Application of cyclobutene amide compound in antiviral drug

Abstract

The invention relates to the field of medicinal chemistry, and discloses application of cyclobutene amide compounds in antiviral drugs. The structural characteristics of the compounds are as shown in a general formula (I), wherein EWG is an electron withdrawing group and is selected from aryl-substituted sulfonyl; r1 is selected from alkyl or aryl; r2 is selected from alkyl or aryl; and n represents the number of carbon atoms and may be 1 or 2. In-vitro pharmacodynamic studies show that the series of compounds have a remarkable inhibition effect on influenza A virus (H1N1 subtype), and the antiviral efficacy of the compounds is obviously superior to that of a clinical common antiviral drug ribavirin. In addition, toxicological evaluation proves that the compounds have ideal safety characteristics. The cyclobuteneamide derivative can be used as an effective component for developing an anti-influenza A virus drug, and a new candidate drug molecule is provided for clinical treatment of influenza virus infection. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a cyclobutenamide compound, a preparation method thereof, and an application thereof in anti-influenza A virus (IAV) drugs. Background Art

[0002] As an important structural unit, cyclobutene and its derivatives are widely present in natural products and drug molecules. Such compounds not only have important application value in organic synthesis due to their unique structural rigidity, but also are favored by researchers due to their diverse biological activities. Among them, cyclobutenamide compounds have become one of the hotspots in current organic chemistry and medicinal chemistry research due to their unique structural features and potential pharmacological activities.

[0003] In the field of synthetic chemistry, although the traditional Ficini reaction ([2+2] thermal cycloaddition of ynamine and cycloalkenone) can construct the cyclobutenamine skeleton, due to the high reactivity of ynamine, its synthesis, storage, and operation all face major challenges. To break through this technical bottleneck, researchers introduced ynamide with an electron-withdrawing group on the nitrogen atom as a stable substitute and successfully developed a variety of improved methods including copper / silver catalytic systems and highly selective asymmetric catalysis. However, these transition metal-catalyzed schemes still face inherent defects such as high cost and metal residue. Existing non-metal systems (such as heat-driven or Lewis acid-catalyzed) generally have deficiencies such as harsh reaction conditions and poor substrate generality. In response to these key technical problems, the applicant's team developed a Tf2NH-catalyzed regioselective Ficini reaction of ynamide and enone. This non-metal catalytic system has significant advantages: mild conditions (without transition metals), good selectivity, high atom economy, and excellent yield (Org. Biomol. Chem. 2024, 22, 4264-4268), providing a new strategy for the synthesis of cyclobutenamide compounds.

[0004] Based on the completion of this synthetic methodology research, the applicant's team further carried out the biological activity research of cyclobutenamide compounds. Through systematic in vitro activity screening, it was first found that such compounds have significant anti-influenza A virus (IAV) activity and excellent cell safety at the same time. This important discovery has laid a solid foundation for the development of such compounds as a new generation of anti-influenza virus lead drugs, and there is no relevant literature report yet. Summary of the Invention

[0005] Based on the highly efficient synthesis technology of cyclobutene amide developed in the early stage, through systematic structure-activity relationship studies, it was found that cyclobutene amide derivatives with specific structures exhibited excellent antiviral activities. Among them, the representative compounds not only showed significant inhibitory effects on IAV, but their selectivity indexes were significantly better than the clinical control drug ribavirin, providing candidate molecules for the development of new anti-influenza A virus drugs.

[0006] For this reason, the purpose of the present invention is to provide a new application of cyclobutene amide compounds as anti-influenza A virus (IAV) drugs, providing important candidate molecules for the development of anti-influenza drugs.

[0007] To achieve the purpose of the present invention, the specific technical solutions are as follows:

[0008] The general structural formula of the cyclobutene amide compounds is as follows:

[0009]

[0010] Among them, EWG is an electron-withdrawing group, selected from sulfonyl group, aryl-substituted sulfonyl group; R 1 is a substituent, selected from alkyl group or aryl group; R 2 is a substituent, selected from alkyl group or aryl group; n represents the number of carbon atoms, which can be 1 or 2.

