CONJUGATE FOR PREVENTING AND TREATING VIRAL INFECTIONS AND USE THEREOF

Abstract

The present invention relates to a conjugate for preventing and treating viral infections and a use thereof, and in particular, to a protein-anti-influenza compound conjugate having a structure of formula I-1 or a pharmaceutically acceptable salt, an ester, an isomer, a solvate, a prodrug or an isotope label thereof. The conjugate has small molecules (D.sup.1 and D.sup.2) linked, by means of linkers (L.sup.1 and L.sup.2) to an Fc monomer, an Fc domain, an Fc linker peptide, an albumin or an albumin linker peptide (E) and having anti-influenza virus activity. The conjugate or the intermediate compound of the present invention has significant anti-influenza virus activity, and meanwhile, has excellent in vitro/in vivo pharmacokinetic property and safety and has good clinical application prospects.

##STR00001##

Claims

1. A conjugate of formula I-1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, ##STR00410## wherein, m is 1 or 2; n is an integer from 1 to 20, preferably an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and two n's are identical; each of D1 and D2 is an anti-influenza virus small molecule drug, and is independently selected from a compound having an anti-influenza activity as shown in formulae D-1-1 to D-1-8: ##STR00411## ##STR00412## R.sub.1 is selected from OH, NH.sub.2, NH(NH)NH.sub.2 and NHC(NH)NHR.sub.6; R.sub.2 and R.sub.3 are each independently selected from H, OH, F, Cl and Br; R.sub.4 is selected from COOH, P(O)(OH).sub.2 and SO.sub.3H; R.sub.5 is selected from COC.sub.1-6 alkyl, COC.sub.1-6 haloalkyl, SO.sub.2C.sub.1-6 alkyl, and SO.sub.2C.sub.1-6 haloalkyl; preferably selected from COCH.sub.3, COCF.sub.3, and SO.sub.2CH.sub.3; X is selected from O and S; R.sub.6 is selected from the following groups: ##STR00413## Y is selected from the following groups, wherein (attached to L.sup.1) specifies the end of the group Y attached to the end of L.sup.1, wherein N denotes an atom within the ring when drawn inside the ring: ##STR00414## ring A is selected from C.sub.3-C.sub.20 cycloalkyl, substituted C.sub.3-C.sub.20 cycloalkyl, C.sub.3-C.sub.20 cycloalkenyl, substituted C.sub.3-C.sub.20 cycloalkenyl, C.sub.6-C.sub.15 aryl, 3- to 20-membered heterocycloalkyl, substituted 3- to 20-membered heterocycloalkyl, substituted C.sub.6-C.sub.15 aryl and substituted 5- to 15-membered heteroaryl; R groups are each independently selected from H, deuterium, optionally substituted C.sub.1-C.sub.20 alkyl, optionally substituted C.sub.2-C.sub.20 streptenyl, optionally substituted C.sub.3-C.sub.20 cycloalkyl, optionally substituted 3- to 20-membered heterocycloalkyl, optionally substituted C.sub.6-C.sub.15 aryl, and optionally substituted 5- to 15-membered heteroaryl; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; L.sup.1 is a linker attached to the drug (D.sup.1 or D.sup.2) and L.sup.2, and includes but not limited to following structures: wherein bonds at left and right ends of the following structures are attached to an oxygen atom of the drug and a bond marked by the wavy line in the middle is attached to L.sup.2, ##STR00415## wherein: W is selected from O, S, NR.sup.b, CH.sub.2 or absent; R.sup.a and R.sup.b are each independently selected from H, optionally substituted C.sub.1-C.sub.20 alkyl, optionally substituted C.sub.2-C.sub.20 alkenyl; y.sup.1 and y.sup.2 are each independently 0, 1, 2, 3, 4, 5 or 6; q.sup.1 and q.sup.2 are each independently selected from 1, 2, 3, 4, 5 and 6; or L.sup.1 is selected from following structures: ##STR00416## ##STR00417## ##STR00418## ##STR00419## ##STR00420## ##STR00421## ##STR00422## ##STR00423## ##STR00424## ##STR00425## ##STR00426## L.sup.2 is a linker attaching L.sup.1 and E (Fc monomer, Fc structural domain, Fc-binding peptide, albumin, or albumin-linking peptide) and having a structure sleeted from formula L2-1 to L2-15, wherein (L.sup.1) and (E) denote ends of L.sup.2 connected to L.sup.1 and E, respectively: TABLE-US-00037 L.sup.2 structure No. Structure L2-1 embedded image L2-2 L2-3 L2-4 L2-5 L2-6 L2-7 L2-8 L2-9 L2-10 L2-11 L2-12 L2-13 L2-14 L2-15 wherein, Z is NR, S or O; R are each independently selected from hydrogen, deuterium, optionally substituted C.sub.1-C.sub.20 alkyl, optionally substituted C.sub.2-C.sub.20 chain alkenyl, optionally substituted C.sub.3-C.sub.20 cycloalkyl, optionally substituted 3- to 20-membered heterocycloalkyl, optionally substituted C.sub.6-C.sub.15 aryl and optionally substituted 5- to 15-membered heteroaryl; s and t are integers from 1 to 20; such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; y is 0 or 1; Q is N or CH; p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; E is an antibody or an antibody fragment thereof, an Fc structural domain monomer, an Fc structural domain, an Fc-binding peptide, an albumin or an albumin-linking peptide, and comprises an amino acid sequence of any one of SEQ ID Nos. 1-68 or an amino acid sequence that is at least 95% identical to any one of SEQ ID Nos. 1-68.

2. The conjugate of claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, ##STR00442## wherein m is 1 or 2; n is an integer from 1 to 10; each of D.sup.1 and D.sup.2 is independently selected from the compound having an anti-influenza activity as shown in the formulae D-1-1 to D-1-8; D.sup.1 and D.sup.2 are covalently attached to L.sup.1 that is covalently attached to L.sup.2, and L.sup.2 is covalently attached to E; E is selected from the Fc structural domain monomer, the Fc structural domain, the Fc-binding peptide, the albumin or the albumin-linking peptide.

3. The conjugate of claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, wherein the conjugate has a structure of formula I-1-1 to I-1-8: TABLE-US-00038 Conjugate No. Structure I-1-1 embedded image I-1-2 I-1-3 I-1-4 I-1-5 I-1-6 I-1-7 I-1-8 wherein each variable is as defined in claim 1.

4. The conjugate of claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, wherein the conjugate has a structure of formula I-1-A to I-1-D: TABLE-US-00039 No. Structure I-1-A embedded image I-1-B I-1-C I-1-D wherein each variable is as defined in claim 1.

5. The conjugate of claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, wherein the conjugate has a structure of formula C-1 to C-115: TABLE-US-00040 Conju- gate No. Conjugate Structure C-1 embedded image C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20 C-21 C-22 C-23 C-24 C-25 C-26 C-27 C-28 C-29 C-30 C-31 C-32 C-33 C-34 C-35 C-36 C-37 C-38 C-39 C-40 C-41 C-42 C-43 C-44 C-45 C-46 C-47 C-48 C-49 C-50 embedded image C-51 C-52 C-53 C-54 C-55 C-56 C-57 C-58 C-59 C-60 C-61 C-62 C-63 C-64 C-65 C-66 C-67 C-68 C-69 C-70 C-71 C-72 C-73 C-74 C-75 C-76 C-77 C-78 C-79 C-80 C-81 C-82 C-83 C-84 C-85 C-86 C-87 C-88 C-89 C-90 C-91 C-92 C-93 C-94 C-95 C-96 C-97 C-98 embedded image C-99 C-100 C-101 C-102 C-103 C-104 C-105 C-106 C-107 C-108 C-109 C-110 C-111 C-112 C-113 C-114 C-115 where n is as defined in claim 1, and protein is E as defined in claim 1.

6. The conjugate of claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, wherein a ratio of n to m is from 1 to 20, preferably from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

7. The conjugate of claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, wherein the conjugate has an average DAR value between 0.5 and 10.0.

8. The conjugate of claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, wherein E comprises an Fc structural domain monomer or an Fc structural domain containing said Fc structural domain monomer, wherein said Fc structural domain monomer comprises or consists of an amino acid sequence of any one of SEQ ID Nos. 1-68 or an amino acid sequence that is at least 95% identical to any one of SEQ ID Nos. 1-68.

9. The conjugate of claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, wherein E is recognizable for viral surface antigens, such as CR6261, CR8020, MEDI8897, Palivizumab, SD38, or the Fc structural domain monomer may be an Fc structural domain monomer of an antibody subtype (such as, IGHG1*01 (such as G1m(za)), IGHG1*07 (such as G1m(zax)), IGHG1*04(such as G1m(zav)), IGHG1*03(G1m(f)), IGHG1*08 (such as G1m(fa)), IGHG2*01, IGHG2*02, IGHG2*06, IGHG3*01, IGHG3*04, IGHG3*05, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*13, IGHG3*03, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18, IGHG3*19, IGHG2*04, IGHG4*01, IGHG4*02, IGHG4*03) of any kind of immunoglobulins.

10. The conjugate of claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, wherein the conjugate has a structure below: ##STR00570## ##STR00571## ##STR00572## wherein n is as defined in claim 1.

11. A compound of formula I-2, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, ##STR00573## wherein L.sup.1, D.sup.1 and D.sup.2 are as defined in formula I-1 of claim 1, L.sup.3 is selected from following structures: TABLE-US-00041 L.sup.3 structure No. Structure L3-1 embedded image L3-2 L3-3 L3-4 L3-5 L3-6 L3-7 L3-8 L3-9 L3-10 L3-11 L3-12 L3-13 L3-14 L3-15 wherein each variable such as Z, R, Q, s, y, p is as defined for L.sup.2 in claim 1.

12. The compound of Formula I-2 of claim 11, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, wherein said compound has a structure of Formula C-Inter-1 to C-Inter-115: TABLE-US-00042 No. Structure C-Inter-1 embedded image C-Inter-2 C-Inter-3 C-Inter-4 C-Inter-5 C-Inter-6 C-Inter-7 C-Inter-8 C-Inter-9 C-Inter-10 C-Inter-11 C-Inter-12 C-Inter-13 C-Inter-14 C-Inter-15 C-Inter-16 C-Inter-17 C-Inter-18 C-Inter-19 C-Inter-20 C-Inter-21 C-Inter-22 C-Inter-23 C-Inter-24 C-Inter-25 C-Inter-26 embedded image C-Inter-27 C-Inter-28 C-Inter-29 C-Inter-30 C-Inter-31 C-Inter-32 C-Inter-33 C-Inter-34 C-Inter-35 C-Inter-36 C-Inter-37 C-Inter-38 C-Inter-39 C-Inter-40 C-Inter-41 C-Inter-42 C-Inter-43 C-Inter-44 C-Inter-45 C-Inter-46 C-Inter-47 C-Inter-48 C-Inter-49 C-Inter-50 embedded image C-Inter-51 C-Inter-52 C-Inter-53 C-Inter-54 C-Inter-55 C-Inter-56 C-Inter-57 C-Inter-58 C-Inter-59 C-Inter-60 C-Inter-61 C-Inter-62 C-Inter-63 C-Inter-64 C-Inter-65 C-Inter-66 C-Inter-67 C-Inter-68 C-Inter-69 C-Inter-70 C-Inter-71 C-Inter-72 C-Inter-73 C-Inter-74 embedded image C-Inter-75 C-Inter-76 C-Inter-77 C-Inter-78 C-Inter-79 C-Inter-80 C-Inter-81 C-Inter-82 C-Inter-83 C-Inter-84 C-Inter-85 C-Inter-86 C-Inter-87 C-Inter-88 C-Inter-89 C-Inter-90 C-Inter-91 C-Inter-92 C-Inter-93 C-Inter-94 C-Inter-95 C-Inter-96 C-Inter-97 C-Inter-98 embedded image C-Inter-99 C-Inter-100 C-Inter-101 C-Inter-102 C-Inter-103 C-Inter-104 C-Inter-105 C-Inter-106 C-Inter-107 C-Inter-108 C-Inter-109 C-Inter-110 C-Inter-111 C-Inter-112 C-Inter-113 C-Inter-114

13. A Pharmaceutical composition comprising the conjugate of formula I-1 as claimed in claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, other small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists), and optionally pharmaceutically acceptable excipients.

14. A method for the prevention or treatment of a subject having a viral infection or at a risk of having a viral infection, comprising administering to the subject, for example by injection, an effective amount of the conjugate of formula I-1 as claimed in claim 1, a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof.

15. The method of claim 14, wherein the viral infection is an infection caused by an influenza virus or a parainfluenza virus; preferably, the viral infection is an infection caused by influenza virus A, B or C, or parainfluenza virus; preferably, the subject having a viral infection or at a risk of having a viral infection may be a subject with an immune system deficiency; preferably, the subject having a viral infection or at a risk of having a viral infection may be a subject who is or will be treated with an immunosuppressive agent, preferably, the subject having a viral infection or at a risk of having a viral infection may be a subject diagnosed with an immunosuppression disease; preferably, the subject diagnosed with an immunosuppression disease has cancer or acquired immunodeficiency syndrome; preferably, the subject diagnosed with an immunosuppression disease has leukemia, lymphoma, humoral immunodeficiency, T-cell deficiency, complement deficiency or multiple myeloma; preferably, the subject is a subject undergoing or about to undergo a hematopoietic stem cell transplant; preferably, the subject is a subject undergoing or about to undergo an organopoietic transplant; and/or preferably, the subject may be at a risk of a secondary infection.

16. A method for preventing a secondary infection of a subject caused by an influenza virus infection, comprising administering to the subject, for example by an injection, an effective amount of the conjugate of formula I-1 as claimed in claim 1, a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, preferably, said secondary infection is a respiratory infection; preferably, said secondary infection is associated with pneumonia; preferably, said secondary infection is a bacterial, or viral, or fungal infection; preferably, said bacterial infection is an infection caused by methicillin-resistant Staphylococcus aureus; preferably, said bacterial infection is an infection caused by Streptococcus pneumoniae; preferably, the conjugate of formula I-1 or the compound of formula I-2, or pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, is administrated by an intramuscular injection, intravenous injection, intradermal injection, intra-arterial injection, intraperitoneal injection, intra-lesional injection, intracranial injection, intra-articular injection, intrapleural injection, intratracheal injection, intraprostatic injection, intranasal injection, intravitreous injection, intravaginal injection, intrarectal injection, local injection, intra-tumor injection, intraperitoneal injection, subcutaneous injection, subconjunctival injection, intracapsular injection, mucosal injection, intrapericardial injection, intra-umbilical injection, intra-ocular injection, oral, local inhalation, injection or infusion, preferably, the conjugate of formula I-1 or the compound of formula I-2, or pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, combines with another therapeutic agent in administration or in the preparation of a medicament; preferably, the another therapeutic agent is an antiviral drug; preferably, the antiviral drug is baloxavir, pimodivir, oseltamivir, zanamivir, peramivir, laninamivir, amantadine, MEDI8852 or rimantadine; preferably, another drug used by the subject is an antiviral vaccine; preferably, the antiviral drug and the conjugate of formula I-1 or the compound of formula I-2, or pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, are sequentially administered, for example, by injection to the subject; and/or preferably, the antiviral drug and the conjugate of formula I-1 or the compound of formula I-2, or pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled derivative thereof, are simultaneously administered, for example by injection, to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0233] FIG. 1: SEC-HPLC of conjugate 1.

[0234] FIG. 2: SEC-HPLC of conjugate 2.

[0235] FIG. 3: SEC-HPLC of conjugate 3.

[0236] FIG. 4: SEC-HPLC of conjugate 4.

[0237] FIG. 5: SEC-HPLC of conjugate 5.

[0238] FIG. 6: SEC-HPLC of conjugate 6.

[0239] FIG. 7: SEC-HPLC of conjugate 7.

[0240] FIG. 8: SEC-HPLC of conjugate 8.

[0241] FIG. 9: SEC-HPLC of conjugate 9.

[0242] FIG. 10: LCMS of conjugate 1.

[0243] FIG. 11: LCMS of conjugate 2.

[0244] FIG. 12: LCMS of conjugate 3.

[0245] FIG. 13: LCMS of conjugate 4.

[0246] FIG. 14: LCMS of conjugate 5.

[0247] FIG. 15: LCMS of conjugate 6.

[0248] FIG. 16: LCMS of conjugate 7.

[0249] FIG. 17: LCMS of conjugate 8.

[0250] FIG. 18: LCMS of conjugate 9.

[0251] FIG. 19: Residual free drug content of conjugate 1.

