Conjugates, compositions, and methods for treating influenza

Abstract

Conjugates comprising ligands, linkers, and multiple haptens that target viruses, such as influenza, are described. Pharmaceutical compositions comprising the conjugates, and methods for treating or preventing viral infections using the conjugates are also described.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to Patent Application No. 63 / 590,999 filed on 17 October 2023 and Patent Application No. 63 / 700,420 filed on 27 September 2024, both of which are incorporated herein by reference as if they were described in their entirety.

[0002] This disclosure includes a conjugate comprising a plurality of haptens linked to a target ligand for a target protein on a virus or a virus-infected cell, and a composition comprising such a conjugate, such as a pharmaceutical composition, and a method for preparing such a conjugate. In addition, this disclosure further includes a method for treating a viral infection. One such viral infection is influenza. [Background technology]

[0003] Influenza is one of the most life-threatening and widespread diseases, causing approximately 3 to 5 million severe illnesses and 250,000 to 500,000 deaths annually. In addition to causing high morbidity and mortality rates, influenza imposes significant socioeconomic burdens resulting from loss of productivity and medical intervention and treatment.

[0004] Because influenza viruses constantly change through discontinuous and continuous mutations of their antigens, vaccines often lose their effectiveness against mutant strains. The application of currently available chemotherapy is also limited. Approved anti-influenza drugs are either M2 ion channel inhibitors or neuraminidase inhibitors. Examples of M2 ion channel inhibitors include amantadine and rimantadine. These drugs work by blocking the acid-activated M2 ion channel of the virus, thereby inhibiting the release of viral ribonucleoproteins from virions into the host cell substrate. However, both H1N1 and H3N2 viruses currently prevalent in humans are resistant to these inhibitors. Therefore, for example, the Centers for Disease Control and Prevention (CDC) recommends limiting their use due to the rapid emergence of drug resistance. [Overview of the project] [Problems that the invention aims to solve]

[0005] Widely used neuraminidase inhibitors include oseltamivir and zanamivir, which act as competitive inhibitors, binding to the active site of neuraminidase in competition with sialic acid. While these inhibitors are effective against both influenza A and B viruses, they have two major limitations. Firstly, only minimal benefits of neuraminidase inhibitors have been observed in reducing symptom severity and shortening disease duration (0.6–0.7 days out of 7). Secondly, drug resistance is a problem with this class of antiviral agents. An increase in the number of oseltamivir-resistant strains has been observed since the 2007–2008 season. Given the limitations of current anti-influenza chemotherapy, there is an urgent need to develop novel anti-influenza drugs with novel mechanisms of action. [Means for solving the problem]

[0006] Formula I: [ka] (In the formula, T is a radical of a ligand for a target protein of an influenza virus or a virus-infected cell; L 1 , L 2 and L 3 are each independently selected linkers; and A 1 and A 2 are each independently selected hapten radicals), and pharmaceutically acceptable salts or solvates thereof, are described in the present specification.

[0007] One or more compounds of Formula I:

Chemical Formula

[0008] A composition for use in the treatment of influenza and influenza infections, comprising one or more compounds of Formula I:

Chemical Formula

[0009] A unit dose for use in the treatment of influenza and influenza infection, comprising one or more formulas I: [ka] (In the formula, T is a ligand radical for the target protein of the influenza virus or virus-infected cell; L 1 , L 2 and L 3 Each of them is an independently selected linker; and A 1 and A 2 Unit doses described herein include a compound of a hapten (each being a radical of a hapten independently selected) and a therapeutically effective amount of its pharmaceutically acceptable salt or solvate, and optionally one or more diluents, carriers, or excipients, or a combination thereof.

[0010] A method for treating influenza and influenza infection in a host animal, comprising one or more formulas I: [ka] (In the formula, T is a ligand radical for the target protein of the influenza virus or virus-infected cell; L 1 , L 2 and L 3 Each of them is an independently selected linker; and A 1 and A 2 Methods described herein include administering a composition thereof, comprising a compound of a hapten (where each is a radical of a hapten independently selected), a pharmaceutically acceptable salt or solvate thereof, optionally one or more diluents, carriers or excipients, or a combination thereof, or a therapeutically effective unit dose thereof, to a host animal having influenza or an influenza infection.

[0011] In the manufacture of a pharmaceutical product for use in the treatment of influenza and influenza infection in a host animal, one or more formulas I: [ka] (In the formula, T is a ligand radical for the target protein of the influenza virus or virus-infected cell; L 1 , L 2 and L 3 Each of them is an independently selected linker; and A 1 and A 2 Uses of compounds of a hapten (each being an independently selected hapten radical) and their pharmaceutically acceptable salts or solvates, optionally including one or more diluents, carriers or excipients, or combinations thereof, are described herein.

[0012] In the first exemplary embodiment, T is a radical of a neuraminidase inhibitor.

[0013] In a second exemplary embodiment, T is a radical of a hemagglutinin inhibitor.

[0014] In another exemplary embodiment, the hapten recruits endogenous antibodies present in the host animal.

[0015] In another exemplary embodiment, the compounds, compositions and unit doses described herein are used in a method for treating influenza and influenza infection in a host animal, wherein the compounds, compositions and unit doses are administered to the host animal, and after administration, A 1 and A 2 One or more antibodies bind to each of these epitopes.

[0016] In another exemplary embodiment, the compounds, compositions and unit doses described herein are used in a method for treating influenza and influenza infection in a host animal, wherein the compounds, compositions and unit doses are administered to the host animal, and after administration, A 1 and A 2 A different antibody binds to each of these epitopes, or any of those epitopes.

[0017] In another exemplary embodiment, L 1 , L 2 and L 3 Each of these is selected independently and may contain a single divalent atom or a chain of atoms. In another exemplary embodiment, L 2 L 1 to A 1 It may be a bond that connects to L. In another exemplary embodiment, L 3 L 1 to A 2 It could be a bond that connects to something.

[0018] The compounds, compositions, unit doses, methods and uses described herein are useful in the treatment of influenza and influenza infections. Such treatments include prophylactic and preventive measures for host animals, including host animals at risk of influenza and influenza infection, or host animals exposed to influenza and influenza infection, but in which no symptoms of influenza and influenza infection are observed. The compounds, compositions, unit doses, methods and uses described herein are useful, for example, in preventing the onset of influenza and influenza infections and / or reducing the severity of symptoms that subsequently occur in association with influenza and influenza infections.

[0019] The embodiments and other configurations, advantages, and aspects disclosed herein, and how to obtain them, will become apparent in light of the detailed descriptions of various exemplary embodiments of this disclosure below. Such detailed descriptions will be better understood in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0020] [Figure 1-1] This figure shows examples of compounds from the present invention. [Figure 1-2] Continuation of Figure 1-1. [Figure 1-3] Continuation of Figure 1-2. [Figure 1-4] Continuation of Figure 1-3. [Figure 2A] This figure shows efficacy plots for compound 24 and other compounds under various administration routes. [Figure 2B] This figure shows efficacy plots for compound 24 and other compounds under various administration routes. [Figure 3] This is a diagram showing the synthesis scheme of compound 11 used in the synthesis of compound 24. [Figure 4-1] This is a diagram showing the synthesis scheme for compound 24. [Figure 4-2] Continuation of Figure 4-1. [Figure 5]This figure shows a plot of fluorescence intensity as a function of zanamivir-rhodamine. The figure shows that zanamivir-rhodamine binds to neuraminidase with a binding affinity of 8.253 nM. [Figure 6] This figure shows plots of fluorescence intensity as a logarithmic function [concentration](nM) for compound 24 and zanamivir. Figure 6 shows the competitive binding of compound 24 (left panel) and zanamivir (right panel) to neuraminidase-expressing HEK cells. The figure shows the maintenance of affinity to neuraminidase after incorporation into the conjugate compound 24. [Figure 7] Figure 7 shows a plot of TCID50 / mL (tissue culture infectious dose) as a function of dose (mg / kg), from the study in Method Example 2. Figure 7 shows viral titers measured by hemagglutination 24 hours after treatment with Tamiflu (oseltamivir phosphate), vehicle (phosphate-buffered saline, "PBS"), or increasing concentrations of compound 24 (showing four measurements reported using "mg / kg"). Mice treated with compound 24 had a 1 / 100 reduction in infectious virus compared to those treated with vehicle, and a reduction of less than 1 / 10 compared to standard treatment. [Figure 8-1] This figure shows the cytokine response in human PBMCs to various treatments including compound 24 (Method Example 6). [Figure 8-2] Continuation of Figure 8-1. [Figure 9] This figure shows a plot of normalized fluorescence (RFU) as a logarithmic function of the concentration of compound 24. Figure 9 shows the neuraminidase inhibitory activity of compound 24 against multiple influenza strains. [Figure 10] This figure shows a plot of normalized fluorescence (RFU) as a logarithmic function of the concentration of compound 41. Figure 10 shows the neuraminidase inhibitory activity of compound 41 against multiple influenza strains (Method Example 7). [Figure 11]This figure, from Method Example 5, shows graphs of survival rate (%) and body weight (%) as a percentage of days after infection for mice (n=5 / group) that were infected with 10LD-50 influenza A H3N2 / Wisconsin / 15 / 2009 and treated with intraperitoneal administration of human IgG (IVIg(GAMUNEX(registered trademark)-C) at 24 hpi (times after infection) and administration of compound 24 at 48 hpi. [Figure 12] This figure from Method Example 5 shows graphs of cytokine and chemokine levels measured in the lungs of mice from the treatment group (compound 24 or Tamiflu) or the control group. [Figure 13] This figure from Method Example 5 shows graphs of cytokine and chemokine levels measured in the serum of mice from the treatment group (compound 24 or Tamiflu) or the control group. [Figure 14A] This figure shows the in vivo efficacy of compound 24 against seasonal influenza strains A / California / 07 / 2009(H1N1)pdm09, B / Brisbane / 60 / 2008, and A / H3N2 / Wisconsin / 15 / 2009 in mice (N=5 / cohort) infected with a 10×LD50 titer, compared to oseltamivir phosphate and vehicle controls. [Figure 14B] This figure shows viral titers measured by hemagglutination against seasonal influenza strains A / California / 07 / 2009(H1N1)pdm09, A / H3N2 / Wisconsin / 15 / 2009, and B / Brisbane / 60 / 2008 in mice (N=5 / cohort) infected with a 10×LD50 titer 24 hours after in vivo treatment with vehicle (PBS), oseltamivir phosphate, or compound 24. Compound 24 shows a statistically significant improvement compared to both vehicle controls and oseltamivir phosphate-positive controls. [Figure 15A]This figure shows the in vivo efficacy of compound 24 against Tamiflu-resistant influenza A / Hong Kong / 2369 / 2009 (H1N1pdm09) in mice infected with a 10×LD50 titer (N=5 / cohort) compared to oseltamivir phosphate and vehicle controls. [Figure 15B] This figure shows the percentage body weight (B) following administration of compound 24, oseltamivir phosphate, or vehicle-treated mice infected with influenza A / Hong Kong / 2369 / 2009 (H1N1pdm09, oseltamivir phosphate (Tamiflu) resistant). [Figure 15C] This figure shows the viral titer present in the lungs of mice treated with compound 24, oseltamivir phosphate, or vehicle, 24 hours after drug administration and 48 hours after infection with influenza A strain A / Hong Kong / 2369 / 2009 (H1N1pdm09, oseltamivir phosphate (Tamiflu) resistant) with a 10×LD50. [Figure 16] This figure shows the prevention of influenza infection in co-fed guinea pigs by compound 24. [Figure 17A] This figure shows the change in efficacy of compound 24 compared to oseltamivir phosphate after 5 cycles of mutation in vivo, as measured by neuraminidase inhibition. [Figure 17B] This figure shows the change in pathogenicity of influenza virus A / PR8 / 34(H1N1) after 5 in vivo mutation cycles, as measured by LD50 in vivo, compared to oseltamivir phosphate. [Figure 18A] This figure shows IVIS imaging of Group 1 (mock-ups) and Group 2 (infected and vehicle-treated mice). [Figure 18B] This figure from Method Example 12 shows IVIS imaging of Group 3 (infected mice treated with oseltamivir phosphate) and Group 4 (infected mice treated with compound 24). [Figure 19A]This figure shows HEK293 cells infected with a virus by antibody-dependent cell-mediated cytotoxicity (ADCC) induction using compound 24. [Figure 19B] This figure shows HEK293 cells infected with a virus by antibody-dependent phagocytosis (ADCP) induction using compound 24. [Figure 19C] This figure shows HEK293 cells infected with a virus by compound 24 through complement-dependent cell damage (CDC) induction. [Figure 20] This figure shows the neuraminidase inhibitory activity of compound 24 administered intranasally ("IN") against seasonal and drug-resistant influenza A and B strains. [Figure 21] This figure shows the in vivo efficacy-time-dependent survival rates of compound 24 against A / Illinois / 37 / 2018 (H1N1, baloxavir resistant). [Modes for carrying out the invention]

[0021] The following enumerated sections further illustrate the invention as described herein. Each section describes various configurations and aspects of the invention, including ligands, linkers, and haptens, as well as various combinations of those configurations and aspects in many embodiments of the invention. The following enumerated sections are understood to illustrate numerous embodiments of the invention and are not to be construed as limiting the scope of the invention in any way.

[0022] 1. Formula [ka] (In the formula, T is a ligand radical for the target protein of the influenza virus or virus-infected cell. L 1 , L 2 and L 3 Each of these is an independently selected linker, and A 1 and A 2Compounds of (where each is an independently selected hapten radical), or pharmaceutically acceptable salts or solvates thereof. 2. The target protein is neuraminidase, as described in item 1. 3. The ligand is a neuraminidase inhibitor, as described in item 1. 4. The ligand is a compound selected from sialic acid and its analogues, as described in item 1. 5. The ligand is selected from zanamivir, peramivir, laninamivir, oseltamivir, and 2,3-dehydro-2-deoxy-n-acetylneuraminic acid, as well as their analogs and derivatives, as described in item 1. 6. The ligand is zanamivir or its analog or derivative, as described in item 1. 7. The ligand is peramivir, or an analog or derivative thereof, of the compound described in item 1.

[0023] 8. The ligand is, formula [ka] A compound of the compound described in item 1, or an analog or derivative thereof. 9. The ligand is laninamivir, or an analog or derivative thereof, as described in item 1.

[0024] 10. The ligand is, formula [ka] The compound described in item 1, which is an analog or derivative thereof. 11. The ligand is oseltamivir, or an analog or derivative thereof, as described in item 1. 12. The target protein is hemagglutinin, as described in item 1. 13. The ligand is a hemagglutinin inhibitor, as described in item 1.

[0025] 14. Ligands are, [ka] , as well as analogs and derivatives thereof, the compounds described in item 1.

[0026] 15. The compounds described in item 1, wherein the ligand is umifenobil, arbidol, tert-butylhydroquinone, flufirvitide 3 (VEDTKIDLWSYNAELL (SEQ ID NO: 1)), cyclo(Ac-YWHKNKYVLTYSC)LFAAG-CONH2 (SEQ ID NO: 2), cyclo(Ac-YRWVWTSFFSEPYFVVC)G-CONH2 (SEQ ID NO: 3), cyclo(Ac-YLKIYWSKIHGLVSEWC)G-CONH2 (SEQ ID NO: 4), or cyclo(Ac-YVLFRWDHGTLATHWVC)G-CONH2 (SEQ ID NO: 5). 16. The ligand is not a folate, and is one of the compounds listed in item 1. 17.A 1 and A 2 The compound is the same hapten, as described in any one of items 1 to 16. 18.A 1 and A 2 The compound is a different hapten, as described in any one of sections 1 to 16. 19. Haptens are selected from rhamnose, nitrophenyl, nitrophenol, nitroaniline, dinitrophenyl (DNP), dinitrophenol, dinitroaniline, trinitrophenyl (TNP), trinitrophenol, trinitroaniline, chloronitrophenyl, chloronitrophenol, chloronitroaniline, iodonitrophenyl, iodonitrophenol, iodonitroaniline, nitrotyrosine, hydroxynitrotyrosine, aminonitrotyrosine, 4-hydroxy-3-nitrophenylacetic acid, α-galactosyl moiety, sulfated Gal, phosphorylcholine, bacterial antigens, and viral antigens. A 1 and A 2A compound according to any one of items 1 to 18, wherein each is independently selected, comprising rhamnose, an α-galactosyl moiety, dinitrobenzene, dinitroaniline, trinitrobenzene, and a radical of dinitroaniline. 20.A 1 and / or A 2 The compound is a radical of dinitrophenyl (DNP), dinitrophenol, or dinitroaniline, as described in any one of items 1 to 19. 21.A 1 and / or A 2 A compound described in any one of items 1 to 20, which is a radical of rhamnose containing L-rhamnose. 22.A 1 and A 2 One of them is the radical of L-rhamnose, A 1 and A 2 The other is a radical of dinitroaniline, a compound as described in any one of items 1 to 21. 23. A hapten is a compound described in any one of items 1 to 22, having an epitope with endogenous Ab, B, or T cells. 24. Haptens are compounds listed in any one of sections 1-23 that are not FITC. 25L 2 and L 3 is L 1 A compound described in any one of items 1 to 24, which is bonded (attached) to the same atom. 26.L 2 and L 3 is L 1 A compound described in any one of items 1 to 25, which is bound to the same atom shown above. 27.L 2 and L 3 is L 1 A compound described in any one of items 1 to 26, bonded to the same non-carbon atom above. 28.L 2 and L 3 is L 1 A compound described in any one of items 1 to 27, which is bound to the same nitrogen atom above. 29.L2 and L 3 is attached to different atoms of L 1 , The compound according to any one of items 1 to 28. 30. L 1 , L 2 and L 3 one or more of is a single divalent atom selected from N, O, P and S, where N and P are optionally substituted, The compound according to any one of items 1 to 29. 31. L 1 , L 2 and L 3 one or more of is a chain of atoms, the length of each chain is independently selected and ranges from about 2 to about 60, The compound according to any one of items 1 to 30. 32. L 1 , L 2 and L 3 one or more of comprises or further comprises ethoxy, ethylamino, ethylene glycol, azaethylene glycol, (PEG) n , or aza-(PEG) n , or a combination thereof, wherein n is in the range of 2 to about 36, The compound according to any one of items 1 to 31. 33. L 1 , L 2 and L 3 one or more of comprises or further comprises (PEG) n , or aza-(PEG) n , or a combination thereof, wherein n is 2 to 36, The compound according to any one of items 1 to 32. 34. L 1 , L 2 and L 3 each independently comprises or further comprises ethoxy, ethylamino, ethylene glycol, azaethylene glycol, (PEG) n , or aza-(PEG) n , or a combination thereof, wherein n is 2 to 36, The compound according to any one of items 1 to 33. 35. L 1 , L 2 and L3 One or more of the compounds described in any one of items 1 to 34 include or additionally include O-alkyl-O, N-alkyl-N, C(O)-alkyl-C(O), or NC(O)-alkyl-C(O)N, or a combination thereof. 36.L 1 , L 2 and L 3 Compounds described in any one of items 1 to 35, comprising or additionally O-alkyl-C(O), N-alkyl-C(O), O-alkyl-N-alkyl-C(O), N-alkyl-O-alkyl-C(O), or C(O)alkyl-C(O), or a combination thereof. 37.L 1 The compounds described in any one of items 1 to 36, comprising or additionally comprising O-alkyl-C(O)N-diyl, O-alkyl-O-alkyl-C(O)N-diyl, or N-alkyl-O-alkyl-O-alkyl-C(O)N-diyl. 38.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 37, each containing one or more amino acids, or additionally containing them. 39.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 38 include, or additionally include, one or more selected hydrophilic amino acids from Arg, Asn, Asp, Cys, Glu, Gln, His, Lys, Met, Orn, Ser, or Thr, including their respective naturally occurring L enantiomers.