[0011] Preferably: EWG is selected from sulfonyl group, aryl-substituted sulfonyl group, and the aryl is preferably phenyl substituted by methyl, halogen or nitro; R 1 is selected from benzyl, C1-4 alkyl group or phenyl; R 2 is selected from C1-6 alkyl group, phenyl or thiophenyl; n represents the number of carbon atoms, which can be 1 or 2.

[0012] The synthesis route of the cyclobutene amide compounds is as follows:

[0013]

[0014] The specific synthesis steps are as follows:

[0015] Place alkynyl amide 1 and cyclopentene ketone or cyclohexene ketone 2 in a dry reaction tube. After replacing nitrogen, successively add dichloroethane and trifluoromethanesulfonic acid, and stir at room temperature. Monitor the reaction process by thin-layer chromatography. After the reaction is complete, separate the cyclobutene amide compound 3 by silica gel column chromatography.

[0016] In the above steps, the molar ratio of alkynyl amide 1, cyclopentene ketone or cyclohexene ketone 2, and trifluoromethanesulfonic acid is 1.2:1:0.2.

[0017] The experimental data further reveal that the cyclobutene amide derivatives prepared by the present invention exhibit excellent antiviral properties. Through systematic in vitro activity evaluation, it is found that this series of compounds show significant inhibitory effects against influenza A virus (H1N1). Cell biology assessment shows that within the effective antiviral concentration range, the compounds have extremely low toxic effects on host cells, showing excellent biocompatibility characteristics. Notably, compared with the clinical first-line drug ribavirin, this series of compounds present a significantly improved activity-safety balance characteristic: its half-maximal effective concentration (EC 50 ) is reduced by 62% compared to ribavirin, while the half-maximal cytotoxic concentration (CC 50 ) is significantly increased. This unique "high efficiency and low toxicity" characteristic, combined with its novel chemical structure, makes this series of compounds ideal candidate molecules for the development of a new generation of anti-influenza drugs, with important clinical translation value. Detailed implementation manners

[0018] To make the technical solutions of the present invention clearer, the present invention will be elaborated in detail through specific embodiments below. It should be particularly noted that the following embodiments are only used to illustrate the technical solutions of the present invention and do not constitute any limitation to the protection scope of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are all conventional technical means in the art.

[0019] Main instruments and chemical reagents

[0020] The main instruments used in the experiment include: Bruker Ascend TM 400 nuclear magnetic resonance spectrometer (for compound structure characterization), Bruker micrOTOF-Q II high-resolution mass spectrometer (for molecular weight determination), Zhiwei Smart Fourier transform infrared spectrometer (Tianjin Gangdong Technology Co., Ltd.), WFH-203B ultraviolet analyzer (Shanghai Huyue Ming Scientific Instrument Co., Ltd.), and XT4A microscopic melting point apparatus (Beijing Keyi Dian Guang Instrument Factory).

[0021] All chemical reagents and raw materials used in the experiments (including the key intermediate alkynylamide 2) are commercially available analytical reagents or synthesized according to the literature methods. The synthesis of the known compound alkynylamide 2 can refer to the following literature methods (Org. Lett. 2016, 18, 5022 - 5025; J. Org. Chem., 2011, 76, 1852 - 1873; Chem. Commun., 2015, 51, 3316 - 3319; Chem. Commun., 2014, 50, 6001 - 6004; Angew. Chem. Int. Ed., 2009, 48, 4381 - 4385). Unless otherwise specified, all reactions are carried out under nitrogen protection, and the solvents are dried by standard methods. The reaction progress is monitored by thin-layer chromatography, and the products are purified by column chromatography. The following examples will detail the specific reaction conditions, purification methods and other key parameters to ensure the reproducibility of the experimental protocol.

[0022] Example 1: Synthesis of target compounds cyclobutenamides 3a - 3l

[0023] Specifically noted that in the compound structure: Ts represents p-toluenesulfonyl, Cs represents p-chlorobenzenesulfonyl, Ns represents p-nitrobenzenesulfonyl, and n-hex represents n-hexyl.

