A selenium-containing triazine derivative, its preparation method and uses

Abstract

The present invention provides a compound represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof, and a preparation method and use thereof. Among them, R1 is a benzyl group substituted by 1 to 5 halogens, R2 is H or a halogen, R3 is a C1-C6 alkyl group optionally substituted by 1 to 3 halogens, R4 is H, a halogen or a C1-C6 alkyl group, and the halogen is selected from fluorine, chlorine, bromine and iodine. By introducing a selenocarbonyl group into the triazine ring in the present invention, the activity of the obtained compound against the novel coronavirus is significantly improved compared with the corresponding unselenized positive control drug. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a selenium-containing triazine derivative, a preparation method thereof, and uses thereof. Background Art

[0002] Coronaviruses are classified in the order Nidovirales, family Coronaviridae, genus Coronavirus. Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with a linear genome and are a large group of viruses widely existing in nature. Coronaviruses have a diameter of about 80-120 nm, with a methylated cap structure at the 5′ end of the genome and a poly(A) tail at the 3′ end. The genome is about 27-32 kb in length, making it the largest genome among known RNA viruses. Coronaviruses only infect vertebrates, such as humans, mice, pigs, cats, dogs, wolves, chickens, cattle, and birds. Coronaviruses were first isolated from chickens in 1937. The diameter of the virus particles is 60-200 nm, with an average diameter of 100 nm, and they are spherical or oval in shape, showing pleomorphism. The virus has an envelope with spikes on it, and the whole virus resembles a corona. The spikes of different coronaviruses are significantly different. Tubular inclusions can sometimes be seen in coronavirus-infected cells.

[0003] The novel coronavirus 2019 (referred to as the COVID-19 virus, 2019-nCoV or SARS-CoV-2, which causes coronavirus disease 2019, COVID-19) is the seventh known coronavirus that can infect humans. The other six are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (which causes severe acute respiratory syndrome), and MERS-CoV (which causes Middle East respiratory syndrome). To date, multiple variants of SARS-CoV-2 have emerged, including Alpha, Beta, Gamma, Delta, Omicron, etc.

[0004] The COVID-19 virus is a positive-sense single-stranded RNA virus. After its spike (S) protein binds to the receptor ACE2 on the human cell membrane, it enters the cell and releases viral RNA. The viral RNA is translated into two polyproteins, pp1a and pp1ab, in the host cell. These two polyproteins are cleaved by 3CLpro and PLpro into various non-structural proteins (NSPs) required for the COVID-19 virus. Subsequently, replication-transcription occurs under the combined action of multiple non-structural proteins such as RNA polymerase (RdRp), and the progeny structural proteins are translated. Then, they combine with the progeny positive-strand RNA and are assembled into new virus particles through the Golgi apparatus and the ERGIC lumen for release.

[0005] At present, the research on COVID-19 drugs mainly focuses on three targets: the spike (S) protein, 3CL protease, and RNA polymerase, which are indispensable in the process of virus invasion and proliferation in the human body. The COVID-19 drug S-217622, jointly developed by Hokkaido University and Shionogi, is a 3CL protease inhibitor. Similar to the COVID-19 drug Paxlovid of Pfizer, S-217622 inhibits the activity of 3CL protease, preventing the cleavage of protein precursors and the formation of mature virions, thus inhibiting the self-replication of the virus. In addition, through structural improvement, S-217622 can get rid of the dependence on P450 enzyme inhibitors (such as ritonavir), and can be used for the treatment of COVID-19 alone, expanding the scope of applicable populations.

[0006]

[0007] However, there is still a need in the art for more effective drugs with anti-coronavirus, especially anti-SARS-CoV-2 activity, to effectively prevent and control infectious diseases such as COVID-19. Summary of the Invention

[0008] Therefore, the object of the present invention is to provide a selenium-containing triazine derivative, its preparation method and use. By structurally modifying S-217622 and its analogs, the present invention significantly improves the anti-coronavirus, especially anti-SARS-CoV-2 activity of S-217622 and its analogs.