[0027] 40L 1 , L 2 and L 3One or more of the compounds described in any one of items 1 to 39 include, or additionally include, one or more selected hydrophilic amino acids from β-NH2-Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Lys, Met, Orn, Ser, or Thr, including their respective naturally occurring L enantiomers. 41.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 40 include, or additionally include, one or more amino acids selected from glycine, serine, proline, ornithine, and lysine. 42.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 41 include ornithine and L-lysine, or additionally include ornithine or lysine. 43.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 42 include L-lysine, or additionally include lysine. 44.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 43 contain proline, including L-proline, or additionally contain proline. 45.L 1 , L 2 and L 3 One or more of these are (L-Pro) n Includes (Pro) n A compound according to any one of items 1 to 44, comprising or additionally comprising, where n is 1 to 6. 46.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 45, including (L-Pro)3, (Pro)3, or additionally including (L-Pro)3. 47.L 1 , L2 and L 3 One or more of the compounds described in any one of items 1 to 46 include (Pro)3-Lys, or additionally (L-Pro)3-(L-Lys). 48.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 47, comprising (L-Pro)6, (Pro)6, or additionally (L-Pro)6. 49.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 48 include (Pro)6-Lys, or additionally (L-Pro)6-(L-Lys). 50L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 49 include serine containing glycine and L-serine, or additionally containing serine. 51.L 1 , L 2 and L 3 One or more of these are [Gly-(L-Ser)] n (Gly-Ser) n A compound comprising or additionally comprising, where n is 1 to 3, any one of the compounds described in item 1 to 50. 52.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 51 include [Gly-(L-Ser)]2, or include (Gly-Ser)2 in addition. 53.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 52 include (Gly-Ser)2-Lys, which contains [Gly-(L-Ser)]2-(L-Lys), or additionally contains (Gly-Ser)2-Lys. 54.L 1 , L 2 and L 3One or more of the compounds described in any one of items 1 to 53 include [Gly-(L-Ser)]3, or include (Gly-Ser)3 in addition. 55.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 54 include (Gly-Ser)3-Lys, which comprises [Gly-(L-Ser)]3-(L-Lys), or additionally includes (Gly-Ser)3-Lys. 56.L 1 , L 2 and L 3 A compound according to any one of items 1 to 55, comprising or additionally comprising one of the following groups: C(O), C(O)O, C(O)NH, OC(O)NH, or NHC(O)NH. 57.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 56 include, or additionally include, 1,2,3-triazole-1,4-diyl, 1,2,3-triazole-1,4-diyl, or a combination thereof. 58.L 1 , L 2 and L 3 One or more of the compounds described in any one of items 1 to 57 include, or additionally include, maleimido-diyl or thiomaleimido-N,S-diyl. 59.L 2 and / or L 3 A compound that is hydrophilic, as described in any one of items 1 to 58. 60L 1 The compound described in any one of items 1 to 59, which includes a region capable of forming an α-helix structure. 61.L 2 and / or L 3 The extended structure of any one of the compounds described in section 1 to 60 has a length of at least 8 Å, 9 Å, 10 Å, 11 Å, 12 Å, 13 Å, 14 Å, 15 Å, 20 Å, 25 Å, 30 Å, 35 Å, or 40 Å. 62.L 2 and / or L 3(PEG) n or ather (PEG) n A compound according to any one of items 1 to 61, comprising or additionally comprising, where n is in the range of approximately 3 to approximately 36, approximately 4 to approximately 36, approximately 5 to approximately 36, approximately 6 to approximately 36, or approximately 7 to approximately 36. 63.L 2 and / or L 3 The compounds described in any one of items 1 to 62, comprising, or additionally comprising, one or more divalent cycloalkyl groups containing an adamantyl group, heterocyclyl groups containing a maleimidyl group, aryl groups, heteroaryl groups containing a triazolyl groups, stilbene groups, oligoproline groups, or oligopiperidine groups. 64.TL 1 The extended structure of the compound is one of the compounds described in any one of sections 1 to 63, having a length of at least 5 Å, 6 Å, 7 Å, 8 Å, 9 Å, 10 Å, 11 Å, or 12 Å. 65L 1 (PEG) n or ather (PEG) n A compound according to any one of items 1 to 64, comprising or additionally comprising, where n is in the range of approximately 3 to approximately 36, approximately 4 to approximately 36, approximately 5 to approximately 36, or approximately 6 to approximately 36. 66.L 1 This refers to any one of the compounds listed in item 1 to 65, which does not contain L-lysine. 67.L 1 This refers to any one of the compounds listed in item 1 to 66, excluding DBCO.

[0028] 68. Conjugates are, [ka] (In the formula, L1, L2, and L3 are linkers, each independently), or [ka] Not any compound listed in any one of items 1 to 67. 69. A compound of any of the formulas shown in Figure 1, or a pharmaceutically acceptable salt or solvate thereof.

[0029] 70. Expression [ka] A compound thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0030] 71. Formula [ka] A compound thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0031] 72. Formula [ka] A compound thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0032] 73. Expression [ka] A compound thereof, or a pharmaceutically acceptable salt or solvate thereof. The exemplary compounds described in sections 1 to 73 may be included in any composition, unit dose, use or method described herein.

[0033] Conjugate The conjugate compounds described herein can be formed from ligands having the ability to target proteins on influenza or virus-infected cells. In many embodiments, proteins on the surface of influenza or virus-infected cells, or those otherwise accessible from the surface of influenza or virus-infected cells, are referred to. The conjugates described herein can also be formed from two or more haptens, each containing an immune system-responsive epitope or antigen. The conjugates described herein can also be formed from polyvalent linkers covalently binding ligands to two or more haptens. Numerous selections of ligands, haptens, and linkers are described herein. Each ligand and hapten described herein may be proteinaceous or small molecules. Each linker described herein may be proteinaceous or small molecules, or a combination thereof. It is understood that any ligand, any hapten, and any linker described herein may be combined to form a conjugate. In addition, the linker itself may be formed from a variety of components, including single atoms, functional groups, and chemical fragments. It is understood that each combination of components for forming the linker contained in the conjugate is described herein.

[0034] In some embodiments, the conjugate has a dual mechanism of action, where it inhibits the virus and further labels or modifies virus-infected cells for intervention by the immune system of a host animal, including humans. The conjugate elicits an immune response in the host animal against the virus or virus-infected cells by recruiting antibodies in the host animal. Exemplarily, the antibodies are endogenous.

[0035] While not strictly theoretical, anti-hapten antibodies are thought to bind to haptens upon recruitment, activating the innate immune system against the target virus and virus-infected cells. The haptens may be identical or different. In conjugates with identical haptens, the host animal's immune system may be amplified. In conjugates with different haptens, the host animal's immune system may respond in multiple ways. The conjugates described herein provide therapeutic delivery systems for selectively or specifically delivering haptens to target viruses and virus-infected cells. In certain embodiments, the haptens are selected to activate the target's innate immune system, recruit immune cells, and / or otherwise leverage the target's own immune system against the virus. Targeting ligands can selectively or specifically recognize target proteins or receptors, such as the virus, the envelope protein of the virus, which may be expressed in large quantities or exclusively on the surface of infected cells, or accessible from the surface of infected cells.

[0036] In other embodiments, the conjugate compound of formula I is L 1 L bonded to the same atom above 2 -A 1 and L 3 -A 2 It may have fragments. In some embodiments, the same atom is not a carbon atom. In some embodiments, the same atom is a nitrogen atom. Alternatively, the conjugate of formula I is L 1 L bonded to different atoms above 2 -A 1 and L 3 -A 2 It may have fragments.

[0037] The compounds, compositions, unit doses, and methods described herein are useful in the treatment of influenza A, influenza B, and avian influenza. Exemplary influenza strains that can be treated with the compounds, compositions, unit doses, and methods described herein include, but are not limited to, H1N1, H1N1pdm09, H3N2, and avian influenza strains H5N1 and H7N9, as well as resistant strains, such as oseltamivir-resistant H1N1pdm09 and baloxavir-resistant H1N1pdm09.

[0038] While not strictly theoretical, the method can induce an immune response that leads to the removal of antibody-coated viruses or antibody-coated virus-infected cells via antibody-dependent phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or complement-dependent cell-mediated cytotoxicity (CDC), which, in conjunction with inhibition of viral germination by neuraminidase inhibition, leads to viral eradication. The method for activating the immune response may further involve administering autologous antibodies, allogeneic IgG antibodies, or human IVIG to the target. In these and other embodiments, the target may be further treated with anti-hapten antibodies.

[0039] In many exemplary embodiments, T is a radical of a neuraminidase inhibitor. Exemplary neuraminidase inhibitors include, but are not limited to, sialic acid and its analogues and derivatives, zanamivir, peramivir, laninamivir, oseltamivir, 2,3-dehydro-2-deoxy-n-acetylneuraminic acid and its analogues and derivatives, compounds disclosed in, for example, U.S. Patent No. 6,340,702, International Publication No. 1991 / 016320, International Publication No. 1996 / 026933, International Publication No. 1999 / 033781, and others.

[0040] In many embodiments, the ligand is zanamivir [ka] or its analog or derivative.

[0041] It is understood that radicals can form on any atom of the aforementioned material to form radical T.

[0042] In many embodiments, T is an expression [ka] It is a radical having any specific stereochemical structure, or having a mixture of two or more stereochemical structures.

[0043] In many embodiments, T is an expression [ka] It is radical.

[0044] In many embodiments, the ligand is peramivir [ka] This includes analogs or derivatives thereof, including compounds described in Chand et al., J Med Chem 44(25):4379-92 (2001), Bai et al., Viruses 13(624):1-13 (2021), etc.

[0045] In many embodiments, T is an expression [ka] It is radical.

[0046] In many embodiments, the ligand is an amino analog of peramivir. [ka] or its analog or derivative.

[0047] In many embodiments, T is an expression [ka] is a radical of.

[0048] In many embodiments, the ligand is laninamivir

Chemical Formula

[0049] In many embodiments, the ligand is a compound of formula

Chemical Formula

[0050] In many embodiments, the ligand is oseltamivir.

[0051] In many embodiments, the ligand is of the formula

Chemical Formula

[0052] In many embodiments, the ligand is of the formula

Chemical Formula

[0053] In many embodiments, the ligand is of the formula

Chemical Formula

[0054] In many embodiments, the ligand is formula [ka] (wherein R is H, Me, Et or a salt; R1 is NH2, azide or guanidino; R2 is C(O)CH3, C(O)CF3, or SO2Me; R3 is alkyl (primary or secondary, C-1 to C-5), or CH2OMe).

[0055] In another exemplary embodiment, T is a radical of a hemagglutinin inhibitor. Exemplary hemagglutinin inhibitors include, but are not limited to, flufirbitide 3, umifenovir, arbidol, and tert-butylhydroquinone.

[0056] In many embodiments, the ligand is formula [ka] This includes compounds such as those described in Kitamura et al., PNAS 121(22):pp. 1-9 (2024), Bai et al., Viruses 13(624):pp. 1-13 (2021), and others.

[0057] It is understood that radicals can be formed on any atom of any of the aforementioned ligands, and thus form radical T.

[0058] In many embodiments, T is an expression VEDTKIDLWSYNAELL (SEQ ID NO: 1) is the radical of fulfillbitide 3. 20. The ligand is cyclo(Ac-YWHKNKYVLTYSC)LFAAG-CONH2 (SEQ ID NO: 2), cyclo(Ac-YRWVWTSFFSEPYFVVC)G-CONH2 (SEQ ID NO: 3), cyclo(Ac-YLKIYWSKIHGLVSEWC)G-CONH2 (SEQ ID NO: 4), or cyclo(Ac-YVLFRWDHGTLATHWVC)G-CONH2 (SEQ ID NO: 5), and includes the compounds described in item 1, including those listed in Pascha et al., ACS Chem Biol 17:24 pp. 25-36 (2022).

[0059] It is understood that radicals can form radical T on the N-terminus, C-terminus, or inner atoms of a peptide chain. It is also understood that other radicals can form radical T on the side chains of amino acids.

[0060] Each hapten described herein can be conjugated by an antibody. After administration to the subject, each hapten (A 1 and A 2 ) can be bound by an antibody. In certain embodiments, two haptens, A 1 and A 2 These can be conjugated by different antibodies after administration to the target. It is understood that any hapten containing a peptide sequence that elicits an immunological response in a host animal can be used to form the conjugates described herein. It is further understood that the immunological response in a host animal can be elicited or supplemented via vaccines as adjunct therapy to the methods and uses described herein.

[0061] In another embodiment, A 1 and A 2is each independently selected radical of an antigen for which endogenous antibodies against each antigen are present in a host animal. Exemplary haptens include, but are not limited to, rhamnose including L-rhamnose, nitrophenyl, nitrophenol, nitroaniline, dinitrophenyl (DNP), dinitrophenol, dinitroaniline, trinitrophenyl (TNP), trinitrophenol, trinitroaniline, chloronitrophenyl, chloronitrophenol, chloronitroaniline, iodonitrophenyl, iodonitrophenol, iodonitroaniline, nitrotyrosine, hydroxynitrotyrosine, aminonitrotyrosine, 4-hydroxy-3-nitrophenylacetic acid, α-galactosyl moiety, sulfated Gal, formula

Chemical Formula

[0062] In many embodiments, the hapten is selected from dinitrophenyl (DNP), dinitrophenol and dinitroaniline. In many embodiments, A 1 and / or A 2 is a radical of dinitrophenyl (DNP), dinitrophenol or dinitroaniline, and comprises a radical of the formula

Chemical Formula

[0063] In many embodiments, the hapten is selected from rhamnose including L-rhamnose. In many embodiments, A 1 and / or A 2 is a radical of rhamnose including L-rhamnose, and comprises a radical of the formula

Chemical Formula

[0064] In current embodiments, A 1 It is a DNP radical, and A 2 is a rhamnose radical, or vice versa. In many embodiments, A 1 It is a DNP radical, and A 2 It is an L-rhamnose radical, or vice versa.

[0065] In another embodiment, A 1 and A 2 None of them contain fluorescein, such as FITC.

[0066] As used herein, the term “linker” generally refers to a chain of atoms that connects two or more functional parts of a molecule to form a conjugate. Exemplarily, the atomic chain is selected from C, N, O, S, Si, and P, or C, N, O, S, and P, or C, N, O, and S. The atomic chain covalently connects the different functional capabilities of the conjugate. Linkers can have a wide range of lengths, for example, from about 2 to about 100 atoms in a continuous main chain. The atoms used in linker formation can be combined by any chemically related means, for example, chains of carbon atoms that form alkylene, alkenylene, and alkylylene groups; chains of carbon and oxygen atoms that form ethers, polyoxyalkylene groups, or, when combined with carbonyl groups, esters and carbonates; chains of carbon and nitrogen atoms that form amines, imines, polyamines, hydrazines, hydrazones, or, when combined with carbonyl groups, amides, ureas, semicarbazides, carbazides; chains of carbon, nitrogen, and oxygen atoms that form alkoxyamines, alkoxylamines, or, when combined with carbonyl groups, urethanes, amino acids, acyloxylamines, hydroxamic acids, etc.; and many others. In addition, the atoms forming the chains in each of the exemplary embodiments described above may be saturated or unsaturated, and thus form single, double, or triple bonds, and it is understood that alkanes, alkenes, alkynes, imines, etc., may be radicals included in the linker. In addition, the (multiple) atoms forming the linker may each form a cyclic structure, or be a part of a cyclic structure forming a divalent cyclic structure that forms the linker, and it is understood that the linker may include cycloalkanes, cyclic ethers, cyclic amines, and other heterocycles, arylenes, heteroarylenes, etc. In this latter configuration, it is understood that the linker length can be defined by any path through one or more cyclic structures. Exemplarily, the linker length is defined by the shortest path through each of the cyclic structures.It is understood that the linker may be optionally substituted with one or more open valencies in the chain of atoms, such as by optional substitution of any of the carbon, nitrogen, silicon, or phosphorus atoms. It is also understood that the linker may form a conjugate by connecting two or more functional parts of a molecule with any open valencies, and that any of the two or more functional parts of the molecules forming the conjugate do not need to be bonded at any apparent end of the linker.

[0067] In many embodiments, L 1 , L 2 and L 3 Each independently contains a chain of atoms ranging in length from 3 to approximately 60 atoms. In many embodiments, L 1 , L 2 and L 3 Each independently contains a chain of atoms with a length of approximately 4 Å to approximately 72 Å. The chain of atoms is the main chain portion of the conjugate of formula I. As used herein, the term “main chain” of linker L refers to T and L. 2 During T and L 3 During A 1 and L 1 During, or A 2 and L 1 This refers to the shortest chain of consecutive atoms that form a covalent bond between them.

[0068] In many embodiments, L 1 , L 2 or L 3 Each independently includes a chain of atoms of at least 3 atoms, at least 7 atoms, at least 10 atoms, at least 14 atoms, or at least 20 atoms. In many embodiments, L 1 , L 2 or L 3 Each independently has a chain of lengths between 3 and 7 atoms, between 7 and 10 atoms, between 10 and 14 atoms, between 14 and 20 atoms, between 20 and 30 atoms, between 30 and 40 atoms, between 40 and 50 atoms, or between 50 and 60 atoms. In many embodiments, L1 , L 2 or L 3 Each of these independently includes a chain with a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 atoms. As used herein, the term "between" includes the endpoints, and chain lengths between 3 and 5 atoms mean including 3, 4, and 5 atoms.

[0069] L 2 and L 3 Both are L 1 When covalently bonding to L, 2 and L 3 Both are L 1 It may bond to the same atom of L 2 and L 3 Each is L 1 It is understood that it may bond to different atoms. In another embodiment, L 2 and L 3 L 1 It is bonded to the same atom above, and in this case, that atom is not a carbon atom.

[0070] In many embodiments, L 1 , L 2 or L 3 Each of these independently contains one or more amino acids or peptide residues.

[0071] As used herein, the term “amino acid” generally refers to beta, gamma, and longer amino acids, including cyclic groups that have both an amino group and an acidic group, from which radicals can be formed. An example of an acyclic amino acid is given by formula: -N(R)-(CR'R'') q -C(O)- (wherein R is hydrogen, alkyl, acyl or a preferred nitrogen protecting group, R' and R'' are hydrogen or substituents, each independently selected in its respective appearance, and q is an integer such as 1, 2, 3, 4 or 5). Exemplaryly, R' and / or R'' independently correspond to, but are not limited to, hydrogen or side chains present in naturally occurring amino acids, such as methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxymethyl, guanidinopropyl, etc., as well as their derivatives and protected derivatives. The above formula includes all stereoisomers. For example, amino acids can be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, etc.

[0072] For example, L 1 , L 2 or L 3 Each of these can independently contain one or more lysine residues, each of which can independently be substituted as it may. For example, L 1 The formula is: [ka] (In the formula, A 1 Or A 2 The lysine residue may include (which is bound to the lysine residue via a linker). In another embodiment, the lysine residue is L-Lys. In one example, L 1 It contains at least one lysine residue, for example, L-Lys.