[0024]

[0025] Taking the synthesis step of compound 3a as an example: Alkynylamide 1a (107.8 mg, 0.36 mmol), cyclohexenone 2a (28.8 mg, 0.30 mmol), dichloroethane (1.5 mL, alkynylamide concentration 0.24 M) and trifluoromethanesulfonic acid (16.9 mg, 0.06 mmol) were successively added to a dried reaction tube and stirred at room temperature for 10 minutes. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, it was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10:1) to obtain cyclobutenamide 3a (110.1 mg, 0.28 mmol) with a yield of 93%.

[0026] Expanded synthesis: Using the same experimental method, the known compound alkynylamide 1 synthesized according to the literature was respectively subjected to cyclization reactions with commercially available cyclic enones to successfully prepare the corresponding target compounds 3b - 3l.

[0027]

[0028] Compound 3a: 93% yield, R f = 0.19 [6:1 petroleum ether:ethyl acetate]; white solid; melting point = 114 - 115 °C; 1HNMR(400MHz,CDCl3)δ7.74(d,2H,J=8.3Hz),7.34 - 7.27(m,7H),4.63,4.56(ABq,2H,J AB =15.0Hz),3.18(dq,1H,J=4.6,2.3Hz),2.75(br,1H),2.45(s,3H),1.81 - 1.77(m,2H),1.68 - 1.67(m,3H),1.45 - 1.39(m,2H),1.33 - 1.22(m,2H); 13 C{ 1 H}NMR(100MHz,CDCl3)δ210.7,144.1,144.0,136.6,136.5,129.9,128.6,127.9,127.7,127.6,126.7,53.9,51.3,40.5,38.6,23.8,21.8,17.1,12.6.

[0029]

[0030] Compound 3b: 83% yield, R f =0.33[5:1 petroleum ether:ethyl acetate]; white solid; melting point = 114–115 °C; 1 H NMR(400MHz,CDCl3)δ7.80(d,2H,J=8.6Hz),7.51(d,2H,J=8.6Hz),7.34 - 7.27(m,5H),4.63,4.56(ABq,2H,J AB =14.9Hz),3.19(dq,1H,J=4.6,2.4Hz),2.76(br,1H),1.86 - 1.77(m,3H),1.66 - 1.65(m,3H),1.50 - 1.43(m,2H),1.31 - 1.22(m,1H); 13 C{ 1 H}NMR(100MHz,CDCl3)δ210.4,144.9,139.7,138.1,136.1,129.5,129.1,128.7,128.0,127.9,126.3,53.7,51.5,40.7,38.6,23.8,17.2,12.6; IR(neat)(cm -1 )2928m,2865w,1686s,1521m,1294w,626s; HRMS(ESI): m / z calcd for C 22 H 23 ClNO3S[M + H] +416.1082, found 416.1080.

[0031]

[0032] Compound 3c: 77% yield, R f = 0.29 [5:1 petroleum ether: ethyl acetate]; white solid; melting point = 115–116 °C; 1 1H NMR (400 MHz, CDCl3) δ 8.39 (d, 2H, J = 8.8 Hz), 8.04 (d, 2H, J = 8.8 Hz), 7.35 - 7.28 (m, 5H), 4.66, 4.58 (ABq, 2H, J AB = 14.8 Hz), 3.19 (br, 1H), 2.79 (br, 1H), 1.96 - 1.79 (m, 3H), 1.67 - 1.66 (m, 3H), 1.55 - 1.47 (m, 2H), 1.32 - 1.25 (m, 1H); 13 13C{ 1 1H}NMR (100 MHz, CDCl3) δ 210.1, 150.4, 146.1, 145.5, 135.6, 128.9, 128.8, 128.2, 128.1, 125.8, 124.5, 53.4, 51.8, 41.0, 38.7, 23.8, 17.2, 12.6.

