Sulfadimidine neuraminidase inhibitors, processes for their preparation and use

Abstract

The present application relates to a kind of sulfadimidine neuraminidase inhibitors and its preparation and application, the preparation method of the inhibitor is specifically (1a) sulfadimidine, monoethyl ester acyl chloride and anhydrous potassium carbonate are reacted, and after treatment, the intermediate of formula (II) is obtained;(2a) the intermediate of formula (II) is dissolved in organic solvent, after adding potassium hydroxide, the intermediate of formula (III) is obtained;(3a) the intermediate of formula (III) is dissolved in organic solvent, after adding substituted aniline, HOBT and EDCI, the inhibitor is obtained after treatment;(1b) sulfadimidine, chloroacetyl chloride and anhydrous potassium carbonate are reacted, and the intermediate of formula (IV) is obtained;(2b) the intermediate of formula (IV) is dissolved in organic solvent, after adding substituted mercaptan or thiophenol and anhydrous potassium carbonate, the inhibitor is obtained after treatment.Compared with prior art, the structure of the inhibitor of the present application is novel, has good neuraminidase inhibitory activity, and can be used for preparing the drug for inhibiting neuraminidase activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a sulfadimidine neuraminidase inhibitor and a preparation method and application thereof. BACKGROUND

[0002] Neuraminidase is a glycoprotein distributed on the envelope of influenza virus, which hydrolyzes the glycosidic bond between sialic acid on the surface of host cells and glycoprotein when the virus infects, promotes the release of mature virus from the surface of infected cells to continue to infect new cells. Meanwhile, it can prevent the aggregation of progeny virus particles released from host cells, and can also hydrolyze sialic acid in respiratory mucosa to prevent the inactivation of progeny virus particles and promote the spread of virus in the respiratory tract, thus playing an important role in the replication and infection cycle of the virus. Therefore, neuraminidase is one of the important targets for the research and design of anti-influenza virus drugs.

[0003] At present, only 6 anti-influenza virus drugs have been approved by the US FDA, including 2 M2 proton channel inhibitors (amantadine and rimantadine), 3 neuraminidase inhibitors (zanamivir, oseltamivir and peramivir), and 1 Cap-dependent endonuclease inhibitor (baloxavir). Anti-influenza virus drugs targeting neuraminidase can be divided into the following categories according to their structures: acylhydrazone, thiophene, dihydrofuran coumarin, oxadiazole, triazole, oxalamide and some natural products.

[0004] In recent years, due to the wide application of neuraminidase inhibitor drugs in clinical practice, drug-resistant virus strains have emerged, such as H5N1-H274Y mutant, which has a tyrosine residue instead of a histidine residue, which interferes with the hydrophobic pocket formed by Glu276 of neuraminidase. This mutation prevents the conformational change required for oseltamivir binding, hindering the hydrophobic binding of oseltamivir acid to the active pocket, thereby greatly reducing the sensitivity of neuraminidase to oseltamivir, affecting the binding of the drug to the enzyme, and drug-resistant virus strains of zanamivir, peramivir and laninamivir have also begun to appear.

[0005] Therefore, the development of new, efficient, safe and economical neuraminidase inhibitors is still a research hotspot in the field of anti-influenza virus. SUMMARY

[0006] The purpose of the present application is to provide a sulfadimidine neuraminidase inhibitor and a preparation method and application thereof to solve the above problems. The inhibitor has a novel structure and good neuraminidase inhibition activity, and can be used for preparing a drug for inhibiting the activity of neuraminidase.

[0007] The purpose of the present application can be achieved by the following technical solutions:

[0008] A sulfanilamide neuraminidase inhibitor having a chemical structure of formula (I) or formula (V):

[0009]

[0010] wherein R1 in formula (I) and formula (V) is selected from any one of the following structural formulae:

[0011] (X=F, Cl, Br or I), (X=F, Cl, Br or I), (X=F, Cl, Br or I),

[0012]

[0013] In the above structural formulae, indicates that the R group is connected to the amide group or the sulfur.

[0014] Preferably, R1 is selected from any one of the following structural formulae:

[0015]

[0016]

[0017] More preferably, the chemical structure of the inhibitor is selected from one of the following structural formulae:

[0018]

[0019] The present application also provides a preparation method of a sulfanilamide neuraminidase inhibitor having a chemical structure of formula (I), comprising the following steps:

[0020] (1a) forming a first reaction system by mixing sulfanilamide, monoethyl ester acyl chloride and anhydrous potassium carbonate, and obtaining a formula (II) intermediate after reaction and first post-treatment;

[0021] (2a) dissolving the formula (II) intermediate obtained in step (1a) in an organic solvent A, adding potassium hydroxide to form a second reaction system, and obtaining a formula (III) intermediate after reaction and second post-treatment;

[0022] (3a) the intermediate of formula (III) obtained in step (2a) is dissolved in an organic solvent B, substituted aniline, 1-hydroxybenzotriazole (HOBT) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) are added to form a third reaction system, and after reaction, a third post-treatment is performed to obtain the sulfonamide dimethyrimidine inhibitor shown in formula (I).

