WO2023155337A1

WO2023155337A1 - Novel deuterated triazine compound, preparation method therefor, composition thereof and use thereof

Info

Publication number: WO2023155337A1
Application number: PCT/CN2022/097499
Authority: WO
Inventors: 黄才古; 孙辉; 曾维霖
Original assignee: 广州谷森制药有限公司
Priority date: 2022-02-18
Filing date: 2022-06-08
Publication date: 2023-08-24
Also published as: CN116655606A

Abstract

Disclosed is a deuterated triazine compound represented by formula I or a pharmaceutically acceptable salt, isomer, prodrug or cocrystal thereof. Also disclosed are a preparation method for the deuterated compound, a composition of the deuterated compound and use of the deuterated compound. The compound, as a 3CL protease inhibitor, on the basis of having similar inhibitory activity against viruses, achieves better pharmacokinetic properties and therapeutic effects and better druggability. The structural formula of formula I is shown on the left.

Description

Novel deuterated triazine compound, its preparation method, composition and application

technical field

The invention belongs to the field of medicine, and in particular relates to a novel deuterated triazine compound, a preparation method, a composition and an application thereof. The above-mentioned pharmaceutical composition is used for preparing a medicine for treating and preventing viral infection.

Background technique

The coronavirus in the human body was first isolated in the UK in the 1960s. The virus got its name from the crown-shaped protrusions on its surface. The SARS virus belongs to the order Nesting Viridae, Coronaviridae, and the genus Coronaviridae, and is a subgroup B coronavirus of the genus Beta. Most of the virus particles are round, with a capsule, and there are coronal-arranged fibrils around the periphery, distributed in the cytoplasm, in a round shape, and the diameter of the virus is between 80 and 120nm. SARS is an infectious disease with rapid onset, rapid spread and high fatality rate. Most of the infected patients have direct or indirect contact with patients, or live in endemic areas. MERS virus is a beta subgroup C coronavirus, the full name of which is Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which causes Middle East Respiratory Syndrome after infection. MERS-CoV was first discovered in Saudi Arabia in September 2012. It was named "SARS-like virus" because of its similar clinical symptoms to SARS. It also became the sixth known human coronavirus and the first isolated in the past 10 years. 3 types. The new coronavirus is a new strain of coronavirus that has never been found in humans before. It was first discovered and reported in 2019. It is still prevalent in many countries around the world and has not been well controlled in many countries and regions. Alipha, Beta, Gamma, Delta, Omicron and other mutant strains appeared.

Common signs after a person is infected with a coronavirus include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. There is no specific treatment for the disease caused by the new coronavirus.

Sho Konno et al reported that the peptidomimetic 3CL protease inhibitor has anti-SARS-CoV virus activity, Bioorg. The inhibitory effect of a series of dipeptide-type compounds with the same target as 3CL protease inhibitors on SARS-CoV was investigated.

In January 2022, Yuto U. et al. from Shionogi (Shionogi) discovered the lead triazine compound 1 through systematic research and high-throughput screening (antiviral activity). Peptide, non-covalent compound 3, namely S-217622. The generic name of the drug is Ensitrelvir Fumarate (Ensitrelvir Fumarate), chemical name: (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imine) -3-((1-methyl-1H-1,2,4-triazol-3-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5- Triazole-2,4-dione fumaric acid eutectic (IUPAC), the structural formula is as follows:

The drug has better druggability, pharmacological activity and safety, and clinical phase I-IIa studies have initially demonstrated its clinical value-oral effectiveness and safety. The clinical indication is the treatment of mild to moderate new coronary pneumonia, and it is expected to achieve single-drug administration (no PK enhancer required). However, the oral absolute bioavailability of the drug in dogs is low (64.7%), which may be related to the oxidative metabolism in the body of the methyl fragment in the molecular structure P1-P1' due to the first-pass effect. The clinical pharmacokinetic properties and metabolic safety of the drug need to be improved.

Therefore, there is still a need in the art to develop novel 3CL protease inhibitor compounds with better inhibitory activity or pharmacokinetic properties. The present invention designs and discloses novel deuterated triazine compounds. On the basis of comparable virus inhibitory activity, It has achieved better pharmacokinetic properties and therapeutic effects than S-217622, and has better druggability. At the same time, the preparation method, pharmaceutical composition and application thereof are disclosed, the production scale-up of the medicine can be realized, the quality is controllable, and the medicine has good clinical value.

