A cyano-containing selenolactam compound, its preparation method and uses

Abstract

The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and a preparation method and use thereof, wherein R1 is selected from (C1-C6 alkyl)amino optionally substituted with 1 to 5 fluorines, C1-C6 alkyl-C(O)NH- optionally substituted with 1 to 5 fluorines, and C1-C6 alkyl-S(O)2NH- optionally substituted with 1 to 5 fluorines. By replacing the oxygen in the butyrolactam ring with selenium, the obtained selenobutyrolactam compound has significantly improved anti-SARS-CoV-2 activity compared with the corresponding butyrolactam positive control drug. #imgabs0#

Description

Technical Field

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

[0002] Coronaviruses are classified into the order Nidovirales, family Coronaviridae, and genus Coronavirus in the system. Viruses of the genus Coronavirus are RNA viruses with an envelope, a linear single-stranded positive-sense genome, and are a large class 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 full-length genome is about 27 - 32 kb, making it the virus with the largest genome among currently known RNA viruses. Coronaviruses only infect vertebrates, such as humans, mice, pigs, cats, dogs, wolves, chickens, cows, 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 inclusion bodies 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 7th known coronavirus that can infect humans. The other 6 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). So far, multiple variants of SARS-CoV-2 have emerged, including Alpha, Beta, Gamma, Delta, Omicron, etc.

[0004] The COVID-19 virus is a single-stranded positive-sense RNA virus. After its spike (S) protein binds to the receptor ACE2 on the human cell membrane, it enters the cell and releases the 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 multiple non-structural proteins (NSPs) required for the COVID-19 virus. Subsequently, under the combined action of multiple non-structural proteins such as RNA polymerase (RdRp), replication-transcription occurs, translating into progeny structural proteins, which then 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. Among the highly sought-after COVID-19 oral drug tracks, Pfizer's Paxlovid is the most prominent. Paxlovid has been approved or authorized for emergency use in more than 10 countries around the world. Paxlovid consists of two antiviral drugs, among which PF-07321332 is Nirmatrelvir, a 3CL protease inhibitor of the novel coronavirus. Nirmatrelvir inhibits the replication of the novel coronavirus, and Ritonavir prolongs its effective blood drug concentration in the body by inhibiting the metabolism of Nirmatrelvir in the body.

[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 cyano-containing selenolactam compound, its preparation method and use. By structurally modifying PF-07321332 and its analogs, the present invention significantly improves the anti-coronavirus, especially anti-SARS-CoV-2 activity of PF-07321332 and its analogs.

[0009] In the context of the present invention, the novel coronavirus includes the original SARS-CoV-2 strain and 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) or a pharmaceutically acceptable salt thereof:

[0012]

[0013] Wherein, R1 is selected from (C1-C6 alkyl) amino optionally substituted with 1 to 5 fluorines, C1-C6 alkyl-C(O)NH- optionally substituted with 1 to 5 fluorines, and C1-C6 alkyl-S(O)2NH- optionally substituted with 1 to 5 fluorines.

[0014] Preferably, R1 is selected from CF3C(O)NH-, CF3S(O)2NH-, CH3C(O)NH-, CH3CH2C(O)NH-, or CF3CH2NH-.

[0015] Preferably, the structure of the compound is as follows:

[0016]

[0017] On the other hand, the present invention provides a method for preparing the compound of formula (I) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0018]

[0019] Mix the compound of formula (II) with a selenium reagent in a solvent and react at 10-40 °C for 1-3 hours to obtain the compound of formula (I); wherein R1 is as defined above.

[0020] Preferably, the solvent is selected from one or more of toluene, benzene, chlorobenzene and xylene; the selenium reagent is Woollins reagent or selenium; the reaction is carried out at room temperature; and / or the reaction is carried out for 1.5-2.5 h.

[0021] 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 is completed, 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.

[0022] On the other hand, the present invention provides a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention, and a pharmaceutically acceptable carrier or excipient.

[0023] Preferably, the pharmaceutical composition further comprises vitamin C. After combining the compound of formula (I) or a pharmaceutically acceptable salt thereof of the present invention with vitamin C, the antiviral therapeutic effect can be further improved.

[0024] On the other hand, the present invention also provides the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating a disease caused by coronavirus infection, preferably, the coronavirus is SARS-CoV-2.