[0023] Preferably, in step (1a), anhydrous K2CO3 is used as an acid-binding agent, and an organic solvent DMF is added to the reaction system.

[0024] Preferably, in step (1a), the temperature of the first reaction system is 0-60°C, preferably 25°C, and the reaction time is 4-8h, preferably 6h.

[0025] Preferably, in step (1a), the first post-treatment is performed by adding the reaction solution after the reaction of the first reaction system dropwise into 500mL distilled water, filtering and washing with saturated brine to obtain the crude product of the intermediate of formula (II), and recrystallizing with 95% aqueous ethanol to obtain the pure intermediate of formula (II).

[0026] Preferably, in step (1a), the addition amount ratio of the sulfonamide dimethyrimidine, monoethyl ester acyl chloride, anhydrous potassium carbonate and organic solvent DMF is (20-30)mmol:(30-45)mmol:(30-45)mmol:(20-200)mL, preferably 20mmol:30mmol:30mmol:40mL.

[0027] Preferably, in step (2a), potassium hydroxide is used for hydrolysis, and the organic solvent A is ethanol.

[0028] Preferably, in step (2a), the temperature of the second reaction system is 0-40°C, preferably 25°C, and the reaction time is 2-6h, preferably 4h.

[0029] Preferably, in step (2a), the second post-treatment is performed by adding 100mL distilled water to the second reaction system, adjusting the pH to 1 with concentrated hydrochloric acid, filtering and washing with saturated brine to obtain the crude product of the intermediate of formula (III), and recrystallizing from water to obtain the pure intermediate of formula (III).

[0030] Preferably, in step (2a), the addition amount ratio of the intermediate of formula (II), potassium hydroxide and organic solvent A is (10-30)mmol:(30-90)mmol:(80-160)mL, preferably 10mmol:30mmol:100mL.

[0031] Preferably, in step (3a), the organic solvent B is ethyl acetate, dichloromethane, methanol, ethanol or DMF, preferably DMF.

[0032] Preferably, in step (3a), the temperature of the third reaction system is 0-60℃, preferably 25℃, and the reaction time is 2-12h, preferably 6h.

[0033] Preferably, in step (3a), the third post-treatment process specifically involves: adding the reaction liquid after the reaction of the third reaction system dropwise to 500 mL of distilled water, filtering and washing with saturated saline to obtain the crude product, and recrystallizing with 95% aqueous ethanol to obtain the pure sulfadiazine inhibitor shown in formula (I).

[0034] Preferably, in step (3a), the ratio of the added substituted aniline, formula (III) intermediate, HOBT, EDCI and organic solvent B is 5 mmol:(6-7.5) mmol:(10-12.5) mmol:(17.5-20) mmol:(20-50) mL, and more preferably 5 mmol:7.5 mmol:10 mL:17.5 mmol:50 mL.

[0035] This invention also provides a method for preparing a sulfadimidine-based neuraminidase inhibitor, wherein the sulfadimidine-based neuraminidase inhibitor has the chemical structural formula shown in formula (V), and the specific steps are as follows:

[0036] (1b) Sulfadimethazine, chloroacetyl chloride and anhydrous potassium carbonate are formed into a first reaction system, and after the reaction, the intermediate of formula (IV) is obtained by a first post-treatment.

[0037] (2b) Dissolve the intermediate of formula (Ⅳ) obtained in step (1b) in an organic solvent, add substituted thiols or thiophenols and anhydrous potassium carbonate to form a second reaction system, and after the reaction, perform a second post-treatment to obtain the sulfadiazine inhibitor shown in formula (Ⅴ).

[0038] Preferably, in step (1b), anhydrous K2CO3 is used as an acid-binding agent, and organic solvent DMF is added to the reaction system.

[0039] Preferably, in step (1b), the temperature of the first reaction system is 0-60℃, preferably 0℃, and the reaction time is 1-6h, preferably 1.5h.

[0040] Preferably, in step (1b), the first post-treatment process specifically involves: adding the reaction liquid after the reaction of the first reaction system dropwise to 500 mL of distilled water, filtering and washing with saturated brine to obtain the crude product of intermediate (IV), and recrystallizing with 95% aqueous ethanol to obtain the pure intermediate (IV).

[0041] Preferably, in step (1b), the ratio of the amount of sulfadiazine, chloroacetyl chloride, anhydrous potassium carbonate and organic solvent DMF added is (20-30) mmol:(30-45) mmol:(30-45) mmol:(40-200) mL, and more preferably 20 mmol:30 mmol:30 mmol:40 mL.

[0042] Preferably, in step (2b), anhydrous K2CO3 is used as the acid-binding agent, and the organic solvent is DMF.

[0043] Preferably, in step (2b), the second reaction system is placed at a temperature of 20-100°C, preferably 60°C, and the reaction time is 8-16 hours, preferably 10 hours.