[0033]

[0034] Compound 3d: 74% yield, R f = 0.32 [5:1 petroleum ether: ethyl acetate]; white solid; melting point = 61–62 °C; 1 1H NMR (400 MHz, CDCl3), δ 7.65 (d, 2H, J = 8.3 Hz), 7.27 (d, 2H, J = 8.6 Hz), 3.48 - 3.33 (m, 2H), 3.31 (br, 1H), 2.90 (br, 1H), 2.42 (s, 3H), 2.12 - 2.06 (m, 1H), 1.98 - 1.89 (m, 2H), 1.75 - 1.74 (m, 3H), 1.64 - 1.60 (m, 2H), 1.52 - 1.44 (m, 2H), 1.37 - 1.25 (m, 3H), 0.91 (t, 3H, J = 7.3 Hz); 13 13C{ 11H NMR (100 MHz, CDCl3) δ 210.4, 143.7, 143.5, 137.1, 129.7, 127.5, 127.0, 53.8, 47.6, 40.5, 38.6, 31.5, 24.0, 21.8, 19.6, 17.4, 13.9, 12.6; IR (neat) (cm -1 ) 2957 s, 2727 m, 1693 s, 1458 m, 1031 m, 815 s, 572 m; HRMS (ESI): m / z calcd for C 20 H 28 NO3S [M + H] + 362.1784, found 362.1784.

[0035]

[0036] Compound 3e: 71% yield, R f = 0.38 [5:1 petroleum ether:ethyl acetate]; white solid; melting point = 152–153 °C; 1 1H NMR (400 MHz, CDCl3), δ 7.58 (d, 2H, J = 8.3 Hz), 7.32 - 7.29 (m, 3H), 7.25 - 7.22 (m, 4H), 3.39 (br, 1H), 2.89 (br, 1H), 2.46 - 2.38 (m, 4H), 2.12 - 2.01 (m, 1H), 1.94 - 1.82 (m, 2H), 1.70 - 1.59 (m, 5H); 13 13C{ 1 1H} NMR (100 MHz, CDCl3) δ 210.9, 144.0, 142.9, 138.4, 136.8, 129.7, 129.5, 129.4, 129.0, 128.4, 128.1, 55.5, 40.1, 39.3, 24.1, 21.8, 17.7, 12.2; IR (neat) (cm -1 ) 2966 m, 2861 w, 1685 s, 1492 m, 1226 w, 1089 s, 542 m; HRMS (ESI): m / z calcd for C 22 H 24 NO3S [M + H] + 382.1471, found 382.1471.

[0037]

[0038] Compound 3f: 85% yield, R f= 0.29 [5:1 petroleum ether:ethyl acetate]; white solid; melting point = 62–63 °C; 1 1H NMR (400 MHz, CDCl3), δ 7.73 (d, 2H, J = 8.3 Hz), 7.33 - 7.23 (m, 7H), 4.70, 4.49 (ABq, 2H, J AB = 14.9 Hz), 3.19 (br, 1H), 2.81 (br, 1H), 2.45 (s, 3H), 2.29 - 2.23 (m, 1H), 1.93 - 1.77 (m, 4H), 1.46 - 1.33 (m, 4H), 1.31 - 1.13 (m, 7H), 0.89 (t, 3H, J = 7.0 Hz); 13 13C{ 1 1H} NMR (100 MHz, CDCl3) δ 210.7, 148.6, 144.0, 136.8, 136.4, 129.8, 128.6, 128.1, 127.8, 127.7, 126.1, 53.6, 51.5, 40.6, 37.0, 31.8, 29.6, 27.1, 26.4, 24.3, 22.8, 21.8, 17.3, 14.3; IR (neat) (cm -1 ) 2925 s, 2853 m, 1594 w, 1347 s, 1070 w, 863 m, 557 s; HRMS (ESI): m / z calcd for C 28 25 36 H + 34

[0039]

