Application of direct C3-pyrroline alkylated derivative of 2-quinolinone in preparation of antiviral drugs

Abstract

The invention discloses an application of a direct C3-pyrroline alkylated derivative of 2-quinolinone in preparation of an antiviral drug. The preparation method comprises the following steps: by taking gamma, delta-unsaturated oxime ester as a raw material, enhancing the electron donating property of a sulfur-containing zwitterionic compound through KHCO3 to form a ternary EDA compound, carrying out charge transfer under the irradiation of visible light to realize photoactivation of oxime ester and generate imine free radicals, and carrying out intramolecular cyclization to generate alkyl free radicals, so as to obtain the compound. Therefore, direct C3-pyrroline alkylation reaction of 2-quinolinone is realized, and a target compound is obtained. The reaction raw materials are easy to obtain, the synthesis steps are simple, and the reaction conditions are mild. The direct C3-pyrroline alkylated derivative of 1, 2-quinolinone can show a certain inhibition effect on influenza virus replication under the condition of 25M concentration, and can be further used in potential antiviral drugs.

Description

Technical Field

[0001] The invention belongs to the field of organic chemistry, and specifically relates to the application of direct C3-pyrroline alkylation derivatives of 2-quinolinone in the preparation of antiviral drugs. Background Art

[0002] Influenza is an acute respiratory infectious disease mainly caused by influenza A virus (IAV), which can cause pandemic epidemics and is the leading cause of death worldwide, affecting 10-30% of the population each year. Influenza virus is a negative-sense single-stranded RNA virus belonging to the family Orthomyxoviridae. Based on the antigenic determinants of their nucleoprotein and matrix proteins, influenza viruses are divided into four subtypes, namely influenza A, B, C and D viruses. As mentioned above, highly contagious diseases are usually caused by influenza A viruses, which are the most important pathogens in clinical practice.

[0003] Influenza A(H3N2) is a respiratory disease caused by influenza A(H3N2) virus, which can be transmitted through the respiratory tract. Patients often show symptoms of common influenza, but it may also lead to death. Currently, influenza vaccination remains the main preventive measure to control the spread of viruses in human and animal populations. However, due to the frequent mutation of influenza viruses, influenza vaccines only have a protective effect on the antigens of the currently circulating influenza virus strains, and can only achieve a protection rate of 60%. Therefore, anti-influenza drugs seem to be more effective in preventing highly contagious viral infections and reducing the prevalence of diseases. Over the years, many anti-influenza drugs targeting viral proteins have been developed. However, in recent years, the emergence of highly invasive virus strains such as H1N1, H5N1 and H7N9, as well as an increasing number of drug-resistant virus strains, have once again emphasized the need for new anti-influenza drugs to destroy these pathogens.

[0004] Influenza virus is an enveloped virus with two glycoproteins on its surface: hemagglutinin (HA) and neuraminidase (NA), which are essential for viral infection. Specifically, NA promotes the release of new virus particles from host cells by catalyzing the cleavage of the α-glycosidic bond in the terminal sialic acid residue of the cellular glycoconjugate bound to the viral HA.

[0005] Currently, small molecule anti-influenza virus drugs are mainly divided into four categories: (I) M2 ion channel protein inhibitors (amantadine, rimantadine, etc.); (II) neuraminidase (NA) inhibitors (zanamivir, oseltamivir, peramivir, lanamivir, etc.); (III) hemagglutinin (HA) inhibitors (arbidol, nitazoxanide, etc.); and (IV) RNA-dependent RNA polymerase (RdRp) inhibitors (favipiravir, baloxavir-maboxil, etc.). So far, only a few anti-influenza virus drugs have been approved for marketing. Although these antiviral drugs are useful, their clinical efficacy is increasingly affected by the emergence and spread of resistant strains. For example, more than 98% of isolates that show resistance to adamantane drugs have the S31N mutation, prompting the US Food and Drug Administration to no longer recommend the use of M2 ion channel blockers. In addition, the I38T mutation in the PA protein has become common, resulting in a significant decrease in susceptibility to BXM, with an observed 30- to 50-fold increase in the EC50 value for influenza A virus.

[0006] The main anti-influenza drugs used clinically today are neuraminidase inhibitors (NAIs). Four types of NAIs have been approved for treatment and have become the main choice for clinical management of influenza. However, the widespread use of NAIs and the continuous emergence of drug-resistant influenza virus strains have not only reduced the efficacy of anti-influenza drugs, but also posed a further threat to public health. Therefore, it is of great significance to develop anti-influenza drugs with new mechanisms. Summary of the invention

[0007] In order to overcome the above technical defects, the present invention provides the application of direct C3-pyrroline alkylated derivatives of 2-quinolinone in the preparation of antiviral drugs, and studies the synthesis method and active application of the compound. Oxime ester and 2-quinolinone are used as raw materials, and direct C3-pyrroline alkylation of oxime ester is carried out under visible light and sulfur-containing zwitterion catalysis, and a one-step reaction is performed to obtain direct C3-pyrroline alkylated derivatives of 2-quinolinone containing biological activity. The derivative has simple synthesis steps and high yield. The activity against H3N2 influenza virus is tested and has obvious inhibitory effect.