[0009] In the context of the present invention, the SARS-CoV-2 includes the original strain of SARS-CoV-2 as well as various SARS-CoV-2 variants such as Alpha, Beta, Gamma, Delta, and Omicron.

[0010] The object of the present invention is achieved by the following technical solutions:

[0011] On the one hand, the present invention provides a compound of formula (I), its tautomer or its pharmaceutically acceptable salt:

[0012]

[0013] Wherein, R1 is a benzyl group substituted by 1 to 5 halogens, R2 is H or a halogen, R3 is a C1-C6 alkyl group optionally substituted by 1 to 3 halogens, R4 is H, a halogen or a C1-C6 alkyl group, and the halogen is selected from fluorine, chlorine, bromine and iodine.

[0014] Preferably, wherein R1 is a benzyl group substituted by 1 to 3 halogens; R3 is a C1-C3 alkyl group optionally substituted by 1 to 3 halogens, and / or R4 is H, a halogen or a C1-C3 alkyl group.

[0015] Preferably, wherein, R1 is selected from:

[0016]

[0017] R2 is H or chlorine;

[0018] R3 is methyl, ethyl, propyl or isopropyl optionally substituted by 1 to 3 fluorines; and / or

[0019] R4 is H or methyl.

[0020] Preferably, the structure of the compound is as follows:

[0021]

[0022] On the other hand, the present invention provides a method for preparing the compound shown in formula (I), its tautomer or its pharmaceutically acceptable salt, comprising the following steps:

[0023]

[0024] Mix the compound shown in formula (II) with a selenium reagent in a solvent and react at 100 - 150 °C to obtain the compound shown in formula (I); wherein R1 - R4 are defined as in any one of claims 1 to 4.

[0025] Preferably, the solvent is selected from one or more of toluene, nitrobenzene, chlorobenzene and xylene; the selenium reagent is Woollins reagent or selenium; the reaction is carried out at 110 - 140 °C; and / or the reaction is carried out for 1 - 10 hours.

[0026] Preferably, the method of the present invention further comprises the steps of adding an organic solvent and an aqueous alkali solution to the reaction mixture after the reaction, stirring, separating the layers, evaporating the organic phase to dryness, and column chromatography separation; more preferably, the organic solvent is ethyl acetate, dichloromethane, methyl tert-butyl ether and / or isopropyl acetate, and / or the aqueous alkali solution is an aqueous solution of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, sodium hydroxide and / or potassium hydroxide.

[0027] On yet another aspect, the present invention provides a pharmaceutical composition comprising the compound shown in formula (I) of the present invention, its tautomer or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

[0028] Preferably, the pharmaceutical composition further comprises vitamin C. After combining the compound shown in formula (I) of the present invention, its tautomer or its pharmaceutically acceptable salt with vitamin C, the antiviral treatment effect can be further improved.

[0029] On the other hand, the present invention also provides the use of the compound represented by the formula (I), its tautomer or its pharmaceutically acceptable salt in the preparation of a drug for preventing and / or treating diseases caused by coronavirus infection. Preferably, the coronavirus is SARS-CoV-2.

[0030] The inventors unexpectedly found that when a selenocarbonyl group is introduced into the triazine ring of S-217622 or its analogs, the resulting compound has significantly improved activity compared to the corresponding non-selenated compound S-217622 or its analogs. For example, the inhibitory activity of the compound Y7301 of the present invention against SARS-CoV-2 3CL protease is about 3 times that of S-217622. The compounds of the present invention have significantly improved anti-SARS-CoV-2 beta coronavirus activity compared to the corresponding non-selenated compounds. The compounds of the present invention can be prepared into drugs for preventing and / or treating diseases and / or symptoms induced by SARS-CoV-2 (including the original SARS-CoV-2 strain and various SARS-CoV-2 variants such as Alpha, Beta, Gamma, Delta, and Omicron) infection, and have significantly improved curative effects. Description of the Drawings

