Baricitinib derivatives, processes for their preparation and use thereof

Abstract

This invention belongs to the technical field of pharmaceutical preparation, specifically relating to baloxavir derivatives, their preparation methods, and applications. The baloxavir derivatives are compounds I, II, or III, with the following general structural formulas: Formula I: where R1 and R2 are H, OCH3, or Br; Formula II: where R1 and R2 are H or F, R3 is H, F, OCH3, or a benzene ring, and A is C or O; Formula III: where A is S or O, B is C or N, and X is CH3, Cl, or a benzene ring. This invention provides a baloxavir derivative for use as a nuclease inhibitor, and also provides a method for its preparation.

Description

Technical Field

[0001] This invention belongs to the technical field of drug preparation, specifically relating to baloxavir derivatives, their preparation methods, and applications. Background Technology

[0002] Viruses are organisms that parasitize host cells and replicate themselves. Common viruses include influenza virus, HIV, hepatitis B virus, and rabies virus. Influenza virus is characterized by high mutation rate and strong infectivity, which leads to its high frequency and wide range of occurrence. Viruses can be transmitted in many ways, such as through droplets, contact, blood, food, vertical transmission, and sexual contact. Among these, droplet transmission and contact transmission are the primary modes of transmission for influenza virus, with droplet transmission being arguably the most widespread and harmful. Influenza virus can cause respiratory illnesses (such as pneumonia or cardiopulmonary failure), resulting in a high mortality rate among infected individuals.

[0003] For viral illnesses, medication remains the primary treatment method. The first 48 hours after the onset of flu symptoms are crucial for treatment; starting medication within this timeframe can alleviate symptoms and shorten the duration of the illness. Currently, commonly used antiviral drugs in clinical practice include: ion channel blockers, neuraminidase inhibitors, and traditional Chinese medicine antiviral drugs.

[0004] In 2015, Shionogi Co., Ltd. of Japan took a different approach, breaking the dominance of neuraminidase inhibitors in antiviral drugs by developing a novel antiviral drug, baloxaviridine (trade name: Xofluza; compound code S-033188 / S-033447), which targets both influenza A and B viruses. Baloxaviridine works by inhibiting cap-dependent endonuclease (CEN) in the influenza virus, interrupting the viral genome replication process and thus blocking viral proliferation.

[0005] Baloxavir ester (BXM), a novel antiviral drug with a completely new mechanism of action targeting influenza A and B viruses, has broad market prospects. Current research on baloxavir ester mainly focuses on its efficacy, safety, pharmacokinetics, pharmacokinetics, and pharmacology. Reports on its derivatives are scarce, and studies on its structure-activity relationship are even rarer. Baloxavir ester is a prodrug; its active ingredient is baloxavir. Baloxavir ester is hydrolyzed in vivo to baloxavir before it can exert its antiviral effect. The pharmacophore of baloxavir is 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione. The triazine structure chelates with manganese ions, serving as a key binding site and playing an irreplaceable role in pharmacological activity. Meanwhile, 7,8-difluorodibenzo[b,e]thiazoline... The pharmacological effects of -11(6H)-ol are not yet clear.

[0006] Therefore, in order to find candidate compounds with comparable or better pharmacological activity, baloxavir was used as a lead compound. While keeping the pharmacophore group of baloxavir unchanged, its structure was modified, and the resulting compound was screened for pharmacological activity in order to obtain candidate drugs with better pharmacological activity and fewer toxic side effects. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing technology and provide a baloxavir derivative for use as a nuclease inhibitor. The present invention also provides a method for its preparation.

[0008] The baloxavir derivatives described in this invention are compound I, compound II, or compound III, and their general structural formulas are general formula I, general formula II, and general formula III.

[0009] General formula I is: Where R1 and R2 are H, OCH3, or Br;

[0010] The N-linking group in the triazine ring of general formula I is:

[0011]

[0012] General formula II is: Where R1 and R2 are H or F, R3 is H, F, OCH3 or a benzene ring, and A is C or O.

[0013] The N-linking group in the triazine ring of general formula II is:

[0014]

[0015] Formula III is: A is S or O, B is C or N, and X is CH3, Cl, or a benzene ring;

[0016] The N-linking group in the triazine ring of general formula III is:

[0017]

[0018] Furthermore, the structural formulas I-1, I-2, I-3, and I-4 of general formula I are as follows:

[0019]

[0020] Furthermore, the structural formulas II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, and II-9 of general formula II are as follows:

[0021]

[0022] Furthermore, the structural formulas III-1, III-2, III-3, III-4, III-5, III-6, III-7, and III-8 of general formula III are as follows:

[0023]

[0024]

[0025] The preparation method of the baloxavir derivative, specifically compound I, involves the following steps: mixing and reacting diphenylmethanol / 4,4'-dimethoxydiphenylmethanol / 4,4'-difluorodiphenylmethanol / (4-bromophenyl)(phenyl)methanol), 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, and 1-propylphosphonic anhydride in a molar ratio of (1.0–1.2):(1.0–1.5):(1.5–2.5) to obtain compound I. The 1-propylphosphonic anhydride is a 50% (w / w) solution prepared from ethyl acetate.

[0026] The preparation steps of compound II are as follows: Dibenzocycloheptenol / dibenzocycloheptenol / 10,11-difluoro-7,12-dihydrobenzo[e]naphtho[1,2-b]oxheptaeno-7-ol / 9,10-difluoro-8,13-dihydrobenzo[e]naphtho[1,2-b]oxheptaeno-13-ol / 7,8-difluoro-6,11-dihydrodibenzo[b,e]oxheptaeno-7-ol / 7,8-difluoro-4-methoxy-6,11-dihydrodibenzo[b,e]oxheptaeno-7-ol / 7,8-difluoro-2-methoxy-6,11-dihydrodibenzo[b] Compound II was obtained by reacting a mixture of oxaheptane-7-ol / 4,7,8-trifluoro-6,11-dihydrodibenzo[b,e]oxaheptane-7-ol / 2,7,8-trifluoro-6,11-dihydrodibenzo[b,e]oxaheptane-7-ol, 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, and 1-propylphosphonic anhydride in a molar ratio of (1.0–1.2):(1.0–1.5):(1.5–2.5). The 1-propylphosphonic anhydride was a 50% (w / w) solution prepared from ethyl acetate.

[0027] The reaction temperature for the preparation of compounds I and II is 90–110 °C.

[0028] The preparation steps of compound III are as follows: 2-chloro-5-chloromethylthiazole / 2-chloro-5-chloromethylpyridine / 4-chloromethyl-5-methyl-2-phenyloxazole / 4-chloromethyl-3,5-dimethylisooxazole / 2-cyano-4'-bromomethylbiphenyl / 2-chloro-5-chloromethylthiophene / 3-chloromethyl-5-methylisooxazole are mixed with 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione in a molar ratio of (1.0~1.2):(1.0~1.5) to obtain compound III.

[0029] The reaction temperature in the preparation of compound III is 50–80 °C.

[0030] The aforementioned baloxavir derivative is used as a nuclease inhibitor.

[0031] On the other hand, this invention relates to a method for studying the bioactivity of a nuclease inhibitor.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] (1) This invention provides a new class of compounds with endonuclease inhibitory activity. More specifically, this invention provides a new class of endonuclease inhibitors prepared using baloxavir as a lead compound. Compared with existing baloxavir, compounds I-4, series II target compounds, and compound III-8 of this invention have similar biological activity to baloxavir.

[0034] (2) The baloxavir derivatives prepared by the present invention are 21 derivatives in three series with baloxavir as the lead compound. They are compounds of formula I, II and III or stereoisomers, tautomers, metabolites, pharmaceutically acceptable salts or their prodrugs, and side chains belonging to the same family of compounds.

