Immunoactivating small molecule compounds conjugated to antibodies and uses thereof

Abstract

The application provides an immune-activating small-molecule compound coupled with an antibody and an application thereof, and a general formula of the immune-activating small-molecule compound coupled with the antibody is shown in the following formula: wherein a linking functional group is a maleimide group, a carboxyl group, an isothiocyanate group, an azide group or an alkyne group, a connecting chain is an alkoxy group or an alkyl chain, the antibody is a PD-L1 antibody, and KX-main body is a main body part of an immune-activating small molecule KX. The application can superimpose the effect of immune-activating activation on the basis of maintaining the original anti-tumor function of the antibody, and greatly improves the anti-tumor effect.

Description

Technical Field

[0001] This invention relates to the field of antibody-drug conjugates (ADCs), and more specifically, to an immune-activating small molecule compound conjugated with an antibody and its applications. Background Technology

[0002] While antibody-drug conjugates (ADCs) improve the effectiveness of targeted anti-tumor therapy, drug resistance still develops with prolonged use; in many cases, although tumors are suppressed for a certain period, recurrence or metastasis still occurs. This is because while targeting and eliminating tumors, they do not stimulate the immune system. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide an immune-activating small molecule compound of antibody-drug conjugate and its application, addressing the problem that the above-mentioned antibody-drug conjugates fail to activate immune function.

[0004] The technical solution of this invention to solve the above-mentioned technical problems is to provide an immunoactivating small molecule compound of an antibody-conjugated compound: the general formula of the immunoactivating small molecule compound of the antibody-conjugated compound is shown below:

[0005]

[0006] The linking functional group is a maleimide group, carboxyl group, isothiocyanate group, azide group, or alkynyl group; the linking chain is an alkoxy or alkyl chain; the KX-body is the main part of the immunostimulating small molecule KX; the antibody is a PD-L1 antibody; and the immunostimulating small molecule compound of the conjugated antibody is one of the following:

[0007]

[0008]

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022] The present invention also provides the application of the immunoactivating small molecule compound of the antibody-conjugated as described above in the preparation of immunomodulatory drugs.

[0023] The present invention also provides the application of the immunoactivating small molecule compound of the conjugated antibody as described above in the preparation of conjugated antibodies.

[0024] The present invention also provides the application of the immunoactivating small molecule compound of the antibody-coupled as described above in the preparation of antitumor drugs.

[0025] This invention can enhance the anti-tumor effect by adding an immune activation effect while maintaining the original antibody's anti-tumor function. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] This invention provides an immunoactivating small molecule compound KX conjugated with an antibody, the general formula of which is shown below:

[0028]

[0029] The linking functional group is maleimide group, carboxyl group, isothiocyanate group, azide group or alkynyl group, the linking chain is alkoxy or alkyl chain, the antibody is IgG1, IgG2, IgG4, IgA, IgM, IgY or nanobody, and KX-body is the main part of the immune-activating small molecule KX.

[0030] The following examples illustrate the synthesis method and application effects of this bifunctional small molecule compound.

[0031] Example 1

[0032] HPLC conditions (the purity of compounds purified by HPLC or silica gel column chromatography reached ≥98.5%).

[0033] Mobile phase: C18 silica, acetonitrile / water with 0.05% trifluoroacetic acid gradient: 45% acetonitrile / 55% water with 0.05% trifluoroacetic acid, isogradient wash, flow rate 5 mL / min.

[0034] UV: 254nm; Instrument model: Agilent 1260 Infinity||; Column model: YMC-Pack ODS-A, 250×20mm, S-5μm.12nm;

[0035] Mass spectrometry conditional mobile phase

[0036] 0 5 95 0.5 5 90 10 0.5 7 90 10 0.5 10 5 5 0.5 12 5 5 0.5

[0037] UV: 254nm; Mass spectrometer for substance identification: Angilent; Liquid chromatography: Angilent 1260 Infinity||; Mass spectrometry: Angilent G6125B; Column model: YMC-Pack ODS-A, 150×4.6mm, S-5μm.12nm; Instrument model for antibody and peptide detection: XevoG2XSQTOF mass spectrometer, manufacturer: Waters Corporation.

