2-aminonicotinonitrile compounds and salts and compositions thereof for use in the preparation of broad-spectrum anti-coronavirus drugs

Abstract

The application provides application of a 2-aminonicotinonitrile compound and a salt and a composition thereof in preparation of a broad-spectrum anti-coronavirus drug, wherein the 2-aminonicotinonitrile compound is a compound shown in the following general formula, wherein R1, R2 and R3 are independently H, Ar, OH, CH3 or OMe; R4 and R5 are independently H, OH, OMe or Ar, Ar is a halogen group; and R6 is H, OH, OMe, Cl, Ph or CH3. The 2-aminonicotinonitrile compound has anti-coronavirus activity, can inhibit replication of various coronaviruses including the new coronavirus, can inhibit coronavirus RNA replication and protein expression, can be used for preparing a broad-spectrum anti-coronavirus drug, has a good application prospect, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of a small molecule compound in the preparation of a broad-spectrum anti-coronavirus drug. Background Technology

[0002] Coronaviruses are single-stranded positive-sense RNA viruses. They are not only diverse in species (α, β, γ, and δ, four genera) and have a broad host spectrum (involving humans, bats, pangolins, pigs, rats, minks, and birds), but can also cause a variety of diseases, including those affecting the respiratory, digestive, and nervous systems. Coronavirus Disease 2019 (COVID-19) is an acute respiratory infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This is the most infectious and deadliest global pandemic outside of the 1918 Spanish flu (H1N1 swine flu), posing a significant challenge to human health and causing a major impact on global political, economic, and social stability. SARS-CoV-2 belongs to the genus β-coronavirus and is the seventh coronavirus discovered that can infect humans, following HCoV-OC43, HCoV-HKU1, HCoV-NL63, HCoV-229E, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Given the diversity of coronavirus species and the high pathogenicity, transmissibility, rapid mutation, long-term epidemicity of different subtypes, as well as the limitations of vaccine efficacy, exploring broad-spectrum anti-coronavirus drug targets and innovative drugs targeting the key common mechanisms in the initial replication process of coronavirus infection is of significant strategic importance for controlling emerging coronavirus epidemics.

[0003] To combat the coronavirus pandemic, identifying broad-spectrum antiviral targets and innovative drugs is crucial to containing the outbreak. Based on the successful experience of existing antiviral drug development, targeted antiviral drugs mainly fall into two categories. The first category targets viral proteins, including inhibitors that inhibit viral invasion, protease inhibitors that inhibit viral protein hydrolysis, polymerase inhibitors that inhibit viral RNA synthesis, and nucleocapsid inhibitors that inhibit viral assembly. While antiviral drugs targeting these viral targets have the advantage of high selectivity and druggability, the rapid mutation of coronaviruses means that antiviral drugs targeting viral proteins constantly face risks such as drug resistance mutations leading to ineffectiveness. The second category targets key host proteins involved in the viral life cycle, including receptor inhibitors, cellular protease inhibitors, and cathepsin inhibitors. However, because a common target mediating coronavirus invasion and replication has not yet been found, no effective broad-spectrum antiviral drugs have been discovered to date.

[0004] Although studies have reported on small molecule active compounds and drugs with different structures that inhibit coronaviruses, very few anti-coronavirus drugs can actually be used for clinical treatment. Therefore, designing and synthesizing a novel small molecule active compound that can effectively inhibit coronaviruses on a broad spectrum is essential for the clinical development of anti-coronavirus drugs.

[0005] Cyanopyridine, a branch of pyridine, is an important organic chemical raw material. Due to its excellent biological and reactive properties, it has always been a research hotspot. Utilizing the chemical reactivity of its cyano group, these compounds are widely used in the synthesis of pharmaceuticals, agrochemicals, food / feed additives, daily chemicals, dyes, and other intermediates, such as nicotinic acid, nicotinamide, isonicotinic acid, isonicotinamide hydrazide, pymetrozine, chromium nicotinate, 2-chloronicotinic acid, and atrazine. Many natural products and synthetic compounds also contain this structure. Literature reports that these compounds possess strong biological activities, such as antibacterial, antihypertensive, cardiovascular disease treatment, and anti-inflammatory properties. Recently, they have also been shown to have IKK-β inhibitory activity and function as GPR54 antagonists. In recent years, their application areas have continued to expand, attracting widespread attention. Recently, derivatives of this class, such as 2-aminonicotinic nitrile compounds, have been found to have strong antihypertensive, anti-inflammatory, analgesic, antipyretic, antitumor, and IKK-β inhibitory activities. However, no studies have been reported on whether such compounds have anti-coronavirus effects. Summary of the Invention

[0006] To overcome the shortcomings of the above-mentioned technologies, one object of the present invention is to provide the application of 2-aminonicotinic nitrile compounds and their salts and compositions in the preparation of broad-spectrum anti-coronavirus drugs. 2-aminonicotinic nitrile compounds have anti-coronavirus activity and can inhibit coronavirus replication, inhibit coronavirus RNA transcription, protein expression and release of new viruses. They have good anti-coronavirus effects and can be used to prepare broad-spectrum anti-coronavirus drugs. They have good application prospects and are suitable for large-scale promotion and application.

[0007] To achieve the above objectives, the present invention provides the use of 2-aminonicotinic nitrile compounds, their salts, and compositions in the preparation of broad-spectrum anticoronavirus drugs, wherein the 2-aminonicotinic nitrile compounds are compounds having the following general formula (I): (I) Among them, R1, R2 and R3 are independently H, Ar, OH, CH3 or OMe; R4 and R5 are independently H, OH, OMe or Ar, where Ar is a halogen group; R6 is H, OH, OMe, Cl, Ph or CH3.

[0008] Preferably, R1 is OH.

[0009] More preferably, R2 and R3 are both OMe, R4 is H, R5 is Ar, and R6 is H.

[0010] Preferably, the 2-aminonicotinic nitrile compound is selected from one of the following combinations of compounds: .

[0011] Preferably, the broad-spectrum anticoronavirus drug is used to inhibit the replication of coronaviruses.

[0012] Preferably, the broad-spectrum anticoronavirus drug is used to inhibit the transcription of coronavirus RNA.

[0013] Preferably, the broad-spectrum anticoronavirus drug is used to inhibit the expression of coronavirus NP protein.

[0014] Preferably, the broad-spectrum anticoronavirus drug is used to inhibit the release of newly generated coronavirus virus.

[0015] Preferably, the broad-spectrum anti-coronavirus drug acts on the replication phase of the coronavirus life cycle.

[0016] Preferably, the coronavirus is selected from one or more of canine coronavirus CCV, porcine epidemic diarrhea virus PEDV, mouse hepatitis virus MHV, chicken infectious bronchitis virus IBV, novel coronavirus SARS-CoV-2, and human common coronaviruses OC43, HCoV-NL63, and HCoV-229E.

[0017] Preferably, the coronavirus is one or more mutants of the novel coronavirus SARS-CoV-2.

[0018] Preferably, the composition comprises two or more compounds of general formula (I), or the composition further comprises other components for enhancing the anticoronavirus activity of the compounds of general formula (I).

[0019] Preferably, the composition further includes a pharmaceutically acceptable carrier or excipient.

[0020] Preferably, the anticoronavirus pharmaceutical composition is used to formulate any of the following dosage forms: tablets, pills, powders, capsules, granules, injections, and oral liquids.

[0021] The beneficial effects of this invention are as follows: 2-Aminonicotinamide compounds possess broad-spectrum anti-coronavirus activity, capable of inhibiting the replication of various coronaviruses, including SARS-CoV-2, and suppressing the transcriptional replication of coronavirus RNA, protein expression, and / or the release of nascent viruses. They exhibit good anti-coronavirus activity and can be used to prepare broad-spectrum anti-coronavirus drugs, showing promising application prospects and suitability for large-scale promotion and application.

[0022] These and other objects, features and advantages of the present invention will be fully apparent from the following detailed description and drawings, and can be achieved by the methods, means and combinations thereof specifically pointed out in the specification. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the cytotoxicity test results of w01-w29 small molecule drugs on Vero cells, where Molnup (Molnupiravir) represents the positive control drug, administered at a dose of 10 μM.

[0024] Figure 2 This is a schematic diagram illustrating the anti-coronavirus efficacy of w01-w29 small molecule drugs on Vero cells. Model represents the virus control, Molnup represents the positive drug control, and *** indicates a comparison with the Model group. P <0.001.

[0025] Figure 3This is a schematic diagram showing the results of detecting the inhibitory effects of compounds w08, w09, w10, w11, w13, w15, and w19 on coronavirus replication in a Vero cell model. Hoechst represents a fluorescence micrograph of Vero cells stained with Hoechst, Anti-NP represents a fluorescence micrograph showing the fluorescence emission of fluorescein isothiocyanate (FITC) from the NP-NP antibody-FITC-labeled goat anti-mouse complex of the coronavirus protein, Merge represents the fusion effect of the Hoechst-stained fluorescence micrograph and the fluorescence micrograph showing the fluorescence emission of fluorescein isothiocyanate (FITC) from the NP-NP antibody-FITC-labeled goat anti-mouse complex of the coronavirus protein, Control represents the blank control, Model represents the virus control, and the image scale bar is 100 μm.

