Neuraminidase inhibitors based on hydrophobic tag technology, methods of making, derivatives, pharmaceutical compositions, and uses thereof

Abstract

The application discloses a neuraminidase inhibitor based on a hydrophobic tag technology and a preparation method, a derivative, a pharmaceutical composition and an application thereof, and the structure of the neuraminidase inhibitor is shown as formula (I). The neuraminidase inhibitor based on the hydrophobic tag technology has enhanced antiviral activity and effective oseltamivir resistance reversal activity, and provides a new drug for the treatment of influenza virus infection, especially oseltamivir-resistant virus infection.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical application technology, and in particular to a neuraminidase inhibitor based on hydrophobic tagging technology, its preparation method, derivatives, pharmaceutical compositions and applications. Background Technology

[0002] Influenza virus is a single-stranded, negative-sense RNA virus, classified into four types—A, B, C, and D—based on their antigenic gene characteristics. Frequent outbreaks of influenza virus infection seriously endanger the lives and health of humans and animals. Currently, three main classes of antiviral drugs are used for the treatment and prevention of influenza: M2 ion channel blockers, neuraminidase inhibitors (NAIs), and cap-dependent endonuclease inhibitors (HAIs) such as baloxavir. Due to widespread drug resistance, M2 ion channel blockers are no longer used clinically. HAIs are prone to drug resistance and face significant therapeutic limitations, potentially weakening their long-term clinical application. NAIs (including oseltamivir, zanamivir, peramivir, and lanimivir) remain the most effective antiviral drugs due to their potent efficacy, better safety profile, and lower side effects. However, the widespread use of oseltamivir has led to the emergence of drug-resistant viral strains, primarily caused by mutations such as H274Y (H1N1 and H5N1), N294S (H7N9), and R292K (H3N2). The emergence of drug-resistant strains has resulted in increased treatment failure rates, increased risk of community transmission, and consequently, greater challenges to public health control. Therefore, developing new antiviral drugs to address influenza drug resistance remains crucial. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies and effectively address the problem of influenza virus drug resistance, this application provides a neuraminidase inhibitor based on hydrophobic tag technology, its preparation method, derivatives, pharmaceutical compositions, and applications. The following technical solutions are adopted in this application:

[0004] The first aspect of this application discloses a neuraminidase inhibitor based on hydrophobic tag technology, the structure of which is shown in formula (Ⅰ):

[0005]

[0006] Linker is selected from the following structures: n is a natural number between 2 and 10, m is 0 or 1, k is a natural number between 0 and 10, h is a natural number between 0 and 10, and s is a natural number between 1 and 5;

[0007] HyT is selected from the following structures: For example, n can be 2, 3, 4, 5, 6, 7, 8, 9 or 10, k can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, h can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and s can be 1, 2, 3, 4 or 5.

[0008] In one implementation of this application, n is a natural number between 6 and 9.

[0009] In one implementation of this application, k is a natural number between 1 and 4.

[0010] In one implementation of this application, h is a natural number between 1 and 4.

[0011] In one implementation of this application, s is a natural number between 1 and 4.

[0012] In one implementation of this application, s is 1.

[0013] In one implementation of this application, HyT is selected from the following structure:

[0014] In one implementation of this application, the neuraminidase inhibitor includes at least one of the following compounds L7 to L19:

[0015]

[0016] The second aspect of this application discloses a derivative of a neuraminidase inhibitor as described in the first aspect of this application, including pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates, or deuterated compounds.

[0017] The third aspect of this application discloses a method for preparing a neuraminidase inhibitor based on hydrophobic tag technology, comprising:

[0018]

[0019] HyT-NH2 was reacted with HATU and DIPEA; hydrochloric acid was then added to remove the Boc protecting group; oseltamivir was then reacted with TEA, CDI and DMF to obtain the neuraminidase inhibitor.

[0020] or, HyT-NH2 was reacted with HATU and DIPEA in the presence of HATU and DIPEA; then lithium hydroxide and water were added and reacted at 50°C; then oseltamivir was reacted with HATU and DIPEA in the presence of HATU and DIPEA to obtain the neuraminidase inhibitor.

[0021] Where n is a natural number between 2 and 10, k is a natural number between 0 and 10, h is a natural number between 0 and 10, s is a natural number between 1 and 5, and HyT is selected from the following structure:

[0022] The fourth aspect of this application discloses a pharmaceutical composition comprising a neuraminidase inhibitor as described in the first aspect of this application or a derivative as described in the second aspect, and comprising one or more pharmaceutically acceptable excipients.

[0023] The fifth aspect of this application discloses the use of a neuraminidase inhibitor as described in the first aspect of this application or a pharmaceutical composition as described in the fourth aspect in the preparation of a medicament for treating and / or preventing diseases related to influenza virus infection.

[0024] The sixth aspect of this application discloses the use of a neuraminidase inhibitor as described in the first aspect of this application or a pharmaceutical composition as described in the fourth aspect in the preparation of a medicament for treating diseases related to oseltamivir resistance.

[0025] The seventh aspect of this application discloses the use of a neuraminidase inhibitor as described in the first aspect of this application or a pharmaceutical composition as described in the fourth aspect in the preparation of a medicament for inhibiting the activity of influenza A virus carrying the H274Y variant, said influenza A virus including at least one of H1N1 and H5N1.

[0026] The beneficial effects of this application are as follows:

[0027] The neuraminidase inhibitor based on hydrophobic tag technology of this application has enhanced antiviral activity and effective oseltamivir resistance reversal activity, providing a new drug for the treatment of influenza virus infection, especially oseltamivir-resistant virus infection. Attached Figure Description

[0028] Figure 1 This is a graph showing the degradation activity of NA protein in 293T cells treated with different compounds involved in Example 2 of this application.

[0029] Figure 2 This is a diagram showing the NP protein expression results of MDCK cells treated with different compounds, as described in Example 4 of this application.

[0030] Figure 3 This is a diagram showing the molecular docking results involved in Embodiment 5 of this application. Detailed Implementation

[0031] The present application will now be described in further detail with reference to specific embodiments and accompanying drawings. In the following embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other materials or methods. In some cases, certain operations related to the present application are not shown or described in the specification. This is to avoid obscuring the core parts of the present application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; the relevant operations can be fully understood based on the description in the specification and general technical knowledge in the art.

[0032] Furthermore, the features, operations, or characteristics described in the specification can be combined in any suitable manner to form various embodiments. Simultaneously, the steps or actions in the method description can be rearranged or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various orders in the specification and drawings are merely for the clear description of a particular embodiment and do not imply a mandatory order, unless otherwise stated that a particular order must be followed. Unless otherwise defined, all scientific and technical terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application relates.

[0033] The term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts.

[0034] The term "stereoisomer" includes enantiomers, diastereomers, and geometric isomers. Some compounds in this application have cyclic hydrocarbon groups that can be substituted on more than one carbon atom, in which case all their geometric forms, including cis and trans, and mixtures thereof, are within the scope of this application.

[0035] The term "solvent" refers to the physical bond between a compound of this application and one or more solvent molecules. This physical bond includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate can be isolated, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Solvents include solution phases and separable solvates. Representative solvates include ethanolides, methanolides, etc.

[0036] The term "ester" refers to a compound formed by the reaction of a carboxylic acid and an alcohol. In medicinal chemistry, esters are often used to modify the solubility, stability, and bioavailability of drugs.

[0037] The term "prodrug" refers to a Formula I compound that is suitable for administration to patients without excessive toxicity, irritation, or allergic reactions, and is effective for its intended purpose. Prodrugs include acetals, esters, and zwitterionic forms. Prodrugs are converted in the body, such as through hydrolysis in the blood, to yield the parent compound.

[0038] The term "solvent" refers to a complex formed when drug molecules and solvent molecules are bonded together by non-covalent bonds (such as hydrogen bonds, van der Waals forces, etc.). A hydrate is an example.

[0039] The term "deuterated compound" refers to a drug molecule in which some or all of the hydrogen atoms (deuterated) are removed. 1 H) is replaced with deuterium ( 2 H, i.e., deuterium.

[0040] The term "treatment" refers to the prevention, cure, reversal, reduction, mitigation, minimization, suppression, cessation, and / or cessation of one or more clinical symptoms of a disease after its onset.

[0041] The term "prevention" refers to the treatment taken before a disease develops to avoid, minimize, or prevent the disease from developing or progressing.

[0042] Currently, existing anti-influenza drugs have limited efficacy against influenza infection due to severe side effects and drug resistance issues. Given the differences between viral target proteins and human proteins, PROTAC (Protein Degradation Targeting Chimera) technology can achieve complete degradation of viral proteins, thereby enhancing antiviral efficacy and reducing the development of drug resistance. In recent years, PROTACs have been used to target antiviral targets such as coronavirus 3CLpro, influenza NA protein, and hepatitis C virus (HCV) NS3 protein. Hydrophobic tag (HyT) technology utilizes ligands with hydrophobic tags to mimic the hydrophobic properties of partially unfolded proteins. These modified proteins are recognized by molecular chaperones such as heat shock proteins (e.g., Hsp70 and Hsp90) and their degradation is promoted via the proteasome. Compared to PROTACs, HyT technology offers an effective method for improving drug properties due to its lower molecular weight and fewer hydrogen bond donor / receptor (HBDs / HBAs) requirements.