[0038] Synthesis of compound K1:

[0039]

[0040]

[0041] First, SZU-115 (52 mg, 0.089 mmol) and VC-PAB-MMAE (100 mg, 0.089 mmol) were dissolved in 2 mL of DMF. DIPEA (46 μL) was added and stirred until homogeneous. Then, PyAOP (48 mg) was added. The resulting mixture was stirred at room temperature for 3 hours. The product was purified by preparative HPLC, and the purified solution was freeze-dried to give compound 115-VC-MMAE (119 mg, yield 79%). ESI-MS: m / z = 844.4 [(M / 2) + H]+.

[0042] Then, 115-VC-MMAE (100 mg, 0.059 mmol) and N3-PEG3-NH2 (13 mg, 0.060 mmol) were dissolved in 2 mL of a DMF-H2O (3:1 volume ratio) mixed solvent, and copper sulfate pentahydrate (37 mg) and sodium L-ascorbate (20 mg) were added. The mixture was stirred at room temperature. The reaction was monitored by LC-MS until completion, and compound 115-VC-MMAE-2 (70 mg, yield 62%) was prepared and purified by HPLC; ESI-MS: m / z = 953.6 [(M / 2)+H]+.

[0043] 115-MC-MMAE-2 (60 mg, 0.03 mmol) and NHS-MC-Ester (12 mg, 0.045 mmol) were dissolved in 2 mL of DMF. The resulting mixture was stirred overnight at room temperature. The product was purified by preparative HPLC, and the purified solution was freeze-dried to give compound K1 (49 mg, yield 79%); ESI-MS: m / z = 686.4 [(M / 3) + H]+.

[0044] Synthesis of compound K2:

[0045]

[0046]

[0047] First, SZU-101 (100 mg, 0.225 mmol) and NHS (26 mg, 0.225 mmol) were dissolved in 2 mL of DMF. DIPEA (40 μL) and EDCI (45 mg) were added, and the mixture was stirred at room temperature for 6 hours. The mixture was lyophilized, and the lyophilized product was poured into cold water to precipitate a solid. The solid was filtered and dried under vacuum to obtain compound 101-NHS (104 mg, 85% yield). Lys-2 (71 mg, 0.15 mmol) and DMAP (10 mg) were dissolved in 2 mL of DMF. 101-NHS (82 mg, 0.15 mmol) was added, and the mixture was stirred at room temperature for 6 hours. The solvent was removed by lyophilization, and TFA / DCM (1:3 mixed volume, 4 mL) was added to the residue under cooling. The mixture was stirred at room temperature for 1 hour. The solvent was removed by vacuum distillation, and the residue was purified by HPLC to obtain compound 101-Lys-2 (85 mg, yield 71%), ESI-MS: m / z = 795.3 [M+H]+. Compound 101-Lys-2 (80 mg, 0.1 mmol) and triethylamine (28 μL) were dissolved in 1.5 mL of dry DMF. PyAOP (52 mg, 0.1 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. DBCO-PEG4-amine TFA salt (52 mg, 0.1 mmol) was then added. The mixture was stirred at room temperature for 3 hours, followed by the addition of piperidine (30 μL) and stirring for another 3 hours. The reaction solution was lyophilized, and the lyophilized product was used.

[0048] Compound 101-Lys-DBCO (49 mg, yield 45%) was prepared and purified by HPLC. ESI-MS: m / z = 1078.5 [M+H]+.

[0049] MC-Val-Cit-PAB-PNP (30 mg) was dissolved in 0.5 mL of dry DMF. 101-Lys-DBCO (44 mg, 0.04 mmol) and DIPEA (10 μL) were mixed in 0.5 mL of DMF and slowly added to the MC-Val-Cit-PAB-PNP solution after cooling to 0°C in an external bath. After the addition was complete, the mixture was shaken at room temperature for 3 hours. The solvent was removed by freeze-drying, and the residue was dissolved in a small amount of methanol. Compound K2 (53 mg, yield 79%) was purified by preparative HPLC. ESI-MS: m / z = 839.0 [(M / 2) + H]+.

[0050] Synthesis of compound K3:

[0051] Following the synthetic method of compound K1, replacing VC-PAB-MMAE with VC-PAB-514 yields compound K3, ESI-MS: m / z = 852.4 [(M / 2)+H]+:

[0052]

[0053] The synthesis steps of VC-PAB-514 were as follows: Boc-VC-PAB-PNP (65 mg) and OTS514 (37 mg) were dissolved in 1 mL of DMF, and DIPEA (52 μL) was added. The mixture was shaken at room temperature for 1 hour. 1 mL of a mixed solvent of DCM-TFA (3:1 volume ratio) was added to the reaction mixture. After stirring at room temperature for 30 minutes, DCM-TFA was removed by vacuum distillation at 30 °C under reduced pressure. The residue was lyophilized, and the lyophilized product was dissolved in a small amount of 95% methanol. Preparative HPLC was used for separation and purification to obtain VC-PAB-514 (51 mg); ESI-MS: m / z = 771.1 [M+H]+.