[0073] In many embodiments, L 1 , L 2 or L 3 Each is independently polyethylene glycol n (PEG n ) may include a portion where n=1 to 36. In many embodiments, the linker includes one or more PEG portions where all carbon and oxygen atoms of one or more PEG portions are part of the linker's main chain.

[0074] In many embodiments, L 1 , L 2 or L 3 Each of these may independently contain one or more alkyl groups.

[0075] In many embodiments, L 1 , L 2 or L 3 Each of these may independently contain one or more sugar moieties, glycan residues, or peptidoglycan residues.

[0076] The bonds connecting the atoms in the chain can be saturated or unsaturated, and it is understood that, for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, and imides can be divalent radicals contained in L. In each of these, the L atoms in the chain may or may not be substituted.

[0077] In addition, the atoms that form the linker are also cyclized into saturated or unsaturated divalent cyclic radicals within the linker, for example, formula: [ka] (In the formula, each 1 Each X is independently CH2, NR', or O, where R' is alkyl or hydrogen, and each X 2 It is understood that these can also independently form radicals of S, O, N, NH, CR'' (where R'' is alkyl or hydrogen). Examples of such radicals include: [ka] These are some examples.

[0078] In some embodiments, L 1 , L 2 or L 3The linker contains suitable substituents that alter the hydrophobicity or hydrophilicity of the linker. Exemplary hydrophobic groups include alkyl, cycloalkyl, aryl, and arylalkyl groups, each of which may be substituted.

[0079] L 1 , L 2 or L 3 Each of these is independent and expressed by the following formula: [ka] (In the formula, x and y are independently 1, 2, 3, 4, or 5, where an asterisk indicates a link to another linker fragment, or T or A) 1 Or A 2 It may include alkylene-amino-alkylene carbonyl and alkylene-thio-(carbonylalkylsuccinimido-3-yl) moieties, which include identifying the binding site to the alkylene-amino-alkylene carbonyl moiety.

[0080] Additional examples of groups that may be present in the linker include 1-alkylsuccinimido-3-yl, carbonyl, thionocarbonyl, alkyl, cycloalkyl, alkylcycloalkyl, alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-alkylsuccinimido-3-yl, 1-(carbonylalkyl)succinimido-3-yl, alkylsulfoxyl, sulfonylalkyl, alkylsulfoxylalkyl, alkylsulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl)succinimido-3-yl, and 1-(carbonyltetrahydrofuranyl)succinimido-3-yl, where each group may be substituted or unsubstituted. In some embodiments, one or more of the aforementioned groups may be used in combination (or more than once) (e.g., -alkyl-C(O)-alkyl), and may further include additional nitrogen (e.g., alkyl-C(O)-NH-, -NH-alkyl-C(O)- or -NH-alkyl-), oxygen (e.g., -alkyl-O-alkyl-) or sulfur (e.g., -alkyl-S-alkyl-). Examples include alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimido-3-yl, and succinimido-3-ylthiol, where each group may be substituted or unsubstituted.

[0081] In some embodiments, L 1 , L 2 or L 3 It can be formed via or derived from click chemistry. For example, L 1 , L 2 or L 3These can be derived from copper-catalyzed azide-alkyne cycloaddition reactions (CuAAC), strain-promoted azide-alkyne cycloaddition reactions (SPAAC), reverse electron-demanded Diels-Alder reactions (IEDDA), and Staudinger ligation (SL). For example, T may be the part of formula T-N3. In that case, T-N3 is as follows: [ka] (Here, T and A 1 / A 2 The wavy lines connected to T and A 1 / A 2 It can react with alkynes, as shown in the linker between them and the groups to which they bond.

[0082] In other embodiments, L 1 , L 2 or L 3 teeth [ka] (In the formula, x is an integer between 0 and 50, and y is an integer between 0 and 50.)

[0083] In other embodiments, L 1 , L 2 or L 3 teeth [ka] (In the formula, R 2 and R 3 Each of these is independently H or C 1~6 It may include (it is alkyl; and z is an integer between 1 and 8).

[0084] In other embodiments, L 1 , L 2 or L 3This may include amides, esters, ureas, carbonates, carbamates, amino acids, amines, ethers, alkyls, alkenes, alkynes, heteroalkylcycloalkyls, aryls, heterocycloalkyls, heteroaryls, carbohydrates, glycans, peptidoglycans, polypeptides, or any combination thereof. In some embodiments, L 1 , L 2 or L 3 This may include glycosylated amino acids. In some embodiments, L 1 , L 2 or L 3 This may include one or more monosaccharides, disaccharides, polysaccharides, glycans, or peptidoglycans. In some embodiments, L 1 , L 2 or L 3 It does not contain glycans. In some embodiments, L 1 , L 2 or L 3 It does not contain sugar.

[0085] In some embodiments, L 1 , L 2 or L 3 It may contain rigid functional groups such as oligoproline or oligopiperidine.

[0086] In some embodiments, the oligoproline or oligopiperidine has about 2 to about 50 (including 50), about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 15, about 2 to about 10, or about 2 to about 6 repeating units (e.g., proline or piperidine).

[0087] In some embodiments, L 1 , L 2 or L 3 (-CH2CH2-O-) n (wherein n is an integer between 1 and 36 and including these (e.g., 1-2, 2-6, 3-8, 6-12, and 4-10)), peptides, alkylamide groups (e.g., C(O)N(H)C2-C) 18 Alkyl- or C2~C 18Alkyl-C(O)N(H)-), alkylamide alkyl group (e.g., C2~C) 18 Alkyl-C(O)N(C2~C 18 Alkyl)2 or C2~C 18 Alkyl-C(O)N(H)-C2~C 18 This may include alkyl groups, such as -CH2CH2C(O)N(CH2CH2)2 or -CH2CH2C(O)N(H)(CH2CH2)- groups, or two or more combinations thereof.

[0088] In some embodiments, L 1 , L 2 or L 3 teeth [ka] (wherein the formula, m may be an integer from 0 to 20, e.g., 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8, or 3 to 9). For example, m may be 0 or 1. Or m may be 5 or 6. Or m may be 7 or 8. In some embodiments, L 1 , L 2 or L 3 teeth [ka] (wherein the formula, p and q may each independently be integers from 0 to 20, e.g., 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8, or 3 to 9). For example, p may be 2 or 3. For example, q may be 2 or 3. In some embodiments, L 1 , L 2 or L 3 teeth [ka] (wherein d can be an integer between 0 and 20, e.g., 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8, or 3 to 9). For example, d could be 1, 2, or 3.

[0089] In some embodiments, L 1 , L 2or L 3 They are all together, [ka] (For example, in the expression, m, p, d, and q can each independently contain integers from 0 to 20, e.g., 1 to 20, 0 to 15, 1 to 10, 2 to 10, 2 to 8, or 3 to 9). For example, m could be 0 or 1. Or m could be 5 or 6. Or m could be 7 or 8. For example, p could be 2 or 3. For example q could be 2 or 3. For example d could be 1, 2, or 3.

[0090] In some embodiments, L 1 , L 2 or L 3 They are all together, [ka] It may include.

[0091] In another embodiment, the following compounds are described. [ka]

[0092] In many embodiments, the target protein may be the influenza envelope protein or the influenza envelope protein on the surface of virus-infected cells. In many embodiments, the target protein may be influenza neuraminidase or influenza hemagglutinin.

[0093] The compounds described herein may contain one or more chiral centers, or otherwise may have the ability to exist as multiple stereoisomers. In one embodiment, it is understood that the inventions described herein are not limited to any particular stereochemical requirements, and the compounds, as well as the compositions, methods, uses, and pharmaceuticals containing them, may be pure or any of various stereoisomeric mixtures, including racemic and other mixtures of enantiomers, mixtures of other diastereomers, etc. It is also understood that such stereoisomeric mixtures may contain a single stereochemical structure at one or more chiral centers, or a mixture of stereochemical structures at one or more other chiral centers.

[0094] Similarly, the compounds described herein may include geometric centers, e.g., cis, trans, E, and Z double bonds, or spatial configurations, e.g., relative configurations on a ring, such as cis, trans, syn, and anti. In another embodiment, it is understood that the inventions described herein are not limited to any specific geometric isomer requirements, and the compounds, as well as compositions, methods, uses, and pharmaceuticals containing them, may be pure or any of a mixture of various geometric isomers. It is also understood that such a mixture of geometric isomers may include a single configuration at one or more double bonds, while also including a mixture of geometric configurations at one or more other double bonds.

[0095] The conjugates described herein can be “deuterated,” that is, one or more hydrogen atoms can be replaced with deuterium. Since deuterium and hydrogen have nearly identical physical properties, deuterium substitution is the smallest possible structural change. Deuteration is well known to those skilled in the art.

[0096] As used herein, the term “solvate” refers to a compound complexed with a solvent molecule as described herein. It is understood that the compounds described herein may form complexes with a solvent by simply mixing the compound with the solvent or by dissolving the compound in the solvent. When a compound is used as a pharmaceutical, it is understood that such a solvent is pharmaceutically acceptable. It is further understood that when a compound is used as a pharmaceutical, the relative amount of solvent forming the solvate should be less than the established guidelines for such pharmaceutical use, for example, less than the International Conference on Harmonisation of Registration of Pharmaceuticals for Human Use (ICH) guidelines. It is understood that solvates can be isolated from excess solvent by evaporation, precipitation, and / or crystallization. In some embodiments, the solvate is amorphous, and in other embodiments, the solvate is crystalline. When the solvent is water, the solvate is called a hydrate.

[0097] Where used herein, the term “composition” generally refers to any product containing specified amounts of specified raw materials, as well as any product directly or indirectly arising from combinations of specified raw materials in specified amounts. Certain functional groups, such as hydroxyl and amino groups, are understood to form complexes and / or coordination compounds with water and / or various solvents in various physical forms of the compounds. The compositions described herein may be prepared from the isolated compounds described herein, or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It is also understood that compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and / or other solid forms of the compounds described herein. Furthermore, compositions may be prepared from various hydrates and / or solvates of the compounds described herein. In addition, compositions may be prepared from various cocrystals of the compounds described herein. Therefore, any pharmaceutical composition referring to the compounds described herein is understood to include each or any combination of the various solid forms and / or solvates or hydrates of the compounds described herein.

[0098] A pharmaceutical composition may be prepared by combining one or more conjugates with one or more pharmaceutically acceptable excipients, carriers, or diluents, or any combination thereof, and optionally with one or more additional pharmaceutically active agents.

[0099] definition In this document, the terms “a,” “an,” or “the” are used to include one or more unless the context clearly indicates otherwise. The term “or” is used to refer to a non-excludative “or” unless otherwise indicated. In addition, any expressions or technical terms adopted herein and not otherwise defined are understood to be for illustrative purposes only and not to limit.

[0100] In each case disclosed herein, any reference to an integer range for any variable is understood to describe the range being described, each of the individual elements within that range, and each of the possible partial ranges of that variable. For example, a reference that n is an integer from 0 to 8 describes the ranges of 0, 1, 2, 3, 4, 5, 6, 7, and 8, which are individual and selectable values, e.g., n is 0, or n is 1, or n is 2, and other ranges. In addition, a reference that n is an integer from 0 to 8 also describes the respective partial ranges that may form the basis for further embodiments, e.g., n is 1 to 8, 1 to 7, 1 to 6, 2 to 8, 2 to 7, 1 to 3, 2 to 4, and other integers.

[0101] Furthermore, unless otherwise indicated, references to numbers are understood to necessarily reflect the relative precision of those numbers. For example, a reference to a number with a certain precision based on significant figures necessarily includes a range of values ​​that, after appropriate rounding, correspond to that number. For instance, a reference to the number 1 with one significant figure is understood to correctly refer to a range of values ​​between 0.5 and 1.4. Similarly, a reference to the number 1.0 with two significant figures is understood to correctly refer to a range of values ​​between 0.95 and 1.04. The relative precision of a number can be further indicated by modifying it with the term "approximately," showing that the modified number has a lower precision.

[0102] Where used herein, the term “approximately” generally means that the number is approximate when used with a number or limit, and is understood to include a range of values ​​when described. For example, a real number mentioned with one significant digit includes, by definition, a so-called rounding range; the number approximately 5 includes a range of at least 4.5 to 5.4, with each of those values ​​rounded to 5. The same is understood for real numbers expressed with additional significant digits, where the corresponding rounding range applies to the last significant digit. An integer is understood to include at least values ​​of ±1 for single-digit numbers, ±10 for two-digit numbers, and so on. Depending on the context and the variable being mentioned, the term “approximately” is also understood to imply a range based on a percentage of the number being mentioned, such as approximately 5 being interpreted as including 5 ± 10% or 5 ± 20%. Regardless of their content, unless otherwise indicated, a range of values ​​should not be understood as including a negative range when referring to a positive number, and vice versa. Furthermore, depending on the context, the numbers mentioned should not be interpreted as including the value of zero when used with additional components, unless otherwise indicated.

[0103] The phrase "approximately X to Y" has the same meaning as "approximately X to approximately Y" unless otherwise indicated. Similarly, the phrase "approximately X, Y, or approximately Z" has the same meaning as "approximately X, approximately Y, or approximately Z" unless otherwise indicated.

[0104] Values ​​expressed in range form should be understood in a flexible manner, including not only the numerical value explicitly mentioned as the limit of the range, but also all individual numerical values ​​or partial ranges contained within that range, as if each numerical value and partial range were explicitly mentioned. For example, the range "approximately 0.1% to approximately 5%" or "approximately 0.1% to approximately 5%" should be interpreted to include not only approximately 0.1% to approximately 5%, but also individual values ​​within the indicated range (e.g., 1%, 2%, 3%, and 4%) as well as partial ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, and 3.3% to 4.4%).

[0105] As used herein, the term “radical” in relation to a ligand for a target protein or hapten means, as described herein, one or more atoms or groups, such as a hydrogen atom or an alkyl group on a heteroatom, that have been removed and are polyvalent linkers L 1 , L 2 and L 3 This refers to a ligand for each target protein or hapten, which provides a radical for covalent linkage or conjugation to the target protein or hapten. When one or more groups are replaced by one or more different groups, the resulting compound is an analog of the ligand. Exemplary analogs include, but are not limited to, compounds that share functional, and in some cases structural, similarities with the compounds described herein. It is also understood that such radicals can be formed on acid, ester, or amide groups, such as carboxy, phosphoryl, and sulfuric acid, by removing OH, ester, or amide groups. It is also understood that such radicals can be formed by removing other fragments, such as halo, alkoxy, amino, heterocyclyl, or heteroaryl groups.

[0106] "Alkyl alkyl groups" are saturated, partially saturated, or unsaturated linear or branched acyclic hydrocarbons, consisting of 1 to 10 carbon atoms (C1-C1). 10The alkyl group has 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 1 to 3 carbon atoms (C1-C3 alkyl), or 2 to 6 carbon atoms (C2-C6 alkyl). In some embodiments, the alkyl group is monovalent. Examples of monovalent alkyl groups include -CH3 and -CH2CH3. Monovalent alkyl groups can be found, for example, as substituents in the linker L chain. In some embodiments, the alkyl group is divalent, as found, for example, in the linker L chain. Examples of divalent alkyl groups include, but are not limited to, -CH2- and -CH2CH2-. In some embodiments, the alkyl group is a saturated alkyl group. In some embodiments, the alkyl group is an unsaturated alkyl group, also referred to as an alkenyl group or alkynyl group.

[0107] The term "heteroalkyl," whether alone or in combination with other terms, means a stable linear or branched chain, or a combination thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, where the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, P, S, and Si can be located at any internal position of the heteroalkyl group or at a position where the alkyl group is bonded to the rest of the molecule. Examples include, without limitation, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH2=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. For example, up to two heteroatoms may be consecutive, as in -CH2-NH-OCH3.

[0108] As used herein, the term "aryl" includes monocyclic and polycyclic aromatic carbocyclic groups, each of which may be optionally substituted. Examples of aromatic carbocyclic groups described herein include, but are not limited to, phenyl and naphthyl. As used herein, the term "heteroaryl" includes aromatic heterocyclic groups, each of which may be optionally substituted. Examples of aromatic heterocyclic groups include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetradinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzoisoxazolyl, and benzoisothiazolyl.

[0109] As used herein, the term “optionally substituted” includes the substitution of a hydrogen atom on an optionally substituted radical with another functional group. Examples of such other functional groups include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylhetalkyl, heteroaryl, heteroarylalkyl, heteroarylhetalkyl, nitro, sulfonic acid and its derivatives, carboxylic acid and its derivatives. Exemplarily, any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylhetalkyl, heteroaryl, heteroarylalkyl, heteroarylhetalkyl, and / or sulfonic acid are optionally substituted.

[0110] Examples of substituents include, but are not limited to, radical-(CH2) x Z X (Here x is an integer from 0 to 6, Z XThis includes halogens, hydroxyls, C1-C6 alkanoyloxys, optionally substituted alloyloxys, alkyls containing C1-C6 alkyls, alkoxys containing C1-C6 alkoxys, cycloalkyls containing C3-C8 cycloalkyls, cycloalkoxys containing C3-C8 cycloalkoxys, alkenyls containing C2-C6 alkenyls, alkynyls containing C2-C6 alkynyls, haloalkyls containing C1-C6 haloalkyls, haloalkoxys containing C1-C6 haloalkoxys, and halocycloalkyls containing C3-C8 halo Selected from rocycloalkyl, halocycloalkoxy including C3-C8 halocycloalkoxy, amino, C1-C6 alkylamino, (C1-C6 alkyl)(C1-C6 alkyl)amino, alkylcarbonylamino, N-(C1-C6 alkyl)alkylcarbonylamino, aminoalkyl, C1-C6 alkylaminoalkyl, (C1-C6 alkyl)(C1-C6 alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N-(C1-C6 alkyl)alkylcarbonylaminoalkyl, cyano, and nitro; or Z X -CO2R 4 and -CONR 5 R 6 Selected from, where R 4 , R 5 , and R 6 Each of these elements is independently selected from hydrogen, C1-C6 alkyl, aryl-C1-C6 alkyl, and heteroaryl-C1-C6 alkyl in their respective appearances.

[0111] The compounds described herein can be used in both human clinical medicine and veterinary applications. Therefore, the host animals treated with the compounds described herein may be humans, or, in the case of veterinary applications, laboratory animals, farm animals, pets, or wild animals. The present invention can be applied to host animals including, but is not limited to, humans, rodents (e.g., mice, rats, hamsters, etc.), laboratory animals such as rabbits, monkeys, and chimpanzees, pets such as dogs, cats, and rabbits, farm animals such as cattle, horses, pigs, sheep, and goats, and captured wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.

[0112] Where used herein, the term “therapeutic dose” refers to the amount of an active compound or drug that elicits a biological or pharmacokinetic response, including the alleviation of symptoms of a disease or disorder being treated, in a histological system, animal, or human, as sought by a researcher, veterinarian, physician, or other clinician. In one embodiment, a therapeutic dose is the amount that can treat or alleviate a disease or symptoms of a disease with a reasonable benefit / risk ratio applicable to any medical treatment. However, it is understood that the total daily dose of the compounds and compositions described herein may be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutic dose level for any particular patient depends on a variety of factors, including the disorder being treated and its severity; the activity of the particular compound being employed; the particular composition being employed; the patient’s age, weight, overall health, sex, and diet; the timing, route of administration, and rate of elimination of the particular compound being employed; the duration of treatment; drugs used in combination with or incidentally with the particular compound being employed; and similar factors known to a researcher, veterinarian, physician, or other clinician of ordinary skill.