Contents of the invention

The invention relates to a novel deuterated triazine compound, a preparation method, a pharmaceutical composition and an application thereof.

In certain embodiments, the present invention relates to a deuterated triazine compound represented by formula I, or a pharmaceutically acceptable salt, isomer, prodrug or co-crystal thereof:

wherein: each of R ₁ to R ₃ is independently hydrogen or deuterium; each of R ₄ to R ₇ is independently hydrogen or deuterium; each of R ₈ to R ₁₀ is independently hydrogen or deuterium; the additional condition is that among R1 to R ₁₀ at least One is deuterium.

The above compound disclosed by the present invention is characterized in that R ₁ to R ₃ are 2-3 deuteriums; R ₄ to R ₇ are 2-4 deuteriums; R ₈ to R ₁₀ are 2-3 deuteriums.

The above compound disclosed in the present invention is characterized in that the compound is a compound selected from the following group or a pharmaceutically acceptable salt thereof:

The invention discloses a preparation method of the above-mentioned compound formula I, which comprises the following steps: (1) in an organic solvent, under the action of a base, the compound I-G of the general formula and the compound I-F undergo a substitution reaction to obtain an intermediate I-E; (2) In an organic solvent, the intermediate I-E undergoes a deprotection reaction (de-tert-butyl) under the action of an acid to obtain an intermediate I-D; (3) in an organic solvent, the compound I-D of the general formula and the compound I-C undergo a substitution reaction under the action of a base , to obtain the intermediate I-B of the general formula; (4) in an organic solvent, the intermediate I-B of the general formula and the compound I-A are condensed under the action of a base to obtain the deuterated triazine compound of the general formula I.

The invention discloses a pharmaceutical composition, which is characterized in that it contains a pharmaceutically acceptable carrier and the above compound, or a pharmaceutically acceptable salt, isomer, prodrug or co-crystal thereof. The pharmaceutical composition is characterized in that it contains another therapeutic drug, and the additional therapeutic drug is an antiviral drug.

The use of the above-mentioned compound formula I or its pharmaceutically acceptable salt disclosed in the present invention, or the pharmaceutical composition is characterized in that it is used to prepare a 3CL protease inhibitor pharmaceutical composition.

The invention discloses the use of the above pharmaceutical composition, which is characterized in that the pharmaceutical composition is used for preparing medicines for treating and preventing virus infection. Preferably, wherein said viral infection is human coronavirus, new coronavirus (SARS-CoV-2), SARS coronavirus and MERS coronavirus.

In various embodiments, the compound preferably has one of the structures shown in Table 1 below.

Table 1 representative deuterated triazine compounds

In certain embodiments, the present invention contemplates pharmaceutical compositions comprising a pharmaceutically acceptable excipient and a compound disclosed herein. In certain embodiments, the pharmaceutical compositions are in the form of tablets, capsules, pills, or aqueous buffers, such as saline or phosphate buffers.

In certain embodiments, the disclosed pharmaceutical compositions may comprise a compound disclosed herein and a propellant. In certain embodiments, the propellant is an aerosolized propellant, such as compressed air, ethanol, nitrogen, carbon dioxide, nitrous oxide, hydrofluoroalkanes (HFAs), 1,1,1,2-tetrafluoroethane , 1,1,1,2,3,3,3-heptafluoropropane or combinations thereof.

In certain embodiments, the present invention contemplates pressurized or non-pressurized containers containing a compound or pharmaceutical composition described herein. In certain embodiments, the container is a hand pump nebulizer, inhaler, metered dose inhaler, dry powder inhaler, nebulizer, vibrating mesh nebulizer, jet nebulizer, or ultrasonic nebulizer.

In certain embodiments, the compound or pharmaceutical composition is administered orally, intravenously or pulmonally, ie, pulmonary.

In certain embodiments, the present invention relates to the use of the compounds described herein in the preparation of a medicament for the treatment or prevention of viral infections, such as human coronavirus, new coronavirus (SARS-CoV-2), SARS Coronavirus and MERS-CoV infection.