[0054] Synthesis of compound K4:

[0055]

[0056] First, 115-VC-514 (80 mg, 0.06 mmol) and N3-PEG3-Acid (15 mg, 0.06 mmol) were dissolved in 2 mL of a DMF-H2O (3:1 volume ratio) mixed solvent, and copper sulfate pentahydrate (37 mg) and sodium L-ascorbate (20 mg) were added. The mixture was stirred at room temperature. The reaction was monitored by LC-MS until completion, and compound K4 (73 mg, yield 77%) was prepared and purified by HPLC; ESI-MS: m / z = 791.4 [(M / 2)+H]+.

[0057] Following the synthetic method of compound K4, replacing 115-VC-514 with 115-VC-MMAE yields compound K5, ESI-MS: m / z = 968.2[(M / 2)+H]+;

[0058]

[0059] Synthetic route of compound K6:

[0060]

[0061] Compound GY102 (500 mg, 1 mmol), N-hydroxysuccinimide (NHS, 120 mg), and 10 mL of DMF solution were added to EDC-HCl (210 mg) and stirred at room temperature for 3 hours. Then, 2 mL of DMF solution containing compound a1 (175 mg, 1 mmol) and DIPEA (350 μL) was slowly added. The resulting mixture was stirred overnight at room temperature. The reaction solution was freeze-dried, and the lyophilized product was dissolved in 20 mL of saturated Na2CO3 solution and stirred at room temperature for 30 minutes. The solution was neutralized to pH 4 with acetic acid, and the neutralized solution was extracted with ethyl acetate (3 x 20 mL). The extract was dried over anhydrous sodium sulfate, the desiccant was removed, and the filtrate was concentrated under reduced pressure to give compound 102-N3 (337 mg, yield 52%). ESI-MS: m / z = 649.3 [M+H]+.

[0062] Compound 102-N3 (130 mg, 0.2 mmol) and VC-PAB-MMAE (225 mg, 0.2 mmol) were dissolved in 5 mL of DMF, and DIPEA (40 μL) and PyAOP (115 mg) were added; the mixture was stirred at room temperature for 3 hours. The reactants were lyophilized, and the lyophilized product was dissolved in a small amount of methanol. Compound 102-VC-MMAE (168 mg, yield 49%) was purified by preparative HPLC; ESI-MS: m / z =

[0063] 878.1[(M / 2)+H]+.

[0064] Compound 102-VC-MMAE (88 mg, 0.05 mmol) and YN-PEG4-MC (16 mg, 0.05 mmol) were dissolved in 4 mL of H2O-DMF (1:3 volume ratio). Sodium L-ascorbate (11 mg) and copper sulfate pentahydrate (14 mg) were added. The mixture was stirred at room temperature for 6 hours. The reactants were lyophilized, dissolved in a small amount of methanol, filtered, and the filtrate was purified by preparative HPLC to give compound K6 (57 mg, yield 55%), ESI-MS: m / z = 689.5 [(M / 3)+H]+.

[0065] Following the synthesis method of compound K2, replacing compound SZU-101 with GY101 yields compound K7, ESI-MS: m / z = 863.9[(M / 2)+H]+.

[0066] Following the synthetic method of compound K6, replacing VC-PAB-MMAE with VC-PAB-514 yields compound K8, ESI-MS: m / z = 856.4 [(M / 2)+H]+.

[0067] Synthesis of compound K9:

[0068]

[0069]

[0070] Compound GY100 (525 mg, 2 mmol) was dissolved in 15 mL of DMF, and AcO-FURAN (525 mg, 4 mmol) and DBU (610 mg, 4 mmol) were added. The resulting mixture was heated to 90 °C and stirred for 24 hours. The solvent was removed by vacuum distillation, and the residue was extracted with ethyl acetate (30 mL x 3). The extracts were combined, and the solvent was removed by vacuum distillation. The residue was recrystallized from ethanol to give FURAN-100 (338 mg, yield 51%), ESI-MS: m / z = 332.1 [M+H]+.