[0113] The conjugate and composition can be delivered by preferred delivery methods, including, but not limited to, intranasal, oral, and intravenous delivery. The method may further include administering autoantibodies or alloimmunoglobulin G (IgG) antibodies.

[0114] In each of the preceding and subsequent embodiments, unless otherwise indicated, the transitional phrase “essentially consisting of” is understood to mean that the corresponding composition, unit dose, method or scope of use does not materially affect the identified compound or the process mentioned, and the fundamental and novel characteristics of the invention described herein. For example, a method described herein consisting essentially of a single compound or a genus of compounds is understood to represent monotherapy for the disease mentioned. Monotherapy may include co-administration of one or more carriers, vehicles, diluents, adjuvants, excipients, etc., and combinations thereof, and / or co-administration of one or more additional pharmaceutical components, but such latter additional active ingredients are understood to be for treating diseases and / or symptoms distinct from the treatment of the underlying disease described herein, such as the treatment of a viral infection itself. Examples of additional active ingredients include, for example, active ingredients for treating pain, inflammation, cough, nasal congestion, etc. [Examples]

[0115] The following examples further illustrate the present invention as described herein. These examples illustrate many embodiments of the present invention and should not be construed as limiting the scope of the invention in any way.

[0116] [Table 1-1] [Table 1-2]

[0117] General. Unless otherwise indicated, all starting compounds and reagents are commercially available. Unless otherwise indicated, reactions are carried out in an ambient atmosphere, at ambient temperature and pressure, and the progress of the reactions is monitored by TLC, LC-MS, or both. Unless otherwise indicated, solutions are concentrated by rotary evaporation under reduced pressure, high vacuum, or both, and compounds are isolated. [Examples]

[0118] (3S,4R)-3-acetamido-2-((14S,40S,41R)-14-(4-(3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanamide)butyl)-41,42-dihydroxy-13,16,38-trioxo-1-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,19,22,25,28,31,34,39-decaoxa-12,15,37-triazadotetracontan-40-yl)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (compound 24) (Figures 3-4) Compound 3: Compounds 1 (Sigma-Aldrich, 0.5 g, 2.47 mmol) and 2 (BroadPharm, 0.33 g, 2.47 mmol) were dissolved in ethyl alcohol (25 mL), to which TEA (1.38 mL, 9.87 mmol) was added. The reaction mixture was heated at 55 °C for 16 hours. After the reaction was complete, the reaction mixture was cooled and concentrated, and the residue was collected in Teledyne CombiFlash Rf + Compound 3 was purified by flash column chromatography on Lumen (silica gel, 12g column, 0-20% MeOH in DCM) and obtained as a yellow solid (90% yield). LC-MS[M+H] + =300.24. Chemistry-An Asian Journal (2012), No. 7, (2), pp. 272-276.

[0119] Compound 5: Compound 3 (0.1 g, 0.33 mmol) in dimethyl sulfoxide (2 mL) was mixed with HATU (0.11 g, 0.28 mmol, 0.85 eq.), followed by DIPEA (0.29 mL, 1.67 mmol, 5.0 eq.) under an argon atmosphere and stirred for 10 minutes. Compound 4 (Chem-Impex International, 0.11 g, 0.27 mmol, 0.8 eq.) was added to the reaction mixture and stirred for 2-3 hours. After the reaction was complete, the reaction mixture was quenched by adding water and extracting with RINKAN. The combined organic layers were washed with brine, dehydrated with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel (4 g) column chromatography on Teledyne CombiFlash Rf+ Lumen (0-10% MeOH in DCM), and the fraction was analyzed by LC-MS to obtain Compound 5 (70% yield). LC-MS[M+H] + = 650.67.

[0120] Compound 7: α-L-rhamnose monohydrate (6, Sigma-Aldrich, 1.0 g, 5.49 mmol) was dissolved in 9.2 mL of anhydrous pyridine. The solution was stirred in an ice bath, purged with nitrogen gas, and then anhydrous acetate (4.15 mL, 43.92 mmol, 8.0 eq.) was added dropwise for 15 minutes while maintaining the internal temperature below 10°C. The reaction mixture was gradually warmed to ambient temperature over 2 hours. After 20 hours, the reaction mixture was poured into siRNA and extracted twice with 1.0 M HCl. The combined organic layers were washed with saturated sodium carbonate solution, water, and brine, dehydrated with anhydrous Na2SO4, and concentrated to obtain compound 7 (yield 98%), which was used without further purification. LC-MS[M+H] + =333.32 and / or LC-MS[M+H2O]=350.32.

[0121] Compound 9: Compound 7 (0.50 g, 1.50 mmol) in DCM (7.5 mL) was mixed with H2N-PEG4-OH (8, BroadPharm, 0.35 g, 1.81 mmol, 1.2 eq.) under an inert atmosphere. The reaction flask was placed in an ice bath, and diethyl boron trifluoride etherate (Sigma-Aldrich, 0.56 mL, 4.51 mmol, 3.0 eq.) was added dropwise over 30 minutes at 4°C. The reaction mixture was stirred for 2 hours, after which the cooling was removed. After complete reaction, the reaction mixture was poured into ice water and extracted using DCM (3 × 10 mL). The combined organic layers were washed twice with saturated sodium bicarbonate, water, and brine, dehydrated with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel (12g) column chromatography on Teledyne CombiFlash Rf+ Lumen (0-10% MeOH in DCM) to obtain compound 9 (yield 90%). LC-MS[M+H] + =466.51. Biomacromolecules (2020), 21, pp. 793-802

[0122] Compound 10: Compounds 5 (0.06 g, 0.09 mmol) and 9 (0.04 g, 0.09 mmol, 1.0 eq.) were added to dimethyl sulfoxide (1.5 mL) under an argon atmosphere with PyBOP (0.05 g, 0.10 mmol, 1.1 eq.), followed by DIPEA (0.081 mL, 0.46 mmol, 5.0 eq.). After the reaction was complete, the reaction mixture was quenched by adding water and extracting with ethyl acetate. The combined organic layer was washed with brine, dehydrated with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel (4 g) column chromatography on Teledyne CombiFlash Rf+ Lumen (0-10% MeOH in DCM) to obtain compound 10 (yield 93%). LC-MS[M+H] + = 1098.15.

[0123] Compound 11: Compound 10 (0.01 g, 0.01 mmol) in dry DCM (0.2 ml) was mixed with DEA ​​(100 uL) under argon. The solution was stirred for 1 hour. Upon completion of the reaction, the DEA was evaporated and the residue was precipitated in diethyl ether to obtain compound 11 as a yellow solid (quantitative yield), which was used without further purification. LC-MS [M+H] + = 875.91.

[0124] Compound 13: Compound 12 (Alfa Chemistry, 5g, 11.0 mmol) in THF (40 mL) was mixed with triphenylphosphine (3.67 g, 14 mmol, 1.27 eq.), and the resulting solution was stirred for 12 hours. Subsequently, water (10 mL) was added, and the solution was stirred for a further 2-4 hours. The reaction mixture was diluted with SiO2 (2 × 30 mL) and then with DCM (3 × 25 mL), and extracted. The combined organic layers were washed with brine, dehydrated with anhydrous Na2SO4, and concentrated. The residue was purified by flash column chromatography on Teledyne CombiFlash Rf+ Lumen (0-100% SiO2 in hexane) using silica gel (24 g) to obtain 2.89 g of compound 13 as a yellow powder (yield 61%). LC-MS[M+H] + = 431.42.

[0125] Compound 15: Compound 13 (2.74 g, 6.37 mmol) and N,N'-bis(Boc)-1H-pyrazole-1-carboxamidine (14, Sigma-Aldrich, 2.57 g, 8.28 mmol, 1.30 eq.) were dissolved in THF (20 mL), to which TEA (1.5 mL) was added. The reaction mixture was stirred overnight. After the reaction was complete, the reaction mixture was quenched with water, diluted with SiO2 (3 × 20 mL), and extracted. The combined organic layer was washed with brine, dehydrated with anhydrous Na2SO4, and evaporated. The residue was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 24 g column, 0-100% SiO2 in hexane) to obtain compound 15 as a white solid (4.14 g, 97% yield). LC-MS[M+H] + = 673.70.

[0126] Compound 16: 0.5 M sodium methoxide in MeOH (2.9 mL, 0.5 M, 1.414 mmol) was added to a stirred solution of Compound 15 (4.225 g, 6.281 mmol) in anhydrous MeOH (70 mL). The reaction mixture was stirred for 1 hour. Dowex 50XW(H + The resin was added to the reaction mixture to lower the pH to approximately 3, filtered, washed with MeOH, and concentrated to obtain compound 16, which was used without further purification. LC-MS[M+H] + = 547.58.

[0127] Compound 17: Compound 16 in anhydrous acetone (70 mL) was mixed with 2,2-dimethoxypropane (7.7 mL, 6.54 g, 62.81 mmol, 10 eq.), followed by p-toluenesulfonic acid (120 mg, 0.628 mmol, 0.1 eq.). The resulting mixture was stirred overnight. The reaction mixture was concentrated, and the residue was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 12 g column, 0-100% siRNA in hexane) to obtain compound 17 as a white solid (2.4 g, 65% yield). LC-MS[M+H] + = 587.64.

[0128] Compound 19: Compounds 17 (1.46 g, 2.49 mmol) and 18 (Sigma-Aldrich, 3.51 g, 17.43 mmol) were added to pyridine (30 mL) with DMAP (2.13 g, 17.43 mmol). The reaction mixture was stirred overnight. After the reaction was complete, the reaction mixture was diluted with SiO2 (3 × 25 mL) and extracted. The combined organic layer was washed with water and brine, dehydrated with anhydrous Na2SO4, concentrated, and the residue was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 24 g column, 0-100% SiO2 in hexane) to obtain compound 19 as a white solid (1.62 g, yield 87%). LC-MS[M+H] + = 752.45.

[0129] Compound 21: Compound 19 (0.168 g, 0.223 mmol) in THF (1.1 mL) was mixed with 20 (BroadPharm, 0.079 g, 0.223 mmol, 1.0 eq.), followed by DIPEA (0.1 mL, 0.56 mmol, 2.5 eq.) under argon, and the mixture was stirred for 12 hours. After the reaction was complete, the reaction mixture was diluted with RINKAN (3 × 25 mL) and extracted. The combined organic layer was washed with water and brine, dehydrated with anhydrous Na₂SO₄, and concentrated. The residue was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 12 g column, 0-10% MeOH in DCM) to obtain 21 as a white solid (0.205 g, 90% yield). LC-MS[M+H] + =967.06.

[0130] Compound 21 (0.025 g, 0.026 mmol) in compound 22:DCM:dimethyl sulfoxide (10:1, 0.3 mL) was mixed with HATU (0.01 g, 0.026 mmol, 1.0 eq.), followed by DIPEA (0.012 μL, 0.065 mmol, 2.5 eq.) under argon, and the mixture was stirred for 3-5 minutes. Compound 11 (0.023 g, 0.026 mmol, 1.0 eq.) in DCM (0.2 mL) was added to the reaction mixture and the mixture was stirred for 2-3 hours. After the reaction was complete, the reaction mixture was evaporated, and the residue was purified by silica gel (4 g) column chromatography on Teledyne CombiFlash Rf+ Lumen (0-10% MeOH in DCM), and the fractions were analyzed by LC-MS. The combined fraction containing compound 22 was evaporated to obtain compound 22 (yield 61%). LC-MS[M+H] + = 1822.94.

[0131] Compound 24: Compound 24 was prepared by deprotection of Compound 22. It is understood that deprotection of Compound 22 can be achieved by first removing the Boc and ketal protecting groups using TFA in DCM, followed by deprotection of the methyl and acetyl protecting groups using LiOH·H2O in MeOH. Alternatively, deprotection of Compound 22 can be achieved by first removing the methyl and acetyl protecting groups using NaOMe in MeOH, followed by deprotection of the Boc and ketal protecting groups using TFA in DCM. Compound 22 (38 mg, 0.021 mmol) was dissolved in MeOH (0.5 mL) and treated dropwise with 0.5 M sodium methoxide in MeOH (190 μL). The reaction mixture was stirred for 1 hour. Upon completion of the reaction, Dowex® 50WX8(H) was used. + The reaction mixture was neutralized by adding a resin, filtered, and concentrated to obtain compound 23, which was used without further purification. LC-MS analysis showed that the unpurified compound 23 also contained compounds resulting from the loss of acetonide protecting groups and Boc protecting groups.

[0132] Compound 24: TFA (0.8 mL) was added to Compound 23 in DCM (0.8 mL) at ice-cold temperature. Cooling was removed and the reaction mixture was stirred for 15 minutes. The TFA was evaporated, and the residue was washed with cold diethyl ether. Compound 24 was purified by preparative HPLC (UPLC) on a C18 column (5-95% B for 60 minutes, flow rate 7 mL / min; B: ACN; A: 20 mM NH4OAc, pH 7 buffer, UV @ 360 nm and 220 nm) to obtain compound 24 (yield 42%). LC-MS[M+H] + =1443.83 (95% purity). [Examples]

[0133] (2R,3R,4S)-3-acetamido-2-((38R,39R)-25-(18-((2,4-dinitrophenyl)amino)-13-oxo-3,6,9,16-tetraoxa-12-azaoctadecyl)-39,40-dihydroxy-13,26,36-trioxo-1-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,16,19,22,29,32,37-nonanooxa-12,25,35-triazadotetracontan-38-yl)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (compound 42).

[0134] [ka] [ka]

[0135] Compound 3. HATU (0.034 g, 0.09 mmol) was added at 0°C to a stirred solution of Compound 1 (Angene, 1.0 g, 1.91 mmol) and Compound 2 (1.07 g, 2.03 mmol) in DMF (15.0 mL). After 10 minutes, ethylbis(propan-2-yl)amine (GLR, 1.77 mL, 9.57 mmol) was added dropwise to the reaction mixture at 0°C. Cooling was stopped, and the mixture was stirred for 12 hours. After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography eluting in 5% MeOH / DCM to obtain Compound 3 as a yellowish liquid. Yield: 1.20 g, 64.65%; LCMS (ELSD) m / z 970.60 [M+1] + .

[0136] Compound 4. To a solution of Compound 3 (1.0 g, 1.03 mmol) in MeOH (50.0 mL), 10% palladium-carbon (Johnson Matthey, 1.00 g) was added, and the mixture was stirred under a hydrogen atmosphere for 6 hours. After the reaction was complete, the reaction mixture was filtered through a Celite bed, washed with MeOH, and the filtrate was evaporated until dry. The residue was triturated with diethyl ether to obtain tert-butyl compound 4 as a pale yellow viscous liquid. Yield: (0.80 g, 82.2%; LCMS (ELSD) m / z 944.65 [M+1]) + .

[0137] Compound 6. Compound 6 was prepared using the same procedure as that used for preparing Compound 3 in this example. Compound 6 was prepared from Compound 5 (0.11 g, 0.37 mmol) and Compound 4 (0.35 g, 0.37 mmol) in DMF (10.0 mL), as well as HATU (0.15 g, 0.40 mmol) and ethylbis(propan-2-yl)amine (0.34 mL, 1.85 mmol). After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography eluting in 10% MeOH / DCM to obtain Compound 6 as a yellowish liquid. Yield: 0.20 g, 44.03%; LCMS (ESI) m / z 1225.30 [M+1] + .

[0138] Compound 7. To a stirred solution of Compound 6 (0.31 g, 0.25 mmol) in anhydrous DCM (5.0 mL), TFA (3.0 mL) was added at 0°C. Cooling was stopped, and the reaction mixture was stirred for 2 hours. After the reaction was complete, the reaction mixture was evaporated to obtain Compound 7 as a bright yellow solution, which was used without further purification. Yield: 0.22 g; LCMS (ESI) m / z 1125.10 [M+1] + .

[0139] Compound (8) was prepared as described in Carbohydrate Research, 342 (2007), pp. 1636-1650.

[0140] Compound 10. Compound 8 (0.21 g, 0.28 mmol) in 5 ml of dry THF was added dropwise to a flask containing DIPEA (GLR, 0.26 ml, 1.44 mmol) under an N2 atmosphere at 25-30°C. After 5 minutes, Compound 9 (Angene, 56 mg, 0.31 mmol) in 2 ml of dry THF was added to the reaction mixture, and the resulting suspension was stirred for a further 24 hours at 25-30°C. The progress of the reaction was monitored by TLC and LC-MS. After the completion of the reaction, the reaction mixture was concentrated, and the residue was purified by column chromatography eluting in 10% MeOH / DCM to obtain compound 10 as a grayish-white solid. Yield: 0.16 g, 70.05%; LC-MS (ESI) m / z 790.10 [M+1] + .

[0141] Compound 11. Compound 11 was prepared using the same procedure as that used for preparing Compound 3 in this example. Compound 11 was prepared from Compound 10 (0.11 g, 0.14 mmol) and Compound 7 (0.17 g, 0.15 mmol) in DMF (5.0 mL), as well as HATU (0.08 g, 0.15 mmol) and ethylbis(propan-2-yl)amine (GLR, 0.13 mL, 0.70 mmol). After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography eluting in 10% MeOH / DCM to obtain Compound 11 as a yellowish liquid. Yield: 0.12 g, 45.42%; LCMS (ESI) m / z 1896.00 [M+1] + .

[0142] Compound 42: Compound 5 (0.11 g, 0.06 mmol) was added to a stirred solution of compound 5 in THF (3 mL) and MeOH (1 mL) over 10 minutes at 25-30°C with a 1.0 M solution of sodium hydroxide (0.1 mL). The homogeneous solution was observed and stirred at 25-30°C for 4 hours. Completion of the reaction was confirmed by TLC and LC-MS, and the pH of the reaction mixture was measured using Dowex 50XW8(H + The solution was gradually adjusted to 7 by using a resin. The reaction mixture was filtered through a Celite bed and evaporated. To the stirred residue solution in anhydrous DCM (3.0 mL), TFA (1.0 mL) was added at 0°C, and the reaction mixture was stirred at 15°C for 2 hours. After the reaction was complete, the solvent was evaporated, and the residue was purified by preparative HPLC using water / ACN in 0.1% TFA to obtain compound 42 as a yellowish solid. Yield: 0.015 g, 37.07%; LCMS (ESI) m / z 1516.35 [M+1] + . 1H NMR (400 MHz, DMSO-d6): 8.86 (d, J = 2.8 Hz, 1H), 8.82 (brs, 1H), 8.25 (dd, J = 9.2 & 2.4 Hz, 1H), 7.93 (m, 2H), 7.86 (d, J = 8.80 Hz, 1H), 7.35 (brs, 1H), 7.27 (d, J = 9.60 Hz, 1H), 7.18 (brs, 3H), 5.27 (s, 1H), 5.13 - 4.84 (m, 5H), 4.72 (brs, 1H), 4.56 (s, 1H), 4.17 (d, J = 10.40 Hz, 1H), 3.93 - 3.85 (m, 2H), 3.67 - 3.35 (m, 60H), 3.21 - 3.14 (m, 6H), 3.02 (d, J = 5.20 Hz, 1H),2.59 (t, J = 8.40 Hz, 1H), 2.54 - 2.51 (m, 16H), 2.36 - 2.29 (m, 4H), 1.77 (s, 3H), 1.11 (d, J = 6.0 Hz, 3H). [Examples]

[0143] (2R,3R,4S)-3-acetamido-2-((14S,45R,46R)-14-(4-(3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanamide)butyl)-46,47-dihydroxy-13,43-dioxo-1-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,18,21,24,27,30,33,36,39,44-dodecaoxa-12,15,42-triazaheptatetracontan-45-yl)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (compound 41)

[0144] [ka] [ka]

[0145] Compound 1 was prepared as described in International Publication No. 2023 / 205669.