[0043] In this application, the amino group of oseltamivir is modified to utilize its additional binding cavity, thereby enhancing antiviral efficacy and combating drug resistance. This application designs oseltamivir-derived HyTTDs (HyT-based protein degraders) by attaching a hydrophobic tag HyT to the amino group of oseltamivir using both flexible and rigid chains (such as alkyl and polyethylene glycol structures). The antiviral efficacy against the H1N1-H274Y virus strain was evaluated in vitro, and their effectiveness was confirmed by immunofluorescence. The ability of these compounds to degrade neuraminidase (NA) proteins was assessed by Western blot analysis. Finally, their binding interactions were predicted through molecular docking studies. The neuraminidase inhibitor based on hydrophobic tag technology in this application exhibits enhanced antiviral activity and effective oseltamivir resistance reversal activity.

[0044] The present application will be further described in detail below through specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the application. In these embodiments, unless otherwise specified, all reagents and instruments used are commercially available, and experimental procedures were performed according to product instructions and standard experimental procedures. Thin-layer chromatography was performed on 0.20 mm thick silica gel plates (purchased from Ocean Company in Qingdao, Shandong, China) equipped with a QF-254 UV lamp. Column chromatography used marine silica gel 60 (300-400 mesh). Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker 600 or 400 MHz spectrometer, using CDCl3 or DMSO-d6 as solvents and tetramethylsilane (TMS) as the internal standard. High-resolution mass spectrometry (HRMS) was recorded using a Thermo-Fisher Orbitrap Fusion equipped with an electron spray ionization probe (ESI). Martin-Dalby canine kidney cells (MDCK) and 293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia) and cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin, both from Gibco, Thermo Fisher Scientific, USA). The H1N1 influenza strain (A / Puerto Rico / 8 / 1934, containing NA-H274Y) was also purchased from ATCC. Virus infection experiments were conducted in BSL-2 laboratories at Guangzhou Medical University and Southern Medical University. All animal experiments strictly followed the "Technical Guidelines for Non-Clinical Pharmacokinetic Studies of Drugs" issued by the National Medical Products Administration (NMPA) and relevant guidelines from the International Council for Harmonisation of Technical Requirements for Drugs (ICH).

[0045] Example 1

[0046] (1) The synthetic routes for compounds L1 to L6 are shown below:

[0047]

[0048] In the above synthetic route, the compound oseltamivir on the left is converted into the compound on the right through reaction a or reaction b. Reaction a: The reaction was carried out in dry dichloromethane (DCM) with the corresponding acids (1-adamantaneacetic acid, (+)-mentholoxyacetic acid, 2,2-diphenylacetic acid, and 9-fluoreneacetic acid, respectively) for 4 hours using HATU (O-(7-azabenzotriazol-1-yl)-N,N',N”-tetramethylurea hexafluorophosphate) and DIPEA (diisopropylethylamine), respectively; the final products were compounds L1, L2, L3, and L4. Reaction b consisted of two steps: the first step was carried out in DMF (N,N-dimethylformamide) with TEA (triethylamine) and CDI (1,1'-carbodiimide dicyclohexyl) for 1 hour; the second step was carried out at room temperature with the corresponding amines (5-norbornene-2-methylamine or 1-adamantanemethylamine, respectively) for 10 hours; the final products were compounds L5 and L6.

[0049] Compound L1—ethyl(3R,4R,5R)-4-acetamido-5-[2-((3R,5R,7R)-adamantane-1-yl)acetamido]-3-(pent-3-oxy)cyclohex-1-ene-1-carboxylic acid ester: Starting with oseltamivir (0.53 g, 1.69 mmol) and 1-adamantaneacetic acid (0.3 g, 1.5 mmol), the final product was a white solid L1 (0.47 g, yield 64.85%). 1 H NMR (600MHz, DMSO-d6) δ7.93(d,J=9.2Hz,1H),7.56(d,J=8.9Hz,1H),6.68(t,J=2.5Hz,1H),4.20–4.14(m,2 H),4.12–4.08(m,1H),3.84–3.77(m,1H),3.76–3.74(m,1H),3.46–3.42(m,1H),2.24–2.17(m,1H),1.98–1. 92(m,3H),1.87(d,J=12.7Hz,1H),1.83(s,1H),1.81(s,3H),1.70(d,J=12.2Hz,3H),1.61(d,J=12.2Hz,6H) ,1.58–1.53(m,3H),1.51–1.38(m,5H),1.26(t,J=7.1Hz,3H),0.88(t,J=7.4Hz,3H),0.81(t,J=7.4Hz,3H). 13C NMR (151MHz, DMSO-d6) δ170.1,169.9,166.0,138.8,129.9,129.0,81.6,75.9,67.9,67.6,60.9,53.6,50.7,47.6,42.6,42.5,40.5,38. 7,38.6,36.9,32.5,31.2,30.4,30.3,28.9,28.6,26.1,25.6,23.9,23.7,23.3,22.9,14.5,14.4,11.4,11.3,9.9,9.4.HRMS(ESI):[M+H] + Calculated for C 28 H 45 N2O5:489.3328, found:489.3321.

[0050] Compound L2—ethyl(3R,4R,5R)-4-acetamido-5-[2-(((1S,2R,5S)-2-isopropyl-5-methylcyclohexyl)oxy)acetamido]-3-(pent-3-oxy)cyclohex-1-ene-1-carboxylic acid ester: Starting with oseltamivir (0.48 g, 1.54 mmol) and 2-[((1S,2R,5S)-2-isopropyl-5-methylcyclohexyl)oxy]acetic acid (0.3 g, 1.4 mmol), the final product was a white solid L2 (0.47 g, yield 69.01%). 1 H NMR(600MHz, CDCl3)δ7.16(d,J=7.6Hz,1H),6.83–6.81(m,1H),5.77(d,J=7.9Hz,1H),4.22–4.18(m,2H),4 .13–4.13(m,2H),4.04–4.01(m,1H),3.99(d,J=15.3Hz,1H),3.86(d,J=15.2Hz,1H),3.84–3.81(m,1H),3. 14–3.11(m,1H),2.77–2.72(m,1H),2.42–2.36(m,1H),2.16–2.10(m,1H),1.99–1.95(m,1H),1.94(s,3H), 1.84(s,1H),1.67–1.61(m,2H),1.54–1.48(m,4H),1.31(s,4H),0.94–0.86(m,15H),0.77(d,J=7.0Hz,3H). 13C NMR (151MHz, CDCl3) δ171.4,170.5,166.0,137.5,129.2,82.5,80.5,75.6,67.7,61.0,53.4,47.8,47.3, 40.0,34.4,31.5,30.3,26.2,25.8,25.7,23.2,23.1,22.2,21.0,16.1,14.2,9.5,9.4.HRMS(ESI):[M+H] + Calculated for C 28 H 49 N2O6:509.3591, found:509.3581.

[0051] Compound L3—ethyl(3R,4R)-4-acetamido-5-[(3,3-diphenylprop-1-en-2-yl)amino]-3-(pent-3-oxy)cyclohex-1-en-1-carboxylic acid ester: Starting from oseltamivir (0.48 g, 1.55 mmol) and 2,2-diphenylacetic acid (0.3 g, 1.41 mmol), a white solid L3 (0.45 g, yield 63.23%) was obtained. 1 H NMR (600MHz, DMSO-d6) δ8.26(d,J=9.1Hz,1H),7.79(d,J=9.3Hz,1H),7.30(dd,J=5.3,2.7Hz,8 H),7.24–7.20(m,2H),6.65–6.63(m,1H),4.88(s,1H),4.16–4.11(m,2H),4.09–4.05(m,1H),4. 00–3.93(m,1H),3.79–3.74(m,1H),2.47(dd,J=17.7,5.2Hz,1H),2.24–2.17(m,1H),1.44–1.37 (m,6H),1.35–1.30(m,1H),1.21(t,J=7.1Hz,4H),0.82(t,J=7.4Hz,3H),0.73(t,J=7.4Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ171.2,169.9,165.9,140.8,140.8,139.1,129.0,128.9,128.9,128.7,128.6,12 7.0,81.6,75.7,60.9,57.1,54.0,47.8,40.5,31.1,26.2,25.6,22.8,14.5,9.9,9.4.HRMS(ESI):[M+Na] + Calculated for C30 H 38 N2O5Na:531.3410,found:531.3403.