In certain embodiments, the present invention is directed to methods of preparing compounds by admixing the starting materials and reagents disclosed herein under conditions that form the compounds disclosed herein.

The invention designs and discloses a novel deuterated triazine compound, which realizes better pharmacokinetic properties and therapeutic effects than S-217622 on the basis of comparable virus inhibitory activity, and better druggability. At the same time, the preparation method, pharmaceutical composition and application thereof are disclosed, the production scale-up of the medicine can be realized, the quality is controllable, and the medicine has good clinical value.

The following detailed descriptions are exemplary and explanatory only, not restrictive.

In the following examples, unless otherwise indicated, all solvents and reagents used were obtained commercially and used as received.

The procedure described below can be used to synthesize compounds 1-11.

This article uses the following abbreviations:

LHMDS: lithium hexamethyldisilazide

ACN: Acetonitrile

THF: Tetrahydrofuran

DMF: N,N-Dimethylformamide

LiAlD ₄ : lithium tetradeuterium aluminum

PBr ₃ : phosphorus tribromide

EtOAc: ethyl acetate

PE: petroleum ether

CD ₃ I: deuteroiodomethane

K ₂ CO ₃ : potassium carbonate

Detailed ways

Preparation Example

Embodiment 1: the synthesis of compound I-F

1) Synthesis of compound I-F-1

Add raw materials I-F-2 (10g, 52.6mmol) and 100ml of tetrahydrofuran into a 250ml three-necked flask, cool to -10°C, add tetradeuterium aluminum lithium (6.3g, 157.8mmol) in 4 batches, after feeding, warm to room temperature 25°C, stirred for 4h. TLC monitored the end point, cooled to 0°C, slowly added water (6g), 15% sodium hydroxide solution (6g), and water (18g) dropwise, raised to room temperature and stirred for 1h, filtered, washed, and the filtrate was concentrated under reduced pressure to obtain yellow Crude oil. Purified by column chromatography (PE:EtOAc=1:1), the compound I-F-1 (6.5 g, yield 75%) was obtained as a pale yellow oil.

2) Synthesis of compound I-F-a

Add the raw material I-F-1 (6.5g, 39.4mmol) and 150ml of tetrahydrofuran into a 500ml three-necked flask, cool to 0°C, slowly add phosphorus tribromide THF solution (21.3g, 78.8mmol, 60ml) dropwise, and the injection is completed. Rise to 40°C and stir for 4h. TLC monitored the end point, cooled to room temperature, slowly added water (100g) dropwise, added ethyl acetate (200ml), the organic phase was washed with water, sodium bicarbonate solution, and brine respectively, and the organic phase was concentrated under reduced pressure to obtain a yellow oil Crude. Purified by column chromatography (PE:EtOAc=2:1), the compound I-F-a (7.3 g, yield 82%) was obtained as a pale yellow oil.

Summary of compound I-F structural formulas:

The preparation method of compound I-F-b is the same as that of I-F-a (using lithium aluminum hydride as reducing agent).

Embodiment 2: the synthesis of compound I-C

1) Synthesis of compound I-C-1

Add raw materials I-C-2 (10g, 70.9mmol) and 100ml of tetrahydrofuran to a 250ml three-necked flask, cool to -10°C, add lithium tetradeuterium aluminum (8.5g, 212.7mmol) in 4 batches, after feeding, warm to room temperature 25°C, stirred for 4h. TLC monitored the end point, cooled to 0°C, slowly added water (8.5g), 15% sodium hydroxide solution (8.5g), and water (26g) dropwise, raised to room temperature and stirred for 1h, filtered, washed, and the filtrate was concentrated under reduced pressure. The crude product was obtained as pale yellow oil. Purified by column chromatography (PE:EtOAc=1:1), the compound I-C-1 (6.4 g, yield 78%) was obtained as an off-white solid.

