Ketoamide derivatives and their use in pharmaceuticals

Abstract

The present invention provides ketoamide derivatives and their use in pharmaceuticals, which belong to the technical field of organic synthetic drugs. Specifically, the present invention provides a compound as shown in Formula I, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof. The compound is capable of inhibiting the M domain of SARS-CoV-2. pro It can effectively inhibit the activity of SARS-CoV-2 M pro The compounds of the present invention can be used to manufacture and administer SARS-CoV-2 M inhibitors, which block the replication and transcription of the SARS-CoV-2 virus in the patient's body. Pro It can be used to manufacture inhibitors, anti-SARS-CoV-2 drugs, and drugs for preventing and / or treating novel coronavirus pneumonia, and its future prospects are very bright. TIFF2025507472000495.tif32170

Description

[Technical field]

[0001] The present invention belongs to the technical field of organic synthetic drugs, specifically, SARS-CoV-2 M pro The present invention relates to ketoamide derivatives having inhibitory activity against ketoacillin-resistant Staphylococcus aureus (Cas9), as well as methods for preparing and using the same. [Background technology]

[0002] In 2019, coronavirus pneumonia (COVID-19, also known as novel coronavirus pneumonia) was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as novel coronavirus), and as of February 2022, it has infected more than 400 million people and killed nearly 6 million people. At present, only two oral anti-SARS-CoV-2 drugs are commercially available. Molnupiravir, which targets RdRp and was developed by Merck & Co., Inc., is a nucleoside analogue that may have side effects and genotoxicity due to mutations, and its safety needs to be proven by more data. The other is PF-07321332, an oral drug developed by Pfizer Inc. that targets the main protease of the virus, but it is easily metabolized by cytochrome CYP enzymes in the human body and needs to be used in combination with ritonavir to achieve its antiviral effect. Therefore, there is currently an urgent need to develop safe, effective, and orally available anti-SARS-CoV-2 drugs.

[0003] The coronavirus genomic RNA is approximately 30 knt in length, has a 5' cap structure and a 3' poly-a tail, and contains at least six open reading frames (ORFs). The first ORF (ORF1a / b), which occupies approximately two-thirds of the genome length, directly translates two polyproteins, pp1a and pp1ab, and there is an a-1 frameshift between ORF1a and ORF1b. These polyproteins are involved in the translation of the main protease (abbreviated M pro , 3C-like protease (3CL pro) and one or two papain-like proteases (PLPs) to convert them into 16 nonstructural proteins. These nonstructural proteins are involved in the production of the subgenomic RNA and complete the process of viral replication and entry by encoding the four major structural proteins (envelope (E), membrane (M), spike (S) and nucleocapsid (N) proteins) and other accessory proteins.

[0004] M pro The PP1a and PP1ab polyproteins are hydrolytically cleaved into functional proteins, which is a critical step in the viral replication process. Enzymes essential for viral replication, such as RdRp and nsp13, cannot be fully functional to complete replication without prior proteolytic release. Thus, the viral M pro Inhibition of this can block the production of infectious viral particles, thereby alleviating the symptoms of the disease.

[0005] M pro is conservative within coronaviruses and M pro The substrates for M share several common features: the amino acids from N- to C-terminus are numbered pairwise (-P4-P3-P2-P1↓P1'-P2'-P3') and the cleavage point is between P1 and P1'. pro has a unique substrate preference for glutamine at the P1 site (Leu-Gln↓(Ser,Ala,Gly)) that is not present in host proteases, which may explain the pro Thus, the absolute dependence of the virus on the correct function of this protease, combined with the lack of a homologous human protease, indicates that high selectivity can be achieved by targeting M pro are ideal antiviral targets.

[0006] Therefore, the M of the SARS-CoV-2 virus proThere is an urgent need to develop oral drugs that can effectively inhibit enzyme activity. Summary of the Invention

[0007] It is an object of the present invention to provide novel ketoamide derivatives and their use in medicine.

[0008] The present invention provides a compound according to formula I, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof. TIFF2025507472000002.tif32170 (wherein Q is a nitrogen-containing heteroaryl ring or a nitrogen-containing fused heteroaryl ring; R 1 is hydrogen, unsubstituted or halogenated C 1~8 Alkyl groups, unsubstituted or halogenated C 1~8 Alkoxy groups, halogens, 5- to 6-membered aryl groups, 5- to 6-membered heteroaryl groups, saturated 3- to 8-membered heterocyclyl groups, saturated 3- to 8-membered cycloalkyl groups, NHCOR 1a Selected from R 1a is C 1~8 an unsubstituted or halogenated saturated 3- to 8-membered heterocyclyl group, an unsubstituted or halogenated saturated 3- to 8-membered cycloalkyl group, an unsubstituted or halogenated 5- to 6-membered aryl group, and an unsubstituted or halogenated 5- to 6-membered heteroaryl group; R 2 is hydrogen, C 1~8 Alkyl group, C 1~8 is selected from an alkoxy group, a halogen atom, a 5- to 6-membered aryl group, a 5- to 6-membered heteroaryl group, a saturated 3- to 8-membered heterocyclyl group, and a saturated 3- to 8-membered cycloalkyl group; R 3 is hydrogen, C 1~8 Alkyl group, C 1~8 Alkoxy groups, halogens, 5- to 6-membered aryl groups, 5- to 6-membered heteroaryl groups, saturated 3- to 8-membered heterocyclyl groups, saturated 3- to 8-membered cycloalkyl groups, CHR 3a Selected from R 3ais selected from an unsubstituted or halogenated saturated 3- to 8-membered heterocyclyl group, an unsubstituted or halogenated saturated 3- to 8-membered cycloalkyl group, an unsubstituted or halogenated 5- to 6-membered aryl group, an unsubstituted or halogenated 5- to 6-membered heteroaryl group, and an unsubstituted or halogenated fused ring aryl group; R 4 is hydrogen or one of the following substituted or unsubstituted groups: 1~8 Alkyl group, C 1~8 is selected from an alkoxy group, a saturated 3- to 8-membered cycloalkyl group, a saturated 3- to 8-membered heterocyclyl group, a 5- to 6-membered aryl group, a 5- to 6-membered heteroaryl group, a bridged ring group, a fused ring aryl group, a fused heteroaryl group, or a saturated 5- to 6-membered heterocyclyl group or a 5- to 6-membered aryl group; The substituent of the substituted group is R 4a , R 4b , R 4c , R 4d and each independently Hydrogen, halogen, phenyl group, cyano group, hydroxy group, ester group, trimethylsilyl group, -(CH2) m -SO2R', -COOR'', or Any one of the following groups, which is unsubstituted or substituted with any one or more of halogen, cyano, haloalkyl, and haloalkoxy groups: 1~8 Alkyl group, C 1~8 Alkoxy group, benzyl group, pyridyl group or saturated 3-6 membered cycloalkyl group wherein R′ and R″ are each independently selected from C 1~8 Alkyl group wherein m is an integer of 0 to 3; Or, the above R 4a , R 4b are bonded to form a halogenated or unsubstituted saturated 3- to 6-membered carbocyclic ring or a halogenated or unsubstituted carboheterocyclic ring, L 1 is none, substituted or unsubstituted -(CH2) n - or substituted or unsubstituted -O-(CH2) n-, -O-, -NHCO-, -NHSO2-, -CONH-, -NHCOO-, -NHCONH-, -NH-, n is an integer of 1 to 3, and the substituent of the substitution is C 1~3 Alkyl group, C 1~3 an alkoxy group or a phenyl group, X is None, CR 5 R 6 , N.R. 5 R 6 , O or SO2; R 5 , R 6 are each independently Hydrogen, halogen, R 5a , R 5b , R 5c is substituted or unsubstituted C 1~8 alkyl group or R 5 , R 6 Combined, R 6a , R 6b forms a substituted or unsubstituted saturated 3- to 8-membered cycloalkyl group or a substituted or unsubstituted saturated 3- to 8-membered heterocyclyl group, R 5a , R 5b , R 5c are each independently hydrogen, an alkynyl group, a hydroxyl group, -CONH2, -N(CH3)2, a halogen, or C 1~8 an alkoxy group, a 5- to 6-membered aryl group, a 5- to 6-membered heteroaryl group, or R 5a , R 5b , R 5c any two of these are bonded to form a saturated 3- to 8-membered cycloalkyl group or a saturated 3- to 8-membered heterocyclyl group; R 6a , R 6b each independently represents a hydrogen atom, an alkenyl group, a halogen atom, a halogen-substituted or unsubstituted C 1~8 alkyl group or R 6a , R 6b are bonded to form a 5- to 6-membered aryl group, L 2 No, nothing. TIFF2025507472000003.tif45170TIFF2025507472000004.tif48170orRa , R b is substituted or unsubstituted Selected from TIFF2025507472000005.tif14170, where R0 is hydrogen, C 1~3 or is an alkyl group, or is bonded to R5 to form a substituted or unsubstituted bridged ring, a substituted or unsubstituted saturated 3- to 6-membered ring, or a substituted or unsubstituted saturated 3- to 6-membered benzene ring structure, and the substituent of the substituted structure is C 1~3 is an alkyl group or a halogen; R a , R b are each independently hydrogen, a cyano group, or C 1~5 alkyl group, 3- to 6-membered cycloalkyl group, or R a , R b are bonded to form a saturated 3- to 6-membered carbon ring, Y is O or S; t is any integer from 0 to 3, q ​​is any integer from 0 to 4, r is any integer from 0 to 3, s is any integer from 0 to 3, k is any integer from 0 to 3, and u is any integer from 0 to 3.

[0009] Furthermore, the above Q is a 5- to 6-membered nitrogen-containing heteroaryl ring, a 5- or 6-membered nitrogen-containing heteroaryl ring, or a 6-membered nitrogen-containing fused heteroaryl ring.

[0010] Furthermore, the above Q TIFF2025507472000006.tif38170TIFF2025507472000007.tif65170or The file is TIFF2025507472000008.tif13170.

[0011] Furthermore, the above Q TIFF2025507472000009.tif13170 or The file is TIFF2025507472000010.tif13170.

[0012] Furthermore, the above R 1is hydrogen, halogen, halogenated or unsubstituted C 1~8 Alkyl group or unsubstituted C 1~8 It is an alkoxy group.

[0013] Furthermore, the above R 1 is hydrogen, halogen, halogenated or unsubstituted C 1~3 Alkyl group or unsubstituted C 1~3 It is an alkoxy group.

[0014] Furthermore, the above R 1 is hydrogen, fluorine, chlorine, a methyl group, a methoxy group, or -CF3.

[0015] Furthermore, the above R 2 is hydrogen or C 1~8 It is an alkyl group.

[0016] Furthermore, the above R 2 is hydrogen or a methyl group.

[0017] Furthermore, the above R 3 is hydrogen or CH2R 3a and R 3a is unsubstituted or halogenated C 1~4 It is selected from an alkyl group, an unsubstituted or halogenated 5- to 6-membered cycloalkyl group, an unsubstituted or halogenated 5- to 6-membered aryl group, an unsubstituted or halogenated 5- to 6-membered heteroaryl group, or an unsubstituted or halogenated fused ring aryl group.

[0018] Furthermore, the above R 3 is hydrogen or CH2R 3a and R 3a is unsubstituted or halogenated C 1~2 It is selected from an alkyl group, an unsubstituted or halogenated 5- to 6-membered cycloalkyl group, an unsubstituted or halogenated 5- to 6-membered aryl group, an unsubstituted or halogenated 5- to 6-membered heteroaryl group, or an unsubstituted or halogenated naphthyl group.

[0019] Furthermore, the above R 3 is hydrogen or CH2R3a and R 3a is selected from a phenyl group, an ethyl group, a cyclohexyl group, a furyl group, a naphthyl group or an F-substituted phenyl group.

[0020] Furthermore, the above R 4 is any one of the following substituted or unsubstituted groups: 1~4 Alkyl group, C 1~4 is selected from an alkoxy group, a saturated 3- to 7-membered cycloalkyl group, a saturated 4- to 6-membered heterocyclyl group, a 5- to 6-membered aryl group, a 5- to 6-membered heteroaryl group, a bridged ring group, a naphthyl group, a 5- to 6-membered aromatic heterocyclyl group phenyl group, or a saturated 5- to 6-membered heterocyclyl group phenyl group; The substituent of the substituted group is R 4a , R 4b , R 4c , R 4d and each independently Hydrogen, halogen, phenyl group, cyano group, hydroxy group, ester group, trimethylsilyl group, -(CH2) m -SO2R', -COOR'', Any one of the following groups, substituted or unsubstituted with halogen: 1~3 Alkyl group, C 1~3 Alkoxy group or saturated 3-4 membered cycloalkyl group wherein R′ and R″ are each independently selected from C 1~4 alkyl group, m is an integer of 0 to 2; Or, the above R 4a , R 4b , R 4c , R 4d Any two of these may be combined to form a halogenated or unsubstituted saturated 3- to 6-membered carbocyclic or heterocyclic ring.

[0021] Furthermore, the above R 4 is any one of the following substituted or unsubstituted groups: 1~4 Alkyl group, C 1~4is selected from an alkoxy group, a saturated 3- to 7-membered cycloalkyl group, a saturated 4- to 6-membered heterocyclyl group, a 5- to 6-membered aryl group, a 5- to 6-membered azaaryl group, a bridged ring group, a naphthyl group, a benzofuryl group, a benzopyridyl group, or a saturated 5- to 6-membered oxygen heterocyclic group phenyl group; The substituent of the substituted group is R 4a , R 4b , R 4c , R 4d and each independently Hydrogen, halogen, phenyl group, cyano group, hydroxy group, ester group, trimethylsilyl group, -(CH2) m -SO2R', -COOR'', Any one of the following groups, substituted or unsubstituted with halogen: 1~3 Alkyl group, C 1~3 Alkoxy group or saturated 3-membered cycloalkyl group wherein R′ and R″ are each independently selected from C 1~4 alkyl group, m is 0 or 1; Or, the above R 4a , R 4b , R 4c , R 4d Any two of these may combine to form a halogenated or unsubstituted saturated 3- to 6-membered carbocyclic or oxygen heterocyclic ring.

[0022] Furthermore, the above R 4 is a fluorine-substituted saturated 4- to 6-membered cycloalkyl group.

[0023] Furthermore, the above R 4 is a saturated 4- to 6-membered cycloalkyl group substituted with two fluorines.

[0024] Furthermore, the above R 4 but TIFF2025507472000011.tif14170, where z is an integer between 1 and 3.

[0025] Furthermore, the above R 4 is selected from any one of the following substituted or unsubstituted groups: -CH3, -OCH3, TIFF2025507472000012.tif81170

[0026] Furthermore, the above R 4 is selected from any one of the following substituted or unsubstituted groups: -CH3, CF3, -OCH3, -OCF3, -OC(CH3)3, TIFF2025507472000013.tif72170TIFF2025507472000014.tif105170TIFF2025507472000015.tif121170TIFF2025507472000016.tif64170

[0027] Furthermore, the above L 1 is substituted or unsubstituted -(CH2) n -, n is an integer of 1 to 3, and the substituted substituent is C 1~3 It is an alkyl group or a phenyl group.

[0028] Furthermore, the above L 1 is substituted or unsubstituted -(CH2) n -, and the substituent of the substitution is a methyl group or a phenyl group.

[0029] Furthermore, there is no X, CR 5 R 6 , or NR 5 R 6 and R 5 , R 6 are each independently Hydrogen, Fluoro, R 5a , R 5b , R 5c is substituted or unsubstituted C 1~5 alkyl group or R 5 , R 6 Combined, R 6a , R 6b forms a substituted or unsubstituted saturated 3- to 6-membered cycloalkyl group or a saturated 4-membered oxygen heterocyclic group, R 5a , R 5b , R 5care each independently selected from hydrogen, an ethynyl group, a hydroxy group, -CONH2, -CONHCH3, -N(CH3)2, fluorine, a methoxy group, a phenyl group, a methylsulfonyl group, an amino group, a carboxy group, a carbomethoxy group, and an azaphenyl group, or R 5a , R 5b , R 5c any two of these combine to form a saturated 3- to 6-membered cycloalkyl group or a saturated 5- to 6-membered oxygen heterocyclic group; R 6a , R 6b are each independently selected from hydrogen, a vinyl group, fluorine, and a fluorine-substituted methyl group, or R 6a , R 6b are bonded to form a phenyl group, L 2 None, TIFF2025507472000017.tif98170 or R a , R b is substituted or unsubstituted TIFF2025507472000018.tif14170, where R0 is hydrogen, C 1~3 It is an alkyl group or, when combined with R5, has the following structure, which may be substituted or unsubstituted: TIFF2025507472000019.tif38170 is formed, and the substituent of the substituted structure is a methyl group or a fluorine group, R a , R b are each independently selected from hydrogen, a cyano group, a butyl group, and a 6-membered cycloalkyl group; or R a , R b are linked to form a three-membered carbon ring, Y is O or S; t is 0 or 1, q is an integer of 0 to 4, r is 0 or 1, s is 0 or 1, k is 0 or 1, and u is 0 or 1.

[0030] Furthermore, the above X is CR 5 R 6 And L 2 but TIFF2025507472000020.tif14170, where q is 0 or 1.

[0031] Furthermore, the above X is none, TIFF2025507472000021.tif65170TIFF2025507472000022.tif81170or TIFF2025507472000023.tif21170, L 2 But, none. TIFF2025507472000024.tif96170TIFF2025507472000025.tif96170or TIFF2025507472000026.tif14170, Or L 2 is bonded to X to form one of the following structures: Forming TIFF2025507472000027.tif70170TIFF2025507472000028.tif46170.

[0032] Furthermore, if the above X TIFF2025507472000029.tif12170 or TIFF2025507472000030.tif21170, L 2 but TIFF2025507472000031.tif14170 or The file is TIFF2025507472000032.tif14170.

[0033] Furthermore, the structure of the compound is as shown in Formula II or Formula III. TIFF2025507472000033.tif39170TIFF2025507472000034.tif32170

[0034] Furthermore, the structure of the above compound is as shown in formula II-A. TIFF2025507472000035.tif39170

[0035] Furthermore, the structure of the compound is as shown in formula II-Aa, formula II-Ab, formula II-Ac, or formula II-Ad. TIFF2025507472000036.tif39170TIFF2025507472000037.tif39170TIFF2025507472000038.tif39170TIFF2025507472000039.tif39170

[0036] Furthermore, the structure of the above compound is as shown in formula II-Aa-1. TIFF2025507472000040.tif39170

[0037] Furthermore, the compound has any one of the following structures: TIFF2025507472000041.tif250170TIFF2025507472000042.tif242170TIFF2025507472000043.tif246170TIFF2025507472000044.t if233170TIFF2025507472000045.tif255170TIFF2025507472000046.tif232170TIFF2025507472000047.tif230170TIFF20255074720 00048.tif241170TIFF2025507472000049.tif243170TIFF2025507472000050.tif243170TIFF2025507472000051.tif214170TIFF202 5507472000052.tif250170TIFF2025507472000053.tif248170TIFF2025507472000054.tif232170TIFF2025507472000055.tif130170

[0038] Furthermore, the structure of the above compound is as shown in formula II-Aa-2. TIFF2025507472000056.tif39170M is fluorine, chlorine, a methyl group, a methoxy group or -CF3.