[0146] Compound 3: Compound 1 (0.0758 g, 0.087 mmol) and N-Boc-PEG8 bromide (2, Broad Pharm, 0.05 g, 0.087 mmol, 1.0 eq.) in DMF (0.87 mL) were mixed with DIPEA (0.038 μL, 0.216 mmol, 2.5 eq.), followed by potassium iodide (0.014 g, 0.087 mmol, 1.0 eq.) under an argon atmosphere and stirred for 1-2 hours. The reaction temperature was raised to 50°C using an oil bath and stirred for 16 hours. After the reaction was complete, the reaction mixture was quenched with water and extracted with SiO2 (3 × 5 mL). The combined organic layer was washed with brine, dehydrated with anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 4g column, 5-10% MeOH in DCM), and 34 was obtained as a yellow solid (0.083g, 65%). LCMS[M+H]+=1371.52.

[0147] Compound 4: To compound 3 (0.083 g, 0.061 mmol) in DCM (2.0 mL), TFA (0.6 mL) was added at ice-cold temperature. Cooling was removed and stirring was continued for 1 hour. After the reaction was complete, the TFA was evaporated, and the residue was washed with cold diethyl ether (3 × 5 mL) and hexane (2 × 3 mL). Compound 4 was dried under vacuum to obtain compound 4 (quantitative yield), which was used without further purification. LCMS[M+H]+=1271.41.

[0148] Compound 6: Compound 5 (0.046 g, 0.061 mmol) in DCM (0.6 mL) was mixed with Compound 4 (0.077 g, 0.061 mmol, 1.0 eq.), followed by TEA (0.025 μL, 0.182 mmol, 3.0 eq.) under an argon atmosphere, and the mixture was stirred for 2-6 hours. After the reaction was complete, the reaction mixture was diluted with water and extracted with toluene (3 × 10 mL). The combined organic layer was washed with brine, dehydrated with anhydrous sodium 2SO4, and concentrated. The residue was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel, 12 g column, 5-10% MeOH in DCM) to obtain compound 6 as a yellow amorphous solid (0.09 g, 79%). LCMS[M+H]+ = 1884.14.

[0149] Compound 7: Compound 6 (0.09 g, 0.048 mmol) was dissolved in DCM (2.1 mL), to which TFA (0.6 mL) was added at ice-cold temperature. Cooling was removed, and stirring was continued for 1 hour. After the reaction was complete, the TFA was evaporated, and the residue was washed with cold diethyl ether (3 × 5 mL) and hexane (2 × 3 mL) to obtain compound 7 (0.079 g, quantitative yield), which was used without further purification. LCMS[M+H]+=1643.74.

[0150] Compound 41: Compound 7 (0.079 g, 0.048 mmol) was dissolved in MeOH (1.2 mL), and 0.5 M sodium methoxide in MeOH (0.5 mL, 3.5 eq.) was added dropwise, and the mixture was stirred for 1 hour. After the reaction was complete, the reaction mixture was neutralized by adding Dowex® 50WX8(H+) resin, filtered, washed with MeOH, and concentrated. The residue was purified by preparative ULC on a C18 column with a 5-95% gradient B for 45 minutes at a flow rate of 15 mL / min (A: 20 mM NH4OAc, pH 7 buffer and B: ACN; UV@360 nm and 240 nm) to obtain compound 41 (3.3 mg, yield 50%). Purity 89%@360 nm and 84%@240 nm. LCMS[M+H]+=1503.62. [Examples]

[0151] (2R,3R,4S)-3-acetamido-2-((14R,58R,59R)-14-(4-(3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanamide)butyl)-59,60-dihydroxy-13,16,56-trioxo-1-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,19,22,25,28,31,34,37,40,43,46,49,52,57-hexadecaoxa-12,15,55-triazahexacontan-58-yl)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (compound 43

[0152] [ka]

[0153] Compound (1) was prepared as described in Carbohydrate Research, 342 (2007), pp. 1636-1650.

[0154] Compound 3. Compound 3 was prepared using the same procedure as for the preparation of Compound 10 in Example 2. Compound 3 was prepared from Compound 1 (0.10 g, 0.13 mmol) in 5 ml of dry THF and 0.12 ml of DIPEA (0.66 mmol), and Compound 2 (Havtech, 82.2 mg, 0.13 mmol) in 2 ml of dry THF. After the reaction was complete, the mixture was concentrated, and the residue was purified by column chromatography eluting in 10% MeOH / DCM to obtain Compound 3 as a grayish-white solid. Yield: 0.08 g, 48.88%; LCMS (ESI) m / z 1230.15 [M+1] + .

[0155] Compound 5. Compound 5 was prepared using the same procedure as for the preparation of Compound 3 in Example 2. Compound 5 was prepared from Compound 3 (0.10 g, 0.08 mmol) and Compound 4 (78.2 g, 0.12 mmol) in DMF (3.0 mL), HATU (0.034 g, 0.09 mmol), and ethylbis(propan-2-yl)amine (GLR, 0.08 mL, 0.41 mmol). After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography eluting in 10% MeOH / DCM to obtain Compound 5 as a yellowish liquid. Yield: 0.09 g, 53.05%; LCMS (ESI) M / 2 1043.85 [M+1] + .

[0156] Compound 43. Compound 43 was prepared using the same procedure as for the preparation of Compound 42 in Example 2. Compound 43 was prepared from Compound 5 (0.09 g, 0.07 mmol) in THF (3 mL), MeOH (1 mL), and 1.0 M aqueous sodium hydroxide solution. After the reaction was complete, the mixture was concentrated and dried, and the residue was purified by preparative HPLC using water / ACN in 0.1% TFA to obtain Compound 43 as a yellowish solid. Yield: 0.016 g, 21.74%; LCMS (ESI) m / z 1706.80 [M+1] + . 11H NMR (400 MHz, DMSO-d6): δ 8.86 (d, J = 2.72 Hz, 1H), 8.82 (brs, 1H), 8.26 - 8.23 (m, 2H), 7.99 (d, J = 8.12 Hz, 1H), 7.94 (t, J = 5.48 Hz, 1H), 7.88 (d, J = 9.12 Hz, 1H), 7.79 (t, J = 5.24 Hz, 1H), 7.40 (d, J = 7.0 Hz, 1H), 7.27 (d, J = 9.72 Hz, 1H), 7.18 - 7.04 (m, 2H), 5.34 (brs, 1H), 4.86 - 4.79 (m, 5H), 4.55 (s, 1H), 4.22 - 4.17 (m, 3H), 3.94 - 3.83 (m, 2H), 3.66 - 3.60 (m, 6H), 3.59 - 3.56 (m, 4H), 3.53 - 3.40 ( m, 56H), 3.39 -3.32 (m, 8H), 3.23 - 3.15 (m, 4H), 3.04 - 3.01 (m, 2H), 2.97 - 2.95 (m, 2H), 2.91 - 2.76 (m, 1H), 2.61 - 2.55 (m, 5H), 2.37 (q, J = 6.52 Hz, 2H), 2.33 - 2.99 (m, 2H), 1.90 (s, 1H), 1.77 - 1.76 (m, 3H), 1.58 - 1.56 (m, 1H), 1.49 -1.43 (m, 1H), 1.35 - 1.29 (m, 2H), 1.28 - 1.19 (m, 3H), 1.16 - 1.11 (m, 5H), 0.99 (t, J = 7.16 Hz, 8H), 0.92 - 0.90 (m, 1H).

Examples

[0157] (2R,3R,4S)-3-acetamido-2-((14R,31R,32R)-14-(4-(3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanamide)butyl)-32,33-dihydroxy-13,16,29-trioxo-1-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,30-tetraoxa-12,15,28-triazatritriacontan-31-yl)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (compound 44)

[0158] [ka]

[0159] Compound (1) was prepared as described in Carbohydrate Research, 342 (2007), pp. 1636-1650.

[0160] Compound 3. Compound 3 was prepared using the same procedure as that used for preparing Compound 10 in Example 2. Compound 10 was prepared from Compound 1 (0.20 g, 0.27 mmol) in dry THF (5 ml), DIPEA (0.25 ml, 1.33 mmol), and 12-aminododecanoic acid (2, 53.7 mg, 0.27 mmol) in dry THF (2 ml). After the reaction was complete, the mixture was concentrated and the residue was purified by column chromatography eluting in 10% MeOH / DCM to obtain Compound 3 as a grayish-white solid. Yield: 0.09 g, 40.86%; LCMS (ESI) m / z 828.20 [M+1] + .

[0161] Compound 5. Compound 5 was prepared using the same procedure as for the preparation of Compound 1 described in Example 2. Compound 5 was prepared from Compound 3 (0.09 g, 0.11 mmol) and Compound 4 (0.10 mL, 0.12 mmol) in DMF (3.0 mL) using HATU (0.045 g, 0.12 mmol) and ethylbis(propan-2-yl)amine (GLR, 0.09 mL, 0.54 mmol). After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography eluting in 10% MeOH / DCM to obtain Compound 5 as a yellowish liquid. Yield: 0.08 g, 43.68%; LCMS (ESI) m / z 1684.30 [M+1] + .

[0162] Compound 44. Compound 44 was prepared using the same procedure as for the preparation of Compound 42 in Example 2. Compound 44 was prepared from Compound 5 (0.09 g, 0.05 mmol) in THF (3 mL), MeOH (1 mL), and 1.0 M NaOH aqueous solution. After the reaction was complete, the mixture was concentrated and dried, and the residue was purified by preparative HPLC using water / ACN in 0.1% TFA to obtain Compound 44 as a yellowish solid. Yield: 0.024 g, 34.44%; LCMS (ESI) m / z 1304.9 [M+1] + . 11H NMR (400 MHz, DMSO-d6): δ 13.15 (brs, 1H), 8.86 (d, J = 2.8 Hz, 1 H), 8.82 (brs, 1H), 8.25 (dd, J = 9.28, 2.32 Hz, 1H), 7.96 (d, J = 9.0 Hz, 1H), 7.87 (t, J = 1.6 Hz, 1H), 7.78 (q, J = 8.36 Hz, 2H), 7.53 (d, J = 8.72 Hz, 1H), 7.26 (d, J = 9.68 Hz, 2H), 7.16 - 7.08 (m, 4H), 5.67 (s, 1H), 4.91 (d, J = 1.8 Hz, 1H), 5.15 (d, J = 1.2 Hz, 1H), 4.85 (dd, J = 3.4, 1.0 Hz, 1H), 4.71 (brs, 2H), 4.56 - 4.55 (m, 2H), 4.52 - 4.12 (m, 6H), 4.01 (q, J = 2.3 Hz, 2H), 3.85 (brs, 2H), 3.66- 3.31 (m, 26H), 3.19 - 3.17 (m, 4H), 2.90 - 2.82 (m, 5H), 2.29 (t, J = 6.36 Hz, 2H), 2.09 - 2.08 (m, 2H), 1.78 (s, 3H), 1.45 - 1.21 (m, 24H), 1.12 (d, J = 6.24 Hz, 3H). Examples

[0163] (2R,3R,4S)-3-acetamido-2-((2R,3R)-15-((S)-2-((S)-2-((R)-23-(4-(3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanamide)butyl)-21,24-dioxo-36-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy )-3,6,9,12,15,18,28,31,34-nonanoxa-22,25-diazahexatriacontyl)carbamoyl)pyrrolidine 1-carbonyl)pyrrolidine 1-carbonyl)pyrrolidine 1-yl)-1,2-dihydroxy-5,15-dioxo-4,9,12-trioxa-6-azapentadecane-3-yl)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (compound 45)

[0164] [ka] [ka]

[0165] Compound 3. Compound 3 was prepared using the same procedure as in Example 2. Compound 3 was prepared from Compound 1 (0.24 g, 0.42 mmol, Habotech) and Compound 2 (0.40 g, 0.42 mmol) in DMF (8.0 mL) using HATU (0.17 g, 0.46 mmol) and ethylbis(propan-2-yl)amine (0.22 mL, 1.25 mmol). After the reaction was complete, the mixture was concentrated, and the residue was purified by silica gel column chromatography eluting in 5% MeOH / DCM to obtain Compound 3 as a yellowish liquid. Yield: 0.35 g, 58.6%; LCMS (ESI) m / z 1449.20 [M+18] + .

[0166] Compound 4. To a stirred solution of Compound 3 (0.30 g, 0.21 mmol) in anhydrous dimethylformamide (3.0 mL), piperidine (0.6 mL) was added, and the reaction mixture was stirred for 30 minutes. Upon completion of the reaction, the solvent was evaporated, and the residue was triturated with diethyl ether to obtain Compound 4 as a bright yellow solution, which was used without further purification. Yield: 0.16 g, 63.13%; LCMS (ESI) m / z 1210.20 [M+1] + .

[0167] Compound 6. Compound 6 was prepared using the same procedure as that used for preparing Compound 3 in Example 2. Compound 6 was prepared from Compound 4 (0.20 g, 0.16 mmol) and Compound 5 (0.09 g, 0.16 mmol) in DMF (5.0 mL) using HATU (0.07 g, 0.18 mmol) and ethylbis(propan-2-yl)amine (0.09 mL, 0.48 mmol). After the reaction was complete, the mixture was concentrated and the residue was purified by silica gel column chromatography eluting in 10% MeOH / DCM to obtain Compound 6 as a yellowish liquid. Yield: 0.11 g, 38.61%; LCMS (ESI) M / Z 1741.48 [M+1] + .

[0168] Compound 7. To a stirred solution of Compound 6 (0.11 g, 0.17 mmol) in dimethylformamide anhydrous (2.0 mL), piperidine (0.4 mL) was added, and the reaction mixture was stirred for 30 minutes. Upon completion of the reaction, the solvent was evaporated, and the residue was triturated with diethyl ether to obtain Compound 7 as a bright yellow solution, which was used without further purification. Yield: 0.085 g, 88.71%; LCMS (ESI) m / z 1500.90 [M+1] + .

[0169] Compound 9. Compound 9 was prepared using the same procedure as that used for preparing Compound 3 in Example 2. Compound 3 was prepared from Compound 7 (0.08 g, 0.05 mmol) and Compound 8 (0.04 g, 0.05 mmol) in DMF (4.0 mL) using HATU (0.02 g, 0.06 mmol) and ethylbis(propan-2-yl)amine (0.03 mL, 0.15 mmol). After the reaction was complete, the mixture was concentrated and the residue was purified by silica gel column chromatography eluting in 10% MeOH / DCM to obtain Compound 9 as a yellowish liquid. Yield: 0.09 g, 74.86%; LCMS (ESI) m / z 1137.60 [M / 2+1] + .

[0170] Compound 45. Compound 45 was prepared using the same procedure as for the preparation of Compound 42 in Example 2. Compound 45 was prepared from Compound 9 (0.09 g, 0.04 mmol) in THF (3 mL) and MeOH (1 mL). After the reaction was complete, the mixture was concentrated and dried, and the residue was purified by preparative HPLC using water / ACN in 0.1% TFA to obtain Compound 45 as a yellowish solid. Yield: 20 mg, 26.69%; HRMS (ESI) m / z 1892.91 [M+1] + . 11H NMR (400 MHz, DMSO-d6): 8.86 (d, J = 2.80 Hz, 1H), 8.83 (brs, 1H), 8.26 (dd, J = 9.2 & 2.4 Hz, 1H), 8.00 - 7.90 (m, 3H), 7.83 - 7.78 (m, 2H), 7.53 (brs, 1H), 7.27 (d, J = 9.6 Hz, 2H), 7.22 - 7.20 (m, 1H), 5.66 (brs, 1H), 5.09 (brs, 1H), 4.84 (dd, J = 10.8 & 2.0 Hz, 1H), 4.76 - 4.73 (m, 2H), 4.61 - 4.51 (m, 4H), 4.36 -4.28 (m, 1H), 4.26 - 4.19 (m, 3H), 3.99 (qt, J = 8.8 Hz, 1H), 3.81 (brs, 1H), 3.65 - 3.58 (m, 10H), 3.53 - 3.24 (m, 64H), 2.49 - 2.33 (m, 4H), 2.25 - 2.1.83 (m, 7H), 1.80 (s, 3H), 1.62 - 1.1.55 (m, 1H), 1.45 - 1.30 (m, 3H), 1.25 - 1.18 (m, 1H), 1.11 (d, J = 6.4 Hz, 3H). [Examples]

[0171] (2R,3R,4S)-3-acetamido-2-((14R,17R,23R,29R,58R,59R)-14-(4-(3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanamide)butyl)-59,60-dihydroxy-17,23,29-tris(hydroxymethyl)-13,16,19,22,25,28,31,34,56-nanooxo-1-(((2R ,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,37,40,43,46,49,52,57-decaoxa-12,15,18,21,24,27,30,33,55-nonazahexacontan-58-yl)-4-guanidino-3,4-dihydro-2H-pyran-6-carboxylic acid (compound 46)

[0172] [ka] [ka]

[0173] Compound 8a. The peptide solid-phase synthesis (SPS) of compound 8 was performed manually in a peptide reactor using 2-CTC resin, under a standard peptide coupling and deprotection protocol using Fmoc and tBu protecting groups.

[0174] General procedure for amide coupling: DMF and an activated amino acid solution prepared by weighing appropriate amounts of Fmoc-Gly-OH (Chemscene, 2.0 eq.) or Fmoc-Ser(OtBu)-OH (BLD Pharma, 2.0 eq.) or 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-oic acid (Habotech; 2.0 eq.) from HATU (2.5 eq.) and DIPEA (3.0 eq.) were added to a reactor containing swollen 2CTC resin. The reactor was shaken for 2 hours.

[0175] General procedure for Fmoc deprotection: The Fmoc group was deprotected by shaking a reactor containing resin in 20% piperidine in DMF for 0.5 hours.

[0176] Compound 8: Compound 6 was added to a reactor containing swollen 2CTC resin. A solution of Compound 7 (2.0 eq.) in dry THF was then added dropwise, followed by DIPEA (GLR, 3 eq.). The reactor was shaken for 24 hours.

[0177] Compound 8a (resin cleavage): Compound 8 was added to a 2CTC resin, which was then suspended in 20% 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in DCM in a peptide reactor. The reaction vessel was shaken for 30 minutes. The resin was filtered and washed three times with 10 mL of 20% HFIP in DCM. Cold diethyl ether was added to the combined filtrate to obtain 8a as a white solid. Yield: 250 mg. LC-MS (ESI) m / z 1567.45 [M+1] + .

[0178] Compound 10. Compound 10 was prepared using the same procedure as for the preparation of Compound 3 in Example 2. Compound 10 was prepared from Compound 8 (0.25 g, 0.10 mmol) and Compound 9 (0.11 g, 0.11 mmol) in DMF (6.0 mL) using HATU (Chempure, 0.043 g, 0.33 mmol) and ethylbis(propan-2-yl)amine (GLR, 0.08 mL, 0.11 mmol). After the reaction was complete, the mixture was concentrated and the residue was purified by silica gel column chromatography eluting in 5% MeOH / DCM to obtain Compound 10 as a yellowish liquid. Yield: 0.16 g, 63.65%; LCMS m / z 1211.85 [M / 2+1] + .