[0071] FURAN-100 (332 mg, 1 mmol) and N3-PEG3-NH2 (220 mg, 1 mmol) were dissolved in 20 mL of H2O-DMF (1:3 volume). Sodium L-ascorbate (220 mg) and copper sulfate pentahydrate (280 mg) were added. The mixture was stirred at room temperature for 6 hours. The reactants were lyophilized, dissolved in a small amount of methanol, filtered, and the filtrate was purified by preparative HPLC to give compound FURAN-102 (286 mg, yield 52%), ESI-MS: m / z = 550.3 [M+H]+.

[0072] Compound FURAN-102 (276 mg, 0.5 mmol) and Fmoc-Val-Cit-PAB-PNP (383 mg, 0.5 mmol) were dissolved in 10 mL of DMF, and DIPEA (105 μL) was added. The reaction mixture was stirred at room temperature, and the reaction was monitored by mass spectrometry until completion. Piperidine (100 μL) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was lyophilized, and the lyophilized product was purified by HPLC. The purified product was then lyophilized to give compound VC-PAB-102 (234 mg, yield 49%). ESI-MS: m / z =

[0073] 955.6[M+H]+.

[0074] Dissolve VC-PAB-102 (192 mg, 0.2 mmol) in 5 mL of DMSO, and add HEX-ANHYDRIDE (26 mg, 0.2 mmol); stir the mixture at room temperature for 2 hours. Add DIPEA (40 μL) and continue stirring at room temperature overnight. Freeze-dry the reaction mixture, and purify the lyophilized product by HPLC. Freeze-dry the purified product to obtain compound K9 (91 mg, yield 42%); ESI-MS: m / z = 542.5 [(M / 2) + H]+.

[0075] Synthesis of compound K10:

[0076]

[0077] Compound SZU-136 (128 mg, 0.3 mmol) and DMAP (81 mg) were mixed thoroughly with 3 mL of dry dichloromethane to obtain reaction solution 1. Compound a2 (60 mg, 0.3 mmol) was mixed with 0.2 mL of dichloromethane and then slowly added dropwise to reaction solution 1. After the addition was complete, the mixture was stirred at room temperature for 3 hours. The reaction solution was freeze-dried, and the lyophilized product was purified by HPLC to obtain PZ-136 (145 mg, yield 88%); ESI-MS: m / z = 551.2 [M+H]+.

[0078] PZ-136 (138 mg, 0.25 mmol) and N3-PEG3-NH2 (55 mg, 0.25 mmol) were dissolved in mL of H2O-DMF (1:3 volume). Sodium L-ascorbate (55 mg) and copper sulfate pentahydrate (70 mg) were added. Following the treatment method of compound FURAN-102, PZ-136-NH2 (119 mg, yield 62%) was obtained. ESI-MS: m / z = 769.4 [M+H]+.

[0079] The compound Mc-Val-Cit-PABC-PNP (111 mg, 0.15 mmol) was dissolved in 2 mL of DMF. PZ-136-NH2 (116 mg, 0.15 mmol) was dissolved in 1 mL of DMF and slowly added dropwise to the Mc-Val-Cit-PABC-PNP solution under 0°C cooling in an external bath. After the addition was complete, the mixture was stirred overnight at room temperature. The reaction solution was lyophilized, and the lyophilized product was purified by preparative HPLC to give compound K10 (98 mg, yield 48%); ESI-MS: m / z = 684.7 [(M / 2) + H]+.

[0080] Synthesis of compound K11:

[0081]

[0082] Compound SZU-136 (170 mg, 0.4 mmol) was dissolved in 3 mL of DMF, and DIPEA (175 μL) was added. Triphosgene (119 mg, 0.4 mmol) was dissolved in 1 mL of dry DMF and slowly added to the SZU-136 DMF solution. After the addition was complete, the reaction was carried out at room temperature for 3 hours. Then, 0.5 mL of DMF solution containing PSSN (75 mg) was added, and the reaction was stirred for another 3 hours. The reactants were lyophilized to remove the solvent, and the residue was separated by silica gel column chromatography (DCM / MeOH = 10:1) to give compound PSSN-136 (92 mg, yield 36%), ESI-MS: m / z = 637.2 [M+H]+.