[0052] Compound L4—ethyl(3R,4R,5R)-5-[2-(9H-fluoren-9-yl)acetamido]-4-acetamido-3-(pent-3-oxy)cyclohex-1-ene-1-carboxylic acid ester: Starting from oseltamivir (0.45 g, 1.46 mmol) and fluoren-9-acetic acid (0.3 g, 1.33 mmol), a white solid L4 (0.45 g, yield 65.78%) was obtained. 1 HNMR(600MHz,DMSO-d6)δ7.96(d,J=9.2Hz,1H),7.88(dd,J=11.9,8.1Hz,2H),7.59(d,J=7.5Hz,1H),7.54(d, J=7.6Hz,1H),7.39(t,J=7.5Hz,2H),7.36–7.31(m,2H),6.68(s,1H),4.33(t,J=7.5Hz,1H),4.21–4.10(m,4H) ,3.82–3.79(m,1H),2.61(dd,J=17.4,5.4Hz,1H),2.26–2.19(m,1H),1.76(s,3H),1.51–1.33(m,5H),1.26–1. 23(m,,4H),1.15(d,J=6.7Hz,1H),1.02(dd,J=12.1,6.6Hz,1H),0.85(t,J=7.3Hz,3H),0.78(t,J=7.3Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ171.0,127.6,127.5,120.4,81.6,75.7,61.0,54.0,48.0,43.9,26.2,25.7,14.6,9.9,9.5.HRMS(ESI):[M+H] + Calculated for C 31 H 39 N2O5:519.2859, found:519.2852.

[0053] Compound L5—ethyl(3R,4R,5R)-4-acetamido-5-[3-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)ureo]-3-(pent-3-oxy)cyclohex-1-en-1-carboxylic acid ester: Starting with oseltamivir (0.5 g, 1.6 mmol) and 5-norbornene-2-methylamine (0.21 g, 1.76 mmol), a pale yellow solid L5 (0.32 g, yield 43.51%) was obtained. 1 HNMR(600MHz,DMSO-d6)δ7.84(dd,J=8.9,4.7Hz,1H),6.67–6.63(m,1H),6.13(dd,J=5.7,3.1Hz,1H),6.11–6.04 (m,1H),5.97–5.91(m,1H),5.66–5.55(m,1H),4.19–4.10(m,2H),4.08–4.01(m,1H),3.78–3.67(m,2H),3.42–3. 37(m,1H),3.33(d,J=4.7Hz,1H),2.80–2.72(m,2H),2.66–2.55(m,2H),2.54–2.44(m,3H),2.17–2.00(m,2H),1. 86–1.79(m,3H),1.76–1.71(m,1H),1.51–1.35(m,4H),1.33–1.28(m,1H),1.26–1.21(m,4H),0.91–0.75(m,6H). 13 CNMR(151MHz,DMSO-d6)δ133.5,82.1,76.4,50.0,42.9,41.0,40.9,40.7,4 0.6,40.4,40.3,40.2,26.7,26.3,23.8,15.1,10.4,10.0.HRMS(ESI):[M+H] + Calculated for C 25 H 40 N3O5 + :462.2968,found:462.2957.

[0054] Compound L6—ethyl(3R,4R,5R)-4-acetamido-5-[3-(((3R,5R,7R)-adamantane-1-yl)methyl)ureo]-3-(pent-3-oxy)cyclohex-1-ene-1-carboxylic acid ester: Starting with oseltamivir (0.5 g, 1.6 mmol) and 1-adamantanemethylamine (0.29 g, 1.76 mmol), a pale yellow solid L6 (0.42 g, yield 53.21%) was obtained. 1H NMR (600MHz, DMSO-d6) δ8.40(d,J=7.9Hz,1H),7.19(t,J=2.5Hz,1H),6.56(t,J=6.2Hz,1H),6.23(d,J=7.7Hz, 1H),4.72–4.62(m,2H),4.61–4.56(m,1H),4.32–4.25(m,2H),3.97–3.92(m,1H),3.88(s,1H),3.34(dd,J=13. 3,6.5Hz,1H),3.19–3.11(m,2H),3.08–3.02(m,2H),2.61–2.53(m,1H),2.35(s,3H),2.22(d,J=12.0Hz,3H),2 .12(d,J=12.0Hz,3H),2.04–1.89(m,10H),1.78(t,J=7.0Hz,3H),1.40(t,J=7.4Hz,3H),1.33(t,J=7.3Hz,3H). 13 CNMR(151MHz,DMSO-d6)δ139.3,82.1,76.5,61.4,54.7,52.1,49.0,41.0,40.8,40.8,40.7,40.6 ,40.4,40.3,40.1,37.7,34.6,32.9,28.8,26.7,26.3,23.8,15.1,10.4,10.0.HRMS(ESI):[M+H] + Calculated for C 28 H 46 N3O5: 504.3437, found: 504.3428.

[0055] (2) Synthesis of compounds L7 to L19

[0056] Overall synthetic route: Using HATU (O-(7-azabenzotriazol-1-yl)-N,N',N”-tetramethylurea hexafluorophosphate) as the condensing agent and DIPEA (diisopropylethylamine) as the base, the corresponding Boc (tert-butyloxycarbonyl) protected aminoalkyl acids (1a-g, i.e., 1a, 1b, 1c, 1d, 1e, 1f, 1g) react with their respective HyT-NH2 to form intermediates 2a-g (i.e., 2a, 2b, 2c, 2d, 2e, 2f, 2g). Subsequently, these intermediates are deprotected with 4M hydrochloric acid to generate 3a-g (i.e., 3a, 3b, 3c, 3d, 3e, 3f, 3g). Simultaneously, oseltamivir reacts with CDI (1...) in the presence of TEA (triethylamine). The methyl esters (1'-carbodiimine dicyclohexyl) were activated and then condensed with the corresponding intermediates 3a-g to give compounds L7, L10-L13, and L17-L19. Under the action of HATU and DIPEA, alkyl carboxylic acid methyl esters 4a-e (i.e., 4a, 4b, 4c, 4d, 4e) reacted with the corresponding HyT-NH2 to generate intermediates 5a-e (i.e., 5a, 5b, 5c, 5d, 5e). These intermediates were then hydrolyzed with lithium hydroxide to remove the methyl ester protecting group, generating intermediates 6a-e (i.e., 6a, 6b, 6c, 6d, 6e). Finally, 6a-e condensed with oseltamivir to synthesize compounds L8-L9 and L14-L16. The specific conditions for each reaction are as follows:

[0057] Reaction c: Starting with compounds 1a-g, the reaction was carried out in dry dichloromethane (DCM) with the corresponding HyT-NH2, HATU, and DIPEA at room temperature for 4 hours to give compounds 2a-g.

[0058] Reaction d: To remove the Boc protecting group, the corresponding Boc compound (1.0 equivalent) was dissolved in a solvent, and then a 4M hydrochloric acid / 1,4-dioxane solution (10.0 equivalent) was added at room temperature. The mixture was stirred at room temperature for 2 hours, and the reaction progress was monitored using thin-layer chromatography (TLC). After removing the dioxane under reduced pressure, no further treatment was required, and the next reaction was carried out directly. Thus, compounds 2a-g reacted to give compounds 3a-g.

[0059] Reaction e: Oseltamivir (1.0 equivalent) and triethylamine (1.0 equivalent) were dissolved in DMF under nitrogen atmosphere. Then, CDI (1.0 equivalent) was added at room temperature, and the mixture was stirred for 1 hour. Next, a DMF solution of the corresponding amine (1.1 equivalent) was added, and stirring continued for 10 hours. After the reaction was complete, the mixture was poured into water and extracted with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain the crude product, which was then purified by column chromatography to obtain the target product. That is, compounds 3a-g were reacted in the following two steps to obtain compounds L7, L10-L13, and L17-L19: 1) Oseltamivir, TEA, and CDI were reacted in DMF (N,N-dimethylformamide) for 1 hour; 2) The reaction was continued at room temperature for 10 hours.

[0060] Reaction f: Starting with compounds 4a-e, 1-iminomethyladamantaneamine, HATU, and DIPEA were reacted in dry DCM for 4 hours to give compounds 5a-e.

[0061] Reaction g: In a single-necked flask, the corresponding ester (1.0 equivalent), lithium hydroxide (5.0 equivalent), and water were added, and the reaction was carried out at 50°C for 4 hours. Thin-layer chromatography (TLC) analysis showed that the reaction was complete. The solution was acidified to pH 1, resulting in a precipitate. The precipitate was filtered and dried to obtain the target product. Thus, compounds 5a-e reacted to give compounds 6a-e.

[0062] Reaction h: In a round-bottom flask, the corresponding carboxylic acid starting material (1.0 equivalent) was dissolved in anhydrous dichloromethane. Then, HATU (1.2 equivalent), DIPEA (2.0 equivalent), and oseltamivir (1.1 equivalent) were added sequentially. The reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction mixture was poured into water, the organic layer was separated, and washed with saturated brine. The solution was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Thus, compounds 6a-e reacted to give compounds L8-L9 and compounds L14-L16.