[0044] Preferably, in step (2b), the second post-treatment process specifically involves: adding the reaction liquid after the reaction of the second reaction system dropwise to 500 mL of distilled water, filtering and washing with saturated saline to obtain a crude product, and recrystallizing with 95% aqueous ethanol to obtain a pure sulfadiazine inhibitor as shown in formula (V).

[0045] Preferably, in step (2b), the ratio of the intermediate of formula (IV), the substituted thiol or thiophenol, the anhydrous potassium carbonate and the organic solvent is (30-45) mmol:(20-30) mmol:(30-45) mmol:(40-200) mL, and more preferably 30 mmol:20 mmol:30 mmol:40 mL.

[0046] Furthermore, the present invention also provides the use of the sulfadiazine-based neuraminidase inhibitor as described above in the preparation of a drug capable of inhibiting neuraminidase activity.

[0047] This invention utilizes a receptor-based molecular docking virtual screening method to screen 600,000 compounds from the ZINC database, obtaining a compound theoretically possessing neuraminidase inhibitory activity. Subsequently, its structure was modified to design more rational compounds, and neuraminidase tests were performed on 10 of these compounds, using Oseltamivircarboxlate (OSC) as a positive control. The IC50 of OSC was... 50 The value is 0.12 μM.

[0048] The IC of the following four compounds synthesized in this invention 50 The values ​​are all close to 0.12 μM:

[0049]

[0050] Among them, the compound with the best inhibitory effect Its IC50 The value was 6.74±0.13μM, indicating good neuraminidase inhibitory activity.

[0051] Compared with the prior art, the present invention provides a neuraminidase inhibitor with a novel scaffold structure, its preparation method and application. The synthesis method is simple, the obtained inhibitor has good neuraminidase inhibitory activity and good neuraminidase inhibitory effect, and can be applied to the preparation of drugs that inhibit neuraminidase activity. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the equation for the preparation method of the sulfadiazine-based neuraminidase inhibitor of the present invention. Detailed Implementation

[0053] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0054] A sulfadiazine-based neuraminidase inhibitor having a chemical structural formula as shown in formula (I) or (V):

[0055]

[0056] In both equations (I) and (V), R1 is selected from... (X = F, Cl, Br or I) (X = F, Cl, Br or I) (X = F, Cl, Br or ...)

[0057]

[0058] Any one of them;

[0059] In the above structural formulas This indicates that the R group is connected to an amide group or sulfur.

[0060] This invention also provides a method for preparing a sulfadiazine-based neuraminidase inhibitor, wherein the sulfadiazine-based neuraminidase inhibitor has the chemical structural formula shown in formula (I), and the preparation method is expressed as follows: Figure 1 As shown, the specific steps are as follows:

[0061] (1a) Sulfadimethinid, monoethyl ester chloride and anhydrous potassium carbonate are formed into a first reaction system, and after the reaction, the intermediate of formula (II) is obtained by first post-treatment.

[0062] (2a) Dissolve the intermediate of formula (II) obtained in step (1a) in organic solvent A, add potassium hydroxide to form a second reaction system, and after the reaction, undergo a second post-treatment to obtain intermediate of formula (III);

[0063] (3a) Dissolve the intermediate of formula (III) obtained in step (2a) in organic solvent B, add substituted aniline, 1-hydroxybenzotriazole (HOBT) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) to form a third reaction system, and after the reaction, the third post-treatment will give the sulfadiazine inhibitor shown in formula (I).

[0064] In step (1a), anhydrous K2CO3 is used as an acid-binding agent, and the organic solvent DMF is added to the reaction system.

[0065] The temperature of the first reaction system is 0-60℃, preferably 25℃, and the reaction time is 4-8h, preferably 6h.

[0066] The first post-treatment process is as follows: the reaction liquid after the first reaction system is added dropwise to 500 mL of distilled water, filtered, and washed with saturated brine to obtain the crude product of intermediate (II). The crude product of intermediate (II) is then recrystallized with 95% aqueous ethanol to obtain the pure intermediate (II).

[0067] The ratio of the amount of sulfadiazine, monoethyl ester chloride, anhydrous potassium carbonate and organic solvent DMF added is (20-30) mmol:(30-45) mmol:(30-45) mmol:(20-200) mL, preferably 20 mmol:30 mmol:30 mmol:40 mL;

[0068] In step (2a), potassium hydroxide is used for hydrolysis, and the organic solvent A is ethanol.

[0069] The temperature of the second reaction system is 0-40℃, preferably 25℃, and the reaction time is 2-6 hours, preferably 4 hours.

[0070] The second post-processing procedure is as follows: 100 mL of distilled water is added to the second reaction system, the pH is adjusted to 1 with concentrated hydrochloric acid, the mixture is filtered and washed with saturated brine to obtain the crude product of intermediate (III). The crude product of intermediate (III) is then recrystallized from water to obtain the pure intermediate (III).