[0026] Figure 4 This is a schematic diagram illustrating the broad-spectrum anti-coronavirus activity of the representative compound w09, where CCV is canine coronavirus; PEDV is porcine epidemic diarrhea virus; MHV is mouse hepatitis virus; IBV is chicken infectious bronchitis virus; OC43, HCoV-NL63, and HCoV-229E are human common coronaviruses; and SARS-CoV-2 is the novel coronavirus.

[0027] Figure 5 This is a schematic diagram showing the effect of the representative compound w09 on the expression of the coronavirus NP protein. In the diagram, Control represents the blank control, Model represents the virus control, Molnupiravir represents the positive control, NP represents the coronavirus protein NP, and ACTB represents the internal reference gene.

[0028] Figure 6 This is a schematic diagram illustrating the effect of the representative compound w09 on the intracellular transcription and replication of the SARS-CoV-2 genome.

[0029] Figure 7 This is a schematic diagram illustrating the effect of the representative compound w09 on the release of newly generated coronavirus virus, where Molnupiravir represents the positive control drug.

[0030] Figure 8 This diagram illustrates the effect of w09 on the potential stages of coronavirus replication lifecycle, where Control represents the blank control and Model represents the virus control. Figure A shows the effect of different drug administration times; Figure B shows the protein expression results of w09 inhibiting the potential stages of coronavirus replication lifecycle; Figure C shows the protein expression results of w09 inhibiting the replication stage of coronavirus replication lifecycle. Detailed Implementation

[0031] In order to find new anti-coronavirus drugs with high activity and good safety, the inventors conducted extensive research and discovered for the first time that 2-aminonicotinic nitrile compounds have good anti-coronavirus activity, with high activity and good safety. Based on this, the present invention was completed.

[0032] This invention first provides the use of 2-aminonicotinic nitrile compounds, their salts, and compositions in the preparation of anticoronavirus drugs, wherein the 2-aminonicotinic nitrile compounds are compounds having the following general formula (I): (I) Among them, R1, R2 and R3 are independently H, Ar, OH, CH3 or OMe; R4 and R5 are independently H, OH, OMe or Ar, where Ar is a halogen group; R6 is H, OH, OMe, Cl, Ph or CH3.

[0033] R1 can be any suitable group, preferably OH. R2, R3, R4, R5 and R6 can each be any suitable group, more preferably R2 and R3 are both OMe, R4 is H, R5 is Ar and R6 is H.

[0034] The anti-coronavirus drug can be used to combat coronaviruses in any suitable manner; preferably, the anti-coronavirus drug is used to inhibit the replication of coronaviruses.

[0035] The anticoronavirus drug can be used to combat coronaviruses in any suitable manner; preferably, the anticoronavirus drug is used to inhibit the expression of coronavirus NP protein.

[0036] The anti-coronavirus drug can be used to combat coronaviruses in any suitable manner; preferably, the anti-coronavirus drug is used to inhibit the transcriptional replication of SARS-CoV-2 RNA.

[0037] The anti-coronavirus drug can act on any stage of the coronavirus life cycle, preferably on the replication stage of the coronavirus.

[0038] The anti-coronavirus drug can be any type of coronavirus, preferably SARS-CoV-2.

[0039] The composition may have any suitable composition. Preferably, the composition comprises two or more compounds of general formula (I) or the composition further comprises other components for enhancing the anticoronavirus activity of the compounds of general formula (I).

[0040] The composition may have any suitable composition. Preferably, the composition includes at least one of a pharmaceutically acceptable carrier, solvent, excipient, adjuvant, and prodrug, wherein the prodrug is a derivative of the compound represented by general formula (I) and is capable of being converted into the compound represented by general formula (I) in vivo by means of in vivo metabolism. The composition may incorporate one or more carriers or excipients to allow for formulation into any pharmaceutically acceptable dosage form. More preferably, the dosage form is selected from one or more of tablets, pills, powders, capsules, granules, injections, and oral liquids.

[0041] The salts described in this invention include pharmaceutically acceptable salts and pharmaceutically unacceptable salts. Pharmaceutically acceptable salts refer to any pharmaceutically acceptable salt, lipid, solvent compound, hydrate, or any other compound that, when administered to a recipient, can (directly or indirectly) provide the compound represented by general formula (I) described in this invention. However, it is understood that pharmaceutically unacceptable salts also fall within the scope of this invention because they can be used to prepare pharmaceutically acceptable salts. The preparation of salts, prodrugs, and derivatives can be carried out by methods known in the art. For example, pharmaceutically acceptable salts of the compounds provided in this invention are synthesized by conventional chemical methods with parent compounds containing basic and acidic groups. Typically, such salts are prepared, for example, by reacting the free acid or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof. Typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Examples of acid addition salts include mineral acid addition salts such as hydrochlorides, hydrobroms, hydroiodates, sulfates, nitrates, and phosphates, and organic acid addition salts such as acetates, trifluoroacetates, maleates, fumarates, citrates, oxalates, succinates, tartrates, malates, mandelates, methanesulfonates, and p-toluenesulfonates. Examples of base addition salts include inorganic salts such as sodium, potassium, calcium, and ammonium salts, and organic base salts such as ethylenediamine, ethanolamine, N,N-dialkylethanolamine, triethanolamine, and basic amino acid salts.

[0042] The compounds and compositions of the present invention can be used with other drugs to provide combination therapy. Other drugs may be formed as part of a unified composition or provided as separate compositions for simultaneous or asynchronous administration.

[0043] To provide a clearer understanding of the technical content of this invention, the following embodiments are provided for detailed description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Example 1: Synthesis method of 2-aminonicotinamide compounds

[0044] The 2-aminonicotinonitrile compounds of the present invention are synthesized according to the method disclosed in Chinese invention patent application CN201210362468.6, but are not limited to this method. The specific synthesis method includes the following steps: Step 1: Add 100 parts by weight of lignin and 10.0–20.0 parts by weight of oxidant to 1 L of 0.5–2.5 mol / L sodium hydroxide solution and stir until homogeneous. React at 160–200℃ for 1–5 h. After cooling, acidify with concentrated sulfuric acid to pH 4.0, then extract with chloroform. Concentrate the extract and recover the distilled chloroform. Separate the concentrated product from the previous step by distillation to obtain purified aromatic aldehyde. The oxidant mentioned above can be either nitrobenzene or CuSO4.

[0045] Step 2: Dissolve 2 mmol of aromatic aldehyde obtained from the oxidative degradation of lignin, 2 mmol of aromatic ketone, and 2 mmol of malononitrile in 1,4-dioxane, then add 4 mmol of ammonium acetate. React at 120°C for 20 min with microwave-assisted heating at 300W. Add ice water to the reaction mixture to precipitate the product, which can then be recrystallized to obtain 2-aminonicotinonitrile.

[0046] Specific examples of compound synthesis are given in Examples 2-28.

[0047] Example 2 4-(4-hydroxy-3,5-dimethoxyphenyl)-6-phenyl-2-amino-3-cyanopyridine (w09)

[0048] Syringaldehyde (2 mmol), acetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120°C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 84% yield. IR (KBr) v / cm -1: 3471, 3395, 3358,3065, 2953, 2111, 1630, 1573, 1588, 1514, 1464, 1379, 1250, 1211, 1109, 770,699; 1 H NMR (300 MHz, DMSO- d 6 ) δ / ppm: 8.88 (s, 1H, OH), 8.12-8.15 (m, 2H, ArH), 7.45-7.56 (m, 3H, ArH), 7.32 (s, 1H, H5), 7.00 (s, 2H, ArH), 6.91 (s, 2H,NH2), 3.86 (s, 6H, 2×OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.9, 158.3,155.0, 147.9, 137.7, 137.3, 130.0, 128.5, 127.2, 126.6, 117.5, 109.1, 106.3,86.5, 56.2; MS (ESI) m / z 348.3 [M+H] + 370.3 [M+Na] + Anal. Calcd for C 20 H 17 N3O3: C, 69.15; H, 4.93; N, 12.10; found: C, 69.03; H, 4.98; N, 12.17. Example 3 4-(4-hydroxy-3-methoxyphenyl)-6-phenyl-2-amino-3-cyanopyridine (w12)

[0049] Vanillin (2 mmol), acetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120°C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 82% yield. IR (KBr) v / cm -1: 3486, 3401, 3308,3196, 2209, 1632, 1577, 1551, 1519, 1469, 1278, 1220, 1126, 764; 1 H NMR (300MHz, DMSO- d 6 ) δ / ppm: 9.53 (s, 1H, OH), 8.11-8.14 (m, 2H, ArH), 7.42-7.56 (m,3H, ArH), 7.24-7.29 (m, 2H, H5, ArH), 7.15 (dd, 1H, ArH, J = 2.1, 8.1 Hz),6.85-6.96 (m, 3H, ArH, NH2), 3.87 (s, 3H, OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ MS (ESI) m / z 318.1 [M+H] + 340.3 [M+Na] + 356.0 [M+K] + Anal. Calculated for C 19 H 15 N3O2: C, 71.91; H, 4.76; N, 13.24; found: C, 71.83; H, 4.70; N, 13.31. Example 4 4-(4-hydroxyphenyl)-6-phenyl-2-amino-3-cyanopyridine (w13)