2) Synthesis of compound I-C-b

Add raw material I-C-1 (6.4g, 55.3mmol) and 150ml tetrahydrofuran into a 500ml three-necked flask, cool to 0°C, slowly add phosphorus tribromide THF solution (29.9g, 110.6mmol, 60ml) dropwise, Raised to 40 ° C, stirred for 6h. TLC monitored the end point, cooled to room temperature, slowly added water (100g) dropwise, added ethyl acetate (200ml), the organic phase was washed with water, sodium bicarbonate solution, and brine respectively, and the organic phase was concentrated under reduced pressure to obtain a light yellow solid Crude. Purified by column chromatography (PE:EtOAc=2:1), the compound I-C-b (7.8 g, yield 79%) was obtained as a white solid.

Summary of Compound I-C Structural Formulas:

The preparation method of compounds I-C-a, I-C-c, and I-C-d is the same as that of I-C-b (using lithium aluminum hydride as reducing agent).

Embodiment 3: the synthesis of compound I-A

1) Synthesis of compound I-A-1

Add raw material I-A-2 (10g, 50.6mmol), DMF80ml and potassium carbonate (20.9g, 151.8mmol) to a 250ml three-necked bottle, and add deuteroiodomethane (14.7g, 101.2mmol) dropwise at room temperature, Rise to 60°C and stir for 8h. The endpoint was monitored by TLC, ethyl acetate (300 ml) was added at room temperature, the organic phase was washed with water and brine, and the organic phase was concentrated under reduced pressure to obtain a crude yellow oil. Purified by column chromatography (PE:EtOAc=3:1), the compound I-A-1 (7.8 g, yield 72%) was obtained as a pale yellow solid.

2) Synthesis of compound I-A-a

Add raw material I-A-1 (7.8g, 36.4mmol), methanol 100ml and palladium carbon 10% (0.39g) in the 200ml three-necked bottle, at room temperature, add ammonium formate (9.2g, 145.6mmol), throw and finish, rise to 60 ° C, stirring for 6h. The endpoint was monitored by TLC, filtered and washed, and the filtrate was concentrated under reduced pressure to obtain a crude yellow solid. Purified by column chromatography (PE:EtOAc=2:1), the compound I-A-a (5.4 g, yield 80%) was obtained as a pale yellow solid.

Compound I-A structural formula summary:

The preparation method of compound I-A-b is the same as I-A-a (using methyl iodide as an alkylating agent).

Embodiment 4: the synthesis of compound I-E

Synthesis of Compound I-E-a

At room temperature, add raw materials I-G (1.0g, 4.3mmol), I-F-b (1.0g, 4.7mmol), DMF10ml and potassium carbonate (1.8g, 12.9mmol) into a 50ml three-necked flask, after the injection is complete, raise to 80°C and stir 4h. The endpoint was monitored by TLC, ethyl acetate (30 ml) was added at room temperature, the organic phase was washed with water and brine, and the organic phase was concentrated under reduced pressure to obtain a crude yellow oil. Purified by column chromatography (PE:EtOAc=3:1), the compound I-E-a (1.1 g, yield 71%) was obtained as a pale yellow oil.

The preparation method of compound I-E-b is the same as that of I-E-a.

Embodiment 5: the synthesis of compound I-D

Synthesis of Compound I-D-a

At room temperature, add raw material I-E-a (1.0 g, 2.7 mmol) and trifluoroacetic acid (2.5 ml) into a 20 ml reaction flask, and stir at room temperature for 24 h after the injection is complete. The end point was monitored by TLC. After the reaction solution was concentrated under reduced pressure, the crude product was slurried with toluene + isopropyl ether, filtered, washed and dried to obtain compound I-D-a (0.8 g, yield 92%), off-white solid.

The preparation method of compound I-D-b is the same as that of I-D-a.

Embodiment 6: the synthesis of compound I-B

Synthesis of Compound I-B-a

At room temperature, add the raw materials I-D-a (0.6g, 1.9mmol), I-C-a (0.4g, 2.3mmol), DMF5ml and potassium carbonate (0.8g, 5.7mmol) into a 20ml reaction flask, after the injection is complete, raise the temperature to 65°C and stir for 4h . The endpoint was monitored by TLC, ethyl acetate (20 ml) was added at room temperature, the organic phase was washed with water and brine, and the organic phase was concentrated under reduced pressure to obtain a crude yellow solid. Purified by column chromatography (PE:EtOAc=2:1), the compound I-B-a (0.47 g, yield 60%) was obtained as a white solid.