[0008] Based on the above purpose, the present invention adopts the following technical solutions:

[0009] The use of a direct C3-pyrroline alkylated derivative of 2-quinolinone in the preparation of an antiviral drug, wherein the structure of the direct C3-pyrroline alkylated derivative of 2-quinolinone is as follows:

[0010]

[0011] Further, the preparation process of the direct C3-pyrroline alkylation derivative of 2-quinolinone is as follows:

[0012]

[0013] (1) Compound A and compound B are used as raw materials, catalyzed by catalyst Z1 and white light under a protective atmosphere, and then alkalized by KHCO3 to obtain the target compound 1a;

[0014] (2) Compound D and Compound E were used as raw materials, catalyzed by catalyst Z1 and white light under a protective atmosphere, and alkalized by KHCO3 to obtain the target compound 2a.

[0015] Furthermore, the molar ratio of compound A (D), compound B (E), catalyst Z1, and KHCO3 is 1:(1-2):(0.02-0.08):(1-2).

[0016] Furthermore, the reaction solvent is dry DMSO, the reaction temperature is room temperature, the visible light is white light of 400-800 nm, the reaction environment is protected by N2 atmosphere, and the concentration of compound A or D in DMSO is 0.1-0.2M.

[0017] Furthermore, in the above technical solution, the virus is an H3N2 subtype influenza virus.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present invention uses 2-quinolinone as a raw material, adopts photocatalysis, and directly alkylates C3-pyrroline with the participation of oxime esters, and obtains direct C3-pyrroline alkylated derivatives containing biologically active 2-quinolinone in a one-step reaction. The problems of lengthy synthetic routes, harsh reaction conditions, and low yields are avoided, and potential safety hazards are reduced.

[0020] 2. The direct C3-pyrroline alkylated derivative of the compound 2-quinolinone of the present invention exhibits a significant inhibitory effect on H3N2 subtype influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Experimental steps for preliminary screening of compounds that inhibit H3N2 subtype influenza virus;

[0022] Figure 2 This is a preliminary screening of the anti-H3N2 subtype IV activity of compounds 1a and 2a at a single concentration (25 μM);

[0023] Figure 3 is the EC of compound 1a 50 (Compound 1a EC 50 =2.085 μM);

[0024] Figure 4 is the CC of compound 1a 50 (Compound 1a CC 50=196.5 μM);

[0025] Figure 5 is the EC of compound 2a 50 (Compound 2a EC 50 =1.59 μM);

[0026] Figure 6 is the CC of compound 2a 50 (Compound 2a CC 50 =97.18 μM). DETAILED DESCRIPTION

[0027] The present invention will be further described below by specific examples. These embodiments should be understood to be only used to illustrate the present invention and not to limit the scope of protection of the present invention. After reading the content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0028] Example 1

[0029] Synthesis of compound 1a:

[0030]

[0031] Add oxime ester A (0.2mmol, 1.0equiv.), 2-quinolinone B (0.3mmol, 1.5equiv.), zwitterionic catalyst Z1 (0.01mmol, 4.19mg, 5mol%) and KHCO3 (0.3mmol, 30.0mg, 1.5equiv.) to a dry Schlenk tube equipped with a stirrer. After N2 replacement 3 times, DMSO (2.0mL) was added with a syringe. The reaction tube was placed in front of a 20W white LED bulb (400-800nm) and stirred at room temperature for 6h. After the reaction was completed, water and ethyl acetate were added, stirred for 10 minutes, separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were then washed with saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography (PE:EtOAc=20:1 to 4:1) to obtain the target compound 1a.

[0032] 1H NMR (600MHz, CDCl3): δ7.97 (dd, J=7.9, 1.5Hz, 1H), 7.76–7.71 (m, 2H), 7.61– 7.56(m,2H),7.50(ddd,J=8.6,7.3,1.6Hz,1H),7.38–7.24(m,7H),7.23–7.19 (m,1H),5.52–5.42(m,1H),4.80(d,J=10.3Hz,1H),3.65(s,3H),2.87(ddd,J =9.2,6.8,2.0Hz,2H),2.12–2.03(m,1H),1.71(dtd,J=13.1,8.8,7.0Hz,1H);

[0033] 13 C NMR (151MHz, CDCl3): (major) δ173.22,161.11,154.85,139.35,134.98,133.23,133.10,130.41,130.2 5,129.71,129.54,128.44,128.28,127.91,127.00,123.39,113.57,75.99,53.42,34.99,29.29,27.77;

[0034] HRMS(ESI)m / z:calculated for C 26 H 24 N3O[M+H] + :394.1914,found:394.1910.