[0035] (3) The method for preparing baloxavir derivatives of the present invention provides a method for studying the bioactivity of a nuclease inhibitor. Attached Figure Description

[0036] Figure 1 This is the high-resolution mass spectrum of compound I-1 of the present invention;

[0037] Figure 2 This is the proton NMR spectrum of compound I-1 of the present invention;

[0038] Figure 3 The carbon spectrum of compound I-1 of this invention;

[0039] Figure 4 This is a high-resolution mass spectrum of compound I-2 of the present invention;

[0040] Figure 5 The hydrogen spectrum of compound I-2 of this invention;

[0041] Figure 6 The carbon spectrum of compound I-2 of this invention;

[0042] Figure 7 This is the high-resolution mass spectrum of compound I-3 of the present invention;

[0043] Figure 8 The hydrogen spectrum of compound I-3 of this invention;

[0044] Figure 9 The carbon spectrum of compound I-3 of this invention;

[0045] Figure 10 This is a high-resolution mass spectrum of compound I-4 of the present invention;

[0046] Figure 11 The hydrogen spectrum of compound I-4 of this invention;

[0047] Figure 12The carbon spectrum of compound I-4 of this invention;

[0048] Figure 13 This is the high-resolution mass spectrum of compound II-1 of the present invention;

[0049] Figure 14 The proton NMR spectrum of compound II-1 of this invention;

[0050] Figure 15 The carbon spectrum of compound II-1 of this invention;

[0051] Figure 16 This is the high-resolution mass spectrum of compound II-2 of the present invention;

[0052] Figure 17 The proton NMR spectrum of compound II-2 of this invention;

[0053] Figure 18 The carbon spectrum of compound II-2 of this invention;

[0054] Figure 19 This is the high-resolution mass spectrum of compound II-3 of the present invention;

[0055] Figure 20 The proton NMR spectrum of compound II-3 of this invention;

[0056] Figure 21 The carbon spectrum of compound II-3 of this invention;

[0057] Figure 22 This is the high-resolution mass spectrum of compound II-4 of the present invention;

[0058] Figure 23 The proton NMR spectrum of compound II-4 of this invention;

[0059] Figure 24 The carbon spectrum of compound II-4 of this invention;

[0060] Figure 25 This is the high-resolution mass spectrum of compound II-5 of the present invention;

[0061] Figure 26 The proton NMR spectrum of compound II-5 of this invention;

[0062] Figure 27 The carbon spectrum of compound II-5 of this invention;

[0063] Figure 28 This is the high-resolution mass spectrum of compound II-6 of the present invention;

[0064] Figure 29 The proton NMR spectrum of compound II-6 of this invention;

[0065] Figure 30 The carbon spectrum of compound II-6 of this invention;

[0066] Figure 31 This is the high-resolution mass spectrum of compound II-7 of the present invention;

[0067] Figure 32 The hydrogen spectrum of compound II-7 of this invention;

[0068] Figure 33 The carbon spectrum of compound II-7 of this invention;

[0069] Figure 34 This is the high-resolution mass spectrum of compound II-8 of the present invention;

[0070] Figure 35 The proton NMR spectrum of compound II-8 of this invention;

[0071] Figure 36 The carbon spectrum of compound II-8 of this invention;

[0072] Figure 37 This is the high-resolution mass spectrum of compound II-9 of the present invention;

[0073] Figure 38 The proton NMR spectrum of compound II-9 of this invention;

[0074] Figure 39 The carbon spectrum of compound II-9 of this invention;

[0075] Figure 40 This is the high-resolution mass spectrum of compound III-1 of the present invention;

[0076] Figure 41 The proton NMR spectrum of compound III-1 of this invention;

[0077] Figure 42 The carbon spectrum of compound III-1 of this invention;

[0078] Figure 43 This is the high-resolution mass spectrum of compound Ⅲ-2 of the present invention;

[0079] Figure 44 The proton NMR spectrum of compound III-2 of this invention;

[0080] Figure 45 The carbon spectrum of compound III-2 of this invention;

[0081] Figure 46 This is the high-resolution mass spectrum of compound Ⅲ-3 of the present invention;

[0082] Figure 47The proton NMR spectrum of compound III-3 of this invention;

[0083] Figure 48 The carbon spectrum of compound III-3 of this invention;

[0084] Figure 49 This is the high-resolution mass spectrum of compound III-4 of the present invention;

[0085] Figure 50 The proton NMR spectrum of compound III-4 of this invention;

[0086] Figure 51 The carbon spectrum of compound III-4 of this invention;

[0087] Figure 52 This is the high-resolution mass spectrum of compound III-5 of the present invention;

[0088] Figure 53 The hydrogen spectrum of compound III-5 of this invention;

[0089] Figure 54 The carbon spectrum of compound III-5 of this invention;

[0090] Figure 55 This is the high-resolution mass spectrum of compound III-6 of the present invention;

[0091] Figure 56 The hydrogen spectrum of compound III-6 of this invention;

[0092] Figure 57 The carbon spectrum of compound III-6 of this invention;

[0093] Figure 58 This is the high-resolution mass spectrum of compound III-7 of the present invention;

[0094] Figure 59 The proton NMR spectrum of compound III-7 of this invention;

[0095] Figure 60 The carbon spectrum of compound III-7 of this invention;

[0096] Figure 61 This is the high-resolution mass spectrum of compound III-8 of the present invention;

[0097] Figure 62 The proton NMR spectrum of compound III-8 of this invention;

[0098] Figure 63 The carbon spectrum of compound III-8 of this invention;

[0099] Figure 64 This is a diagram showing the inhibitory effect of the target compound of the present invention on endonucleases. Detailed Implementation

[0100] The above description only outlines certain aspects of the invention and is not limited to these aspects. These and other aspects will be described in more detail below.

[0101] Example 1

[0102] (R)-12-diphenylmethyl-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅰ-1), with the following structural formula:

[0103]

[0104] 2.02 g (11 mmol) of diphenylmethanol and 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask. 20 mL of 50% T3P ethyl acetate solution and 20 mL of ethyl acetate were added, and the mixture was purged with nitrogen. The reaction was carried out at 100 °C for 12 h. After the reaction was complete, 60 mL of ice water was added to the reaction mixture, and the mixture was extracted with 100 mL × 2 dichloromethane. The combined organic phases were washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to give 6.27 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) yielded 1.95 g of a pale yellow solid, with a yield of 39.56%.

[0105] 1.95 g of the above solid was added to 15 mL of DMAc, followed by 1.66 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The solution was extracted with 100 mL × 2 dichloromethane, and the organic phases were combined. The mixture was washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to obtain 5.89 g of an oily substance. Column chromatography (DCM:CH3OH = 30:1) yielded 680 mg of a pale yellow solid, with a yield of 42.66% and a purity of 96.18%. After purification by preparative liquid chromatography, the purity was 99.91%.

[0106] HRMS(ESI): M / z Calcd for C 23 H 21 N3O4:404.4305.Found:404.1615[M+H] + .

[0107] 1H NMR (600MHz, DMSO-d6, 25℃, TMS): δ3,02-3.08(m,1H),3.38-3.43(m,2H),3.58-3.66(m,2H),3.94(dd,1H),4.39(d,1H),4.56-4.58(m ,1H),5.44(t,1H),5.67-5,71(m,1H),7.19(d,2H),7.23(d,1H),7.29(d,2H),7.36(dd,1H),7.45(t,2H),7.66(d,2H),11.64(s,1H). 13 C-NMR (150MHz, DMSO): δ45.21,65.56,67.87,71.28,72.34,115.75,127.51,128.25,128.32,128.50,129.17,129.28,152.68,160.92,170.95.

[0108] Example 2

[0109] (R)-12-(bis(4-methoxyphenyl)methyl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅰ-2), with the following structural formula:

[0110]

[0111] 2.68 g (11 mmol) of 4,4'-dimethoxydiphenylmethanol and 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask. 20 mL of 50% T3P ethyl acetate solution and 20 mL of ethyl acetate were added, and the mixture was purged with nitrogen. The reaction was carried out at 100 °C for 12 h. After the reaction was complete, 60 mL of ice water was added to the reaction mixture, and the mixture was extracted with 100 mL × 2 dichloromethane. The combined organic phases were washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to give 6.02 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) yielded 3.93 g of a pale yellow solid, with a yield of 71.07%.