[0083] PSSN-136 (64 mg, 0.1 mmol) and NH2-PEG3-N3 (22 mg, 0.1 mmol) were dissolved in 2 mL of DMF, and 0.5 mL of pure water, CuSO4·5H2O (5 mg), and L-sodium ascorbate (5 mg) were added. The mixture was stirred at room temperature for 6 hours. The reaction solution was freeze-dried, and the lyophilized product was purified by HPLC to obtain NH2-PEG3-136-PSSN (47 mg, yield 55%), ESI-MS: m / z = 856.3 [M+H]+.

[0084] BP-23368 (37 mg) was dissolved in 0.5 mL of DMSO. A DMSO solution (0.5 mL) of compound NH2-PEG3-136-PSSN (40 mg, 0.047 mmol) and DIPEA (10 μL) was slowly added to the BP-23368 solution, and the mixture was reacted with shaking at room temperature for 8 hours. The reactants were lyophilized, and the lyophilized product was purified by HPLC to obtain compound K11 (48 mg, yield 69%), ESI-MS: m / z = 745.8 [(M / 2) + H]+.

[0085] Synthesis of compound K12: Following the synthetic route of compound K9, replacing GY100 with SZU-136 yields K12. ESI-MS: m / z = 624.2[(M / 2)+H]+.

[0086] Synthesis of compound K13:

[0087]

[0088] Compound PMQ-1 (106 mg, 0.3 mmol) was dissolved in 1 mL of DMF; MPF (62 mg, 0.3 mmol), DIPEA (52 μL), and condensing agent PyAOP (157 mg, 0.3 mmol) were dissolved in 2 mL of DMF. These solutions were mixed thoroughly under external cooling at 10 °C and slowly added dropwise to the PMQ-1 solution. After the addition was complete, the mixture was stirred at room temperature for 3 hours. The reaction mixture was freeze-dried, and compound PMQ-2 (100 mg, 62% yield) was purified by preparative HPLC. ESI-MS: m / z = 540.2 [M+H]+. Compound PMQ-2 (55 mg, 0.1 mmol) and compound Mal-PEG4-Val-Cit-PAB-PNP (88 mg, 0.1 mmol) were mixed in 2 mL of DMF and reacted overnight at room temperature with shaking. The compound K13 (90 mg, 71% yield) was obtained by freeze-drying using the same method and purified by HPLC. ESI-MS: m / z = 636.8 [(M / 2) + H] +.

[0089] Synthesis of compound K14:

[0090]

[0091] Compound SZU-136 (216 mg, 0.5 mmol) was dissolved in 5 mL of DMF, and CARBO-NCO (58 mg) was added. The reaction was monitored by mass spectrometry until completion. The reaction solution was freeze-dried to remove the solvent, and the product was separated by silica gel column chromatography (DCM / MeOH = 10:1) to obtain compound U-136 (237 mg, yield 88%), ESI-MS: m / z = 540.2 [M+H]+.

[0092] Compound U-136 (108 mg, 0.2 mmol) and N3-PEG3-MC (74 mg, 0.2 mmol) were dissolved in 5 mL of DMF, and 1 mL of pure water, 8 mg of copper sulfate pentahydrate, and 8 mg of sodium L-ascorbate were added. The mixture was stirred at room temperature for 10 hours. The reaction solution was filtered, the filtrate was freeze-dried, and the freeze-dried solution was purified by HPLC to obtain compound K14 (129 mg, yield 71%). ESI-MS: m / z

[0093] =909.3[M+H]+.

[0094] Referring to the synthesis method of compound K14, using Replacing CARBO-NCO yields compound K15 (ESI-MS: m / z = 925.3 [M+H]+).

[0095] Synthesis of compound K16:

[0096]

[0097] Compound KJ-008 (239 mg, 0.3 mmol) and K2CO3 (42 mg) were uniformly mixed in 3 mL of DMF, and Clo-CARBO-IP (46 mg, 0.3 mmol) was added. The mixture was stirred at room temperature for 10 hours. The reaction solution was filtered, the filtrate was freeze-dried, and compound K16 (150 mg, yield 55%) was purified by HPLC. ESI-MS: m / z = 910.4 [M+H]+.