[0063] Compounds L7–L19 were synthesized according to the overall synthetic route. Specifically, the synthetic methods for each compound or its intermediates are as follows:

[0064] Compound 2a—tert-butyl(5-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-5-oxopentyl)carbamate: Starting from Boc-5-aminopentanoic acid (2.0 g, 9.21 mmol) and 5-norbornen-2-methylamine (1.24 g, 10.13 mmol), a pale yellow solid 2a (1.22 g, yield 41.23%) was obtained.

[0065] Compound 3a—5-amino-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)pentanamide: gave a pale yellow oil 3a (0.81 g, yield 96.36%).

[0066] Compound L7—ethyl(3R,4R,5R)-4-acetamido-5-[3-(5-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-5-oxopentyl)ureo]-3-(pent-3-oxy)cyclohex-1-en-1-carboxylic acid ester: Starting with oseltamivir (0.77 g, 2.47 mmol) and 3a (0.5 g, 2.25 mmol), a white solid L7 (0.68 g, yield 54.34%) was obtained. 1 HNMR(600MHz,DMSO-d6)δ7.82(d,J=8.4Hz,1H),7.73(t,J=5.7Hz,1H),6.63(t,J=2.5Hz,1H),6.14(dd,J=5.7,3.0Hz,1H),6.06 (t,J=5.6Hz,1H),5.95(dd,J=5.8,2.8Hz,1H),5.58(d,J=7.7Hz,1H),4.16–4.09(m,2H),4.06–4.00(m,1H),3.76–3.66(m,2H),3 .41–3.96(m,1H),3.01–2.88(m,2H),2.82–2.77(m,1H),2.75(d,J=13.7Hz,1H),2.66–2.58(m,2H),2.18–2.11(m,1H),2.07–1. 99(m,3H),1.79(s,3H),1.54–1.28(m,10H),1.28–1.19(m,5H),1.14–1.00(m,1H),0.84(t,J=7.4Hz,3H),0.77(t,J=7.4Hz,3H). 13 CNMR(151MHz,DMSO-d6)δ171.2,171.0,169.0,165.0,157.2,137.6,136.3,135.9,13 5.8,131.9,129.1,128.1,80.5,74.7,59.8,53.0,48.3,47.6,44.0,43.1,43.1,43.0, 42.0,41.3,40.6,39.5,38.4,38.2,37.8,37.3,34.5,31.0,30.6,29.8,29.2,29.0,2 8.0,26.0,25.1,24.6,22.2,22.2,20.5,19.6,13.5,13.3,8.8,8.4.HRMS(ESI):[M+H]+ calculated for C 30 H 49 N4O6:561.3652, found:561.3638.

[0067] Compound 5a—methyl 8-[((3r,5r,7r)-adamantane-1-yl)methylamino]-8-oxooctanoate: Starting from monomethyl octanoate (2.0 g, 10.6 mmol) and 1-adamantane methylamine (1.92 g, 11.66 mmol), a pale yellow solid 5a (1.71 g, yield 48.23%) was obtained.

[0068] Compound 6a—8-[((3r,5r,7r)-adamantane-1-yl)methylamino]-8-oxooctanoic acid: 6a (1.39 g, 84.96%) was obtained as a pale yellow oil.

[0069] Compound L8—ethyl(3R,4R,5R)-4-acetamido-5-[8-[((3r,5r,7r)-adamantane-1-yl)methylamino]-8-oxooctamido]-3-(pent-3-oxy)cyclohex-1-ene-1-carboxylic acid ester: Starting from 6a (0.5 g, 1.55 mmol) and oseltamivir (0.53 g, 1.7 mmol), a white solid L8 (0.49 g, yield 51.43%) was obtained. 1 HNMR (600MHz, DMSO-d6) δ7.84(dd,J=9.4,5.8Hz,1H),7.73(t,J=5.7Hz,1H),7.64(t,J=8.5Hz,1H),6.64(t,J=2.5Hz,1H),6.19–6.08(m, 1H),5.95–5.95(m,1H),4.16–4.10(m,2H),4.09–4.08(m,1H),3.95–3.88(m,1H),3.79–3.72(m,1H),3.41–3.36(m,1H),3.35–3.29(m,2H ),2.83–2.78(m,1H),2.78–2.72(m,2H),2.67–2.59(m,1H),2.50–2.45(m,1H),2.24–2.13(m,2H),2.07–1.98(m,4H),1.79–1.72(m,4H), 1.51–1.39(m,8H),1.38–1.33(m,1H),1.31–1.28(m,1H),1.26–1.17(m,8H),0.86–0.81(m,4H),0.77(t,J=7.4Hz,3H),0.46–0.42(m,1H). 13C NMR(151MHz,DMSO-d6)δ172.4,172.3,172.2,169.8,165.9,138.9,137.3,137 .0,136.8,133.0,129.0,81.6,75.6,60.9,54.2,49.4,47.6,45.1,44.2,44.1 ,44.1,43.0,42.4,41.6,40.5,39.3,38.9,36.2,35.9,35.9,30.8,30.2,29.2 ,29.1,29.1,26.2,25.8,25.8,25.7,23.3,14.5,9.9,9.4.HRMS(ESI):[M+Na] + calculated for C 35 H 57 N3O6Na: 638.4145, found: [M+Na] + :638.4132.

[0070] Compound 5b—methyl 8-[(bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino]-8-oxooctanoate: Starting from monomethyl octanoate (2.0 g, 10.6 mmol) and 5-norbornen-2-methylamine (1.43 g, 11.66 mmol), a pale yellow solid 5b (1.54 g, yield 49.73%) was obtained.

[0071] Compound 6b—8-[(bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino]-8-oxooctanoic acid: 6b (1.3 g, 88.46%) was obtained as a pale yellow oil.

[0072] Compound L9—ethyl(3R,4R,5R)-4-acetamido-5-[8-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-8-oxooctamido]-3-(pent-3-oxy)cyclohex-1-en-1-carboxylic acid ester: Starting from 6b (0.5 g, 1.79 mmol) and oseltamivir (0.53 g, 1.7 mmol), a white solid L9 (0.49 g, yield 51.43%) was obtained. 1HNMR (600MHz, DMSO-d6) δ7.84(d,J=9.3Hz,1H),7.64(d,J=9.0Hz,1H),7.59(t,J=6.3Hz,1H),6.64(t,J=2.6Hz,1H),4.17–4.1 1(m,2H),4.09–4.06(m,1H),3.95–3.88(m,1H),3.77–3.72(m,1H),3.40–3.36(m,1H),2.75(d,J=6.2Hz,2H),2.50–2.44(m,1H) ,2.24–2.17(m,1H),2.09(t,J=7.4Hz,2H),2.02–1.98(m,2H),1.90(dd,J=6.0,3.2Hz,3H),1.77(s,3H),1.67–1.62(m,3H),1. 60–1.54(m,3H),1.48–1.44(m,4H),1.41(d,J=3.0Hz,6H),1.22(t,J=7.1Hz,5H),0.84(t,J=7.4Hz,3H),0.77(t,J=7.4Hz,3H). 13 C NMR (151MHz, DMSO-d6) δ172.7,172.3,169.8,165.9,138.9,129.0,81.6,75.6,60.9,54.2,54.1,50.5,47.7,40.5,40.3,38.7,3 7.0,36.2,35.9,34.1,30.8,29.0,28.9,28.2,26.8,26.2,25.9,25.8,25.7,23.2,18.5,17.2,14.5,9.9,9.4.HRMS(ESI):[M+H] + Calculated for C 32 H 52 N3O6: 574.3856, found: 574.3847.

[0073] Compound 2b—tert-butyl(9-(((1r,3R,5S,7r)-3,5-dimethyladamantane-1-yl)amino)-9-oxonyl)carbamate: Starting from Boc-9-aminononanoic acid (2.0 g, 7.32 mmol) and memantine (1.44 g, 8.05 mmol), a pale yellow solid 2b (1.31 g, yield 41.73%) was obtained.

[0074] Compound 3b—9-amino-N-((1r,3R,5S,7r)-3,5-dimethyladamantane-1-yl)nonanoamide: 0.96 g of pale yellow oily substance 3b was obtained (yield 96.46%).

[0075] Compound L10—ethyl(3R,4R,5R)-4-acetamido-5-(8-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-8-oxooctamido)-3-(3-pentoxy)cyclohex-1-en-1-carboxylic acid ester: L10 was obtained as a white solid (0.41 g, 41.43% yield) from oseltamivir phosphate (0.51 g, 1.63 mmol) and 3b (0.5 g, 1.49 mmol). 1 HNMR(600MHz,DMSO-d6)δ7.88(d,J=7.6Hz,1H),7.23(s,1H),6.62(s,1H),6.07(s,1H),5.61(d,J=6.8Hz,1H),4.18– 4.08(m,2H),4.05(d,J=6.5Hz,1H),3.71(d,J=6.9Hz,2H),3.40–3.35(m,1H),2.99–2.94(m,1H),2.92–2.85(m,1H), 2.62–2.55(m,1H),2.04(s,2H),1.97(t,J=7.3Hz,2H),1.78(s,3H),1.74–1.70(m,2H),1.55(t,J=9.1Hz,4H),1.46– 1.37(m,6H),1.34–1.30(m,3H),1.21(t,J=6.5Hz,12H),1.07(s,2H),0.83(t,J=7.3Hz,4H),0.78(d,J=13.4Hz,10H). 13 C NMR(151MHz,DMSO-d6)δ172.1,170.0,166.1,158.2,138.7,129.2,81.6,75.8,60.9,54.1,52.5,50.8,48.6,47.6,42.8,4 0.5,36.6,32.3,32.1,30.6,30.4,30.0,29.3,29.2,29.0,26.8,26.2,25.9,25.7,23.3,14.5,9.9,9.5.HRMS(ESI):[M+H] + Calculated for C 38 H 65 N4O6:673.4904, found:673.4985.