[0039] Furthermore, the compound has any one of the following structures: TIFF2025507472000057.tif55170TIFF2025507472000058.tif56170TIFF2025507472 000059.tif70170TIFF2025507472000060.tif55170TIFF2025507472000061.tif47170

[0040] Furthermore, the structure of the above compound is as shown in formula II-Ab-1. TIFF2025507472000062.tif39170

[0041] Additionally, the compound has the following structure: TIFF2025507472000063.tif28170

[0042] Furthermore, the structure of the above compound is as shown in formula II-Ac-1. TIFF2025507472000064.tif39170

[0043] Additionally, the compound has the following structure: TIFF2025507472000065.tif28170

[0044] Furthermore, the structure of the above compound is as shown in formula II-Ad-1. TIFF2025507472000066.tif39170

[0045] Additionally, the compound has the following structure: TIFF2025507472000067.tif28170

[0046] Furthermore, the structure of the compound is as shown in formula II-Ae, II-Af, II-Ag, II-Ah, II-Ai, II-A-i1, II-A-i2, II-A-i3, II-A-i4, or II-A-i5. TIFF2025507472000068.tif39170TIFF2025507472000069.tif39170TIFF2025507472000070. tif39170TIFF2025507472000071.tif39170TIFF2025507472000072.tif39170TIFF202550747 2000073.tif39170TIFF2025507472000074.tif39170TIFF2025507472000075.tif39170TIFF2025507472000076.tif39170TIFF2025507472000077.tif39170Here, U, V, U', and V' are independently hydrogen, C 1~3 It is selected from an alkyl group or a halogen, preferably hydrogen, a methyl group or chlorine; W is O or S.

[0047] Furthermore, the structure of the above compound is as shown in formula II-Ae-1. TIFF2025507472000078.tif39170

[0048] Furthermore, the structure of the compound is as shown in any one of the following: TIFF2025507472000079.tif24170

[0049] Furthermore, the structure of the above compound is as shown in formula II-Ae-2. TIFF2025507472000080.tif39170

[0050] Furthermore, the structure of the above compound is as shown below. TIFF2025507472000081.tif22170

[0051] Furthermore, the structure of the compound is as shown in any one of the following: TIFF2025507472000082.tif71170TIFF2025507472000083.tif82170TIFF2025507472000084.tif64170

[0052] Furthermore, the structure of the above compound is as shown in formula II-Ah-1. TIFF2025507472000085.tif39170

[0053] Furthermore, the structure of the compound is as shown in any one of the following: TIFF2025507472000086.tif80170TIFF2025507472000087.tif63170TIFF2025507472 000088.tif73170TIFF2025507472000089.tif64170TIFF2025507472000090.tif37170

[0054] Furthermore, the structure of the above compound is as shown in formula II-Ah-2. TIFF2025507472000091.tif39170

[0055] Furthermore, the structure of the compound is as shown in any one of the following: TIFF2025507472000092.tif224170TIFF2025507472000093.tif242170TIFF2025507472000094.tif224170 TIFF2025507472000095.tif230170TIFF2025507472000096.tif231170TIFF2025507472000097.tif238170

[0056] Furthermore, the structure of the above compound is as shown in formula II-A-I'-2. TIFF2025507472000098.tif39170

[0057] Furthermore, the compound has any one of the following structures: TIFF2025507472000099.tif38170

[0058] Further, the compound has the structure shown in formula II-Aj. TIFF2025507472000100.tif42170 where z is an integer between 1 and 3, q ​​is an integer between 0 and 4, and R 5 , R 6 At least one of the is not hydrogen, Preferably, z is 1, 2 or 3 and q is 0 or 1.

[0059] Furthermore, the compound has the structure shown in formula II-Aj-1. TIFF2025507472000101.tif40170

[0060] Furthermore, the compound has any one of the following structures: TIFF2025507472000102.tif63170TIFF2025507472000103.tif63170TIFF2025507472 000104.tif63170TIFF2025507472000105.tif62170TIFF2025507472000106.tif70170

[0061] Furthermore, the structure of the compound is as shown in Formula II-B, Formula II-C, or Formula II-D. TIFF2025507472000107.tif39170TIFF2025507472000108.tif41170TIFF2025507472000109.tif39170

[0062] Furthermore, the structure of the above compound is as shown in formula II-Ba. TIFF2025507472000110.tif39170

[0063] Furthermore, the structure of the above compound is as shown in formula II-Ba-1. TIFF2025507472000111.tif39170

[0064] Additionally, the compound has the following structure: TIFF2025507472000112.tif25170

[0065] Furthermore, the structure of the above compound is as shown in formula II-Ca. TIFF2025507472000113.tif41170

[0066] Furthermore, the structural formula of the above compound is as shown in formula II-Ca-1. TIFF2025507472000114.tif41170

[0067] Additionally, the compound has the following structure: TIFF2025507472000115.tif25170

[0068] Furthermore, the structure of the above compound is as shown in formula II-Da. TIFF2025507472000116.tif39170

[0069] Furthermore, the structure of the above compound is as shown in formula II-Da-1. TIFF2025507472000117.tif39170

[0070] Additionally, the compound has the following structure: TIFF2025507472000118.tif28170

[0071] Furthermore, the structure of the compound is as shown in formula III-A, III-B, III-C, III-D, III-E, or III-F. TIFF2025507472000119.tif45170TIFF2025507472000120.tif33170TIFF2025507472000121.tif39170TIFF2025507472000122.tif38170TIFF2025507472000123.tif47170TIFF2025507472000124.tif33170Here, T 1 , T 2 , T 3 are each independently selected from hydrogen or halogen, and at least one is halogen.

[0072] Furthermore, the above T is fluorine.

[0073] Furthermore, the above R 1 is chlorine or hydrogen.

[0074] Furthermore, the structure of the compound is as shown in formula III-Aa, III-Ab, III-Ba, III-Ca, III-Cb, III-Da, III-Db, III-Ea, or III-Fa. TIFF2025507472000125.tif44170TIFF2025507472000126.tif44170TIFF202 5507472000127.tif37170TIFF2025507472000128.tif39170TIFF2025507472 000129.tif39170TIFF2025507472000130.tif37170TIFF2025507472000131. tif37170TIFF2025507472000132.tif47170TIFF2025507472000133.tif37170

[0075] Furthermore, the compound has any one of the following structures: TIFF2025507472000134.tif63170TIFF2025507472000135.tif73170TIFF2025507472 000136.tif65170TIFF2025507472000137.tif81170TIFF2025507472000138.tif37170

[0076] The present invention further provides a pharmaceutical composition, which is a formulation containing the above compound, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof as an active ingredient, together with pharma- ceutically acceptable auxiliary materials.

[0077] The present invention further provides the use of the above compound, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, in the manufacture of a medicament for preventing and / or treating a coronavirus-associated disease.

[0078] Furthermore, the drug for preventing and / or treating the above coronavirus-related disease is an anti-coronavirus drug.

[0079] Furthermore, the anti-coronavirus drug is a drug that inhibits coronavirus infection of cells.

[0080] Furthermore, the anti-coronavirus drug is a coronavirus protease inhibitor, preferably a coronavirus main protease inhibitor.

[0081] Furthermore, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HcoV-229E, HcoV-NL63, HcoV-HKU1 or HcoV-OC43, preferably SARS-CoV-2.

[0082] Furthermore, the drug for preventing and / or treating the above-mentioned coronavirus-related disease is a drug for preventing and / or treating novel coronavirus pneumonia COVID-19.

[0083] Furthermore, the above coronavirus main protease inhibitors have been shown to be effective against SARS-CoV-2M pro It is an inhibitor.

[0084] The definitions of terms used in the present invention are as follows: Unless otherwise specified, the initial definition provided by a group, radical or term in the text applies to the group or term throughout the entire specification, and any term not specifically defined in the text shall represent the meaning that a person skilled in the art may have based on the disclosure and context.

[0085] The minimum and maximum carbon atom content in the hydrocarbon group are represented by prefixes. For example, the prefix C a~b The alkyl group represents an alkyl group containing any number of carbon atoms, from "a" to "b". For example, C 1~8 Alkyl group means a straight or branched chain alkyl group containing 1 to 8 carbon atoms.

[0086] An "alkylene group" refers to a group in which one atom is missing from an alkyl group. For example, a C1-3 alkylene group is a group in which one atom is missing from a C1-3 alkyl group.

[0087] As used herein, "substituted" means that one, two or more hydrogen atoms in a molecule have been replaced with other, different atoms or molecules, including one, two or more substitutions on isotopes or isomers in the molecule.

[0088] A "deuterated compound" refers to a compound in which one or more hydrogens have been replaced with deuterium.

[0089] By "pharmaceutical acceptable" is meant that the carrier, vehicle, diluent, auxiliary material and / or salt formed is chemically or physically compatible with the other ingredients that normally make up the drug dosage form and physiologically compatible with the receptor.

[0090] "Salt" refers to acid and / or base salts formed by a compound or its stereoisomer with inorganic and / or organic acids and / or bases, including zwitterionic salts (internal salts) and further including quaternary ammonium salts, such as alkylammonium salts. These salts may be obtained directly in the final isolation and purification of the compound. They may also be obtained by mixing the compound or its stereoisomer with a certain amount of acid or base appropriately (e.g., equal equivalents). These salts may also be obtained by forming a precipitate in a solution and collecting it by filtration, or by recovering it after evaporation of the solvent, or by reacting in an aqueous medium and then lyophilizing it.

[0091] A "pharmaceutically acceptable salt" may be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, butanedioate, oxalate, malate, succinate, fumarate, maleate, tartrate, or trifluoroacetate salt of a compound.

[0092] "Halogen" is fluorine, chlorine, bromine or iodine.

[0093] By "aryl group" is meant an all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated pi-electron system, such as, for example, a phenyl group. The aryl group does not contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent must be on a carbon atom in the ring having a conjugated pi-electron system. The aryl group may be substituted or unsubstituted. By "5- to 6-membered aryl group" is meant an aryl group having 5 or 6 carbon atoms in the ring.

[0094] The term "heteroaryl group" refers to a heteroaromatic group containing one or more heteroatoms. The heteroatoms referred to here include oxygen, sulfur, and nitrogen. Examples include a furyl group, a thienyl group, a pyridyl group, and a pyrazolyl group. The heteroaryl group can be arbitrarily selected from substituted and unsubstituted groups. The term "5- to 6-membered heteroaryl group" refers to a heteroaryl group having 5 or 6 atoms in the ring.

[0095] The term "cycloalkyl group" refers to a saturated or unsaturated cyclic hydrocarbon substituent. For example, the term "saturated 3- to 8-membered cycloalkyl group" refers to a saturated cycloalkyl group having 3 to 8 carbon atoms in the ring.

[0096] The term "heterocyclyl group" refers to a saturated or unsaturated cyclic hydrocarbon substituent, and the cyclic hydrocarbon has at least one ring heteroatom (including, but not limited to, O, S, or N). For example, a "saturated 3-8 membered heterocyclyl group" refers to a saturated heterocyclyl group having 3 to 8 ring atoms. An "oxygen heterocycle" refers to a heterocycle in which the heteroatom is O, and does not include S or N. The same applies below.

[0097] "Bridged ring group" means a polycyclic cycloalkyl group in which two rings share two adjacent carbon atoms.

[0098] The term "fused ring aryl group" refers to a polycyclic aromatic group in which two rings share two adjacent carbon atoms, and examples thereof include a naphthyl group (i.e., a six-membered aromatic ring, or a benzene ring), an anthracenyl group, and a phenanthrenyl group.

[0099] "Fused heteroaryl ring / group" means a polycyclic heteroaryl ring / group containing at least one heteroatom (O, N or S), and two of the rings share two adjacent carbon atoms or heteroatoms. For example, a 5-membered aryl or heteroaryl ring (e.g., furan, thiophene, pyrrole, pyridine ring), a 6-membered aryl or heteroaryl ring (e.g., benzene, pyridine, pyridazine, pyrimidine, pyrazine ring), such as indole, or a 6-membered aryl or heteroaryl ring, such as quinoline, or two or more fused rings, such as acridine. "Nitrogen-containing fused heteroaryl ring" means that at least one of the heteroatoms in the above "fused heteroaryl ring" is N.

[0100] The term "saturated 5- to 6-membered heterocyclyl group or 5- to 6-membered aryl group" refers to a group formed by a "saturated 5- to 6-membered heterocycle" and a "5- to 6-membered aryl ring" sharing two adjacent carbon atoms or heteroatoms.

[0101] "CD3" refers to a methyl group substituted with three deuteriums.

[0102] "The substituent is an ester group" means that a methylene group CH2 is replaced with =O to form C=O.

[0103] "Saturated heterocycle" means a saturated ring in which at least one carbon atom in a saturated carbocyclic ring is replaced with O, N and / or S. "Oxygen heterocycle" refers to a ring in which at least one carbon atom in the carbocyclic ring is replaced with O.

[0104] Unless otherwise specified, the ring "bonded to form a ring" described in the present invention includes unsubstituted rings and rings substituted with substituents. For example, "R 4a , R 4b are bonded to form a ring," "R 2a , R 2b are bonded to form a ring."

[0105] As a result of the test, the present invention is a novel coronavirus main protease M pro The present invention provides a compound that can effectively inhibit the activity of SARS-CoV-2, which can effectively inhibit the replication and transcription of SARS-CoV-2 virus in the patient's body, inhibit SARS-CoV-2 infection in cells, and provide strong support for combating SARS-CoV-2.

[0106] At the same time, the compounds provided by the present invention also have good internal safety and pharmacokinetic properties, low cardiotoxicity, and low likelihood of inducing acute arrhythmia or even sudden death after administration.

[0107] The compounds of the present invention are proIt can effectively inhibit the activity of SARS-CoV-2 and has antiviral activity in vitro and in vivo against SARS-CoV-2 wild-type virus strains (in vitro) and mutant virus strains (in vivo and in vitro).

[0108] The compounds of the present invention are Pro It can be used to manufacture inhibitors, anti-SARS-CoV-2 drugs, and drugs for preventing and / or treating novel coronavirus pneumonia, and its future prospects are very bright.

[0109] Naturally, based on the above content of the present invention, various other forms of modifications, substitutions or changes can be made in light of general technical knowledge and conventional means in this field, on the premise that they do not deviate from the above basic technical idea of ​​the present invention.

[0110] The above content of the present invention will be described in more detail below by way of specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention. Any technology realized based on the above content of the present invention belongs to the scope of the present invention. [Brief description of the drawings]

[0111] [Figure 1] FIG. 1 shows the enzyme inhibitory activity of compound 126 against SARS-COV-2 MPro. [Diagram 2] FIG. 2 shows the enzyme inhibitory activity of compound 275 against SARS-COV-2 MPro. [Diagram 3] FIG. 3 shows the enzyme inhibitory activity of compound 289 against SARS-COV-2 MPro. [Figure 4] FIG. 4 shows the enzyme inhibitory activity of compound 296 against SARS-COV-2 MPro. [Diagram 5] FIG. 5 shows the enzyme inhibitory activity of compound 398 against SARS-COV-2 MPro. [Figure 6]FIG. 6 shows the antiviral activity of some compounds of the present invention at the cellular level. [Figure 7] FIG. 7 shows the antiviral activity of compound 398 at the cellular level. [Figure 8] FIG. 8 shows the results of measuring the viral load in an in vivo antiviral test of compound 398. [Figure 9] FIG. 9 shows the results of measuring the viral titer of compound 398 in an in vivo antiviral test. [Figure 10] FIG. 10 shows the results of immunohistochemical staining and histopathological staining in an in vivo antiviral test of compound 398. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0112] All the materials and equipment used in the present invention are conventional and purchased from commercial sources.

[0113] The handling conditions for the preparation of the ketoamide derivatives shown in formula I above include the following:

[0114] Example 1 Preparation of Compound 1 TIFF2025507472000139.tif95170

[0115] Step a: Preparation of intermediate 1 (tert-butyl ((2R,3S)-hydroxy-4-oxy-1-phenyl-4-(pyridin-2-ylmethyl)amino)butan-2-yl)carbamate). TIFF2025507472000140.tif23170 The raw material (2S,3R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-4-phenylbutyric acid (590mg, 2mmol) was dissolved in dichloromethane, and pyridin-2-ylmethylamine (217mg, 2mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (1.14g, 3mmol), and N,N-diisopropylethylamine (769mg, 6mmol) were added in sequence and reacted at room temperature overnight. After detecting the completion of the reaction by TLC, it was extracted with saturated ammonium chloride and saturated sodium bicarbonate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 616mg of white solid intermediate 1. Yield 80%, MS(ESI) m / z: 386.2 [M+H] + .

[0116] Step b: Preparation of intermediate 2 ((2S,3R)-3-amino-2-hydroxy-4-phenyl-N-(pyridin-2-ylmethyl)butyramide hydrochloride). TIFF2025507472000141.tif21170 Intermediate 1 (384 mg, 1 mmol) was dissolved in dichloromethane, protected with nitrogen gas, and added hydrochloric acid dioxane (1.25 mL, 5 mmol, 4 M) under ice bath conditions. After the addition was completed, the mixture was transferred to room temperature and stirred for 1 to 2 h. After the completion of the reaction was detected by TLC, the reaction solution was concentrated to obtain 358 mg of white solid intermediate 2, which was used directly in the next reaction (step e).

[0117] Step c: Preparation of intermediate 3 ((R)-2-(benzyloxy)propionic acid methyl ester). TIFF2025507472000142.tif13170 The raw materials (R)-2-hydroxypropionic acid methyl ester (105 mg, 1 mmol), silver oxide (290 mg, 1.25 mmol), and benzyl bromide (214 mg, 1.25 mmol) were dissolved in dichloromethane solution and stirred at room temperature for 48 h. After detecting the completion of the reaction by TLC, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated and purified by column chromatography to obtain 155 mg of intermediate 3 as a colorless oily liquid. Yield 80%, MS (ESI) m / z: 195.1 [M+H] + .