[0098] Synthesis of compound K17:

[0099]

[0100] Compound SZU-115 (582 mg, 1 mmol), DIPEA (180 μL), and PyAOP (522 mg, 1 mmol) were dissolved in DMF (10 mL) and stirred until homogeneous. NH2-PEG2-OH (150 mg, 1 mmol) was then added, and the mixture was stirred at room temperature for 3 hours. The solution was concentrated under reduced pressure and purified by silica gel column chromatography (DCM:MeOH = 10:1) to give compound 115-1 (471 mg, 66% yield), ESI-MS: m / z = 713.3 [M+H]+. Compound 115-1 (357 mg, 0.5 mmol) and N3-PEG3-MC (185 mg, 0.5 mmol) were dissolved in DMF-H2O = 4:1 (5 mL). Copper sulfate pentahydrate (12 mg) and sodium L-ascorbate (12 mg) were added; the resulting mixture was stirred at room temperature for 6 hours. The reactants were concentrated under reduced pressure and separated by silica gel column chromatography (DCM / MeOH = 10:1) to give compound 115-2 (254 mg, yield 47%); ESI-MS: m / z = 1082.5 [M+H]+.

[0101] Compound DPDC (20 μL, 0.1 mmol) was dissolved in 0.5 mL of dry DMF; compound 115-2 (110 mg, 0.1 mmol), imidazole (15 mg), and DMAP (25 mg) were dissolved in 1 mL of dry DMF and slowly added to the DPDC solution. The mixture was stirred and reacted for 60 minutes. Compound Dxd (50 mg, 0.1 mmol) was dissolved in 1 mL of DMF and slowly added to the reaction mixture. After the reaction was complete as monitored by mass spectrometry, the solvent was evaporated under reduced pressure, and the residue was separated by silica gel column chromatography (DCM:MeOH = 10:1) to give compound K17 (106 mg, yield 63%), ESI-MS: m / z = 844.4 [(M / 2) + H]+.

[0102] Synthesis of compound K18:

[0103]

[0104] Compound OTS514 (365 mg, 1 mmol) and B-PEG3-N3 (283 mg, 1 mmol) were mixed and dissolved in 5 mL of DMF, and K2CO3 (150 mg) was added; the mixture was stirred at room temperature for 6 hours. The reaction mixture was lyophilized, and the lyophilized product was subjected to silica gel column chromatography (DCM / MeOH = 5:1). The solution containing the product was concentrated and dried to give compound 514-1 (175 mg, yield 31%). Compound 514-1 (147 mg, 0.26 mmol) and GY100 (68 mg, 0.26 mmol) were dissolved in 2 mL of DMF, and 0.5 mL of water, copper sulfate pentahydrate (7 mg), and sodium L-ascorbate (7 mg) were added. The mixture was stirred and reacted for 6 hours. The reactants were freeze-dried, and the freeze-dried product was separated by silica gel column chromatography (DCM:MeOH = 5:1) to give compound 514-100 (128 mg, yield 58%), ESI-MS: m / z = 827.4 [M+H]+.

[0105] Dissolve 514-100 (100 mg, 0.12 mmol) in 1.5 mL of DMF, add DMAP (15 mg, 0.123 mmol), mix and stir well, then add MC-VC-PAB-PNP (89 mg, 0.12 mmol). The mixture is stirred at room temperature for 3 hours. Remove the solvent by vacuum distillation, and purify the residue using preparative HPLC. Concentrate the purified solution under reduced pressure and freeze-dry to give compound K18 (104 mg, yield 61%), ESI-MS: m / z = 713.8 [(M / 2) + H]+.

[0106] Synthesis of compound K19:

[0107]

[0108] Compounds GY155 (506 mg, 1 mmol) and YN-514 (445 mg, 1 mmol) were dissolved in 15 mL of DMF. 3 mL of pure water, copper sulfate pentahydrate (25 mg), and sodium L-ascorbate (25 mg) were added. The mixture was stirred under nitrogen protection for 6 hours. The resulting solution was purified by silica gel column chromatography (DCM:MeOH = 5:1) to obtain compound GY191 (551 mg, yield 58%), ESI-MS: / z = 950.4 [M+H]+. GY191 (100 mg, 0.1 mmol) was dissolved in 2 mL of DMF, and DIPEA (34 μL) was added. After stirring thoroughly, the mixture was slowly added to a DMF solution of MC-VC-PAB-PNP (75 mg, 0.1 mmol) in a 0°C external bath under cooling. The reaction was allowed to proceed at room temperature, and mass spectrometry was used to detect the reaction until completion. The reaction mixture was freeze-dried, and the freeze-dried product was purified by preparative HPLC to obtain compound K19 (68 mg, yield 44%), ESI-MS: m / z = 775.4 [(M / 2) + H] +.