[0071] The ratio of the intermediate of formula (II), potassium hydroxide and organic solvent A is (10-30) mmol:(30-90) mmol:(80-160) mL, preferably 10 mmol:30 mmol:100 mL;

[0072] In step (3a), the organic solvent B is ethyl acetate, dichloromethane, methanol, ethanol, or DMF, preferably DMF.

[0073] The temperature of the third reaction system is 0-60℃, preferably 25℃, and the reaction time is 2-12h, preferably 6h.

[0074] The third post-treatment process is as follows: the reaction liquid after the reaction in the third reaction system is added dropwise to 500 mL of distilled water, filtered, and washed with saturated brine to obtain the crude product. The crude product is then recrystallized with 95% aqueous ethanol to obtain the pure sulfadiazine inhibitor shown in formula (I).

[0075] The ratio of the added substituted aniline, formula (III) intermediate, HOBT, EDCI and organic solvent B is 5 mmol:(6-7.5) mmol:(10-12.5) mmol:(17.5-20) mmol:(20-50) mL, preferably 5 mmol:7.5 mmol:10 mL:17.5 mmol:50 mL.

[0076] This invention also provides a method for preparing a sulfadiazine-based neuraminidase inhibitor, wherein the sulfadiazine-based neuraminidase inhibitor has the chemical structural formula shown in formula (V), and the preparation method is expressed as follows: Figure 1 As shown, the specific steps are as follows:

[0077] (1b) Sulfadimethazine, chloroacetyl chloride and anhydrous potassium carbonate are formed into a first reaction system, and after the reaction, the intermediate of formula (IV) is obtained by a first post-treatment.

[0078] (2b) Dissolve the intermediate of formula (IV) obtained in step (1b) in an organic solvent, add substituted thiols or thiophenols and anhydrous potassium carbonate to form a second reaction system, and after the reaction, perform a second post-treatment to obtain the sulfadiazine inhibitor shown in formula (V).

[0079] In step (1b), anhydrous K₂CO₃ is used as an acid-binding agent, and the organic solvent DMF is added to the reaction system.

[0080] The temperature of the first reaction system is 0-60℃, preferably 0℃, and the reaction time is 1-6h, preferably 1.5h.

[0081] The first post-treatment process is as follows: the reaction liquid after the first reaction system is added dropwise to 500 mL of distilled water, filtered, and washed with saturated brine to obtain the crude product of intermediate (IV). The crude product is then recrystallized with 95% aqueous ethanol to obtain the pure intermediate (IV).

[0082] The ratio of the amount of sulfadiazine, chloroacetyl chloride, anhydrous potassium carbonate and organic solvent DMF added is (20-30) mmol:(30-45) mmol:(30-45) mmol:(40-200) mL, preferably 20 mmol:30 mmol:30 mmol:40 mL;

[0083] In step (2b), anhydrous K2CO3 is used as the acid-binding agent, and the organic solvent is DMF.

[0084] The second reaction system is placed at a temperature of 20-100℃, preferably 60℃, for a reaction time of 8-16 hours, preferably 10 hours.

[0085] The second post-treatment process is as follows: the reaction liquid after the second reaction system is added dropwise to 500 mL of distilled water, filtered, and washed with saturated brine to obtain the crude product. The crude product is then recrystallized with 95% aqueous ethanol to obtain the pure sulfadiazine inhibitor shown in formula (V).

[0086] The ratio of the intermediate of formula (IV), the substituted thiol or thiophenol, the anhydrous potassium carbonate and the organic solvent is (30-45) mmol:(20-30) mmol:(30-45) mmol:(40-200) mL, preferably 30 mmol:20 mmol:30 mmol:40 mL.

[0087] Performance testing:

[0088] The prepared inhibitors were tested for their inhibitory activity against neuraminidase. The specific test method is as follows:

[0089] 1. Experimental instruments and materials

[0090] Multifunctional fluorescent microplate reader, SP-Max 3500FL, Shanghai Flash Spectrum Biotechnology Co., Ltd.;

[0091] Clean bench;

[0092] TopPette manual single-channel adjustable pipettes, 0.5-10μL, 10-100μL, 20-200μL, 100-1000μL, purchased from Titan Technologies;

[0093] 96-well microplate (black), sterilized, Corning;

[0094] H5N1 neuraminidase was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; the fluorescent substrate 2'-(4-methylumbelliferone)-α-D-acetylneuraminidase sodium hydrate (4-MUNANA) (Sigma, M8639) used in the enzyme inhibition experiment was purchased from Sigma-Aldrich; 2-(N-morpholine) ethanesulfonic acid (MES), calcium chloride, sodium hydroxide, and anhydrous ethanol were purchased from Titan Technology.

[0095] Positive control drug: Oseltamivir carboxlate (OSC), Shanghai Hekang Biotechnology Co., Ltd.

[0096] 2. Experimental Methods

[0097] The positive control drug and the target compound prepared in the examples were dissolved in DMSO and the initial concentration was prepared to be 1000 μM. It was then serially diluted to seven concentration gradients: 200 μM, 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0.064 μM. Three sets of each concentration gradient were prepared in sequence.