[0118] Step d: Preparation of intermediate 4 ((R)-2-(benzyloxy)propionic acid). TIFF2025507472000143.tif13170 Intermediate 3 (155 mg, 0.8 mmol) was dissolved in 2 mL of ethanol solution, and an aqueous solution (2 mL) of lithium hydroxide (19 mg, 0.8 mmol) was added dropwise under ice bath conditions, and the reaction was continued for 30 min. After the completion of the reaction was detected by TLC, ice water was added, and the mixture was extracted with ethyl acetate. The pH of the aqueous phase was adjusted to 2-3 with dilute hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate. The organic phases were combined and concentrated to obtain 100 mg of intermediate 4 as a colorless oily liquid, which was used as is in the next reaction.

[0119] Step e: Preparation of intermediate 5 ((2S,3R)-3-((R)-2-(benzyloxy)propionamido)-2-hydroxy-4-phenyl-N-(pyridin-2-ylmethyl)butyramide). TIFF2025507472000144.tif25170 Intermediate 4 (90 mg, 0.5 mmol) was dissolved in dichloromethane, and intermediate 2 (180 mg, 0.5 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (286 mg, 0.75 mmol), and N,N-diisopropylethylamine (258 mg, 2 mmol) were added in order, and the mixture was reacted at room temperature overnight. After detecting the completion of the reaction by TLC, the mixture was extracted with saturated ammonium chloride and saturated sodium bicarbonate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 180 mg of yellow solid intermediate 5. Yield 80%, MS (ESI) m / z: 448.2 [M+H] + .

[0120] Step f: Preparation of compound 1 ((R)-3-((R)-2-(benzyloxy)propionamido)-2-oxo-4-phenyl-N-(pyridin-2-ylmethyl)butyramide). TIFF2025507472000145.tif25170 Intermediate 5 (180 mg, 0.4 mmol) was dissolved in 10 mL of dichloromethane, stirred under ice bath, and Dess-Martin oxidant (212 mg, 0.5 mmol) was added in several portions. After the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After the reaction was completed, it was diluted with dichloromethane (5 mL), and then extracted with saturated sodium thiosulfate (5 mL) and saturated sodium bicarbonate (5 mL) in sequence. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and then separated by column chromatography to obtain 116 mg of white solid product 1. Yield 65%. 1 H NMR (400 MHz, DMSO-d6) δ 9.28 (t, J = 6.2 Hz, 1H), 8.57-8.46 (m, 1H), 8.20 (d, J = 8.3 Hz, 1H), 7.82-7.67 (m, 1H), 7.42-7.15 (m, 13H), 5.34-5.23 (m, 1H), 4.44 (s, 2H), 4.42-4.35 (m, 1H), 4.31-4.23 (m, 1H), 3.87-3.80 (m, 1H), 3.25-3.18 (m, 1H), 2.94 (dd, J = 13.8, 9.3 Hz, 1H), 1.15 (d, J = 12.3, 6.7 Hz, 3H).

[0121] Example 2 Preparation of Compound 137 TIFF2025507472000146.tif111170

[0122] Step a: Same as step a in Example 1. Step b: Same as step b in the first embodiment.

[0123] Step c: Preparation of intermediate 3 (tert-butyl ((R)-1-(((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)amino)-1-oxopropan-2-yl)carbamate). TIFF2025507472000147.tif24170 Intermediate 2 (358 mg, 1 mmol) was dissolved in dichloromethane, (tert-butoxycarbonyl)-D-alanine (189 mg, 1 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (570 mg, 1.5 mmol), and N,N-diisopropylethylamine (513 mg, 4 mmol) were added in sequence, and the mixture was allowed to react overnight at room temperature. After detecting the completion of the reaction by TLC, the mixture was extracted with saturated ammonium chloride and saturated sodium bicarbonate, respectively, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 365 mg of white solid intermediate 3. Yield 80%, MS (ESI) m / z: 457.2 [M+H] + .

[0124] Step d: Preparation of intermediate 4 ((2S,3R)-3-((R)-2-aminopropionamido)-2-hydroxy-4-phenyl-N-(pyridin-2-ylmethyl)butyramide hydrochloride). TIFF2025507472000148.tif28170 Intermediate 3 (229 mg, 0.5 mmol) was dissolved in dichloromethane, protected with nitrogen gas, and added hydrochloric acid dioxane (0.625 mL, 2.5 mmol, 4 M) under ice bath conditions. After the addition was completed, the mixture was transferred to room temperature and stirred for 1 to 2 hours. After the completion of the reaction was detected by TLC, the reaction solution was concentrated to obtain 214 mg of white solid intermediate 4, which was used directly in the next reaction.

[0125] Step e: Preparation of intermediate 5 (N-((R)-1-(((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)amino)-1-oxopropan-2-yl)benzamide). TIFF2025507472000149.tif24170 Intermediate 4 (214 mg, 0.5 mmol) was dissolved in dichloromethane, and stirred for 10 min under ice bath conditions. N,N-diisopropylethylamine (194 mg, 1.5 mmol) was added, and after uniform stirring, a dichloromethane solution in which benzoyl chloride (71 mg, 0.5 mmol) was dissolved was added dropwise. The reaction was continued for 0.5 to 1 h. After the completion of the reaction was detected by TLC, ice water was added and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 207 mg of white solid intermediate 5. Yield 90%, MS (ESI) m / z: 461.2 [M + H] + .

[0126] Step f: Preparation of product 137 (N-((R)-1-(((R)-3,4-dioxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)amino)-1-oxopropan-2-yl)benzamide). TIFF2025507472000150.tif24170 Intermediate 5 (207 mg, 0.45 mmol) was dissolved in 10 mL of dichloromethane, stirred under ice bath, and Dess-Martin oxidant (212 mg, 0.5 mmol) was added in several portions. After the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After the reaction was completed, the mixture was diluted with dichloromethane (5 mL), and then extracted with saturated sodium thiosulfate (5 mL) and saturated sodium bicarbonate (5 mL) in sequence. The organic phase was collected, dried, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 134 mg of white solid product 137. Yield 65%. 1 H NMR (400 MHz, DMSO-d6) δ 9.23 (t, J = 27.6, 6.1 Hz, 1H), 8.53-8.47 (m, 1H), 8.46-8.32 (m, 2H), 7.93-7.82 (m, 2H), 7.77-7.70 (m, 1H), 7.57-7.50 (m, 1H), 7.49-7.41 (m, 2H), 7.31-7.14 (m, 7H), 5.28-5.17 (m, 1H), 4.58-4.48 (m, 1H), 4.43 (s, 2H), 3.22-3.09 (m, 1H), 2.94-2.79 (m, 1H), 1.23 (s, 3H).

[0127] Example 3 Preparation of Compound 331 TIFF2025507472000151.tif111170

[0128] Step a: Same as step a in Example 2. Step b: Same as step b in Example 2. Step c: Same as step c in the second embodiment. Step d: Same as step d in the second embodiment.

[0129] Step e: Preparation of intermediate 5 ((2S,3R)-3-((R)-2-((4-fluorophenyl)sulfonamido)propionamido)-2-hydroxy-4-phenyl-N-(pyridin-2-ylmethyl)butyramide). TIFF2025507472000152.tif30170 Intermediate 4 (180 mg, 0.5 mmol) was dissolved in 5 mL of dichloromethane, pyridine (118 mg, 1.5 mmol) and 4-fluoro-benzenesulfonyl chloride (100 mg, 0.5 mmol) were added, and the mixture was allowed to react at room temperature overnight. After detecting the completion of the reaction by TLC, the mixture was extracted with saturated ammonium chloride and saturated sodium bicarbonate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by column chromatography to obtain 174 mg of yellow solid intermediate 5. Yield 68%, MS (ESI) m / z: 515.2 [M+H] + .

[0130] Step f: Preparation of compound 331 ((R)-3-((R)-2-((4-fluorophenyl)sulfonamido)propionamido)-2-oxo-4-phenyl-N-(pyridin-2-ylmethyl)butyramide). TIFF2025507472000153.tif28170 Intermediate 5 (154 mg, 0.3 mmol) was dissolved in dichloromethane and stirred under ice bath, Dess-Martin oxidant (170 mg, 0.4 mmol) was added in several portions, and after the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react, and after the reaction was completed, it was diluted with dichloromethane, and then extracted with saturated sodium thiosulfate and saturated sodium bicarbonate in sequence, and the organic phase was collected, dried, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 92 mg of white solid product 331. Yield 60%. 1 H NMR (400 MHz, DMSO-d6) δ 9.34-9.22 (m, 1H), 8.54-8.47 (m, 1H), 8.45-8.34 (m, 2H), 8.08-7.99 (m, 1H), 7.83-7.73 (m, 2H), 7.72-7.62 (m, 2H), 7.29-7.16 (m, 7H), 5.20-4.92 (m, 1H), 4.44 (d, J = 18.7, 6.4 Hz, 2H), 3.94-3.84 (m, 1H), 3.16-3.05 (m, 1H), 2.82-2.65 (m, 1H), 0.97 (d, 3H).

[0131] Example 4 Preparation of Compound 296 TIFF2025507472000154.tif113170

[0132] Step a: Same as step a in Example 2. Step b: Same as step b in Example 2. Step c: Same as step c in the second embodiment. Step d: Same as step d in the second embodiment.

[0133] Step e: Preparation of intermediate 5 (4,4-difluoro-N-((R)-1-(((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)amino)-1-oxopropan-2-yl)piperidine-1-formamide). TIFF2025507472000155.tif341704,4-Difluoropiperidine (50mg, 0.42mmol) was dissolved in tetrahydrofuran, and after adding triethylamine (163μL, 1.27mmol) at 0℃, a tetrahydrofuran solution of triphosgene (124mg, 0.42mmol) was added dropwise, and the reaction was allowed to proceed while keeping warm for 30 minutes. The reaction solution was then added dropwise to a tetrahydrofuran solution of intermediate 4 (180mg, 0.42mmol) and triethylamine (163μL, 1.27mmol) in an ice bath, and the reaction was allowed to proceed overnight at room temperature. After detecting the completion of the reaction by TLC, the reaction solution was concentrated, extracted with ethyl acetate and water, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and separated and purified by column chromatography to obtain 70mg of a yellow solid intermediate 5. Yield 33%, MS (ESI) m / z: 504.2 [M+H] + .

[0134] Step f: Preparation of product 296 (N-((R)-1-(((R)-3,4-dioxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)amino)-1-oxopropan-2-yl)-4,4-difluoropiperidine-1-formamide). TIFF2025507472000156.tif34170 Intermediate 5 (70 mg, 0.14 mmol) was dissolved in 10 mL of dichloromethane, stirred under ice bath, Dess-Martin oxidant (61 mg, 0.14 mmol) was added, and after the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After detecting the completion of the reaction by TLC, it was diluted with dichloromethane (5 mL), and further extracted with saturated sodium thiosulfate (5 mL) and saturated sodium bicarbonate (5 mL) in sequence. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and then separated by column chromatography to obtain 36 mg of white solid product 58. Yield 50%. 1 H NMR (400 MHz, DMSO-d6) δ 9.30-9.16 (m, 1H), 8.55-8.49 (m, 1H), 8.50-8.43 (m, 1H), 7.79-7.73 (m, 1H), 7.30-7.17 (m, 7H), 6.76-6.66 (m, 1H), 4.46-4.42 (m, 1H), 4.40-4.35 (m, 1H), 4.22-4.15 (m, 1H), 3.94-3.87 (m, 1H), 3.43-3.41 (m, 4H), 3.16-3.09 (m, 1H), 2.94-2.83 (m, 1H), 1.92-1.83 (m, 4H), 1.17 (d, J = 7.2 Hz, 1.5H), 1.04 (d, J = 7.2 Hz, 1.5H).

[0135] Example 5 Preparation of Compound 313 TIFF2025507472000157.tif113170

[0136] Step a: Same as step a in Example 2. Step b: Same as step b in Example 2.

[0137] Step c: Preparation of intermediate 3 (1-(((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)carbamoyl)cyclobutyl)carbamic acid tert-butyl ester). TIFF2025507472000158.tif29170 Intermediate 2 (358 mg, 1 mmol) was dissolved in dichloromethane, and (tert-butoxycarbonyl)-1-aminocyclobutane carboxylic acid (215 mg, 1 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (570 mg, 1.5 mmol), and N,N-diisopropylethylamine (513 mg, 4 mmol) were added in sequence and reacted at room temperature overnight. After detecting the completion of the reaction by TLC, it was extracted with saturated ammonium chloride and saturated sodium bicarbonate, respectively, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and separated and purified by column chromatography to obtain 385 mg of white solid intermediate 3. Yield 80%, MS (ESI) m / z: 483.3 [M+H] + .

[0138] Step d: Preparation of intermediate 4 (1-amino-N-((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)cyclobutane-1-formamide) hydrochloride. TIFF2025507472000159.tif30170 Intermediate 3 (385 mg, 0.8 mmol) was dissolved in dichloromethane, protected with nitrogen gas, and added with hydrochloric acid dioxane (1 mL, 4 mmol, 4 M) under ice bath conditions. After the addition was completed, the mixture was transferred to room temperature and stirred for 1 to 2 hours. After the completion of the reaction was detected by TLC, the reaction solution was concentrated to obtain 360 mg of white solid intermediate 4, which was used directly in the next reaction.

[0139] Step e: Preparation of intermediate 5 (4,4-difluorocyclohexyl) 1-(((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)carbamoyl)cyclobutyl)carbamate). TIFF2025507472000160.tif37170 (4,4-difluorocyclohexyl)methanol (63 mg, 0.42 mmol) was dissolved in tetrahydrofuran, and after adding triethylamine (163 μL, 1.27 mmol) at 0 ° C, a tetrahydrofuran solution of triphosgene (124 mg, 0.42 mmol) was added dropwise, and the mixture was allowed to react for 30 minutes while keeping the temperature. Then, the reaction solution was added dropwise to a tetrahydrofuran solution of intermediate 4 (180 mg, 0.42 mmol) and triethylamine (163 μL, 1.27 mmol) under ice bath, and the mixture was allowed to react overnight at room temperature. After detecting the completion of the reaction by TLC, the reaction solution was concentrated, extracted with ethyl acetate and water, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and separated and purified by column chromatography to obtain 70 mg of a yellow solid intermediate 5. Yield 30%, MS (ESI) m / z: 559.3 [M+H] + .

[0140] Step f: Synthesis of compound 313 ((4,4-difluorocyclohexyl)methyl (R)-(1-((3,4-dioxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)carbamoyl)cyclobutyl)carbamate). TIFF2025507472000161.tif36170 Intermediate 5 (70 mg, 0.13 mmol) was dissolved in 10 mL of dichloromethane, stirred under ice bath, Dess-Martin oxidant (84 mg, 0.2 mmol) was added, and after the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After detecting the completion of the reaction by TLC, it was diluted with dichloromethane (5 mL), and further extracted using saturated sodium thiosulfate (5 mL) and saturated sodium bicarbonate (5 mL) in sequence. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and then separated by column chromatography to obtain 38 mg of white solid compound 313. Yield 55%. 1 H NMR (400 MHz, DMSO-d6) δ 9.29-9.18 (m, 1H), 8.51 (d, 1H), 7.82-7.73 (m, 1H), 7.75-7.61 (m, 1H), 7.42-7.11 (m, 8H), 5.27-5.10 (m, 1H), 4.46 (d, J = 6.2 Hz, 2H), 3.87-3.74 (m, 1H), 3.19-3.10 (m, 1H), 3.02-2.85 (m, 1H), 2.43-2.29 (m, 2H), 2.08-1.96 (m, 3H), 1.86-1.66 (m, 5H), 1.28-1.20 (m, 2H).

[0141] Example 6 Preparation of Compound 52 TIFF2025507472000162.tif93170

[0142] Step a: Preparation of intermediate 1 ((R)-methyl 2-amino-3-(4-fluorophenyl)propionic acid methyl ester hydrochloride). TIFF2025507472000163.tif191704-Fluoro-D-phenylalanine (1.83 g, 10 mmol) was added to a round-bottom flask, dissolved in methanol, and the reaction system was stirred in an ice bath. Then, 10 mL of thionyl chloride was slowly added dropwise to the reaction system, and after the addition was completed, the temperature was raised to 65 ° C and the reaction was allowed to proceed overnight. The next day, the reaction system was cooled to room temperature, the solvent was distilled off under reduced pressure, and the resulting white solid was used in the next reaction without further purification.

[0143] Step b: Preparation of intermediate 2 ((R)-2-((tert-butoxycarbonyl)amino)-3-(4-fluorophenyl)propionic acid methyl ester). TIFF2025507472000164.tif15170 Intermediate 1 (2.34g, 10mmol) was placed in a flask, dissolved in water, and potassium carbonate (4.14g, 30mmol) was added. Dicarbonic acid di-tert-butyl ester (2.85g, 13mmol) was then dissolved in 20mL of THF and slowly added dropwise to the reaction system. After the addition was completed, the reaction was allowed to proceed at room temperature for 2h. After the reaction was completed, the reaction system was diluted with 20mL of water, extracted twice with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a colorless oily liquid, which was used as is in the next reaction.

[0144] Step c: Preparation of intermediate 3 ((R)-(1-(4-fluorophenyl)-3-hydroxypropan-2-yl)carbamic acid tert-butyl ester). TIFF2025507472000165.tif12170Add intermediate 2 (2.97g, 10mmol) to a flask, dissolve in methanol, and then stir the reaction system in an ice bath. Sodium borohydride (1.89g, 50mmol) was then slowly added to the reaction system. After the addition was complete, the temperature was raised to room temperature and the reaction was allowed to proceed overnight. The next day, water was added to the reaction system to quench the reaction, the reaction solution was concentrated, the residue was diluted with water, extracted twice with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and then separated and purified by column chromatography to obtain 1.25g of white solid intermediate 3. Yield 47%. MS (ESI) m / z: 270.2 [M+H] + .

[0145] Step d: Preparation of intermediate 4 ((R)-(1-(4-fluorophenyl)-3-oxopropan-2-yl)carbamic acid tert-butyl ester). TIFF2025507472000166.tif13170 Intermediate 3 (2.69g, 10mmol) was dissolved in 100mL of dichloromethane, stirred under ice bath, and Dess-Martin oxidant (5.08g, 12mmol) was added in several portions. After the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After the reaction was completed, the mixture was diluted with dichloromethane, and then extracted with saturated sodium thiosulfate and saturated sodium bicarbonate in sequence. The organic phase was collected, dried, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 1.87g of white solid intermediate 4. Yield 70%. MS (ESI) m / z: 268.2 [M+H] + .