The preparation methods of compounds I-B-b, I-B-c, I-B-d, I-B-e, I-B-f are the same as I-B-a.

Embodiment 7: the synthesis of compound I

Synthesis of compound I-1

At room temperature, add raw materials I-B-a (0.3g, 0.72mmol), I-A-b (0.17g, 0.94mmol), THF6ml into a 20ml reaction flask, and add LHMDS (1M, 1.46mmol) dropwise at 0°C. Stir at ℃ for 2h, then rise to room temperature and stir for 1h. The end point was monitored by TLC, quenched by adding saturated ammonium chloride dropwise, and ethyl acetate (20 ml) was added, the organic phase was washed with water and brine, and the organic phase was concentrated under reduced pressure to obtain a light yellow solid crude product. Purified by column chromatography (DCM:MeOH=10:1), the compound I-1 (0.18 g, yield 46%) was obtained as a white solid.

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.36(s,1H),8.43(s,1H),7.75(s,1H),7.51-7.66(m,2H),7.42(m,1H ), 5.27(s,2H), 5.02(s,2H), 4.17(s,3H). MS (ESI): 536 ([M+H] ⁺ ).

Synthesis of Compound I-2

The preparation method of compound I-2 is the same as that of I-1 (using reaction materials I-B-f and I-A-a).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.33(s,1H),8.38(s,1H),7.70(s,1H),7.52-7.67(m,2H),7.45(m,1H ), 5.23(s,2H), 5.07(s,2H), 3.95(s,3H). MS (ESI): 536 ([M+H] ⁺ ).

Synthesis of Compound I-3

The preparation method of compound I-3 is the same as that of I-1 (using reaction materials I-B-a and I-A-a).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.31(s,1H),8.41(s,1H),7.72(s,1H),7.51-7.67(m,2H),7.46(m,1H ), 5.24(s,2H), 5.06(s,2H). MS (ESI): 539 ([M+H] ⁺ ).

Synthesis of compound I-4

The preparation method of compound I-4 is the same as that of I-1 (using reaction materials I-B-b and I-A-b).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.35(s,1H),8.39(s,1H),7.72(s,1H),7.51-7.66(m,2H),7.43(m,1H ), 5.25(s,2H), 4.14(s,3H), 3.96(s,3H). MS (ESI): 535 ([M+H] ⁺ ).

Synthesis of compound I-5

The preparation method of compound I-5 is the same as that of I-1 (using reaction materials I-B-e and I-A-b).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.36(s,1H),8.38(s,1H),7.74(s,1H),7.53-7.66(m,2H),7.41(m,1H ), 5.07(s,2H), 4.16(s,3H), 3.95(s,3H). MS (ESI): 535 ([M+H] ⁺ ).

Synthesis of compound I-6

The preparation method of compound I-6 is the same as that of I-1 (using reaction materials I-B-c and I-A-a).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.37(s,1H),8.39(s,1H),7.76(s,1H),7.52-7.65(m,2H),7.43(m,1H ), 5.28(s,2H). MS (ESI): 541 ([M+H] ⁺ ).

Synthesis of compound I-7

The preparation method of compound I-7 is the same as that of I-1 (using reaction materials I-B-d and I-A-a).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.38(s,1H),8.39(s,1H),7.75(s,1H),7.52-7.64(m,2H),7.42(m,1H ), 5.08(s,2H). MS (ESI): 541 ([M+H] ⁺ ).

Synthesis of compound I-8

The preparation method of compound I-8 is the same as that of I-1 (using reaction materials I-B-c and I-A-b).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.35(s,1H),8.41(s,1H),7.76(s,1H),7.52-7.62(m,2H),7.43(m,1H ), 5.25(s,2H), 4.16(s,3H). MS (ESI): 538 ([M+H] ⁺ ).

Synthesis of compound I-9

The preparation method of compound I-9 is the same as that of I-1 (using reaction materials I-B-d and I-A-b).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.38(s,1H),8.40(s,1H),7.75(s,1H),7.55-7.62(m,2H),7.44(m,1H ), 5.07(s,2H), 4.15(s,3H). MS (ESI): 538 ([M+H] ⁺ ).