[0112] 3.93 g of the above solid was added to 20 mL of DMAc, followed by 2.97 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered to obtain a pale yellow solid. Recrystallization from methanol yielded 2.04 g of a pale yellow solid, with a yield of 88.70% and a purity of 93.92%. After preparative liquid chromatography purification, the purity was 99.94%.

[0113] HRMS(ESI): M / z Calcd for C 25 H 25 N3O6:463.4825.Found:487.3614[M+Na+H] + .

[0114] 1 H NMR (600MHz, DMSO-d6, 25℃, TMS): δ2.87(s,1H),3.12-3.18(m,1H),3.21(s,1H),3.50-3.65(m,2H),3.74(dd,6H),3.83-3.87(m,1H) ),4.35-4.37(m,1H),5.10(s,1H),6.74(dd,2H),6.85(dd,2H),6.93(dd,2H),7.15-7.20(m,2H),7.43-7.45(m,2H),11.53(s,1H). 13 C-NMR (150MHz, DMSO): δ46.19,54.90,54.99,55.07,55.10,113.36,113.66 ,114.53,128.33,128.61,128.65,128.84,157.24,157.54,158.87,159.08.

[0115] Example 3

[0116] (R)-12-(di(4-fluorophenyl)methyl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridinyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅰ-3), with the following structural formula:

[0117]

[0118] 2.42 g (11 mmol) of 4,4'-difluorodiphenylmethanol and 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask. 20 mL of 50% T3P ethyl acetate solution and 20 mL of ethyl acetate were added, and the mixture was purged with nitrogen. The reaction was carried out at 100 °C for 12 h. After the reaction was complete, 60 mL of ice water was added to the reaction mixture, and the mixture was extracted with 100 mL × 2 dichloromethane. The combined organic phases were washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to give 7.13 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) yielded 2.63 g of a pale yellow solid, with a yield of 49.56%.

[0119] 2.63 g of the above solid was added to 15 mL of DMAc, followed by 2.07 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The solution was extracted with 100 mL × 2 dichloromethane. The combined organic phases were washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to obtain 5.45 g of an oily substance. Column chromatography (DCM:CH3OH = 30:1) yielded 873 mg of a pale yellow solid, with a yield of 40.04% and a purity of 97.44%. After preparative liquid chromatography purification, the purity was 99.98%.

[0120] HRMS(ESI): M / z Calcd for C 23 H 19 F2N3O4:439.1344.Found:440.1425[M+H] + .

[0121] 1 H NMR (600MHz, DMSO-d6, 25℃, TMS): δ3.12(s,1H),3.40(td,1H),3.62-3.68(m,2H),3.93(dd,1H),4.37(d,1H),4 .54-4.57(m,1H),5.52(d,1H),7.04(t,2H),7.13-7.14(m,1H),7.25-7.34(m,5H),7.71(dd,2H),11.60(s,1H). 13C-NMR (150MHz, DMSO): δ45.14,65.66,67.84,70.44,71.08,115.13,116.00,129.57,129.62,131.40,131.45,139.32,152.68,160.85,171.00.

[0122] Example 4

[0123] (12aR)-12-((4-bromobenzene)(benzene)methyl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridinyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅰ-4), with the following structural formula:

[0124]

[0125] 2.62 g (11 mmol) of (4-bromophenyl)(phenyl)methanol and 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask. 20 mL of 50% T3P ethyl acetate solution and 20 mL of ethyl acetate were added, and the mixture was purged with nitrogen. The reaction was carried out at 100 °C for 12 h. After the reaction was complete, 60 mL of ice water was added to the reaction mixture, and the mixture was extracted with 100 mL × 2 dichloromethane. The combined organic phases were washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to give 9.03 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) yielded 1.37 g of a pale yellow solid, with a yield of 24.00%.

[0126] 1.37 g of the above solid was added to 15 mL of DMAc, followed by 1.01 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The solution was extracted with 100 mL × 2 dichloromethane. The combined organic phases were washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to obtain 5.08 g of an oily substance. Column chromatography (DCM:CH3OH = 30:1) yielded 860 mg of a pale yellow solid, with a yield of 74.78% and a purity of 95.86%. After preparative liquid chromatography purification, the purity was 99.24%.

[0127] HRMS(ESI): M / z Calcd for C 23 H 20 BrN3O4:481.0637 / 483.0617.Found:482.0636[M+H] + / 484.1425[M+H] + .

[0128] 1 H NMR (600MHz, DMSO-6, 25℃, TMS): δ0.67(d,2H),0.85(d,1H),1.04-1.07(m,1H),1.23(d,2H),4.02(dd,1H),4.54-4 .58(m,1H)5.73(ddd,1H),6.20(s,1H),7.20(s,2H),7.28-7.29(m,3H),7.45(s,2H),7.63(dd,2H),11.61(s,1H). 13 C-NMR (150MHz, DMSO): δ46.19,62.89,67.91,71.49,73.52,119.70,126.21,126.40,126.60,126.87 ,128.02,128.15,128.32,128.43,128.64,128.83,130.93,137.66,142.55,145.16,145.23,169.59.

[0129] Example 5

[0130] (R)-12-(5H-dibenzo[a,d][7]cycloheptene-5-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridinyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅱ-1), with the following structural formula:

[0131]

[0132] 2.29 g (11 mmol) of dibenzocycloheptenol and 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask. 20 mL of 50% T3P ethyl acetate solution and 20 mL of ethyl acetate were added, and the mixture was purged with nitrogen. The reaction was carried out at 100 °C for 12 h. After the reaction was complete, 50 mL of ice water was added to the reaction mixture, and the mixture was extracted with 120 mL × 2 ethyl acetate solutions. The combined organic phases were washed successively with 50 mL of saturated sodium bicarbonate aqueous solution and 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and distilled to give 4 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) yielded 2.4 g of a pale yellow solid, with a yield of 46.42%.

[0133] 2.4 g of the above compound was added to 15 mL of DMAc, followed by 1.90 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with 10 mL of water and dried to obtain a pale yellow solid. Recrystallization yielded 1.98 g of a pale yellow solid, compound II-1, with a yield of 100% and a purity of 94.49%. After preparative liquid chromatography purification, the purity was 99.93%.

[0134] HRMS(ESI): M / z Calcd for C 25 H 21 N3O4:427.4519.Found:428.3532[M+H] + .

[0135] 1 H NMR (600MHz, DMSO-d6, 25℃, TMS): δ2.82(d,1H),3.08(dd,1H),3.54(s,1H),3.95-3.97(m,2H),4.00(d,1H),5.01(d,1H),5.10(s,1H),6 .69(dd,1H),7.10(dd,1H),7.23(d,1H),7.28(dd,1H),7.34-7.42(m,3H),7.47(dd,1H),7.55-7.62(m,3H),7.69(t,1H),11.11(s,1H). 13 C NMR (150MHz, DMSO): δ50.15,50.53,66.02,68.09,73.27,126.32,127.91,128.66,130. 11,131.03,131.42,132,56,133.82,134,11,134,47,135.66,142.24,161.14,170.08.

[0136] Example 6

[0137] (R)-12-(5H-dibenzo[a,d][7]cycloheptane-5-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridinyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅱ-2), with the following structural formula:

[0138]

[0139] 2.10 g (10 mmol) of dibenzocycloheptanol and 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask. 20 mL of ethyl acetate solution (50% by mass) and 20 mL of ethyl acetate were added, and the mixture was purged with nitrogen. The reaction was carried out at 100 °C for 12 h. After the reaction was complete, 50 mL of ice water was added to the reaction mixture, and the mixture was extracted with 120 mL × 2 ethyl acetate solutions. The combined organic phases were washed successively with 50 mL of saturated sodium bicarbonate aqueous solution and 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and distilled to give 2.72 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) yielded 3.14 g of a pale yellow solid, with a yield of 60.62%.