[0040] Compound 3g: 90% yield, R f = 0.20 [5:1 petroleum ether:ethyl acetate]; white solid; melting point = 151–152 °C; 1 1H NMR (400 MHz, CDCl3) δ 7.84 (d, 2H, J = 8.3 Hz), 7.37 - 7.34 (m, 7H), 7.18 - 7.17 (m, 3H), 7.02 - 7.00 (m, 2H), 4.55, 4.42 (ABq, 2H, J AB = 14.4 Hz), 3.29 (s, 2H), 2.46 (s, 3H), 2.18 - 1.93 (m, 3H), 1.61 - 1.48 (m, 3H); 13 13C{ 11H NMR (100 MHz, CDCl3) δ 210.8, 144.7, 144.3, 136.3, 135.5, 132.1, 130.0, 128.9, 128.6, 128.52, 128.48, 128.1, 128.0, 127.4, 126.8, 55.5, 51.2, 40.6, 37.4, 24.9, 21.8, 17.5; IR (neat) (cm -1 ) 2924 m, 2869 w, 1693 s, 1368 s, 1143 m, 662 s, 654 s; HRMS (ESI): m / z calcd for C 28 H 28 NO3S [M + H] + 458.1784, found 458.1784.

[0041]

[0042] Compound 3h: 91% yield, R f = 0.23 [5:1 petroleum ether:ethyl acetate]; white solid; melting point = 91–92 °C; 1 1H NMR (400 MHz, CDCl3), δ 7.77 (d, 2H, J = 8.3 Hz), 7.43 - 7.40 (m, 2H), 7.37 - 7.30 (m, 5H), 3.54 (d, 1H, J = 4.3 Hz), 3.48 (br, 1H), 2.97 (s, 3H), 2.71 - 2.64 (m, 1H), 2.43 (s, 3H), 2.25 - 2.16 (m, 2H), 2.07 - 1.95 (m, 1H), 1.77 - 1.67 (m, 2H); 13 13C{ 1 1H} NMR (100 MHz, CDCl3) δ 210.9, 144.1, 141.8, 135.5, 131.7, 129.9, 128.8, 128.6, 127.9, 127.3, 55.0, 40.2, 37.4, 35.7, 24.8, 21.7, 17.8, where there is a carbon signal overlap at 128.8 ppm; IR (neat) (cm -1 ) 2937 m, 2875 w, 1692 s, 1494 m, 1229 w, 1157 m, 676 s; HRMS (ESI): m / z calcd for C 22 H 24 NO3S [M + H] + 382.1471, found 382.1470.

[0043]

[0044] Compound 3i: 82% yield, R f = 0.39 [5:1 petroleum ether: ethyl acetate]; white solid; melting point = 140–141 °C; 1 H NMR (400 MHz, CDCl3), δ 7.62 - 7.58 (m, 2H), 7.55 (d, 2H, J = 8.3 Hz), 7.37 - 7.27 (m, 5H), 7.24 - 7.19 (m, 5H), 3.64 (d, 1H, J = 4.1 Hz), 3.49 - 3.44 (m, 1H), 2.72 - 2.66 (m, 1H), 2.39 (s, 3H), 2.25 - 2.07 (m, 3H), 1.80 - 1.71 (m, 2H); 13 C{ 1 H}NMR (100 MHz, CDCl3) δ 211.2, 145.0, 144.2, 138.5, 136.1, 131.2, 129.5, 129.4, 129.1, 128.7, 128.5, 128.2, 127.7, 127.3, 56.1, 40.5, 37.2, 24.9, 21.8, 17.8, where there is a carbon signal overlap at 128.7 ppm; IR (neat) (cm -1 ) 2924 m, 2858 w, 1688 s, 1447 m, 1236 w, 1054 m, 583 s; HRMS (ESI): m / z calcd for C 27 H 26 NO3S [M + H] + 444.1628, found 444.1628.

[0045]