[0252] FIG. 20: Residual free drug content of conjugate 2.

[0253] FIG. 21: Residual free drug content of conjugate 3.

[0254] FIG. 22: Residual free drug content of conjugate 4.

[0255] FIG. 23: Residual free drug content of conjugate 5.

[0256] FIG. 24: Residual free drug content of conjugate 6.

[0257] FIG. 25: Residual free drug content of conjugate 7.

[0258] FIG. 26: Residual free drug content of conjugate 8.

[0259] FIG. 27: Residual free drug content of conjugate 9.

DETAILED DESCRIPTION

[0260] These and other aspects and embodiments of the present disclosure are exemplified in the following embodiments. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present disclosure. The following embodiments further illustrate the present disclosure, however, it should be understood that the embodiments are described in an illustrative and not limiting manner and that a variety of modifications may be made by the skilled in the art.

Example 1: Preparation of Fc Portion

[0261] The amino acid sequences of the protein (SEQ ID Nos:1-68) were reverse-translated to synthesize corresponding nucleotide sequences. The nucleotide sequence with appropriate cleavage sites (XbaI+SalI) at its two ends was cloned into the pWX4.1 expression vector (WuXi Biotechnology Co., Ltd., Shanghai, China). Each constructed vector carries either the signal peptide sequence of human interleukin 2 or the signal peptide sequence of human serum albumin. The pWX4.1 plasmid was transfected into E. coli Top10 strain (Life Technologies) for DNA amplification and purified using the PURELINK HiPURE Plasmid Filter Maxiprep Kit (Life Technologies). Then, the plasmids were then transfected into CHO cells (ATCC) using the EXPIFECTAMINE 293 Transfection Kit (Life Technologies). The transfected cells were cultured for 7 days, then centrifuged and precipitated, and filtered. The supernatant was collected and purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA) to obtain purified h-IgG Fc. The purified samples were subjected to 4-12% Bis Tris SDS-PAGE electrophoresis and 1-2 g of the sample were used, and then stained with Thomas Brilliant Blue. Each sample was subjected to both reducing (R) and non-reducing (NR) treatments.

Example 2: General Synthesis Method of h-IgG1 Fc-PEG4-azides

##STR00306##

[0262] At 0 C., PEG-azide-NHS ester (1 to 20 equivalents) was dissolved in a solution of DMF/PBS, which was then added to a PBS solution of h-IgG1 Fc prepared in Example 1. The reaction was stirred at room temperature for 1 to 10 h. The reaction system was concentrated by centrifugation, and the PBS solution was used for washing. The target h-IgG1 Fc-PEG4-azide was prepared by preparative chromatography.

Example 3: Generalized Synthesis Method of Conjugates

##STR00307##

[0263] The h-IgG1 Fc-PEG4-azide dissolved in PBS solution was added to the PBS solution of terminal alkynyl compounds, copper sulfate, tris(3-hydroxypropyltriazolylmethyl)-amine, and sodium ascorbate. The reaction solution was reacted for at least 12 hours, then diluted with PBS, and concentrated by centrifugation. Then, the PBS solution was used for washing, and the target conjugate was prepared by preparative chromatography. The average DAR (drug/antibody ratio) of the conjugate was analyzed by MS.

Example 4: Synthesis of Intermediate Drug (INT-DRUG)

##STR00308##

Step One: Synthesis of INT-DRUG-2

##STR00309##

[0264] INT-DRUG-1 (3.0 g, 6.57 mmol) was added to the reaction flask, followed by the additions of the tetrahydrofuran (30 mL), triphenylphosphine (1.9 g, 7.23 mmol), and the reaction was stirred at 30 C. for 2 h. after this reaction ends, lithium hydroxide monohydrate (28 mg) and water (1.5 mL) were added, and the reaction continues to stir at 30 C. for 16 h. Once TLC monitors the end of the reaction, N,N-di-Boc-1H-1-guanidinium pyrazole (2.1 g, 6.91 mmol) was added and stirred at 30 C. for 48 h. After the reaction finishes, the reaction solution was concentrated, and separated and purified by column chromatography on silica gel (ethyl acetate/petroleum ether-1:1). The colorless oil INT-DRUG-2 (2.2 g) was obtained after purification. LCMS: [M+1].sup.+ found 673.40

Step Two: Synthesis of INT-DRUG-3

##STR00310##

[0265] Under nitrogen protection, INT-DRUG-2 (2.19 g, 3.25 mmol) was added to a reaction flask, and then a methanol (8 mL) solution of dried sodium methanolate (0.65 mmol). The reaction solution was stirred at room temperature for 10 min, and quenched with HCl (0.4N, 1,4-dioxane solution, 2.5 mL). The reaction solution was concentrated, separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether, 70%100%) to give a white solid INT-DRUG-3 (1.15 g). LCMS: [M+1]+ found 547.33.

Step Three: Synthesis of INT-DRUG-4

##STR00311##

[0266] INT-DRUG-3 (386.9 mg, 0.71 mmol), acetonitrile (10 mL), CDI (160.8 mg, 0.99 mmol), and DMAP (302.7 mg, 2.47 mmol) were added to the reaction flask. The reaction was stirred at room temperature for 16 hours. At the end of the reaction, the reaction solution was concentrated and separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether, 0%45%) to obtain a white solid INT-DRUG-4 (242.8 mg). LCMS: [M+1]+ found 573.35.

Step Four: Synthesis of INT-DRUG-5

##STR00312##

[0267] INT-DRUG-4 (403 mg, 0.7 mmol), DMAP (1.37 g, 11.2 mmol), pyridine (60 mL), and phenyl p-nitrochloroformate (2.11 g, 10.5 mmol) were added to the reaction flask and stirred at 40 C. for 4 h after nitrogen displacement. The reaction was quenched (can not be left for a long time) with water (20 mL) when the rection conversion detected by LCMS was to be >90%. The reverse column was used for separation (acetonitrile/water 0%90%), and the white solid INT-DRUG (173 mg) was obtained by freeze-drying. LCMS: [M+1]+ found 738.33.

Example 5: Synthesis of Intermediate a (Inter-A)

##STR00313##

Step One: Synthesis of Intermediate 2

##STR00314##

[0268] Compound 1 (150 g, 1.43 mol) and triethylamine (289 g, 2.85 mol) were added to the reaction flask followed by addition of tetrahydrofuran (1 L), which was then stirred at 0 C. after nitrogen displacement. The CbzCl (243 g, 1.43 mol) dissolved in tetrahydrofuran (500 mL) was added dropwise to the reaction solution. The reaction solution was stirred at room temperature overnight. At the end of the reaction, the reaction was quenched by the addition of water (3 L) and extracted with ethyl acetate (300 mL2). The combined organic phases were washed with saturated saline, dried with anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane/methanol, 20/1) to give the yellow oil compound 2 (255 g).

Step Two: Synthesis of Intermediate 3

##STR00315##

[0269] Compound 2 (245 g, 1.02 mol) and carbon tetrabromide (509 g, 1.54 mol) were added to the reaction flask followed by addition of the dichloromethane (2 L), which was then stirred at 0 C. after nitrogen displacement. Triphenylphosphine (403 g, 1.54 mol) dissolved in dichloromethane (500 mL) was added dropwise to the reaction solution. The reaction solution was stirred at room temperature for 1.5 h and concentrated to obtain the crude product at the end of the reaction. The crude product was purified by silica gel column chromatography (dichloromethane/methanol, 20/1) to give the yellow oil compound 3 (200 g, crude).

Step Three: Synthesis of Intermediate 4

##STR00316##

[0270] Compound 3 (200 g, crude) and N,N-dimethylformamide (2 L) was added to the reaction flask. Byramine (28.3 g, 264 mmol) and sodium carbonate (73.0 g, 528 mmol) were added to the reaction solution. The reaction solution was stirred at 60 C. overnight. At the end of the reaction, the temperature was reduced to room temperature and the reaction solution was added with water (3 L) and extracted with ethyl acetate (300 mL2). The combined organic phases were washed with saturated saline, then dried with anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane/methanol, 20/1) to give the yellow oil compound 4 (45 g).

Steps Four and Five: Synthesis of Intermediate 6

##STR00317##

[0271] Compound 4 (43.0 g, 78.2 mmol) and tetrahydrofuran (450 mL) were added to the reaction flask and stirred at 0 C. after nitrogen displacement. LiAlH.sub.4 (23.7 g, 62.6 mmol) was added to the reaction solution. The reaction solution was stirred at room temperature for 1.5 h, and then warmed up to reflux for 6 h with continuous stirring. The temperature was lowered to 0 C. at the end of the reaction and sodium sulfate decahydrate (90 g) was added to quench the reaction. The reaction solution was stirred at room temperature for 1 h. Triethylamine (31.7 g, 31.3 mmol) and Boc anhydride (68.3 g, 31.3 mmol) were added to the reaction solution and stirred at room temperature overnight. After the reaction completed, water (2 L) was added and ethyl acetate (300 mL2) was used for extractions. The combined organic phases were washed with saturated saline, then dried with anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane/methanol, 30/1) to give yellow oil compound 6 (20 g). LCMS: [M+1]+ found 510.2.

Step Six: Synthesis of Inter-A

##STR00318##

[0272] Under nitrogen atmosphere, compound 6 (19 g, 3.73 mmol), palladium carbon (2 g) and methanol (400 mL) were added to the reaction flask. Replacement of nitrogen with hydrogen were performed several times. The reaction solution was stirred overnight at room temperature. At the end of the reaction, the reaction solution was filtered, and the filter cake was washed with methanol (50 mL), and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane/methanol, 15/1) to give a yellow oil intermediate A (14 g). LCMS: [M+1]+ found 420.2.

Example 6: Synthesis of Linker 1

##STR00319##

Step One: Synthesis of Compound 9

##STR00320##

[0273] Compound 8 (1.5 g, 6.51 mmol) and triethylamine (1.97 g, 19.5 mmol) were dissolved in dichloromethane (30 mL) and stirred at 0 C. after nitrogen displacement. Phenyl p-nitrochloroformate (1.31 g, 6.51 mol) dissolved in dichloromethane (3 mL) was added dropwise into the reaction solution with a syringe. The reaction solution was stirred at room temperature for 6 h. The crude compound 9 was obtained by concentration at the end of the reaction.

Step Two: Synthesis of Compound 10

##STR00321##

[0274] Crude compound 9 (1.47 mmol) and triethylamine (1.97 g, 19.5 mmol) were dissolved in dichloromethane (20 mL) and stirred at room temperature. Intermediate A (2.71 g, 1.47 mol) dissolved in dichloromethane (2 mL) was added to the reaction solution. The reaction solution was stirred at room temperature overnight. The crude product was obtained by concentration at the end of the reaction. The crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 1/1) to give the yellow oil compound 10 (1.6 g).

Step Three: Synthesis of Linker 1

##STR00322##

[0275] To solution of Compound 10 (1.8 g, 2.66 mmol) dissolved in 1,4-dioxane (10 mL) was added HCl (4M, 1,4-dioxane solution, 10 mL), which was stirred at room temperature for 2 h. The reaction solution was concentrated at the end of the reaction to give the crude, yellow oil linker 1 (1.6 g). LCMS: [M+1].sup.+ found 478.2.

[0276] .sup.1H NMR (400 MHz, CDCl.sub.3): 9.50 (s, 4H), 4.23 (s, 4H), 3.86-3.62 (m, 18H), 3.17 (s, 3H), 2.81 (s, 5H), 2.48 (s, 1H), 1.73 (s, 6H)

Example 7: Synthesis of Linker-2

##STR00323##

Step One: Synthesis of Compound L2-2

##STR00324##

[0277] Compound L2-1 (2.00 g, 8.6 mmol) and triethylamine (2.61 g, 26 mmol) were dissolved in dichloromethane (20 mL) and stirred at 0 C. after nitrogen displacement. Phenyl p-nitrochloroformate (1.73 g, 8.6 mol) dissolved in dichloromethane (3 mL) was dropped into the reaction solution with a syringe. The reaction solution was stirred at 30 C. for 6 h. Water was added at the end of the reaction, and ethyl acetate was used for extraction. The combined organic phases were dried with anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 100/120/1) to obtain the yellow oil compound L2-2 (1.3 g).

Step Two: Synthesis of Compound L2-3

##STR00325##

[0278] Compound 2 (1.3 g, 3.2 mmol) and DIEA (0.83 g, 6.4 mmol) were dissolved in acetonitrile (15 mL) and stirred at room temperature. Intermediate A (1.34 g, 3.2 mmol) dissolved in acetonitrile (5 mL) was added to the reaction solution. The reaction solution was stirred at 80 C. overnight. Water (20 mL) was added at the end of the reaction and ethyl acetate (320 mL) was used for extraction. The combined organic phases were dried with anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane/methanol, 20/1) to give the yellow oil compound L2-3 (1.2 g).

Step Three: Synthesis of Linker-2

##STR00326##

[0279] Compound L2-3 (1.2 g, 1.8 mmol) dissolved in 1,4-dioxane (10 mL) was added with HCl (4M, 1,4-dioxane solution, 10 mL), which was stirred at room temperature for 2 h. The reaction solution was concentrated at the end of the reaction to give the crude brown oil Linker 2 (0.93 g). LCMS: [M+1]+ found 477.3.

[0280] .sup.1H NMR (400 MHz, DMSO_d.sub.6) 8.94 (s, 4H), 6.41 (m, 1H), 4.14 (s, 2H), 3.65-3.67 (m, 4H), 3.54 (m, 6H), 3.51 (m, 4H), 3.44-3.34 (m, 11H), 3.16-3.18 (m, 2H), 3.06 (m, 4H), 2.54 (s, 6H).

Example 8: Synthesis of Linker-4

##STR00327##

Step One: Synthesis of L4-2

##STR00328##

[0281] Trichloromethyl chloroformate (539 mg, 1.02 mol) dissolved in dry tetrahydrofuran (5 mL) was stirred at 0 C. after nitrogen displacement. Compound L4-1 (500 mg, 2.27 mmol) and DIEA that were dissolved in tetrahydrofuran (5 mL) was added dropwise to the reaction solution. The reaction solution was stirred at 0 C. for 1 h. At the end of the reaction, the filtrate obtained from filtration through diatomaceous earth was concentrated to give the crude compound L4-2. The crude compound L4-2 was used directly in the next step without purification.

Step Two: Synthesis of L4-3

##STR00329##

[0282] Intermediate A (300 mg, 0.715 mmol) and DIEA (277 mg, 2.15 mmol) that were dissolved in dichloromethane (2 mL) was stirred at 0 C. The crude compound L4-2 dissolved in dichloromethane (1 mL) was added dropwise to the reaction solution. The reaction solution was stirred overnight at room temperature. At the end of the reaction, the pH was adjusted to 7 with 1N hydrochloric acid and dichloromethane (21 mL) was used for extraction. The organic phases were combined and concentrated to obtain the crude product. The crude product was purified by preparative silica gel plate to give the yellow oil compound L4-3 (150 mg). LCMS: [M+1]+ found 666.4.

Step Three: Synthesis of L4-4

##STR00330##

[0283] Compound L4-3 (3 g, 4.51 mmol), palladium/carbon hydroxide (0.3 g) and methanol (60 mL) were added to the reaction flask under nitrogen atmosphere. Hydrogen was used to displace nitrogen several times. The reaction solution was stirred overnight at room temperature. At the end of the reaction, the reaction solution was filtered, and the filter cake was washed with methanol (10 mL). The filtrate was concentrated to give the crude yellow oil compound L4-4 (2.4 g). LCMS: [M+1]+ found 532.3

Step Four: Synthesis of L4-5

##STR00331##

[0284] Compound L4-4 (2.4 g, 4.51 mmol), DIEA (1.74 g, 13.4 mmol) and Intermediate C (3.08 g, 9.02 mmol) were dissolved in DMF (20 mL) and stirred at 80 C. overnight after the nitrogen displacement. At the end of the reaction, the solution was reduced to room temperature, added with water (100 mL) and extracted with ethyl acetate (220 mL). The combined organic phases were dried with anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane/methanol, 20/1) to obtain the crude product. The crude product was purified three more times with preparative silica gel plates to give the yellow oil compound L4-5 (350 mg). LCMS: [M+1]+ found 702.3.

Step Five: Synthesis of Linker 4

##STR00332##

[0285] Compound L4-5 (350 mg, 2.66 mmol) dissolved in 1,4-dioxane (3 mL) was added with HCl (4M, 1,4-dioxane solution, 5 mL) and stirred at room temperature for 2 h. The reaction was concentrated at the end of the reaction to give the crude yellow oil Linker 4 (310 mg).