[0140] 3.14 g of the above compound was added to 20 mL of DMAc, followed by 1.90 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with 10 mL of water and dried to obtain a pale yellow solid. Recrystallization yielded 2.59 g of a pale yellow solid, with a yield of 100% and a purity of 92.08%. After preparative liquid chromatography purification, the purity was 99.11%.

[0141] HRMS(ESI): M / z Calcd for C 25 H 23 N3O4:429.4678.Found:430.1771[M+H] + .

[0142] 1 H NMR (600MHz, DMSO-d6, 25℃, TMS): δ2.84-2.89(m,2H),3.59(t,1H),3.76(t,2H),3.98(dd,3H),4.27(d,1H),4.29(s,1H),5.44( dd,2H),6.85(dd,1H),6.91(d,1H),7.18-7.21(m,2H),7.34(s,1H),7.37-7.38(m,2H),7.43(s,2H),8.66(d,1H),11.80(s,1H). 13C NMR (150MHz, DMSO): δ30.98,31.87,45.39,65.57,68.24,69.89,74.72,110.13,115.50,125.83,128.64,128.8 3,129.17,129.51,130.89,131.00,131.10,131.36,133.63,135.03,139.92,140.29,153.13,161.38,171.00.

[0143] Example 7

[0144] (12aR)-12-(10,11-difluoro-7,12-dihydrobenzo[e]naphtho[1,2-b]oxepane-7-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridinyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅱ-3), with the following structural formula:

[0145]

[0146] 1.29 g (4.3 mmol) of 10,11-difluoro-7,12-dihydrobenzo[e]naphtho[1,2-b]oxaconitine-7-ol and 1.40 g (4.3 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask, followed by the addition of 10 mL of 50% ethyl acetate solution and 10 mL of ethyl acetate. The mixture was then purged with nitrogen and reacted at 100 °C for 12 h. After the reaction was complete, 50 mL of ice water was added to the reaction solution, and the mixture was extracted with 120 mL × 2 ethyl acetate. The organic phases were combined and washed successively with 50 mL of saturated sodium bicarbonate aqueous solution and 50 mL of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and distilled to give 2.66 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) yielded 0.64 g of a pale yellow solid, with a yield of 24.43%.

[0147] 0.64 g of the above compound was added to 10 mL of DMAc, followed by 0.44 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with 10 mL of water and dried to obtain a pale yellow solid. Recrystallization yielded 0.5 g of a pale yellow solid, with a yield of 100% and a purity of 93.44%. After preparative liquid chromatography purification, the purity was 99.85%.

[0148] HRMS(ESI): M / z Calcd for C28 H 21 F2N3O5:517.4802.Found:518.1528[M+H] + .

[0149] 1 H NMR (600MHz, DMSO-d6, 25℃, TMS): δ3.56(s,1H),3.67(d,1H),3.86(dd,1H),4.00(dd,1H),4.46-4.53(m,2H),5.16-5.25(m,1H),5.55(dd, 2H),5.72(d,1H),7.37-7.40(m,2H),7.44(d,1H),7.48(d,1H),7.51-7.57(m,2H),7.61(dd,2H),7.90(d,1H),8.25(dd,1H),11.81(s,1H). 13 C NMR (150MHz, DMSO): δ46.66,61.79,65.95,68.58,71.09,113.51,114.33,121.66,123.03,126.69, 126.89,127.85,128.22,128.80,129.11,129.31,130.12,130.58,134.13,135.10,152.93,162.11.

[0150] Example 8

[0151] (12aR)-12-(9,10-difluoro-8,13-dihydrobenzo[e]naphtho[2,1-b]oxepane-13-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridinyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅱ-4), with the following structural formula:

[0152]

[0153] 1.49 g (5 mmol) of 9,10-difluoro-8,13-dihydrobenzo[e]naphtho[1,2-b]oxaconitine-13-ol and 1.64 g (5 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask, followed by the addition of 15 mL of T3P ethyl acetate solution (50% by mass) and 15 mL of ethyl acetate. The mixture was then purged with nitrogen and reacted at 100 °C for 12 h. After the reaction was complete, 50 mL of ice water was added to the reaction solution, and the mixture was extracted with 120 mL × 2 ethyl acetate. The organic phases were combined and washed successively with 50 mL of saturated sodium bicarbonate aqueous solution and 50 mL of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and distilled to give 3.07 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) gave 1.7 g of a pale yellow solid, with a yield of 56.67%.

[0154] 1.7 g of the above compound was added to 10 mL of DMAc, followed by 1.2 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with 10 mL of water and dried to obtain a pale yellow solid. Recrystallization yielded 1.1 g of a pale yellow solid, with a yield of 100% and a purity of 91.26%. After preparative liquid chromatography purification, the purity was 100%.

[0155] HRMS(ESI): M / z Calcd for C 28 H 21 F2N3O5:517.4802.Found:518.1528[M+H] + .

[0156] 1 H NMR (600MHz, DMSO-d6, 25℃, TMS): δ2.81(d,1H),3.02(dd,1H),3.32(d,2H),3.94(d,1H),4.85-4.87(m,1H),5.08(d,1H),5. 63-5.69(m,1H),6.34(m,1H),7.24(m,3H),7.57(dd,2H),7.84(d,2H),8.14-8.23(m,1H),8.58-8.67(m,3H),11.67(s,1H). 13C NMR (150MHz, DMSO): δ53.23,63.93,64.40,65.70,65.98,67.77,122.23,124.10,125.15,126.89,127.34,129.64,1 30.55,131.34,132.56,133.61,133.85,134,15,155.38,165.06,165.20,165.94,166.06,166.20,169.41,170.07.

[0157] Example 9

[0158] 12-(7,8-difluoro-6,11-dihydrodibenzo[b,e]oxaheptane-11-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅱ-5), with the following structural formula:

[0159]

[0160] 1.45 g (5.84 mmol) of 7,8-difluoro-6,11-dihydrodibenzo[b,e]oxaconitine-7-ol and 1.91 g (5.84 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask, followed by the addition of 15 mL of T3P ethyl acetate solution (50% by mass) and 15 mL of ethyl acetate. The mixture was then purged with nitrogen and reacted at 100 °C for 12 h. After the reaction was complete, 50 mL of ice water was added to the reaction solution, and the mixture was extracted with 120 mL × 2 ethyl acetate. The organic phases were combined and washed successively with 50 mL of saturated sodium bicarbonate aqueous solution and 50 mL of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and distilled to give 3.55 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) yielded 0.84 g of a pale yellow solid, with a yield of 25.85%.

[0161] 0.84 g of the above compound was added to 10 mL of DMAc, followed by 0.63 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with 10 mL of water and dried to obtain a pale yellow solid. Recrystallization yielded 0.49 g of a pale yellow solid, with a yield of 70% and a purity of 92.02%. After preparative liquid chromatography purification, the purity was 100%.

[0162] HRMS(ESI): M / z Calcd for C 24 H19 F2N3O5:467.4216.Found:468.1376[M+H] + . 1 HNMR (600MHz, DMSO-d6, 25℃, TMS): δ2.76(d,1H),2.95(d,1H),3.38(s,1H),3.66(s,1H),3.81(dd,1H),3.87-3.88(m,1H),4.46-4.50(m,2H),5.24 -5.28(m,1H),5.32(s,1H),6.07-6.12(m,1H),6.77-6.85(m,1H),6.97(t ,1H),7.07(dt,2H),7.23-7.26(m,1H),7.33-7.44(m,2H),11.78(s,1H). 13 C NMR (150MHz, DMSO): δ46.66,61.08,65.97,68.57,70.11,71.16,117.48,118.16,120.29,120.31,120.61,1 21.36,122.35,124.97,126.84,127.39,128.80,129.12,129.31,132.24,133.62,134.06,157.13,157.58.