[0050] p-Hydroxybenzaldehyde (2 mmol), acetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120°C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 80% yield. IR (KBr)v / cm -1 : 3469, 3382,3314, 3200, 2212, 1632, 1612, 1575, 1547, 1518, 1432, 1284, 1213, 824, 768; 1 HNMR (300 MHz, DMSO- d 6 ) δ / ppm: 8.10-8.12 (m, 2H, ArH), 7.56 (d, 2H, ArH, J = 8.4Hz), 7.46-7.55 (m, 3H, ArH), 7.22 (s, 1H, H5), 6.86-6.97 (m, 4H, ArH, NH2); 13 CNMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.9, 158.9, 158.3, 154.7, 137.7, 129.8,128.6, 127.4, 127.2, 117.4, 115.5, 108.9, 85.9; MS (ESI) m / z 288.1 [M+H] + 310.1 [M+Na] + Anal. Calculated for C 18 H 13 N3O: C, 75.25; H, 4.56; N, 14.63; found: C, 75.41; H, 4.41; N, 14.51. Example 5 4-(4-hydroxy-3,5-dimethoxyphenyl)-5-methyl-6-p-chlorophenyl-2-amino-3-cyanopyridine (w10)

[0051] Syringaldehyde (2 mmol), 4-chlorophenylacetone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 76% yield. IR (KBr) v / cm -1: 3463, 3364,3239, 2203, 1642, 1611, 1551, 1520, 1415, 1332, 1125, 831; 1 H NMR (300 MHz, DMSO- 6d ) δ / ppm: 8.69 (s, 1H, OH), 7.51-7.59 (m, 4H, ArH), 6.66-6.70 (m, 4H, ArH, NH2), 3.80 (s, 6H, 2×OCH3), 1.91 (s, 3H, CH3); 13 C NMR (75.5 MHz, DMSO- 6d ) δ / ppm: 159.8, 158.0, 155.8, 147.9, 138.9, 136.0, 133.1, 130.7, 128.0, 126.7,117.1, 116.8, 106.3, 89.8, 56.2, 16.9; MS (ESI) m / z 396.1 [M+H] + , 418.1 [M+Na] + Anal. Calculated for C 21 H 18 ClN3O3: C, 63.72; H, 4.58; N, 10.62; found: C, 63.83; H, 4.50; N, 10.51. Example 6 4-(4-hydroxy-3-methoxyphenyl)-5-methyl-6-p-chlorophenyl-2-amino-3-cyanopyridine (w22)

[0052] Vanillin (2 mmol), 4-chlorophenylacetone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 71% yield. IR (KBr) v / cm -1 : 3462, 3338,3211, 2228, 1634, 1556, 1516, 1276, 1242, 1092, 1032, 824; 1H NMR (300 MHz, DMSO- 6d ) δ / ppm: 9.32 (s, 1H, OH), 7.50-7.58 (m, 4H, ArH), 6.95 (d, 1H, ArH, J =1.8 Hz), 6.90 (d, 1H, ArH, J = 8.1 Hz), 6.78 (dd, 1H, ArH, J = 1.8, 8.1 Hz), 6.67 (s, 2H, NH2), 3.79 (s, 3H, OCH3), 1.88 (s, 3H, CH3); 13 C NMR (75.5 MHz, DMSO- 6d ) δ / ppm: 160.3, 158.5, 156.2, 147.9, 147.5, 139.4, 133.6, 131.2,128.4, 128.2, 121.7, 117.6, 117.3, 116.0, 113.3. 90.3, 56.3, 17.3; MS (ESI) m / z 366.0 [M+H] + Anal. Calculated for C 20 H 16 ClN3O2: C, 65.67; H, 4.41; N, 11.49; found: C, 65.76; H, 4.55; N, 11.40. Example 7 4-(4-hydroxyphenyl)-5-methyl-6-p-chlorophenyl-2-amino-3-cyanopyridine (w23)

[0053] p-Hydroxybenzaldehyde (2 mmol), 4-chlorophenylacetone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 72% yield. IR (KBr) v / cm -1 : 3457,3360, 3241, 2217, 1634, 1550, 1516, 1249, 1167, 1092, 842;1 H NMR (300 MHz, DMSO- 6d ) δ / ppm: 9.75 (s, 1H, OH), 7.48-7.55 (m, 4H, ArH), 7.19 (d, 2H, ArH, J =7.5 Hz), 6.89 (d, 2H, ArH, J = 7.5 Hz), 6.67 (s, 2H, NH2), 1.85 (s, 3H, CH3); 13 C NMR (75.5 MHz, DMSO- 6d ) δ / ppm: 159.8, 158.0, 157.8, 155.4, 133.1, 130.9,130.7, 129.8, 127.9, 127.2, 117.1, 116.8, 115.3, 89.9, 16.7; MS (ESI) m / z 336.1 [M+H] + , 358.1 [M+Na] + Anal. Calculated for C 19 H 14 ClN3O: C, 67.96; H, 4.20; N, 12.51; found: C, 67.85; H, 4.29; N, 12.44. Example 8 4-(4-hydroxy-3,5-dimethoxyphenyl)-6-p-fluorophenyl-2-amino-3-cyanopyridine (w19)

[0054] Syringaldehyde (2 mmol), 4-fluoroacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 85% yield. IR (KBr) v / cm -1 : 3513, 3401,2945, 2211, 1609, 1576, 1548, 1512, 1459, 1376, 1257, 1215, 1117, 834; 1 H NMR (300 MHz, DMSO-) d6 ) δ / ppm: 8.88 (s, 1H, OH), 8.18-8.23 (m, 2H, ArH), 7.30-7.37(m, 3H, ArH, H5), 6.98 (s, 2H, ArH), 6.92 (s, 2H, NH2), 3.85 (s, 6H, 2×OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.9, 157.2, 155.1, 147.9, 137.3, 129.6,129.5, 126.6, 115.6, 115.3, 108.9, 106.3, 86.2, 56.2; MS (ESI) m / z 366.1 [M+H] + , 388.1 [M+Na] + Anal. Calculated for C 20 H 16 FN3O3: C, 65.75; H, 4.41; N, 11.50; found: C, 65.84; H, 4.34; N, 11.43. Example 9 4-(4-hydroxy-3-methoxyphenyl)-6-p-fluorophenyl-2-amino-3-cyanopyridine (w20)

[0055] Vanillin (2 mmol), 4-fluoroacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 81% yield. IR (KBr) v / cm -1 : 3495, 3382,3349, 3236, 2213, 1643, 1601, 1577, 1554, 1512, 1460, 1360, 1276, 1219, 1159,1034, 829; 1 H NMR (300 MHz, DMSO- d 6 ) δ / ppm: 9.48 (s, 1H, OH), 8.16-8.22 (m, 2H,ArH), 7.26-7.35 (m, 4H, ArH, H5), 7.15 (dd, 1H, ArH, J = 1.5, 8.1 Hz), 6.80-6.94 (m, 3H, ArH, NH2), 3.86 (s, 3H, OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm:161.4, 157.7, 155.4, 148.8, 18.0, 134.7, 130.1, 130.0, 128.2, 121.9, 117.9,116.1, 115.8, 113.1, 109.3, 86.8, 56.3; MS (ESI) m / z 336.0 [M+H] + Anal.Calcd for C 19 H 14 FN3O2: C, 68.05; H, 4.21; N, 12.53; found: C, 68.21; H, 4.11; N, 12.45. Example 10 4-(4-hydroxyphenyl)-6-p-fluorophenyl-2-amino-3-cyanopyridine (w21)

[0056] p-Hydroxybenzaldehyde (2 mmol), 4-fluoroacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 82% yield. IR (KBr) v / cm -1 : 3513,3411, 3359, 2211, 1610, 1595, 1575, 1551, 1512, 1370, 1283, 1217, 1163, 823; 1 H NMR (300 MHz, DMSO- d 6 ) δ / ppm: 9.91 (s, 1H, OH), 8.15-8.21 (m, 2H, ArH), 7.55(d, 2H, ArH, J = 8.7 Hz), 7.31 (t, 2H, ArH, J = 9.0 Hz), 7.22 (s, 1H, H5), 6.85-6.96 (m, 4H, ArH, NH2); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.9, 158.9,157.2, 154.8, 134.2, 129.8, 129.5, 129.4, 127.4, 117.3, 115.6, 115.5, 108.7,86.2; MS (ESI) m / z 306.0 [M+H] + Anal. Calculated for C 18 H 12 FN3O: C, 70.81; H, 3.96; N, 13.76; found: C, 70.72; H, 3.90; N, 13.72. Example 11 4-(4-hydroxy-3,5-dimethoxyphenyl)-6-p-chlorophenyl-2-amino-3-cyanopyridine (w16)