[0286] LCMS: [M+1].sup.+ found 502.3, .sup.1H NMR (400 MHz, CDCl.sub.3): 12.19-12.17 (m, 1H), 9.53-9.48 (m, 4H), 4.26-4.22 (m, 2H), 4.06-4.15 (m, 31H), 3.07-2.70 (m, 9H), 2.53-2.27 (m, 6H), 2.18 (s, 1H), 1.27-1.24 (m, 4H).

Example 9: Synthesis of Linker 5

##STR00333##

Step One: Synthesis of L5-2

##STR00334##

[0287] N-Boc-4-hydroxypiperidine (5.0 g, 14.6 mmol) and DMF (50 mL) were added to the reaction flask and stirred at 0 C. after the nitrogen displacement. Sodium hydrogen (0.64 g, 16 mmol) was added to the reaction solution. The reaction solution was stirred at room temperature for 0.5 h. Compound 1 (3.2 g, 16 mmol) was then added to the reaction solution and continued to be stirred at room temperature for 2 h. At the end of the reaction, the reaction solution was added to ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried with anhydrous sodium sulfate and concentrated to obtain the crude compound L5-2 (4.5 g).

Step Two: Synthesis of L5-3

##STR00335##

[0288] Compound L5-2 (4.5 g, 12.1 mmol) dissolved in ethyl acetate (30 mL) was added with HCl (4M, ethyl acetate, 30 mL) and stirred at room temperature for 2 h. After the reaction was completed, reaction solution was concentrated to give the crude compound L5-3 (5.0 g). LCMS: [M+1]+ found 272.2.

Step Three: Synthesis of L5-4

##STR00336##

[0289] Compound L5-3 (5.00 g, crude) and triethylamine (5.6 g, 55.2 mmol) that were dissolved in dichloromethane (50 mL) was stirred at room temperature after the nitrogen displacement. Phenyl p-nitrochloroformate (5.6 g, 27.6 mmol) dissolved in dichloromethane (10 mL) was dropped into the reaction solution with a syringe. The reaction solution was stirred at room temperature for 2 h. At the end of the reaction, ice water was added and dichloromethane was used for extraction. The combined organic phases were dried with anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the yellow oil compound L5-4 (5.2 g, crude). LCMS: [M+1]+ found 437.1.

Step Four: Synthesis of L5-5

##STR00337##

[0290] Compound L5-4 (5.2 g, crude) and potassium carbonate (1.91 g, 13.8 mmol) were dissolved in DMF (20 mL) and stirred at room temperature. Intermediate A (1.94 g, 4.6 mmol) was dissolved in DMF (5 mL) and added to the reaction solution. The reaction solution was stirred at 130 C. for 48 h. At the end of the reaction, water (200 mL) was added and ethyl acetate (3100 mL) was used for extraction. The combined organic phases were dried with anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the yellow oil compound L5-5 (0.8 g). LCMS: [M+1]+ found 717.5.

Step Five: Synthesis of Linker 5

##STR00338##

[0291] Compound L5-5 (0.8 g, 1.1 mmol) dissolved in ethyl acetate (10 mL) was added with HCl (4M, ethyl acetate, 10 mL) and stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated to give the crude compound Linker 5 (0.61 g). LCMS: [M+1]+ found 517.3.

[0292] .sup.1H NMR (400 MHz, CDCl.sub.3): 9.63 (s, 4H), 4.23 (d, 2H), 3.95 (s, 4H), 3.75 (s, 3H), 3.73-3.65 (m, 17H), 3.26-3.14 (m, 6H), 2.79 (m, 9H), 2.48 (s, 1H), 2.02 (d, 2H), 1.72 (s, 2H).

Example 10: Synthesis of Intermediate Inter B

##STR00339##

Step One: Synthesis of IB-2

##STR00340##

[0293] 2-(2-BOC-aminoethoxy)ethanol (IB-1, 9.03 g, 44.0 mmol) and imidazole (6.58 g, 96.8 mmol) were dissolved in dichloromethane (100 mL), which was added drop-wise with a solution of tert-butyldimethylchlorosilane (7.30 g, 48.4 mmol) dissolved in dichloromethane (30 mL) and was stirred for 16 h at room temperature. After the reaction was completed, dichloromethane (100 mL) was added, and dilute hydrochloric acid (0.5 M, 200 mL*2) and saturated sodium bicarbonate (200 mL*2) was used sequentially for washing. The organic phase was dried with anhydrous sodium sulfate, concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA=3:1) to give the white solid IB-2 (13.5 g).

Step Two: Synthesis of IB-3

##STR00341##

[0294] IB-2 (13.5 g, 42.3 mmol) was dissolved in DMF (100 mL), and sodium hydrogen (6.77 g, 169 mmol) was added under an ice bath. After stirring for 20 min, iodomethane (13 mL, 212 mmol) was added followed by stirring for 16 h at room temperature. After the reaction was completed, water (100 mL) and ethyl acetate (200 mL) were added, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude IB-3 (17.0 g), which was directly used in the next step.

Step Three: Synthesis of IB-4

##STR00342##

[0295] IB-3 (17.0 g, calculated as 42.3 mmol) and tetrabutylammonium fluoride trihydrate (29.34 g, 93.0 mmol) were dissolved in tetrahydrofuran (100 mL) and stirred at room temperature for 1 hour. After the reaction was completed, water (200 mL) was added and ethyl acetate (100 mL*3) was used for extraction, followed by washing with saturated brine (100 mL*2). The organic phase was dried with anhydrous sodium sulfate, concentrated under reduced pressure and then purified by silica gel column chromatography (PE:EA=2:1) to give the brown oil IB-4 (5.6 g). LCMS[M-100]+: 120.15

Step Four: Synthesis of Inter B

##STR00343##

[0296] IB-4 (13.0 g, 59.32 mmol) and carbon tetrabromide (29.5 g, 88.98 mmol) were dissolved in dichloromethane (50 mL), to which the solution of triphenylphosphine (23.34 g, 88.98 mmol) in dichloromethane (20 mL) was added dropwise under an ice bath. The reaction solution was stirred at room temperature for 24 hours. After the reaction was completed, concentration under reduced pressure and purification by silica gel column chromatography (PE:EA=2:1) were performed to give the colorless oil IB-4 (6.5 g). LCMS[M-100]+:182.17.

Example 11: Synthesis of Linker 8

##STR00344##

Step One: Synthesis of L8-2

##STR00345##

[0297] After propynyl-triethylene glycol (L8-1, 5.0 g, 26.56 mmol) was dissolved in dichloromethane (50 mL), N,N-diisopropylethylamine (6.87 g, 53.13 mmol) and p-toluenesulphonyl chloride (5.57 g, 29.22 mmol) were added, and the reaction was stirred at room temperature for 10 hours. After the reaction completed, water (50 mL) was added for washing, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude L8-2 (11.0 g), which was directly used in the next step. LCMS[M+1]+:343.23.

Step Two: Synthesis of L8-3

##STR00346##

[0298] After L8-2 (11.0 g, calculated as 26.56 mmol) was dissolved in tetrahydrofuran (50 mL), 4-tert-butoxycarbonylaminopiperidine (5.3 g, 26.56 mmol) and N,N-diisopropylethylamine (6.9 g, 53.12 mmol) were added, and stirred at 70 C. for 24 h. After the reaction was completed, concentration under reduced pressure and purification by silica gel column chromatography (EA) were performed to give the brown oil L8-3 (5.1 g). LCMS[M+1]+: 371.40

Step Three: Synthesis of L8-4

##STR00347##

[0299] L8-3 (3.43 g, 9.27 mmol) placed in the solution (30 ml) of ethyl acetate and hydrochloride was stirred at room temperature for 1 h. At the end of the reaction, concentration was performed to give the crude L8-4 (2.5 g) that was directly used in the next step. LCMS[M+1]+: 271.30

Step Four: Synthesis of L8-6

##STR00348##

[0300] Crude L8-4 (2.5 g, calculated as 9.27 mmol) was dissolved in DMF (50 ml), and then L8-5 (6.5 g, 23.1 mmol) and DIEA (2.4 g, 18.5 mmol) were added and stirred at 75 C. for 24 h. Concentration under reduced pressure, silica gel column chromatography (DCM:MeOH=10:1) and reverse HPLC were performed to give the L8-6 (90 mg). LCMS[M+1]+:673.60.

Step Five: Synthesis of Linker 8

##STR00349##

[0301] L8-6 (90 mg, 0.15 mmol) was placed in 1,4-dioxane hydrochloride solution (5 ml) and stirred at room temperature for 1 h. At the end of the reaction, concentration was performed to give the crude Linker 8 (80 mg).

Example 12: Synthesis of Linker 9

##STR00350##

Step One: Synthesis of L9-2

##STR00351##

[0302] L9-1 (3.2 g, 9.17 mmol) was added to the reaction flask, followed by the addition of methanol solution of methylamine (10 mL) and KI (304 mg, 1.83 mmol). The solution was stirred at room temperature overnight. At the end of the reaction, the solvent was removed by rotary evaporation to obtain the crude L9-2, which was used directly in the next step.

Step Two: Synthesis of L9-3

##STR00352##

[0303] The crude compound L9-2 (1.9 g, 9.18 mmol) was dissolved in a mixed solvent of tetrahydrofuran:water (20 mL:10 mL), followed by the additions of sodium bicarbonate (1.5 g, 18.36 mmol) and di-tert-butyl dicarbonate (2.2 g, 10.1 mmol), which was stirred at room temperature overnight. At the end of the reaction, the reaction solution was quenched by addition of water (30 mL), extracted with ethyl acetate (50 mL*3), and dried with anhydrous sodium sulfate. The organic phase was concentrate by column purification (PE:EA, EA=30%) to give the colorless liquid compound L9-3 (1.5 g).

Step Three: Synthesis of L9-4

##STR00353##

[0304] Compound L9-3 (1.5 g, 4.88 mmol) was dissolved in tetrahydrofuran (15 mL) followed by addition of sodium hydride (290 mg, 7.33 mmol, 60%) at 0 C. The reaction solution was stirred at 0 C. for 30 min. Bromopropargyl (700 mg, 5.86 mmol) was added, and the reaction was carried out overnight at room temperature. At the end of the reaction, the reaction solution was quenched by addition of water (20 mL), extracted with ethyl acetate (20 mL), extracted with ethyl acetate (50 mL*3), dried with anhydrous sodium sulfate, concentrated, and purified by column (PE:EA, EA=40%) to give the sticky, colorless product L9-4 (1.0 g).

Step Four: Synthesis of L9-5

##STR00354##

[0305] Compound L9-4 (600 mg, 1.73 mmol) was dissolved in dichloromethane (5 mL), and a solution of hydrochloride in dioxane (5 mL, 4M in dioxane) was added. The reaction was carried out at room temperature for 1 h. At the end of the reaction, a direct concentration was performed to give the crude L9-5 in the form of hydrochloride, which was used directly in the next step.

Step Five: Synthesis of L9-6

##STR00355##

[0306] Triphosgene (300 mg, 0.98 mmol) was dissolved in tetrahydrofuran (15 mL), to which pyridine (250 mg, 2.94 mmol) in tetrahydrofuran solution (2 mL) was added in ice-water bath under nitrogen atmosphere. The solution was stirred in ice-water bath for 30 min. Then, compound L9-5 (550 mg, 1.96 mmol), triethylamine (300 mg, 2.94 mmol) in tetrahydrofuran solution (2 mL) were added, which reacted at room temperature for 3 h. After the reaction completed, the reaction was quenched with water (10 mL), extracted with ethyl acetate (30 mL*3), dried with anhydrous sodium sulfate and concentrated to obtain the crude L9-6, which was directly used in the next step.

Step Six: Synthesis of L9-7

##STR00356##

[0307] Compound L9-6 was dissolved in tetrahydrofuran (15 mL), followed by addition of Inter-A (500 mg, 1.17 mmol) in tetrahydrofuran (2 mL), triethylamine (300 mg, 2.94 mmol), and 4-dimethylaminopyridine (20 mg, 0.16 mmol) in tetrahydrofuran (2 mL), and the reaction was carried out for 4 h at 60 C., and stirred at room temperature overnight. After the reaction completed, the reaction was quenched with water (10 mL), extracted with ethyl acetate (30 mL*3), dried with anhydrous sodium sulfate and concentrated to give the compound L9-7 (530 mg).

Step Seven: Synthesis of L9-8

##STR00357##

[0308] Compound L9-7 (100 mg, 0.14 mmol) was dissolved in dichloromethane (5 mL), followed by addition of hydrochloride in dioxane solution (5 mL, 4M in dioxane). The reaction was carried out at room temperature for 1 h. A direct concentration was performed to give the crude Linker 9 in the form of hydrochloride, which was used directly in the next step. LCMS[M+1]+: 491.3

Example 13: Synthesis of Linker 10

##STR00358##

Step One: Synthesis of L10-2

##STR00359##

[0309] Chlorosulfonyl isocyanate (400 mg, 2.83 mmol) was dissolved in dichloromethane (10 mL) and stirred at 0 C. under nitrogen protection. A solution of bromoethanol (353 mg, 2.83 mmol) in dichloromethane (5 mL) was added dropwise and stirred at 0 C. for 1 h. Then, a solution of L10-1 (2.83 mmol) in dichloromethane (5 mL) was added dropwise and stirred at 0 C. for 1 h. After the reaction was completed, dichloromethane (10 mL) was added, and the diluted hydrochloric acid (1M, 20 mL) was used for washing. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude yellow oil L10-2 (800 mg) was obtained by purification of silica gel column chromatography (PE:EA=1:1).

Step Two: Synthesis of L10-3

##STR00360##

[0310] The crude L10-2 (800 mg) was dissolved in dichloromethane (10 mL), and TEA (2 mL) was added. The reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, dichloromethane (10 mL) was added, and dilute hydrochloric acid (1M, 20 mL) was used for washing. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The yellow oil L10-3 (670 mg) was obtained by purification of the silica gel column chromatography (PE:EA=1:3).

Step Three: Synthesis of L10-4

##STR00361##

[0311] L10-3 (300 mg, 0.79 mmol), Inter-A (330 mg, 0.79 mmol) and TEA (160 mg, 1.60 mmol) were dissolved in acetonitrile (10 mL) and stirred for 10 h at 80 C. After the reaction was completed, the solvent was removed by rotary evaporation and purification by the reversed-phase column chromatography (acetonitrile/water) was performed to give the yellow oil L10-4 (190 mg).

Step Four: Synthesis of Linker 10

##STR00362##

[0312] L10-4 (107 mg, 0.15 mmol) was placed in 1,4-dioxane hydrochloride solution (5 ml) and stirred at room temperature for 1 h. At the end of the reaction, concentration was performed to obtain the crude Linker 10 that was directly used in the next step. MS[M+1].sup.+: 513.51.

Example 14: Synthesis of Linker 11

##STR00363##

Step One: Synthesis of L11-4

##STR00364##

[0313] L11-1 (360 mg, 0.86 mmol), L11-2 (238 mg, 0.86 mmol), L11-3 (230 mg, 0.9 mmol) and triethylamine (93 mg, 0.9 mmol) were dissolved in dichloromethane (10 mL) under nitrogen protection and were stirred for 3 h at room temperature. After the reaction completed, the solvent was removed by rotary evaporation and purification by silica gel column chromatography (PE:EA=1:1) was obtained to give colorless oil L11-4 (830 mg).

Step Two: Synthesis of L11-6

##STR00365##

[0314] L11-4 (406 mg, 0.62 mmol), L11-5 (150 mg, 0.80 mmol), and triphenylphosphine (241 mg, 0.92 mmol) were dissolved in tetrahydrofuran (10 mL) under nitrogen protection and were stirred at 0 C. for 10 min. Then, the solution of DIAD (250 mg, 1.24 mmol) in tetrahydrofuran solution (2 mL) was added dropwise. The reaction solution was warmed up to 80 C. and stirred for 12 hours. After the reaction completed, the solvent was removed by rotary evaporation and purification by silica gel column chromatography (PE:EA=1:1) was obtained to give the colorless oil L11-6 (190 mg).