Synthesis of compound I-10

The preparation method of compound I-10 is the same as that of I-1 (using reaction materials I-B-b and I-A-a).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.39(s,1H),8.41(s,1H),7.75(s,1H),7.53-7.64(m,2H),7.43(m,1H ), 5.26(s,2H), 3.95(s,3H). MS (ESI): 538 ([M+H] ⁺ ).

Synthesis of compound I-11

The preparation method of compound I-11 is the same as that of I-1 (using reaction materials I-B-e and I-A-a).

¹ H NMR(400MHz,DMSO-d6)δH(ppm):9.38(s,1H),8.39(s,1H),7.74(s,1H),7.55-7.64(m,2H),7.44(m,1H ), 5.08(s,2H), 3.96(s,3H). MS (ESI): 538 ([M+H] ⁺ ).

Embodiment 8:

This example is the antiviral activity of compound S-217622, compound I-1, compound I-2, compound I-4 and compound I-5 to SARS coronavirus, MERS coronavirus and HCoV human coronavirus.

The antiviral effects of compound S-217622, compound I-1, compound I-2, compound I-4 and compound I-5 on SARS coronavirus, MERS coronavirus and HCoV human coronavirus were studied on Vero E6 and Vero76 cell lines active. The neutral red assay was used to determine virus-induced and compound-induced cytopathic inhibition (CPE), and the EC ₅₀ and CC ₅₀ values of the compounds were calculated. For the neutral red test, the test compound was dissolved in DMSO at a concentration of 10 mg/mL and serially diluted with 8 half-log diluents up to a maximum test concentration of 50 μg/mL (86 μM). Each dilution was added to 5 wells of a 96-well plate containing 80-100% confluent Vero E6 or Vero76 cells. 3 wells of each dilution were infected with virus, and 2 wells were not infected as toxicity control. 6 wells were untreated and infected as virus control. For SARS, MERS, and HCoV human coronaviruses, the viruses were diluted to MOIs of 0.003, 0.002, 0.001, and 0.03 50% cell culture infectious dose per cell, respectively. Plates were incubated at 37°C in an incubator with 5% CO2. On day 5 (HCoV) or day 7 (SARS, MERS) after infection, when the untreated virus control wells reached the maximum CPE, the plates were stained with neutral red dye for approximately 2 hours. The supernatant dye was removed, each well was washed with phosphate buffered saline (PBS), and the dye was washed in a 1:1 mixture of citrate buffer and ethanol for 30 min. The optical density at 540 nm was read on a spectrophotometer and converted to a percentage of the control. The CPE 50% ( _EC50 ) against virus and the concentration of test compound required to cause 50% cell death ( _CC50 ) in the absence of virus were calculated.

Effect Example

Embodiment 9:

This embodiment is the detection of inhibition of protease activity targeting SARS-CoV-2 virus M ^Pro

Detection principle: 3-chymotrypsin-like protease (3-chymotrypsin-like Protease), the main protease (M ^Pro , also known as 3CL ^Pro ), is encoded by ORF1 (located in nsp5), located in the central region of the replicase gene, It is a key protein in the replication of the new coronavirus RNA. Its mechanism of action is: after the new coronavirus invades cells, it will use the host cells to synthesize two ultra-long replicase polypeptides (ppla and pplab) necessary for its own replication. The replicase polypeptide needs to be further cut into multiple proteins (such as RdRp, helicase, etc.), and further assembled into the replication transcription machinery required for the virus to initiate the replication of its own genetic material. M ^Pro has at least 11 cleavage sites on the replicase polypeptide, and only when these sites on the replicase polypeptide are cut normally, it is assembled into a replication transcription machine and starts virus replication. In view of the fact that M ^Pro protease is very important in the process of virus replication, and there is no similar protein in the human body, the main protease M ^Pro becomes a potential key drug target against the new coronavirus. The inhibitory activity of deuterated triazines against SARS-CoV-2-M ^Pro protease was evaluated by fluorescence resonance energy transfer method.