[0146] Step e: Preparation of intermediate 5 (((2R)-1-cyano-3-(4-fluorophenyl)-1-hydroxypropan-2-yl)carbamic acid tert-butyl ester). TIFF2025507472000167.tif19170Add intermediate 4 (2g, 7.48mmol) and cesium fluoride (568mg, 3.74mmol) to a flask, dissolve in 50mL of methanol, and then stir the reaction system under ice bath. Then, trimethylsilyl cyanide (890mg, 8.98mmol) was slowly added to the reaction system. After the addition was completed, the mixture was stirred at room temperature for 5 hours. After detecting the completion of the reaction by TLC, the reaction solution was concentrated, diluted with water, and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain intermediate 5 as a yellow oily liquid, which was used as is in the next reaction.

[0147] Step f: Preparation of intermediate 6 ((3R)-3-amino-4-(4-fluorophenyl)-2-hydroxybutyric acid hydrochloride). TIFF2025507472000168.tif20170 Intermediate 5 (2.21 g, 7.5 mmol) was placed in a flask and dissolved in 12 mL of dioxane, and then 27 mL of 6N hydrochloric acid was added. The reaction system was stirred at a temperature of 100 ° C for 12 hours. After the completion of the reaction was detected by TLC, the system was cooled to room temperature and the reaction solution was concentrated to obtain intermediate 6 as a brown solid, which was used as it is in the next reaction.

[0148] Step g: Preparation of intermediate 7 ((3R)-3-amino-4-(4-fluorophenyl)-2-hydroxybutyric acid methyl ester hydrochloride). TIFF2025507472000169.tif14170Add intermediate 6 (1.8g, 7.5mmol) to a flask, dissolve in methanol, and then stir in an ice bath and add 5mL of thionyl chloride dropwise. After the addition was complete, remove the ice bath and heat to reflux overnight to react. After detecting the completion of the reaction by TLC, the reaction solution was concentrated to obtain intermediate 7 as a brown solid, which was used as it is in the next reaction (step j).

[0149] Step h: Same as step c in the first embodiment. Step i: Same as step d in Example 1.

[0150] Step j: Preparation of intermediate 10 ((3R)-3-((R)-2-(benzyloxy)propionamido)-4-(4-fluorophenyl)-2-hydroxybutyric acid methyl ester). TIFF2025507472000170.tif30170 Intermediate 9 (180 mg, 1 mmol) was dissolved in dichloromethane, and intermediate 7 (263 mg, 1 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (456 mg, 1.2 mmol), and N,N-diisopropylethylamine (387 mg, 3 mmol) were added in order, and the mixture was reacted at room temperature overnight. After detecting the completion of the reaction by TLC, the mixture was extracted with saturated ammonium chloride and saturated sodium bicarbonate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 316 mg of yellow solid intermediate 10. Yield 81%, MS (ESI) m / z: 390.2 [M+H] + .

[0151] Step k: Preparation of intermediate 11 ((3R)-3-((R)-2-(benzyloxy)propionamido)-4-(4-fluorophenyl)-2-hydroxybutyric acid). TIFF2025507472000171.tif32170 Intermediate 10 (310 mg, 0.8 mmol) was dissolved in 5 mL of ethanol solution, and lithium hydroxide (168 mg, 4 mmol) was added in several portions while stirring in an ice bath. After the addition was completed, the reaction was continued at room temperature for 2 h. After the completion of the reaction was detected by TLC, ice water was added, and the mixture was extracted with ethyl acetate. The pH of the aqueous phase was adjusted to 2-3 with dilute hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate. The organic phases were combined and concentrated to obtain intermediate 4 as a yellow solid, which was used directly in the next reaction.

[0152] Step 1: Preparation of intermediate 12 ((3R)-3-((R)-2-(benzyloxy)propionamido)-N-((5-chloropyridin-2-yl)methyl)-4-(4-fluorophenyl)-2-hydroxybutyramide). TIFF2025507472000172.tif30170 Intermediate 11 (300 mg, 0.8 mmol) was dissolved in dichloromethane, and 5-chloro-pyridine-2-methylamine (114 mg, 0.8 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (456 mg, 1.2 mmol), and N,N-diisopropylethylamine (387 mg, 3 mmol) were added in sequence and reacted at room temperature overnight. After detecting the completion of the reaction by TLC, it was extracted with saturated ammonium chloride and saturated sodium bicarbonate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 286 mg of yellow solid intermediate 12. Yield 72%, MS (ESI) m / z: 500.2 [M+H] + .

[0153] Step m: Preparation of compound 52 ((R)-3-((R)-2-(benzyloxy)propionamido)-N-((5-chloropyridin-2-yl)methyl)-4-(4-fluorophenyl)-2-oxobutyramide). TIFF2025507472000173.tif30170 Intermediate 12 (250 mg, 0.5 mmol) was dissolved in 10 mL of dichloromethane, stirred under ice bath, and Dess-Martin oxidant (297 mg, 0.7 mmol) was added in several portions. After the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After the reaction was completed, it was diluted with dichloromethane, and then extracted with saturated sodium thiosulfate and saturated sodium bicarbonate in sequence. The organic phase was collected, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 120 mg of white solid product 52. Yield 50%. 1 H NMR (400 MHz, DMSO-d6) δ 9.39-9.28 (m, 1H), 8.55-8.52 (m, 1H), 8.32-8.23 (m, 1H), 7.91-7.82 (m, 1H), 7.33-7.21 (m, 5H), 7.18-7.04 (m, 4H), 5.27-5.15 (m, 1H), 4.48-4.40 (m, 2H), 4.35-4.21 (m, 3H), 3.86-3.79 (m, 2H), 3.21-3.16 (m, 1H), 2.96-2.86 (m, 1H), 1.19-1.10 (m, 3H).

[0154] Example 7 Preparation of Compound 324 TIFF2025507472000174.tif110170

[0155] Step a: Same as step a in Example 2. Step b: Same as step b in Example 2.

[0156] Step c: Preparation of intermediate 3 (methyl 3-(((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)amino)-2,2-dimethyl-3-oxopropionate). TIFF2025507472000175.tif281702,2-Dimethylmalonic acid monomethyl ester (146 mg, 1 mmol) was dissolved in dichloromethane, and intermediate 2 (356 mg, 1 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (456 mg, 1.2 mmol), and N,N-diisopropylethylamine (516 mg, 4 mmol) were added in order, and the mixture was reacted at room temperature overnight. After detecting the completion of the reaction by TLC, the mixture was extracted with saturated ammonium chloride and saturated sodium bicarbonate, respectively, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 310 mg of yellow solid intermediate 3. Yield 75%, MS (ESI) m / z: 414.3 [M+H] + .

[0157] Step d: Preparation of intermediate 4 (3-((((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)amino)-2,2-dimethyl-3-oxopropionic acid). TIFF2025507472000176.tif28170 Intermediate 3 (310 mg, 0.75 mmol) was dissolved in 5 mL of ethanol solution, and lithium hydroxide (147 mg, 3.5 mmol) was added in several portions while stirring in an ice bath. After the addition was completed, the reaction was continued at room temperature for 2 h. After the completion of the reaction was detected by TLC, ice water was added, and the mixture was extracted with ethyl acetate. The pH of the aqueous phase was adjusted to 3-4 with dilute hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate. The organic phases were combined and concentrated to obtain intermediate 4 as a yellow solid, which was used directly in the next reaction.

[0158] Step e: Intermediate 5(N 1 -((4,4-difluorocyclohexyl)methyl)-N 3 Preparation of -((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)-2,2-dimethylpropanediamide). TIFF2025507472000177.tif28170 Intermediate 4 (300 mg, 0.75 mmol) was dissolved in dichloromethane, and 4,4-difluorocyclohexylmethylamine hydrochloride (139 mg, 0.75 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (342 mg, 0.9 mmol), and N,N-diisopropylethylamine (349 mg, 2.7 mmol) were added in sequence and reacted at room temperature overnight. After detecting the completion of the reaction by TLC, it was extracted with saturated ammonium chloride and saturated sodium bicarbonate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 259 mg of yellow solid intermediate 5. Yield 65%, MS (ESI) m / z: 531.3 [M+H] + .

[0159] Step f: Compound 324 ((R)-N 1 -((4,4-difluorocyclohexyl)methyl)-N 3 Preparation of -(3,4-dioxo-1-phenyl-4-((pyridin-2-ylmethyl)amino)butan-2-yl)-2,2-dimethylpropanediamide). TIFF2025507472000178.tif23170 Intermediate 5 (212 mg, 0.4 mmol) was dissolved in 10 mL of dichloromethane, stirred under ice bath, and Dess-Martin oxidant (297 mg, 0.7 mmol) was added in several portions. After the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After the reaction was completed, it was diluted with dichloromethane, and then extracted with saturated sodium thiosulfate and saturated sodium bicarbonate in sequence. The organic phase was collected, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 109 mg of white solid product 324. Yield 52%. 1 H NMR (400 MHz, Chloroform-d) δ 8.62-8.55 (m, 1H), 8.12-8.03 (m, 1H), 7.75-7.66 (m, 1H), 7.31-7.26 (m, 3H), 7.26-7.22 (m, 2H), 7.14-7.08 (m, 2H), 6.86-6.76 (m, 2H), 5.57-5.49 (m, 1H), 4.63 (d, J = 5.4 Hz, 2H), 3.41-3.33 (m, 1H), 3.20-3.11 (m, 1H), 3.09-2.98 (m, 2H), 2.07-2.04 (m, 1H), 1.78-1.72 (m, 2H), 1.67-1.49 (m, 4H), 1.40-1.37 (m, 3H), 1.35 (s, 3H), 1.26 (s, 2H).

[0160] Example 8 Preparation of Compound 326 TIFF2025507472000179.tif85170

[0161] Step a: Same as step a in Example 2. Step b: Same as step b in Example 2.

[0162] Step c: Preparation of intermediate 3 (2-(4-bromo-1H-pyrazol-1-yl)-2-methylpropionic acid ethyl ester). TIFF2025507472000180.tif14170 The raw material 4-bromo-1H-pyrazole (300 mg, 2.04 mmol) was dissolved in anhydrous DMF, and NaH (98 mg, 2.45 mmol) was added in several portions under ice bath conditions, and then the mixture was stirred at room temperature for 30 minutes. 2-Bromo-2-methylpropionic acid ethyl ester (478 mg, 2.45 mmol) was slowly added dropwise to the mixture under ice bath, and after the addition was completed, the mixture was reacted at room temperature overnight. After the completion of the reaction was detected by TLC, the mixture was quenched with methanol, the reaction solution was concentrated, and the mixture was separated and purified by column chromatography to obtain 320 mg of intermediate 3 as a yellow solid. Yield 60%, MS (ESI) m / z: 261.0 [M+H] + .

[0163] Step d: Preparation of intermediate 4 (2-methyl-2-(4-phenyl-1H-pyrazol-1-yl)propionic acid ethyl ester). TIFF2025507472000181.tif13170 Intermediate 3 (320 mg, 1.23 mmol), phenylboronic acid (165 mg, 1.35 mmol), 1,1-bis(diphenylphosphino)ferrocenedichloropalladium(II) (90 mg, 0.123 mmol), potassium carbonate (339 mg, 2.45 mmol) were dissolved in dioxane / water solution (20 mL: 1 mL), protected with nitrogen gas, and reacted at 95 °C for 12 h. After detecting the completion of the reaction by TLC, it was filtered with diatomaceous earth, and then separated and purified by column chromatography to obtain 220 mg of intermediate 4. Yield 70%, MS (ESI) m / z: 259.3 [M+H] + .

[0164] Step e: Preparation of intermediate 5 (2-methyl-2-(4-phenyl-1H-pyrazol-1-yl)propionic acid). TIFF2025507472000182.tif13170 Intermediate 4 (220 mg, 0.86 mmol) was dissolved in 10 mL of ethanol solution, and an aqueous solution (2 mL) of lithium hydroxide (41 mg, 1.7 mmol) was added dropwise under ice bath conditions, and the reaction was continued for 30 min. After the completion of the reaction was detected by TLC, ice water was added and the mixture was extracted with ethyl acetate. The pH of the aqueous phase was adjusted to 2-3 with dilute hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate. The organic phases were combined and concentrated to obtain intermediate 5 as a colorless oily liquid, which was used as is in the next reaction.

[0165] Step f: Preparation of intermediate 6 ((2S,3R)-2-hydroxy-3-(2-methyl-2-(4-phenyl-1H-pyrazol-1-yl)propionamido)-4-phenyl-N-(pyridin-2-ylmethyl)butyramide). TIFF2025507472000183.tif28170 Intermediate 5 (198 mg, 0.86 mmol) was dissolved in dichloromethane, and intermediate 2 (307 mg, 0.86 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (380 mg, 1.0 mmol), and N,N-diisopropylethylamine (444 mg, 3.44 mmol) were added in sequence and reacted at room temperature overnight. After detecting the completion of the reaction by TLC, it was extracted with saturated ammonium chloride and saturated sodium bicarbonate, respectively, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 248 mg of yellow solid intermediate 6. Yield 60%, MS (ESI) m / z: 484.2 [M+H] + .

[0166] Step g: Preparation of compound 326 ((R)-3-(2-methyl-2-(4-phenyl-1H-pyrazol-1-yl)propionamido)-2-oxo-4-phenyl-N-(pyridin-2-ylmethyl)butyramide). TIFF2025507472000184.tif28170 Intermediate 6 (242 mg, 0.5 mmol) was dissolved in 10 mL of dichloromethane, stirred under ice bath, and Dess-Martin oxidant (297 mg, 0.7 mmol) was added in several portions. After the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After the reaction was completed, the mixture was diluted with dichloromethane, and then extracted with saturated sodium thiosulfate and saturated sodium bicarbonate in sequence. The organic phase was collected, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 132 mg of white solid product 326. Yield 56%. 1 H NMR (400 MHz, DMSO-d6) δ 9.23 (t, J = 6.3 Hz, 1H), 8.50 (d, J = 4.9 Hz, 1H), 8.26 (s, 1H), 7.97 (s, 1H), 7.76 (t, J = 7.9 Hz, 1H), 7.69-7.53 (m, 3H), 7.37 (t, J = 7.6 Hz, 2H), 7.32-7.04 (m, 8H), 5.23-5.10 (m, 1H), 4.51-4.37 (m, 2H), 3.18-3.06 (m, 1H), 2.98-2.85 (m, 1H), 1.70 (s, 6H).

[0167] Example 9 Preparation of Compound 58 TIFF2025507472000185.tif94170

[0168] Step a: Same as step a in Example 2. Step b: Same as step b in Example 2.

[0169] Step c: Preparation of intermediate 3 ((benzyloxy)(methyl)carbamic acid tert-butyl ester). TIFF2025507472000186.tif19170 The raw material N-(benzyloxy)carbamic acid tert-butyl ester (1000 mg, 4.48 mmol) was dissolved in DMF, and NaH (269 mg, 6.73 mmol) was added in an ice bath. After stirring for 30 min, CHI (mg, 5.38 mmol) was added. After detecting the completion of the reaction by TLC, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated and purified by column chromatography to obtain 752 mg of intermediate 3 as a colorless oily liquid. Yield 80%, MS (ESI) m / z: 238.1 [M+H] + .

[0170] Step d: Preparation of intermediate 4 (O-benzyl-N-methylhydroxylamine). TIFF2025507472000187.tif19170 Intermediate 3 (752 mg, 3.17 mmol) was dissolved in 10 mL of dichloromethane, 10 mL (4 M) of hydrochloric acid in dioxane was added, and the reaction was continued for 30 min. After the completion of the reaction was detected by TLC, the mixture was dried by rotation and used as it was in the next reaction.

[0171] Step e: Preparation of intermediate 5 ((2S,3R)-3-(3-(benzyloxy)-3-methylurea)-2-hydroxy-4-phenyl-N-(pyridin-2-ylmethyl)butyramide). TIFF2025507472000188.tif32170 Intermediate 4 (100 mg, 0.42 mmol) was dissolved in tetrahydrofuran, and triethylamine (163 μL, 1.27 mmol) was added at 0 ° C., followed by dropwise addition of a tetrahydrofuran solution of triphosgene (124 mg, 0.42 mmol), and the reaction was carried out while keeping the temperature for 30 minutes. At 0 ° C., the reaction solution was dropped into a tetrahydrofuran solution containing triethylamine (163 μL, 1.27 mmol) of intermediate 2 (119.7 mg, 0.42 mmol), and the reaction was carried out at room temperature overnight. After detecting the completion of the reaction by TLC, the mixture was concentrated, and then extracted with EA and water, and the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 60 mg of a yellow solid intermediate 5. Yield 32%, MS (ESI) m / z: 449.2 [M+H] + .

[0172] Step f: Preparation of product 58 ((R)-3-(3-(benzyloxy)-3-methylurea)-2-oxo-4-phenyl-N-(pyridin-2-ylmethyl)butyramide). TIFF2025507472000189.tif30170 Intermediate 5 (60 mg, 0.13 mmol) was dissolved in 10 mL of dichloromethane, stirred under ice bath, Dess-Martin oxidant (56 mg, 0.13 mmol) was added, and after the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After the reaction was completed, it was diluted with dichloromethane (5 mL), and further extracted with saturated sodium thiosulfate (5 mL) and saturated sodium bicarbonate (5 mL) in sequence. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and then separated by column chromatography to obtain 26 mg of white solid product 58. Yield 45%. MS (ESI) m / z: 447.2028 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.33 (d, J = 5.6 Hz, 1H), 8.51 (d, J = 4.2 Hz, 1H), 7.99 (m, 1H), 7.74 (dt, J = 14.1, 6.4 Hz, 2H), 7.46 (dd, J = 7.6, 1.6 Hz, 1H), 7.38 (m, 3H), 7.28 (dd, J = 10.3, 5.1 Hz, 4H), 7.22 (m, 2H), 6.48 (s, 1H), 4.79 (s, 2H), 4.48 (m, 1H), 4.45 (s, 2H), 3.41 (m, 1H), 3.04 (m, 1H), 2.89 (s, 3H).

[0173] Example 10 Preparation of Compound 398 TIFF2025507472000190.tif118170

[0174] Step a: Preparation of intermediate 1 (tert-butyl ((2R,3S)-hydroxy-4-oxy-1-phenyl-4-(pyridin-2-ylmethyl)amino)butan-2-yl)carbamate). TIFF2025507472000191.tif23170 The raw material (2S,3R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-4-phenylbutyric acid (590mg, 2mmol) was dissolved in DCM, and 2-thiazolemethanamine (228mg, 2mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (1.14g, 3mmol), and N,N-diisopropylethylamine (769mg, 6mmol) were added in that order and reacted at room temperature overnight. The mixture was extracted with saturated ammonium chloride and saturated sodium bicarbonate, the reaction solution was concentrated, and the mixture was separated and purified by column chromatography to obtain 623mg of intermediate 1. Yield 80%, MS (ESI) m / z: 392.2 [M+H] + .