[0098] 2.1 Sample preparation for testing

[0099] a. Add 70 μL of buffer (33 mM MES, 4 mM CaCl2) to each well of a 96-well microplate;

[0100] b. Add 10 μL of neuraminidase to each well;

[0101] c. Add 10 μL of the prepared neuraminidase inhibitor sample or positive control sample of the desired concentration to each well, and set up three blank control groups at the same time;

[0102] d. Add 10 μL of neuraminidase substrate (100 μM·4-MUNANA) to each well.

[0103] 2.2 Detection

[0104] a. Place the 96-well microplate in a multi-functional fluorescent microplate reader and shake to mix for 1 minute;

[0105] b. Set the temperature to 37℃ and incubate for 5 minutes to allow the neuraminidase to mix thoroughly with the sample to be tested and interact with it.

[0106] c. Remove the 96-well microplate and add 10 μL of neuraminidase fluorescent substrate to each well;

[0107] d. Place it back in the multi-functional fluorescent microplate reader and shake to mix for 1 minute;

[0108] e. Incubate at 37°C for 30 minutes, remove, add 150 μL of stop solution (14 mM NaOH in 83% ethanol aqueous solution) to each well, place again in a multi-functional fluorescent microplate reader, shake to mix for 1 minute, set the excitation wavelength to 355 nm and the emission wavelength to 460 nm, and start fluorescence intensity (RFU) measurement after incubation.

[0109] f. Repeat the above steps to perform 3 sets of parallel experiments.

[0110] Note: The first well in the 96-well microplate is used as the blank group. No test sample is added, but 10 μL LDMSO solution is added.

[0111] Calculate the average inhibition rate of the sample at each gradient concentration in each parallel experiment, and then fit the corresponding IC50 using Origin. 50 value.

[0112] Both the positive control drug and the target compound were prepared into a mixed solution with an initial concentration of 1000 μM using DMSO solution. These two mixed solutions were then serially diluted to seven concentration gradients: 200 μM, 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0.064 μM, with three sets prepared for each concentration gradient. 70 μL of neuraminidase buffer, 10 μL of neuraminidase, and the positive control drug sample at each gradient concentration were added to a 96-well black fluorescent microplate. Three blank control experiments were also conducted. The plate was shaken for 1 minute to mix and incubated at 37°C for 5 minutes. The 96-well plate was then removed, and 10 μL of neuraminidase substrate was added to each well. The plate was shaken for 1 minute to mix and incubated at 37°C for 30 minutes. After incubation, 150 μL of stop solution (14 mM NaOH in 83% ethanol) was added to each well. The plate was then placed back into the multi-functional fluorescence microplate reader, shaken for 1 minute to mix, and the excitation wavelength was set to 355 nm and the emission wavelength to 460 nm. After incubation, fluorescence intensity (RFU) was measured. Three parallel experiments were performed, and the average inhibition rate of the sample at each concentration gradient was calculated for each parallel experiment. The corresponding IC50 was then fitted using Origin. 50 The inhibition rate of each sample was calculated, and then the corresponding IC50 was obtained by fitting the data using Origin. 50 value.

[0113] The above-described embodiments will be described in more detail below with reference to specific examples.

[0114] Example 1

[0115] N1-(3,4-dimethoxyphenyl)-N4-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)succinamide, the structural formula of which is shown below:

[0116]

[0117] The specific synthesis steps are as follows:

[0118] (1) Accurately weigh 5.57 g (20 mmol) of sulfadiazine and add it to a 150 mL round-bottom flask. Add 4.15 g (30 mmol) of anhydrous potassium carbonate and 40 mL of DMF. Slowly add 4.18 mL (30 mmol) of succinate monoethyl ester chloride using a constant pressure dropping funnel under ice bath conditions. Stir the mixture at 25 °C for 6 hours. After the reaction is complete, add the reaction solution dropwise to 500 mL of distilled water, filter, and wash with saturated brine to obtain the crude product. Recrystallize from 95% aqueous ethanol to obtain the pure intermediate of formula (II).

[0119] (2) Accurately weigh 4.06 g (10 mmol) of intermediate (II) and add it to a 250 mL round-bottom flask. Add 100 mL of ethanol, and add a solution of 1.68 g (30 mmol) of potassium hydroxide dissolved in 5 mL of distilled water to the round-bottom flask under ice bath conditions. Stir the mixture at 25 °C for 4 hours. After the reaction is complete, add 100 mL of distilled water to the system, adjust the pH to 1 with concentrated hydrochloric acid, filter, and wash with saturated brine to obtain the crude product. Then recrystallize from water to obtain the pure intermediate (III).