Specific detection method: the volume of the entire enzymatic reaction system is 120 μL, the final concentration of protease is 30 nM, the final concentration of substrate is 20 μM, and the buffer solution of the reaction system includes 50 mM Tris, pH 7.3 1 mM EDTA. Add SARS-CoV-2-M ^Pro protease and different concentrations of target compounds to the 96-well plate, incubate at 30°C for 10 minutes, add the substrate and quickly put it into a microplate reader for reading. Excitation light and emission light are 340nm and 405nm, respectively. The test time is 10min, and the fluorescence value is read every 30s. For the final result, the reading value of the first 2 minutes was used to fit the reaction rate, and compared with the control group (DMSO), the inhibition rate was calculated. Use Graghpad prism 7 to plot the _IC50 value of the anti-SARS-CoV-2 virus compound at the corresponding time point. The specific values are shown in Table 3.

Table 3: _IC50 values of SARS-CoV-2-M ^Pro protease

化合物compound	IC ₅₀(μΜ) _IC50 (μΜ)
化合物I-1Compound I-1	0.022±0.007μΜ0.022±0.007μΜ
化合物I-2Compound I-2	0.025±0.008μΜ0.025±0.008μΜ
S-217622S-217622	0.018±0.005μΜ0.018±0.005μΜ

Example 10:

This embodiment is a pharmacokinetic study of the compound on rats

Pharmacokinetic studies were performed in male Wistar-Hannover rats at 7-10 weeks of age. All animals were housed individually during the pharmacokinetic studies. Food and water were given ad libitum (animals were dosed in the fed state). Animals were fasted overnight and fed 4 hours after dosing. Blood samples were collected through jugular vein cannulation at predetermined time points after intragastric administration (30 mg/kg). At study completion, animals were euthanized by overdose of inhalational anesthesia followed by exsanguination. Blood samples were collected into tubes containing _K2EDTA and stored on ice until centrifuged to obtain plasma, which was stored in a -20°C freezer.

LC-MS/MS Analysis of Plasma Samples: Plasma samples were processed using a protein precipitation method using acetonitrile:methanol containing 500:50, containing propranolol (50ng/ml) as an internal standard, and then according to a standard curve prepared in blank plasma ( 0.1-2500ng/ml) for quantification. Analytes in plasma samples were quantified using LC-MS/MS. Briefly, a Waters ACQUITY Ultra Performance Liquid Chromatography System coupled to a Sciex 6500 Triple Quadrupole Mass Spectrometer was used. Chromatographic separation was accomplished using a Waters Acquity UPLC BEH C18 column (1.7m, 2.150mm). Optimize mobile phases to achieve good separation between analytes. Typically, solvent A consisted of 0.025% formic acid and 1 mM ammonium acetate in water/acetonitrile (95:5 v/v) and solvent B consisted of 0.025% formic acid and 1 mM ammonium acetate in water/acetonitrile (5:95 v/v). The gradient generally starts at 3-30% B until approximately 1.2 minutes, then increases to 50-65% B to 1.6 minutes, then decreases to 10-30% B until approximately 1.7-1.9 minutes. Analyst 1.7 software was used for peak integration and standard curve regression.

Pharmacokinetic analysis: Pharmacokinetic parameters were calculated using non-compartmental analysis (Watson v.7.5, Thermo Scientific). As shown in Table 4, it can be seen from the results that compound I-1 and compound I-2 have a better half-life in animals, and thus have better therapeutic effects.

Table 4: Pharmacokinetic parameters of compounds

化合物compound	T _1/2(h) T _1/2 (h)
化合物I-1Compound I-1	3.53.5
化合物I-2Compound I-2	3.53.5

S-217622

2.5

Application example

Embodiment 11:

For the oral tablet preparation method of deuterated compound (taking compound I-1 as an example)

The pharmaceutical carriers used in oral tablets include regulators, fillers, binders, disintegrants, additives, glidants, lubricants, film coating materials, plasticizers, coloring agents, and the like.