[0175] Step b: Preparation of intermediate 2 ((2S,3R)-3-amino-2-hydroxy-4-phenyl-N-(thiazol-2-ylmethyl)butyramide hydrochloride). TIFF2025507472000192.tif21170 Intermediate 1 (391 mg, 1 mmol) was dissolved in dichloromethane, protected with nitrogen gas, and added with hydrochloric acid dioxane (1.25 mL, 5 mmol, 4 M) under ice bath conditions, transferred to room temperature and stirred, and after monitoring the completion of the reaction by TLC, the reaction solution was concentrated to obtain 327 mg of intermediate 2, which was used directly in the next reaction.

[0176] Step c: Preparation of intermediate 3 (tert-butyl ((R)-1-(((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((thiazol-2-ylmethyl)amino)butan-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)carbamate). TIFF2025507472000193.tif22170 Intermediate 2 (327 mg, 1 mmol) was dissolved in dichloromethane, and N-tert-butoxycarbonyl-O-methyl-D-serine (189 mg, 1 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (570 mg, 1.5 mmol), and N,N-diisopropylethylamine (513 mg, 4 mmol) were added in that order, and the mixture was reacted at room temperature overnight. The mixture was extracted with saturated ammonium chloride and saturated sodium bicarbonate, and the reaction solution was concentrated and separated and purified by column chromatography to obtain 394 mg of intermediate 3. Yield 80%, MS (ESI) m / z: 493.2 [M+H] + .

[0177] Step d: Preparation of intermediate 4 ((2S,3R)-3-((R)-2-amino-3-methoxypropionamido)-2-hydroxy-4-phenyl-N-(thiazol-2-ylmethyl)butyramide hydrochloride). TIFF2025507472000194.tif23170 Intermediate 3 (246 mg, 0.5 mmol) was dissolved in dichloromethane, protected with nitrogen gas, and added with hydrochloric acid-dioxane (0.625 mL, 2.5 mmol, 4 M) under ice bath conditions. The mixture was transferred to room temperature and stirred for 1 to 2 h. The reaction solution was then rotary dried to obtain 214 mg of intermediate 4, which was used as is in the next reaction.

[0178] Step e: Preparation of intermediate 5 (3,3-difluoro-N-((R)-1-(((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((thiazol-2-ylmethyl)amino)butan-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)cyclohexane-1-formamide). TIFF2025507472000195.tif29170 Intermediate 4 (214 mg, 0.5 mmol) was dissolved in dichloromethane and stirred for 10 minutes under ice bath conditions, and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (570 mg, 1.5 mmol), N,N-diisopropylethylamine (194 mg, 1.5 mmol), 3,3-difluorocyclohexane-1-carboxylic acid (82 mg, 0.5 mmol) were added and reacted at room temperature overnight. After the reaction was completed, the mixture was extracted with saturated ammonium chloride and saturated sodium bicarbonate, the reaction solution was concentrated, and the mixture was separated and purified by column chromatography to obtain 242 mg of intermediate 5. Yield 90%, MS (ESI) m / z: 539.2 [M+H] + .

[0179] Step f: Preparation of product 398 (N-((R)-1-(((R)-3,4-dioxo-1-phenyl-4-((thiazol-2-ylmethyl)amino)butan-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-3,3-difluorocyclohexane-1-formamide). TIFF2025507472000196.tif28170 Intermediate 5 (242 mg, 0.45 mmol) was dissolved in 10 mL of dichloromethane, stirred under ice bath, and Dess-Martin oxidant (212 mg, 0.5 mmol) was added in several portions. After the addition was completed, the mixture was transferred to room temperature and stirred for 2 hours to react. After the reaction was completed, it was diluted with dichloromethane (5 mL), and then extracted with saturated sodium thiosulfate (5 mL) and saturated sodium bicarbonate (5 mL) in sequence. The organic phases were combined, dried, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 241 mg of white solid product 398. Yield 65%. 1 H NMR (400 MHz, DMSO-d6) δ 9.59 (dt, J = 26.7, 6.3 Hz, 1H), 8.48-8.30 (m, 1H), 8.10 (td, J = 9.1, 8.4, 5.7 Hz, 1H), 7.78-7.71 (m, 1H), 7.68-7.62 (m, 1H), 7.35-7.17 (m, 5H), 5.29-5.16 (m, 1H), 4.68-4.59 (m, 2H), 4.58-4.46 (m, 1H), 3.48-3.38 (m, 1H), 3.28-3.21 (m, 2H), 3.19-3.10 (m, 3H), 2.95-2.78 (m, 1H), 2.53-2.51 (m, 1H), 2.09-1.92 (m, 2H), 1.86-1.60 (m, 4H), 1.51-1.35 (m, 1H), 1.33-1.21 (m, 1H).

[0180] Example 11 Preparation of Compound 490 TIFF2025507472000197.tif95170

[0181] Step a: Same as step a in Example 10. Step b: Same as step b in Example 10.

[0182] Step c: Preparation of intermediate 3 (1-(2,4,5-trifluorobenzyl)-1H-pyrazole-4-carboxylic acid ethyl ester). TIFF2025507472000198.tif13170 Raw material 1H-pyrazole-4-carboxylic acid ethyl ester (70 mg, 0.5 mmol) was dissolved in 10 ml of ultra-dehydrated tetrahydrofuran, and sodium hydride (36 mg, 1.5 mmol) was slowly added under ice bath conditions, followed by 1-(bromomethyl)-2,4,5-trifluorobenzene (135 mg, 0.6 mmol), reacted for 30 minutes, and then reacted at room temperature overnight. After detecting the completion of the reaction by TLC, it was concentrated, then extracted with EA and water, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and separated and purified by column chromatography to obtain 114 mg of yellow solid intermediate 3. Yield 80%, MS (ESI) m / z: 285.0 [M+H] + .

[0183] Step d: Preparation of intermediate 4 (1-(2,4,5-trifluorobenzyl)-1H-pyrazole-4-carboxylic acid). TIFF2025507472000199.tif13170 Intermediate 3 (114 mg, 0.4 mmol) was dissolved in 2 mL of ethanol solution, and an aqueous solution (2 mL) of lithium hydroxide (19 mg, 0.8 mmol) was added dropwise under ice bath conditions, and the reaction was continued for 30 min. After the completion of the reaction was detected by TLC, the mixture was dried by rotation, and then water was added and the pH was adjusted to 2-3 with dilute hydrochloric acid (1 M). The mixture was then extracted three times with an appropriate amount of ethyl acetate. The organic phases were combined and concentrated to obtain intermediate 4, which was used directly in the next reaction.

[0184] Step e: Preparation of intermediate 5 (N-((2R,3S)-3-hydroxy-4-oxo-1-phenyl-4-((thiazol-2-ylmethyl)amino)butan-2-yl)-1-(2,4,5-trifluorobenzyl)-1H-pyrazole-4-formamide). TIFF2025507472000200.tif29170 Intermediate 4 (38 mg, 0.3 mmol) was dissolved in dichloromethane, and intermediate 2 (108 mg, 0.3 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (172 mg, 0.45 mmol), and N,N-diisopropylethylamine (155 mg, 1.2 mmol) were added in sequence and reacted at room temperature overnight. After detecting the completion of the reaction by TLC, the mixture was extracted with saturated ammonium chloride and saturated sodium bicarbonate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by column chromatography to obtain 127 mg of intermediate 5. Yield 80%, MS (ESI) m / z: 530.1 [M+H] + .

[0185] Step f: Preparation of compound 490 ((R)-N-(3,4-dioxo-1-phenyl-4-((thiazol-2-ylmethyl))amino)butan-2-yl)-1-(2,4,5-trifluorobenzyl)-1H-pyrazole-4-formamide). TIFF2025507472000201.tif30170 Intermediate 5 (106 mg, 0.2 mmol) was dissolved in 10 mL of dichloromethane and stirred under ice bath, Dess-Martin oxidant (127 mg, 0.3 mmol) was added in several portions, and the reaction was continued in an ice bath for 2 hours after the addition was completed. After the reaction was completed, it was diluted with dichloromethane (5 mL), and saturated sodium thiosulfate (5 mL) and saturated sodium bicarbonate (5 mL) were added in sequence, and the reaction solution was maintained at 0 ° C and stirred for 5 min. The organic phase was separated, and the aqueous phase was extracted twice with DCM, and the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and then separated by column chromatography to obtain 82 mg of white solid compound 490. Yield 78%. 1 H NMR (400 MHz, DMSO-d6) δ 9.56 (t, J = 6.2 Hz, 1H), 8.56 (d, J = 7.2 Hz, 1H), 8.25 (s, 1H), 7.88 (s, 1H), 7.71 (d, J = 3.3 Hz, 1H), 7.65-7.60 (m, 1H), 7.58 (d, J = 3.2 Hz, 1H), 7.52-7.45 (m, 1H), 7.31-7.14 (m, 5H), 5.38 (s, 2H), 5.32-5.25 (m, 1H), 4.62 (d, J = 6.2 Hz, 2H), 3.23-3.15 (m, 1H), 2.94-2.85 (m, 1H). HRMS (ESI-TOF) m / z calcd. for C 25 H 20 F3N5O3S [M+H] + 528.1312, found 533.1313.

[0186] Example 12 Preparation of Compound 511 TIFF2025507472000202.tif93170

[0187] Step a: Same as step a in Example 10. Step b: Same as step b in Example 10.

[0188] Step c: Preparation of intermediate 3 (2-(2-(4,4-difluorocyclohex-1-en-1-yl)thiazol-4-yl)acetic acid ethyl ester). TIFF2025507472000203.tif12170 The raw materials 2-bromo-4-thiazole ethyl acetate (249 mg, 1.0 mmol), 4,4-difluorocyclohexa-1-alkenylboronic acid pinacol ester (162 mg, 1.0 mmol), 1,1'-bis(diphenylphosphino)ferrocenedichloropalladium(II) (73 mg, 0.1 mmol), and potassium carbonate (176 mg, 2.0 mmol) were dissolved in 10 ml of dioxane, 0.5 ml of water was added, and the mixture was protected with nitrogen gas and refluxed overnight. After the reaction was completed, the reaction solution was spun to dryness under reduced pressure, extracted with ethyl acetate / water system, the organic phase was washed with brine, the sample was stirred, and intermediate 3 (247 mg, 86%) was obtained after column chromatography. MS (ESI) m / z: 288.1 [M+H]+.

[0189] Step d: Preparation of intermediate 4 (2-(2-(4,4-difluorocyclohexyl)thiazol-4-yl)acetic acid ethyl ester). TIFF2025507472000204.tif12170The intermediate 3 from the previous step was dissolved in 10 mL of methanol, and palladium carbon with a mass fraction of 10% was added. The mixture was stirred overnight at room temperature under a hydrogen gas atmosphere to completely react, and then filtered under reduced pressure through diatomaceous earth and eluted with an appropriate amount of methanol. The filtrate was dried by spinning to obtain 236 mg of intermediate 4, which was used directly in the next step without purification. MS (ESI) m / z: 290.1 ​​[M+H]+.

[0190] Step e: Preparation of intermediate 5 (2-(2-(4,4-difluorocyclohexyl)thiazol-4-yl)acetic acid). TIFF2025507472000205.tif13170 The whole of intermediate 4 was dissolved in MeOH / H2O (5ml:5ml) solution, lithium hydroxide solution (1M; 2.45mL, 2.45mmol) was added, and the reaction system was stirred at 0°C for 3 hours. After the reaction was completed, 5mL of water was added to the reaction solution, and the pH was adjusted to 1 with hydrochloric acid, and then extracted with ethyl acetate (10mL x 3). The organic phase was combined and dried over anhydrous Na2SO4, and the filtrate was concentrated under reduced pressure to obtain 210mg of crude product, which was used directly in the next step without further purification. MS (ESI) (m / z): 262.1 [M+H] + .

[0191] Step f: Preparation of intermediate 6 ((2S,3R)-3-(2-(2-(4,4-difluorocyclohexyl)thiazol-4-yl)acetamido)-2-hydroxy-4-phenyl-N-(thiazol-2-ylmethyl)butyramide). TIFF2025507472000206.tif28170 Intermediate 5 (210 mg, 0.79 mmol), intermediate 2 (238 mg, 0.82 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (373 mg, 0.95 mmol), N,N-diisopropylethylamine (427 μL, 2.37 mmol) were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 hours, then diluted with dichloromethane, washed with saturated ammonium chloride, saturated sodium bicarbonate and saturated sodium chloride in turn, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, the sample was stirred, and subjected to column chromatography to obtain intermediate 6 (482 mg, 87%) as a white oil. 1 H NMR (400 MHz, DMSO-d6) δ 8.57 (t, J = 6.2 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.69 (d, J = 3.3 Hz, 1H), 7.57 (d, J = 3.3 Hz, 1H), 7.25 (dd, J = 8.1, 6.7 Hz, 2H), 7.21-7.14 (m, 3H), 7.08 (s, 1H), 6.20 (d, J = 5.8 Hz, 1H), 4.60-4.45 (m, 2H), 4.35-4.24 (m, 1H), 3.94 (dd, J = 5.8, 2.6 Hz, 1H), 3.57-3.45 (m, 2H), 3.23-3.17 (m, 1H), 2.92-2.82 (m, 1H), 2.72-2.61 (m, 1H), 2.20-1.89 (m, 6H), 1.82-1.68 (m, 2H). MS (ESI) m / z: 535.2 [M+H] + .

[0192] Step f: Preparation of compound 511 ((R)-3-(2-(2-(4,4-difluorocyclohexyl)thiazol-4-yl)acetamido)-2-oxo-4-phenyl-N-(thiazol-2-ylmethyl)butyramide). TIFF2025507472000207.tif28170 Intermediate 6 (53 mg, 0.1 mmol) was dissolved in 10 mL of dehydrated dichloromethane, and then Dess-Martin oxidant (51 mg, 0.12 mmol) was added in several portions. After stirring at 0°C for 2 hours, the mixture was quenched with sodium thiosulfate solution. The mixture was extracted with dichloromethane (30 mL x 2). The organic layers were combined and extracted with saturated NaHCO3 and saturated brine, and further dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was separated and purified by column chromatography to obtain product 511 (37 mg, 70%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.55 (t, J = 6.2 Hz, 1H), 8.51 (d, J = 7.1 Hz, 1H), 7.73 (d, J = 3.3 Hz, 1H), 7.64 (d, J = 3.3 Hz, 1H), 7.30-7.16 (m, 5H), 7.13 (s, 1H), 5.30-5.20 (m, 1H), 4.62 (dd, J = 6.2, 2.8 Hz, 2H), 3.58 (s, 2H), 3.21-3.09 (m, 2H), 2.84 (dd, J = 14.0, 9.0 Hz, 1H), 2.14-2.03 (m, 4H), 2.04-1.88 (m, 2H), 1.79-1.62 (m, 2H). HRMS (ESI-TOF) m / z calcd. for C 25 H 26 F2N4O3S2 [M+H] + 533.1487, found 533.1493.

[0193] Example 13 Preparation of Compound 516 TIFF2025507472000208.tif92170

[0194] Step a: Same as step a in Example 10. Step b: Same as step b in Example 10.

[0195] Step c: Preparation of intermediate 3 (3-(hydroxyamino)-3-iminopropionic acid ethyl ester). TIFF2025507472000209.tif13170 Ethyl cyanoacetate (113 mg, 1.0 mmol), hydroxylamine hydrochloride (70 mg, 1.0 mmol) and sodium carbonate (212 mg, 2.0 mmol) were dissolved in ethanol and refluxed for 3 to 4 hours. After the reaction was completed, the reaction solvent was dried by rotating under reduced pressure, water was added and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was subjected to column chromatography to obtain intermediate 3 (73 mg, 50%). 1 H NMR (400 MHz, CDCl3) δ 5.04 (s, 2H), 4.20 (m, 2H), 3.19 (s, 2H), 1.30 (t, J = 4.6 3H). MS (ESI) m / z: 147.1 [M+H] + .

[0196] Step d: Preparation of intermediate 4 (2-(5-(3,4-difluorophenyl)-1,2,4-oxadiazol-3-yl)acetic acid ethyl ester). TIFF2025507472000210.tif14170 Intermediate 3 (73 mg, 0.5 mmol) from the previous step and 3,4-difluorobenzoic acid chloride (96 mg, 0.55 mmol) were dissolved in 10 mL of pyridine and reacted at 90 ° C for 20 h. After the reaction was completed, the solvent was dried by rotating, diluted with water, and extracted with ethyl acetate three times. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by column chromatography to obtain intermediate 4 (94 mg, 0.35 mmol, 70%). MS (ESI) m / z: 269.1 [M+H] + .

[0197] Step e: Preparation of intermediate 5 (2-(5-(3,4-difluorophenyl)-1,2,4-oxadiazol-3-yl)acetic acid). TIFF2025507472000211.tif14170 Intermediate 4 (94 mg, 0.35 mmol) obtained in the previous step was dissolved in MeOH / H2O (2 mL:2 mL), lithium hydroxide solution (1 M; 1.05 mL, 1.05 mmol) was added, and the reaction system was stirred at 0 °C for 3 hours. After the reaction was completed, 5 mL of water was added to the reaction solution, and the pH was adjusted to 1 with hydrochloric acid, and then extracted with ethyl acetate. The organic phases were combined and dried over anhydrous Na2SO4, and the filtrate was concentrated under reduced pressure to obtain 72 mg of crude product, which was used directly in the next step without further purification.

[0198] Step f: Preparation of intermediate 6 ((2S,3R)-3-(2-(5-(3,4-difluorophenyl)-1,2,4-oxadiazol-3-yl)acetamido)-2-hydroxy-4-phenyl-N-(thiazol-2-ylmethyl)butyramide). TIFF2025507472000212.tif29170 Intermediate 5 (72 mg, 0.3 mmol), intermediate 2 (87 mg, 0.3 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (136 mg, 0.36 mmol), N,N-diisopropylethylamine (156 μL, 0.9 mmol) were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 hours, then diluted with dichloromethane, washed with saturated NH4Cl, saturated NaHCO3 and saturated NaCl in sequence, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain white oily intermediate 6 (126 mg, 82%). MS (ESI) m / z: 514.1 [M+H] + .