[0120] (3) Accurately weigh 0.77 g (5 mmol) of 3,4-dimethoxyaniline, 2.84 g (7.5 mmol) of intermediate of formula (III), 1.35 g (10 mmol) of HOBT and 3.35 g (17.5 mmol) of EDCI and add them to a 100 mL round bottom flask. Add 50 mL of DMF and stir at 25 °C for 6 hours under nitrogen flow. After the reaction is complete, add the reaction solution dropwise to 500 mL of distilled water, filter and wash with saturated brine to obtain crude product. Recrystallize with 95% aqueous ethanol to obtain the inhibitor shown in formula (I).

[0121] Experimental results

[0122] N1-(3,4-dimethoxyphenyl)-N4-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)succinamide, white solid, yield 43.7%, IC50 50 The value was 6.74±0.13μM, the melting point was 203.7-205.2℃, the purity was 99.90%, and the IC50 of the positive control drug was... 50 The value is 0.12 μM.

[0123] 1H NMR(401MHz,DMSO-d6)δ11.20(s,1H),10.93(s,1H),9.03(s,1H),8.07–7.93(m,4H),6 .89–6.81(m,1H),6.78–6.71(m,3H),3.73(d,J=9.0Hz,6H),2.78(m,4H),2.26(s,6H). 13 C NMR (101MHz, DMSO) δ159.95,159.59,156.64,149.09,147.78,141.61,131.96,129.53,129.46,127 .08,120.97,120.84,120.29,120.12,112.98,112.35,55.99,55.87,34.57,23.32.HRMS(ESI)calcd for C 24 H 27 N5O6S[M+Na] + :536.1574; Found:536.1569.

[0124] Example 2

[0125] 2-((4,6-dihydroxypyrimidin-2-yl)thio)-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)acetamide, with the following structural formula, was prepared using a method similar to that in Example 1.

[0126]

[0127] White solid, yield 25.6%, IC 50 The value is 10.71±0.51μM, the melting point is 250.9-252.3℃, and the purity is 99.80%.

[0128] 1 H NMR (401MHz, DMSO-d6) δ8.86(s,1H),7.99–7.89(m,2H),7.85–7.71(m,2H),6.73(d,J=7.8Hz,1H),5.32(s,1H),4.09(s,2H),2.25(s,6H). 13 CNMR(101MHz,DMSO-d6)δ168.29,167.87,162.90,156.62,152.18,142.97,135.32,129.69,118.71,113.98,86.20,36.34,34.59.HRMS(ESI)calcd for C 18 H18 N6O5S2[M+Na] + :485.0672; Found:485.0673.

[0129] Example 3

[0130] N1-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)-N2-(4-fluorophenyl)oxalamide, with the following structural formula, was prepared using a method similar to that in Example 1.

[0131]

[0132] White solid, yield 43.8%, IC 50 The value is 13.56±3.77μM, the melting point is 240.4-241.6℃, and the purity is 99.72%.

[0133] 1 H NMR (401MHz, DMSO-d6) δ11.16(s,1H),10.97(s,1H),8.08–7.97(m,4H),7.91–7.85(m,2H),7.47–7.15(m,2H),6.74(d,J=0.8Hz,1H),2.25(s,6H). 13 C NMR(101MHz,DMSO-d6)δ160.57,159.54,159.24,158.65,158.17,156.61,141.62,136.79,134.46,1 29.54,127.09,122.98,122.90,120.85,120.28,116.03,115.81,113.85,23.28.HRMS(ESI)calcdfor C 20 H 18 FN5O4S[M+Na] + :466.0956; Found:466.0957.

[0134] Example 4

[0135] N1-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)-N4-(4-fluorophenyl)succinamide, with the following structural formula, was prepared using a method similar to that in Example 1.

[0136]

[0137] White solid, yield 47.8%, IC 50The value is 15.33±1.05μM, the melting point is 187.3-190.0℃, and the purity is 99.32%.

[0138] 1 H NMR (400MHz, DMSO-d6) δ10.93(s,1H),9.10(s,1H),8.02–7.98(m,4H),7.32(d,J=7.2Hz,1H),7.14–6.95(m,4H),2.88(d,J=7.0Hz,4H),2.25(s,6H). 13 C NMR(100MHz,DMSO-d6)δ161.48,160.02,159.47,156.68,142.58,142.51,141.60,136.63,130.69 ,130.61,129.47,125.32,120.12,115.92,115.71,113.58,113.37,34.59,23.31.HRMS(ESI)calcd for C 22 H 22 FN5O4S[M+Na] + :494.1269;Found:494.1260.

[0139] Example 5

[0140] N1-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)-N2-(4-nitrophenyl)oxalamide, with the following structural formula, was prepared using a method similar to that in Example 1.

[0141]

[0142] Yellow solid, yield 38.8%, IC 50 The value is 16.23±0.98μM, the melting point is 199.5-201.2℃, and the purity is 98.78%.

[0143] 1 H NMR(401MHz,DMSO-d6)δ11.45(s,1H),11.23(s,1H),8.33–8.25(m,2H),8.19–8 .12(m,2H),8.05–7.90(m,4H),6.77(d,J=0.8Hz,1H),2.26(s,6H),2.00(s,1H). 13C NMR(101MHz,DMSO-d6)δ161.60,159.08,159.07,155.87,143.30,141.90,141 .85,132.49,128.77,125.51,119.30,118.01,116.17,23.35.HRMS(ESI)calcd for C 20 H 18 N6O6S[M+Na] + :493.0901; Found:493.0896.