Step Three: Synthesis of Linker 11

##STR00366##

[0315] L11-6 (85 mg, 0.1 mmol) was placed in TFA (5 ml) and stirred at room temperature for 3 h. After the reaction completed, concentration was performed to give the Linker 11 that was directly used in the next step. LCMS[M+1].sup.+: 432.41

Example 15: Synthesis of Conjugate Intermediate C-Inter-1

##STR00367##

Step One: Synthesis of C-Inter-1-1

##STR00368##

[0316] Linker 1 (65 mg, 0.13 mmol) was added to the reaction flask followed by addition of DMF (10 mL) and DIEA (2 mL), which were stirred at room temperature. The solution of INT-DRUG (200 mg, 0.27 mmol) dissolved in dichloromethane (5 mL) was added dropwise to the reaction solution that was stirred at room temperature overnight. After the reaction completed, the solvent was removed by rotary evaporation to give the crude and purification by reverse column (water/acetonitrile) and freeze-drying were performed to give the C-Inter-1-1 (216 mg).

Step Two: Synthesis of C-Inter-1-2

##STR00369##

[0317] Compound C-Inter-1-1 (216 mg, 0.13 mmol) was dissolved in methanol (2 mL) followed by addition of aqueous lithium hydroxide (0.65 mmol, 2 mL), which were stirred at room temperature for 1 hour. After the reaction completed, the solvent was removed by rotary evaporation to give the crude compound C-Inter-1-2, which was used directly in the next step.

Step Three: Synthesis of C-Inter-1

##STR00370##

[0318] Compounds C-Inter-1-2 were dissolved in trifluoroacetic acid, and stirred at room temperature for 10 min. The solvent was removed by rotary evaporation to give the crude C-Inter-1. Purification by reverse column chromatography (0.1% TFA aqueous solution/0.1% TFA acetonitrile) and freeze dried were performed to give the C-Inter-1 (47 mg). LCMS: [M+1]+ found 1166.53.

Example 16: Synthesis of Conjugate Intermediate C-Inter-2

##STR00371## ##STR00372##

Step One: Synthesis of C-Inter-2-1

##STR00373##

[0319] Linker 2 (100 mg, 0.20 mmol) dissolved in DMF (1 ml) was added with DIEA (0.1 g, 1.23 mmol), to which the INT-DRUG (300 mg, 0.41 mmol) dissolved in DCM (2 ml) was added dropwise. The mixture was stirred at room temperature for 16 h, and then concentrated under reduced pressure followed by the reversed-column chromatography to give C-Inter-2-1 (230 mg).

Step Two: Synthesis of C-Inter-2-2

##STR00374##

[0320] Compound C-Inter-2-1 (230 mg, 0.14 mmol) was dissolved in methanol (5 mL) followed by addition of aqueous lithium hydroxide (30 mg, 1 ml), which were stirred at room temperature for 1 hour. Then, concentration was directly performed to give the crude compound C-Inter-1-2, which was used directly in the next step.

Step Three: Synthesis of C-Inter-2

##STR00375##

[0321] The crude C-Inter-2-2 (300 mg, calculated as 0.14 mmol) was dissolved in TFA (5 ml), and stirred at room temperature for 5 min. The concentration under reduced pressure, and purification by reversed-phase preparative HPLC were performed to give C-Inter-2 (72 mg). LCMS[M+1]+:1194.00.

Example 17: Synthesis of Conjugate Intermediate C-Inter-3

##STR00376## ##STR00377##

Step One: Synthesis of C-Inter-3-1

##STR00378##

[0322] The crude Linker 8 (80 mg, calculated as 0.15 mmol) dissolved in DMF (1 ml) was added with DIEA (0.6 g, 4.5 mmol), to which the INT-DRUG (210 mg, 0.3 mmol) dissolved in DCM (2 ml) was added dropwise. The mixture was stirred at room temperature for 16 h. Subsequently, concentration under reduced pressure was performed to give the crude C-Inter-3-1 (300 mg), which was directly used in the next step.

Step Two: Synthesis of C-Inter-3-2

##STR00379##

[0323] Crude C-Inter-3-1 (300 mg, calculated as 0.15 mmol) was dissolved in methanol (5 mL) followed by addition of aqueous lithium hydroxide (30 mg, 1 ml), which were stirred at room temperature for 1 hour. Then, concentration under reduced pressure was performed to give the crude C-Inter-3-2 (350 mg), which was used directly in the next step.

Step Three: Synthesis of C-Inter-3

##STR00380##

[0324] The crude C-Inter-3-2 (350 mg, calculated as 0.15 mmol) was dissolved in TFA (5 ml), and stirred at room temperature for 5 min. Then, concentration under reduced pressure and reverse preparative HPLC were performed to give the C-Inter-3 (20 mg). LCMS[M+1]+:1189.50.

Example 18: Synthesis of Conjugate Intermediate C-Inter-4

##STR00381##

Step One: Synthesis of C-Inter-4-1

##STR00382##

[0325] Linker 4 (70 mg, 0.14 mmol) was added to the reaction flask followed by addition of DMF (10 mL) and DIEA (2 mL), which were stirred at room temperature. The solution of INT-DRUG (180 mg, 0.24 mmol) dissolved in dichloromethane (5 mL) was added dropwise to the reaction solution and stirred at room temperature overnight. After the reaction completed, the solvent was removed by rotary evaporation to give the crude compound, and the purification by reverse column (water/acetonitrile) and freeze-drying were performed to give the product C-Inter-4-1 (210 mg).

Step Two: Synthesis of C-Inter-4-2

##STR00383##

[0326] Compound C-Inter-4-1 (210 mg, 0.12 mmol) dissolved in methanol (2 mL) was added with aqueous lithium hydroxide (0.65 mmol, 2 mL), which was stirred at room temperature for 1 hour. After the reaction completed, the solvent was removed by rotary evaporation to give the crude compound C-Inter-4-2, which was used directly in the next step.

Step Three: Synthesis of C-Inter-4

##STR00384##

[0327] The crude C-Inter-4-2 (350 mg, calculated as 0.15 mmol) was dissolved in TFA (5 ml), and stirred at room temperature for 5 min. Then concentration under reduced pressure and preparative HPLC (P-HPLC) chromatography were performed to give the C-Inter-4 (20 mg). LCMS[M+1]+:1189.50.

Example 19: Synthesis of Conjugate Intermediate C-Inter-5

##STR00385##

Step One: Synthesis of C-Inter-5-1

##STR00386##

[0328] The crude Linker 5 (86 mg) dissolved in DMF (1 ml) was added with DIEA (0.5 mL), to which INT-DRUG (230 mg, 0.32 mmol) dissolved in DCM (2 ml) was added dropwise. The mixture was stirred for 16 h at room temperature, and then concentrated under reduced pressure to obtain the crude C-Inter-5-1 crude. Purification by reverse column chromatography was performed to give the target product (170 mg).

Step Two: Synthesis of C-Inter-5-2

##STR00387##

[0329] C-Inter-5-1 (170 mg) dissolved in methanol (5 ml) was added with aqueous lithium hydroxide (21 mg, 1 ml), stirred at room temperature for 1 h and concentrated under reduced pressure to give the crude C-Inter-5-2, which was used directly in the next step.

Step Three: Synthesis of C-Inter-5

##STR00388##

[0330] The crude C-Inter-5-2 (350 mg) was dissolved in TFA (5 ml), stirred at room temperature for 5 min, concentrated under reduced pressure and subjected to preparative HPLC (P-HPLC) chromatography to give the C-Inter-5 (72 mg). LCMS [M/2+1]+: 617.45.

Example 20: Synthesis of C-Inter-6

##STR00389##

Step One: Synthesis of C-Inter-6-2

##STR00390##

[0331] Compound Linker 9 (80 mg, 0.15 mmol) was added to the reaction flask followed by DMF (4 mL) and DIEA (2 mL), which was then stirred at room temperature. INT-DRUG (220 mg, 0.3 mmol) dissolved in dichloromethane (2 mL) was added dropwise to the reaction solution that was then stirred at room temperature overnight. After the reaction completed, the solvent was removed by rotary evaporation to give the crude compound, and purification by reverse column (water/acetonitrile) and freeze-drying were performed to give the product C-Inter-6-2 (190 mg).

Step Two: Synthesis of C-Inter-6-3

##STR00391##

[0332] Compound C-Inter-6-2 (110 mg, 0.065 mmol) dissolved in methanol (5 mL) was added with the aqueous solution (1.5 mL) of lithium hydroxide monohydrate (12 mg, 0.28 mmol), which was then stirred for 4 hours at room temperature. After the reaction completed, the solvent was removed by rotary evaporation to give the crude compound C-Inter-6-3 that was directly used in the next step.

Step Three: Synthesis of C-Inter-6-3

##STR00392##

[0333] Compound C-Inter-6-3 was dissolved in trifluoroacetic acid (3 mL) and stirred at room temperature for 15 min. Then, the solvent was removed by rotary evaporation to give the crude compound. The crude compound was purified by reverse column chromatography (0.1% TFA aqueous solution/0.1% TFA acetonitrile) and freeze dried to give C-Inter-6 (53 mg). LCMS: [M+1].sup.+ found 1207.2.

Example 21: Synthesis of C-Inter-7

##STR00393##

Step One: Synthesis of C-Inter-7-1

##STR00394##

[0334] Linker 10 (calculated as 0.15 mmol) dissolved in DMF (2 ml) was added with DIEA (0.6 g, 4.5 mmol), to which INT-DRUG (210 mg, 0.3 mmol) dissolved in DCM (2 ml) was added dropwise. The mixture was stirred at room temperature for 16 h, then concentrated under reduced pressure to obtain the crude product. The crude product was purified by reversed-phase column chromatography (acetonitrile/water) and freeze dried to give the white solid C-Inter-7-1 (180 mg). LCMS[M12+1]+: 855.5.

Step Two: Synthesis of C-Inter-7-2

##STR00395##

[0335] C-Inter-7-1 (180 mg, 0.11 mmol) dissolved in methanol (5 ml) was added with aqueous lithium hydroxide (0.55 mmol, 1 ml), which was stirred at room temperature for 1 h. After addition of acetic acid (0.3 mL), rotary evaporation was performed to obtain the crude product C-Inter-7-2, which was directly used in the next step.

Step Three: Synthesis of C-Inter-7

##STR00396##

[0336] The crude C-Inter-7-2 placed in trifluoroacetic acid (1 ml) was stirred at room temperature for 30 min. After the reaction completed, rotary evaporation was performed to remove the solvent and give the crude product. The crude product was purified by reverse column purification (acetonitrile/0.1% TFA aqueous solution) and freeze dried to give the C-Inter-7 (77 mg). LCMS [M+1]+:1229.5.

Example 22: Synthesis of C-Inter-10

##STR00397##

Step One: Synthesis of C-Inter-10-1

##STR00398##

[0337] Linker 11 (calculated as 0.1 mmol) dissolved in DMF (2 ml) was added with DIEA (0.4 g, 3 mmol), to which INT-DRUG (147 mg, 0.2 mmol) dissolved in DCM (2 ml) was added dropwise. The mixture was stirred for 16 h at room temperature, and then concentrated under reduced pressure to obtain the crude product. The crude product was purified by reversed-phase column chromatography (acetonitrile/water), and freeze dried to obtain the white solid C-Inter-10-1 (70 mg). LCMS[M/2+1]+: 865.4.

Step Five: Synthesis of C-Inter-10-2

##STR00399##

[0338] C-Inter-10-1 (70 mg, 0.043 mmol) dissolved in methanol (2 ml) was added with aqueous lithium hydroxide (0.22 mmol, 1 ml), which was stirred at room temperature for 1 h. After addition of acetic acid (0.3 mL), rotary evaporation was performed to give the crude product C-Inter-10-2 that was used directly in the next step.

Step Six: Synthesis of C-Inter-10

##STR00400##

[0339] The crude C-Inter-10-2 was placed in trifluoroacetic acid (1 ml) and stirred at room temperature for 30 min. After the reaction completed, rotary evaporation was performed to remove the solvent and give the crude product. The crude product was purified by reversed-column purification (acetonitrile/0.1% TFA aqueous solution) and freeze dried to give the C-Inter-10. LCMS[M+1]+:1148.5.

Example 23: In Vitro Anti-Influenza Virus (Neuraminidase Inhibition Assay) Activity

1. Experimental Purpose:

[0340] To verify the effect of different neuraminidase inhibitors on the activity of different neuraminidases

2. Experimental Reagents and Consumables:

TABLE-US-00008 TABLE 1 Experimental reagents and consumables Product Company Cat.No. 96-well microtiter plate Greiner 655077 HTS black 4-Methylumbelliferyl- Sigma-Aldrich M8639-25MG N-acetyl--D-neuraminic acid sodium salt hydrate Tris Sigma 93362-500G CaCl.sub.2 Greagent 10043-52-4 NaCl Greagent 7647-14-5

TABLE-US-00009 TABLE 2 Instruments Product Company Type Microplate Reader Tecan infinite 200Pro pH Meter METTLER Seven Compact TOLEDO

TABLE-US-00010 TABLE 3 Neuraminidase Product Company Cat.No. Batch No. Influenza A H1N1 Sino 11058- LC14OC2611 (A/California/04/2009) Biological VNAHC Neuraminidase/NA (Active) Influenza A H3N2 Sino 40017- LC05AU2801 Neuraminidase/NA (Active) Biological VNAHC

TABLE-US-00011 TABLE 4 Positive control compounds (neuraminidase inhibitors) Molecular Product Company Cat.No. Batch No. weight Zanamivir Adamas 139110-80-8 P1695633 332.31

3. Experimental Preparation

[0341] 1) Assay buffer configuration: 50 mM Tris, 5 mM CaCl.sub.2), 200 mM NaCl, pH 7.5. [0342] 6.05 g of Tris, 0.56 g of CaCl.sub.2 and 11.69 g of NaCl were weighed and dissolved in 800 ml of ultrapure water. The pH was adjusted to 7.5, and 1 L of water was added followed by filtration through a 0.22 micron membrane. The solution was packaged at 50 ml/tube and stored at 4 C. [0343] 2) Neuraminidase substrate storage solution: Neuraminidase substrate 4-Methylumbelliferyl-N-acetyl--D-neuraminic acid sodium salt hydrate was dissolved in water at the concentration of 20 mM, which was packaged at 100 ml/tube and stored at 20 C. in shade. [0344] 3) Different neuraminidases were dissolved according to the reagent instructions, which were packaged at 10 U/tube and stored at 80 C. [0345] 4) The neuraminidase inhibitor to be tested was dissolved in sterile water at the concentration of 5 mM, which was packaged at 50 l/tube and stored at 80 C.

4. Experimental Procedure

[0346] 1) The different neuraminidases were diluted to 4 U/ml (4) with Assay buffer, with a final concentration of 1 U/ml. [0347] 2) 25 L of diluted neuraminidase solution was added to columns 2 to 12 of the 96-well plate, and 25 L of Assay buffer was added to column 1. [0348] 3) The different neuraminidase inhibitors were diluted to 4 M, with a final concentration of 1 M. Then gradient dilutions for the diluted solution of each inhibitor were performed according to the following table.

TABLE-US-00012 TABLE 5 Gradient dilution for each neuraminidase inhibitor 4 1 use CON final CON CON Configuration Method (nM) (nM) 1 600 L 40000 nM zanamivir 4000.00 1000.00 2 300 L CON-1 + 1333.33 333.33 600 L assay buffer 3 300 L CON-2 + 444.44 111.11 600 L assay buffer 4 300 L CON-3 + 148.15 37.04 600 L assay buffer 5 300 L CON-4 + 49.38 12.35 600 L assay buffer 6 300 L CON-5 + 16.46 4.12 600 L assay buffer 7 300 L CON-6 + 5.49 1.37 600 L assay buffer 8 300 L CON-7 + 1.83 0.46 600 L assay buffer 9 300 L CON-8 + 0.61 0.15 600 L assay buffer 10 300 L CON-9 + 0.20 0.05 600 L assay buffer [0349] 4) 25 L of neuraminidase inhibitors at concentration (CON) 1-10 in Table 4 was added to columns 211 respectively, and 25 l Assay buffer was added to columns 1 and 12. [0350] 5) The 96-well plate was covered with a sealing touch and incubated for 20 min at room temperature. [0351] 6) The Microplate Reader was turned on and warmed up to 37 C. Relevant parameters are set: excitation wavelength at 365 nm with a wave width of 9 nm; the emission wavelength of 450 nm with a wave width of 20 nm; Gain at 60. [0352] 7) Neuraminidase substrate diluted to 400 M was added to 96-well plate at 50 l/well, incubated at 37 C. for 20 min and then put into the Microplate Reader for reading. 8) Data analysis was performed using GraphPad Prism 8

5. Data Analysis:

[0353] 1) Calculation of background value: average fluorescence value of BACKGROUND in column 1. [0354] 2) Calculation of neuraminidase enzyme activity: subtraction of the background value from the average fluorescence value of different neuraminidases at 1 U/ml in Column 12 subtracts the background value. [0355] 3) Calculation of enzyme activity for neuraminidase inhibitors at different concentrations: subtraction of the background value from the fluorescence value of each well in columns 2 to 11. [0356] 4) Effect of different concentrations of neuraminidase inhibitors on enzyme activity: enzyme activity of different concentrations of neuraminidase inhibitors/neuraminidase enzyme activity*100% [0357] 5) Calculation of IC50 using non-linear curve fitting, wherein the horizontal coordinate is the inhibitor concentration, and the vertical coordinate is the effect of using different concentrations of neuraminidase inhibitor on enzyme activity, in GraphPad Prism 8 software.