[0025] The present inventors unexpectedly found that when oxygen in the butyrolactam ring of compound PF-07321332 or its analogs was replaced by selenium, the resulting compound had significantly improved activity compared to the corresponding non-selenated compound PF-07321332 or its analogs. For example, the inhibitory activity of compound Y7201 of the present invention against SARS-CoV-2 3CL protease was more than about 2 times that of PF-07321332. 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 infection with the novel coronavirus (including the original strain of SARS-CoV-2 and various SARS-CoV-2 variants such as Alpha, Beta, Gamma, Delta, and Omicron), and have significantly improved therapeutic effects. Brief Description of the Drawings

[0026] Figure 1 is Nirmatrelvir, a 3CL protease inhibitor of the novel coronavirus;

[0027] Figure 2 is the general structural formula of the cyano-containing selenated butyrolactam compound;

[0028] Figure 3 is the structure of Y7201;

[0029] Figure 4 is the reaction formula of the cyano-containing selenated butyrolactam compound;

[0030] Figure 5 is the reaction formula of the compound for synthesizing Y7201;

[0031] Figure 6 For the data obtained in Example 2, processed using the software GraphPad Prism 7.0, the calculated IC 50 value;

[0032] Figure 7 is the measurement result of quantifying virus replication by measuring the yield of infectious virus in the Vero16 apical wash fluid through plaque assay on a monolayer of Vero16 cells and by measuring the yield of viral RNA from total cellular RNA through real-time PCR;

[0033] Figure 8 is the general structural formula of the cyano-containing selenated butyrolactam compound. Detailed Description of the Invention

[0034] 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.

[0035] In the following examples, the reagents and raw materials used, unless otherwise specified, are all commercially available.

[0036] Among them, PF-07321332 was purchased from NetChem (Shandong) Chemical Technology Co., Ltd.;

[0037] The selenium reagent (Woollins reagent) was purchased from WuXi Labex (Wuhan) Chemical Technology Co., Ltd.;

[0038] EV71 3Cpro was purchased from Sigma-Aldrich Reagent Company.

[0039] Example 1

[0040]

[0041] PF-07321332 (500 mg) was added to toluene (10 mL), and Woollins reagent (600 mg) was added. The reaction was carried out at room temperature for 2 hours under nitrogen protection.

[0042] Ethyl acetate and aqueous sodium bicarbonate solution were added and stirred for 15 minutes. The layers were separated, the organic phase was dried and concentrated by evaporation, and column chromatography was used to obtain 1350 mg of solid Y7201, with a yield of 62.2%. MS (m / z): 564.2 (M+H) + 。

[0043] 1 1H NMR (400 MHz, DMSO-d6) δ 9.43 - 9.30 (m, 1H), 8.96 (d, J = 8.3 Hz, 1H), 7.22 (s, 1H), 5.02 - 4.90 (m, 1H), 4.44 - 4.35 (m, 1H), 4.13 (s, 1H), 3.96 - 3.82 (m, 1H), 3.65 (d, J = 10.8 Hz, 1H), 3.03 - 2.89 (m, 1H), 2.89 - 2.68 (m, 1H), 2.35 - 2.23 (m, 1H), 2.19 - 1.99 (m, 2H), 1.71 - 1.59 (m, 2H), 1.59 - 1.48 (m, 1H), 1.30 (d, J = 7.7 Hz, 1H), 1.02 (s, 3H), 0.98 (s, 9H), 0.85 (s, 3H).

[0044] Example 2 : Evaluation of the inhibitory activity of the compound against SARS-CoV-2 3CL protease

[0045] Inhibitory activity test of the compound against the 3CL protease of SARS-CoV-2. The enzymatic level inhibitory activity of the inhibitor against 3CL protease was determined using fluorescence resonance energy transfer (FRET) technology.

[0046] <Operation procedure>

[0047] 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 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 remaining operations were the same.

[0048] <Calculation of each measured item value>

[0049] 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.

[0050] Table 1

[0051] Compound <![CDATA[IC 50 (nM)]]> Y7201 8.5 PF-07321332 19.5

[0052] From the above results, it can be seen that Y7201 shows significantly improved inhibitory activity against the 3CL protease of SARS-CoV-2, which is more than twice that of PF-07321332. Since the 3CL protease of SARS-CoV-2 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.

[0053] Example 3: Determination of anti-SARS-CoV-2 beta-coronavirus activity

[0054] 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 β-coronavirus (SARS-CoV-2βRFP) expressing green fluorescent protein at a multiplicity of infection of 0.1 pfu / cell. The apical wash solution 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 compound of the example 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.

[0055] Table 2

[0056] Compound <![CDATA[CPEEC 50 (nM)]]> <![CDATA[CC 50 (nM)]]> Y7201 32 Greater than 20000 PF-07321332 85 Greater than 20000

[0057] As can be seen from the results in Table 2, the compounds of the present invention have outstanding anti-SARS-CoV-2 β-coronavirus activity, and compared with the positive control drug PF-07321332, the anti-SARS-CoV-2 β-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 10 - 40 °C for 1 - 3 hours to obtain the compound of formula (I’); wherein the selenium reagent is Woollins reagent or selenium.

3. The method according to claim 2, wherein the solvent is selected from one or more of toluene, benzene, chlorobenzene and xylene; the reaction is carried out at room temperature; and / or the reaction is carried out for 1.5 - 2.5 h.

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 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.

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’) as defined in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating a disease caused by coronavirus infection, wherein, The coronavirus is the novel coronavirus.

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