[0057] Syringaldehyde (2 mmol), 4-chloroacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 84% yield. IR (KBr) v / cm -1 : 3462, 3369,3228, 2208, 1614, 1554, 1520, 1377, 1311, 1219, 1117, 807; 1 H NMR (300 MHz, DMSO- d 6 ) δ / ppm: 8.89 (s, 1H, OH), 8.17 (d, 2H, ArH, J= 8.4 Hz), 7.57 (d, 2H,ArH, J = 8.4 Hz), 7.33 (s, 1H, H5), 6.99 (s, 2H, ArH), 6.95 (s, 2H, NH2), 3.85(s, 6H, 2×OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.9, 156.9, 155.2, 147.9,137.3, 136.5, 134.8, 129.0, 128.6, 126.5, 117.4, 109.0, 106.3, 86.3, 56.2; MS(ESI) m / z 382.3 [M+H] + , 404.3 [M+Na] + Anal. Calculated for C 20 H 16 ClN3O3: C, 62.91; H, 4.22; N, 11.01; found: C, 62.99; H, 4.10; N, 11.04. Example 12 4-(4-hydroxy-3-methoxyphenyl)-6-p-chlorophenyl-2-amino-3-cyanopyridine (w17)

[0058] Vanillin (2 mmol), 4-chloroacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 83% yield. IR (KBr) v / cm -1 : 3472, 3368,3234, 3073, 2207, 1640, 1580, 1552, 1516, 1284, 1222, 1127, 1092, 809; 1 H NMR (300 MHz, DMSO-) d 6 ) δ / ppm: 9.49 (s, 1H, OH), 8.16 (d, 2H, ArH, J= 8.7 Hz), 7.56(d, 2H, ArH, J = 8.7 Hz), 7.26-7.29 (m, 2H, ArH, H5), 7.15 (dd, 1H, ArH, J =1.8, 8.1 Hz), 6.86-6.95 (m, 3H, ArH, NH2), 3.86 (s, 3H, OCH3); 13 C NMR (75.5MHz, DMSO- d 6 ) δ / ppm: 160.9, 156.9, 155.0, 148.3, 147.6, 136.5, 134.8, 129.0,128.6, 127.6, 121.4, 117.3, 115.5, 112.7, 109.0, 86.7, 55.8; MS (ESI) m / z :352.1 [M+H] + Anal. Calculated for C 19 H 14 ClN3O2: C, 64.87; H, 4.01; N, 11.94; found: C, 64.80; H, 4.11; N, 11.99. Example 13 4-(4-hydroxyphenyl)-6-p-chlorophenyl-2-amino-3-cyanopyridine (w18)

[0059] p-Hydroxybenzaldehyde (2 mmol), 4-chloroacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 84% yield. IR (KBr) v / cm -1 : 3507,3401, 3369, 2210, 1609, 1572, 1552, 1519, 1367, 1283, 1263, 1090, 818; 1 H NMR (300 MHz, DMSO-) d 6 ) δ / ppm: 9.92 (s, 1H, OH), 8.15 (d, 2H, ArH,J = 8.7 Hz),7.50-7.59 (m, 4H, ArH), 7.25 (s, 1H, H5), 6.87-6.94 (m, 4H, ArH, NH2); 13 C NMR (75.5 MHz, DMSO-) d 6 ) δ / ppm: 160.9, 159.0, 157.0, 154.9, 136.5, 130.5, 129.9,129.1, 128.6, 127.3, 117.3, 115.5, 108.9, 86.6; MS (ESI) m / z 332.1 [M+H] + Anal. Calcd for C 18 H 12 ClN3O: C, 67.19; H, 3.76; N, 13.06; found: C, 67.11; H, 3.70; N, 13.14. Example 14 4-(4-hydroxy-3,5-dimethoxyphenyl)-6-p-bromophenyl-2-amino-3-cyanopyridine (w24)

[0060] Syringaldehyde (2 mmol), 4-bromoacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120°C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 85% yield. IR (KBr) v / cm -1 : 3484, 3460,3373, 2206, 1614, 1568, 1552, 1521, 1377, 1312, 1219, 1118, 805; 1 H NMR (300MHz, DMSO- d 6 ) δ / ppm: 8.86 (s, 1H, OH), 8.10 (d, 2H, ArH, J = 8.4 Hz), 7.70 (d,2H, ArH, J= 8.4 Hz), 7.33 (s, 1H, H5), 6.99 (s, 2H, ArH), 6.92 (s, 2H, NH2), 3.85 (s, 6H, 2×OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 161.4, 157.5, 155.7,148.4, 137.8, 137.4, 132.0, 129.8, 127.0, 124.1, 117.9, 109.5, 106.8, 87.4,56.7; MS (ESI) m / z 426.0 [M+H] + Anal. Calculated for C 20 H 16 BrN3O3: C, 56.35; H, 3.78; N, 9.86; found: C, 56.30; H, 3.71; N, 9.92. Example 15 4-(4-hydroxy-3-methoxyphenyl)-6-p-bromophenyl-2-amino-3-cyanopyridine (w25)

[0061] Vanillin (2 mmol), 4-bromoacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 81% yield. IR (KBr) v / cm -1 : 3470, 3368,3235, 2208, 1640, 1579, 1552, 1516, 1259, 1220, 1127, 810; 1 H NMR (300 MHz, DMSO- d 6 ) δ / ppm: 9.50 (s, 1H, OH), 8.09 (d, 2H, ArH, J = 8.7 Hz), 7.69 (d, 2H,ArH, J= 8.7 Hz), 7.26-7.29 (m, 2H, ArH, H5), 7.15 (dd, 1H, ArH, J = 2.1, 8.1Hz), 6.91-6.94 (m, 3H, ArH, NH2), 3.86 (s, 3H, OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.9, 157.0, 155.0, 148.3, 147.5, 136.9, 131.5, 129.2, 127.6,123.6, 121.4, 117.4, 115.5, 112.7, 108.9, 86.8, 55.8; MS (ESI) m / z 396.0 [M+H] + Anal. Calculated for C 19 H 14 BrN3O2: C, 57.59; H, 3.56; N, 10.60; found: C, 57.51; H, 3.51; N, 10.66. Example 16 4-(4-hydroxyphenyl)-6-p-bromophenyl-2-amino-3-cyanopyridine (w26)

[0062] p-Hydroxybenzaldehyde (2 mmol), 4-bromoacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 82% yield. IR (KBr) v / cm -1 : 3503,3395, 3367, 2210, 1607, 1584, 1570, 1549, 1519, 1366, 1282, 1262, 1009, 816; 1 H NMR (300 MHz, DMSO- d 6 ) δ / ppm: 9.91 (s, 1H, OH), 8.08 (d, 2H, ArH, J= 8.1Hz), 7.68 (d, 2H, ArH, J = 8.1 Hz), 7.55 (d, 2H, ArH, J = 8.4 Hz), 7.25 (s, 1H,H5), 6.90-6.93 (m, 4H, ArH, NH2); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 161.4,159.5, 157.5, 155.4, 137.4, 132.0, 130.4, 129.7, 127.8, 124.1, 117.8, 116.0,109.3, 87.1; MS (ESI) m / z 366.0 [M+H] + Anal. Calculated for C 18 H 12 BrN3O: C, 59.03; H, 3.30; N, 11.47; found: C, 59.15; H, 3.37; N, 11.41. Example 17 4-(4-hydroxy-3,5-dimethoxyphenyl)-6-p-hydroxyphenyl-2-amino-3-cyanopyridine (w5)

[0063] Syringaldehyde (2 mmol), 4-hydroxyacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120°C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 80% yield. IR (KBr) v / cm -1 : 3483, 3366,3222, 2216, 1625, 1556, 1518, 1448, 1414, 1244, 1114, 773, 706; 1 H NMR (300MHz, DMSO- d 6 ) δ / ppm: 9.92, 8.88 (2s, 2H, 2×OH), 8.01 (d, 2H, ArH, J= 8.1 Hz),7.19 (s, 1H, H5), 6.95 (s, 2H, ArH), 6.85 (d, 2H, ArH, J = 8.1 Hz), 6.80 (s,2H, NH2), 3.85 (s, 6H, 2×OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.9, 159.4,158.3, 154.6, 147.8, 137.1, 128.9, 128.5, 126.8, 117.8, 115.3, 108.0, 106.1,85.1, 56.1; MS (ESI) m / z 364.0 [M+H] + Anal. Calculated for C 20 H 17 N3O4: C, 66.11; H, 4.72; N, 11.56; found: C, 66.00; H, 4.78; N, 11.61. Example 18 4-(4-hydroxy-3-methoxyphenyl)-6-p-hydroxyphenyl-2-amino-3-cyanopyridine (w6)