[0076] Compound L11—(3R,4R,5S)-4-acetamido-5-[3-(9-(((1r,3R,5S,7r)-3,5-dimethyladamantane-1-yl)amino)-9-oxonyl)ureo]-3-(3-pentoxy)cyclohex-1-ene-1-carboxylic acid: L11 was obtained as a white solid (0.21 g, 41.43%), starting from L10 (0.5 g, 1.49 mmol). 1 HNMR (600MHz, DMSO-d6) δ12.53(s,1H),7.81(d,J=8.6Hz,1H),7.22(s,1H),6.61(t,J=2.5Hz,1H),6.03(t,J=5.7Hz,1H),5.5 5(d,J=7.9Hz,1H),4.04–3.99(m,1H),3.75–3.66(m,2H),3.40–3.36(m,1H),3.01–2.96(m,1H),2.91–2.86(m,1H),2.58(dd,J =17.1,4.5Hz,1H),2.06–2.02(m,1H),2.02–1.95(m,3H),1.79(s,3H),1.73(d,J=3.1Hz,2H),1.56(t,J=9.2Hz,4H),1.47–1. 40(m,5H),1.35–1.28(m,4H),1.23(d,J=9.4Hz,11H),1.09(s,2H),0.84(t,J=7.4Hz,3H),0.80(s,6H),0.78(t,J=7.4Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ172.1,170.0,167.8,158.3,138.2,129.8,81.5,75.9,54.3,52.5,52.4,50.8,48.7,47.6,47.5,4 2.8,40.5,36.6,32.3,32.2,30.6,30.4,30.0,29.3,29.2,29.0,26.8,26.2,25.9,25.7,23.3,9.9,9.5.HRMS(ESI):[M+H] + Calculated for C 36 H 61 N4O6:645.4591, found:645.4578.

[0077] Compound 2c—tert-butyl(9-((((3r,5r,7r)-adamantane-1-yl)methyl)amino)-9-oxonyl)carbamate: 2c was obtained as a pale yellow solid (1.42 g, 44.37%), starting from Boc-9-aminononanoic acid (2.0 g, 7.32 mmol) and 1-adamantanemethylamine (1.2 g, 8.05 mmol).

[0078] Compound 3c—N-((((3r,5r,7r)-adamantane-1-yl)methyl)-9-aminononanoamide: 3c was obtained as a pale yellow oil (1.05 g, 96.78%).

[0079] Compound L12—ethyl(3R,4R,5R)-4-acetamido-5-[3-(9-((((3r,5r,7r)-adamantane-1-yl)methyl)amino)-9-oxonyl)carbamoyl]-3-(pent-3-oxy)cyclohex-1-ene-1-carboxylic acid ester: L12 was obtained as a white solid (0.41 g, 40.63%) from oseltamivir (0.53 g, 1.71 mmol) and 3c (0.5 g, 1.56 mmol). 1 H NMR (600MHz, DMSO-d6) δ8.41(t,J=9.0Hz,1H),8.18(t,J=6.3Hz,1H),7.23(d,J=2.5Hz,1H),6.62(t,J=5.7Hz,1H),6.1 6(d,J=7.7Hz,1H),4.76–4.68(m,2H),4.64–4.59(m,1H),4.35–4.24(m,2H),4.01–3.95(m,1H),3.91(d,J=14.2Hz,2H), 3.61–3.45(m,2H),3.34(d,J=6.2Hz,2H),3.20(dd,J=17.3,4.4Hz,1H),3.12–3.04(m,3H),2.70–2.56(m,3H),2.39(d,J =17.2Hz,3H),2.28–2.12(m,5H),2.11–1.88(m,14H),1.86–1.78(m,10H),1.43(t,J=7.4Hz,3H),1.37(t,J=7.3Hz,3H). 13C NMR (151MHz, DMSO-d6) δ171.8,169.1,165.2,157.3,137.8,128.3,80.7,74.9,60.0,53.2,49.6,47.7,3 6.1,35.0,33.2,31.2,29.5,28.2,27.3,25.9,25.3,25.1,24.8,22.3,13.6,9.0,8.6.HRMS(ESI):[M+H] + Calculated for C 37 H 63 N4O6:659.4748, found:659.4740.

[0080] Compound 2d—tert-butyl(9-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-9-oxonyl)carbamate: Starting from Boc-9-aminononanoic acid (2.0 g, 7.32 mmol) and 5-norbornen-2-methylamine (0.99 g, 8.05 mmol), 2d was obtained as a pale yellow solid (1.39 g, 45.89%).

[0081] Compound 3d—9-amino-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)nonanoamide: 3d was obtained as a pale yellow oil (1.0 g, 97.45%).

[0082] Compound L13—ethyl(3R,4R,5R)-4-acetamido-5-[3-(9-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-9-oxonyl)ureo]-3-(pent-3-oxy)cyclohex-1-en-1-carboxylic acid ester: Starting with oseltamivir (0.53 g, 1.97 mmol) and 3d (0.5 g, 1.79 mmol), a white solid L13 (0.42 g, 38.63%) was obtained. 1HNMR(600MHz,DMSO-d6)δ8.37–8.12(m,2H),7.17–7.06(m,1H),6.58–6.40(m,1H),6.05( d,J=8.1Hz,1H),4.68–4.57(m,2H),4.56–4.44(m,2H),4.27–4.12(m,2H),3.92–3.85(m, 1H),3.84–3.75(m,3H),3.51–3.43(m,1H),3.42–3.28(m,2H),3.12–3.05(m,1H),3.03–2 .91(m,3H),2.84–2.67(m,2H),2.42–2.14(m,5H),2.07–1.61(m,20H),1.39–1.20(m,6H). 13 C NMR (151MHz, DMSO-d6) δ174.0,170.0,166.0,158.2,140.0,139.5,138.7,129.1,129.1,127.4,81.5,75.7,70.4,70.0,7 0.0,69.4,60.8,54.2,48.6,44.9,44.7,39.0,31.9,30.0,26.1,25.7,23.2,22.6,19.0,14.5,9.8,9.4.HRMS(ESI):[M+H] + Calculated for C 34 H 57 N4O6:617.4278, found:617.4262.

[0083] Compound 5c—methyl 9-[((3r,5r,7r)-adamantane-1-yl)methyl]amino-9-oxononanoic acid ester: Starting from 9-methoxy-9-oxononanoic acid (2.0 g, 9.9 mmol) and 1-adamantanemethylamine (1.79 g, 10.89 mmol), a pale yellow solid 5c (1.61 g, 46.82%) was obtained.

[0084] Compound 6C—9-[((3r,5r,7r)-adamantane-1-yl)methyl]amino-9-oxononanoic acid: gave a pale yellow oil 6C (1.31 g, 84.68%).

[0085] Compound L14—ethyl(3R,4R,5R)-4-acetamido-5-[9-((((3r,5r,7r)-adamantane-1-yl)methyl)amino)-9-oxononanoylamino]-3-(pent-3-oxy)cyclohex-1-ene-1-carboxylic acid ester: Starting from 6c (0.5 g, 1.49 mmol) and oseltamivir (0.53 g, 1.64 mmol), a white solid L14 (0.37 g, 39.73%) was obtained. 1 H NMR (400MHz, DMSO-d6) δ7.83 (dd, J=9.2, 2.5Hz, 1H), 7.66–7.56 (m, 2H), 6.64 (d, J=2.4Hz, 1H), 4.20–4 .04(m,3H),3.97–3.86(m,1H),3.79–3.69(m,1H),3.42–3.36(m,1H),3.34(s,2H),2.75(d,J=6.2Hz,2 H),2.24–2.15(m,1H),2.09(t,J=7.3Hz,2H),2.01(t,J=7.3Hz,2H),1.94–1.87(m,3H),1.76(s,3H),1 .69–1.53(m,6H),1.51–1.38(m,13H),1.26–1.20(m,9H),0.84(t,J=7.4Hz,3H),0.77(t,J=7.3Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ172.7,172.3,165.9,138.9,129.0,81.6,75.6,60.9,54.2,47.7,35.8, 34.1,30.8,29.0(d,J=4.2Hz),28.2,26.2,26.0,25.8,25.7,23.3,14.5,9.4.HRMS(ESI):[M+H] + Calculated for C 36 H 60 N3O6: 630.4482, found: 630.4470.