[0163] Example 10

[0164] (12aR)-12-(7,8-difluoro-4-methoxy-6,11-dihydrodibenzo[b,e]oxaheptane-11-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅱ-6), with the following structural formula:

[0165]

[0166] 1.39 g (5 mmol) of 7,8-difluoro-4-methoxy-6,11-dihydrodibenzo[b,e]oxaconitine-7-ol and 1.64 g (5 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask, followed by the addition of 15 mL of T3P ethyl acetate solution (50% by mass) and 15 mL of ethyl acetate. The mixture was then purged with nitrogen and reacted at 100 °C for 12 h. After the reaction was complete, 50 mL of ice water was added to the reaction solution, and the mixture was extracted with 120 mL × 2 ethyl acetate. The organic phases were combined and washed successively with 50 mL of saturated sodium bicarbonate aqueous solution and 50 mL of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and distilled to give 2.88 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) gave 1.83 g of a pale yellow solid, with a yield of 62.35%.

[0167] 1.83 g of the above compound was added to 15 mL of DMAc, followed by 1.32 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with 10 mL of water (10 mL total volume). After drying, a pale yellow solid was obtained. Recrystallization yielded 1.49 g of a pale yellow solid, with a yield of 96.13% and a purity of 90.93%. After preparative liquid chromatography purification, the purity was 99.28%.

[0168] HRMS(ESI): M / z Calcd for C 25 H 21 F2N3O6:497.4475.Found:498.1641[M+H] + .

[0169] 1 H NMR (600MHz, DMSO-d6, 25℃, TMS): δ3.03(dd,1H),3.07-3.11(m,1H),3.26(d, 1H),3.39(dd,1H),3.68(dd,1H),3.72(d,3H),3.82-3.88(m,1H),5.24(dd,1 H),5.53(s,1H),5.67(dd,1H),5.72-5.79(m,1H),7.02-7.14(m,1H),7.56(d d,1H),7.60(dd,2H),7.66(dd,1H),7.79(d,1H),7.84(dd,1H),11.66(s,1H). 13C NMR (150MHz, DMSO): δ41.63,52.28,54.25,65.12,68.57,69.52,88.22,113.11,125.86,126.25,126.57,1 28.45,128.61,128.73,131.71,132.27,132.92,140.06,145.56,148.75,150.45,153.94,164.05,172.46.

[0170] Example 11

[0171] (12aR)-12-(7,8-difluoro-2-methoxy-6,11-dihydrodibenzo[b,e]oxaheptane-11-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridinyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅱ-7), with the following structural formula:

[0172]

[0173] 1.39 g (5 mmol) of 7,8-difluoro-2-methoxy-6,11-dihydrodibenzo[b,e]oxaconitine-7-ol and 1.64 g (5 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask, followed by the addition of 15 mL of T3P ethyl acetate solution (50% by mass) and 15 mL of ethyl acetate. The mixture was then purged with nitrogen and reacted at 100 °C for 12 h. After the reaction was complete, 50 mL of ice water was added to the reaction solution, and the mixture was extracted with 120 mL × 2 ethyl acetate. The organic phases were combined and washed successively with 50 mL of saturated sodium bicarbonate aqueous solution and 50 mL of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and distilled to give 2.67 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) gave 1.72 g of a pale yellow solid, with a yield of 58.60%.

[0174] 1.72 g of the above compound was added to 15 mL of DMAc, followed by 1.24 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with 10 mL of water and dried to obtain a pale yellow solid. Recrystallization yielded 1.23 g of a pale yellow solid, with a yield of 84.83% and a purity of 91.66%. After preparative liquid chromatography purification, the purity was 99.84%.

[0175] HRMS(ESI): M / z Calcd for C25 H 21 F2N3O6:497.4475.Found:498.1631[M+H] + .

[0176] 1 H NMR (600MHz, DMSO-d6, 25℃, TMS): δ2.92(dd,1H),3.27(dd,1H),3.38(dt,1H),3 .72(d,3H),3.85(dd,1H),4.30-4.41(m,1H),4.44-4.51(m,1H),5.16(dd,1H), 5.66(dd,1H),5.74(d,1H),5.81(d,1H),5.99(dd,1H),6.81(d,1H),6.93(d,1H ),6.98-7.03(m,1H),7.17(d,1H),7.24(dd,1H),7.44(ddd,1H),11.58(s,1H). 13 C NMR (150MHz, DMSO): δ45.62,54.57,55.40,65.54,69.05,70.79,91.10,113.57,115.91,116.45,117.79,121.3 7,122.10,126.85,127.75,131.70,132.42,141.12,148.91,149.21,150.02,153.40,154.03,154.54,172.99.

[0177] Example 12

[0178] 12-(4,7,8-trifluoro-6,11-dihydrodibenzo[b,e]oxaheptane-11-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅱ-8), with the following structural formula:

[0179]

[0180] 1.33 g (5 mmol) of 4,7,8-trifluoro-6,11-dihydrodibenzo[b,e]oxaconitine-7-ol and 1.64 g (5 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask, followed by the addition of 15 mL of T3P ethyl acetate solution (50% by mass) and 15 mL of ethyl acetate. The mixture was then purged with nitrogen and reacted at 100 °C for 12 h. After the reaction was complete, 50 mL of ice water was added to the reaction solution, and the mixture was extracted with 120 mL × 2 ethyl acetate. The organic phases were combined and washed successively with 50 mL of saturated sodium bicarbonate aqueous solution and 50 mL of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and distilled to give 3.2 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) gave 0.91 g of a pale yellow solid, with a yield of 31.71%.

[0181] 0.91 g of the above compound was added to 10 mL of DMAc, followed by 0.67 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with 10 mL of water and dried to obtain a pale yellow solid. Recrystallization yielded 0.6 g of a pale yellow solid, with a yield of 78.95% and a purity of 94.96%. After preparative liquid chromatography purification, the purity was 99.89%.

[0182] HRMS(ESI): M / z Calcd for C 24 H 18 F3N3O5:485.4120.Found:486.1279[M+H] +

[0183] 1 H NMR (600MHz, 600MHz, DMSO-d6, 25℃, TMS): δ3.54(s,1H),3.66(d,1H),3.87(dd,1H),3.95-4.07(m,1H),4.46(dd,2H),5.37(dd,2H),5.52(d, 1H),5.71(dd,1H),6.18(t,1H),7.06(dd,1H),7.16-7.18(m,1H),7.29(dd,1H),7.35-7.38(m,1H),7.44(d,1H),7.55(dd,1H),11.78(s,1H). 13C NMR (150MHz, DMSO): δ45.88,46.65,61.78,65.95,68.54,70.54,118.08,118.47,121.10,122.78,126.96,1 28.43,128.80,129.11,129.31,133.15,133.55,145.21,145.90,149.68,151.64,154.07,161.83,166.11.

[0184] Example 13

[0185] 12-(2,7,8-trifluoro-6,11-dihydrodibenzo[b,e]oxaheptane-11-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c]pyridyl[2,1-f][1,2,4]triazine-6,8-dione (Ⅱ-9), with the following structural formula:

[0186]

[0187] 1.33 g (5 mmol) of 2,7,8-trifluoro-6,11-dihydrodibenzo[b,e]oxaconitine-7-ol and 1.64 g (5 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione were added to a 100 mL thick-walled pressure-resistant flask, followed by the addition of 15 mL of T3P ethyl acetate solution (50% by mass) and 15 mL of ethyl acetate. The mixture was then purged with nitrogen and reacted at 100 °C for 12 h. After the reaction was complete, 50 mL of ice water was added to the reaction solution, and the mixture was extracted with 120 mL × 2 ethyl acetate. The combined organic phases were washed successively with 50 mL of saturated sodium bicarbonate aqueous solution and 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and distilled to give 3.1 g of a pale yellow solid. Column chromatography (DCM:CH3OH = 30:1) gave 1.35 g of a pale yellow solid, with a yield of 47.04%.