[0064] Vanillin (2 mmol), 4-hydroxyacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 78% yield. IR (KBr) v / cm -1 : 3496, 3409,2208, 1607, 1577, 1544, 1513, 1278, 1259, 1224, 1170, 835; 1 H NMR (300 MHz, DMSO- d 6 ) δ / ppm: 9.90, 9.50 (2s, 2H, 2×OH), 8.90 (d, 2H, ArH, J= 8.7 Hz), 7.24(s, 1H, H5), 7.11-7.16 (m, 2H, ArH), 6.79-6.93 (m, 5H, ArH, NH2), 3.86 (s,3H, OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.9, 159.4, 158.3, 154.4, 148.1,147.5, 128.9, 128.5, 128.0, 121.3, 117.7, 115.5, 115.3, 112.5, 107.9, 85.0,55.7; MS (ESI) m / z 334.0 [M+H] + Anal. Calculated for C 19 H 15 N3O3: C, 68.46; H, 4.54; N, 12.61; found: C, 68.41; H, 4.58; N, 12.67. Example 19 4,6-Di-p-hydroxyphenyl-2-amino-3-cyanopyridine (w7)

[0065] p-Hydroxybenzaldehyde (2 mmol), 4-hydroxyacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 82% yield. IR (KBr) v / cm -1 : 3482,3382, 2212, 1638, 1611, 1578, 1549, 1518, 1450, 1374, 1280, 1230, 1171, 823; 1 H NMR (300 MHz, DMSO- d 6 ) δ / ppm: 9.86 (s, 2H, 2×OH), 7.98 (d, 2H, ArH, J = 8.7Hz), 7.52 (d, 2H, ArH, J= 8.7 Hz), 7.10 (s, 1H, H5), 6.90 (d, 2H, ArH, J = 8.7Hz), 6.84 (d, 2H, ArH, J = 8.7 Hz), 6.75 (s, 2H, NH2); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.9, 159.4, 158.8, 158.4, 154.3, 129.7, 128.8, 128.5, 127.6,117.6, 115.4, 115.3, 107.8, 84.9; MS (ESI) m / z 304.2 [M+H] + Anal. Calcd for C 18 H 13 N3O2: C, 71.28; H, 4.32; N, 13.85; found: C, 71.21; H, 4.37; N, 13.77. Example 20 4-(4-hydroxy-3,5-dimethoxyphenyl)-6-(4-hydroxy-3-methoxyphenyl)-2-amino-3-cyanopyridine (w1)

[0066] Syringaldehyde (2 mmol), 4-hydroxy-3-methoxyacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 79% yield. IR (KBr) v / cm -1 :3504, 3482, 3316, 2204, 1630, 1577, 1514, 1313, 1256, 1217, 1116, 813; 1 H NMR (300 MHz, DMSO-) d 6 ) δ / ppm: 7.71 (s, 1H, ArH), 7.64 (d, 1H, ArH, J= 8.4 Hz),7.24 (s, 1H, H5), 6.95 (s, 2H, ArH), 6.88 (d, 1H, ArH, J = 8.4 Hz), 3.85, 3.86(3s, 9H, 3×OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.6, 158.3, 148.6,147.7, 147.5, 136.8, 128.9, 126.8, 120.7, 117.7, 115.3, 111.1, 108.2, 106.1,85.1, 56.1, 55.7; MS (ESI) m / z 394.2 [M+H] + Anal. Calculated for C 21 H 19 N3O5: C, 64.12; H, 4.87; N, 10.68; found: C, 64.19; H, 4.82; N, 10.61. Example 21 4,6-Di(4-hydroxy-3-methoxyphenyl)-2-amino-3-cyanopyridine (w2)

[0067] Vanillin (2 mmol), 4-hydroxy-3-methoxyacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 82% yield. IR (KBr) v / cm -1 :3506, 3448, 3365, 2206, 1628, 1581, 1544, 1515, 1275, 1258, 1134, 827; 1 H NMR (300 MHz, DMSO-) d 6 ) δ / ppm: 7.71 (s, 1H, ArH), 7.63 (d, 1H, ArH, J= 8.1 Hz),7.25 (s, 1H, H5), 7.22 (s, 1H, ArH), 7.14 (d, 1H, ArH, J = 8.4 Hz), 7.94 (d, 1H, ArH, J = 8.1 Hz), 6.88 (d, 1H, ArH, J = 8.4 Hz), 3.86 (s, 6H, 2×OCH3); 13 CNMR (75.5 MHz, DMSO- d 6 ) δ / ppm: 160.6, 158.3, 154.4, 148.6, 147.8, 147.5,147.4, 128.9, 127.9, 121.2, 120.7, 117.7, 115.4, 115.3, 112.4, 111.1, 108.2,85.1, 55.7; MS (ESI) m / z 364.2 [M+H] + 386.2 [M+Na] + Anal. Calcd for C 20 H 17 N3O4: C, 66.11; H, 4.72; N, 11.56; found: C, 66.23; H, 4.64; N, 11.51. Example 22 4-(4-hydroxyphenyl)-6-(4-hydroxy-3-methoxyphenyl)-2-amino-3-cyanopyridine (w3)

[0068] p-Hydroxybenzaldehyde (2 mmol), 4-hydroxy-3-methoxyacetophenone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 81% yield. IR (KBr) v / cm -1 : 3336, 3198, 2211, 1633, 1575, 1514, 1361, 1260, 1229, 1039, 825; 1 H NMR (300 MHz, DMSO-) d6 ) δ / ppm: 7.69 (d, 1H, ArH, J = 1.5 Hz), 7.62 (d, 1H, ArH, J =8.4 Hz), 7.54 (d, 2H, ArH, J = 8.7 Hz), 7.17 (s, 1H, H5), 6.93 (d, 2H, ArH, J =8.7 Hz), 6.87 (d, 1H, ArH, J = 8.4 Hz), 3.86 (s, 3H, OCH3); 13 C NMR (75.5 MHz, DMSO- d 6 ) δ MS (ESI) m / z 334.2 [M+H] + 356.2 [M+Na] + Anal. Calculated for C 19 H 15 N3O3: C, 68.46; H, 4.54; N, 12.61; found: C, 68.42; H, 4.61; N, 12.68. Example 23 4-(4-hydroxy-3,5-dimethoxyphenyl)-5-methyl-6-phenyl-2-amino-3-cyanopyridine (w27)

[0069] Syringaldehyde (2 mmol) obtained from the oxidative degradation of lignin was dissolved in 1,4-dioxane with phenylacetone (2 mmol) and malononitrile (2 mmol). Ammonium acetate (4 mmol) was then added, and the mixture was heated to 120°C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to give 4-(4-hydroxy-3,5-dimethoxyphenyl)-5-methyl-6-phenyl-2-amino-3-cyanopyridine in 75% yield. IR (KBr) v / cm -1: 3483, 3366, 3222,2970, 2938, 1626, 1604, 1556, 1518, 1448, 1245, 1114, 723, 706; 1 H NMR (300MHz, DMSO- 6d ) δ / ppm: 7.40-7.54 (m, 5H, ArH), 6.67 (s, 2H, ArH), 3.80 (s, 6H, 2×OCH3), 1.92 (s, 3H, CH3); 13 C NMR (75.5 MHz, DMSO- 6d ) δ / ppm: 161.7, 158.4,156.1, 148.3, 140.5, 136.2, 129.2, 128.8, 128.4, 127.3, 117.7, 117.4, 106.7,90.0, 56.7, 17.4; MS (ESI) m / z 362.1 [M+H] + , 384.1 [M+Na] + Anal. Calcd for C 21 H 19 N3O3: C, 69.79; H, 5.30; N, 11.63; found: C, 69.71; H, 5.35; N, 11.74. Example 244 - (4-hydroxy-3-methoxyphenyl)-5-methyl-6-phenyl-2-amino-3-cyanopyridine (w28)

[0070] Vanillin (2 mmol) obtained from the oxidative degradation of lignin was dissolved in 1,4-dioxane with acetone (2 mmol) and malononitrile (2 mmol). Ammonium acetate (4 mmol) was then added, and the mixture was heated to 120°C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to give 4-(4-hydroxy-3-methoxyphenyl)-5-methyl-6-phenyl-2-amino-3-cyanopyridine in 72% yield. IR (KBr) v / cm -1 : 3477, 3349, 3226,2222, 1631, 1558, 1515, 1448, 1271, 1244, 1216, 1121, 776;1 H NMR (300 MHz, DMSO- 6d ) δ / ppm: 7.42-7.52 (m, 5H, ArH), 6.96 (d, 1H, ArH, J = 1.8 Hz), 6.91(d, 1H, ArH, J = 7.8 Hz), 6.91 (dd, 1H, ArH, J = 1.8, 7.8 Hz), 3.80 (s, 3H,OCH3), 1.89 (s, 3H, CH3); 13 C NMR (75.5 MHz, DMSO- 6d ) δ / ppm: 161.7, 158.4,155.9, 147.9, 147.3, 140.5, 129.2, 128.8, 128.4, 128.3, 121.7, 117.7, 117.4,115.8, 113.2, 90.0, 56.3, 17.3; MS (ESI) m / z 332.0 [M+H] + , 354.0 [M+Na] + 370.0 [M+K] + Anal. Calculated for C 20 H 17 N3O2: C, 72.49; H, 5.17; N, 12.68; found: C, 72.41; H, 5.29; N, 12.60. Example 25 4-(4-hydroxyphenyl)-5-methyl-6-phenyl-2-amino-3-cyanopyridine (w29)