6. Summary of Data:

TABLE-US-00013 H1N1 H3N2 (1 U/ml) (1 U/ml) Compound IC.sub.50(nM) IC.sub.50(nM) Zanamivir 1.00 4.06 C-Inter-1 10.42 23.04 C-Inter-2 14.18 44.33 C-Inter-3 21.30 36.71 C-Inter-4 48.67 158.60 C-Inter-5 36.99 115.10 C-Inter-6 11.95 24.94 C-Inter-7 7.64 20.01 C-Inter-10 10.76 19.15

[0358] The tested compounds all demonstrated high neuraminidase inhibitory activity without much activity loss due to the inclusion of the linker moiety. The inhibitory activities against H1N1 and H3N2 strains also reached the nM level, showing the worth of further development.

Example 24: Influenza Virus-Mediated Cytopathic Inhibitory Activity

1. Experimental Method

[0359] In vitro cytopathic-based anti-IAV/IBV activity assay refers to quantify the effect of the tested compounds on IAV/IBV-induced cytopathic using MDCK cell lines.

2. Experimental Material

TABLE-US-00014 Material Origin Batch No. H1N1(A/PR/8/34) virus ATCC VR1469 MDCK cell ATCC CCL-34 96-well plate Corning 3917 Fetal Bovine Serum (FBS) Hyclone SH30406.05 100 Pen-Strep solution (P/S) Hyclone SV30010 DMEM Hyclone SH30243.01 TPCK treated trypsin Sigma T1426 Cell-titer Glo Promega G7571

3. Experimental Procedure

[0360] 1) MDCK cells were digested and diluted to 210.sup.5/mL with DME medium containing 2% FBS and 100 P/S, which was inoculated into 96-well plate at 50 L per well and placed in an incubator for overnight culture. [0361] 2) The tested compounds were diluted to 2 M that was then triple diluted with a total of 8 dilution ratios. 5 L of diluted compound was added to each well and placed in the incubator for 1 h. [0362] 3) Influenza virus was diluted to 2,222 pfu/mL with DMEM medium containing 2% FBS, 1% P/S and 4 g/mL TPCK treated trypsin. 45 L of virus was added to each well. A virus control group without compounds but with viruses and a cell control group without compounds and viruses were also configured. The cells were incubated in an incubator for 4 days. [0363] 4) 50 L of Cell-titer Glo was added to each well, and the chemiluminescence was detected by a Microplate Reader.

4. Data Analysis: Calculate the Viral Inhibition Rate of the Compounds.

[0364]
Inhibition rate=(value of tested compoundsaverage value of virus control)/(average value of cell controlaverage value of virus control) [0365] The calculated inhibition rate was used to calculate the EC50 value by fitting the inhibition curve using the log(inhibitor) vs. responseVariable slope (four parameters) in the Nonlinear regression (curve fit) function of GraphPad Prism 8 software.

5. Summary of Results

TABLE-US-00015 Compound EC50 (nM) Zanamivir 39.69 C-Inter-1 0.28 C-Inter-2 0.42 C-Inter-3 0.39 C-Inter-5 0.45 C-Inter-6 0.25 C-Inter-7 0.57 C-Inter-10 0.39

[0366] The test compounds demonstrated excellent in vitro cellular anti-influenza activity, all of which showed nearly 100-fold or more than 100-fold improvement in activity compared to Zanamivir positive molecule. The in vitro cellular anti-influenza activity demonstrates the strong anti-influenza activity of the listed molecules, showing the huge worth of further development.

Example 25: Cytotoxicity Assay of Compounds

1. Experimental Method

[0367] Toxicity of the tested compounds to MDCK cells was quantified by MDCK cell lines.

2. Experimental Material

TABLE-US-00016 Material Origin Batch No. MDCK Cell ATCC CCL-34 96-well plate Corning 3917 Fetal Bovine Serum (FBS) Hyclone SH30406.05 100 Pen-Strep solution Hyclone SV30010 (P/S) DMEM Hyclone SH30243.01 TPCK treated trypsin Sigma T1426 Cell-titer Glo Promega G7571

3. Experimental Procedure

[0368] 1) MDCK cells were digested and diluted to 210.sup.5/mL with DME medium containing 2% FBS and 1% P/S, which was inoculated into 96-well plate at 50 L per well and placed in an incubator for overnight culture. [0369] 2) The tested compounds were diluted to 2 mM that was then triple diluted with a total of 8 dilution ratios. 5 L of diluted compound was added to each well and placed in the incubator for 1 h. [0370] 3) Each well was supplemented with 45 L of DMEM medium containing 2% FBS and 1% P/S, and 4 g/mL TPCK treated trypsin. The cell control without compound was also configured. The cells were incubated in an incubator for 4 days. [0371] 4) 50 L of Cell-titer Glo was added to each well, and the chemiluminescence was detected by a Microplate Reader.

4. Data Analysis: Inhibition Rate of Compounds on Cell Growth

[0372]
Inhibition rate=(average value of cell controlvalue of tested compounds)/average value of cell control

[0373] The calculated inhibition rate was used to calculate the EC50 value by fitting the inhibition curve with the log(inhibitor) vs. responseVariable slope (four parameters) in the Nonlinear regression (curve fit) function of GraphPad Prism 8 software.

5. Summary of Results

TABLE-US-00017 Compound CC50 (M) Zanamivir >100 C-Inter-1 >100 C-Inter-2 >100 C-Inter-3 >100 C-Inter-5 >100 C-Inter-6 >100 C-Inter-7 >100 C-Inter-10 >100

[0374] The tested compounds demonstrated excellent in vitro cellular safety. The cytotoxicity of tested compounds was above 100 M, which is similar to the that of Zanamivir positive molecule. The in vitro cytotoxicity assay demonstrated the excellent safety profile of the listed molecules, showing great value for further development.

Example 26: Expression and Purification of hIgG1-Fc

1. Experimental Material

TABLE-US-00018 Material Producer Batch No. ExpiCHO-S cell Thermo A29127 ExpiFectamine Thermo A29130 CHO Transfection kit OptiPRO SFM Thermo 12309019 reduced-serum medium
2. Expression of hIgG1-Fc [0375] 1) The day before transfection (day 1), the cells were diluted to a final density of 3-4106 viable cells/mL and growed overnight. [0376] 2) On the day of transfection, the cell density should be 5-6106 cells/mL, and cell viability must be >95%. [0377] 3) Transfect 0.8 g of pcDNA3.1 plasmid expressing hIgG1-Fc per mL of cells, 0.8 g plasmids were diluted in 40 L of OptiPRO SFM Medium (4 C.) and mixed gently as 0.8 g of pcDNA3.1 plasmid expressing hIgG1-Fc was transfected in each mL of cells [0378] 4) 3.2 L of ExpiFectamine CHO was diluted with 37 L of OptiPRO SFM medium (4 C.) and mixed gently. [0379] 5) The diluted ExpiFectamine CHO reagent was added to the diluted DNA, which was mixed gently and incubated for 1-5 min at room temperature. [0380] 6) The above complexes were added slowly to each shake flask. [0381] 7) The cells were placed in a shaker at 37 C. with 8% CO2 and the speed of 125 rpm. [0382] 8) After incubating the cells for 20 hours, for per mL of cells, 10 L of ExpiCHO Transfection Enhancer and 400 L of ExpiCHO feed were mixed at this ratio, which was then added to each culture flask. [0383] 9) The cell supernatant was harvested after eight days of incubation.
3. Purification of hIgG1 Fc

1) Collection of Supernatant

[0384] The Fc protein fermentation broth that has been transfected to harvest conditions was transferred to the 50 ml centrifuge tube marked in order and name, and centrifuged at 10000 rpm/min for 5 min. The supernatant after centrifugation was poured into a new 50 ml centrifuge tube marked in name until the same Fc fermentation broth has been centrifuged and the supernatant has been collected.

2) Pretreatment

[0385] The gravity column was treated by immersion in 0.5 M NaOH solution. Pro A packing solution was added to the empty gravity column. When the solution in the gravity column was dripped completely, 0.1M NaOH solution was taken and added to the column. When the solution in the gravity column was dripped completely, pure water was taken and added to the column.

3) Equilibration Sampling

[0386] The column was equilibrated by adding 5-10 column volumes of Pro A Equilibration Buffer to the Pro A column. The supernatant to be purified from the centrifugation process was added to the Pro A column and the flow-through supernatant was collected into a clean 250 ml shake flask. After all the supernatant has passed through the column, 5-10 column volumes of Pro-A Equilibration Buffer was added until all the Equilibration Buffer has flowed through at the bottom.

4) Protein Elution

[0387] The washed column was sealed at the bottom with a clean plug, into which 2-3 column volumes of Pro-A/G elution buffer was added, and the packing material was suspended using a pipette and then kept still for 5 min. For preparation of elution, a new collection tube was prepared, to which a certain proportion of Tris neutralizer was added to prevent protein denaturation. The plug was removed to collect the liquid into the collection tube and the protein concentration was measured. The protein was stored in 80 C. refrigerator.

3. Characterization of hIgG1 Fc

[0388] According to the sequence information of the Fc described herein, the target Fc is obtained according to the expression purification steps of this embodiment.

TABLE-US-00019 Average SEQ Expression Final Final Final Purity Molecular ID Volume Production Concentration Volume Vehicle (SEC_HPLC) Weight Isoelectric NO (L) (mg) (mg/L) (mL) (PBS) (%) (Da) Point 64 5 3919.5 26.13 150.0 PH 7.4 99.66 55266.6 8.93 67 5 2810.8 23.62 119.0 PH 7.4 99.68 55176.4 8.93

Example 27: Synthesis and Characterization of Conjugate 1

1) Synthesis and Characterization of Conjugate 1

##STR00401##

[0389] Step 1: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64) (45.4 mg) was pipetted into a 50 mL test tube. Reaction buffer (PBS, pH 7.4) was added to the test tube to give a final concentration of Fc of 14.97 mg/mL. Then, the active ester (azide-PEG4-C2-PFP ester) was added to give an active ester/Fc ratio of 9.95. The reaction vials were placed in a shaker incubator and reacted for 2 hours at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was run through an Amicon ultra centrifugal filter (10K, 15 mL) to remove excess active ester, and the modified Fc (SEQ ID NO: 64)-Azide was displaced into buffer (PBS, pH 7.4)

[0390] Step 2: Original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64)-Azide was pipetted into a 50 mL tube. The reaction buffer (20 mM histidine, pH 5.5), 20 equivalents (equivalents refer to molar equivalents when not explicitly indicated herein and not inconsistent with the context) of THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (100 mM), 30 equivalents of sodium ascorbate (100 mM), 20 equivalents of copper sulfate (101 mM) and 12 equivalents of C-Inter-1 (20 mg/mL) were added to the test tube to give a final concentration of Fc (SEQ ID NO: 64)-Azide of 11.99 mg/mL. The reaction vials were placed in a shaker incubator and reacted for 30 minutes at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was purified using an Amicon ultra centrifugal filter (10K, 15 mL) to obtain conjugate 1, with a concentration of 16.37 mg/mL as determined by UV, an actual weight of 24.5 mg, a DAR value of 4.34 as determined by LC-MS, a monomer content of 99.25% as determined by SEC-HPLC, a residual free drug content of less than 2%, and a endotoxin content of less than 0.098 EU/mg.

2) Characterization Method

Amicon Ultra Centrifugal Filter Purification Method

[0391] Amicon Ultra centrifugal filter was used according to the following steps: [0392] 1) Wash the Amicon tube with sodium hydroxide solution (0.5 M). Allow the tubes to stand for 15 minutes. [0393] 2) Rinse the tubes three times with double-distilled water followed by addition of formulated buffer (20 mM histidine, pH 5.5). [0394] 3) Centrifuge and discard the filtered liquid. [0395] 4) Add the sample to the centrifuge tube, then centrifuge and discard the filtered liquid which were repeated 5 times. [0396] 5) Transfer the remaining solution in the Amicon tube to a new collection tube.

UF/DF System Purification Method

[0397] UF/DF system purification method was performed according to the following steps: [0398] 1) Clean the system with sodium hydroxide solution (0.2 M). Allow the sodium hydroxide solution to flow for 5 minutes. Repeat this step once. [0399] 2) Rinse the collector twice with double-distilled water. [0400] 3) Rinse the collector twice with the prepared buffer until the pH of the filtrate is consistent with that of the buffer. [0401] 4) Add the sample to the collector and replace the buffer by at least 10 times the permeate volume. [0402] 5) Collect the solution in the collector.

Concentration Determination of Conjugates

[0403] The concentration of the sample and final conjugate during the process were determined by the Nanodrop spectrophotometer in UV-Vis mode. [0404] 1) The baseline is configured at 750 nM. [0405] 2) Calculations based on Beer-Lambert law

[00001] $\begin{matrix} A = E * c * 1 A_{280} = E_{280}^{Fc} * [mAb] * 1 + E_{280}^{LD} * [LD] * 1 \\ A_{252} = E_{252}^{Fc} * [mAb] * 1 + E_{252}^{LD} * [LC] * 1 \end{matrix}$ [0406] E: molar absorption coefficient; [0407] c: molar concentration; [0408] l: Light path (Nanodrop: 0.1 cm)

Polymerization Determined by SEC-HPLC

[0409] Volume exclusion chromatography was performed at 25 C. using an Agilent 1260 series HPLC system and a TSK gel G3000SWXL volume exclusion column (7.8300 mm, 5 m). The mobile phase is consisted of 78 mM KH2PO4, 122 mM K2HPO4, 250 mM KCl and 15% IPA, at a pH of 7.00.1. The flow rate was set at 0.75 mL/min. Each sample volume was 40-50 g. The samples were detected at 280 nm with a UV detector. The retention time of the aggregation peak was recorded based on its relative molecular weight. The aggregation level was determined by the relative area of the peaks.

DAR Value Determined by LC-MS

[0410] LC-MS was run at 25 C. using an Agilent 6224 series HPLC system, TOF mass spectrometer and an Agilent PLRP-S 1000A column (8 m, 502.1 mm). A double-distilled water solution of 0.05% trifluoroacetic acid was used as mobile phase A, and acetonitrile with 0.05% trifluoroacetic acid was used as mobile phase B. The flow rate was set at 0.5-0.4 mL/min. The sample volume was 2 g. DAR values were calculated based on peak abundance of deconvolution quality.

LC-MS Method for Determining DAR Value:

TABLE-US-00020 Gas Temp. 350 C. Drying Gas 13 L/min Nebulizer 45 psig VCap 5000 V Fragmentor 170 V Mass Range 300-8000 m/z Acquisition Rate 3 spectra/s Equipment Agilent Technologies 6224 TOF LC/MS Column Agilent PLRP-S 1000A, 8 m, 50 2.1 mm Column Temp. 80 C. Mobile phase Phase A: 0.05% TFA in H.sub.2O; Phase B: 0.05% in Acetonitrile Flow rate 0.4 mL/min Injection amount 2 g Detection wavelength 280 nm, 214 nm, 650 nm Flow Gradient Time A (%) B (%) (mL/min) 0.00 75 25 0.500 0.70 66 34 0.400 5.00 55 45 0.400 6.00 10 90 0.400 7.00 10 90 0.400 7.10 75 25 0.400 10.00 75 25 0.400

Residual Free Drugs Determined by LC-MS

[0411] Residual free drug levels (mol/mol, free drug/bound drug) were determined by LC-MS. Two standards (2% & 50%) respectively containing free drug and Fc were prepared. The percentage of free drug was determined by comparing the EIC peak area of the residual free drug in the conjugate sample with that of standards.

[0412] Two standards were prepared according to the following steps: [0413] C.sub.Fc (1 mg/mL), DAR, MW.sub.Fc, MW.sub.Drug were known.

