic phases were combined and dried over anhydrous sodium sulfate, the organic phase was concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA=1:2) to obtain the target compound as a white solid (34.5 mg, yield 51.2%). Product detection data: MS (ESI, pos.ion) m / z: 564.2 [M+H] + ; 1 H NMR (500 MHz, CDCl 3):δ9.95(s,1H),9.43(s,1H),8.12(s,1H)4.90-4.89(m,1H),4.54-4.52(m,1H),4.33-4.23(m,1H),4.07-3.95(m,1H),3.80-3.76(m,1H),3.38-3.24(m,2H),2.46-2.30(m,2H),2.20-2.18(m,1H),1.89-1.80(m,3H),1.55-1.51(m,1H),1.35-1.30(m,2H),0.96(s,9H),0.90(s,6H). It can be seen that the structure of the product prepared in this embodiment is correct.

[0142] Experimental example

[0143] 1. In vitro enzyme activity test:

[0144] This method refers to and uses the Novel Coronavirus (2019-nCoV) Mpro / 3CLpro Inhibitor Screening Kit (Cat. No.: PS0312M) produced by Bio-Tech for inhibitor activity screening. This product uses the principle of fluorescence resonance energy transfer (FRET).

[0145] Experimental method: Take a black-bottom 96-well plate, add the recombinant main protease (3C-like protease) enzyme solution or the test substance or solvent to each well in accordance with the instructions of the kit reaction system, incubate at 37°C for 15 minutes, then add the substrate and incubate at 37°C for 1 hour. Among them, 8 concentrations are set for each test substance, 2 replicates are set for each concentration, and a positive control group, a negative control group (OSU), and a blank control group (NSU) with only the substrate added are set at the same time. The specific sample addition information of each reagent is detailed in Table 1 below.

[0146] Table 1 Inhibition rate determination test reagent addition amount

[0147]

[0148] Kit related information: (Name: Novel Coronavirus Mpro / 3CLpro Inhibitor Screening Kit; Reagent source: Bio-Tech; Specification: 100T; Storage condition: -20℃.)

[0149] The ELISA instrument detected the signal at a wavelength of EX / EM = 325 / 393 nm within 1 min and 1 h after adding the substrate, and the inhibition rate was calculated according to the following formula. The half-maximal inhibitory concentration (IC) was calculated using the GraphPad Prism fitting curve. 50 ).

[0150] Inhibition rate % = 1-[(OSU FI值 - Drug administration group FI值 ) / (OSU FI值 -NSU FI值 )]×100%.

[0151] According to the above method steps, the biological data of the compounds described in Examples 1-3 of the present invention and the reference compound (PF-07321332) were tested respectively, wherein the reference compound PF-07321332 was synthesized according to the method disclosed in WO2021 / 250648, and its structural formula is as follows:

[0152]

[0153] The above biological data test results are shown in Table 2 below.

[0154] Table 2 In vitro enzymatic activity data

[0155] Compound No. <![CDATA[IC 50 (nM)]]> Example 1 Compound 12.7 Example 2 Compound 24.5 Example 3 Compound 20.3 Reference compound PF-07321332 26.2

[0156] From the above test results, it can be seen that the compound synthesized in Example 1 of the present invention has a significant and good inhibitory effect on hexokinases, while the compounds described in Examples 2-3 and the reference compound PF-07321332 have similar technical effects, and both have good inhibitory effects.

[0157] 2. Pharmacokinetic evaluation of the compound of the present invention after intravenous injection or oral administration

[0158] This experimental example evaluated the pharmacokinetic study of the compounds in Examples 1-3 and the control compound in SD rats, dogs or monkeys.

[0159] The compounds of Examples 1-3 of the present invention were administered in a physiological saline solution of 5% DMSO + 5% Kolliphor HS 15 + 90% Saline.

[0160] For intravenous (iv) dosing, animals were dosed with 1 mg / kg and blood (0.3 mL) was collected at time points of 0.083, 0.25, 0.5, 1.0, 2.0, 5.0, 7.0 and 24 hours and centrifuged at 3000 or 4000 rpm for 10 minutes.

[0161] For oral (po) dosing, animals were dosed with 5 mg / kg and blood (0.3 mL) was collected at time points of 0.25, 0.5, 1.0, 2.0, 5.0, 7.0 and 24 hours and centrifuged at 3000 or 4000 rpm for 10 minutes.

[0162] The plasma solution was collected and stored at -20°C or -70°C until the above-mentioned LC / MS / MS analysis. The results are shown in Table 3 below.

[0163] Table 3 Pharmacokinetic data of some compounds in SD rats

[0164]

[0165]

[0166] As can be seen from the data in Table 3, the synthetic compound of the present invention has a larger exposure in animals, good absorption, good pharmacokinetic properties, and better performance than the control compound.

[0167] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A peptidomimetic compound and a pharmaceutically acceptable salt, tautomer or isotope-labeled substance thereof, characterized in that: The compound has a structure shown in the following formula (I): in, The R 1 and R 2 is -CH3; The R 3 and R 4 Independently selected from hydrogen, -F, -COCH3, -CF2CH3.

2. The peptidomimetic compound and its pharmaceutically acceptable salt, tautomer or isotope-labeled substance according to claim 1, characterized in that: The R 3 is selected from hydrogen, -F; The R 4 Selected from hydrogen, -F, -COCH3, -CF2CH3.

3. The peptidomimetic compound and pharmaceutically acceptable salt, tautomer or isotope-labeled substance thereof according to claim 1 or 2, characterized in that: The compound is selected from one of the following compounds:

4. A method for preparing the peptidomimetic compound according to any one of claims 1 to 3 and its pharmaceutically acceptable salt, tautomer or isotope-labeled substance, characterized in that: The method comprises the steps of introducing aliphatic, heterocyclic or aromatic substituents of selected structures into the rings in the compound molecules represented by formula (I).

5. Use of the peptidomimetic compound according to any one of claims 1 to 3 and its pharmaceutically acceptable salt, tautomer or isotope label for preparing a 2019-nCoV main protease inhibitor.

6. Use of the peptidomimetic compound according to any one of claims 1 to 3 and its pharmaceutically acceptable salts, tautomers or isotope labels for the preparation of drugs for treating novel coronavirus pneumonia caused by 2019 novel coronavirus infection.

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