[0188] 1.35 g of the above compound was added to 10 mL of DMAc, followed by 0.72 g of anhydrous lithium chloride. The mixture was heated to 70 °C under nitrogen protection and reacted for 8 h. 20 mL of water was added dropwise to the reaction solution, and the pH was adjusted to approximately 6 with 1 M dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with 10 mL of water and dried to obtain a pale yellow solid. Recrystallization yielded 0.78 g of a pale yellow solid, with a yield of 69.03% and a purity of 94.54%. After preparative liquid chromatography purification, the purity was 99.87%.

[0189] HRMS(ESI): M / z Calcd for C 24 H 18F3N3O5:485.4120.Found:486.1280[M+H] + .

[0190] 1 H NMR (600MHz, DMSO-d6, 25℃, TMS): δ3.54(d,1H),3.65-3.67(m,1H),3.85-3.87(m,1H),3.98-4.05(m,1H),4.33-4.35(m,2H),5.18-5.24(m ,2H),5.54(s,1H),5.64(s,1H),6.84-6.90(m,1H),7.03-7.11(m,2H),7.24(dd,2H),7.35-7.44(m,1H),7.51-7.55(m,1H),11.78(s,1H). 13 CNMR (150MHz, DMSO): δ45.58,60.26,63.93,64.90,67.82,69.16,107.56,111.98,117.27,118.10,121.22, 122.19,125.96,127.09,127.72,128.04,128.24,132.03,137.06,152.45,156.66,160.94,161.34,165.05.

[0191] Example 14

[0192] 12-((2-chlorothiazo-5-yl)-methyl))-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]-oxazinyl[3,4-c[pyridinyl[2,1-f][1,2,4]triazine-6,8-dione(Ⅲ-1), with the following structural formula:

[0193]

[0194] 2.34 g (14 mmol) of 2-chloro-5-chloromethylthiazole, 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, 1.93 g (14 mmol) of potassium carbonate, and 30 mL of DMF were added sequentially to a reaction flask. The mixture was stirred and heated to 50 °C for 8 h. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with 70 mL × 2 dichloromethane solutions. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to give 6.27 g of a pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 1.46 g of a pale yellow solid.

[0195] The above compound, 816 mg of lithium chloride, and 10 mL of DMAc were added sequentially to a reaction flask. Stirring was started, and the mixture was heated to 70 °C for 8 h. After the reaction was complete, 50 mL of water was added, and the pH was adjusted to 6 with 1 M hydrochloric acid. The mixture was extracted with 70 mL × 2 dichloromethane solutions, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 2.92 g of a pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 180 mg of a pale yellow solid, with a yield of 4.89% and a purity of 90.95%. Preparative liquid chromatography purification yielded a purity of 99.48%.

[0196] HRMS(ESI):C 14 H 13 ClN4O4S,M / z[M+Na] + 391.0248;

[0197] 1 H-NMR (600MHz, DMSO-d6): δ3.15-3.16(m,1H),3.25(d,1H),3.40-3.45(m,1H),3.63(s,1H),3.83(s,1 H),3.92(s,1H),4.36-4.40(m,1H),4.46(d,1H),5.89(d,1H),7.26(d,1H),7.48(t,2H),11.61(s,1H). 13 C-NMR (150MHz, DMSO): δ65.91,67.67,67.74,70.25,72.81,129.14,132.01,135.54,136.38,138.47,142.65,142.81,143.40,152.16.

[0198] Example 15

[0199] 12-((6-chloropyridin-3-yl)-methyl))-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]-oxazinyl[3,4-c[pyridinyl[2,1-f][1,2,4]triazine-6,8-dione(Ⅲ-2), with the following structural formula:

[0200]

[0201] 2.29 g (14 mmol) of 2-chloro-5-chloromethylpyridine, 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, 1.93 g (14 mmol) of potassium carbonate, and 30 mL of DMF were added sequentially to a reaction flask. The mixture was stirred and heated to 50 °C for 8 h. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with 70 mL × 2 dichloromethane. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to give 8.01 g of a pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 2.47 g of a pale yellow solid.

[0202] The above solid, 2.29 g of lithium chloride, and 15 mL of DMAc were added sequentially to a reaction flask. Stirring was started, and the mixture was heated to 70 °C for 8 h. After the reaction was complete, 50 mL of water was added, and the pH was adjusted to 6 with 1 M hydrochloric acid. The mixture was extracted with 70 mL × 2 dichloromethane solutions, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 7.10 g of a pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 1 g of a pale yellow solid, with a yield of 27.62% and a purity of 96.79%. After preparative liquid chromatography purification, the purity was 99.79%.

[0203] HRMS(ESI):C 16 H 15 ClN4O4,M / z[M+H] + 363.0860;

[0204] 1 H-NMR (600MHz, DMSO-d6): δ3.26(t,1H),3.51(d,2H),3.84-3.93(m,2H),4.37(dd,3H),5.0 8(s,1H),5.82(d,1H),7.16(d,1H),7.50(dd,1H),7.80(dd,1H),8.28(t,1H),11.67(s,1H). 13 C-NMR (150MHz, DMSO): δ65.35,111.22,115.69,124.09,130.15,137.95,141.15,150.10,150.87,152.80,170.96.

[0205] Example 16

[0206] 7-Hydroxy-12-((5-methyl-2-phenyloxazol-4-yl)-methyl))-3,4,12,12a-tetrahydro-1H-[1,4]-oxazinyl[3,4-c[pyridyl[2,1-f][1,2,4]triazine-6,8-dione(Ⅲ-3), with the following structural formula:

[0207]

[0208] 2.89 g (14 mmol) of 4-chloromethyl-5-methyl-2-phenyloxazole, 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, 1.93 g (14 mmol) of potassium carbonate, and 30 mL of DMF were added sequentially to a reaction flask. The mixture was stirred and heated to 50 °C for 8 h. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with 70 mL × 2 dichloromethane. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to give 4.27 g of a pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 2.23 g of a pale yellow solid.

[0209] The above solid, 1.88 g of lithium chloride, and 15 mL of DMAc were added sequentially to a reaction flask. Stirring was started, and the mixture was heated to 70 °C for 8 h. After the reaction was complete, 50 mL of water was added, and the pH was adjusted to 6 with 1 M hydrochloric acid. The mixture was extracted with 70 mL × 2 dichloromethane solutions, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 6.95 g of a pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 1.02 g of a pale yellow solid, with a yield of 25% and a purity of 95.16%. After purification by preparative liquid chromatography, the purity was 98.54%.

[0210] HRMS(ESI):C 21 H 20 N4O5, M / z[M+H] + :409.1511;

[0211] 1 H-NMR (600MHz, DMSO-d6): δ2.07(s,3H),3.22(t,1H),3.53(d,2H),3.84-3.98(m,2H),4.25(d d,3H),5.18(d,1H),5.90(d,1H),7.18(d,1H),7.51(dd,3H),7.89-7.99(m,2H),11.67(s,1H). 13C-NMR (150MHz, DMSO): δ9.37,65.37,111.33,125.61,126.60,129.01,129.75,130.38,137.92,148.43,152.59,158.94,169.46,171.00.

[0212] Example 17

[0213] 12-((3,5-dimethylisoxazol-4-yl)-methyl))-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]-oxazinyl[3,4-c[pyridinyl[2,1-f][1,2,4]triazine-6,8-dione(Ⅲ-4), with the following structural formula:

[0214]

[0215] 2.04 g (14 mmol) of 4-chloromethyl-3,5-dimethylisoxazole, 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, 1.93 g (14 mmol) of potassium carbonate, and 30 mL of DMF were added sequentially to a reaction flask. The mixture was stirred and heated to 50 °C for 8 h. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with 70 mL × 2 dichloromethane. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to give 8.20 g of a pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 4.22 g of a pale yellow solid.