[0046] Compound 3j: 43% yield, R f = 0.22 [5:1 petroleum ether: ethyl acetate]; white solid; melting point = 153–154 °C; 1 H NMR (400 MHz, CDCl3), δ 7.84 (d, 2H, J = 8.3 Hz), 7.36 - 7.34 (m, 3H), 7.21 - 7.18 (m, 3H), 7.13 - 7.11 (m, 2H), 7.03 - 7.01 (m, 2H), 4.57, 4.52 (ABq, 2H, J AB= 14.4 Hz), 3.41 (d, 1H, J = 4.2 Hz), 3.27 - 3.24 (m, 1H), 2.46 (s, 3H), 2.17 - 2.13 (m, 1H), 2.01 - 1.85 (m, 2H), 1.65 - 1.56 (m, 1H), 1.47 - 1.36 (m, 2H); 13 C{ 1 H} NMR (100 MHz, CDCl3) δ 209.9, 144.3, 138.2, 136.3, 135.5, 134.2, 130.1, 128.6, 128.5, 128.04, 128.02, 127.5, 127.3, 127.2, 124.4, 55.4, 50.9, 40.7, 38.3, 24.9, 21.8, 17.2; IR (neat) (cm -1 ) 2922 m, 2850 w, 1690 s, 1453 m, 1353 s, 930 m, 557 s; HRMS (ESI): m / z calcd for C 26 H 26 NO3S2 [M + H] + 464.1349, found 464.1349.

[0047]

[0048] Compound 3k: 96% yield, R f = 0.17 [5:1 petroleum ether:ethyl acetate]; white solid; melting point = 100–101 °C; 1 1H NMR (400 MHz, CDCl3), δ 7.72 (d, 2H, J = 8.3 Hz), 7.35 - 7.24 (m, 7H), 4.51, 4.44 (ABq, 2H, J AB = 15.0 Hz), 2.94 (br, 1H), 2.90 (br, 1H), 2.45 (s, 3H), 1.94 - 1.78 (m, 2H), 1.74 - 1.73 (m, 3H), 1.70 - 1.64 (m, 2H); 13 C{ 1 H} NMR (100 MHz, CDCl3) δ 216.0, 144.3, 144.2, 136.5, 136.2, 130.0, 129.2, 128.6, 127.9, 127.8, 127.5, 54.3, 51.3, 40.2, 34.1, 21.8, 19.9, 13.1.

[0049] Example 2: Study on the antiviral activity of the cyclobutenamide compounds synthesized in Example 1 of the present invention

[0050] The experimental reagents, experimental instruments and their sources used in the antiviral activity research of compounds 3a - 3k are shown in Tables 1 and 2.

[0051] Table 1 Experimental reagents and manufacturers

[0052]

[0053] Table 2 Experimental instruments and manufacturers

[0054]

[0055] Detection of cytotoxicity and antiviral activity of compounds 3a - 3k

[0056] Take MDCK cells in good growth state and inoculate them evenly into 96 - well plates at a density of 1.4×10⁴ cells / well. After the cells adhere for 24 hours, aspirate and discard the culture medium, and gently rinse 3 times with pre - cooled PBS buffer. The test compounds are first prepared into a 20 mM stock solution with DMSO, and then serially diluted with virus maintenance medium (concentration range from 800 μM to 6.25 μM, a total of 8 concentration gradients). Add 200 μL of culture medium containing different concentrations of compounds to each well in the experimental group. At the same time, set up a blank control group (containing only virus maintenance medium), and set 6 parallel replicates for each group. After the cells are cultured at 37 °C and 5% CO₂ (volume percentage) for 72 hours, add 20 μL of MTT solution to each well and continue to incubate in the dark for 4 hours to form formazan crystals. After carefully aspirating and discarding the supernatant, add 160 μL of DMSO to each well and place it on a horizontal shaker to shake for 15 minutes to completely dissolve the crystals. Finally, measure the absorbance value at a wavelength of 492 nm with an enzyme - linked immunosorbent assay (ELISA) reader. The formula for calculating cell viability is: (OD value of the experimental group / OD value of the control group) × 100%. Curve fitting is performed using IBM SPSS27 statistical software to calculate the half - cytotoxic concentration (CC 50 ) of each compound, and the specific data are shown in Table 3.

[0057] Table 3 Detection results of cytotoxicity and antiviral activity of compounds 3a - 3k

[0058]

[0059] Note: a represents the 50% cytotoxic concentration for MDCK cells, b represents the 50% effective concentration against H1N1, c represents the therapeutic index SI, d represents no anti - IAV activity between 0.625 μM and 100 μM.