[0179] Compound 46. Compound 46 was prepared using the same procedure as for the preparation of Compound 42 in Example 2. Compound 46 was prepared from Compound 10 (0.12 g, 0.05 mmol) in THF (3 mL) and MeOH (1 mL). After the reaction was complete, the mixture was concentrated and dried, and the residue was purified by preparative HPLC using water / ACN in 0.1% TFA to obtain Compound 46 as a yellowish solid. Yield: 0.019 g, 20.47%; HRMS (ESI) m / z 1874.82 [M+1] + . 1 1H NMR (400 MHz, DMSO-d6): 8.86 (d, J = 2.80 Hz, 1H), 8.83 (brs, 1H), 8.27 - 8.24 (m, 3H), 8.14 (brs, 1H), 8.02 - 7.92 (m, 4H), 7.84 - 7.79 (m, 2H), 7.51 (brs, 1H), 7.27 (d, J = 10.6 Hz, 2H), 7.07 - 7.01 (m, 3H), 5.60 (brs, 1H), 5.16 - 5.06 (m, 3H), 4.83 (d, J = 10.6 Hz, 1H), 4.74 - 4.73 (m, 2H), 4.56 (s, 2H), 4.48 (brs, 1H), 4.33 - 4.20 (m, 5H), 4.15 - 4.14 (m, 1H), 3.96 (qt, J = 9.2 Hz, 1H), 3.84 - 3.72 (m, 7H), 3.66 - 3.55 (m, 15H), 3.54 - 3.45 (m, 30 H), 3.44 - 3.36 (m, 7H), 3.23 - 3.16 (m, 4H), 3.05 - 2.5 (4H), 2.38 (t, J = 8.4 Hz, 1H), 2.30 (t, J = 8.4 Hz, 1H), 1.78 (s, 3H), 1.70 - 1.60 (m, 1H), 1.55 - 1.45 (m, 1H), 1.38 - 1.23 (m, 4H), 1.12 (d, J = 6.4 Hz, 3H). [Examples]

[0180] Ethyl(3R,4R,5S)-4-acetamido-5-(3-(25-(18-((2,4-dinitrophenyl)amino)-13-oxo-3,6,9,16-tetraoxa-12-azaoctadecyl)-13,26-dioxo-1-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3,6,9,16,19,22,29,32-octaoxa-12,25-diazatetratriacontan-34-yl)ureido)-3-(pentan-3-yloxy)cyclohexa-1-ene-1-carboxylate (compound 47)

[0181] [ka]

[0182] Compound 3. Compound (3) was prepared using commercially available oseltamivir (1, BLD) and amino-PEG2-t-butyl ester (2, BLD) as described in Eur. J. Med. Chem., 2020, 200, 112423.

[0183] Compound 4. To a stirred solution of Compound 3 (0.10 g, 0.17 mmol) in anhydrous DCM (3.0 mL), TFA (1.0 mL) was added at 0°C. Cooling was stopped, and the reaction mixture was stirred for 12 hours. After the reaction was complete, the solvent was evaporated to obtain Compound 4 as a bright yellow solution, which was used in the next reaction. Yield: 0.08 g; LCMS (ESI) m / z 516.20 [M+1] + .

[0184] Compound 6. Compound 6 was prepared using the same procedure as for the preparation of Compound 3 in Example 2. Compound 6 was prepared from Compound 4 (0.15 g, 0.29 mmol) and Compound 5 (0.36 g, 0.32 mmol) in DMF (8.0 mL) using HATU (0.17 g, 0.32 mmol) and ethylbis(propan-2-yl)amine (GLR, 0.27 mL, 1.45 mmol). After the reaction was complete, the mixture was concentrated and the residue was purified by silica gel column chromatography eluting in 10% MeOH / DCM to obtain Compound 6 as a yellowish liquid. Yield: 0.095 g, 22.05%; LCMS (ESI) m / z 1622.65 [M+1] + .

[0185] Compound 47. Sodium ethoxide (0.41 mg, 0.002 mmol) was added at 0°C to a stirred solution of compound 6 (0.10 g, 0.061 mmol) in ethanol (5.0 mL). Cooling was stopped and the reaction mixture was stirred for 1 hour. After the reaction was complete, the reaction mixture was diluted with acid (Dowex 50, H) until the pH was approximately 7. + The mixture was treated with [a specific method], filtered through a calcined funnel, and washed with MeOH. The filtrate was concentrated, and the residue was purified by preparative HPLC to obtain compound 47 as a yellowish solid. Yield: 25 mg, 27.11%; LCMS (ESI) m / z 1496.80 [M+1] + . 1H NMR (400 MHz, DMSO-d6): δ 8.86 (d, J = 2.80 Hz, 1H), 8.83 (brs, 1H), 8.25 (dd, J = 9.2 & 2.4 Hz, 1H), 7.90 (m, 2H), 7.80 (d, J = 8.8 Hz, 1H), 7.27 ((d, J = 9.6 Hz, 1H), 6.10 (brs, 1H), 5.77 (d, J = 7.2 Hz, 1H), 4.55 (d, J = 1.6 Hz, 1H), 4.16 - 4.10 (m, 2H), 4.08 - 4.03 (m, 1H), 3.76 - 3.55 (m, 22H), 3.53 - 3.33 (m, 51H), 3.20 - 3.13 (m, 8H), 2.61 - 2.57 (m, 4H), 2.34 - 2.29 (m, 5H), 1.78 (s, 3H), 1.47 - 1.32 (m, 4H), 1.21 (d, J = 7.2 Hz, 3H), 1.11 (d, J = 6.0 Hz, 3H), 0.83 (t, J = 7.2 Hz, 3H), 0.76 (t, J = 7.2 Hz, 3H).

[0186] Method Example 1: Binding affinity of compound 24 to HEK expressing neuraminidase. K when it competes with zanamivir-rhodamine. i (Nature Communications 11, article number: 5597(2020)) was determined using binding and competition assays. First, the K of zanamivir-rhodamine binding to neuraminidase was determined. dThe following was determined. Human fetal kidney cells (HEK239) transfected with influenza virus neuraminidase were suspended in staining buffer (2% fetal bovine serum in phosphate-buffered saline) at a density of 2 million cells / mL and then seeded in triplicate on a U-bottom plate (50 μL / well). Next, two sets of serial dilutions of zanamivir-rhodamine were prepared in staining buffer. Nonspecific binding was determined in one set, including 100-fold zanamivir. The diluted compounds were added to the cells (50 μL / well) and incubated on a shaker at room temperature for 1 hour. The cells were washed twice with 200 μL of phosphate-buffered saline and resuspended in 150 μL of staining buffer. Fluorescence was measured using an Atuune NxT flow cytometer (Fisher), and K d This was calculated using Graph Pad Prism, one site-total, and non-specific binding.

[0187] Next, compound 24 and zanamivir K i We were able to determine the K. Cells were seeded as described above. Compound 24 was subjected to a series of serial dilutions, and zanamivir was subjected to a separate dilution. Each dilution was mixed with an equal volume of 80 nM zanamivir-rhodamine. The compound mixture was added to the cells (50 μL / well) and incubated on a shaker at room temperature for 1 hour. The cells were washed and resuspended as described above. Fluorescence was measured using an Atuune NxT flow cytometer (Fisser). The K calculated above was used. d And using a final concentration of 20 nM of zanamivir-rhodamine, and using One site-Fit Ki on Graph Pad Prism, K i We were able to calculate it.

[0188] Figure 5 shows that zanamivir-rhodamine binds to neuraminidase with a binding affinity of 8.253 nM. The binding of zanamivir to neuraminidase is well-established in the literature, and excessive fluorescence blockade by zanamivir indicates that zanamivir-rhodamine is binding to neuraminidase. The inhibition constants of compound 24 and zanamivir were quantified using this same rhodamine conjugate (Figure 6). Zanamivir has a Ki of 0.496 nM. The Ki of compound 24 was 7.152 nM, which is in the low nanomolar concentration range.

[0189] Method Example 2: Dose Range Exploration Study - Viral Titer. A group of 6-8 week old female BALB / c mice (n=5 / group) were infected with 10LD50 H3N2 / Wisconsin / 15 / 2009 influenza virus on day 0 of the experiment. At 24 hpi, mice were administered 6 g / kg of human IVIg by intraperitoneal injection, achieving human antibody titers against DNP and rhamnose at the time of test substance administration. At 48 hpi, mice were treated with the test substance. Compound 24 was administered as a single intranasal dose of 2.2 mg / kg, 0.72 mg / kg, 0.24 mg / kg, or 0.081 mg / kg in a predetermined cohort. Mice in two control groups received Tamiflu (oseltamivir phosphate) as a positive control (5 mg / kg, twice daily for 5 days) and a vehicle (PBS) as a placebo. Viral titers in mouse lungs were measured in two ways: live infectious virus was measured using a hemagglutination assay, and total viral RNA was measured using reverse transcription polymerase chain reaction (rt-PCR). However, rt-PCR cannot distinguish between live and dead viruses and is therefore not useful for determining efficacy.

[0190] First, mouse lungs were homogenized with 5 μL of cold phosphate-buffered saline per milligram of tissue. 100 μL of lysate was set aside for RNA isolation, and the remaining lysate was used for hemagglutination. All samples were stored at -80°C.

[0191] For the hemagglutination assay, MDCK-London (Madinderby canine kidney) cells were seeded in a 96-well plate and incubated until confluence. On the day of the assay, lung lysates were serially diluted 3-fold with DMEM / F12 (1:1) supplemented with 0.3% bovine serum albumin. The MDCK-London cells were washed, and 50 μL of the diluted lysate was added to each well so that each dilution contained 4 copies. The cells were incubated at 37°C and 5% CO2 for 1 hour, and then 200 μL of DMEM / F12 (1:1) supplemented with 0.3% bovine serum albumin and 1 μg / ml trypsin was added to each well. The cells were incubated at 37°C and 5% CO2 for 48 hours. Each well was tested for hemagglutination by incubation in a V-bottom plate with 50 μL of tissue culture supernatant containing 50 μL of 0.5% turkey erythrocytes in phosphate-buffered saline. After 30 minutes at room temperature, the plate was tilted 45 degrees and the negative or positive wells were counted. Negative wells had red dots that rolled when the plate was tilted, similar to the negative control. Positive wells were cloudy and did not have red dots at the bottom of the well. For each lung sample, TCID was performed using the Reed-Münch method. 50 The values ​​were calculated and plotted using GraphPad Prism.

[0192] Figure 7 shows that most of the live infectious viruses were eliminated after treatment with compound 24. This is supported by the high survival rate and rapid weight recovery of mice treated with compound 24, as seen in Method Example 5 below and the corresponding figure.

[0193] Method Example 3: Neuraminidase Inhibition Assay: To evaluate the neuraminidase inhibitory activity of compound 24, a standard neuraminidase inhibitory assay was performed using the NA-Fluor® influenza neuraminidase assay kit (catalog number 4457091, Invitrogen®). The assay was performed according to the manufacturer's protocol. Briefly, a standard curve was first created using 4-methylumbelliferone sodium salt (4-MU(SS)), and the linear range of substrate turnover detection was determined on a Synergy Neo2 HTS multimode microplate reader (Biotek). Relative fluorescence unit (RFU) values ​​were identified on the instrument within the linear range of fluorescence detection. For each virus strain, the dilution factor best corresponding to the selected relative fluorescence unit (RFU) value to be used in the assay was determined from the 4-methylumbelliferone sodium salt (4-MU(SS)) standard curve. The virus stock solution was then titrated by the neuraminidase activity assay. For this assay, serial dilutions of the virus stock solution were prepared in black, 96-well flat-bottom plates. Then, 50 μL of 200 μM NA-Fluor® substrate (MUNANA, 4-(methylumbelliferone sodium)-N-acetylneuraminic acid) was added to each well. The plate was protected from light and incubated at 37°C for 60 minutes. The plate was placed in a shaker inside the incubator. The reaction was then terminated by adding 100 μL of NA-Fluor® reaction stop solution (0.2 M sodium carbonate) to each well. Fluorescence intensity was then measured using excitation wavelength 350 nm and emission wavelength 440 nm. Relative fluorescence units (RFU) values ​​were plotted for each virus dilution and each virus strain, and dilution factors producing RFUs selected from the 4-methylumbelliferone sodium salt (4-MU(SS)) standard curve were selected and used for normalization of neuraminidase activity in the neuraminidase inhibition assay. Finally, for the neuraminidase inhibition assay, 10-fold serial dilutions of the test compound were prepared in NA-Fluor® assay buffer (66.6 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 8 mM CaCl2, pH 6.5).Next, 25 μL of a 4-fold serial dilution of compound 24 was added to all rows of a 96-well plate, followed by 25 μL of 1x assay buffer added to the virus-free control wells. 25 μL of diluted virus sample was added to the designated wells and mixed with the diluted compound 24. The plate was incubated at 37°C for 30 minutes with shaking. Next, 50 μL of diluted 200 μM NA-Fluor® substrate was added to each well and incubated at 37°C for 60 minutes, protected from light. The reaction was terminated by adding 100 μL of NA-Fluor® reaction stop solution to all wells. For fluorescence intensity measurement, the plate was read using an excitation wavelength of 350 nm and an emission wavelength of 440 nm. The data was plotted using GraphPad Prism 10.0.0 software to create sigmoid dose-response curves. The graphs were then analyzed using the built-in nonlinear regression curve fitting program to determine the half-number inhibitory concentration (IC). 50 ) was decided. Lower IC 50 A higher value indicates the strength of the compound.

[0194] Figure 9 illustrates the sigmoid dose-response curves for each virus strain, and the table within it shows the respective median inhibitory concentrations (IC). 50 The values ​​shown are. These data illustrate that zanamivir, which targets the ligand portion in compound 24, not only binds to the target enzyme but also inhibits its function, acting as a direct antiviral agent across many strains. Neuraminidase inhibition suppresses viral particle budding from infected cells and limits the spread of infection to nearby healthy cells.

[0195] Method Example 4. A group of 6-8 week old female BALB / c mice (n=5 / group) were given 10 LD on day 0 of the experiment. 50The mice were infected with the A / H1N1 / PR8 / 1934 influenza virus. 24 hours prior to drug administration, the mice were given 6 g / kg of human IVg by intraperitoneal injection to achieve human antibody titers against DNP and rhamnose at the time of test substance administration. The mice were treated with the test substance at 48 hpi (Figure 2A) or 96 hpi (Figure 2B). Compound 24 was administered in a designated cohort as a single intranasal dose of 1.5 μmol / kg, a single intravenous dose of 1.5 μmol / kg, or a single oral dose of 13.5 μmol / kg. Three control groups of mice received PBS with either Tamiflu (oseltamivir phosphate), Xofluza (baloxavir marboxil), or placebo.

[0196] To evaluate the efficacy of the drug, mice were weighed and monitored daily for 14 days after infection. A death was counted if the mouse lost 25% of its body weight or was diagnosed as critically ill.

[0197] The results of this experiment are plotted against body weight, illustrating the ratio of days after infection to survival rate (%) and days after infection to body weight (%) for mice (n=5 / group).

[0198] In the initial treatment phase (48 hpi), mice treated with a single intranasal dose of compound 24 at 1.5 μmol / kg had a 100% survival rate, as did mice treated with a single intravenous dose of compound 24 at 1.5 μmol / kg. The cohort treated with a single oral dose had an 80% survival rate. Mice treated with 5 mg / kg of Tamiflu twice daily for 5 days had a 60% survival rate, while mice treated with a single oral dose of 10 mg / kg of Xofluza had an 80% survival rate. On the other hand, in the later treatment phase (96 hpi), mice treated with a single intranasal dose of compound 24 at 1.5 μmol / kg had a 100% survival rate, as did mice treated with a single intravenous dose of compound 24 at 1.5 μmol / kg. The cohort treated with a single oral dose had a 60% survival rate. Mice treated with 5 mg / kg of Tamiflu twice daily for 5 days had a survival rate of 20%, while mice treated with a single oral dose of 10 mg / kg of Xofluza had a survival rate of 60%.

[0199] This demonstrates that compound 24 is more effective than Tamiflu and Xofluza in both the early and late stages of infection, and that all three routes of administration are effective, with intranasal administration at a dose of 1.5 μmol / kg being the best route of administration.

[0200] Method Example 5: Dose Range Exploration Study - Efficacy / Toxicity. In the study of Method Example 2, survival and cytokine response were also measured. Regarding weight gain, mice were weighed and monitored daily for 14 days post-infection, and were counted as dead if they lost 25% of their body weight or were diagnosed as mortally wounded.

[0201] The results of this experiment are shown in Figure 11. Figure 11 is a graph of the survival rate (%) and the survival rate (%) of days after infection and body weight (%) for mice (n=5 / group) that were infected with 10LD50 influenza A H3N2 / Wisconsin / 15 / 2009 and administered intraperitoneally with human IgG (IVIg(GAMUNEX(registered trademark)-C) at 24 hpi and a conjugate at 48 hpi.

[0202] Mice treated with single intranasal doses of compound 24 at 2.2 mg / kg, 0.72 mg / kg, and 0.24 mg / kg achieved a 100% survival rate, while a cohort treated with a single intranasal dose of 0.081 mg / kg achieved a 60% survival rate. Mice treated with 5 mg / kg of Tamiflu twice daily for 5 days also achieved a 60% survival rate. This demonstrates the superior efficacy of compound 24 compared to Tamiflu and suggests that a dose of 0.24 mol / kg may be the minimum effective dose for compound 24 to achieve 100% survival in mice. While all cohort mice experienced gradual weight loss, those that survived were able to recover from the weight loss induced by the viral infection. This data indicates that compound 24 is 100% effective, and that even at one-ninth the dose, a single dose of compound 24 is equivalent to 10 doses of Tamiflu administered over a 5-day period.

[0203] Cytokines from both the treatment and control groups were measured in lung tissue samples (n=3 / group) using BioLegend's LEGENDplex® bead-based immunoassay, employing a standard protocol provided by the manufacturer (BioLegend, San Diego, CA).

[0204] Figures 12 (lung) and 13 (serum) show graphs of specific cytokine expression levels in the lung and serum, respectively, measured for each group 24 hours after drug administration. Compound 24 administered intranasally did not induce a cytokine storm in the lung or serum. A dose-dependent decrease in IFN-γ was observed in the lung. Levels of TNF-α and 12 other inflammatory cytokines, which are not reported here but are included in BioLegend's LEGENDplex kit, did not change in either the lung or serum. Regarding IL-6, the data shown were not statistically significant. For comparison, the decrease in IFN-γ in the lung indicates a reduction in systemic inflammation, resulting from more rapid elimination of the infection during treatment with compound 24. In serum, IFN-γ showed an increase, but with large, statistically insignificant error bars. These data indicate that compound 24 does not induce a cytokine storm.

[0205] Method Example 6. A cytokine release assay was performed for compound 24 (results shown in Figure 8) to assess the risk of causing a cytokine storm in humans. For this assay, human PBMCs pooled from at least four donors were washed and seeded at 5 × 10⁵ cells / well. 1000 nM of compound 24 was added to PBMCs with or without 10 mg / mL IVIG until a final volume of 200 uL was reached. A 1000 nM TLR7 agonist was used as a positive control, and the culture medium was used as a negative control. PBMCs were incubated with the compound at 37C and 5% CO₂, and supernatant samples were collected at 2, 6, and 24 hours. Cells were removed from the supernatant by centrifugation at 450 × g for 10 minutes, and the supernatant was stored at -80C.