[0086] Compound 5d—methyl 9-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-9-oxononanoic acid ester: Starting from 9-methoxy-9-oxononanoic acid (2.0 g, 9.9 mmol) and 5-norbornene-2-methylamine (1.33 g, 10.89 mmol), a pale yellow solid 5d (1.38 g, 45.96%) was obtained.

[0087] Compound 6d—9-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-9-oxononanoic acid: gave a pale yellow oil 6d (1.06 g, 80.68%).

[0088] Compound L15—ethyl(3R,4R,5R)-4-acetamido-5-(9-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-9-oxononamido)-3-(pent-3-oxy)cyclohex-1-en-1-carboxylic acid ester: Starting from 6d (0.5 g, 1.7 mmol) and oseltamivir (0.53 g, 1.64 mmol), a white solid L15 (0.24 g, 41.73%) was obtained. 1 HNMR (600MHz, DMSO-d6) δ7.83(dd,J=9.3,4.8Hz,1H),7.72(t,J=5.7Hz,1H),7.63(t,J=9.5Hz,1H),6.63(d,J=2.5Hz,1H),6.14(dd,J=5.7,3.0Hz,1 H),5.94(dd,J=5.7,2.8Hz,1H),4.18–4.11(m,2H),4.07(dd,J=7.3,3.5H z,1H),3.94–3.87(m,1H),3.77–3.71(m,1H),3.40–3.36(m,1H),2.82–2.7 8(m,1H),2.75(d,J=11.1Hz,1H),2.65–2.58(m,1H),2.47(dd,J=17.5,5. 4Hz,1H),2.24–2.13(m,2H),2.08–1.95(m,4H),1.76(d,J=2.4Hz,4H),1.5 0–1.40(m,7H),1.38–1.33(m,1H),1.30(dd,J=8.1,2.0Hz,1H),1.27–1.17 (m,11H),1.15–1.07(m,1H),0.84(t,J=7.4Hz,3H),0.76(t,J=7.4Hz,3H). 13C NMR (151MHz, DMSO-d6) δ171.2,171.1,168.7,164.9,137.8,136.3,135.9,135.8,131. 9,128.0,80.5,80.5,74.6,59.8,53.2,53.2,48.3,46.6,44.0,43.1,43.1,43.0,41.9 ,41.3,40.6,39.5,38.2,37.9,35.1,34.8,33.0,29.8,29.2,28.0,28.0,27.9,27.8,2 5.1,24.8,24.8,24.7,24.6,23.9,22.2,20.5,19.6,13.5,8.8,8.4.HRMS(ESI):[M+Na] + Calculated for C 33 H 53 N3O6Na:610.3832,found:610.3823.

[0089] Compound 5e-methyl 10-((((3r,5r,7r)-adamantane-1-yl)methyl)amino)-10-oxodecanoate: Starting from 10-oxo-10-methoxydecanoic acid (2.0 g, 9.25 mmol) and 1-adamantanemethylamine (1.68 g, 10.18 mmol), a pale yellow solid 5e (1.67 g, 49.96%) was obtained.

[0090] Compound 6e-10-((((3r,5r,7r)-adamantane-1-yl)methyl)amino)-10-oxodecanoic acid: gave a pale yellow oil 6e (1.28 g, 79.68%).

[0091] Compound L16—ethyl(3R,4R,5R)-4-acetamido-5-(10-((((3r,5r,7r)-adamantane-1-yl)methyl)amino)-10-oxodecanoylamino)-3-(pent-3-oxy)cyclohex-1-ene-1-carboxylic acid ester: Starting from 6e (0.5 g, 1.43 mmol) and oseltamivir (0.49 g, 1.57 mmol), a white solid L16 (0.42 g, 45.73%) was obtained. 1HNMR (600MHz, DMSO-d6) δ7.83(dd,J=9.5,3.6Hz,1H),7.63(t,J=7.3Hz,1H),7.59(t,J=6.3Hz,1H),6.64(t,J=2.5Hz,1H),4. 17–4.11(m,2H),4.09–4.06(m,1H),3.95–3.88(m,1H),3.78–3.72(m,1H),3.41–3.36(m,1H),3.33(s,2H),2.75(d,J=6.3Hz,2 H),2.49–2.44(m,1H),2.23–2.16(m,1H),2.09(t,J=7.3Hz,2H),2.00–1.97(m,2H),1.93–1.89(m,3H),1.76(s,3H),1.65(d, J=12.0Hz,3H),1.57(d,J=11.7Hz,3H),1.48–1.40(m,12H),1.29–1.19(m,12H),0.84(t,J=7.4Hz,3H),0.77(t,J=7.4Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ172.7,172.3,169.8,138.9,129.0,81.6,75.6,60.9,54.2,50.4,47.7,40.3,37.0,3 6.2,35.9,34.1,30.8,29.3,29.1,29.1,28.2,26.2,26.0,25.9,25.7,23.2,14.5,9.9,9.4.HRMS(ESI):[M+H] + Calculated for C 37 H 62 N3O6:644.4639, found:644.4629.

[0092] Compound 2e—tert-butyl(2-(2-(2-(((((3r,5r,7r)-adamantane-1-yl)methyl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamate: Starting from 8-tert-butoxycarbonylamino-3,6-dioxooctanoic acid (2.0 g, 7.6 mmol) and 1-adamantanemethylamine (1.68 g, 8.36 mmol), a pale yellow solid 2e (1.67 g, 53.69%) was obtained.

[0093] Compound 3e-N-((((3r,5r,7r)-adamantane-1-yl)methyl)-2-(2-(2-aminoethoxy)ethoxy)acetamide: yielded a pale yellow oil 3e (1.23 g, 97.68%).

[0094] Compound L17—ethyl(3R,4R,5R)-4-acetamido-5-(3-(2-(2-(2-((((3r,5r,7r)-adamantane-1-yl)methyl)amino)-2-oxoethoxy)ethoxy)ethyl)ureo)-3-(pent-3-oxy)cyclohex-1-en-1-carboxylic acid ester: Starting from oseltamivir (0.5 g, 1.6 mmol) and 3e (0.54 g, 1.76 mmol), a white solid L17 (0.43 g, 41.73%) was obtained. 1 H NMR (600MHz, DMSO-d6) δ7.93(d,J=6.9Hz,1H),7.51(t,J=6.3Hz,1H),6.61(s,1H),6.20(s,1H),5.83(d,J=6.6Hz ,1H),4.16–4.03(m,3H),3.90(s,2H),3.73–3.66(m,2H),3.61–3.52(m,4H),3.39(s,3H),3.16–3.09(m,2H),2.81 (d,J=6.2Hz,2H),2.64–2.56(m,1H),2.03(dd,J=17.8,7.5Hz,1H),1.89(s,3H),1.78(s,3H),1.63(d,J=11.9Hz, 3H),1.55(d,J=11.9Hz,3H),1.48–1.31(m,10H),1.24–1.21(m,3H),0.82(t,J=7.3Hz,3H),0.76(t,J=7.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ170.0,169.8,166.0,158.3,138.8,129.1,81.5,75.7,70.8,70.6,70.3,69.8,6 0.9,54.2,50.0,48.6,40.3,37.0,34.2,32.0,28.1,26.2,25.7,23.3,14.6,9.9,9.5.HRMS(ESI):[M+H] + Calculated for C 34 H 57 N4O8:649.4176, found:649.4167.

[0095] Compound 2f—tert-butyl(2-(2-(2-(((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamate: Starting from 8-tert-butoxycarbonylamino-3,6-dioxooctanoic acid (2.0 g, 7.6 mmol) and 5-norbornene-2-methylamine (1.02 g, 8.36 mmol), a pale yellow solid 2f (1.55 g, 55.69%) was obtained.

[0096] Compound 3f—starting from 8-tert-butoxycarbonylamino-3,6-dioxooctanoic acid (2.0 g, 7.6 mmol) and 5-norbornene-2-methylamine (1.02 g, 8.36 mmol)—gave a pale yellow solid 2f (1.55 g, 55.69%). Compound 3f—gave a pale yellow oil 3f (1.06 g, 93.78%).