[00002] $\begin{matrix} M_{Fc} = 1 / {MW}_{Fc} \\ M_{Total drug} DAR / {MW}_{Fc} \\ a) Moles of 2 % standards DAR * M_{Fc} * 2 % \\ b) Moles of 5 % standards DAR * M_{Fc} * 5 % \end{matrix}$ [0414] C=Concentration as determined by spectrophotometer (mg/mL) [0415] DAR=Molar ratio of drug to antibody as determined by LC-MS [0416] MW=molar weight

[0417] Taking 20% standard of the conjugate 5 as an example.

[0418] C.sub.Fc: 1 mg/mL; DAR: 4.13; MW.sub.mAb: 58160 g/mol; MW.sub.Drug: 1150.2 g/mol

[00003] $\begin{matrix} M_{Fc} = 1 / 58160 mol / L \\ M_{Total drug} 1 / 58160 * DAR mol / L = 4.13 / 58160 mol / L \\ M_{2 % standard drug} = 2 % * 4.13 / 58160 mol / L = 1.42 u mol / L \end{matrix}$

[0419] LP amount of 100 uL 2% standard: 1.42 umol/L*10.sup.4 L*1150.2 g/mol=0.1633 ug C-Inter-7

[0420] The 200 standard was prepared by dissolving 0.1633 ug C-Inter-7 in 100 L of 1 mg/mL Fc (SEQ ID NO: 64).

LC-MS Method for Determination of Residual Free Drug:

TABLE-US-00021 Gas Temp. 250 C. Drying Gas 13 L/min Nebulizer 45 psig VCap 3500 V Fragmentor 250 V Mass Range 150-7000 m/z Acquisition Rate 1 spectra/s Equipment Agilent Technologies 6224 TOF LC/MS Column Agilent PLRP-S 1000A, 8 m, 50 2.1 mm Column Temp. 80 C. Mobile phase Phase A: 0.05% TFA in H.sub.2O; Phase B: 0.05% in Acetonitrile Flow rate 0.4 mL/min Injection amount 2 g Detection 280 nm, 214 nm, 650 nm wavelength Flow Gradient Time A (%) B (%) (mL/min) 0.00 95 25 0.400 0.50 95 34 0.400 2.00 70 45 0.400 8.00 20 90 0.400 8.50 10 90 0.400 10.00 10 25 0.400 10.01 95 25 0.400 13.00 95 25 0.400

Endotoxin Determination

[0421] The endotoxin levels of samples were determined by Endosafe-PTS (Charles River, MCS150K) endotoxin detector. A 25 L sample was pipetted into each of the four reservoirs of the PTS detector. In addition to the LAL reagent plus the positive reference control, the reader extracts the sample in the sample channel and mixes the sample with the LAL reagent. The samples combined with the colorimetric substrate and then were incubated. After mixing, the optical density of the wells is measured and analyzed based on an internally archived standard curve.

[0422] The endotoxin data for conjugates of embodiments in present application are shown in the following table.

TABLE-US-00022 Endo UV Conc. Endo Sample (EU/mL) (mg/mL) (EU/mg) Conjugate 1 <1.60 16.37 <0.098 Conjugate 2 6.18 18.6 0.332 Conjugate 3 <1.28 8.56 <0.150 Conjugate 4 <1.5 21.69 <0.069 Conjugate 5 4.05 21.74 0.186 Conjugate 6 <1.8 19.67 <0.092 Conjugate 7 <1.1 15.53 <0.071 Conjugate 8 1.2 13.59 0.088 Conjugate 9 <1.95 16.77 <0.116

Example 28: Synthesis and Characterization of Conjugate 2

##STR00402##

[0423] Step 1: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64) (140.0 mg) was pipetted into a 50 mL test tube. Reaction buffer (PBS, pH 7.4) was added to the test tube to give a final concentration of Fc of 14.97 mg/mL. Then, the active ester (azide-PEG4-C2-PFP ester) was added to give an active ester/Fc ratio of 9.4. The reaction vials were placed in a shaker incubator and reacted for 2 hours at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was run through an Amicon ultra centrifugal filter (10K, 15 mL) to remove excess active ester, and the modified Fc (SEQ ID NO: 64)-Azide was displaced into buffer (PBS, pH 7.4)

[0424] Step 2: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64)-Azide was pipetted into a 50 mL tube. Reaction buffer (20 mM histidine, pH 5.5), 20 equivalents of THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (100 mM), 30 equivalents of sodium ascorbate (100 mM), 20 equivalents of copper sulfate (101 mM), and 12 equivalents of C-Inter-2 (20 mg/mL) were added to the test tube to make the Fc (SEQ ID NO: 64)-Azide to a final concentration of 11.99 mg/mL. The reaction vials were placed in a shaker incubator and reacted for 30 minutes at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was purified by Amicon ultra centrifugal filter (10K, 15 mL) to obtain the conjugate 2.

[0425] Referring to the assay and analysis method of Example 27, the following data was measured for conjugate 2: [0426] concentration of 18.6 mg/mL measured by UV, actual weight of 37.2 mg, DAR value of 4.04 determined by LC-MS, monomer content of 99.07% determined by SEC-HPLC, residual free drug content of less than 2%, and endotoxin content of less than 0.098 EU/mg.

Example 29: Synthesis and Characterization of Conjugate 3

##STR00403##

[0427] Step 1: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64) (46.5 mg) was pipetted into a 50 mL test tube. Reaction buffer (PBS, pH 7.4) was added to the test tube to give a final concentration of Fc of 14.97 mg/mL. Then the active ester (azide-PEG4-C2-PFP ester) was added to give an active ester/Fc ratio of 5.4/6.2. The reaction vials were placed in a shaking incubator and reacted for 2 hrs at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was run through an Amicon ultra centrifugal filter (10K, 15 mL) to remove excess active ester, and the modified Fc (SEQ ID NO: 64)-Azide was displaced into buffer (PBS, pH 7.4).

[0428] Step 2: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64)-Azide was pipetted into a 50 mL tube. Reaction buffer (20 mM histidine, pH 5.5), 20 equivalents of THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (100 mM), 30 equivalents of sodium ascorbate (100 mM), 20 equivalents of copper sulfate (101 mM), and 12 equivalents of C-Inter-3 (20 mg/mL) were added to the test tube to make the Fc (SEQ ID NO: 64)-Azide to a final concentration of 11.99 mg/mL. the reaction vials were placed in a shaker incubator and reacted for 30 minutes at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was purified using an Amicon ultra centrifugal filter (10K, 15 mL) to obtain the conjugate 3.

[0429] Referring to the assay and analysis method of Example 27, the following data was measured for conjugate 3: [0430] concentration of 8.56 mg/mL measured by UV, actual weight of 30.7 mg, DAR value of 4.31 determined by LC-MS, monomer content of 99.10% determined by SEC-HPLC, residual free drug content of less than 5%, and endotoxin content of less than 0.150 EU/mg.

Example 30: Synthesis and Characterization of Conjugate 4

##STR00404##

[0431] Step 1: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64) (234.2 mg) was pipetted into a 50 mL test tube. Reaction buffer (PBS, pH 7.4) was added to the test tube to give a final concentration of Fc of 14.97 mg/mL. Then, the active ester (azide-PEG4-C2-PFP ester) was added to give an active ester/Fc ratio of 5.7. The reaction vials were placed in a shaker incubator and reacted for 2 hrs at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was run through an Amicon ultra centrifugal filter (10K, 15 mL) to remove excess active ester, and the modified Fc (SEQ ID NO: 64)-Azide was displaced into buffer (PBS, pH 7.4).

[0432] Step 2: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64)-Azide was pipetted into a 50 mL tube. Reaction buffer (20 mM histidine, pH 5.5), 20 equivalents of THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (100 mM), 30 equivalents of sodium ascorbate (100 mM), 20 equivalents of copper sulfate (101 mM), and 12 equivalents of C-Inter-6 (20 mg/mL) were added to the test tubes to make Fc (SEQ ID NO: 64)-Azide to a final concentration of 11.99 mg/mL. The reaction vials were placed in a shaker incubator and reacted for 30 minutes at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was purified by Amicon ultra centrifugal filter (10K, 15 mL) to obtain conjugate 4.

[0433] Referring to the assay and analysis method of Example 27, the following data was measured for conjugate 4: [0434] concentration of 21.69 mg/mL measured by UV, actual weight of 39.0 mg, DAR value of 4.24 determined by LC-MS, monomer content of 99.32% determined by SEC-HPLC, residual free drug content of less than 2%, and endotoxin content of less than 0.069 EU/mg.

Example 31: Synthesis and Characterization of Conjugate 5

##STR00405##

[0435] The original buffer of Fc (SEQ ID NO: 64)-Azide was obtained by referring to step 1 of conjugate synthesis in Example 27.

[0436] The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64)-Azide was pipetted into a 50 mL test tube. Reaction buffer (20 mM histidine, pH 5.5), 20 equivalents of THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (100 mM), 30 equivalents of sodium ascorbate (100 mM), 20 equivalents of copper sulfate (101 mM), and 12 equivalents of C-Inter-7 (20 mg/mL) were added to the test tube to make the Fc (SEQ ID NO: 64)-Azide to a final concentration of 11.99 mg/mL. The reaction vials were placed in a shaker incubator and reacted for 30 minutes at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was purified by Amicon ultra centrifugal filter (10K, 15 mL) to obtain conjugate 5.

[0437] Referring to the assay and analysis method of Example 27, the following data was measured for conjugate 5: [0438] concentration of 21.74 mg/mL measured by UV, actual weight of 47.8 mg, DAR value of 4.16 determined by LC-MS, monomer content of 99.24% determined by SEC-HPLC, residual free drug content of less than 2%, and endotoxin content of 0.186 EU/mg EU/mg.

Example 32: Synthesis and Characterization of Conjugate 6

##STR00406##

[0439] The original buffer of Fc (SEQ ID NO: 64)-Azide was obtained by referring to step 1 of conjugate synthesis in Example 27.

[0440] The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 64)-Azide was pipetted into a 50 mL tube. Reaction buffer (20 mM histidine, pH 5.5), 20 equivalents of THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (100 mM), 30 equivalents of sodium ascorbate (100 mM), 20 equivalents of copper sulfate (101 mM), and 12 equivalents of C-Inter-10 (20 mg/mL) were added to the test tubes to make Fc (SEQ ID NO: 64)-Azide to a final concentration of 11.99 mg/mL. The reaction vials were placed in a shaker incubator and reacted for 30 minutes at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was purified using an Amicon ultra centrifugal filter (10K, 15 mL) to obtain the conjugate 6.

[0441] Referring to the assay and analysis method of Example 27, the following data was measured for conjugate 6: [0442] concentration of 19.67 mg/mL measured by UV, actual weight of 39.3 mg, DAR value of 4.21 determined by LC-MS, monomer content of 99.31% determined by SEC-HPLC, residual free drug content of less than 2%, and endotoxin content of less than 0.092 EU/mg.

Example 33: Synthesis and Characterization of Conjugate 7

##STR00407##

[0443] Step 1: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 67) (378.9 mg) was pipetted into a 50 mL test tube. Reaction buffer (PBS, pH 7.4) was added to the test tube to make the final concentration of mAb 14.97 mg/mL. Then, the active ester (azide-PEG4-C2-PFP ester) was added to make the ratio of active ester/Fc 8.8. The reaction vials were placed in a shaker incubator and reacted for 2 hrs at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was run through an Amicon ultra centrifugal filter (10K, 15 mL) to remove excess active ester, and the modified Fc (SEQ ID NO: 67)-Azide was displaced into buffer (PBS, pH 7.4).

[0444] Step 2: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 67)-Azide was pipetted into a 50 mL tube. Reaction buffer (20 mM histidine, pH 5.5), 20 equivalents of THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (100 mM), 30 equivalents of sodium ascorbate (100 mM), 20 equivalents of copper sulfate (101 mM), and 18 equivalents of C-Inter-1 (20 mg/mL) were added to the test tubes to make Fc (SEQ ID NO: 67)-Azide to a final concentration of 11.99 mg/mL. The reaction vials were placed in a shaker incubator and reacted for 30 minutes at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was purified using Amicon ultra centrifugal filter (10K, 15 mL) to obtain conjugate 7.

[0445] Referring to the assay and analysis method of Example 27, the following data was measured for conjugate 7: [0446] concentration of 15.53 mg/mL measured by UV, actual weight of 74.0 mg, DAR value of 5.79 determined by LC-MS, monomer content of 98.77% determined by SEC-HPLC, residual free drug content of less than 2%, and endotoxin content of less than 0.071 EU/mg.

Example 34: Synthesis and Characterization of Conjugate 8

##STR00408##

[0447] The original buffer of Fc (SEQ ID NO: 67)-Azide was obtained by referring to step 1 of conjugate synthesis in Example 33.

[0448] The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 67)-Azide was pipetted into a 50 mL tube. Reaction buffer (20 mM histidine, pH 5.5), 20 equivalents of THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (100 mM), 30 equivalents of sodium ascorbate (100 mM), 20 equivalents of copper sulfate (101 mM), and 18 equivalents of C-Inter-10 (20 mg/mL) were added to the test tubes to make Fc (SEQ ID NO: 67)-Azide to a final concentration of 11.99 mg/mL. The reaction vials were placed in a shaker incubator and reacted for 30 minutes at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was purified using an Amicon ultra centrifugal filter (10K, 15 mL) to obtain the conjugate 8.

[0449] Referring to the assay and analysis method of Example 27, the following data was measured for conjugate 8: [0450] concentration of 13.59 mg/mL measured by UV, actual weight of 54.0 mg, DAR value of 5.92 determined by LC-MS, monomer content of 98.88% determined by SEC-HPLC, residual free drug content of less than 2%, and endotoxin content of less than 0.088 EU/mg.

Example 35: Synthesis and Characterization of Conjugate 9

##STR00409##

[0451] Step 1: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 67) (3240 mg) was pipetted into a 150 mL reaction vial. Reaction buffer (PBS, pH 7.4) was added to the reaction vial to give a final concentration of Fc of 14.97 mg/mL. Then, the active ester (azide-PEG4-C2-PFP ester) was added to give an active ester/Fc ratio of 8.0/8.2. The reaction vial was placed in a shaker incubator and reacted for 2 hrs at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was filtered through a UF/DF membrane filtration system to remove excess active ester, and the modified Fc (SEQ ID NO: 67)-Azide was displaced into buffer (PBS, pH 7.4).

[0452] Step 2: The original buffer (PBS, pH 7.4) containing Fc (SEQ ID NO: 67)-Azide was pipetted into a 150 mL tube. Reaction buffer (20 mM histidine, pH 5.5), 20 equivalents of THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (100 mM), 30 equivalents of sodium ascorbate (100 mM), 20 equivalents of copper sulfate (101 mM), and 18 equivalents of C-Inter-7 (20 mg/mL) were added to the test tubes to make Fc (SEQ ID NO: 67)-Azide to a final concentration of 11.99 mg/mL. The reaction vials were placed in a shaker incubator and reacted for 30 minutes at 22 C. and 60 revolutions per minute. At the end of the reaction, the reaction solution was purified by UF/DF membrane filtration system to obtain conjugate 9.

[0453] Referring to the assay and analysis method of Example 27, the following data was measured for conjugate 9: [0454] concentration of 16.77 mg/mL measured by UV, actual weight of 3220 mg, DAR value of 5.86 determined by LC-MS, monomer content of 98.75% determined by SEC-HPLC, residual free drug content of less than 2%, and endotoxin content of less than 0.116 EU/mg.

Example 36: In-Vitro Anti-Influenza Virus (Influenza Virus H1N1-Mediated Cytopathic Inhibition Assay) Activity of the Conjugate

1. Experimental Method

[0455] The in vitro cytopathic-based anti-IAV/IBV activity assay was used, which uses the MDCK cell line to quantify the effects of tested conjugates on IAV/IBV-induced cytopathies.

2. Experimental Material

TABLE-US-00023 Material Origin Batch No. H1N1(A/PR/8/34) ATCC VR1469 MDCK cell ATCC CCL-34 96-well plate Corning 3917 Fetal Bovine Serum (FBS) Hyclone SH30406.05 100 Pen-Strep solution (P/S) Hyclone SV30010 DMEM Hyclone SH30243.01 TPCK treated trypsin Sigma T1426 Cell-titer Glo Promega G7571 Zanamivir Adamas P1695633

3. Experimental Procedure

[0456] 1) MDCK cells were digested and diluted to 210.sup.5/mL with DME medium containing 2% FBS and 1% P/S, which was inoculated into 96-well plate at 50 L per well and placed in an incubator for overnight culture [0457] 2) The tested conjugates were diluted to 2 M that was then triple diluted with a total of 8 dilution ratios. 5 L of diluted compound was added to each well and placed in the incubator for 1 h. [0458] 3) Influenza virus was diluted to 2,222 pfu/mL with DMEM medium containing 2% FBS, 1% P/S and 4 g/mL TPCK treated trypsin. 45 L of virus was added to each well. A virus control group without compounds but with viruses and a cell control group without compounds and viruses were also configured. The cells were incubated in an incubator for 4 days. [0459] 4) 50 L of Cell-titer Glo was added to each well, and the chemiluminescence was detected by a Microplate Reader.