[0031] Figure 1 is S-217622, which is an inhibitor of SARS-CoV-2 3CL protease;

[0032] Figure 2 is the general structural formula of a compound of a selenium-containing triazine derivative;

[0033] Figure 3 is the chemical group of R1;

[0034] Figure 4 is the structure of Y7301;

[0035] Figure 5 is the reaction formula of a selenium-containing triazine derivative compound;

[0036] Figure 6 is the reaction formula for synthesizing the Y7301 compound;

[0037] Figure 7 For the data obtained in Example 2, it was processed using the software GraphPad Prism 7.0 to calculate the IC 50 value;

[0038] Figure 8 is the measurement result of quantifying virus replication by measuring the yield of infectious virus in the Vero16 apical wash fluid by plaque assay on a monolayer of Vero16 cells and by measuring the yield of viral RNA from total cellular RNA by real-time PCR;

[0039] Figure 9 It is the general chemical structure formula of a selenium-containing triazine derivative. Specific embodiments

[0040] The present invention will be further described in detail below in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more comprehensively. The specific embodiments are only used to illustrate the technical solutions of the present invention and do not limit the present invention in any way.

[0041] Unless otherwise specified, the reagents and raw materials used in the following examples are all commercially available.

[0042] Among them, S-217622 was purchased from Jinan Hongkangda Chemical Co., Ltd.;

[0043] The selenium reagent (Woollins reagent) was purchased from Wuxi Lanbo (Wuhan) Chemical Technology Co., Ltd.;

[0044] EV71 3Cpro was purchased from Sigma-Aldrich Reagent Company.

[0045] Example 1

[0046]

[0047] Add S-217622 (530 mg) to xylene (10 mL), add Woollins reagent (1.06 g), and reflux overnight under nitrogen protection.

[0048] Cool the reaction solution to room temperature, add ethyl acetate and aqueous sodium bicarbonate solution, stir for 15 minutes, separate the layers, dry the organic phase and evaporate to dryness, and obtain 170 mg of solid Y7301 by column chromatography, with a yield of 28.6%. MS (m / z): 596.2 (M+H) + 。

[0049] 1 1H-NMR (DMSO-d6) δ 9.35 (s, 1H), 8.45 (s, 1H), 7.75 (s, 1H), 7.69 (m, 1H), 7.59 (m, 1H), 7.45 (m, 1H), 5.25 (s, 2H), 5.01 (s, 2H), 4.12 (s, 3H), 3.93 (s, 3H).

[0050] Example 2 : Evaluation of the inhibitory activity of the compound against SARS-CoV-2 3CL protease

[0051] Inhibitory activity test of the compound against the 3CL protease of SARS-CoV-2. The enzyme-level inhibitory activity of the inhibitor against 3CL protease was determined using fluorescence resonance energy transfer (FRET) technology.

[0052] <Operation procedure>

[0053] In a 96-well plate, 27.5 μL of enzyme buffer (20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.3) was added to each well, along with 2.5 μL of compounds at different concentrations and 5 μL of EV71 3Cpro (final concentration 3 μM). Incubate together at 37 °C for 20 min. Then, 15 μL of the fluorescent substrate diluted with buffer (final concentration 20 μM) was added. The fluorescence parameters were measured using a Gen5 fluorometer, with the excitation wavelength and emission wavelength being 340 nm and 490 nm, respectively. Keep at 37 °C and read the data after 10 min. For the negative control, no compound was added, and the rest of the operations were the same.

[0054] <Calculation of each measured item value>

[0055] The obtained data was processed using the software GraphPad Prism 7.0 to calculate the IC 50 value. The experimental results are shown in Table 1.