[0109] Synthesis of compound K20: Following the synthesis method of compound K19, compound GY100 was replaced with SZU-136 to obtain compound K20. ESI-MS: m / z = 787.4 [(M / 2)+H]+:

[0110]

[0111] Synthesis of compound K21:

[0112]

[0113] Dissolve 667 mg of 101-NHS in 2 mL of anhydrous DMF, add 300 mg of N3-PEG3-NH2, and stir overnight at room temperature. After the reaction is complete, pour the reaction solution into water, and a white solid precipitates out. Dry the solid to obtain 101-PEG3-N3.

[0114] The 101-PEG3-N3 obtained in the previous step was dissolved in 10 mL of anhydrous THF, and triphenylphosphine (484 mg) was added under ice bath conditions. The mixture was stirred overnight at room temperature, and the reaction was monitored by LC-MS. After the reaction was complete, 20 mL of water was added, and the mixture was stirred for another 30 min. The mixture was then filtered under reduced pressure, washed twice with water, and dried to obtain 101-PEG3-NH2 (625 mg, combined yield of 79%). ESI-MS: m / z = 619.3 [M+H]+.

[0115]

[0116] ARS1620 (108 mg, 0.25 mmol) and 4-bromobutyne (24 μL) were dissolved in DMF (2 mL), and K2CO3 (35 mg) was added. The mixture was stirred at room temperature for 12 hours. The reaction solution was filtered, and 0.4 mL of pure water, N3-NH2 (25 mg, 0.25 mmol), CuSO4·5H2O (8 mg), and L-sodium ascorbate (8 mg) were added to the filtrate. The resulting mixture was reacted at room temperature for 6 hours. The reaction solution was filtered, and the filtrate was lyophilized. The lyophilized product was purified by HPLC to obtain 1620-NH2 (77 mg, yield 53%), ESI-MS: m / z = 584.2 [M+H]+.

[0117]

[0118]

[0119] TCT (185 mg, 1 mol) was dissolved in 5 mL of dry DMF and slowly added to DIPEA (175 μL) under external bath cooling at 0 °C. Compound 101-PEG3-NH2 (619 mg, 1 mmol) was dissolved in 3 mL of dry DMF and slowly added to the TCT solution under external bath cooling at 0 °C. The reaction was continued for 3 hours after the addition was complete. The reactants were lyophilized, and the lyophilized product was separated and dried by HPLC. The separated product was lyophilized again to obtain lyophilized product 1 (276 mg, yield 36%), ESI-MS: m / z = 767.2 [M+H]+. Following the same method used to synthesize lyophilized compound 1, lyophilized compound 1 was reacted with 1620-NH2 to obtain lyophilized compound 2 (ESI-MS: m / z = 657.6 [(M / 2)+H]+); lyophilized compound 2 was reacted with AMPIP to obtain lyophilized compound 3 (ESI-MS: m / z = 696.4 [(M / 2)+H]+).

[0120] Dissolve lyophilized product 3 (139 mg, 0.1 mmol) in 3 mL of DMF and add DIPEA (175 μL). Dissolve N3-PEG3-VC-PAB-PNP (77.4 mg, 0.1 mmol) in 0.5 mL of DMF and slowly add it to the solution of lyophilized product 3 while stirring at 0 °C. After the addition is complete, react at room temperature for 6 hours. Freeze-dry the reaction solution, and purify the lyophilized product by preparative HPLC. Freeze-dry the purified product solution to obtain compound K21 (135 mg, yield 67%), ESI-MS: m / z = 676.2 [(M / 3) + H]+.

[0121] Synthesis of compound K22:

[0122]

[0123] Compounds BP-23736 (129 mg, 0.15 mmol) and Val-Cit-PAB (114 mg, 0.30 mmol) were dissolved in 1 mL of DMF and reacted at room temperature with stirring for 8 hours. The reaction solution was lyophilized and purified by semi-preparative HPLC to obtain compound MC-DPEG4-VC-PAB (108 mg, yield 52%), ESI-MS: m / z = 694.0 [(M / 2) + H]+. NPC (43 mg, 0.14 mmol) was dissolved in 0.5 mL of anhydrous THF. 1 mL of DMF containing compound MC-DPEG4-VC-PAB (100 mg, 0.07 mmol) was dissolved and slowly added dropwise to the NPC solution. After the addition was complete, the reaction solution was continued to react at room temperature with shaking for 3 hours. Separately, 0.5 mL of a solution of compound KJ-032 (50 mg, 0.14 mmol) was added dropwise to the reaction solution, and the reaction was continued at room temperature overnight. The reaction solution was freeze-dried, and the freeze-dried product was purified by HPLC to obtain compound K22 (55 mg, yield 36%), ESI-MS: m / z = 712.0 [(M / 3) + H] +.