[0216] The above solid, 4.06 g of lithium chloride, and 25 mL of DMAc were added sequentially to a reaction flask. Stirring was started, and the mixture was heated to 70 °C for 8 h. After the reaction was complete, 50 mL of water was added, and the pH was adjusted to 6 with 1 M hydrochloric acid. The mixture was extracted with 70 mL × 2 dichloromethane solutions, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 10.24 g of a pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 1.16 g of a pale yellow solid, with a yield of 33.52% and a purity of 96.93%. After purification by preparative liquid chromatography, the purity was 99.85%.

[0217] HRMS(ESI):C 16 H 18 N4O5, M / z[M+H] + :347.1353;

[0218] 1H-NMR (600MHz, DMSO-d6): δ2.05(s,3H),2.14(s,3H),3.25(s,1H),3.58(d,2H),3.84-3.8 6(m,2H),4.13(s,2H),4.42(s,1H),5.04(s,1H),5.89(s,1H),7.07(s,1H),11.76(s,1H). 13 C-NMR (150MHz, DMSO): δ9.95,10.65,49.92,62.08,66.05,73.15,82.55,1 01.00,108.75,111.98,138.69,153.60,160.11,168.98,171.61,174.39.

[0219] Example 18

[0220] 4'-((7-hydroxy-6,8-dione-3,4-dihydro-1H-[1,4]-oxazinyl[3,4-c[pyridyl[2,1-f][1,2,4]triazine-12(6H,8H,12aH)-yl)methyl)-[1,1'-biphenyl]-4-carboxynitrile (Ⅲ-5), with the following structural formula:

[0221]

[0222] 3.80 g (14 mmol) of 2-cyano-4'-bromomethylbiphenyl, 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, 1.93 g (14 mmol) of potassium carbonate, and 30 mL of DMF were added sequentially to a reaction flask. The mixture was stirred and heated to 50 °C for 8 h. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with 70 mL × 2 dichloromethane. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to give 8 g of pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 3.60 g of pale yellow solid.

[0223] The above solid, 2.93 g of lithium chloride, and 30 mL of DMAc were added sequentially to a reaction flask. Stirring was started, and the mixture was heated to 70 °C for 8 h. After the reaction was complete, 50 mL of water was added, and the pH was adjusted to 6 with 1 M hydrochloric acid. The mixture was extracted with 70 mL × 2 dichloromethane solutions, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 14.72 g of a pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 1.72 g of a pale yellow solid, with a yield of 40.18% and a purity of 91.42%. After purification by preparative liquid chromatography, the purity was 99.65%.

[0224] HRMS(ESI):C 24 H 20 N4O4, M / z[M+H] + :429.1563;

[0225] 1 H-NMR (600MHz, DMSO-d6): δ3.32(s,1H),3.57(d,2H),3.84-3.95(m,2H),4.38(s,3H),5.06(s,1H),5.8 1(d,1H),7.13(d,1H),7.46(d,2H),7.54(d,2H),7.61(dd,2H),7.80(t,1H),7.96(d,1H),11.69(s,1H). 13 C-NMR (150MHz, DMSO): δ65.39,110.15,118.32,128.24,128.75,129.97,133.44,133.74,137.71,143.96,152.83,170.93.

[0226] Example 19

[0227] 12-((5-chlorothiophene-2-yl)-methyl))-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]-oxazinyl[3,4-c[pyridinyl[2,1-f][1,2,4]triazine-6,8-dione(Ⅲ-6), with the following structural formula:

[0228]

[0229] 2.33 g (14 mmol) of 2-chloro-5-chloromethylthiophene, 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, 1.93 g (14 mmol) of potassium carbonate, and 30 mL of DMF were added sequentially to a reaction flask. The mixture was stirred and heated to 50 °C for 8 h. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with 70 mL × 2 dichloromethane. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 6.27 g of a pale yellow oil.

[0230] The above-mentioned oily substance, 3.81 g of lithium chloride, and 30 mL of DMAc were added sequentially to a reaction flask. Stirring was started, and the mixture was heated to 70 °C for 8 h. After the reaction was complete, 50 mL of water was added, and the pH was adjusted to 6 with 1 M hydrochloric acid. Extraction was performed with 70 mL × 2 dichloromethane solutions. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 6.74 g of a pale yellow oily substance. Column chromatography (DCM:CH3OH = 30:1) yielded 1.16 g of a pale yellow solid, with a yield of 16.35% and a purity of 99.16%. Purification by preparative liquid chromatography yielded a purity of 99.95%.

[0231] HRMS(ESI):C 15 H 14 ClN3O4S, M / z[M+H] + 368.0469;

[0232] 1 H-NMR (600MHz, DMSO-d6): δ3.21(td,1H),3.49-3.83(m,2H),3.90(dd,2H),4.27(s,1H),4. 47(dd,2H),5.07(d,1H),5.88(t,1H),6.79(d,1H),6.97(d,1H),7.21(d,1H),11.66(s,1H). 13 C-NMR (150MHz, DMSO): δ54.81,65.34,111.24,115.43,126.69,129.38,129.53,136.09,137.71,152.80,170.96.

[0233] Example 20

[0234] 7-Hydroxy-12-((5-methylisoxazol-3-yl)-methyl))-3,4,12,12a-tetrahydro-1H-[1,4]-oxazinyl[3,4-c[pyridinyl[2,1-f][1,2,4]triazine-6,8-dione(Ⅲ-7), with the following structural formula:

[0235]

[0236] 1.84 g (14 mmol) of 3-chloromethyl-5-methylisoxazole, 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, 1.93 g (14 mmol) of potassium carbonate, and 30 mL of LDMF were added sequentially to a reaction flask. The mixture was stirred and heated to 50 °C for 8 h. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with 70 mL × 2 dichloromethane. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 6.27 g of a pale yellow oil.

[0237] The above-mentioned oily substance, 2.11 g of lithium chloride, and 15 mL of DMAc were added sequentially to a reaction flask. Stirring was started, and the mixture was heated to 70 °C for 8 h. After the reaction was complete, 50 mL of water was added, and the pH was adjusted to 6 with 1 M hydrochloric acid. Extraction was performed with 70 mL × 2 dichloromethane solutions. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 4.46 g of a pale yellow oily substance. Column chromatography (DCM:CH3OH = 30:1) yielded 830 mg of a pale yellow solid, with a yield of 25% and a purity of 97.23%. Preparative liquid chromatography purification yielded a purity of 99.85%.

[0238] HRMS(ESI):C 15 H 16 N4O5, M / z[M+H] + 333.1190;

[0239] 1 H-NMR (600MHz, DMSO-d6): δ2.50(d,3H),3.21(td,1H),3.52(dt,2H),3.83-3.94(m,2 H),4.38(dt,3H),5.09(d,1H),5.92(d,1H),6.31(s,1H),7.30(d,1H),11.58(s,1H). 13 C-NMR (150MHz, DMSO): δ65.28, 102.31, 111.52, 115.14, 137.75, 152.67, 158.73, 170.11, 171.02.

[0240] Example 21

[0241] ((4'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazinyl[3,4-c[pyridinyl[2,1-f][1,2,4]triazine-6,8-dione(Ⅲ-8), with the following structural formula:

[0242]

[0243] 3.80 g (14 mmol) of 2-cyano-4'-bromomethylbiphenyl, 3.27 g (10 mmol) of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazol[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, 1.93 g (14 mmol) of potassium carbonate, and 30 mL of DMF were added sequentially to a reaction flask. The mixture was stirred and heated to 50 °C for 8 h. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with 70 mL × 2 dichloromethane. The extract was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to give 8 g of pale yellow oil. Column chromatography (DCM:CH3OH = 30:1) yielded 3.60 g of pale yellow solid.