[0206] Cytokines in the supernatant were measured using a human antiviral response panel (biolegend). This kit measures 13 different proteins using fluorescently encoded beads and fluorescently labeled antibodies: IL-1β, IL-6, IL-8, IL-10, IL-12p70, IFN-α2, IFN-β, IFN-λ1, IFN-λ2 / 3, IFN-γ, TNF-α, IP-10, and GM-CSF. Fluorescence was measured using an Attune NxT flow cytometer (Fisher), and the concentration of each protein was quantified using LEGENDplex™ software (biolegend). Finally, the data were graphed using GraphPad prism.

[0207] For all cytokines tested, compound 24 was at a similar level to the negative control and did not exhibit intrinsic immunogenicity from the molecule alone. Because compound 24 could potentially bind to two antibodies, IVIG was included to determine the risk of agglutination that could trigger an immune response. IVIG was administered at a physiologically appropriate concentration of 10 mg / mL, which increased the background for some of the proteins tested. Considering this, compound 24 again exhibited a similar level to the negative control and did not show immunogenicity from the molecule when bound to antibodies. This suggests that compound 24 is unlikely to induce cytokine release unless it recruits immune cells in circulation and binds to neuraminidase-expressing cells or viruses.

[0208] Method Example 7. To evaluate the neuraminidase inhibitory activity of compound 41, a standard neuraminidase inhibitory assay was performed using the NA-Fluor™ influenza neuraminidase assay kit (catalog number 4457091, Invitrogen™). The assay was performed according to the manufacturer's protocol. Briefly, a standard curve was first created using 4-methylumbelliferone sodium salt (4-MU(SS)), and the linear range of substrate turnover detection was determined on a Synergy Neo2 HTS multimode microplate reader (Biotek). Relative fluorescence unit (RFU) values ​​were identified within the linear range of fluorescence detection on the instrument. For each virus strain, the dilution factor best corresponding to the selected relative fluorescence unit (RFU) value to be used in the assay was determined from the 4-methylumbelliferone sodium salt (4-MU(SS)) standard curve. The virus stock solution was then titrated by the neuraminidase activity assay. For this assay, serial dilutions of the virus stock solution were prepared in black, 96-well flat-bottom plates. Then, 50 μL of 200 μM NA-Fluor™ substrate (MUNANA, 4-(methylumbelliferone sodium)-N-acetylneuraminic acid) was added to each well. The plates were protected from light and incubated at 37°C for 60 minutes. The plates were placed in a shaker inside the incubator. The reaction was then terminated by adding 100 μL of NA-fluor™ reaction stop solution (0.2 M sodium carbonate) to each well. Fluorescence intensity was then measured using excitation wavelength 350 nm and emission wavelength 440 nm. Relative fluorescence units (RFU) values ​​were plotted for each virus dilution and each virus strain, and dilution factors producing RFUs selected from the 4-methylumbelliferone sodium salt (4-MU(SS)) standard curve were selected and used for normalization of neuraminidase activity in the neuraminidase inhibition assay. Finally, for the neuraminidase inhibition assay, the test compound was serially diluted 10-fold in NA-Fluor™ assay buffer (66.6 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 8 mM CaCl2, pH 6.5).Next, 25 μL of a 4-fold serial dilution of compound 41 was added to all rows of a 96-well plate, followed by 25 μL of 1-fold assay buffer added to the virus-free control wells. 25 μL of diluted virus sample was added to the designated wells and mixed with the diluted compound 41. The plate was incubated at 37°C for 30 minutes with shaking. Then, 50 μL of diluted 200 μM NA-Fluor™ substrate was added to each well and incubated at 37°C for 60 minutes, protected from light. The reaction was terminated by adding 100 μL of NA-Fluor™ reaction stop solution to all wells. For fluorescence intensity measurement, the plate was read using an excitation wavelength of 350 nm and an emission wavelength of 440 nm. The data were plotted using GraphPad Prism 10.0.0 software to create sigmoid dose-response curves. The graphs were then analyzed using the built-in nonlinear regression curve fitting program to determine the half-percentage inhibitory concentration (IC50). A lower IC50 value indicates a higher strength of the compound.

[0209] Figure 10 illustrates the sigmoid dose-response curves for each virus strain, and the table within it shows the median inhibitory concentrations (IC) for each compound 41. 50 The values ​​shown illustrate that zanamivir, which targets the ligand portion of compound 41, not only binds to the target enzyme but also inhibits its function, acting as a direct antiviral agent across many strains. Neuraminidase inhibition suppresses viral particle budding from infected cells and limits the spread of infection to nearby healthy cells.

[0210] Method Example 8. Efficacy of compound 24IN against multiple influenza strains (Figures 14A and 14B). The efficacy of an intranasal formulation of compound 24 (0.6% n-dodecyl β-D-maltoside in PBS, "DDM" and 2% Avicel 591), referred to as "compound 24IN") was evaluated against three influenza strains: A / H3N2 / Wisconsin / 15 / 2009, A / California / 07 / 2009(H1N1)pdm09 (a pandemic strain of H1N1 that accounts for a large portion of the seasonal influenza burden each year), and B / Brisbane / 60 / 2008). In Figures 14A and 14B, compound 24IN was compared with oseltamivir phosphate (oral) and vehicle controls (0.6% DDM and 2% Avicel 591 in PBS and intranasally). In each study, mice were infected with one of three seasonal influenza strains with a 10 × LD50 dose, and 48 hours after infection, they were administered either 2.2 mg / kg of compound 24IN, oseltamivir phosphate orally, or a vehicle control intranasally. A partial cohort was sacrificed 24 hours after infection, and viral titers in the lungs were detected. The remainder of the group were observed for the following 14 days as part of a survival study, with daily weight recordings.

[0211] As shown in Figure 14A, compound 24IN showed a 100% survival rate for A / H3N2 / Wisconsin / 15 / 2009, in contrast to the group receiving oseltamivir phosphate (60% survival rate) and the vehicle control (0% survival rate). The Mantelcox log-rank test showed that the survival curve for compound 24 was statistically significant for influenza A / Winconsin / 15 / 2009(H3N2) with a p-value of 0.0013. Compound 24IN showed a 100% survival rate for A / California / 07 / 2009(H1N1)pdm09, in contrast to 40% in the oseltamivir phosphate group and 0% in the vehicle control group. The Mantelcox log-rank test showed that the survival curve for compound 24 was statistically significant for the A / California / 07 / 2009(H1N1pdm09) strain with a p-value of 0.0006. Compound 24 showed a 100% survival rate against influenza B, in contrast to 0% in the vehicle control group. In the oseltamivir phosphate treatment group, 40% lost more than 25% of their initial body weight during the two-week study period and were euthanized before the end of the study. The Mantelcox log-rank test showed that the survival curve for compound 24 was statistically significant (p-value 0.0065) for the B / Brisbane / 60 / 2008 strain.

[0212] In addition, compound 24IN appeared to result in a better quality of life in infected mice, as no significant changes in body weight were detected. Weight loss was observed in all other treatment groups. Figure 14A shows the corresponding lung viral titers.

[0213] Method Example 9. Efficacy against influenza A / Hong Kong / 2369 / 2009 (H1N1pdm09, Tamiflu resistant) (Figures 15A and 15B). The efficacy of compound 24IN was tested in a mouse model against A / Hong Kong / 2369 / 2009 (H1N1pdm09, oseltamivir (Tamiflu) resistant, an H1N1 pandemic strain, which accounts for a large portion of the seasonal influenza burden each year and is also Tamiflu resistant). All mice were first given 10 × LD50 Infection was carried out using the influenza A / Hong Kong / 2369 / 2009 (H1N1pdm09, oseltamivir [Tamiflu] resistant) strain. IVIG (6 g / kg) was administered intraperitoneally (IP) to compound 24IN and vehicle groups to induce human-level anti-DNP and anti-rhamnose antibody titers.

[0214] The procedure was initiated at 24 hpi (1 dpi). Mice that received either 2.2 mg / kg of compound 24IN or a vehicle control were first anesthetized with isoflurane. Each mouse was then given either 0.25 mL / kg of the vehicle intranasally or 0.25 mL / kg of compound 24IN into its nostril, and then returned to its cage. Mice receiving Tamiflu (oseltamivir phosphate) were force-administered 5 mg / kg orally, and then returned to their cage.

[0215] Twenty-four hours after the procedure, three mice were selected from each group, euthanized, and their lungs were collected and flash-frozen in liquid nitrogen. The lungs were homogenized, and a triple TCID50 assay was performed on MDCK cells to assess the cytopathic effect. Viral titers were calculated using the Reid-Münch method.

[0216] The remaining mice were observed and weighed daily during the two-week study period, at which point they were euthanized. Mice that had lost 25% of their original body weight at any point during the study were euthanized according to the IACUC protocol.

[0217] As shown in Figure 15A, compound 24IN demonstrated protection against influenza A strain A / Hong Kong / 2369 / 2009 (H1N1pdm09, oseltamivir resistant) by complete elimination of detectable viral titers and survival of all mice until the completion of the study (day 14). As shown in Figure 15C, oseltamivir and vehicle-treated mice were found to contain similarly high viral titers in lung samples collected 24 hours after drug administration (approximately 2 × 10⁶ TCID50), and all mice in these groups (except the compound 24IN group) were sacrificed by day 7 due to a weight loss of more than 25%, as shown in Figure 15B. The Mantelcox log-rank test showed that the survival curve for compound 24 was statistically significant (p-value 0.0009) against influenza A strain A / Hong Kong / 2369 / 2009 (H1N1pdm09, oseltamivir resistant).

[0218] Method Example 10. Prevention of H1N1 infection by compound 24 (Figure 16). The ability of compound 24IN to prevent influenza infection was evaluated. A single dose of compound 24IN was found to outperform the vehicle control. For this study, guinea pigs were infected with human H1N1 influenza and administered 4 g / kg of IVIG intraperitoneally. The guinea pigs were then treated with either a vehicle or 4 mg / kg of compound 24IN intranasally 24 hours after infection. 24 hours after treatment, the infected guinea pigs were co-fed with untreated guinea pigs for 14 days. Nasal swabs were collected every other day, and viral RNA was measured using PCR. At the end of the study, influenza exposure was assessed by measuring anti-neuraminidase and anti-hemagglutinin antibody titers.

[0219] The results are shown in Figure 16. In the group treated with the vehicle, all untreated co-fed animals were infected by day 4, and infection was confirmed by an increase in antibody titer. In the group treated with compound 24IN, only one out of 10 untreated co-fed guinea pigs became infected, and this occurred on day 8. Infection was confirmed by positive serological reactions to anti-hemagglutinin and anti-neuraminidase. The data suggest that compound 24IN not only protected 90% of the guinea pigs in this long-term exposure infection model, but also delayed infection in one case of vehicle-related infection.

[0220] In public health interventions, reducing viral shedding through infection reduction is considered as important as managing symptoms. While symptom reduction can improve patient well-being, it can lead to a premature resumption of normal activities during the infection period. These data suggest that a single dose treatment with compound 24 reduced the rate of secondary infection with human H1N1 by 90%.

[0221] Method Example 11. Compound 24 Mutation Development (Figures 17A and 17B). Compound 24IN was evaluated for its ability to induce drug-resistant variants of influenza A virus (IAV) after successive passage in the presence of compound 24. Traditionally, drug resistance development studies are performed in vitro to simplify experimental design and to rapidly identify mutations. However, due to the immunologically dependent nature of compound 24, the mutation development studies described here were performed in vivo to more rigorously test whether compound 24 induces drug-resistant IAV in conjunction with a functioning immune system. Furthermore, due to compound 24's efficacy in removing almost all traces of IAV, it was necessary to use suboptimal doses to maintain a measurable viral load and promote mutations during passage. A dose of 81 μg / kg of compound 24 was able to reduce IAV infection in mice but did not eliminate it, and was therefore selected as a suboptimal dose.

[0222] Three groups of five mice each were used for each passage cycle. All groups received 10 LD50. 50 Influenza A / PR8 / 34(H1N1) virus was administered. Group 1 received a single dose of compound 24 (81 μg / kg) intranasally 48 hours after infection. Group 2 received a single intranasal administration of vehicle (20 mg / ml Avicel, 6 mg / ml DDM, 1×PBS, pH 7.4) 48 hours after infection. Group 3 received 5 mg / kg of oseltamivir phosphate orally twice daily for 2 days, starting 48 hours after infection. 24 hours after infection, IVIG (6 g / kg) was administered intraperitoneally (IP) to the compound 24IN and vehicle groups to induce human-level anti-DNP and anti-rhamnose antibody titers. Body weight and survival monitoring were recorded daily, and each group was sacrificed 4 days after drug administration for TCID (Thorough Care Inspection). 50 The viral titer was determined, and RNA was extracted from lung tissue. The titer was determined, and 10 LD50 was used. 50 After maintaining a constant level of infection across passages, the process was repeated for each consecutive cycle.

[0223] As shown in Figure 17B, compound 24IN did not produce drug-resistant mutants that survived after 5 passages, and as observed in the neuraminidase inhibition assay (NIA), IC 50It was found that compound 24 maintained efficacy with only a slight decrease of about 1.5 times, from approximately 12 nM to 20 nM. In contrast, oseltamivir phosphate treatment during IAV passage resulted in a decrease in NAI efficacy of less than 1.500 times, from approximately 2 nM to 3,000 nM (Hong, BT et al., Oseltamivir hydroxamate and acyl sulfonamide derivatives as influenza neuraminidase inhibitors. Bioorganic & medicinal chemistry 2014, 22(23), pp. 6647-6654). These results suggest that compound 24, even at doses below the optimal dose, does not promote the emergence of drug resistance mutations in vivo to the same extent as oseltamivir. Furthermore, compound 24 showed maintenance of NIA efficacy throughout the study period, while the efficacy of oseltamivir, the industry standard, deteriorated significantly. As shown in Figure 17A, when an additional LD50 study was performed on the mutants after 5 cycles, the LD50 of mice treated with Tamiflu showed a 4000-fold increase in virulence compared to a 4-fold increase in mice treated with compound 24. Overall, this study suggests that long-term use of compound 24 does not promote the emergence of drug-resistant IAV mutants.

[0224] Method Example 12. Efficacy of compound 24IN for avian influenza (Figures 18A and 18B). Three groups of mice (n=4 / cohort) were infected with 10E2 PFU / mouse using the labeled avian influenza strain rH5N1 Nluc A / Texas / 37 / 2024, currently spreading in Texas. A fourth group was a mock mouse, used as a negative control and infected using a virus-free virus-infecting medium. Mice were treated with either 2 mg / kg of compound 24IN intranasally, or 5 mg / kg of Tamiflu orally twice daily for 5 days, or compound 24 vehicle intranasally. Mice were sequentially imaged using an IVIS imaging system, and viral fluorescence was tracked to quantify the viral load. On day 6, mice treated with compound 24IN had almost no residual virus in their bodies after a single dose, while those treated with Tamiflu had significantly higher levels. Mice were sacrificed, and viral load was quantified in each via the TCID50 assay. Viral load in the lungs of Tamiflu-treated mice was found to be significantly higher in mice treated with compound 24IN, suggesting that compound 24 is effective in clearing avian influenza infections, including highly pathogenic avian influenza infections, even at very low doses. Figure 18A shows images of groups 1 and 2, and Figure 18B shows images of groups 3 and 4.

[0225] Method Example 13. Pharmacological mechanism of action of compound 24. The mechanism of action of compound 24 was evaluated by its ability to mobilize sufficient naturally occurring anti-hapten antibodies and mediate the killing of virus-infected cells by inducing antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cell-mediated cytotoxicity (CDC). Human fetal kidney cells (HEK293) were modified to express influenza neuraminidase (HEK293-NA) to mimic influenza-infected cells. Compound 24 was evaluated for its ability to activate specific immune system components (FcγRIIIa, FcγRIIa) in effector cells and the complement system. Upon activation, these components are expected to trigger ADCC, ADCP, and CDC, ultimately killing HEK293-NA cells. A control group using unmodified HEK293 cells (HEK293-WT) was used for comparison. HEK293-N1 neuraminidase cell cultures were supplemented with or without human anti-DNP antibody and incubated with serial dilutions of compound 24.

[0226] As shown in Figure 19, compound 24 is effective in mediating ADCC (Figure 19A) and ADCP (Figure 19B) in HEK293 cells transduced with neuraminidase, exhibiting the expected bell-shaped dependence on compound 24 concentration, detected by fluorescence changes upon activation of human FcgRIIIa expressed in effector cells using the Promega ADCC kit. Cell death can be blocked by either IVIG removal on the target cell surface or the absence of neuraminidase, suggesting that ADCC-mediated and ADCP-mediated death depend on two key factors: 1) the presence of anti-hapten antibodies, and 2) the expression of influenza neuraminidase on the cell surface. The dependence on the expression of influenza neuraminidase on the cell surface is an important safety feature, suggesting that uninfected cells should not be damaged by compound 24.

[0227] As shown in Figure 19C, compound 24 was also found to promote CDC-mediated cell death in target cells expressing neuraminidase. Cell death was blocked by introducing a 100-fold excess of free zanamivir, suggesting that the activation of CDC-mediated cell death mediated by compound 24 should also be limited to cells / virions expressing viral neuraminidase.

[0228] The data in Figure 19 validates the immunoactivity of compound 24, demonstrating that compound 24 mobilizes naturally occurring anti-hapten antibodies to activate ADCC-mediated (Panel A), ADCP-mediated (Panel B), and CDC-mediated (Panel C) influenza-infected cells. Compound 24-mediated cell death depends on the presence of both anti-hapten antibodies and viral neuraminidase on the target cell surface, which is a desirable safety feature.

[0229] Method Example 14. Neuraminidase inhibitory activity of compound 24IN against seasonal and drug-resistant influenza A and B strains (Figure 20).

[0230] The neuraminidase inhibitory activity of compound 24IN was evaluated against four seasonal influenza strains (A / PR / 8 / 1934 (H1N1), A / California / 07 / 2009 (H1N1pdm09), A / Wisconsin / 15 / 2009 (H3N2), and B / Brisbane / 60 / 2008 (Victoria lineage)) and two drug-resistant influenza virus strains (A / Hong Kong / 2369 / 2009 (H1N1pdm09, oseltamivir resistant) and A / Illinois / 37 / 2018 (H1N1pdm09, baloxavir resistant). Neuraminidase inhibitory activity was measured by the IC50 of the relative fluorescence units (RFU) of the enzyme product derived from the neuraminidase activity of the virus particles. 50 It was measured based on the following criteria.

[0231] IC for all 6 strains 50This is shown in Figure 20. Compound 24 reduced the neuraminidase activity of three seasonal influenza A strains in the IC25 range of 12.7 to 16.4 nM. 50 Compound 24 was found to inhibit the neuraminidase activity of seasonal influenza B strain (Victoria lineage) at 34.5 nM IC2. 50 Compound 24 was found to inhibit the neuraminidase activity of oseltamivir-resistant and baloxavir-resistant influenza strains, respectively, at IC25 levels of 17.9 and 27.2 nM. 50 It was found that this effectively inhibits the process.