[0035] Example 2

[0036] Synthesis of compound 2a:

[0037]

[0038] Add oxime ester D (0.2mmol, 1.0equiv.), 2-quinolinone E (0.3mmol, 1.5equiv.), zwitterionic catalyst Z1 (0.01mmol, 4.19mg, 5mol%) and KHCO3 (0.3mmol, 30.0mg, 1.5equiv.) to a dry Schlenk tube equipped with a stirrer. After N2 replacement three times, DMSO (2.0mL) was added with a syringe. The reaction tube was placed in front of a 20W white LED bulb (400-800nm) and stirred at room temperature for 6h. After the reaction was completed, water and ethyl acetate were added, stirred for 10 minutes, separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were then washed with saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated by rotary evaporation. The residue was purified by silica gel preparation plate (PE:EtOAc=5:1) to obtain the target compound 2a.

[0039] 1 H NMR (600MHz, CDCl3): δ7.89–7.80(m,2H),7.40(qd,J=5.7,3.1Hz,4H),7.19(d,J=7.4H z,1H),7.13(d,J=8.4Hz,1H),4.96(dtd,J=9.4,7.1,4.8Hz,1H),3.69(s,3H),3.62(dd, J=16.0,4.9Hz,1H),3.13–3.03(m,2H),2.95(dddd,J=17.1,9.5,7.3,1.8Hz,1H),2.68 (s,3H),2.38(dddd,J=12.9,9.9,7.7,5.0Hz,1H),1.79(ddt,J=13.3,10.0,7.0Hz,1H);

[0040] 13 C NMR (151MHz, CDCl3): δ172.66,157.00,155.06,138.66,134.84,133.26,131.38,130.40,129.48,12 8.46,127.84,124.88,111.58,71.05,40.67,35.14,29.29,29.00,17.69; HRMS(ESI)m / z:calculated for C21H22N3O[M+H]+:332.1757,found:332.1754.

[0041] Example 3

[0042] This example is a preliminary test process and results of the inhibition of H3N2 subtype influenza virus cell level by compounds 1a and 2a described in the example. The materials and consumables listed in this example can be obtained from commercial channels unless otherwise specified, and the cells and viruses are from the cell and microbial resource library of CTCC or other relevant institutions. The experimental method of this example is a standard molecular biology, cell biology or virology operation procedure, which can be easily understood and operated by researchers in the field. The specific steps are as follows:

[0043] 1. Cells and viruses:

[0044] Dog kidney cells MDCK, culture medium is DMEM, containing 10% bovine serum (FBS) and 1% streptomycin and penicillin (Penicillin-Streptomycin), 37°C, 5% CO2. H3N2 subtype influenza virus A / Swine / Guangdong / SQQ MA / 2024 (H3N2) (abbreviated as: H3N2-SQQ MA), the hemagglutination titer after chicken embryo proliferation is 2 10 HAU / mL.

[0045] 2. Main reagents and sources:

[0046] DMEM (Gibco, cat: C11995500BT); fetal bovine serum FBS (Gibco, cat: 10270-106); double anti-Penicillin-Streptomycin (10000U / mL) (M&C gene biotechnology, cat: G2723M3); 4-MUNANA (C 21 H 24 NNaO 11 , Sigma, cat: M8639); MTT: (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (tetramethylthiazolium salt) (MCE, cat: HY-15924); DMSO (Solarbio, cat: D8370).

[0047] 3. Detection of compound inhibition of H3N2 influenza virus activity:

[0048] like Figure 1 As shown, MDCK cells were grown at 3×10 5cells / well were inoculated in 24-well plates, and the cells were plated one day in advance, and the cell confluence was 90%-95%; the original culture medium was discarded, and 450 μL DMEM and 50 μL of the compound to be tested (250 μM) or the positive control drug (Oseltamivir phosphate, 100 μM) were added to make the final concentration of the compound 25 μM and the final concentration of the positive control drug 10 μM; incubated at 37°C for 4 hours, the culture medium was discarded, and 225 μL H3N2-SQQ MA virus solution and 25 μL of the compound to be tested (250 μM) or the positive control drug (Oseltamivir phosphate, 100 μM) were added according to the multiplicity of infection (MOI) of 0.01; incubated at 37°C for 2 hours, the free virus was washed off, and 450 μL of cell maintenance medium DMEM and 50 μL of the compound to be tested (250 μM) or the positive control drug (Oseltamivir phosphate, 100 μM); cultured at 37° C. for 48 hours, and the cell culture supernatant was collected; the cell culture with only DMEM was used as a blank control (Mock), and the inhibition rate of the compound on influenza virus was determined by NA activity.