[0144] Example 6

[0145] N1-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)-N3-(4-fluorophenyl)malondiamide, with the following structural formula, was prepared using a method similar to that in Example 1.

[0146]

[0147] White solid, yield 39.9%, IC 50 The value is 17.56±4.24μM, the melting point is 213.4-215.5℃, and the purity is 98.06%.

[0148] 1 H NMR(401MHz,DMSO-d6)δ11.20(s,1H),10.97(s,1H),9.62(s,1H),8.04–7.95(m,4H ),7.37(m,2H),7.20–7.09(m,2H),6.76(d,J=3.1Hz,1H),4.40(s,2H),2.25(s,6H). 13 CNMR(101MHz,DMSO-d6)δ162.99,160.57,160.28,159.53,159.26,156.65,141.61,135.26,130.03,129.95 ,129.53,129.46,127.08,120.84,120.29,120.15,115.64,115.43,115.31,42.46,23.31.HRMS(ESI)calcd for C 21 H 20 FN5O4S[M+Na] + :480.1112;Found:480.1109.

[0149] Example 7

[0150] N1-(4-acetylphenyl)-N2-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)oxalamide, with the following structural formula, was prepared using a method similar to that in Example 1.

[0151]

[0152] White solid, yield 39.0%, IC 50 The value is 20.33±4.72μM, the melting point is 193.5-194.3℃, and the purity is 97.94%.

[0153] 1 H NMR (401MHz, DMSO-d6) δ11.20 (d, J = 8.5Hz, 2H), 8.25–7.83 (m, 8H), 6.76 (s, 1H), 2.57 (s, 3H), 2.26 (s, 6H). 13 C NMR(101MHz,DMSO-d6)δ197.25,159.31,159.23,156.63,142.34,141.59,133.42,129.79 ,129.54,127.03,124.59,120.45,120.29,119.39,110.54,27.07,23.31.HRMS(ESI)calcd for C 22 H 21 N5O5S[M+Na] + :490.1156; Found:490.1155.

[0154] Example 8

[0155] N1-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)-N4-(4-methoxyphenyl)succinamide, with the following structural formula, was prepared using a method similar to that in Example 1.

[0156]

[0157] White solid, yield 41.6%, IC 50 The value is 20.74±2.63μM, the melting point is 190.9-193.3℃, ​​and the purity is 97.48%.

[0158] 1H NMR(401MHz,DMSO-d6)δ10.94(s,1H),10.54(s,1H),9.07(s,1H),8.01–7.88(m,4H),7 .22(d,J=7.9Hz,1H),6.88–6.62(m,5H),3.73(s,3H),2.96–2.80(m,4H),2.25(s,6H). 13 C NMR(101MHz,DMSO-d6)δ159.98,159.78,159.54,156.69,141.62,141.12,136.62,129.87,129.47,12 6.37,124.28,121.35,120.12,119.25,114.72,112.17,110.69,55.39,35.02,23.32.HRMS(ESI)calcd for C 23 H 25 N5O5S[M+Na] + :506.1469; Found:506.1463.

[0159] Example 9

[0160] N1-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)-N4-phenylsuccinamide, with the following structural formula, was prepared using a method similar to that in Example 1.

[0161]

[0162] White solid, yield 46.3%, IC 50 The value is 31.80±4.37μM, the melting point is 188.2-190.5℃, and the purity is 96.38%.

[0163] 1 H NMR (401MHz, DMSO-d6) δ10.93(s,1H),10.70(s,1H),8.03–7.95(m,4H),7.32(d,J=7.8Hz,1H),7.08–6.98(m,5H),2.89(d,J=7.2Hz,4H),2.25(s,6H). 13 C NMR (101MHz, DMSO) δ171.17,171.12,165.86,156.70,142.30,140.06,133.33,128. 91,128.83,124.15,121.23,120.07,114.18,31.87,31.82,23.43.HRMS(ESI)calcd for C22 H 23 N5O4S[M+Na] + :476.1363; Found:476.1363.

[0164] Example 10

[0165] N1-(4-chlorophenyl)-N3-(4-(N-(4,6-dimethylpyrimidin-2-yl)aminosulfonyl)phenyl)malonamide, with the following structural formula, was prepared using a method similar to that in Example 1.

[0166]

[0167] Pale yellow solid, yield 44.4%, IC50 50 The value is 32.94±5.66μM, the melting point is 214.8-216.9℃, and the purity is 95.23%.