[0071] p-hydroxybenzaldehyde (2 mmol) obtained from the oxidative degradation of lignin was dissolved in 1,4-dioxane with acetone (2 mmol) and malononitrile (2 mmol). Ammonium acetate (4 mmol) was then added, and the mixture was heated to 120°C for 20 min using microwave-assisted heating (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to give 4-(4-hydroxyphenyl)-5-methyl-6-phenyl-2-amino-3-cyanopyridine in 73% yield. IR (KBr) v / cm -1: 3447, 3330, 3224, 2212,1644, 1557, 1486, 1408, 1301, 770, 712; 1 H NMR (300 MHz, DMSO- 6d ) δ / ppm: 7.67-7.71 (m, 2H, ArH), 7.42-7.58 (m, 3H, ArH), 7.38 (d, 2H, ArH, J = 8.1 Hz), 7.16(d, 2H, ArH, J = 8.1 Hz), 1.90 (s, 3H, CH3); 13 C NMR (75.5 MHz, DMSO- 6d ) δ / ppm:165.4, 161.9, 159.4, 151.0, 138.2, 130.5, 129.0, 128.9, 128.6, 128.5, 117.2,115.5, 115.4, 81.5, 16.9; MS (ESI) m / z 302.1 [M+H] + , 324.0 [M+Na] + 340.0 [M+K] + Anal. Calculated for C 19 H 15 N3O: C, 75.73; H, 5.02; N, 13.94; found: C, 75.79; H, 5.21; N, 14.00. Example 26 4-(4-hydroxy-3,5-dimethoxyphenyl)-5,6-diphenyl-2-amino-3-cyanopyridine (w8)

[0072] Syringaldehyde (2 mmol), phenylethyl ketone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120°C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 64% yield. IR (KBr) v / cm -1: 3480, 3406,3282, 3136, 2211, 1627, 1548, 1516, 1453, 1414, 1227, 1118, 717, 705; 1 H NMR (300 MHz, DMSO-) 6d ) δ / ppm: 8.53 (s, 1H, OH), 6.36-7.17 (m, 14H, ArH, NH2), 3.53(s, 6H, 2×OCH3); 13 C NMR (75.5 MHz, DMSO- 6d ) δ / ppm: 160.0, 159.0, 154.8, 147.2,140.0, 137.4, 135.4, 131.4, 129.3, 127.7, 127.4, 127.3, 126.1, 124.1, 116.8,107.4, 84.8, 55.8; MS (ESI) m / z 424.1 [M+H] + , 446.2 [M+Na] + Anal. Calcd for C 26 H 21 N3O3: C, 73.74; H, 5.00; N, 9.92; found: C, 73.86; H, 5.15; N, 9.80. Example 27 4-(4-hydroxy-3-methoxyphenyl)-5,6-diphenyl-2-amino-3-cyanopyridine (w14)

[0073] Vanillin (2 mmol), phenylethyl ketone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 66% yield. IR (KBr) v / cm -1 : 3471, 3382,3290, 3124, 2216, 1633, 1547, 1518, 1460, 1279, 1234, 1028, 707; 1 H NMR (300MHz, DMSO- 6d )δ / ppm: 9.12 (s, 1H, OH), 6.54-7.17 (m, 15H, ArH, NH2), 3.56 (s,3H, OCH3), 1.89 (s, 3H, CH3); 13 C NMR (75.5 MHz, DMSO- 6d ) δ / ppm: 160.5, 159.5,155.3, 147.1, 146.9, 140.5, 137.8, 132.0, 129.8, 128.2, 127.9, 127.8, 126.6,124.7, 122.5, 117.2, 115.4, 114.2, 89.9, 55.8; MS (ESI) m / z 394.1 [M+H] + 416.1 [M+Na] + Anal. Calculated for C 25 H 19 N3O2: C, 76.32; H, 4.87; N, 10.68; found: C, 76.41; H, 4.80; N, 10.63. Example 28 4-(4-hydroxyphenyl)-5,6-diphenyl-2-amino-3-cyanopyridine (w15)

[0074] p-Hydroxybenzaldehyde (2 mmol), phenylethyl ketone (2 mmol), and malononitrile (2 mmol) were dissolved in 1,4-dioxane, and then ammonium acetate (4 mmol) was added. The mixture was heated to 120 °C for 20 min with microwave assistance (300 W). Ice water was added to the reaction mixture to precipitate the product, which was then recrystallized to obtain the product in 63% yield. IR (KBr) v / cm -1 : 3475,3360, 3206, 2212, 1611, 1550, 1513, 1450, 1273, 1230, 1110, 865, 703; 1 H NMR (300 MHz, DMSO-) 6d ) δ / ppm: 9.51 (s, 1H, OH), 6.80-7.22 (m, 14H, ArH, NH2), 6.60 (d, 2H, ArH, J = 8.4 Hz);13 C NMR (75.5 MHz, DMSO- 6d ) δ / ppm: 160.0, 158.9,157.1, 155.0, 140.0, 137.2, 131.5, 130.3, 129.3, 127.7, 127.3, 127.2, 127.1,126.1, 124.4, 116.6, 114.6, 89.5; MS (ESI) m / z 364.3 [M+H] + Anal. Calcd for C 24 H 17 N3O: C, 79.32; H, 4.72; N, 11.56; found: C, 79.21; H, 4.79; N, 11.50. Example 29 Comparison of the in vitro anticoronavirus efficacy of the compounds of the present invention 1. Cells and Reagents: Vero cells were purchased from Wuhan Institute of Cell Biology; Molnupiravir (S2597) was purchased from Selleck Biotechnology Co., Ltd. The SARS-CoV-2 strain was the Omeprone XBB isolate. DMEM medium (Catalog No.: KGM12800N-500, 500mL) was purchased from Nanjing Kaiji Biotechnology Co., Ltd.; FBS (Catalog No.: FSP500, 500mL) was purchased from Suzhou Excell Biotechnology Co., Ltd.; penicillin-streptomycin solution (100×) (Catalog No.: C0222, 100mL) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; NP (Catalog No.: #33336, 100μL) was purchased from Cellsignaling Technology Co., Ltd.; FITC-labeled goat anti-mouse (Catalog No.: 33207ES60, 100μL) was purchased from Cellsignaling Technology Co., Ltd. 100 μL of the following reagents were purchased from Shanghai Yisheng Biotechnology Co., Ltd.: Hoechst staining kit (Catalog No.: C0003, specification: 100 times), β-Actin (ACTB) primary antibody (Catalog No.: AA128-1, specification: 100 μL), RIPA lysis buffer (medium) (Catalog No.: P0013C, specification: 100 mL), and SDS-PAGE protein loading buffer (5X) (Catalog No.: P0015L, specification: 15 mL) were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.: HRP-goat anti-mouse IgG secondary antibody (Catalog No.: BF03001, specification: 100 μL) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.: ECL chemiluminescence kit (Catalog No.: E422-02, specification: 500 mL) were purchased from Nanjing Novizan Biotechnology Co., Ltd.: Vero cell culture medium: 10% FBS DMEM medium + 1% penicillin-streptomycin (100×) solution. Vero cell maintenance medium: 2% FBS in DMEM / F12 medium + 1% penicillin-streptomycin (100×) solution.

[0075] 2. Methods 2.1 MTT Testing Method Vero cells in the logarithmic growth phase were divided into groups of 2.0 × 10⁻⁶. 4 Cells were evenly seeded per well in 96-well plates and cultured overnight. The test drug (w01-w29) was diluted to a working concentration of 10 μM with cell maintenance medium. Cells without the drug served as a negative control. After drug administration, cells were cultured for another 48 h, followed by incubation with 5 mg / mL MTT for 2-4 h. The supernatant was then removed, and 150 μL of DMSO was added to dissolve the MTT metabolic crystals. The OD value of each well was measured at 490 nm using a microplate reader. The inhibition rate of cell proliferation at a 10 μM concentration of the drug was calculated based on the OD values.