[0056] Table 1

[0057] Compound <![CDATA[IC 50 (nM)]]> Y7301 11 S-217622 35

[0058] From the above results, it can be seen that Y7301 showed significantly improved inhibitory activity against SARS-CoV-2 3CL protease, about 3 times that of S-217622. Since the SARS-CoV-2 3CL protease is common to the original SARS-CoV-2 strain and various SARS-CoV-2 variants including Alpha, Beta, Gamma, Delta, and Omicron, the compounds of the present invention have inhibitory effects on both the original SARS-CoV-2 strain and its variants. The compounds of the present invention can be prepared into drugs for preventing and / or treating diseases and / or symptoms induced by SARS-CoV-2 infection, and have significantly improved curative effects.

[0059] Example 3 : Determination of anti-SARS-CoV-2 beta-coronavirus activity

[0060] In a 12-well disposable cell culture plate, a confluent monolayer of cell culture was prepared. Vero16 cells were maintained in DMEM supplemented with 10% FBS and supplemented with 1% penicillin / streptomycin. The apical surface of the Vero16 cell culture was washed 24 hours and 1 hour before infection with 1×PBS, and then infected with 1×PBS at 37°C for 1.5 hours. Vero16 cells were infected with a recombinant SARS-CoV-2 beta-coronavirus (SARS-CoV-2βRFP) expressing green fluorescent protein at a multiplicity of infection of 0.1 pfu / cell. The apical wash was removed, the virus inoculum was added, and the inoculated culture was incubated at 37°C, 5% CO2 for 3 hours. The inoculum was removed, and the apical surface of the Vero16 cells was washed 3 times with 500 μL of 1×PBS to remove residual virus. A 3-fold serial dilution of the example compound was prepared starting at 10 μM, in triplicate, and added to the Vero16 cell medium outside the basal side of the culture approximately 30 minutes before infection. Viral replication was evaluated by fluorescence imaging of the cell culture after 48 hours of incubation. In addition, viral replication was quantified by measuring the yield of infectious virus in the Vero16 apical wash by plaque assay on a Vero16 cell monolayer and by quantifying viral RNA production from total cellular RNA by real-time PCR assay. The assay results are shown in Table 2.

[0061] Table 2

[0062] Compound <![CDATA[CPEEC 50 (nM)]]> <![CDATA[CC 50 (nM)]]> Y7301 115 Greater than 20000 S-217622 330 Greater than 20000

[0063] As can be seen from the results in Table 2, the compounds of the present invention have prominent anti-SARS-CoV-2 beta-coronavirus activity, and compared with the positive control drug S-217622, the anti-SARS-CoV-2 beta-coronavirus activity of the compounds of the present invention is significantly improved.

Claims

1. A compound of formula (I') or a pharmaceutically acceptable salt thereof:

2. A method for preparing the compound of formula (I') or a pharmaceutically acceptable salt thereof according to claim 1, comprising the following steps: Mixing the compound of formula (II') with a selenium reagent in a solvent and reacting at 100 - 150 °C to obtain the compound of formula (I').

3. The method according to claim 2, wherein the solvent is selected from one or more of toluene, nitrobenzene, chlorobenzene and xylene; the selenium reagent is Woollins reagent or selenium; the reaction is carried out at 110 - 140 °C; or the reaction is carried out for 1 - 10 hours.

4. The method according to claim 2, further comprising the steps of adding an organic solvent and an aqueous alkali solution to the reaction mixture after the reaction is completed, stirring, separating the layers, evaporating the organic phase to dryness, and separating by column chromatography.

5. The method according to claim 4, wherein the organic solvent is ethyl acetate, dichloromethane, methyl tert-butyl ether or isopropyl acetate, or the aqueous alkali solution is an aqueous solution of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, sodium hydroxide or potassium hydroxide.

6. A pharmaceutical composition comprising the compound of formula (I') or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, further comprising vitamin C.

8. Use of the compound of formula (I') or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating a disease caused by coronavirus infection.

9. The use according to claim 8, wherein the coronavirus is SARS-CoV-2.

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