[0232] Method Example 15. Neuraminidase inhibitory activity against influenza A / PR / 8 / 1934 (H1N1). Tenfold serial dilutions of the test compound were prepared in 1× NA-Fluor™ assay buffer. Fourfold serial dilutions and diluted viral samples were added to the corresponding wells in a black, 96-well transparent flat-bottom plate. The plate was incubated on a plate shaker at 37°C for 30 minutes. 200 μM NA-Fluor™ substrate working solution was added, and the plate was incubated on a plate shaker at 37°C for a further 1 hour to detect viral neuraminidase activity. The reaction was terminated with a reaction stop solution (60% NA-Fluor™ reaction stop solution / 40% ethanol). The fluorescence signal was detected using a BioTek Synergy Neo2 HTS multimode microplate reader with an excitation wavelength of 350 nm and an emission wavelength of 440 nm. The data were analyzed using a GraphPad Prism 10 to determine the median inhibitory concentration (IC50) of the test compound. The activity in this assay demonstrated that the test conjugate retained the direct antiviral activity of the ligand after conjugation to the linker and two haptens. The IC50 values ​​of the test compounds are shown in the table below.

[0233] [Table 2]

[0234] Method Example 16. Inhibitory activity of compound 24 on viral cytopathic effects. In the presence of viral infection, host cells change at the cellular level in response to the infection. This phenomenon is known as cytopathic effects (CPE). The efficacy of compound 24 in preventing CPE and associated cytotoxicity caused by influenza virus infection was determined through a viral CPE inhibition assay. The titer of the viral stock was set to TCID. 50 The measurement was performed to determine the required viral inoculation for the CPE inhibition assay.

[0235] Cell viability from viral CPE inhibition assays was quantified using neutral red staining, and EC24 was tested against four wild strains of compound 24 (A / PR / 8 / 1934 (H1N1), A / Wisconsin / 629-D00015 / 2009 (H1N1pdm09), A / Hong Kong / H090-756-V1(0) / 2009 (H3N2), and B / Brisbane / 60 / 2008 (Victoria lineage)) and one drug-resistant influenza strain (A / Hong Kong / 2369 / 2009 (H1N1pdm09, oseltamivir resistant)). 50 The result was calculated.

[0236] The following table summarizes the inhibition of viral CPE by compound 24 against wild-type and drug-resistant strains.

[0237] [Table 3]

[0238] Compound 24 effectively prevented CPE induced by H1N1 and oseltamivir-resistant strains (EC). 50 Compound 24 also contains H1N1pdm09, H3N2, and influenza B compounds. 50 Inhibition was achieved at <8 μM. It should be noted that these experiments consider only the activity of the zanamivir portion, and if the immune system can be mobilized in vivo, compound 24 is expected to have more than 1,000-fold activity.

[0239] Method Example 17. Efficacy against avian influenza. The efficacy of compound 24 against avian influenza strains A / Duck / MN / 1525 / 81(H5N1), A / HK / 61 / 2016(H7N9), and A / Vietnam / 1203 / 2004(H5N1) was evaluated in vitro. Cells were cultured and then exposed to various dilutions of compound 24 and avian influenza strains. As shown in the table below, the cytopathic effect was observed under a microscope and measured using a red dye, and the EC was measured. 50 The value of the (50% inhibitory concentration of CPE) was calculated.

[0240] [Table 4] Compound 24 was found to inhibit the cytopathic effects and in vitro nascent virion production induced by H5N1 and H7N9 at single-digit micromolar concentrations. In addition, these results confirmed the direct antiviral properties of compound 24 against avian influenza strains. The direct antiviral activity of compound 24 was also verified in vivo, with very low doses providing 100% protection against isolated H5N1 from Texas 2024.

[0241] Method Example 18. Efficacy against A / Illinois / 37 / 2018 (H1N1, baloxavir resistant). Baloxavir treatment has a very high mutagenesis rate, presenting clinical problems regarding the use of baloxavir. In a preliminary study of efficacy against baloxavir variants, 10LD adapted to mice was used. 50 Mice were infected with influenza A / Illinois / 37 / 2018 (H1N1pdm09, baloxavir resistant). As shown in Figure 21, mice treated with compound 24 had a 100% survival rate. Vehicle controls and baloxavir-treated groups had similar survival rates, suggesting that baloxavir was ineffective. Compound 24 may be useful in treating baloxavir-resistant strains of influenza.

[0242] Method Example 19. Complement-dependent cell-mediated cytotoxicity (CDC) assay. Neuraminidase-expressing HEK293 cells (HEK293-NA) were seeded at 10,000 cells per well in a 96-well poly-D lysine-coated, white-walled plate. Cells were incubated overnight at 37°C, aiming for 50% confluence on the day of the assay. Serial dilutions of the compound were prepared in DMEM to four times the target concentration. Cells were washed with 200 μL of PBS, followed by the addition of 25 μL of DMEM per well. 25 μL of the serially diluted compound was added to each well, and the plate was incubated at 37°C for 30 minutes. Rabbit anti-DNP (Invitrogen, catalog number: A6430) or rabbit anti-alpha-gal (Enzo m86) was diluted to 40 μg / mL, and rabbit complement (Sigma Aldrich, catalog number: S7764) was diluted to 20%. 50 μL of this mixture was added to the cells to a final concentration of 20 μg / mL rabbit anti-DNP and 10% rabbit complement. The cells were incubated at 37°C for 4 hours. After the incubation period, the cells were washed twice with 200 μL of PBS. Subsequently, 200 μL of Cell Titer-Glo reagent (Promega, catalog number: G7570) in PBS was added to each well. Fluorescence was measured using a plate reader with glow-type fluorescence reading capability. The percentage of cell death was calculated and plotted, and a bell-shaped curve was drawn. In the low concentration range, the compound stimulates cell killing by complement proteins. In the high concentration range, the compound blocks complement activity by filling receptors on target proteins and antibodies. The activity in this assay demonstrates the effective ability of the complete conjugate to bind to the target viral protein and mobilize the immune system using the other domain of the molecule. The EC50 values ​​for both the low and high concentration ranges of the test compound are shown in the table below.

[0243] [Table 5]

[0244] Method Example 20. Overview of Nonclinical Pharmacokinetics and Metabolism. The pharmacokinetics (PK) of compound 24 were characterized across four administration routes in BALB / c mice: intravenous (IV), subcutaneous (SC), oral, and in. IV and SC showed the highest levels of drug absorption, but in was selected as the preferred route of administration (ROA) due to its ease of administration in human patients. PK studies were also conducted in mice to determine the minimum pharmacological dose (PAD).

[0245] In preclinical PK studies, compound 24 showed rapid absorption across various parameters after IN administration, and the time to reach peak blood concentration (T) was observed. max The mean elimination half-life (t) ranged from approximately 0.42 to 1.10 hours. The bioavailability (F) of compound 24 was dose- and formulation-dependent, and was found to be highest at clinically reasonable dose levels in rodents. At similar doses and across formulations, the mean F was moderate to high (21% to approximately 100%) in mice, low to moderate (approximately 20% to approximately 50%) in rats, and low (4% to approximately 20%) in dogs. In each case, higher F was at a concentration of £10% of the maximum allowable dose (MFD), and lower F was within the MFD range. 1 / 2 The time ranged from 0.603 to 6.81 hours (excluding one outlier, from 0.603 to 4.2 hours), and in rats, the IN dose was increased in stages. These data suggested that absorption of compound 24 became rate-limited during in vivo efflux (i.e., flip-flop dynamics). In dogs, the estimated mean t 1 / 2 At doses up to approximately 20 mg / kg, this occurred approximately 1 hour after IV and IN administration. Day 14 / 1 C max and AUC 0~24 The ratio was close to 1 in rats and dogs, indicating no accumulation following once-daily administration. Compound 24 was found to have low cell permeability and is not a substrate of human P-glycoprotein (P-gp).

[0246] Compound 24 showed low to moderate plasma protein binding. The percentage of unbound compound 24 ranged from 74.0% to 89.4% across mouse, rat, dog, monkey, and human populations, with human plasma showing the highest binding. Splitting operations indicated that the blood-to-plasma ratio of compound 24 was less than 0.72, suggesting limited fractionation into red blood cells. Compound 24 was metabolically stable in vitro across various species. Profiling of pooled rat plasma detected a clearly small amount of circulating metabolite (0.71%) resulting from amide hydrolysis (M1160), with intact compound 24 being the main circulating component.

[0247] Compound 24 did not directly (reversibly) inhibit the major human cytochrome P450 (CYP) groups 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4 / 5. Furthermore, co-incubation with compound 24 did not result in time-dependent inhibition (TDI) of these same CYP groups. Therefore, compound 24 is not considered to induce CYP-mediated drug-drug interactions.

[0248] Method Example 21. Summary of nonclinical safety, pharmacological effects, and toxicity. Safety pharmacological studies were conducted to identify any unintended effects on organ systems of critical importance to life, including the central nervous system (CNS), cardiovascular system, and respiratory system. Compound 24 had no effect on these organ systems. In addition, no changes in electrocardiogram (ECG) parameters were observed in dogs administered compound 24 for 14 days in a GLP toxicity study.

[0249] Compound 24 was not mutagenic in bacterial reverse mutation (Ames) tests or in vitro micronucleus assays. Compound 24 absorbed light between 290 and 700 nm, with a maximum absorption at 360 nm.

[0250] To support dose selection for a 14-day GLP toxicity study, non-GLP dose tolerability studies were conducted in rats and dogs using MFD. In rats, the MFD dose via the IN route, the intended route of clinical administration, was 100 mg / kg. The 100 mg / kg dose was well tolerated in the single-dose tolerability confirmation phase. The 100 mg / kg dose was selected for further evaluation during a 3-day tolerability study administered daily. There was no effect of the treatment on standard pre- and post-mortem assessments supporting the selection of 100 mg / kg as the high dose for the 14-day GLP study. In the 14-day study, rats were administered either by vehicle or doses up to 100 mg / kg via the IN route. There was no effect of the treatment on any pre-mortem evaluations, including disease or death, clinical observation, body weight, diet intake, ophthalmology, CNS (Comprehensive Observational Assessment (FOB)), or clinicopathological parameters (hematology, coagulation, chemistry, urinalysis), nor on post-mortem evaluations, including autopsy, organ weight, and general tissue microscopy. Similarly, the NOAEL in male and female rats administered daily via the IN route for 14 days was 100 mg / kg / day.

[0251] In dogs, the MFD via the IN pathway was 10 mg / kg. Therefore, exposure was increased by supplementing the IN dose with a 10 mg / kg SC dose administered concurrently to assess the potential systemic toxicity of compound 24. The combined 20 mg / kg dose was well tolerated in the single-dose tolerability confirmation phase. The combined 20 mg / kg dose was selected for further evaluation during a 3-day tolerability study administered daily. There was no effect of the treatment on pre-mortem and post-mortem standard assessments, supporting the selection of 20 mg / kg as a high dose for a 14-day GLP study. In the 14-day study, dogs were administered a vehicle or doses up to 20 mg / kg / day. There was no effect of the treatment on any pre-mortem assessments, including disease or death, clinical observations, body weight, diet intake, ophthalmology, ECG, or clinicopathological parameters (hematology, coagulation, chemistry, urinalysis), nor on post-mortem assessments, including autopsy, organ weight, and general tissue microscopy. Similarly, the NOAEL in male and female dogs administered daily via a combined IN and SC route for 14 days was 20 mg / kg / day.

[0252] Method Example 22. Overview of Chemistry, Manufacturing, and Control. Compound 24 is a low molecular weight neuraminidase inhibitor covalently conjugated to dinitrophenyl (DNP) and α-L-rhamnose by a PEG-based linker. Compound 24 was manufactured from the main starting materials in a four-step process and in batches up to 100 g. The structure, including the absolute stereochemistry of the 11 chiral centers, was confirmed by analysis by infrared spectroscopy (IR), ultraviolet-visible absorption spectroscopy, high-resolution mass spectrometry (HRMS), elemental analysis, and 1D / 2D nuclear magnetic resonance (NMR) spectroscopy. Compound 24 was packaged in light-protected HDPE plastic bottles and stored at -20°C ± 5°C for up to 12 months.

[0253] Each publication cited herein is incorporated herein in its entirety for reference.

Claims

1. formula 【Chemistry 1】 (In the formula, T is a ligand radical for the target protein of the influenza virus or virus-infected cell; L １ , L ２ and L ３ Each of them is an independently selected linker; and A １ and A ２ A compound of a hapten (where each is a radical of a hapten independently selected), or a pharmaceutically acceptable salt or solvate thereof.

2. The compound according to claim 1, wherein the ligand is a neuraminidase inhibitor.

3. The compound according to claim 1, wherein the ligand is selected from sialic acid and its analogues.

4. The compound according to claim 1, wherein the ligand is selected from zanamivir, peramivir, laninamivir, oseltamivir, and 2,3-dehydro-2-deoxy-n-acetylneuraminic acid, as well as their analogs and derivatives.

5. The compound according to claim 1, wherein the ligand is zanamivir or an analog or derivative thereof.

6. The compound according to claim 1, wherein the ligand is peramivir, or an analog or derivative thereof.

7. The ligand is, 【Chemistry 2】 The compound according to claim 1, which is a compound or an analog or derivative thereof.

8. The compound according to claim 1, wherein the ligand is laninamivir, or an analog or derivative thereof.

9. The ligand is, 【Transformation 3】 The compound according to claim 1, which is an analog or derivative thereof.

10. The compound according to claim 1, wherein the ligand is oseltamivir, or an analog or derivative thereof.

11. The compound according to claim 1, wherein the ligand is a hemagglutinin inhibitor.

12. The ligand is 【Chemistry 4】 The compound according to claim 1, as well as its analogs and derivatives.

13. The hapten is a compound according to any one of claims 1 to 12, having an epitope that has endogenous Ab, B, or T cells.

14. Each hapten is independently selected from rhamnose, nitrophenyl, nitrophenol, nitroaniline, dinitrophenyl (DNP), dinitrophenol, dinitroaniline, trinitrophenyl (TNP), trinitrophenol, trinitroaniline, chloronitrophenyl, chloronitrophenol, chloronitroaniline, iodonitrophenyl, iodonitrophenol, iodonitroaniline, nitrotyrosine, hydroxynitrotyrosine, aminonitrotyrosine, 4-hydroxy-3-nitrophenylacetic acid, α-galactosyl moiety, sulfated gal, phosphorylcholine, bacterial antigen, and viral antigen, as described in any one of claims 1 to 12.

15. A １ and A ２ one of which is a radical of L-rhamnose; A １ and A ２ the other of which is a radical of dinitrophenyl, the compound according to any one of claims 1 to 12.

16. L ２ and L ３ is L １ The compound according to any one of claims 1 to 12, bonded to the same noncarbon atom above.

17. L ２ and L ３ is L １ The compound according to any one of claims 1 to 12, wherein it is bonded to the same nitrogen atom above.

18. L ２ and L ３ is L １ The compound according to any one of claims 1 to 12, wherein it is bonded to different atoms.

19. L １ , L ２ and L ３ The compound according to any one of claims 1 to 18, wherein one or more of the atoms are chains of atoms, the length of each chain being independently selected and in the range of about 2 to about 60.

20. L １ , L ２ and L ３ One or more of the following are: ethoxy, ethylamino, ethylene glycol, azaethylene glycol, (PEG) ｎ , or Azer (PEG) ｎ A compound according to any one of claims 1 to 19, comprising, or a combination thereof, wherein n is in the range of 2 to about 36.

21. L １ , L ２ and L ３ One or more of these are (PEG) ｎ , or Azer (PEG) ｎ A compound according to any one of claims 1 to 20, comprising, or a combination thereof, wherein n is 2 to 36.

22. L １ , L ２ and L ３ Each of these is ethoxy, ethylamino, ethylene glycol, azaethylene glycol, (PEG) ｎ , or Azer (PEG) ｎ The compound according to any one of claims 1 to 21, comprising, or a combination thereof, wherein n is 2 to 36.

23. L １ , L ２ and L ３ The compound according to any one of claims 1 to 22, wherein one or more of the compounds include or additionally include O-alkyl-O, N-alkyl-N, C(O)-alkyl-C(O), or NC(O)-alkyl-C(O)N, or a combination thereof.

24. L １ , L ２ and L ３ The compound according to any one of claims 1 to 23, wherein one or more of the compounds include or additionally include O-alkyl-C(O), N-alkyl-C(O), O-alkyl-N-alkyl-C(O), N-alkyl-O-alkyl-C(O), or C(O)alkyl-C(O), or a combination thereof.

25. L １ The compound according to any one of claims 1 to 24, comprising or additionally comprising O-alkyl-C(O)N-diyl, O-alkyl-O-alkyl-C(O)N-diyl, or N-alkyl-O-alkyl-O-alkyl-C(O)N-diyl.

26. L １ , L ２ and L ３ One or more of the compounds according to any one of claims 1 to 25, comprising or additionally comprising one or more amino acids.

27. L １ , L ２ and L ３ One or more of these are β-NH ２ A compound according to any one of claims 1 to 26, comprising, or additionally comprising, one or more selected hydrophilic amino acids from Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Lys, Met, Orn, Ser, or Thr, including their respective naturally occurring L enantiomers.

28. L １ , L ２ and L ３ The compound according to any one of claims 1 to 27, wherein one or more of the compounds include, or additionally include, one or more amino acids selected from glycine, serine, proline, ornithine, and lysine.

29. L １ , L ２ and L ３ The compound according to any one of claims 1 to 28, wherein one or more of the compounds include or additionally ornithine or lysine.

30. L １ , L ２ and L ３ One or more of these are (Pro) ｎ The compound according to any one of claims 1 to 29, comprising or additionally comprising, where n is 1 to 6.

31. L １ , L ２ and L ３ One or more of these are (Pro) ３ A compound according to any one of claims 1 to 30, comprising or additionally comprising -Lys.

32. L １ , L ２ and L ３ The compound according to any one of claims 1 to 31, wherein one or more of the groups include or additionally include a C(O), C(O)O, C(O)NH, OC(O)NH, or NHC(O)NH group.

33. L ２ and / or L ３ The compound according to any one of claims 1 to 32, wherein the compound is hydrophilic.

34. L １ The compound according to any one of claims 1 to 18, wherein the compound comprises a region having the ability to form an α-helix structure.

35. L ２ and / or L ３ The compound according to any one of claims 1 to 34, wherein the extended structure has a length of at least 15 Å.

36. L １ The compound according to any one of claims 1 to 35, wherein DBCO is not included.

37. formula 【Transformation 5】 A compound thereof, or a pharmaceutically acceptable salt or solvate thereof.

38. formula 【Transformation 6】 A composition comprising a compound thereof, or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more diluents, carriers, or excipients, or a combination thereof.

39. A composition for use in the treatment of influenza and influenza infections, comprising the formula 【Transformation 7】 A composition comprising a compound thereof, or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more diluents, carriers, or excipients, or a combination thereof.

40. A unit dose for use in the treatment of influenza and influenza infections, and the formula 【Transformation 8】 A unit dose comprising the compound, or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more diluents, carriers, or excipients, or a combination thereof.

41. A method for treating influenza and influenza infection in a host animal, the formula 【Chemistry 9】 A method comprising administering a compound thereof, a pharmaceutically acceptable salt or solvate thereof, or a composition containing said compound, or a unit dose of a therapeutically effective amount thereof.

42. Formula in the manufacture of pharmaceuticals for use in the treatment of influenza and influenza infections in host animals 【Chemistry 10】 Use of the compound, or a pharmaceutically acceptable salt or solvate thereof, or a composition or unit dose containing said compound.

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