[0076] 2.2 CPE Inhibition Assay (CPE) (1) Vero cells in the logarithmic growth phase were seeded into 96-well plates. When the cell density reached 80%, infection was performed. A normal control group, a SARS-CoV-2 infection control group, a test drug group (w01-w29), and a positive drug Molnupiravir control group were set up. (2) Except for the normal control group, each well was filled with 100 TCID45. 50 (3) 200 μL of cell maintenance medium was added to the normal control group, 200 μL of cell maintenance medium containing 1 / 1000 DMSO was added to the virus infection control group, 200 μL of cell maintenance medium containing 1 / 1000 DMSO was added to each well of the test drug group, and 200 μL of cell maintenance medium containing 2 μM Molnupiravir was added to each well of the positive drug control group. After 72 hours of drug treatment, the morphological changes of cells were observed and photographed under an inverted microscope. (4) An appropriate amount of paraformaldehyde or formalin was added to fix the cell culture plate to inactivate the virus. After drying, the plate was stained with crystal violet and photographed to observe the cytopathic effect. Finally, an appropriate volume of acetic acid was added to dissolve the crystal violet, and the OD value of each well was measured at a wavelength of 490 nm. The inhibition rate of the drug against the replication of the novel coronavirus was calculated based on the OD value. The inhibition rate test of other coronaviruses was also carried out in accordance with this protocol, and the virus infection dose was 100 TCID. 50 .

[0077] 2.3 Immunofluorescence detection method (A) Cell Culture: Vero cells in logarithmic growth phase were seeded in 96-well plates. When the cell density reached 80%, infection was initiated. The following groups were set up: a normal control group, a SARS-CoV-2 infection control group, a test drug group (w01-w29), and a positive drug Molnupiravir control group. Except for the normal control group, each well contained 100 mg TCID50. 50(A) Adsorption of coronavirus dilution solution at 4℃ for 1 h, followed by discarding the virus solution after 1 h. 200 μL of cell maintenance medium was added to the normal control group, 200 μL of cell maintenance medium containing 1 / 1000 DMSO was added to the virus infection control group, 200 μL of cell maintenance medium diluted to a working concentration of 10 μM was added to each well of the test drug group, and 200 μL of cell maintenance medium containing 2 μM Molnupiravir was added to the positive drug control group; (B) Fixation: After culturing cells from step (A) for 23 h, 100 μL of formalin was added to each well for fixation for 30 min; (C) Permeabilization: 100 μL of 0.5% Triton X-100 was added to each well for permeabilization at room temperature for 1 h; (D) Blocking: 50 μL of 5% FBS was added to each well for blocking at room temperature for 1 h, followed by washing the cells 3 times with PBS; (E) Primary antibody incubation: 50 μL of primary antibody was added to each well. NP antibody, incubated overnight at 4°C, and washed 3 times with PBS; (F) Secondary antibody incubation: FITC-labeled goat anti-mouse antibody, 50 μL per well, incubated at 4°C in the dark for 2 h; (G) Add 100 μL of Hoechst to each well and stain for 10-15 minutes; (H) Observe under a fluorescence microscope.

[0078] 3. Experimental Results 3.1 Cytotoxicity test results of the compounds described in this invention (Examples 2-28) on Vero cells like Figure 1 As shown, the MTT assay results indicate that, except for w17 and w25, the compounds w01-w29 described in this invention showed no significant cytotoxicity to Vero cells at a concentration of 10 μM, indicating that these compounds do not have significant cytotoxicity to Vero cells at this concentration.

[0079] 3.2 Results of in vitro anti-SARS-CoV-2 tests of the compounds of the present invention (Examples 2-28) like Figure 2 As shown, CPE results indicate that, compared to the virus control group, the compounds described in Examples 2-28 of this invention exhibit better resistance to 100 TCID50 at a concentration of 10 μM. 50 The compounds exhibit a certain protective effect against cytopathic effects caused by coronavirus infection, with w08, w09, w10, w11, w15, w19, w27, w28, and w29 showing the best protective effects, with a protective efficiency of over 50% against coronavirus-induced cytopathic effects. These results indicate that the compounds in the embodiments of this invention possess anti-SARS-CoV-2 pharmacological activity.

[0080] 3.3 Results of the in vitro inhibition of SARS-CoV-2 replication by the compounds of the present invention (Examples 2-28) Immunofluorescence results as follows Figure 3As shown, compared with the normal group, the number of green positive cells in the virus-infected control group was significantly increased. Compared with the virus-infected control group, the number of green positive cells in the compounds of the present invention (Examples 2-28) at a concentration of 10 μM was significantly reduced, indicating that the above compounds have anti-SARS-CoV-2 activity.

[0081] Example 30: Study on the mechanism of action of the preferred compound w09 against coronaviruses 1. Cells and reagents RNA isolater (R401-01) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; formaldehyde aqueous solution (CAS: 50-00-0, batch number: 20190606) was purchased from Sinopharm Chemical Reagent Co., Ltd. Other cells and reagents used in this example are the same as in Example 29.

[0082] 2. Test Methods 2.1 Western blot experiment (A) Cell culture: Vero cells were cultured at a rate of 1 × 10⁻⁶. 6 Cells were seeded in 6-well plates and infected when the cell density reached 80%. The plates included a normal control group, a SARS-CoV-2 infection control group, high-, medium-, and low-dose w09 groups (10 μM, 2 μM, and 0.5 μM), and a Molnupiravir positive control group. Except for the normal control group, each well contained 100 TCID50. 50 The virus dilution solution was incubated at 4℃ for 1 h, and the virus solution was discarded after 1 h. 2 mL of cell maintenance medium was added to the normal control group, 2 mL of cell maintenance medium containing 1 / 1000 DMSO was added to the virus infection control group, 2 mL of cell maintenance medium containing 1 / 1000 DMSO was added to the high, medium and low dose groups of w09, respectively, and 2 mL of cell maintenance medium containing 10 μM, 2 μM and 0.5 μM of w09 were added to the positive control group of Molnupiravir, respectively. 2 mL of cell maintenance medium containing 2 μM Molnupiravir was added to the Molnupiravir positive control group. After culturing for 23 h, the cell supernatant of each group was collected for virus titer detection, and then total protein was extracted.

[0083] (B) Protein extraction: Wash the 6-well plate once with pre-cooled PBS, then add 100 μL of RIPA lysis buffer and 25 μL of SDS-PAGE protein loading buffer (5×) to each well. Place the plate on ice to allow the cells to fully lyse, collect the lysis buffer into EP tubes, boil in a metal bath at 100°C for 10 min, and store in a -20°C freezer for later use.

[0084] (C) Preparation of SDS-PAGE gel: Prepare the upper and lower gels according to the following plan, let them stand at room temperature for 1 hour, and then load the sample after the gel has solidified.

[0085] Name 12% Separating Gel 5% Stacking Gel <![CDATA[H2O]]> 2.64 mL 2.1 mL 30% Acrylamide 3.2 mL 0.5 mL 1M Tris-HCl, pH6.8 0 mL 0.38 mL 1M Tris-HCl, pH8.8 2 mL 0 mL 10% SDS 80 μL 30 μL 10% APS 80 μL 30 μL TEMED 3.2 μL 4 μL (D) Add the protein sample extracted in step (B) to an SDS-PAGE gel containing 12% to separate the proteins. Run electrophoresis at 80V for 30 minutes, then change to 120V until the target bands are completely separated before transferring to a membrane.

[0086] (E) After electrophoresis, cut off the excess gel and cut a nitrocellulose membrane (NC membrane) of the same size. Soak the NC membrane and the transfer filter paper in the wet transfer solution for a few minutes to fully wet them. Gently remove the air bubbles between the filter paper and the NC membrane. Transfer at a constant current of 400mA for 30 minutes. After the transfer is completed, remove the NC membrane and seal it with 5% skim milk for 2 hours.

[0087] (F) Primary antibody incubation: The membrane was then incubated overnight at 4°C with NP or ACTB primary antibody dilution (1:1000 dilution).

[0088] (G) Secondary antibody incubation: After the primary antibody incubation, the membrane was washed three times with TBST for 10 min each time, and then incubated at room temperature for 1 h with HRP-goat anti-mouse IgG secondary antibody dilution solution (1:2000 dilution).

[0089] (H) Development: After incubation with secondary antibody, the protein was washed three times with TBST, and finally developed using an ECL chemiluminescence kit. The obtained protein bands were scanned in grayscale using Quantity One image analysis software (version Quantity One 4.6.2, Bio-Rad) to obtain the relative expression level of the target protein.

[0090] 2.2 RNA-seq detection To further clarify the mechanism of action of w09 against coronavirus, the effect of w09 in inhibiting intracellular SARS-CoV-2 genome transcription and replication was detected using RNA-seq. The specific operation method is as follows: (A) Cell Culture: Vero cells were cultured according to Experimental Method 2.1(A) of Example 30. (B) Cell Lysis: 1 mL of RNA isolater was added to each well, and the cells were lysed by pipetting. The cell lysate was collected into RNA-free EP tubes. (C) Sample Submission. (D) Data Analysis.

[0091] 2.4 TCID 50 test Vero cells in the logarithmic growth phase were seeded into 96-well plates. When the cell density reached 80%, infection was performed. The plates were washed twice with PBS, and the cell supernatants collected in the Western blot experiment of Example 30 were diluted 10-fold with DMEM serum-free medium. -110 -2 …10 -8 50 μL of virus solution was added to each well of a 96-well plate, with three parallel wells per group, and incubated at 4°C. After adsorption with 5% CO2 for 1 h, the virus solution was discarded, and 200 μL of Vero cell maintenance medium was added to each well. The plate was then incubated at 37°C with 5% CO2 for another 72 h. Cytopathic effects were observed and recorded daily. After 72 h, the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The viral load in the supernatant was calculated using the Reed-Muench method.