4. Data Analysis: Calculate the Viral Inhibition Rate of the Conjugate.

[00004] $Inhibition rate = (value of tested conjugate - average value of virus control) / (average value of cell control - average value of virus control) * 100 %$

[0460] The calculated inhibition rate was used to calculate the EC50 value by fitting the inhibition curve using the log(inhibitor) vs. responseVariable slope (four parameters) in the Nonlinear regression (curve fit) function of GraphPad Prism 8 software.

[0461] Reference molecule A and reference molecule B were respectively prepared according to the specific methods of Example 156 (Conjugate 45b) and Example 188 (Conjugate 46) in patent WO2021046549A1.

[0462] Reference molecule A has a purity (SEC-HPLC) of 99.2% and a DAR of 4.26.

[0463] Reference molecule B has a purity (SEC-HPLC) of 98.8% and a DAR of 5.87.

4. Summary of Results

TABLE-US-00024 Compounds DAR custom-character EC50 (nM) Zanamivir / 45.7 Reference molecule A 4.26 0.6 Conjugate 1 4.34 0.3 Conjugate 2 4.04 0.2 Conjugate 3 4.31 0.4 Conjugate 4 4.24 0.2 Conjugate 5 4.16 0.1 Conjugate 6 4.21 0.2 Antiviral activity data for different Fc and higher conjugating DAR values Reference molecule B 5.87 0.4 Conjugate 7 5.79 0.07 Conjugate 8 5.91 0.2 Conjugate 9 5.86 0.07

[0464] As can be seen from the data, the tested conjugates showed a 76- to 652-fold improvement in antiviral activity compared to Zanamivir, demonstrating the superior in vitro anti-H1N1 viral activity.

[0465] At DAR values around 4.2, all the compounds in present application reflect better antiviral activity with lower EC50 values. Conjugate 5 showed a 6-fold improvement in antiviral activity as compared to reference molecule A.

[0466] At DAR values around 5.8, all the compounds in present application reflect better antiviral activity with lower EC50 values. Conjugates 7 and 9 showed nearly 6-fold improvement in antiviral activity as compared to reference molecule B.

[0467] The higher in vitro antiviral activity indicates better in vivo antiviral activity of conjugates in animals and in clinical humans.

Example 37: In-Vitro Anti-Influenza Virus (H5N1, H7N9, Yamagata and Victoria Influenza Viruses) Activity of the Conjugate

1. Experimental Material

TABLE-US-00025 Material Origin Batch No. A/Chicken/Jiangsu/k0402/2010 Yangzhou / (H5N1) University A/Chicken/Eastern/JTC11/2013 Yangzhou / (H7N9) University B/Jiangsu/YZ08/2018 Yangzhou / (Yamagata) University B/YZ29/2019 (Victoria) Yangzhou / University MDCK cell ATCC Cat: CCL-34 96-well plate Corning Cat: 3917 Fetal Bovine Serum (FBS) Hyclone Cat: SH30406.05 100 Pen-Strep solution (P/S) Hyclone Cat: SV30010 DMEM Hyclone Cat: SH30243.01 TPCK treated trypsin Sigma Cat: T1426 Oseltamivir acid MCE HY-13318

2. Experimental Procedure

[0468] 1) MDCK cells were digested and 2.510.sup.4 MDCK cells was inoculated into 96-well plate with DME medium containing 2% FBS and 1% P/S, which was cultured overnight. [0469] 2) The cell supernatant was discarded and the cells were infected with a dose of 0.0025 MOI of virus and placed in the incubator for 1 hour. [0470] 3) The virus not adsorbed to the cells was discarded, and each compound (the starting concentrations are shown in the table below) was triple diluted with eight gradients, and the diluted compound was added and incubated for 72 hours. The viral control group without compound was configured at the same time.

TABLE-US-00026 Influenza strain Conjugate 5 Conjugate 9 H5N1 30 nM 30 nM H7N9 30 nM 30 nM B/Yamagata 10 M 10 M B/Victoria 10 M 10 M [0471] 4) The effect of viral replication in each well was detected by hemagglutination assay and the viral titer was calculated according to the Reed-Muench method. The EC50 of each compound was calculated by the viral titer. The hemagglutination assay and the determination criteria were performed with reference to the WHO diagnostic criteria for influenza viruses.

3. Data Processing: Calculation of the Virus Inhibition Rate of Compounds

[00005] $1) Inhibition rate = (1 - viral titer of the tested sample group / average value of the viral control group * 100 %)$ [0472] 2) The calculated inhibition rate was used to calculate the EC50 value by fitting the inhibition curve with the log(inhibitor) vs. responseVariable slope (four parameters) in the Nonlinear regression (curve fit) function of GraphPad Prism 8 software.

4. Summary of Results

TABLE-US-00027 Oseltamivir Conjugate Conjugate Influenza strain acid Zanamivir 5 9 EC.sub.50 (nM) A/Chicken/Jiangsu/ 210.0 untested 1.1 0.7 k0402/2010 (H5N1) A/Chicken/Eastern/ 540.0 untested 0.8 0.4 JTC11/2013 (H7N9) B/Jiangsu/YZ08/2018 780.0 90.0 18.9 12.3 (Yamagata) B/YZ29/2019 710.0 59.0 58.9 10.7 (Victoria)

[0473] From the results, it can be seen that conjugate 5 and conjugate 9 each show a higher activity than the positive reference molecules in the tested influenza strains. It also demonstrated that the tested conjugates are broad-spectrum against influenza virus, which is of great clinical application value.

Example 38: In-Vitro Anti-Influenza Virus (Clinically Resistant and Other Resistant Strains) Activity of the Conjugate

1. Experimental Material

TABLE-US-00028 Material Origin Batch No. A/California/2/2014 ATCC VR-1938 (H3N2) IFV B/Lee/40 ATCC VR-1535 Oseltamivir-resistant Constructed by / A/Weiss/43 (H1N1) Wuxi New Drug Development Co., Ltd. Shanghai VX-787-resistant Constructed by / A/PR/8/34 (H1N1) Wuxi New Drug Development Co., Ltd. Shanghai Baloxavir-resistant Constructed by / A/PR/8/34 (HIN1) Wuxi New Drug Development Co., Ltd. Shanghai MDCK cell ATCC Cat: CCL-34 96-well plate Corning Cat: 3917 Fetal Bovine Serum Hyclone Cat: SH30406.05 (FBS) 100 Pen-Strep solution Hyclone Cat: SV30010 (P/S) DMEM Hyclone Cat: SH30243.01 TPCK treated trypsin Sigma Cat: T1426 Cell-titer Glo Promega Cat: G7571

1. Experimental Procedure

[0474] MDCK cells were inoculated into 96-well cell culture plates at a density of 15,000 cells per well, a volume of 100 L per well and incubated overnight in a 5% CO2, 37 C. incubator. The next day, 50 L of gradient diluted compounds (3-fold serial dilution, 8 concentration points, double replicate wells) and 50 L of virus were added to each well. For virus infection, trypsin at a final concentration of 2.5 g/ml was added to the experimental culture solution. The total volume of culture medium was 200 L per well. Cells were continued to be cultured at 5% CO2, 35 C. or 37 C. for 5 days until the obvious cellular pathology occurred in the virus-infected control wells without compounds. Then, cell viability was measured in each well using CellTiter-Ge, a cell viability assay reagent. If the cell viability of the compound-tested wells is higher than that of the virus-infected control wells, i.e., the CPE is attenuated, indicating the inhibitory effect of compound on the tested virus.

TABLE-US-00029 Starting concentration of test Oseltamivir Conjugate Conjugate Tested virus strain Zanamivir Baloxavir VX-787 acid 5 9 IFV B/Lee/40 10 M 100 nM untested 100 M 100 nM 100 nM IFV A/California/2/ 30 nM 100 nM untested 100 M 30 nM 30 nM 2014 (H3N2) Oseltamivir-resistant 30 nM 100 nM untested 100 M 30 nM 30 nM A/Weiss/43 (H1N1) Baloxavir-resistant 30 nM 1000 nM untested 100 M 30 nM 30 nM A/PR/8/34 (H1N1) VX-787-resistant 30 nM 100 nM 10 M 100 M 30 nM 30 nM A/PR/8/34 (H1N1)

3. Data Processing:

[0475] The viral inhibition rate of the compounds was first calculated.

[00006] $Inhibition rate (%) = (reading of test well - average value of virus - infected control) / (average value of cell control - average value of virus - infected control) 100$

[0476] The calculated inhibition rate was used to calculate the EC50 value by fitting the inhibition curve using the log(inhibitor) vs. responseVariable slope (four parameters) in the Nonlinear regression (curve fit) function of GraphPad Prism 8 software.

4. Summary of Results

TABLE-US-00030 Influenza strain Oseltamivir acid Zanamivir Baloxavir Conjugate 5 Conjugate 9 EC.sub.50 (nM) IFV B/Lee/40 801.1 57.5 1.47 1.13 0.63 IFV >100,000 >10,000 0.39 0.92 0.66 A/California/2/2014 (H3N2) Oseltamivir-resistant >100,000 24.4 0.69 8.61 8.51 A/Weiss/43 (H1N1) Baloxavir-resistant 2384.0 1391.0 95.0 4.17 2.57 A/PR/8/34 (H1N1) VX-787-resistant 1062.0 166.8 0.58 0.91 0.59 A/PR/8/34 (H1N1)

[0477] From the results, it can be seen that conjugate 5 and conjugate 9 each exhibited a high antiviral activity in the tested influenza strains, with a EC50 value within 10 nM. Meanwhile, it also demonstrated that the tested conjugates are broad-spectrum against influenza virus, especially the maintained high antiviral activity for the clinically resistant strains, indicating the great clinical application value.

Example 39: Pharmacokinetic Study of Conjugate 5 in Mice

1. Experimental Information

[0478] Pharmacokinetic studies were performed using male CD-1 (ICR) mice. The ELISA assay was performed for blood samples, and the specific experimental information and design are shown in the table below.

TABLE-US-00031 Test compounds Conjugate 5 Administration Intravenous Subcutaneous intramuscular route (IV) injection (SC) injection (IM) Dosage 5.0 5.0 5.0 (mg/kg) Vehicle 1 mg/mL 1 mg/mL 2 mg/mL dissolved in dissolved in dissolved in 1 PBS (pH 7.4) 1 PBS (pH 7.4) 1 PBS (pH 7.4) Assay ELISA ELISA ELISA Blood 0, 0.083, 1, 3, 0, 0.25, 1, 3, 0, 0.25, 1, 3, 5, collection 5, 24, 48, 72, 5, 24, 48, 72, 24, 48, 72, 96, point design 96, 168 h 96, 168 h 168 h No. of animals 6 6 6

2. Critical Experimental Material

TABLE-US-00032 Classification Material Batch No. Provider Capture agent Conjugate capture 40017-VNAHC Sino agent biological HRP anti-human-IgG- 151886 Jackson HRP Wash buffer 0.1% PBST WB-20220314-GKS Wuxi App Tec Coating CBS CB-20220314-GKS Wuxi App Buffer Tec Blocking 5% BSA in 0.1% BB-20220316-GKS Wuxi App buffer (BSA) PBST Tec Assay Buffer 1% BSA in 0.1% AB-20220314-SWZ Wuxi App PBST Tec TMB TMB Substrate Kit 10537557 SeraCare Termination 2N H.sub.2SO.sub.4 SS-20220219-YJC Wuxi App solution Tec

3. Detection Steps

[0479] Method description: The conjugate concentration in plasma of CD-1 mouse was determined by ELISA. The lower limit of quantification (LLOQ) of the conjugate was 50.0 ng/mL, while the upper limit of quantification (ULOQ) was 3000 ng/mL. The procedure is described below: [0480] 1) Blood was taken at the designed time points, and the corresponding test samples were prepared with EDTA-K2 as the anticoagulant. [0481] 2) 100 L of coating solution was added to a 96-well microtiter plate that was sealed and incubated overnight at 2-8 C. before use. [0482] 3) The plate was washed 5 times with wash buffer. (300 L/well). [0483] 4) The plate was blocked by adding 300 L of blocking buffer to each well. then sealed and incubated at room temperature for 2 hours without shaking. [0484] 5) Step 2 was repeated. [0485] 6) 100 L of standard solution and assay sample were added to each well. The plate was sealed and incubated at room temperature oscillation of 450 RPM for 2 hours10 minutes. [0486] 7) Step 2 was repeated. [0487] 8) 100 L of HRP solution was added to each well. The plate was sealed and incubated at room temperature oscillation of 450 RPM for 1 hour10 minutes. [0488] 9) Step 2 was repeated. [0489] 10) 100 L of TMB was added to each well. The plate was incubated at room temperature for 5-20 minutes. [0490] 11) 100 L of termination solution was added to each well. [0491] 12) Plates were read within 30 minutes at 450 nm and 630 nm using a SpectraMax M5e/M5/Plus 384 microplate reader. [0492] 13) The reading of the standard solution was converted to the corresponding concentration of the conjugate. [0493] 14) Data processing is performed using Watson LIMS V7.6 or SoftMax Pro GxP 7.0.3 and Microsoft Excel 2007 or higher.

4. Summary of Results

TABLE-US-00033 PK Parameters of Conjugate 5 IV SC IM PK Parameters Mean plasma Mean plasma Mean plasma C.sub.0 (ng/ml) 122965 C.sub.max (ng/ml) 26633 32233 T.sub.max (h) 48.0 24.0 T.sub.1/2 (h) 181 155 109 Cl(mL/min/kg) 0.00889 T.sub.last (h) 169 168 168 AUC.sub.0-last (ng .Math. h/mL) 4572892 3381394 3836941 AUC.sub.0-inf (ng .Math. h/mL) 9378125 6629459 6044639 MRT.sub.0-last (h) 68.4 82.9 75.3 MRT.sub.0-inf (h) 254 234 167 AUC.sub.Extra (%) 51.2 49.0 36.5 AUMC.sub.Extra (%) 86.9 81.9 71.3 Bioavailability (%) 73.9 83.9

[0494] Pharmacokinetic data from mice showed that conjugate 5 has a long half-life and all three routes of administration reached the half-life of more than 100 hours. Meanwhile, all three routes of administration had excellent in vivo exposure. The above data suggests a long half-life and high exposure in the clinic for conjugate 5, enabling the excellent antiviral activity.

Example 40: Pharmacokinetic Study of Conjugate 9 in Rat

[0495] Pharmacokinetic studies of the conjugate were performed using male SD rats. The ELISA assay was performed for blood samples, and the specific experimental information and design are shown in the table below.

TABLE-US-00034 Test compounds Conjugate 9 Administration route Intravenous (IV) Dosage (mg/kg) 15 50 Vehicle 3 mg/mL sample 10 mg/mL sample dissolved in 1 volume dissolved in 1 volume of PBS (pH 7.4) of PBS (pH 7.4) Assay ELISA ELISA Blood collection 0, 0.083, 0.25, 0.5, 1, 4, 8, 24, 72, 120, 168, 336 h.sub. point design No. of animals 6 6

2. Critical Experimental Material

TABLE-US-00035 Classification Material Batch No. Provider Capture agent Conjugate capture 40017-VNAHC Sino agent biological HRP anti-human-IgG-HRP 151886 Jackson Titration plate 96-well micro titration 468667 Thermo plate Buffer Phosphate Buffer PBS-0061 MXB Powder Biotechnologies Reagent Bovine serum albumin BSAS1.0 BovoGen (BSA)

[0496] Materials for which detailed information is not provided can be referred to Example 39.