[0097] Compound L18—ethyl(3R,4R,5R)-4-acetamido-5-[3-(2-(2-(2-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamoyl]-3-(pentane-3-oxy)cyclohex-1-en-1-carboxylic acid ester: Starting from oseltamivir (0.5 g, 1.86 mmol) and 3f (0.55 g, 2.05 mmol), a white solid L18 (0.55 g, 49.43%) was obtained. 1 H NMR (600MHz, DMSO-d6) δ7.84(d,J=8.2Hz,1H),6.63(s,1H),6.14(t,J=5.6Hz,1H),5.79(d,J=7.4Hz,1H),4.16–4.09(m,J=10 .9,4.9Hz,2H),4.08–3.97(m,2H),3.86(d,J=5.3Hz,2H),3.79–3.69(m,2H),3.55(dd,J=9.7,4.8Hz,4H),3.43–3.29(m,4H), 3.13(h,J=7.4Hz,2H),2.61(dd,J=17.2,4.2Hz,1H),2.31–2.24(m,1H),2.20(t,J=3.9Hz,1H),2.08–1.94(m,2H),1.83–1.66 (m,5H),1.53(dd,J=11.9,5.4Hz,1H),1.49–1.31(m,6H),1.22(t,J=7.0Hz,4H),0.83(t,J=7.6Hz,4H),0.77(t,J=7.3Hz,3H). 13C NMR(151MHz,DMSO-d6)δ170.0,169.6,169.4,166.0,158.3,138.8,129.1,81.6,81.5,75.9,75.8,70 .7,70.7,70.5,70.4,70.4,69.8,62.8,62.5,60.9,54.2,53.9,48.8,48.6,48.6,46.6,46.2,43.6,4 2.9,42.8,40.8,39.7,39.2,38.9,38.4,36.9,36.5,34.7,33.6,32.1,32.0,31.8,31.5,29.0,26.8, 26.2,26.1,25.7,25.7,25.3,23.3,23.3,23.0,21.1,19.7,14.5,11.7,9.9,9.5.HRMS(ESI):[M+Na] + Calculated for C 31 H 50 N4O8:629.3526, found:629.3513.

[0098] Compound 2 g – tert-butyl(2-(2-(2-(((1r,3R,5S,7r)-3,5-dimethyladamantane-1-yl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamate: Starting from 8-tert-butoxycarbonylamino-3,6-dioxooctanoic acid (2.0 g, 7.6 mmol) and memantine (1.49 g, 8.36 mmol), 2 g (1.94 g, 60.29%) of a pale yellow solid was obtained.

[0099] Compound 3g—2-(2-(2-aminoethoxy)ethoxy)-N-((1r,3R,5S,7r)-3,5-dimethyladamantane-1-yl)acetamide: yielded 3g (1.44 g, 97.28%) of a pale yellow oil.

[0100] Compound L19—ethyl(3R,4R,5R)-4-acetamido-5-(3-(2-(2-(2-(((1r,3R,5S,7r)-3,5-dimethyladamantane-1-yl)amino)-2-oxoethoxy)ethoxy)ethyl)ureo)-3-(pent-3-oxy)cyclohex-1-ene-1-carboxylic acid ester: Starting from oseltamivir (0.5 g, 1.86 mmol) and 3 g (0.66 g, 2.05 mmol), a white solid L19 (0.56 g, 45.73%) was obtained. 1H NMR (600MHz, DMSO-d6) δ7.81(d,J=8.5Hz,1H),6.87(s,1H),6.64(t,J=2.6Hz,1H),6.12(t,J=5.7Hz,1H),5.81(d,J=7.9Hz,1 H),4.17–4.10(m,2H),4.05–4.02(m,1H),3.76(s,2H),3.75–3.70(m,2H),3.57–3.50(m,4H),3.42–3.36(m,3H),3.19–3.09( m,2H),2.62(dd,J=16.8,4.0Hz,1H),2.06–2.01(m,1H),1.79(d,J=6.3Hz,5H),1.62(d,J=11.7Hz,2H),1.57(d,J=11.8Hz,2H ),1.45–1.40(m,2H),1.34–1.30(m,2H),1.27–1.20(m,6H),1.11(d,J=2.4Hz,2H),0.87–0.80(m,9H),0.78(t,J=7.4Hz,3H). 13 C NMR (151MHz, DMSO-d6) δ169.9,168.7,166.0,158.2,138.8,129.1,81.6,75.8,70.7,70.6,70.4,69.7,60.9,54.2,52.7,50.6,48.6,47.5,4 2.7,40.5,39.9,32.3,32.1,30.4,30.0,29.1,27.0,26.2,25.7,25.0,23.3,22.6,14.5,14.4,9.9,9.5.ESI-MS:m / z606.36HRMS(ESI):[M+H] + Calculated for C 35 H 59 N4O8:663.4333, found:663.4322.

[0101] The specific structures of compounds L1 to L19 are as follows:

[0102]

[0103]

[0104] Example 2

[0105] In vitro anti-influenza virus assay in MDCK cells:

[0106] The anti-influenza virus activity of the compound was assessed by infecting MDCK cells with influenza A virus H1N1 (A / Puerto Rico / 8 / 1934, containing NA-H274Y, where NA-H274Y refers to a specific mutation in the neuraminidase (NA) gene, specifically the substitution of histidine (His, H) at position 274 with tyrosine (Tyr, Y). MDCK cells (2.5 × 10^4 cells / well) were added to 96-well plates and incubated overnight at 37°C and 5% CO2. Cells were then infected with 50% tissue culture infectious dose (TCID50) of influenza virus for 1 hour. After discarding the virus solution, serially diluted versions of the compound were added, and incubation continued for 48 hours. At the end of incubation, 100 μL of Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies) reagent solution (composed of 90 μL culture medium and 10 μL CCK-8) was added to each well. After incubation at 37°C for 90 minutes, absorbance was read at 450 nm using a microplate reader. EC was determined by fitting a curve of cytopathic effect (CPE) percentage versus NA inhibitor concentration using GraphPad Prism6 software. 50 Value. CC 50 The method for determining the value is the same as for EC50, but without viral infection. The entire assay was performed at the BSL-2 facility. Test results are shown in Table 1, where NA indicates inactivity, EC50 indicates... 50 The value represents the concentration of a compound that inhibits 50% of plaque formation, CC. 50 The value represents the concentration of a compound that reduces cell viability by 50%.

[0107] Table 1. Anti-H1N1-H274Y virus activity and cytotoxicity of each compound in MDCK cells.

[0108]

[0109]

[0110] The H274Y mutation significantly reduces the efficacy of oseltamivir, posing challenges to clinical treatment. Therefore, we investigated the antiviral activity of compounds L1–L19 against the oseltamivir-resistant H1N1-H274Y virus strain in MDCK cells, using oseltamivir (OSP) as a control. As shown in Table 1, compounds L1–L6 introduced various hydrophobic tags via amide or urea bonds, but exhibited limited activity against the H1N1-H274Y virus strain. To enhance antiviral activity, compounds L7–L16 with alkyl chains were synthesized by extending the carbon chain length linking oseltamivir and the hydrophobic tag. These compounds exhibited significant antiviral efficacy, with the adamantane-tagged derivative showing superior activity compared to oseltamivir; in particular, compound L12, with a nine-carbon chain linking a hydrophobic tag, showed 157 times greater efficacy than oseltamivir, with an EC50 of [missing value]. 50 The concentration was 0.68 μM; L14 also connects to the hydrophobic tag via a nine-carbon chain, exhibiting 57 times the potency of oseltamivir, with its EC50... 50 The concentration was 1.87 μM, indicating that the linker chain length has a crucial impact on antiviral activity. Furthermore, this application is the first to use ammonium nitrate as a hydrophobic tag, demonstrating effective resistance reversal activity in compounds L10, L11, and L19. In addition, the PEG linker (HO-CC-OH) incorporated as a partial linker structure into compounds L17–L19 also exhibited excellent antiviral activity. Cytotoxicity tests using the CCK-8 assay showed that none of the tested compounds exhibited significant cytotoxicity at concentrations up to 50 μM.

[0111] Example 3

[0112] The degradative activity of the compounds against influenza virus neuraminidase (NA) protein was evaluated by Western blot analysis. A recombinant plasmid containing the full-length NA fragment with a flag tag was transfected into 293T cells. 293T cells were incubated at a rate of 6 × 10⁻⁶ cells / year. 5Cells were seeded at a density of cells / well in 6-well plates and cultured at 37°C and 5% CO2 for 12 hours. Subsequently, 1 μg of plasmid DNA was transfected into the cells. After 8 hours, the medium was replaced with fresh medium, and the compound was added and incubated for 24 hours, using DMSO as a solvent control. NA protein levels were then assessed by Western blot: 293T cells were washed twice with pre-chilled PBS and then lysed on ice with RIPA lysis buffer containing a mixture of protease inhibitors for 15 minutes. The lysate was centrifuged at 15,000g for 30 minutes at 4°C, and the supernatant was collected for BCA protein quantification. Protein samples were separated by 4–12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with PBST containing 5% skim milk powder for 1 hour at room temperature and then incubated overnight with primary antibody at 4°C. The primary antibody dilutions were as follows: Flag (1:1000, Proteintech), GAPDH (1:1000, Cell Signaling Technology). After washing with PBST, the membrane was incubated with HRP-bound secondary antibody for 1 hour, followed by washing with PBST again. Bands were visualized using the NcmECLUltra chemiluminescent substrate kit. Chemiluminescence-based blots were captured using an iBright 1500 imaging system (Invitrogen) and analyzed using ImageJ software. Figure 1 This is a graph showing the degradation activity of NA protein in 293T cells treated with different compounds involved in Example 2 of this application. Figure 1 The vertical axis of the bar chart represents the fold change in degradation activity of NA protein; the fold change in degradation activity for the DSMO group is 1. Figure 1 As shown, HyTTDs primarily exert their antiviral effects by degrading the NA protein. At a concentration of 10 μM, compounds L7, L8, L10, L12, L14, and L17 exhibited significant degradation activity against the NA protein. Among them, L12 showed the most significant degradation effect, which is consistent with its potent antiviral activity against H1N1-H274Y virus in vitro.