[0199] Step g: Preparation of final product 516 ((R)-3-(2-(5-(3,4-difluorophenyl)-1,2,4-oxadiazol-3-yl)acetamido)-2-oxo-4-phenyl-N-(thiazol-2-ylmethyl)butyramide). TIFF2025507472000213.tif29170 Intermediate 6 (52 mg, 0.1 mmol) was dissolved in 10 mL of dichloromethane, and then Dess-Martin oxidant (51 mg, 0.12 mmol) was added in several portions. After stirring at 0°C for 2 hours, the mixture was directly quenched with sodium thiosulfate solution, and the mixture was extracted with dichloromethane. The organic layers were combined and extracted with saturated sodium bicarbonate and brine in sequence, and then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was separated and purified by column chromatography to obtain product 516 (29 mg, 57%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.60 (t, J = 6.3 Hz, 1H), 8.85 (d, J = 7.1 Hz, 1H), 8.21-8.10 (m, 1H), 8.00-7.92 (m, 1H), 7.78-7.67 (m, 2H), 7.65-7.62 (m, 1H), 7.33-7.18 (m, 5H), 5.32-5.22 (m, 1H), 4.68-4.55 (m, 2H), 3.79 (s, 2H), 3.21-3.12 (m, 1H), 2.91-2.80 (m, 1H). HRMS (ESI-TOF) m / z calcd. for C 24 H 19 F2N5O4S [M+H] + 512.1199, found 521.1199.

[0200] Example 14 Preparation of Compound 518 TIFF2025507472000214.tif91170

[0201] Step a: Same as step a in Example 10. Step b: Same as step b in Example 10.

[0202] Step c: Preparation of intermediate 3 (2-(2,5-dioxoimidazolidin-1-yl)acetic acid ethyl ester). TIFF2025507472000215.tif16170 The raw materials imidazolidine-2,4-dione (100 mg, 1.0 mmol), ethyl bromoacetate (165 mg, 1.0 mmol) and potassium carbonate (276 mg, 2.0 mmol) were dissolved in acetonitrile solvent, heated under reflux for 6 to 8 hours and monitored by TLC. After the reaction was completed, the acetonitrile was dried by rotary evaporation under reduced pressure, then diluted with ethyl acetate and washed with water and saturated sodium chloride solution. The organic phase was rotary dried to obtain 167 mg of intermediate 3 crude product. 1 H NMR (400 MHz, CDCl3) 6.24 (s, 1H), 4.25 (s, 2H), 4.22 (m, 2H), 4.06 (s, 2H), 1.28 (t, J = 8.0, 3H). MS (ESI) (m / z): 187.0 [M+H] + .

[0203] Step d: Preparation of intermediate 4 (2-(3-(3-cyano-5-fluorophenyl)-2,5-dioxoimidazolidin-1-yl)acetic acid ethyl ester). TIFF2025507472000216.tif16170167mg of intermediate 3 crude product, 3-bromo-5-fluorobenzonitrile (178mg, 0.9mmol), tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (102mg, 0.1mmol), cesium carbonate (650mg, 2.0mmol) were dissolved in toluene and refluxed overnight under the protection of nitrogen gas. After the reaction was completed, it was concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed with water, the organic phase was dried with anhydrous sodium sulfate, the sample was stirred, and separated and purified by column chromatography to obtain intermediate 4 (238mg, 87%). MS (ESI) m / z: 306.1 [M+H] + .

[0204] Step e: Preparation of intermediate 5 (2-(3-(3-cyano-5-fluorophenyl)-2,5-dioxoimidazolidin-1-yl)acetic acid). TIFF2025507472000217.tif20170 Intermediate 4 (238 mg, 0.783 mmol) obtained in the previous step was dissolved in MeOH / H2O (5 mL:5 mL), lithium hydroxide solution (1 M; 2.35 mL, 2.35 mmol) was added, and the reaction system was stirred at 0 ° C for 3 hours. After the reaction was completed, 5 mL of water was added to the reaction solution, and the pH was adjusted to 1 with hydrochloric acid, and then extracted with ethyl acetate (10 mL × 3), the organic phases were combined, dried over anhydrous Na2SO4, and the filtrate was concentrated under reduced pressure to obtain 220 mg of crude product, which was used directly in the next step without further purification. MS (ESI) (m / z): 278.0 [M+H] + .

[0205] Step f: Preparation of intermediate 6 ((2S,3R)-3-(2-(3-(3-cyano-5-fluorophenyl)-2,5-dioxoimidazolidin-1-yl)acetamide)-2-hydroxy-4-phenyl-N-(thiazol-2-ylmethyl)butyramide). TIFF2025507472000218.tif25170 Intermediate 5 (220 mg, 0.79 mmol), intermediate 2 (230 mg, 0.79 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (360 mg, 0.95 mmol), N,N-diisopropylethylamine (412 μL, 2.37 mmol) were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 hours, then diluted with dichloromethane, washed with saturated NH4Cl, saturated NaHCO3 and saturated NaCl in turn, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, the sample was stirred and subjected to column chromatography to obtain intermediate 6 (334 mg, 77%) as a white oil. MS (ESI) m / z: 551.2 [M+H] + .

[0206] Step f: Preparation of final product 518 ((R)-3-(2-(3-(3-cyano-5-fluorophenyl)-2,5-dioxoimidazolidin-1-yl)acetamide)-2-oxo-4-phenyl-N-(thiazol-2-ylmethyl)butyramide). TIFF2025507472000219.tif29170 Intermediate 6 (55 mg, 0.1 mmol) was dissolved in 10 ml of ultra-dehydrated CH2Cl2, and then Dess-Martin oxidant (51 mg, 0.12 mmol) was added in several portions. After stirring at 0°C for 2 hours, the mixture was directly quenched with sodium thiosulfate solution, and the mixture was extracted with dichloromethane (30 ml x 2). The organic layers were combined and extracted with saturated NaHCO3 and brine, and further dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography to obtain product 518 (33 mg, 60%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.60 (t, J = 6.3 Hz, 1H), 8.78 (d, J = 7.1 Hz, 1H), 8.21 (d, J = 1.9 Hz, 1H), 7.73 (d, J = 3.2 Hz, 1H), 7.63 (d, J = 3.3 Hz, 1H), 7.50-7.44 (m, 1H), 7.34-7.15 (m, 6H), 5.29-5.21 (m, 1H), 4.65-4.60 (m, 2H), 4.12 (s, 2H), 3.89 (s, 2H), 3.21-3.11 (m, 1H), 2.92-2.83 (m, 1H). HRMS (ESI-TOF) m / z calcd. for C 26 H 21 FN6O5S [M+H] + 549.1351, found 549.1355.

[0207] Example 15 Preparation of Compound 532 TIFF2025507472000220.tif108170

[0208] Step a: Same as step a in Example 10. Step b: Same as step b in Example 10.

[0209] Step c: Preparation of intermediate 3 (ethyl 2-(4-(2-fluorophenyl)-1H-1,2,3-triazol-1-yl)acetate). TIFF2025507472000221.tif14170Ethyl bromoacetate (166mg, 1.0mmol), 1-ethynyl-2-fluorobenzene (120mg, 1.0mmol), sodium azide (72mg, 1.1mmol), sodium ascorbate (40mg, 0.2mmol) and copper sulfate pentahydrate (50mg, 0.2mmol) were weighed into a 25ml eggplant flask, tert-butanol / water (10ml / 5ml) was added, and the reaction was carried out at room temperature for 24h. The reaction was monitored by TLC. After the reaction was completed, appropriate water was added, and the mixture was extracted with ethyl acetate, concentrated and passed through a column to obtain 175mg of intermediate 3. Yield 70%. MS (ESI) m / z: 250.1 [M+H] + .

[0210] Step d: Preparation of intermediate 4 (2-(4-(2-fluorophenyl)-1H-1,2,3-triazol-1-yl)acetic acid). TIFF2025507472000222.tif14170 Intermediate 3 (125 mg, 0.5 mmol) obtained in the previous step was dissolved in MeOH / H2O (2 mL:2 mL), lithium hydroxide solution (1 M; 1.05 mL, 1.05 mmol) was added, and the reaction system was stirred at 0 °C for 3 hours. After the reaction was completed, 5 mL of water was added to the reaction solution, and the pH was adjusted to 1 with hydrochloric acid, and then extracted with ethyl acetate. The organic phases were combined and dried over anhydrous Na2SO4, and the filtrate was concentrated under reduced pressure to obtain 120 mg of crude product, which was used directly in the next step without further purification.

[0211] Step e: Preparation of intermediate 5 ((2S,3R)-3-(2-(4-(2-fluorophenyl)-1H-1,2,3-triazol-1-yl)acetamido)-2-hydroxy-4-phenyl-N-(thiazol-2-ylmethyl)butyramide). TIFF2025507472000223.tif30170 Intermediate 4 (75 mg, 0.3 mmol), intermediate 2 (87 mg, 0.3 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (136 mg, 0.36 mmol), N,N-diisopropylethylamine (156 μL, 0.9 mmol) were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 hours, then diluted with dichloromethane, washed with saturated NH4Cl, saturated NaHCO3 and saturated NaCl in sequence, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was separated and purified by column chromatography to obtain white solid intermediate 5 (117 mg, 79%). MS (ESI) m / z: 495.1 [M+H] + .

[0212] Step e: Preparation of compound 532 ((R)-3-(2-(4-(2-fluorophenyl)-1H-1,2,3-triazol-1-yl)acetamido)-2-oxo-4-phenyl-N-(thiazol-2-ylmethyl)butyramide). TIFF2025507472000224.tif30170 Intermediate 5 (49 mg, 0.1 mmol) was dissolved in 10 mL of dichloromethane, and then Dess-Martin oxidant (51 mg, 0.12 mmol) was added in several portions. After stirring at 0°C for 2 hours, the mixture was directly quenched with sodium thiosulfate solution, and the mixture was extracted with dichloromethane (30 mL x 2). The organic layers were combined and extracted with saturated NaHCO3 and brine, and further dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was separated and purified by column chromatography to obtain product 532 (24 mg, 49%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.63 (t, J = 6.2 Hz, 1H), 8.97 (d, J = 7.1 Hz, 1H), 8.31 (d, J = 3.8 Hz, 1H), 8.13 (td, J = 7.6, 1.7 Hz, 1H), 7.73 (d, J = 3.3 Hz, 1H), 7.62 (d, J = 3.3 Hz, 1H), 7.44-7.39 (m, 1H), 7.38-7.27 (m, 4H), 7.27-7.19 (m, 3H), 5.30 (dq, J = 11.7, 4.0 Hz, 1H), 5.24 (d, J = 3.7 Hz, 2H), 4.62 (dd, J = 6.2, 2.1 Hz, 2H), 3.25-3.13 (m, 1H), 2.96-2.82 (m, 1H). MS (ESI) m / z: 493.1 [M+H] + .

[0213] Other target compounds of the present invention were prepared with reference to the methods of Examples 1 to 15. The structures and property data of the obtained target compounds are shown in Table 1.

[0214] Table 1. Structure and properties of the compounds of the present invention [Table 1] TIFF2025507472000226.tif237170TIFF2025507472000227.tif237170TIFF2025507472000228.tif254170TIFF2025507472000229.tif254170TIFF2025507472000230.tif245170TIFF2025507472000231.tif236170TIFF2025507472000232.tif237170TIFF2025507472000233.tif254170TIFF2025507472000234.tif237170TIFF2025507472000235.tif254170TIFF2025507472000236.tif237170TIFF2025507472000237.tif237170TIFF2025507472000238.tif245170TIFF2025507472000239.tif245170TIFF2025507472000240.tif245170TIFF2025507472000241.tif236170TIFF2025507472000242.tif245170TIFF2025507472000243.tif253170TIFF2025507472000244.tif245170TIFF2025507472000245.tif253170TIFF2025507472000246.tif253170TIFF2025507472000247.tif236170TIFF2025507472000248.tif236170TIFF2025507472000249.tif245170TIFF2025507472000250.tif228170TIFF2025507472000251.tif253170TIFF2025507472000252.tif245170TIFF2025507472000253.tif236170TIFF2025507472000254.tif236170TIFF2025507472000255.tif236170TIFF2025507472000256.tif236170TIFF2025507472000257.tif236170TIFF2025507472000258.tif245170TIFF2025507472000259.tif236170TIFF2025507472000260.tif253170TIFF2025507472000261.tif245170TIFF2025507472000262.tif228170TIFF2025507472000263.tif236170TIFF2025507472000264.tif236170TIFF2025507472000265.tif253170TIFF2025507472000266.tif253170TIFF2025507472000267.tif245170TIFF2025507472000268.tif245170TIFF2025507472000269.tif245170TIFF2025507472000270.tif253170TIFF2025507472000271.tif244170TIFF2025507472000272.tif253170TIFF2025507472000273.tif237170TIFF2025507472000274.tif237170TIFF2025507472000275.tif228170TIFF2025507472000276.tif245170TIFF2025507472000277.tif253170TIFF2025507472000278.tif253170TIFF2025507472000279.tif236170TIFF2025507472000280.tif253170TIFF2025507472000281.tif228170TIFF2025507472000282.tif253170TIFF2025507472000283.tif253170TIFF2025507472000284.tif236170TIFF2025507472000285.tif245170TIFF2025507472000286.tif228170TIFF2025507472000287.tif245170TIFF2025507472000288.tif228170TIFF2025507472000289.tif228170TIFF2025507472000290.tif227170TIFF2025507472000291.tif252170TIFF2025507472000292.tif252170TIFF2025507472000293.tif252170TIFF2025507472000294.tif228170TIFF2025507472000295.tif227170TIFF2025507472000296.tif253170TIFF2025507472000297.tif253170TIFF2025507472000298.tif228170TIFF2025507472000299.tif253170TIFF2025507472000300.tif236170TIFF2025507472000301.tif227170TIFF2025507472000302.tif236170TIFF2025507472000303.tif253170TIFF2025507472000304.tif253170TIFF2025507472000305.tif228170TIFF2025507472000306.tif244170TIFF2025507472000307.tif252170TIFF2025507472000308.tif253170TIFF2025507472000309.tif236170TIFF2025507472000310.tif253170TIFF2025507472000311.tif253170TIFF2025507472000312.tif244170TIFF2025507472000313.tif253170TIFF2025507472000314.tif253170TIFF2025507472000315.tif253170TIFF2025507472000316.tif228170TIFF2025507472000317.tif219170TIFF2025507472000318.tif227170TIFF2025507472000319.tif252170TIFF2025507472000320.tif236170TIFF2025507472000321.tif227170TIFF2025507472000322.tif252170TIFF2025507472000323.tif253170TIFF2025507472000324.tif253170TIFF2025507472000325.tif253170TIFF2025507472000326.tif253170 TIFF2025507472000327.tif253170TIFF2025507472000328.tif253170TIFF202550 7472000329.tif253170TIFF2025507472000330.tif227170TIFF2025507472000331 .tif252170TIFF2025507472000332.tif253170TIFF2025507472000333.tif69170.

[0215] The beneficial effects of the compounds of the present invention will now be demonstrated by test examples.

[0216] Test Example 1: M of the compound of the present invention pro Testing the level of inhibition of enzyme activity (1) Test method Recombinant SARS-CoV-2 M pro (final concentration 750 nM) was mixed with serial dilutions of various compounds in 25 μL of assay buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM DTT) and incubated for 10 min. The reaction was initiated by adding 25 μL of fluorescent substrate (MCA-AVLQ↓SGFR-Lys(Dnp)-Lys-NH2) at a final concentration of 20 μM, and the fluorescent signal was measured at 320 nm (excitation) / 405 nm (emission) using a microplate reader. The Vmax of the reaction with different concentrations of compounds and the Vmax of the reaction with DMSO were calculated and used to calculate the IC 50 Curves were generated for each compound at nine concentrations and three independent replicates to determine the anti-SARS-CoV-2 M pro IC 50 All experimental data were analyzed using GraphPad Prism software.

[0217] (2) Test results

[0218] Table 2 Compounds of SARS-COV-2 M Pro Enzyme inhibitory activity against [Table 2] TIFF2025507472000335.tif250170TIFF2025507472000336.tif250170TIFF202 5507472000337.tif250170TIFF2025507472000338.tif250170TIFF20255074720 00339.tif250170TIFF2025507472000340.tif250170TIFF2025507472000341.t if250170TIFF2025507472000342.tif250170TIFF2025507472000343.tif165170

[0219] As can be seen from Table 2 and Figures 1 to 5, the compounds of the present invention inhibit SARS-CoV-2 M Pro The compounds of the present invention can effectively inhibit the activity of SARS-CoV-2 M Pro The inhibitors can be used to prepare drugs against the new coronavirus, and drugs for the prevention and / or treatment of new coronavirus pneumonia.

[0220] Test Example 2: Toxicity test of compounds against Vero E6 cells (1) Test method The cytotoxicity of the compounds was evaluated using Vero E6 cells. The specific test plan was as follows: Vero E6 cells were cultured in a 96-well plate at 2 × 10 4Cells were seeded at a cell density of 100 μL / well and incubated overnight at 37°C in a 5% CO2 incubator. The next day, 100 μL of drug-containing medium was added to each well, and the compounds were diluted in a 2-fold gradient starting from an initial concentration of 500 μM, for a total of six gradients, with three duplicate wells for each concentration, and a drug-free negative control and a blank control in each set of tests. After 72 h of drug treatment, the cell viability was measured using the MTT method, and the median cytotoxic concentration (CC) of the compounds for Vero E6 cells was determined. 50 ) values ​​were calculated. All experimental data were analyzed using GraphPad Prism software.

[0221] (2) Test results

[0222] Table 3. Toxicity of compounds of the present invention against Vero E6 cells [Table 3]

[0223] As can be seen from Table 3, the compounds of the present invention have very low toxicity to Vero E6 cells.

[0224] Test Example 3: In vivo pharmacokinetic characterization of compounds in rats (1) Dosage regimen Male Sprague-Dawley (SD) rats weighing 200-230 g were used. All animals were randomly divided into groups of 3 animals each, and a series of test compounds were administered orally (po) or intravenously (iv) according to the schedule in Table 4 below. The animals were fasted for 12 h before the test and allowed free access to water. Food was provided uniformly 4 h after administration.

[0225] Solutions for oral gavage and intravenous administration were prepared with DMSO / HS15 / PEG400 / NaCl (5 / 3 / 40 / 52, v / v / v). Drugs were administered according to the dosages shown in Table 4, and the administration times were recorded. At the above-mentioned set time points, approximately 0.20 mL of each sample was collected by jugular vein blood collection or other appropriate method, anticoagulated with sodium heparin, and placed on ice after collection. Then, plasma was centrifuged within 1 hour (centrifugation conditions: 6800 g, 6 min, 2-8°C). Plasma samples were stored in a -80°C freezer until analysis. Grouping and blood collection time points are shown in Table 4. Three animals were used at each time point.

[0226] Table 4. Study plan for in vivo pharmacokinetic characterization of compounds in rats [Table 4] TIFF2025507472000346.tif27170

[0227] Table 5. Main pharmacokinetic parameters of the compounds [Table 5] TIFF2025507472000348.tif19170

[0228] The test results show that the compounds of the present invention have good pharmacokinetics in SD rats.