3. Detection Steps

[0497] 1) Blood was taken at the designed time points, and the corresponding test samples were prepared with EDTA-K2 as the anticoagulant. [0498] 2) 100 L of coating solution was added to a 96-well microtiter plate that was sealed and incubated overnight at 2-8 C. before use. [0499] 3) The plate was washed 3 times with wash buffer. (300 L/well) [0500] 4) The plate was blocked by adding 300 L of blocking buffer to each well. then sealed and incubated at room temperature for 2 hours without shaking. [0501] 5) Step 2 was repeated. [0502] 6) 100 L of standard solution/QC and assay sample were added to each well. The plate was sealed and incubated at room temperature oscillation of 450 RPM for 1 hour10 minutes. [0503] 7) Step 2 was repeated. [0504] 8) 100 L of HRP solution was added to each well. The plate was sealed and incubated at room temperature oscillation of 450 RPM for 1 hour10 minutes. [0505] 9) Step 2 was repeated. [0506] 10) 100 L of TMB was added to each well. The plate was incubated at room temperature for 5-20 minutes. [0507] 11) 100 L of 2N H.sub.2SO.sub.4 was added to each well. [0508] 12) Plates were read within 30 minutes at 450 nm and 630 nm using a SpectraMax M5e/M5/Plus 384 microplate reader. [0509] 13) The reading of the standard solution was converted to the corresponding concentration of the conjugate [0510] 14) Data processing is performed using Watson LIMS V7.6 or SoftMax Pro GxP 7.0.3 and Microsoft Excel 2007 or higher.

4. Summary of Results

TABLE-US-00036 PK Parameters of Conjugate 9 IV (15 mg/kg) IV (50 mg/kg) PK Parameters Mean plasma Mean plasma C.sub.0 (ng/mL) 277333 1295190 T.sub.1/2 (h) 177 183 V.sub.dss (L/kg) 0.161 0.138 Cl(mL/min/kg) 0.0108 0.00930 T.sub.last (h) 336 336 AUC.sub.0-last (ng .Math. h/mL) 16995540 65853090 AUC.sub.0-inf (ng .Math. h/mL) 23076300 89619410 MRT.sub.0-last (h) 125 120 MRT.sub.0-inf (h) 248 247 AUC.sub.Extra (%) 26.4 26.5 AUMC.sub.Extra (%) 63.0 64.4

[0511] Pharmacokinetic data from rat showed that conjugate 9 has a long half-life and the IV administration achieved the half-life of more than 150 hours under both dosages. Meanwhile, the exposure increased in a dose-dependent manner at the two dosages. The above data suggests a long half-life and high exposure in the clinic for conjugate 9, enabling the excellent antiviral activity

Example 41. Serum Half-life in Mice (t.SUB.1/2.)

[0512] Pharmacokinetic (PK) studies were performed using CD-1 mice (Charles River Laboratories), weighing between 20-22 g. Each mouse was injected with 50 mg/kg of the compound to be tested (10 ml/kg dose volume) intravenously in the tail. All animals were under IACUC standard experimental conditions. At various time after administration, the mice were executed and blood was taken (EDTA anticoagulated tubes). Plasma was taken after centrifugation (2000g, 10 min) of whole blood to analyze the concentration of the compound to be tested.

[0513] Plasma concentrations of the compound to be tested were measured by sandwich ELISA: ELISA plates were coated with viral surface proteins (influenza neuraminidase, respiratory syncytial virus F protein, and HIV surface glycoprotein gp120/gp41) targeted by the AVC compound, and were sealed in 2% bovine serum PBS for 1 hr at room temperature, and then added with the plasma samples diluted in serial. The horseradish peroxidase-labeled secondary antibody against human IgG-Fc is used for detection, and TMB substrate was added for color development for 20 min. Then, the same volume of reaction termination solution was added, and the absorbance value at OD450 nm was read. The concentration of the compound to be tested was calculated by fitting an S-curve drawn using a 4-parameter equation.

Example 42: Mouse Prophylactic Model for Lethal Dose Influenza Virus

[0514] H1N1 Model: Female BALB/c mice aged 6-8 weeks were infected using a lethal dose of influenza virus strain H1N1 A/Texas/36/91. On day 0, inoculation with nasal drops was performed at a dose of 1LD.sub.90. Intravenous administration was performed 4 hours prior to inoculation. Oral Oseltamivir administration was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. On the fourth day after infection, lung tissues from selected mice were taken, homogenized, and tested for virus titers in lung. Body weight change and survival of mice were monitored daily for 15 days after infection.

[0515] H3N2 Model: Female BALB/c mice aged 6-8 weeks were infected using a lethal dose of influenza virus strain H3N2 A/HongKong/1/68. On day 0, inoculation with nasal drops was performed at a dose of 1LD.sub.90. Intravenous administration was performed 4 hours prior to inoculation. Oral Oseltamivir administration was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. On the fourth day after infection, lung tissues from selected mice were taken, homogenized, and tested for virus titers in lung. Body weight change and survival of mice were monitored daily for 15 days after infection.

[0516] B Model: Female BALB/c mice aged 6-8 weeks were infected using a lethal dose of influenza virus strain B/Malaysia/2506/04. On day 0, inoculation with nasal drops was performed at a dose of 1LD.sub.90. Intravenous administration was performed 4 hours prior to inoculation. Oral Oseltamivir administration was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. On the fourth day after infection, lung tissues from selected mice were taken, homogenized, and tested for virus titers in lung. Body weight change and survival of mice were monitored daily for 15 days after infection.

Example 43: Mouse Therapeutic Model for Lethal Dose Influenza Virus

[0517] H1N1 Model: Female BALB/c mice aged 6-8 weeks were infected using a lethal dose of influenza virus strain H1N1 A/Texas/36/91. On day 0, inoculation with nasal drops was performed at a dose of 1LD.sub.90. Intravenous administration was performed 4 hours prior to inoculation. Oral Oseltamivir administration was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. On the fourth day after infection, lung tissues from selected mice were taken, homogenized, and tested for virus titers in lung. Body weight change and survival of mice were monitored daily for 15 days after infection.

[0518] H3N2 Model: Female BALB/c mice aged 6-8 weeks were infected using a lethal dose of influenza virus strain H3N2 A/HongKong/1/68. On day 0, inoculation with nasal drops was performed at a dose of 1LD.sub.90. Intravenous administration was performed 4 hours prior to inoculation. Oral Oseltamivir administration was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. On the fourth day after infection, lung tissues from selected mice were taken, homogenized, and tested for virus titers in lung. Body weight change and survival of mice were monitored daily for 15 days after infection.

[0519] B Model: Female BALB/c mice aged 6-8 weeks were infected using a lethal dose of influenza virus strain B/Malaysia/2506/04. On day 0, inoculation with nasal drops was performed at a dose of 1LD.sub.90. Intravenous administration was performed 4 hours prior to inoculation. Oral Oseltamivir administration was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. On the fourth day after infection, lung tissues from selected mice were taken, homogenized, and tested for virus titers in lung. Body weight change and survival of mice were monitored daily for 15 days after infection.

Example 44: Activity of Compounds to be Tested in Fatal Influenza Virus Infected Immunodeficient Mouse Model

[0520] Immunodeficient (SCID) mice aged 6-8 weeks were infected using a lethal dose of influenza virus strain A/Puerto Rico/08/1934. On day 0, inoculation with nasal drops was performed at a dose of 3LD.sub.95. Intravenous administration (a dosage of 0.3, 1.0, 3.0 mg/kg) was performed prior to inoculation. Oral Oseltamivir administration was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. On the fourth day after infection, lung tissues from selected mice were taken, homogenized, and tested for virus titers in lung. Body weight change and survival of mice were monitored daily for 35 days after infection.

Example 45. In-Vivo Toxicity of the Compound to be Tested

[0521] 14-day rat dose-ranging study was performed. On days 0 and 7, rats were injected intravenously with 5 mpk, 20 mpk, or 50 mpk of the compound to be tested. Changes in body weight, organ weights, and food intake for rats were monitored and compared to that of the control group. Blood exposure of the compound to be tested is measured by sandwich ELISA as described in Example 39 above.

Example 46. In-Vivo Efficacy of the Compound to be Tested Against the Oseltamivir-Resistant Influenza Virus Strain

[0522] Influenza strain H1N1/A/Perth/261/2009 passaged by mouse is oseltamivir resistant and contains the H275Y mutation. At day 0, inoculation with nasal drops was performed at a dose of 1LD.sub.90. Intravenous administration was performed 4 hours prior to inoculation. Oral Oseltamivir administration was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. Body weight change and survival of mice were monitored daily for 15 days after infection.

Example 47: Fatal Influenza Virus Infected Mouse Model with Delayed Treatment Time

[0523] The compounds to be tested were injected into mice via intravenous administration at various time after infection (2 hrs before infection, 2 hrs after infection, 24 hrs, 48 hrs, 72 hrs, 96 hrs after infection). The time of infection was marked as day 0. All mice were inoculated with 2LD.sub.95 of H1N1 A/Texas/36/91 by nasal drip. Oral Oseltamivir was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. Body weight change and survival of mice were monitored daily for 15 days after infection.

Example 48: Combined Efficacy of Compounds and Other Antiviral Drugs

[0524] Influenza virus: the compound to be tested is diluted in a 10-fold series at a concentration range of 0.001-100 nM and another anti-influenza virus compound is a known clinical or approved drug such as baloxivir, pimodivir, oseltamivir, zanamivir, peramivir, laninamivir, amantadine, MEDI8852 or rimantadine, which were cross-mixed in the concentration range of 1-1000 nM and then added to MDCK cells overnight. The Influenza virus H1N1/A/PR8/34 with an infection index of 0.001-1 was added at the next day. After 4 days of infection, the cells were stained with crystal violet dye and the absorbance at 595 nm was read. The experimental data of drug synergistic effect was analyzed by MacSynergy.

Example 49: 28-day Rat Pharmacokinetic Study in Intravenous or Subcutaneous Administration

[0525] Rats are administered 5 mg/kg of the compound to be tested intravenously or subcutaneously (5 ml/kg dose volume). Blood (EDTA anticoagulated tubes) is taken at various time points (24 hours, 3 days, 7 days, 14 days, 28 days) after administration. After centrifugation (2000g, 10 min) of the whole blood, plasma was taken to analyze the concentration of the compound to be tested. Blood exposure of the compound to be tested was assayed using the ELISA as described in Example 39 above.

Example 50: 28-Day Non-Human Primate Pharmacokinetic Study (Intravenous Administration)

[0526] Macaca fascicularis aged 4.5-8 years weighing 2.5-6.5 kg were administered 5 mg/kg or 20 mg/kg of the compound to be tested intravenously (5 ml/kg dose volume). All animals were subjected to IACUC standard experimental conditions. Blood (EDTA anticoagulated tubes) was taken at different time points (24 h, 3 days, 7 days, 14 days, 28 days) after administration. After centrifugation (2000g, 10 min) of whole blood, plasma was taken to analyze the concentration of the compound to be measured. Blood exposure of the compound to be tested was assayed using the ELISA as described in Example 39 above.

Example 51: Pharmacokinetic Study on the Distribution of Lung Tissue in Mice (Intravenous Administration)

[0527] The CD-1 mice aged 6 weeks were intravenously injected with 10 mg/kg (5 ml/kg dose volume) via tail. All animals were under standard IACUC experimental conditions. At various time after administration, mice were euthanized, and blood (EDTA anticoagulated tubes) and lungs were taken. After centrifugation (2000g, 10 min) of whole blood, plasma was taken to analyze the concentration of the compound to be measured. Lungs were placed in a centrifuge tube, weighed, homogenized to 100 L that was adjusted to 1 mL, placed on ice for 20 minutes and mixed. The homogenate was centrifuged (8000g for 10 minutes) and the supernatant was taken to analyze the concentration of the compound to be measured. The ELISA assay as described in Example 39 above was performed.

Example 52: Single-Dose In Vivo Toxicity Study in Rats

[0528] Sprague-Dawley rats were subcutaneously administered at a dose of 100 mg/kg, 200 mg/kg, or 400 mg/kg, in a 5 ml/kg administration volume. After the drug administration, the health status, feeding, body weight changes, and physiological indicators of the animals were observed for 15 days. On the 15th day, blood was taken to assay the blood biochemical indicators and various histopathological examinations.

Example 53: In Vitro Stability

[0529] Metabolic stability studies in freshly extracted plasma and liver microsomes from mice and human were performed. After co-incubating the compounds to be tested with the plasma or liver microsomes at 37 C. for 24 hours, the changes in DAR of the compounds to be tested were detected by MALDI-TOF mass spectrometry. This method identifies the structural parts of the compounds to be tested that have poor metabolic stability.

Example 54: In Vitro FcR Binding Activity Studies

[0530] Recombinant FcR (I, IIA, IIC, III) protein (1 g/mL) was encapsulated in an ELISA plate overnight and was blocked with 2% bovine serum PBS for 1 hour at room temperature on the next day. The compounds to be tested (0.01-1000 nM) that have been diluted in serial were added and incubated for 1 hour at room temperature. The horseradish peroxidase-labeled secondary antibody against human IgG-Fc was used for assay in conjunction with color development performed by adding TMB substrate for 20 min and addition of an equal volume of reaction termination solution. The absorbance value was read at OD450 nm.

Example 55: Antibody-Dependent Cytotoxicity

[0531] Influenza virus AVC compounds: MDCK cells were infected with A/PR/8/1934 (H1N1), A/HK/1/1968 (H3N2) or B/Malaysia/2506/2004 (Victoria) that have an infection factor in the range of 0.001-10, for 18-24 hrs at 37 C. with 5% CO2. Then, the compounds to be tested were added and ADCC activity was assayed using the PROMEGA kit. Gedivumab (Genentech) was used as a positive control and Fc-N297A was used as a negative control.

Example 56: Antibody-Dependent Cell Phagocytosis

[0532] Influenza virus AVC compounds: MDCK cells were infected with A/PR/8/1934 (H1N1), A/HK/1/1968 (H3N2) or B/Malaysia/2506/2004 (Victoria) that have an infection factor in the range of 0.001-10, for 18-24 hrs at 37 C. with 5% CO2. Then, the compounds to be tested were added and ADCP activity was assayed using the PROMEGA kit. Gedivumab (Genentech) was used as a positive control and Fc-N297A was used as a negative control.

Example 57: Prevention of Secondary Infections Caused by Viral Infections

[0533] Influenza virus infection induced methicillin-resistant Staphylococcus aureus (MRSA) infection model: 6-8 week old BALB/c mice were intranasally inoculated (sublethal dose) with H1N1 A/CA/07/2009pdm influenza virus. The compound to be tested was injected intravenously 2 hours after infection (0.3-3 mg/kg). On day 6 after infection, mice were intranasally inoculated with a sublethal dose (510.sup.7 CFU) of methicillin-resistant Staphylococcus aureus (MRSA) TCH1516. 24 hrs later, lung tissues were taken for bacterial load testing. Lung tissues were homogenized in PBS solution with 1 mm diameter of glass beads and then diluted in a 10-fold series, coated on LA plates and incubated for 1 day at 37 C. CFU were finally converted to bacterial load per g of lung tissue weight. Body weight change and survival of mice were monitored daily for 14 days after infection.

[0534] Influenza virus infection induced Streptococcus pneumoniae infection model: 6-8 week old BALB/c mice were intranasally inoculated (sublethal dose) with H1N1 A/CA/07/2009pdm influenza virus. The compound to be tested was injected intravenously 2 hours after infection (0.3-3 mg/kg). On day 6 after infection, mice were inoculated intranasally with a sublethal dose (110.sup.5 CFU) of Streptococcus pneumoniae 6301. 24 hrs later, lung tissues were taken for bacterial load testing. Lung tissues were homogenized in PBS solution with 1 mm diameter of glass beads and then diluted in a 10-fold series, coated on LA plates and incubated for 1 day at 37 C. CFU were finally converted to bacterial load per g of lung tissue weight. Body weight change and survival of mice were monitored daily for 14 days after infection.

Example 58. Human FcRn Transgenic Mouse Viral Infection Model

[0535] Influenza virus infection: 6-8 week old female B6.Cg-Fcgrttml Dcr Tg(GCGRT)32Dcr/DcrJ mice (Jackson Labs #014565) express the human fetal FcRn receptor. Compounds to be tested having YTE Fc enables to show their extended half-life in this model. Intranasal inoculation with 3LD95 lethal dose of H1N1/A/CA/07/2009 influenza virus were performed. Mice were administered the compound to be tested intravenously (0.01, 0.03, 0.1, 0.3, 1.0 mg/kg, 5 ml/kg dose volume) at 7 days prior to infection. Oral Oseltamivir was used as a positive control for the experiment (Administered 8 hours after infection, 50 mg/kg, twice a day for 5 days) and human Fc fragments were used as a negative control for the experiment. Body weight change and survival of mice were monitored daily for 15 days after infection. Compared to compounds without YTE Fc, compounds with YTE was more effective in preventing viral infection and animal death at the same dose.

CONJUGATE FOR PREVENTING AND TREATING VIRAL INFECTIONS AND USE THEREOF

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