[0244] 1.80 g (3.5 mmol) of the above solid and 20 mL of diethylene glycol dimethyl ether were added to a reaction flask. The mixture was stirred until dissolved and clear. Zinc chloride and sodium azide were then added. Under nitrogen protection, the mixture was heated to 125 °C and reacted for 35 h. After the reaction was completed, the temperature was lowered to 10 °C, and 30 mL of NaNO2 aqueous solution and 30 mL of water were added dropwise. The mixture was filtered and dried to obtain 1.5 g of a pale yellow solid.

[0245] The above solid, 1.27 g (30 mmol) of lithium chloride, and 30 mL of DMAc were added sequentially to a reaction flask. Stirring was started, and the mixture was heated to 70 °C for 8 h. After the reaction was complete, 50 mL of water was added, and the pH was adjusted to 6 with 1 M hydrochloric acid. The mixture was extracted with 70 mL × 2 dichloromethane, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 1.07 g of a pale yellow oil. Column chromatography yielded compound III-8, with a yield of 26.12% and a purity of 92.60%. Preparative liquid chromatography purification yielded a purity of 99.91%.

[0246] HRMS(ESI):C 15 H 16 N4O5, M / z[M+H] + :333.1190;

[0247] 1 H-NMR (600MHz, DMSO-d6): δ3.26(s,1H),3.52(s,1H),3.67(s,1H),3.84(s,1H),3.95(d,1H),4.38(t,3H),5.90(s,1 H),6.38(s,1H),7.13(d,1H),7.46(d,2H),7.55(d,2H),7.60-7.62(m,2H),7.80(t,1H),7.96(d,1H),11.69(s,1H).13 C-NMR (150MHz, DMSO): δ65.39,110.15,118.32,128.24,128.75,129.97,133.44,133.74,135.41,137.71,143.96,152.83,169.46,170.93.

[0248] The pharmacological activities of the compounds prepared above were tested: the inhibitory effect on endonuclease activity and the affinity constant of the endonuclease were tested.

[0249] Experimental materials and instruments:

[0250] (1) Reagents and instruments required for endonuclease inhibitor activity assay

[0251] PA protein sample: Recombinant influenza A virus polymerase acidic protein (PA), molecular weight 26.6KD, expression host Yeast, purchased from Wuhan Huamei Biotechnology Co., Ltd.; fluorescence detection was performed using Ensight (PerkinElmer, Singapore).

[0252] Fluorescent probe sample: sequence and label 5'-6FAM-TGGCAATATCAGCTCCACA-mgBNFQ-3', purchased from Jinruis Biotechnology, 5'-fluorescent tag is 6-carboxyfluorescein (6-FAM) (492nm / 518nm), 3'-fluorescent tag is MGB (Minorgroove binder) non-fluorescent quencher group.

[0253] (2) Reagents and instruments required for affinity constant testing

[0254] Reagents: Amino-coupled assay kit (catalog number BR-1000-50), coupling buffer (10 mm sodium acetate, pH 4.5), buffer (10x PBS-P+), analytical grade DMSO, S-series CM5 chip; enzyme purchased from Wuhan Huamei Biotechnology Co., Ltd. (100 μg CSB-YP395880ILR1), species Influenza A virus (strain A / USA: Huston / AA / 1945h1N1), expression region 1-209 amino acids, protein tag N-terminal 6x His-tagged, expression host yeast, molecular weight 26.6 kDa, amino acid sequence MEDFVRQCFNPMIVELAEKAMKEYGEDLKVETNKFAAICTH LEVCFMYSDFHFINEQGESIIVELGDPNALLKHRFEIIEGRDRTMAWTIVNSICNTTGAEKPK FLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKSEKTHIHIFSFTGEEMATKADYTLDEES RARIKTRLFTIRQEMASRGLWDSFRQSERGEETIEERFEITG; Affinity was tested using an SPR molecular interaction analyzer (Biacore T200, GEhealthcare).

[0255] Experimental methods:

[0256] (1) Establishment of a method for testing endonuclease inhibitory activity

[0257] Preparation of cap-dependent endonucleases: Cloning of endonuclease genes (obtaining gene fragments by PCR or RT-PCR, digestion, electrophoresis, and recovery); Construction of prokaryotic expression plasmids (plasmid digestion, electrophoresis, recovery, ligation with endonuclease gene fragments, transformation, screening and identification of positive clones); Extraction of positive plasmids, transformation into expression-type E. coli, and screening for positive clones; Induction and optimization of expression conditions for positive clone strains (induction concentration, temperature, and time); Protein purification; Protein purity determination and structural identification.

[0258] Assay for the inhibitory effect of compounds on endonuclease activity: Preparation of fluorescently labeled substrate RNA sequence fragments; reaction of endonuclease protein, substrate RNA fragments, and test compounds; optimization of reaction conditions, including optimal reaction temperature, time, and concentration, to determine the reaction system; determination of enzyme-catalyzed reaction time (preliminary experiment); enzyme activity analysis; data analysis (using GraphPad Prism 8 and Origin 2018 to process data and calculate IC50). 50 ).

[0259] The enzyme-catalyzed reaction time was determined in two main groups: a reaction group and a control group. The reaction group used PA protein as the catalytic enzyme, while the control group used bovine serum albumin (BSA) as the catalytic enzyme. The reaction system was established according to Table 1. Fluorescence signals were collected every 1 minute from 0 min to 180 min. The fluorescence signal values ​​were fitted to a logarithmic growth curve. The time point when the fluorescence intensity first exceeded or equaled the cut-off threshold (maximum value) was determined as the start of the reaction plateau. Each group contained 3 replicate tests.

[0260] Table 1. Components and concentrations of the reaction group and control group

[0261]

[0262] The enzyme activity analysis method involved establishing an optimal enzyme-catalyzed reaction time and setting up a concentration gradient for enzyme activity comparison. The experimental groups included compound groups (a total of 8 compound concentration gradients were established, with final concentrations of 1 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM), a positive control, and a negative control. The reaction system was established according to the components listed in Table 2.

[0263] Table 2. Components and concentrations of the reaction group and control group

[0264]

[0265]

[0266] During the system construction process, all components except the probe were added first, and the system was incubated at 37°C for 30 minutes before adding the probe. Then, the fluorescence change was monitored by timing. The signal collection time was the optimal reaction time described in the previous step, and each group contained 3 replicate tests.

[0267] The test was conducted in a 37°C light-protected water bath; Ensight was used to measure and collect data. A 488nm wavelength laser was used as the excitation light for fluorescence signal measurement, and fluorescence intensity readings at a wavelength of 518nm were collected.

[0268] (2) Compound affinity test for endonuclease

[0269] Optimized conditions were used to couple the restriction enzyme to the chip: 100 μg of PA protein was dissolved in 1 mL of deionized water to obtain a 100 μg / mL PA protein solution. The 100 μg / mL PA protein solution was diluted to 40 μg / mL with 10 mM sodium acetate coupling buffer at pH 4.5, the contact time was 600 s, and the restriction enzyme was coupled to the CM5 chip using an SPR molecular interaction analyzer (Biacore T200).

[0270] Because the compound is insoluble in water, the DMSO solvent data needs to be corrected.

[0271] The response values ​​of the compound at different concentrations were determined using an SPR molecular interaction analyzer (Biacore T200).

[0272] The ka and kd values ​​(ka is the affinity constant, kd is the dissociation constant) of the compound to the endonuclease were determined using an SPR molecular interaction analyzer, and then the affinity KD was calculated. KD = Kd / Ka, and the unit of KD is M (mol / L). The results are shown in Table 3.

[0273] Table 3. Affinity of the target compound to endonucleases

[0274]

[0275]

[0276] Of course, the above description is merely a preferred embodiment of the present invention and should not be considered as limiting the scope of the embodiments of the present invention. The present invention is also not limited to the examples described above.

Claims

1. A baloxavir derivative, characterized in that: The compounds are III-5 and III-8, with the following structural formulas: 、 。 2. The use of the baloxavir derivative of claim 1 in the preparation of a nuclease inhibitor.

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