[0049] 4. NA activity detection:

[0050] 50 μL of cell culture supernatant was drawn from each well and added to a 96-well black microplate. Each compound was tested at least twice. 50 μL of influenza virus NA substrate 4-MUNANA at a final concentration of 20 μM was added to each well in the dark. The well was incubated in a 37°C incubator for 30 minutes in the dark. The fluorescence value was measured on a multifunctional enzyme label reader: the excitation light was 355 nm, the emission wavelength was 485 nm, and the fluorescence value was read. Compound inhibition rate (%) = [1-(OD compound-OD blank) / (OD virus control-OD blank)]*100. The initial screening of anti-H3N2 subtype IV activity at a single concentration (25 μM) was obtained, such as Figure 2 Where indicated, compound concentration was 25 μM and control drug concentration was 10 μM.

[0051] 5. Replace the 250 μM compound in step 3 with the compound to be tested at final concentrations of 0.8 μM, 4 μM, 20 μM, 100 μM, and 500 μM, respectively. According to steps 3 and 4, first calculate the inhibition rate of the compound at different concentrations on influenza virus, and then use GraphPad Prism software to obtain the EC value for inhibiting virus replication based on the inhibition rate. 50 , the results are shown in Figure 3 and Figure 5 ,Depend on Figure 3 It can be seen that the EC of compound 1a 50 =2.085μM, by Figure 5 It can be seen that the EC of compound 2a 50 =1.59μM.

[0052] 6. MTT colorimetric assay for cytotoxicity:

[0053] MDCK cells were cultured at 5×10 4 Each cell was inoculated in a 96-well cell culture plate containing 100 μL of cell culture medium, the culture medium was discarded, and 100 μL of compound solution (0.8 μM, 4 μM, 20 μM, 100 μM, 500 μM) was added; 3 replicates were performed for each concentration; a control without compound was also set up; after 72 hours of cell culture, 100 μL of supernatant was discarded, 20 μL of MTT (5 mg / mL) was added, and cultured at 37°C for 4 hours; centrifugation was performed, 100 μL of supernatant was discarded, 100 μL of DMSO was added, and the culture was kept at room temperature and away from light, and shaken for 15 minutes until the blue formazen dissolved; OD595 was detected by an enzyme reader, and OD630 was used as the reference wavelength to calculate the cell survival rate, and CC was obtained based on the cell survival rate using GraphPad Prism software 50 , the results are shown in Figure 4 and Figure 6 ,Depend on Figure 4 It can be seen that the CC of compound 1a 50 =196.5μM; Figure 6 It can be seen that the CC of compound 2a 50 =97.18μM.

[0054] 7. Calculation of the selectivity index of drug anti-influenza virus activity:

[0055] Selective Index (SI) = CC 50 / EC 50 .

[0056] 8. Test results:

[0057] The above results show that at a concentration of 25 μM, compounds 1a and 2a have a significant inhibitory effect on H3N2 subtype viruses. The drug antiviral activity selectivity index SI (Selective Index) is 94.24 and 61.11 respectively (the control drug Oseltamivir phosphate selectivity index is 5143.2).

[0058] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. The use of a direct C3-pyrroline alkylated derivative of 2-quinolinone in the preparation of an antiviral drug, characterized in that: The structural formula of the direct C3-pyrroline alkylation derivative of 2-quinolinone is as follows: 、 。 2. The use of the direct C3-pyrroline alkylated derivative of 2-quinolinone according to claim 1 in the preparation of antiviral drugs, characterized in that: The direct C3-pyrroline alkylation derivatives of 2-quinolinone were prepared as follows: (1) Compound A and compound B are used as raw materials, catalyzed by catalyst Z1 and white light under a protective atmosphere, and then alkalized by KHCO3 to obtain the target compound 1a; (2) Compound D and Compound E were used as raw materials, catalyzed by catalyst Z1 and white light under a protective atmosphere, and alkalized by KHCO3 to obtain the target compound 2a.

3. The use of the direct C3-pyrroline alkylated derivative of 2-quinolinone according to claim 1 in the preparation of antiviral drugs, characterized in that: The molar ratio of compound A or D, compound B or E, catalyst Z1, and KHCO3 is 1:(1~2):(0.02~0.08):(1~2).

4. The use of the direct C3-pyrroline alkylated derivative of 2-quinolinone according to claim 1 in the preparation of antiviral drugs, characterized in that: The reaction solvent is dry DMSO, the reaction temperature is room temperature, and the white light wavelength range is 400~800 nm.

5. The use of the direct C3-pyrroline alkylated derivative of 2-quinolinone according to claim 1 in the preparation of antiviral drugs, characterized in that: The virus is an H3N2 subtype influenza virus.

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