[0092] 2.5 CPE inhibition assay (cell pathogenic effect, CPE): (1) Vero cells in logarithmic growth phase were seeded into 96-well plates. When the cell density reached 80%, infection was performed. Normal control group, virus infection control group (CCV, PEDV, MHV, IBV, OC43, SARS-CoV-2), w09 10 μM drug administration group, and positive drug Molnupiravir control group were set up. (2) Except for the normal control group, each well was filled with 100 TCID35. 50 CCV, PEDV, MHV, IBV, OC43, and SARS-CoV-2 coronavirus dilutions were prepared, with each virus having 3 replicates. The virus was adsorbed at 4°C for 1 h, and then the virus solution was discarded. Subsequent procedures are described in (3) and (4) of Experimental Method 2.2 in Example 29.

[0093] 3. Experimental Results 3.1 Broad-spectrum anti-coronavirus effect of w09 like Figure 4 As shown, w09 at a concentration of 10 μL exhibited inhibition rates of over 80% against coronaviruses such as CCV, PEDV, MHV, IBV, OC43, and SARS-CoV-2, indicating that w09 has the potential to have broad-spectrum anti-coronavirus activity.

[0094] 3.2 The role of w09 in inhibiting SARS-CoV-2 NP protein expression like Figure 5 As shown, in a virus-infected Vero cell model, w09 can dose-dependently inhibit the expression of coronavirus NP protein, indicating that w09 has the effect of inhibiting coronavirus NP protein expression, further demonstrating the anti-coronavirus potential of w09.

[0095] 3.3 w09 inhibits the transcriptional replication of the SARS-CoV-2 genome in cells. like Figure 6As shown, RT-qPCR was used to quantitatively detect and analyze the viral genome copy number in the virus-infected group and the w09-treated group. The results showed that the w09 treatment effectively inhibited the transcriptional replication of SARS-CoV-2 viral RNA in a dose-dependent manner, indicating that w09 may inhibit the replication of SARS-CoV-2 at the RNA level.

[0096] 3.4 WO9 inhibits the release of newly generated coronavirus virus like Figure 7 As shown, compared with the virus control group, the w09 treatment group was able to inhibit the release of newly generated virus in a dose-dependent manner, indicating that w09 has the effect of inhibiting the release of newly generated SARS-CoV-2 virus, which further verifies the above results. Example 31: Mechanism of action of w09 in inhibiting coronavirus replication

[0097] 1. Cells and reagents The cells, virus strains, and reagents used in this embodiment are the same as in Example 30.

[0098] 2. Western blot experiments with different drug administration times To investigate the mechanism of action of w09 against coronavirus, different dosing regimens were used to explore the potential stages of w09's inhibition of coronavirus replication lifecycle. Based on the results of Example 30, w09 at doses of 10 μM and 2 μM showed strong inhibitory effects on coronavirus. Therefore, using these doses, three dosing regimens were designed to explore the potential stages of w09's inhibition of coronavirus replication lifecycle: administration 2 hours before infection (adsorption), drug treatment 1 hour after infection followed by replacement with drug-free cell maintenance medium (entry), and administration 2 hours after infection (replication). The specific experimental procedures are as follows: Cells were loaded at a rate of 1×10 6Cells were seeded in 6-well plates. When the cell density reached 80%, the following three administration methods were used: Adsorption: Before cell infection, WO9 was added for pretreatment for 2 h. Then, WO9 and virus dilution were applied to the cells simultaneously, and adsorption was carried out at 4°C for 1 h. The drug-containing medium was then discarded, and cell maintenance medium without drugs was added for 24 h of further culture. Entry: Virus dilution was added to the cells, and adsorption was carried out at 4°C for 1 h. The virus solution was discarded, and culture medium containing WO9 was added for 1 h of further culture. Then, cell maintenance medium without drugs was added for 23 h of further culture. Replication: An appropriate amount of virus dilution was added, and adsorption was carried out at 4°C for 1 h. The virus solution was discarded, and after 2 h, cell maintenance medium containing WO9 was added for 22 h of further culture. Subsequently, the cells were pretreated for 6 h before reinfection and then treated for 2 h, 4 h, and 6 h after infection, respectively. After 24 h of further culture, total protein was extracted. Subsequent protein extraction and development procedures were performed in accordance with the method described in Example 30.

[0099] 3. Experimental Results Experimental results: Western blot results are as follows Figure 8 As shown in the results of different drug administration times, although the expression levels of viral NP proteins during the adsorption and entry phases were significantly lower than those in the viral infection group, the effect of administration during the replication phase was significantly stronger than that of administration during the adsorption and entry phases, suggesting that w09 may mainly exert its efficacy during the viral replication phase. To further verify the above results, four modes were used to investigate the effect of w09 on inhibiting SARS-CoV-2 viral replication: pretreatment administration for 6 hours followed by reinfection, and administration at 2 hours, 4 hours, and 6 hours post-infection. The results showed that w09 could still effectively inhibit SARS-CoV-2 replication 6 hours post-infection, again indicating that w09 mainly plays a role during the viral replication phase.

[0100] Thus, this invention provides the first systematic demonstration that compounds having general formula (I) possess anti-coronavirus activity, primarily through inhibiting viral RNA transcription, viral protein expression, and the release of newly generated viruses.

[0101] Therefore, unlike previous reports, this invention has found that 2-aminonicotinic nitrile compounds with general formula (I) can significantly inhibit coronavirus replication, RNA transcription, viral NP protein expression, and the release of new viruses, especially showing significant efficacy against the Omeprone variant. Mechanistic studies indicate that this compound mainly acts on the viral invasion and replication / translation stages. Therefore, this invention proposes the use of 2-aminonicotinic nitrile compounds to prepare novel anti-coronavirus drugs, which has broad application prospects.

[0102] In summary, the application of the 2-aminonicotinic nitrile compounds, their salts, and compositions of the present invention in the preparation of anti-coronavirus drugs demonstrates that the 2-aminonicotinic nitrile compounds possess anti-coronavirus activity, inhibiting coronavirus replication, suppressing RNA transcription and protein expression, and inhibiting the release of newly generated viruses. Furthermore, they primarily act on the translation and replication stages of the virus, making them suitable for the preparation of anti-COVID-19 drugs and demonstrating promising application prospects, making them suitable for large-scale promotion and application.

[0103] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, the specification and drawings should be considered illustrative rather than restrictive.

Claims

1. The use of 2-aminonicotinic nitrile compounds, their salts, and compositions in the preparation of broad-spectrum anticoronavirus drugs, wherein the 2-aminonicotinic nitrile compounds are compounds having the following general formula (I): （I） in, R1, R2, and R3 are independently H, Ar, OH, CH3, or OMe; R4 and R5 are independently H, OH, OMe, or Ar, where Ar is a halogen group; R6 is H, OH, OMe, Cl, Ph, or CH3.

2. The application according to claim 1, characterized in that, R1 is preferably OH.

3. The application according to claim 2, characterized in that, R2 and R3 are preferably OMe, R4 is preferably H, R5 is preferably Ar, and R6 is preferably H.

4. The application according to claim 1, characterized in that, The 2-aminonicotinonitrile compound is selected from one of the following combinations of compounds: 。 5. The application according to claim 1, characterized in that, The aforementioned broad-spectrum anti-coronavirus drug is used to inhibit the transcriptional replication of coronavirus RNA and / or the expression of coronavirus proteins.

6. The application according to claim 1, characterized in that, The aforementioned broad-spectrum anti-coronavirus drug is used to inhibit the release of newly generated coronavirus virus.

7. The application according to claim 1, characterized in that, The coronavirus is selected from one or more of canine coronavirus CCV, porcine epidemic diarrhea virus PEDV, mouse hepatitis virus MHV, chicken infectious bronchitis virus IBV, novel coronavirus SARS-CoV-2, and human common coronaviruses OC43, HCoV-NL63, and HCoV-229E.

8. The use of the composition of any one of the 2-aminonicotinic nitrile compounds according to claims 1-4 in the preparation of a broad-spectrum anticoronavirus drug, characterized in that, The composition comprises two or more compounds of general formula (I) or the composition further comprises other components for enhancing the anticoronavirus activity of the compounds of general formula (I).

9. The use of the composition of the 2-aminonicotinic nitrile compound according to claim 8 in the preparation of a broad-spectrum anticoronavirus drug, characterized in that, The composition also includes a pharmaceutically acceptable carrier or excipient.

10. The use of the composition of the 2-aminonicotinic nitrile compound according to claim 8 in the preparation of a broad-spectrum anticoronavirus drug, characterized in that, The anticoronavirus pharmaceutical composition is intended to be formulated into any of the following dosage forms: tablets, pills, powders, capsules, granules, injections, and oral liquids.

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