[0229] Test Example 4: Toxicity test of compounds against human-derived BEAS-2B cells and HUVEC cells (1) Test method Cytotoxicity evaluation of the compound against human-derived cells was performed using BEAS-2B cells (human normal bronchial epithelial cells) and HUVEC cells (human umbilical vein endothelial cells). The specific test plan is as follows: BEAS-2B cells or HUVEC cells were placed in a 96-well plate at 2 × 10 4Cells / well were seeded at a cell density of 100 μL / well and incubated overnight in a 37°C, 5% CO2 incubator. The next day, 100 μL of drug-containing medium was added to each well, and the compounds were diluted in a 2-fold gradient starting from an initial concentration of 500 μM, for a total of six gradients, with three duplicate wells for each concentration, and a drug-free negative control and a blank control were included in each set of tests. After 72 h of drug treatment, the cell viability was measured using the MTT method, and the median cytotoxic concentration (CC) of the compounds for BEAS-2B cells or HUVEC cells was determined. 50 ) values ​​were calculated. All experimental data were analyzed using GraphPad Prism software.

[0230] (2) Test results

[0231] Table 6. Toxicity of compounds to human-derived BEAS-2B and HUVEC cells [Table 6]

[0232] As can be seen from Table 6, the toxicity of the compounds of the present invention to the human-derived cells BEAS-2B cells or HUVEC cells tested is very low.

[0233] Test Example 5: Stability test of compounds against metabolism by human liver microsomes (1) Test method 5 μL of 10 mM test compound stock solution was added to 95 μL of methanol to prepare 500 μM test compound solution. 0.1 M potassium phosphate buffer (K-buffer) at pH 7.40 ± 0.01 was pre-heated in a 37°C water bath. 1.5 μL of 500 μM test compound solution and 18.75 μL of 20 mg / mL human liver microsomes were added to 479.75 μL of pre-heated K-buffer to obtain a 1.5 μM test compound solution containing 0.75 mg / mL microsome solution. 3×NADPH stock solution (6 mM, 5 mg / mL) was prepared by dissolving NADPH in NADPH buffer. 30 μL of the 1.5 μM solution containing 0.75 mg / mL microsome solution was dispensed into the designated test plate used at each time point (0, 5, 15, 30, 45, 60 min). For 0 min, 150 μL of methanol containing an internal standard was added to the wells of the 0 min plate in advance. All other test plates were pre-incubated at 37°C for 5 min. 15 μL of NADPH stock solution (6 mM) was added to all test plates to start the reaction and time calculation. At 5 min, 15 min, 30 min, 45 min, and 60 min, 150 μL of methanol containing an internal standard was added to the wells of the corresponding plate to stop the reaction. After stopping the reaction, the test plate was shaken (600 rpm) for 10 min on a shaker and centrifuged at 4000 rpm for 15 min. 80 μL of the supernatant from each well was transferred to a 96-well sample plate containing 120 μL of water and used for LC / MS analysis.

[0234] (2) Test results

[0235] Table 7. Stability of the compounds of the present invention against metabolism by human liver microsomes [Table 7]

[0236] The test results in Table 7 show that the compounds of the present invention have good stability against metabolism by human liver microsomes. PF-07321332, a commercially available novel coronavirus main protease inhibitor developed by Pfizer, has a elimination half-life (T 1 / 2 ) was 21.18 min, and the compounds of the present invention have superior stability to human liver microsomal metabolism to PF-07321332.

[0237] Test Example 6: Measurement of biochemical activity of compounds against human proteases (1) Test method The biochemical activity of the compounds against human proteases was measured using Biovision's Human Cathepsin K Inhibitor Screening Kit (K150-100), Human Cathepsin B Inhibitor Screening Kit (K147-100), Human Thrombin Activity Test Kit (K373-100), Human Caspase-2 Inhibitor Screening Kit (K152-100), and Human Cathepsin D Inhibitor Screening Kit (K148-100) according to the kit instructions. All tests were performed in triplicate. All test data was analyzed using GraphPad Prism software.

[0238] (2) Test results

[0239] Table 8. Biochemical activity of the compounds of the present invention against human proteases [Table 8] As can be seen from the test results in Table 8, the compounds of the present invention have almost no activity against several common human-derived proteases with similar structures, namely, human cathepsin K, human cathepsin B, human thrombin, human caspase 2 and human cathepsin D, indicating that the compounds of the present invention have good selectivity against the main protease of the new coronavirus.

[0240] Test Example 7: Test of the effect of Compound 398 on the hERG potassium ion channel (1) Purpose of the test Rapid activation of the human delayed rectifier outward potassium current (IKr) is mainly mediated by the hERG ion channel and is involved in the repolarization of human cardiomyocytes. When this current is blocked by drugs, it can lead to clinical long QT syndrome, which can easily induce acute arrhythmias and sudden death. In this test example, the manual patch clamp technique is applied to test the effect of compound 398 on the hERG potassium current in a stable cell line transfected with the hERG potassium channel, thereby confirming whether the test substance has an inhibitory effect on the hERG ion channel.

[0241] (2) Test method After subculture of HEK-293-hERG cells to an appropriate state, they were digested and separated with trypsin and placed in a centrifuge tube. After centrifugation, the supernatant was discarded and the cells were resuspended in extracellular fluid for use. Before patch clamp recording, the cells were dropped onto a culture dish to ensure that the cells had a certain density and were in a single, separated state.

[0242] hERG currents were recorded using the whole-cell patch clamp technique. The cell suspension was placed in a Petri dish and placed on an inverted microscope stage. After the cells attached to the wall, they were perfused with extracellular solution at a flow rate of 1–2 mL / min. Glass microelectrodes were drawn in two steps by a micropipette puller, and the inlet resistance after filling the electrode with the solution was 2–5 MΩ.

[0243] After establishing the whole-cell recording mode, the clamp potential was held at -80 mV. hERG tail currents were evoked by applying a depolarizing voltage to +60 mV for 850 ms, followed by a repolarization to -50 mV for 1275 ms. This series of pulse programs was repeated every 15 s throughout the study.

[0244] After the current was stabilized, extracellular continuous perfusion administration from low to high concentration was used. Starting from low concentration, perfusion was continued until the drug effect was stabilized, i.e., the change in the last 5 stimulation strip current values ​​of each concentration administration step was less than 10% (if the current was 200 pA or more) or less than 30% (if the current was less than 200 pA) of the mean value, and then perfusion was performed with the next concentration. The blocking effect of each test substance (0.3, 1, 3, 10, 30 μM) and the positive control Cisapride on the hERG tail current was tested. Stimulation discharge and signal acquisition were performed by PatchMaster or Clampex 10.6 software, and the signal was amplified by a patch clamp amplifier. Further data analysis and curve fitting were performed using FitMaster or Clampfit 10.6, EXCEL, Graphpad Prism, SPSS 21.0, etc. Data were presented as mean value and standard deviation.

[0245] (3) Test results Under the conditions of this test, the inhibitory effect of the positive control substance cisapride on the hERG current was concentration-dependent, and the inhibition rate on the hERG current at a concentration of 0.1 μM was 86.36%. This indicates that the test results are reliable. Under the conditions of this test, the average inhibition rate on the hERG current at the maximum concentration of 30 μM was 1.28%. That is, the IC 50 It has a relatively low cardiac toxicity and is unlikely to induce acute arrhythmia or sudden death after administration.

[0246] Table 9. Inhibitory effect of the compounds of the present invention on hERG current [Table 9]

[0247] Test Example 8: Measurement of antiviral activity of the compound of the present invention at the cellular level by RT-qPCR method (1) Test method Vero E6-TMPRSS2 cells were infected with SARS-CoV-2 (B.1.1.529) BA.1 mutant strain at an MOI of 0.1. The cells were treated with 5-fold dilutions of the compounds of the present invention from 20 μM to 0.0013 μM, and PF-07321332 was used as a positive control compound. Cell lysates were collected at 24 hpi for RT-qPCR analysis. All experiments were performed in triplicate. All experimental data were analyzed using GraphPad Prism software.

[0248] (2) Test results The test results are shown in Figure 6. The results show that the compounds of the present invention are effective in inhibiting the replication of mutant SARS-CoV-2 (B.1.1.529) in Vero E6-TMPRSS2 cells. Here, under the conditions of this test, compounds 398 and 395 had better inhibitory activity against the replication of SARS-CoV-2 mutant (B.1.1.529) than the positive control compound PF-07321332.

[0249] Test Example 9: Measurement of antiviral activity of compound 398 at the cellular level by plaque assay (1) Test method Vero E6-TMPRSS2 cells were infected in 12-well plates with 50–70 PFU / well of each of MERS-CoV, SARS-CoV-1, SARS-CoV-2, Alpha (B.1.1.7) variant, Beta (B.1.351) variant, and Omicron (B.1.1.529) BA.2 and BA.5 variants. The cells were washed with PBS, covered with 2% agarose / PBS, and mixed 1:1 with 2×DMEM / 2%FBS. At 2 hpi, the cells were treated with WSK-S02 and the positive compound Nirmatrelvir, diluted 5-fold from 0.0013 μM to 20 μM. At 72 hpi, the cells were fixed and stained with 0.5% crystal violet in 25% ethanol / distilled water for 10 min, and the stained plaques were quantified (n=3). EC 50 (Median Effective Concentration) values ​​were calculated using a dose-response model in GraphPad Prism 8.0 software.

[0250] (2) Test results The test results are shown in Figure 7. As the results show, compound 398 can reduce the cytopathic effect caused by MERS-CoV, SARS-CoV-1, SARS-CoV-2 B.1.1.7 (Alpha), SARS-CoV-2 B.1.351 (Beta), SARS-CoV-2 Omicron BA.2 and BA.5 strains infected with Vero E6-TMPRSS2, and the antiviral activity is 1.79, 17.14, 40.95, 10.16, 2.82 and 4.00 times higher than the positive control compound nilmatrellvir (PF-07321332), respectively, which indicates that compound 398 has higher antiviral activity against mutant strains such as SARS-CoV-2 Omicron and other coronaviruses compared to nilmatrellvir.

[0251] Test Example 10: Pharmacodynamic evaluation of the in vivo antiviral activity of compound 398 in K18-hACE2 transgenic mice (1) Test method K18-hACE2 transgenic mice (6–8 weeks old) were purchased from Jackson Laboratory and animals were used in accordance with all relevant ethical regulations and approved by the Committee on the Use of Live Animals in Teaching and Research at the University of Hong Kong. Female or male K18-hACE2 transgenic mice were inoculated intranasally (in) with 2000 PFU of the BA.2-CoV-2 Omicron (B.1.1.529) mutant strain. For early treatment, mice were orally administered 150 mg / kg WSK-S02 Compound 398 twice daily from 1 hpi to day 4 (dpi) on the day of infection.

[0252] For late treatment, mice were orally administered Compound 398 (150 mg / kg) or Compound 398 (150 mg / kg) / ritonavir (RTV, 10 mg / kg), nilmatrellvir (150 mg / kg) or nilmatrellvir (150 mg / kg) / ritonavir (RTV, 10 mg / kg) twice daily from 1 dpi until 4 dpi. Mice treated with vehicle (5% DMSO / 3% Solutol HS-15 / 40% PEG400 / saline) served as control. Mice were monitored daily for survival during the study period, and sacrificed at 4 dpi for organ tissue sampling and virological and histopathological analysis.

[0253] Measurement of viral gene copy number: K18-hACE2 transgenic mouse models infected with SARS-CoV-2 Omicron (B.1.1.529) BA.2 were treated until 4 dpi and sacrificed, and lung tissue samples were obtained from each group of mice. Tissue samples from transgenic mice were lysed using RLT buffer (Qiagen) and extracted using the RNeasy Mini kit. After RNA extraction, RT-qPCR analysis was performed using the Transcriptor First Strand cDNA Synthesis Kit, QuantiNova SYBR Green RT-PCR kit, or QuantiNova Probe RT-PCR kit.

[0254] Measurement of viral titer: Vero E6-TMPRSS2 cells were seeded in 12-well plates one day before infection. K18-hACE2 transgenic mouse models infected with SARS-CoV-2 Omicron (B.1.1.529) BA.2 were treated until 4 dpi and sacrificed, and lung tissue samples were obtained from each group of mice. The supernatants of the collected tissue samples were serially diluted in a gradient and seeded on the cells for 1 h at 37°C. After seeding, the cells were washed three times with PBS and mixed with 2% agarose / PBS and 2xDMEM / 2%FBS in a 1:1 ratio. After 48 h, the cells were fixed and stained with 0.5% crystal violet in 25% ethanol / distilled water for 10 min and used for plaque quantification.

[0255] Histopathological studies: Nasal turbinates and lung tissues of transgenic mice immersed in formic acid were fixed overnight in 10% formalin. Fixed samples were then embedded in paraffin and sectioned at 5 μm using a TP1020 Leica benchtop semi-closed tissue processor. Tissue sections were removed and dried and fixed overnight on anti-peeling slides at 37°C. After deparaffinization and dehydration, sections were treated with antigen blocking agent, and the sections were heated at 85°C for 90s to expose antigens, followed by blocking with 0.3% hydrogen peroxide for 30 min and then with 1% BSA for 30 min. The primary antibody was rabbit anti-SARS-CoV-2-N immune serum, and the secondary antibody was goat anti-rabbit IgG antibody. Signals were generated using a DAB (3,3'-diaminobenzidine) substrate kit. Cell nuclei were labeled with Gill's hematoxylin. Slides were mounted with antifade mounting medium with DAPI. For H&E staining, sections of infected tissues were stained with Gill's hematoxylin and eosin Y. Analysis was performed by taking images using an Olympus BX53 light microscope.

[0256] (2) Test results As shown in Figure 8, in a study using an established model of K18-hACE2 transgenic mice infected with SARS-CoV-2 Omicron (B.1.1.529) BA.2, administration of compound 398 in early treatment significantly reduced viral genomic RNA (vRNA) by 179-fold in the lungs of infected mice at 4 dpi. In the same treatment regimen, production of viral subgenomic mRNA (sgRNA) was inhibited by 337-fold. Even when treatment with compound 398 was delayed until 24 hpi, vRNA and sgRNA copies in the lungs of infected mice were reduced by 16-fold and 12-fold, respectively, at 4 dpi. Compared to mice treated with nilmatrervir, both viral vRNA and sgRNA copies were reduced by 4-fold in the lungs of mice treated with compound 398.

[0257] The infectious viral load in the lungs was measured by plaque assay, and as shown in Figure 9, treatment with compound 398 also effectively inhibited the production of pulmonary infectious viral particles. Studies have reported that when coadministered with RTV, compound 398 delays the clearance of the compound by liver microsomes, thereby enhancing the pharmacokinetics and antiviral efficacy in vivo. Therefore, in this study, we also considered the synergistic therapeutic effect of coadministration of compound 398 and RTV. It should be noted that treatment with compound 398 / RTV was initiated at 24 hpi. Compared to single-agent treatment, the viral load in the lungs of infected mice was further reduced by 15-fold (vRNA), 43-fold (sgRNA), and 6-fold (infectious viral titer). Importantly, the infectious viral titer in the lungs of mice treated with compound 398 / RTV was significantly reduced by approximately 90% compared to mice treated with nilmatoruvir / RTV.

[0258] As shown in Figure 10, the results of immunohistochemical staining for SARS-CoV-2 nucleocapsid (N) protein (Figure 10A) showed that the expression of viral antigens (black arrows) in the lungs of mice was highest in the vehicle control group, followed by treatment with nilmatrellvir or compound 398 alone, and that both nilmatrellvir / RTV and compound 398 / RTV combination treatments similarly restricted N protein expression to very low levels in the lung tissue of infected mice. In addition, the analysis results of H&E staining of tissues (Figure 10B) showed that the most prominent lung pathological feature in the vehicle control group was multifocal inflammatory infiltration in the alveolar septa, peribronchiolar areas, and perivascular areas. In contrast, scattered inflammatory cell infiltration was rarely observed in the alveolar interstitium of mice treated with compound 398. Compared to single drug treatment, the combination administration of compound 398 / RTV further improved the lung tissue structure.

[0259] Test Example 11: In vivo pharmacokinetic characterization of compound 398 in mice, beagle dogs, and cynomolgus monkeys (1) Dosage regimen Male ICR mice (6-8 weeks old, weighing 16-25g), male beagle dogs (1-2 years old, weighing 9-12kg), or male cynomolgus monkeys (6-7 years old, weighing 6-8kg) were randomly assigned to groups of 3 each, and administered a series of test compounds by oral gavage (po) or intravenously (iv) according to the schedule in Table 10 below.

[0260] Solutions for oral gavage and intravenous administration were prepared with DMSO / HS15 / PEG400 / NaCl (5 / 3 / 40 / 52, v / v / v). Drugs were administered according to the dosages shown in Table 4, and the administration times were recorded. At the above-mentioned set time points, approximately 0.20 mL of each sample was collected by jugular vein blood collection or other appropriate method, anticoagulated with sodium heparin, and placed on ice after collection. Then, plasma was centrifuged within 1 hour (centrifugation conditions: 6800 g, 6 min, 2-8°C). Plasma samples were stored in a -80°C freezer until analysis. Grouping and blood collection time points are shown in Table 4. Three animals were used at each time point.

[0261] Table 10. Study plan for evaluation of in vivo pharmacokinetic properties of Compound 398 in mice, beagle dogs, and cynomolgus monkeys [Table 10]

[0262] Table 11. Main pharmacokinetic parameters of the compound [Table 11]

[0263] As shown in Table 11, compound 398 of the present invention has good pharmacokinetic properties in all of mice, beagle dogs and cynomolgus monkeys.

[0264] Test Example 12: Initial evaluation of the in vivo safety of Compound 398 in mice (1) Test method Compounds were dissolved in DMSO / HS15 / PEG400 / NaCl (5 / 3 / 40 / 52, v / v / v). ICR rats (age: 6-8 weeks), half female (16-22 grams) and half male (body weight 17-25 grams), were used. Compound 398 was tested according to the dosing schedule in Table 12, and clinical observations were made on all animals. At the end of the study, samples of the heart, liver, spleen, lungs, kidneys, and administration site were collected. The test results are shown in Table 12.

[0265] (2) Test results

[0266] Table 12. Initial evaluation of in vivo safety of Compound 398 in rats [Table 12]

[0267] The test results show that the compound 398 of the present invention has good internal safety in mice.