[0113] Example 4

[0114] To confirm the potent NA degradation and antiviral effects of the compounds against the H1N1-H274Y virus strain, compounds L12 and L14 were analyzed by immunofluorescence to evaluate their in vitro antiviral activity. MDCK cells (2 × 10^4 cells / well) were seeded in 96-well plates and incubated overnight at 37°C and 5% CO2. MDCK cells were infected with the virus at a multiplicity of infection (MOI) of 0.1 for 1 hour. Subsequently, the virus was discarded, and the appropriate concentration of the compound was added and incubated for 24 hours. Afterward, the cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, followed by blocking with 10% BSA for 1 hour. The cells were then incubated overnight at 4°C with influenza virus nucleoprotein (NP) antibody (GeneTex, GTX1236). Next, the cells were incubated with FITC-labeled secondary antibody for 1 hour (Santa Cruz, CA, USA). DAPI nuclear dye was added for staining for 5 minutes. Fluorescence was observed using a fluorescence microscope (Nikon, Japan). Cells were treated with OSP (100 μM) or the test compound at concentrations of 2.5 μM, 5 μM, and 10 μM for 24 hours. Figure 2 This is a diagram showing the NP protein expression results of MDCK cells treated with different compounds, as described in Example 4 of this application. Figure 2 As shown, compounds L12 and L14 exhibited dose-dependent inhibition of viral nucleoprotein (NP) expression. Compared with 100 μM oseltamivir, compounds L12 and L14 showed significantly enhanced inhibitory effects, further confirming their effectiveness against oseltamivir-resistant influenza.

[0115] Example 5

[0116] To investigate the binding modes of these compounds with neuraminidase (NA), molecular docking studies were performed using the Schrodinger software Maestro (PDB:3CL0

[29] ). Molecular docking studies were performed using the Schrodinger Software Maestro. The H274Y-NA crystal structure of the H5N1 strain and the complex bound to the ligand oseltamivir (PDB ID:3CL0) were obtained from the protein data bank. First, the protonation state of the complex was adjusted to be consistent with pH 7.4. The structure of the endonuclease was pretreated, including the addition of missing hydrogen atoms, bond assignment, amino acid residue state generation, removal of water molecules, and optimization of hydrogen bonds. Next, a grid file containing oseltamivir was created by receptor grid generation. Finally, grid docking was performed to assess the binding energy and predict the interaction modes of the compounds, while PyMOL was used for mapping. Figure 3This is a molecular docking result diagram related to Example 5 of this application, using the H5N1 structure (PDB:3CL0) to illustrate the binding mode of L12 and L14 with the H274Y mutant. The H5N1 protein is presented in a white cartoon form, oseltamivir is shown as a green rod model, and hydrogen bonds are represented by dashed lines. Figure 3 In the diagram, part A is the surface view of L12; part B is the surface view of L14; part C is the 3D representation of L12; and part D is the 3D representation of L14. For example... Figure 3 As shown, the oseltamivir moiety of compounds L12 and L14 occupies the active pocket of the NA protein, while the HyT side chain extends into the region of the protein exposed to the solvent at the amino group extension. This modification enhances the hydrophobicity of the protein surface, mimicking misfolding and promoting degradation. Compounds L12 and L14 bind to the NA protein in a similar pattern, where the oxygen atom in the ether bond acts as a hydrogen bond acceptor, forming a hydrogen bond with Asn294. The ester group of the side chain interacts with the hydrophilic pocket formed by Arg118, Arg371, and Arg292, while the amide group of the side chain forms a hydrogen bond with Arg152.

[0117] Example 6

[0118] Pharmacokinetic studies of L12:

[0119] Given that compound L12 exhibits significant inhibitory activity against influenza virus proliferation and can degrade norepinephrine (NA) in vitro, this application further evaluated its pharmacokinetic (PK) characteristics. The study tested three CD1 mice via intravenous injection at a dose of 1.5 mg / kg, with three mice per group (n=3). At the start of the experiment, 1.85 mg of the test compound L12 was placed in a 5 mL glass vial, and 0.352 mL of dimethyl sulfoxide (DMSO) was added to dissolve it, preparing a 5 mg / mL stock solution. Subsequently, 0.342 mL of this stock solution was mixed with 1.026 mL of Solutol, dissolved by stirring, and then 5.472 mL of physiological saline was added to ensure uniform distribution. A colorless, transparent solution was obtained using sonication and vibration techniques, ultimately preparing a 0.25 mg / mL intravenous formulation. Plasma samples were collected at multiple time points after administration: 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours. Blood was collected using EDTA-K2 anticoagulant tubes and incubated on wet ice for at least 15 minutes. Plasma samples were separated by centrifugation at 6000 rpm for 3 minutes at 2–8 °C. Sample analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS), and pharmacokinetic parameters were calculated using WinNonlin 8.2 software. Key parameters are summarized in Table 2, showing the pharmacokinetic parameters of L12 after a single intravenous injection (dose of 1.5 mg / kg) in CD-1 mice. Results are presented as mean ± standard deviation (SD), n = 3. According to Table 2, after a single intravenous injection, L12 reached its maximum plasma concentration (Cmax) of 5910 ng / mL at 4.9 minutes, with a half-life of 0.246 hours and a clearance rate of 939 mL / hr / kg. These moderate pharmacokinetic parameters suggest that this lead compound has the potential to improve its bioavailability through further optimization.

[0120] Table 2. Pharmacokinetics of compound L12 after intravenous injection in CD-1 mice.

[0121]

[0122]

[0123] In this application, a series of oseltamivir-based HyTTDs were screened, and their activity against the oseltamivir-resistant H1N1-H274Y strain was evaluated. Most compounds showed significant activity at submicromolar concentrations, with compounds L12 and L14 exhibiting the strongest effects (EC). 50The effective values ​​were 0.68 μM and 1.87 μM, respectively, significantly superior to oseltamivir. Western blot experiments confirmed that these compounds could induce the degradation of NA protein. Immunofluorescence studies further revealed dose-dependent inhibition of viral nucleoprotein (NP) expression, confirming its mechanism of action through targeting protein degradation. L12 exhibited moderate pharmacokinetic properties.

[0124] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A neuraminidase inhibitor based on hydrophobic tag technology, characterized in that, The structure is shown in equation (Ⅰ): （Ⅰ）； Linker is selected from the following structures: , , n is a natural number between 2 and 10, m is 0 or 1, k is a natural number between 1 and 4, h is a natural number between 1 and 4, and s is 1; HyT is selected from the following structures: , , .

2. The neuraminidase inhibitor according to claim 1, characterized in that, n is a natural number between 6 and 9.

3. The neuraminidase inhibitor according to claim 1, characterized in that, The neuraminidase inhibitor includes at least one of the following compounds L7 to L19: 。 4. A derivative of a neuraminidase inhibitor as described in any one of claims 1 to 3, comprising a pharmaceutically acceptable salt or a deuterated compound.

5. A method for preparing a neuraminidase inhibitor based on hydrophobic tag technology as described in claim 1, characterized in that, include: , or HyT-NH2 was reacted with HATU and DIPEA; hydrochloric acid was then added to remove the Boc protecting group; oseltamivir was then reacted with TEA, CDI and DMF to obtain the neuraminidase inhibitor. or, , or The reaction was carried out with HyT-NH2 under the condition of adding HATU and DIPEA; then lithium hydroxide and water were added and reacted at 50°C; then oseltamivir was reacted with it under the condition of adding HATU and DIPEA to obtain the neuraminidase inhibitor. Where n is a natural number between 2 and 10, k is a natural number between 1 and 4, h is a natural number between 1 and 4, s is 1, and HyT is selected from the following structure: , , .

6. A pharmaceutical composition, characterized in that, It includes the neuraminidase inhibitor as described in any one of claims 1 to 3 or the derivative as described in claim 4, and includes one or more pharmaceutically acceptable excipients.

7. The use of a neuraminidase inhibitor as described in any one of claims 1 to 3 or a pharmaceutical composition as described in claim 6 in the preparation of a medicament for treating and / or preventing influenza virus infection-related diseases, wherein the influenza virus includes influenza A virus H1N1.

8. The use of a neuraminidase inhibitor as described in any one of claims 1 to 3 or a pharmaceutical composition as described in claim 6 in the preparation of a medicament for inhibiting the activity of an influenza A virus carrying the H274Y variant, wherein the influenza A virus includes H1N1.

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