[0168] 1 H NMR(401MHz,DMSO-d6)δ11.61(s,1H),11.19(s,1H),8.06–7.93(m,4H),7.88–7.80(m, 2H),7.40–7.30(m,2H),7.31(d,J=8.5Hz,1H),4.04(s,2H),2.27(s,6H),2.00(s,1H). 13 C NMR (101MHz, DMSO) δ162.99,162.90,162.77,160.57,159.26,156.65,141.61,135.26,130.03,129.95,1 29.53,129.46,128.06,127.66,127.08,120.84,120.15,115.64,115.43,45.14,23.31.HRMS(ESI)calcd for C 21 H 20 ClN5O4S[M+Na] + :496.0816; Found:496.0865.

[0169] Performance testing

[0170] This invention utilizes a receptor-based molecular docking virtual screening method to screen 600,000 compounds from the ZINC database, obtaining a compound theoretically possessing neuraminidase inhibitory activity. Subsequently, its structure was modified to design more rational compounds, and neuraminidase tests were performed on 10 of these compounds, using Oseltamivircarboxlate (OSC) as a positive control. The IC50 of OSC was... 50 The value is 0.12 μM.

[0171] The IC50 values ​​of the following four compounds synthesized in Examples 1, 2, 3, and 4 are as follows: 50 The values ​​are all close to 0.12 μM:

[0172]

[0173] In Example 1, the inhibitor generated was the compound with the best inhibitory effect, and its structural formula was [structural formula would be inserted here]. Its IC 50 The value was 6.74±0.13μM, indicating good neuraminidase inhibitory activity.

[0174] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A sulfadiazine-based neuraminidase inhibitor, characterized in that, The inhibitor has the chemical structural formula shown in formula (Ⅰ): (Ⅰ)， The inhibitor of formula (I) is specifically selected from any one of the following structural formulas: 、 、 、 、 、 、 or 。 2. A method for preparing a sulfadiazine-based neuraminidase inhibitor as described in claim 1, characterized in that, The specific steps are as follows: (1a) Sulfadiazine, monoethyl ester chloride and anhydrous potassium carbonate are formed into a first reaction system, and after the reaction, the intermediate of formula (II) is obtained by first post-treatment; The chemical structural formula of intermediate (II) is as follows: n is 0, 1, or 2; (2a) Dissolve the intermediate of formula (II) obtained in step (1a) in organic solvent A, add potassium hydroxide to form a second reaction system, and after the reaction, undergo a second post-treatment to obtain intermediate of formula (III); The chemical structural formula of intermediate (III) is as follows: n is 0, 1, or 2; (3a) The intermediate of formula (III) obtained in step (2a) is dissolved in organic solvent B, and substituted aniline, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added to form a third reaction system. After the reaction, a third post-treatment is performed to obtain a sulfadiazine neuraminidase inhibitor.

3. The method for preparing a sulfadiazine-based neuraminidase inhibitor according to claim 2, characterized in that, In step (1a), anhydrous K2CO3 is used as an acid-binding agent, and the organic solvent DMF is added to the reaction system. The temperature of the first reaction system is 0-60 °C, and the reaction time is 4-8 h. The ratio of the amount of sulfadiazine, monoethyl ester chloride, anhydrous potassium carbonate and organic solvent DMF added is (20-30) mmol : (30-45) mmol : (30-45) mmol : (20-200) mL; In step (2a), potassium hydroxide is used for hydrolysis, and the organic solvent A is ethanol. The temperature of the second reaction system is 0-40 °C, and the reaction time is 2-6 h. The ratio of the intermediate of formula (II), potassium hydroxide and organic solvent A is (10-30) mmol : (30-90) mmol : (80-160) mL; In step (3a), the organic solvent B is ethyl acetate, dichloromethane, methanol, ethanol, or DMF. The third reaction system is characterized by a temperature of 0-60 ℃ and a reaction time of 2-12 h. The ratio of the added substituted aniline, formula (III) intermediate, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and organic solvent B is 5 mmol : (6-7.5) mmol : (10-12.5) mmol : (17.5-20) mmol : (20-50) mL.

4. The method for preparing a sulfadiazine-based neuraminidase inhibitor according to claim 2, characterized in that, In step (1a), the first post-treatment process is as follows: the reaction liquid after the reaction of the first reaction system is added dropwise to 500 mL of distilled water, filtered and washed with saturated brine to obtain the crude product of intermediate (II), and recrystallized with 95% aqueous ethanol to obtain the pure intermediate (II). In step (2a), the second post-processing process is as follows: 100 mL of distilled water is added to the second reaction system, the pH is adjusted to 1 with concentrated hydrochloric acid, the mixture is filtered and washed with saturated brine to obtain the crude product of intermediate (III), and the pure intermediate (III) is recrystallized from the water. In step (3a), the third post-treatment process is as follows: the reaction liquid after the reaction of the third reaction system is added dropwise to 500 mL of distilled water, filtered and washed with saturated saline to obtain crude product, and recrystallized with 95% aqueous ethanol to obtain pure sulfadiazine neuraminidase inhibitor.

5. The application of the compound in the preparation of neuraminidase inhibitors, characterized in that, The compound is selected from any one of the following structural formulas: 、 、 、 、 、 、 or 。

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