Claims

1. A compound of formula I, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof. (wherein Q is a nitrogen-containing heteroaryl ring or a nitrogen-containing fused heteroaryl ring; R １ is hydrogen, unsubstituted or halogenated C １～８ Alkyl groups, unsubstituted or halogenated C １～８ Alkoxy group, halogen, 5- to 6-membered aryl group, 5- to 6-membered heteroaryl group, saturated 3- to 8-membered heterocyclyl group, saturated 3- to 8-membered cycloalkyl group, NHCOR １ａ is selected from R １ａ is C １～８ an unsubstituted or halogenated saturated 3- to 8-membered heterocyclyl group, an unsubstituted or halogenated saturated 3- to 8-membered cycloalkyl group, an unsubstituted or halogenated 5- to 6-membered aryl group, an unsubstituted or halogenated 5- to 6-membered heteroaryl group; R ２ is hydrogen, C １～８ Alkyl group, C １～８ selected from an alkoxy group, a halogen, a 5- to 6-membered aryl group, a 5- to 6-membered heteroaryl group, a saturated 3- to 8-membered heterocyclyl group, and a saturated 3- to 8-membered cycloalkyl group; R ３ is hydrogen, C １～８ Alkyl group, C １～８ Alkoxy groups, halogens, 5- to 6-membered aryl groups, 5- to 6-membered heteroaryl groups, saturated 3- to 8-membered heterocyclyl groups, saturated 3- to 8-membered cycloalkyl groups, CH ２ R ３ａ is selected from R ３ａ is selected from an unsubstituted or halogenated saturated 3- to 8-membered heterocyclyl group, an unsubstituted or halogenated saturated 3- to 8-membered cycloalkyl group, an unsubstituted or halogenated 5- to 6-membered aryl group, an unsubstituted or halogenated 5- to 6-membered heteroaryl group, an unsubstituted or halogenated fused ring aryl group; R ４ is hydrogen or any one of the following substituted or unsubstituted groups: １～８ Alkyl group, C １～８ is selected from an alkoxy group, a saturated 3- to 8-membered cycloalkyl group, a saturated 3- to 8-membered heterocyclyl group, a 5- to 6-membered aryl group, a 5- to 6-membered heteroaryl group, a bridged ring group, a fused ring aryl group, a fused heteroaryl group, or a saturated 5- to 6-membered heterocyclyl group or a 5- to 6-membered aryl group; The substituent of the substituted group is R ４ａ , R ４ｂ , R ４ｃ , R ４ｄ and each independently Hydrogen, halogen, phenyl group, cyano group, hydroxy group, ester group, trimethylsilyl group, -(CH ２ ) ｍ -SO ２ R', -COOR'', or Any one of the following groups, which is unsubstituted or substituted with any one or more of halogen, cyano group, haloalkyl group, and haloalkoxy group: １～８ Alkyl group, C １～８ Alkoxy group, benzyl group, pyridyl group or saturated 3- to 6-membered cycloalkyl group wherein R′ and R″ are each independently selected from C １～８ alkyl group, m is an integer from 0 to 3; Or, the R ４ａ , R ４ｂ are linked to form a halogenated or unsubstituted saturated 3- to 6-membered carbocyclic ring or a halogenated or unsubstituted carboheterocyclic ring; L １ is none, substituted or unsubstituted -(CH ２ ) ｎ - or substituted or unsubstituted -O-(CH ２ ) ｎ -, -O-, -NHCO-, -NHSO ２ -, -CONH-, -NHCOO-, -NHCONH-, -NH-, n is an integer of 1 to 3, １ The substituent of １～３ Alkyl group, C １～３ an alkoxy group or a phenyl group, X is None, CR ５ R ６ , N.R. ５ R ６ , O or SO ２ is selected from R ５ , R ６ are each independently Hydrogen, halogen, R ５ａ , R ５ｂ , R ５ｃ is substituted or unsubstituted C １～８ alkyl group or R ５ , R ６ is bonded to R ６ａ , R ６ｂ form a substituted or unsubstituted saturated 3- to 8-membered cycloalkyl group or a substituted or unsubstituted saturated 3- to 8-membered heterocyclyl group, R ５ａ , R ５ｂ , R ５ｃ each independently represents a hydrogen atom, an alkynyl group, a hydroxyl group, or -CONH ２ , -N(CH ３ ) ２ , halogen, C １～８ alkoxy group, 5- to 6-membered aryl group, 5- to 6-membered heteroaryl group, or R ５ａ , R ５ｂ , R ５ｃ any two of the following may be combined to form a saturated 3- to 8-membered cycloalkyl group or a saturated 3- to 8-membered heterocyclyl group; R ６ａ , R ６ｂ each independently represents a hydrogen atom, an alkenyl group, a halogen atom, a halogen-substituted or unsubstituted C １～８ alkyl group or R ６ａ , R ６ｂ are linked to form a 5- to 6-membered aryl group, L ２ No, nothing. Or R ａ , R ｂ is substituted or unsubstituted is selected from Here, R ０ is hydrogen, C １～３ is an alkyl group or R ５ to form a substituted or unsubstituted bridged ring, a substituted or unsubstituted saturated 3- to 6-membered ring, or a substituted or unsubstituted saturated 3- to 6-membered benzene ring structure, and the substituent of the substituted structure is C １～３ is an alkyl group or a halogen; R ａ , R ｂ are each independently a hydrogen atom, a cyano group, １～５ alkyl group, 3- to 6-membered cycloalkyl group, or R ａ , R ｂ are linked to form a saturated 3- to 6-membered carbocyclic ring, Y is O or S; t is any integer from 0 to 3, q ​​is any integer from 0 to 4, r is any integer from 0 to 3, s is any integer from 0 to 3, k is any integer from 0 to 3, and u is any integer from 0 to 3.

2. The compound according to claim 1, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, wherein Q is a 5- to 6-membered nitrogen-containing heteroaryl ring, a 5- or 6-membered nitrogen-containing fused heteroaryl ring, or a 6- or 6-membered nitrogen-containing fused heteroaryl ring.

3. Q or 3. The compound according to claim 2, wherein:

4. Q or 4. The compound according to claim 3, wherein:

5. R １ is hydrogen, halogen, halogenated or unsubstituted C １～８ Alkyl group or unsubstituted C １～８ 2. The compound according to claim 1, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that it is an alkoxy group.

6. R １ is hydrogen, halogen, halogenated or unsubstituted C １～３ Alkyl group or unsubstituted C １～３ 6. The compound according to claim 5, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that: R is an alkoxy group.

7. R １ is hydrogen, fluorine, chlorine, a methyl group, a methoxy group, or -CF ３ 7. The compound according to claim 6, wherein:

8. R ２ is hydrogen or C １～８ 2. The compound according to claim 1, characterized in that it is an alkyl group, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof.

9. R ２ 9. The compound according to claim 8, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that: is hydrogen or a methyl group.

10. R ３ is hydrogen or CH ２ R ３ａ and R ３ａ is unsubstituted or halogenated C １～４ 2. The compound according to claim 1, characterized in that the aryl group is selected from the group consisting of an alkyl group, an unsubstituted or halogenated 5- to 6-membered cycloalkyl group, an unsubstituted or halogenated 5- to 6-membered aryl group, an unsubstituted or halogenated 5- to 6-membered heteroaryl group, and an unsubstituted or halogenated fused ring aryl group, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof.

11. R ３ is hydrogen or CH ２ R ３ａ and R ３ａ is unsubstituted or halogenated C １～２ 11. The compound according to claim 10, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the aryl group is selected from the group consisting of an alkyl group, an unsubstituted or halogenated 5- to 6-membered cycloalkyl group, an unsubstituted or halogenated 5- to 6-membered aryl group, an unsubstituted or halogenated 5- to 6-membered heteroaryl group, and an unsubstituted or halogenated naphthyl group.

12. R ３ is hydrogen or CH ２ R ３ａ and R ３ａ is selected from a phenyl group, an ethyl group, a cyclohexyl group, a furyl group, a naphthyl group or an F-substituted phenyl group, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, according to claim 11.

13. R ４ is any one of the following substituted or unsubstituted groups: １～４ Alkyl group, C １～４ is selected from an alkoxy group, a saturated 3- to 7-membered cycloalkyl group, a saturated 4- to 6-membered heterocyclyl group, a 5- to 6-membered aryl group, a 5- to 6-membered heteroaryl group, a bridged ring group, a naphthyl group, a 5- to 6-membered aromatic heterocyclyl phenyl group, or a saturated 5- to 6-membered heterocyclyl phenyl group; The substituent of the substituted group is R ４ａ , R ４ｂ , R ４ｃ , R ４ｄ and each independently Hydrogen, halogen, phenyl group, cyano group, hydroxy group, ester group, trimethylsilyl group, -(CH ２ ) ｍ -SO ２ R', -COOR'', Any one of the following groups, which may be substituted or unsubstituted with halogen: １～３ Alkyl group, C １～３ Alkoxy group or saturated 3- to 4-membered cycloalkyl group wherein R′ and R″ are each independently selected from C １～４ alkyl groups, m is an integer from 0 to 2; Or, the R ４ａ , R ４ｂ , R ４ｃ , R ４ｄ any two of the following may be combined to form a halogenated or unsubstituted saturated 3- to 6-membered carbocyclic or heterocyclic ring:

14. R ４ is any one of the following substituted or unsubstituted groups: １～４ Alkyl group, C １～４ is selected from an alkoxy group, a saturated 3- to 7-membered cycloalkyl group, a saturated 4- to 6-membered heterocyclyl group, a 5- to 6-membered aryl group, a 5- to 6-membered azaaryl group, a bridged ring group, a naphthyl group, a benzofuryl group, a benzopyridyl group, or a saturated 5- to 6-membered oxygen heterocyclic group, a phenyl group; The substituent of the substituted group is R ４ａ , R ４ｂ , R ４ｃ , R ４ｄ and each independently Hydrogen, halogen, phenyl group, cyano group, hydroxy group, ester group, trimethylsilyl group, -(CH ２ ) ｍ -SO ２ R', -COOR'', Any one of the following groups, which may be substituted or unsubstituted with halogen: １～３ Alkyl group, C １～３ Alkoxy group or saturated 3-membered cycloalkyl group wherein R′ and R″ are each independently selected from C １～４ alkyl group, m is 0 or 1; Or, the R ４ａ , R ４ｂ , R ４ｃ , R ４ｄ any two of the following may be combined to form a halogenated or unsubstituted saturated 3- to 6-membered carbocyclic or oxygen heterocyclic ring:

15. R ４ The compound according to claim 14, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, wherein is a fluorine-substituted saturated 4- to 6-membered cycloalkyl group.

16. R ４ is a difluorine-substituted saturated 4- to 6-membered cycloalkyl group, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, according to claim 15.

17. R ４ but and z is an integer from 1 to 3, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, according to claim 16.

18. R ４ The compound according to claim 14, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that: is selected from any one of the following groups, substituted or unsubstituted: -CH ３ 、-OCH ３ 、 、

19. R ４ 19. The compound according to claim 18, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that: -CH ３ 、CF ３ 、-OCH ３ 、-OCF ３ ,-OC(EH ３ ) ３ 、 、

20. L １ is a substituted or unsubstituted -(CH ２ ) ｎ -, n is an integer of 1 to 3, and the substituent of the substitution is C １～３ 2. The compound according to claim 1, characterized in that it is an alkyl group or a phenyl group, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof.

21. L １ is a substituted or unsubstituted -(CH ２ ) ｎ -, and the substituent of the substitution is a methyl group or a phenyl group, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, according to claim 20.

22. No X, CR ５ R ６ Or NR ５ R ６ and R ５ , R ６ are each independently Hydrogen, Fluoro, R ５ａ , R ５ｂ , R ５ｃ is substituted or unsubstituted C １～５ alkyl group or R ５ , R ６ is bonded to R ６ａ , R ６ｂ forms a substituted or unsubstituted saturated 3- to 6-membered cycloalkyl group or a saturated 4-membered oxygen heterocyclic group, R ５ａ , R ５ｂ , R ５ｃ each independently represents a hydrogen atom, an ethynyl group, a hydroxyl group, or -CONH ２ , -CONHCH ３ , -N(CH ３ ) ２ , fluorine, a methoxy group, a phenyl group, a methylsulfonyl group, an amino group, a carboxy group, a carbomethoxy group, an azaphenyl group, or R ５ａ , R ５ｂ , R ５ｃ any two of these combine to form a saturated 3- to 6-membered cycloalkyl group or a saturated 5- to 6-membered oxygen heterocyclic group; R ６ａ , R ６ｂ are each independently selected from hydrogen, a vinyl group, fluorine, and a fluorine-substituted methyl group, or R ６ａ , R ６ｂ are bonded to form a phenyl group, L ２ But, none. Or R ａ , R ｂ is substituted or unsubstituted and Here, R ０ is hydrogen, C １～３ is an alkyl group or R ５ and bonded to the following substituted or unsubstituted structure: wherein the substituent of the substituted structure is a methyl group or a fluorine group; R ａ , R ｂ are each independently selected from hydrogen, a cyano group, a butyl group, and a 6-membered cycloalkyl group, or R ａ , R ｂ are linked to form a three-membered carbocyclic ring, Y is O or S; The compound according to claim 1, characterized in that t is 0 or 1, q is any integer from 0 to 4, r is 0 or 1, s is 0 or 1, k is 0 or 1, and u is 0 or 1, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof.

23. X is CR ５ R ６ And L ２ but and q is 0 or 1, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, according to claim 22.

24. X is none, or and L ２ But, none. or and Or L ２ is bonded to X to form one of the following structures:

23. The compound according to claim 22, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that

25. X is or And L ２ but or 25. The compound according to claim 24, wherein:

26. 26. The compound according to any one of claims 1 to 25, characterized in that the structure of the compound is as shown in Formula II or Formula III, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof.

27. 27. The compound of claim 26, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, wherein the structure of the compound is as shown in Formula II-A.

28. 28. The compound according to claim 27, wherein the structure of the compound is as shown in formula II-A-a, II-A-b, II-A-c or II-A-d, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof.

29. 29. The compound according to claim 28, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-A-a-1.

30. 30. The compound of claim 29, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the compound has any one of the following structures:

31. The structure of the compound is as shown in formula II-A-a-2: M is fluorine, chlorine, a methyl group, a methoxy group, or -CF ３ 29. The compound according to claim 28, wherein:

32. 32. The compound of claim 31, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the compound has any one of the following structures:

33. 28. The compound according to claim 27, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-A-b-1.

34. 34. The compound of claim 33, wherein the compound has the structure:

35. 28. The compound according to claim 27, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-A-c-1.

36. 36. The compound of claim 35, wherein the compound has the structure:

37. 28. The compound according to claim 27, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-A-d-1.

38. 38. The compound of claim 37, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the compound has the structure:

39. The compound according to claim 27, characterized in that the structure of the compound is as shown in formula II-A-e, II-A-f, II-A-g, II-A-h, II-A-i, II-A-i1, II-A-i2, II-A-i3, II-A-i4 or II-A-i5, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof. (wherein U, V, U', and V' are each independently hydrogen, C １～３ and W is O or S. Preferably, W is selected from the group consisting of an alkyl group or a halogen, and is preferably hydrogen, a methyl group or chlorine.

40. 40. The compound of claim 39, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-A-e-1.

41. 41. The compound of claim 40, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in any one of the following:

42. 40. The compound of claim 39, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-A-e-2.

43. 43. The compound of claim 42, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown below:

44. 40. The compound of claim 39, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in any one of the following:

45. 40. The compound of claim 39, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-A-h-1.

46. 46. ​​The compound of claim 45, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in any one of the following:

47. 40. The compound of claim 39, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-A-h-2.

48. 48. The compound of claim 47, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in any one of the following:

49. 40. The compound according to claim 39, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-A-I'-2.

50. 50. The compound of claim 49, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the compound has any one of the following structures:

51. 28. The compound according to claim 27, characterized in that the compound has the structure shown in formula II-A-j, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof. (wherein z is any integer from 1 to 3, q ​​is any integer from 0 to 4, and R ５ , R ６ At least one of the is not hydrogen, Preferably, z is 1, 2 or 3 and q is 0 or 1.

52. 52. The compound of claim 51, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, wherein the compound has a structure as shown in formula II-A-j-1:

53. 53. The compound of claim 52, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the compound has any one of the following structures:

54. 27. The compound according to claim 26, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in Formula II-B, Formula II-C, or Formula II-D.

55. 55. The compound according to claim 54, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-B-a.

56. 56. The compound according to claim 55, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-B-a-1.

57. 57. The compound of claim 56, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the compound has the structure:

58. 55. The compound according to claim 54, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-C-a.

59. 59. The compound according to claim 58, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-C-a-1.

60. 60. The compound of claim 59, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the compound has the structure:

61. 55. The compound according to claim 54, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-D-a.

62. 62. The compound according to claim 61, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the structure of the compound is as shown in formula II-D-a-1.

63. 63. The compound of claim 62, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the compound has the structure:

64. The compound according to claim 26, characterized in that the structure of the compound is as shown in formula III-A, III-B, III-C, III-D, III-E or III-F, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof. (Here, T １ , T ２ , T ３ are each independently selected from hydrogen or halogen, and at least one is halogen.

65. 65. The compound according to claim 64, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that T is fluorine.

66. R １ 65. The compound according to claim 64, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that is chlorine or hydrogen.

67. 65. The compound according to claim 64, wherein the structure of the compound is as shown in formula III-Aa, III-A-b, III-Ba, III-Ca, III-C-b, III-Da, III-D-b, III-E-a, or III-F-a, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof.

68. 68. The compound of claim 67, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that the compound has any one of the following structures:

69. A pharmaceutical composition, characterized in that it is a formulation produced using a compound according to any one of claims 1 to 68, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof as an active ingredient, together with pharma- ceutically acceptable auxiliary materials.

70. 69. Use of a compound according to any one of claims 1 to 68, or a pharma- ceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, in the manufacture of a medicament for the prophylaxis and / or treatment of a coronavirus-associated disease.

71. 71. The use according to claim 70, characterized in that the drug for preventing and / or treating coronavirus-related diseases is an anti-coronavirus drug.

72. 72. The use according to claim 71, characterized in that the anti-coronavirus drug is a drug that inhibits coronavirus infection of cells.

73. 72. The use according to claim 71, characterized in that the anti-coronavirus drug is a coronavirus proteolytic enzyme inhibitor, preferably a coronavirus main protease inhibitor.

74. Use according to any one of claims 70 to 73, characterised in that the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HcoV-229E, HcoV-NL63, HcoV-HKU1 or HcoV-OC43, preferably SARS-CoV-2.

75. The use according to claim 74, characterized in that the drug for preventing and / or treating a coronavirus-related disease is a drug for preventing and / or treating new coronavirus pneumonia COVID-19.

76. The coronavirus main protease inhibitor is ｐｒｏ 75. The use according to claim 74, characterized in that it is an inhibitor.

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