[0060] Adjust the suspension of MDCK cells in good growth state to a density of 1.4×104 cells / well and inoculate it into a 96-well culture plate. After the cells adhere to the wall for 24 hours, remove the culture medium and wash the cells 3 times with pre-cooled PBS buffer. Subsequently, add 100 μL of A / Weiss / 43(H1N1) influenza virus suspension containing 100 TCID50 to each well for infection. At the same time, set up blank control wells containing only virus adsorption solution, and set 6 parallel wells in each group. Place the infected cell culture plate in an incubator at 37°C and gently shake it every 15 minutes (4 times in total), and continue to culture for 1 hour to ensure sufficient virus adsorption. After removing the virus solution, wash the cells with PBS again. Prepare a 20 mM stock solution of the test compound with DMSO, and then perform serial dilution with virus maintenance medium (concentration gradient from 100 μM to 0.78125 μM, a total of 8 concentrations), and add 200 μL of the compound solution with the corresponding concentration to each well. At the same time, set up virus control groups and normal cell control groups, and each group has 6 replicate wells. After culturing for 72 hours, use the MTT method to detect cell viability and measure the absorbance value at a wavelength of 492 nm. The virus inhibition rate calculation formula is: [(OD value of the experimental group - OD value of the virus control group) / (OD value of the normal control group - OD value of the virus control group)]×100%. Use IBM SPSS 27.0 statistical software for data analysis, calculate the half-maximal effective concentration (EC 50 ), and evaluate the antiviral activity of the compound according to the therapeutic index formula (SI = CC 50 / EC 50 ). The specific data are shown in Table 3.

[0061] In this invention, a systematic anti-influenza A virus activity screening and safety evaluation of cyclobutenamide compounds 3a-3k were carried out through a standardized in vitro antiviral evaluation platform. The MDCK cell model was used in the experiment, and the clinical isolate A / Weiss / 43(H1N1) was used as the research object, and the antiviral effect of the compound was determined by the MTT method. The research data show that compounds 3e (EC 50 = 2.01±0.47 μM) and 3k (EC 50 = 13.25±1.35 μM) both showed good antiviral activity. Among them, the in vitro antiviral efficacy of the lead compound 3e was significantly better than that of the clinical first-line drug ribavirin (EC 50 = 5.28±1.22 μM), and the activity improvement amplitude reached 2.6 times. In terms of safety, 3e showed outstanding cell compatibility (CC 50= 341.38 ± 9.83 μM), and its therapeutic index (SI = 169.84) far exceeds the development standard of conventional antiviral drugs (SI > 10), showing an excellent therapeutic safety window. The comprehensive evaluation results indicate that compound 3e not only has potent antiviral activity but also possesses ideal safety characteristics, fully meeting the screening requirements for innovative drug candidate molecules. These important findings provide a solid experimental basis for the development of novel anti-IAV drugs based on the cyclobutenamide backbone and demonstrate broad clinical application prospects.

Claims

1. The pharmaceutical use of cyclobutene amide compounds with the following general structural formula, characterized in that, For use as an active ingredient in the preparation of an anti-influenza A virus drug: EWG is selected from a sulfonyl group and an aryl-substituted sulfonyl group, and the aryl is selected from a phenyl group substituted with a methyl group, a halogen or a nitro group; R 1 selected from benzyl, C1-4 alkyl or phenyl; R 2 selected from C1-6 alkyl, phenyl or thienyl; n represents the number of carbon atoms and is 1 or 2.

2. The pharmaceutical use of the cyclobutenamide compound according to claim 1, characterized in that, EWG is selected from p-toluenesulfonyl, p-chlorobenzenesulfonyl or p-nitrobenzenesulfonyl; R 1 is selected from benzyl, methyl, butyl or phenyl; R 2 is selected from phenyl, methyl, n-hexyl or thienyl; n represents the number of carbon atoms and is 1 or 2.

3. The pharmaceutical use of the cyclobutene amide compound according to claim 2, characterized in that, EWG is selected from tosyl; R 1 is selected from benzyl or phenyl; R 2 is selected from methyl; n represents the number of carbon atoms and is 1 or 2.

4. The pharmaceutical use of the cyclobutene amide compound according to claim 3, characterized in that, Selected from the following compounds:

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