组分components	作用effect	含量(mg/片)Content (mg/tablet)
化合物I-1Compound I-1	活性成分active ingredient	200200
淀粉starch	填充剂、崩解剂fillers, disintegrants	100100
磷酸氢钙Calcium hydrogen phosphate	填充剂filler	2020
预胶化淀粉pregelatinized starch	填充剂filler	4040
枸橼酸citric acid	调节剂Regulator	22
亚硫酸氢钠sodium bisulfite	添加剂additive	0.50.5
10％淀粉浆10% starch slurry	粘合剂Adhesive	适量Appropriate amount
硬脂酸镁Magnesium stearate	润滑剂lubricant	1.51.5
白色欧巴代White Opadry	包衣预混料Coating Premix	约4about 4
水、乙醇water, ethanol	溶剂solvent	适量Appropriate amount

Operation method:

According to the above formula, compound I-1 was ground and sieved respectively, and then mixed evenly with filler, disintegrant, regulator and additives that had been ground and sieved, and then 10% starch slurry was added to make a soft material in a blender. , The soft material is made into wet granules on a swinging machine, dried in an oven, mixed evenly with a lubricant, and pressed into tablet cores. The tablet core is made into a film-coated tablet with Opadry.

For the capsule preparation method of deuterated compound (taking compound I-1 as example)

The pharmaceutical carriers used in capsules include fillers, binders, disintegrants, additives, lubricants and the like.

组分components	作用effect	含量(mg/粒)Content (mg/grain)
化合物I-1Compound I-1	活性成分active ingredient	200200
乳糖一水合物lactose monohydrate	填充剂filler	8282
预胶化淀粉pregelatinized starch	填充剂、粘合剂fillers, binders	3838
羧甲基淀粉钠Sodium carboxymethyl starch	崩解剂disintegrant	12.512.5
硬脂酸镁Magnesium stearate	润滑剂lubricant	1.51.5

Operation method:

According to the above formula, the compound I-1 and the auxiliary materials are respectively ground and sieved, mixed with fillers, binders and disintegrants in a certain proportion, added to the dry granulator and pressed into strips, and then passed through the crusher Crushed into granules. The granules are evenly mixed with an appropriate amount of lubricant and disintegrant, and then filled into capsules.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the scope of the disclosed patents. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims

A deuterated triazine compound as shown in formula I, or a pharmaceutically acceptable salt, isomer, prodrug or cocrystal thereof:

in:

R 1 to R 3 are each independently hydrogen or deuterium;

R 4 to R 7 are each independently hydrogen or deuterium;

R 8 to R 10 are each independently hydrogen or deuterium;

The additional proviso is that at least one of R 1 to R 10 is deuterium.
The compound according to claim 1, characterized in that R 1 to R 3 are 2-3 deuteriums.
The compound according to claim 1, characterized in that R 4 to R 7 are 2-4 deuteriums.
The compound according to claim 1, wherein R 8 -R 10 are 2-3 deuterium.
The compound according to claim 1, wherein the compound is a compound selected from the following group or a pharmaceutically acceptable salt thereof:
A method for preparing a compound of formula I according to any one of claims 1-5, comprising the following steps:

(1) In an organic solvent, under the action of a base, the compound I-G of the general formula and the compound I-F undergo a substitution reaction to obtain an intermediate I-E;

(2) In an organic solvent, the intermediate I-E undergoes a deprotection reaction under the action of an acid to obtain the intermediate I-D;

(3) In an organic solvent, under the action of a base, the compound I-D of the general formula is subjected to a substitution reaction with the compound I-C to obtain the intermediate I-B of the general formula;

(4) In an organic solvent, under the action of a base, the intermediate I-B of the general formula is condensed with the compound I-A to prepare the deuterated triazine compound described in the formula I.
A pharmaceutical composition, characterized in that it contains a pharmaceutically acceptable carrier and the compound of any one of claims 1-5, or a pharmaceutically acceptable salt, isomer, prodrug or co-crystal thereof.
The pharmaceutical composition according to claim 7, characterized in that it contains another therapeutic drug, and the additional therapeutic drug is an antiviral drug.
A compound or pharmaceutically acceptable salt thereof as claimed in claim 1-5, or the purposes of the pharmaceutical composition as claimed in claim 7, it is characterized in that, for the preparation of 3CL protease inhibitor pharmaceutical composition .
The use according to claim 9, characterized in that the pharmaceutical composition is used for the preparation of medicines for treating and preventing viral infections.
The use according to claim 10, wherein the virus infection is human coronavirus, new coronavirus SARS-CoV-2, SARS coronavirus and MERS coronavirus.