[0124] Compound KJ-032 can be obtained by the Click reaction of GY100 and compound 2-azidoethylamine (ESI-MS: m / z = 348.2 [M+H]+).

[0125] Synthesis of compound K23:

[0126]

[0127] Compounds BP-23311 (78 mg, 0.15 mmol) and SZU-136 (128 mg, 0.3 mmol) were dissolved in a 1:4 H₂O-DMF solvent (2 mL). CuSO₄·5H₂O (10 mg) and sodium L-ascorbate (10 mg) were added, and the mixture was stirred overnight at room temperature under nitrogen protection. The reaction solution was filtered, and the filtrate was lyophilized. A 1:3 TFA-DCM solvent (1:3, 2 mL) was added to the lyophilized product, and the mixture was stirred for 1 hour. The solvent was removed by vacuum distillation, and the residue was purified by HPLC to obtain compound Bis-136 (105 mg, 55% yield). ESI-MS: m / z = 635.1 [(M / 2) + H]⁺.

[0128] Bis-136 (101 mg, 0.08 mmol) was dissolved in 1.5 mL of DMF, and N3-PEG3-VC-PAB-PNP (62 mg, 0.08 mmol) and DIPEA (18 μL) were added and reacted for 6 hours. The reaction solution was freeze-dried, and the lyophilized product was purified by HPLC to obtain compound K23 (59 mg, yield 39%), ESI-MS: m / z = 952.0 [(M / 2)+H]+.

[0129] Synthesis of compound K24:

[0130]

[0131] Compound SZU-115 (100 mg, 0.17 mmol) and BP22468 (64 mg, 0.17 mmol) were dissolved in 2 mL of DMF-H2O (4:1 volume ratio), and CuSO4·5H2O (5 mg) and sodium L-ascorbate (5 mg) were added. The reaction was carried out overnight at room temperature under nitrogen protection. The reaction solution was filtered, the filtrate was freeze-dried, and then purified by HPLC to obtain compound MC-PEG3-115 (63 mg, yield 39%), ESI-MS: m / z = 951.4 [M+H]+. Compound MC-PEG3-115 (60 mg, 0.063 mmol), DIPEA (12 μL), and PyAOP (35 mg, 0.067 mmol) were dissolved in 1 mL of DMF, stirred until homogeneous, and then BP-23958 (33 mg, 0.063 mmol) was added. The mixture was stirred at room temperature for 10 hours. The reaction product was purified by semi-preparative HPLC to obtain compound K24 (48 mg, yield 52%), ESI-MS: m / z = 729.1 [(M / 2) + H] +.

[0132] Synthesis of compound K25:

[0133]

[0134]

[0135] Compound BP-22435 was dissolved in water, neutralized to pH 8 with saturated sodium carbonate solution, and extracted with dichloromethane to obtain the free base of BP-22435. Following the method for preparing K24 from compounds MC-PEG3-115 and BP-23958, compound K25 (ESI-MS: m / z = 648.8 [(M / 2)+H]+) was obtained.

[0136] Synthesis of compound K26:

[0137]

[0138] Following the synthetic method of compound K24, replacing BP-23958 with compound TCO-PEG3-amine yields compound K26 (ESI-MS: m / z = 639.0 [(M / 2)+H]+).

[0139] Synthesis of compound K27:

[0140]

[0141]

[0142] Compound NH2-PIP-N3 (120 mg, 0.5 mmol) and DI-IMICS (89 mg, 0.5 mmol) were dissolved in 2 mL of dry DMSO, and the mixture was stirred at 40 °C for 6 hours. IBRU-1 (194 mg, 0.5 mmol) was then added, and the mixture was stirred at room temperature for another 6 hours. The reactants were lyophilized, and IBRU-2 (164 mg, 49% yield) was purified by HPLC. ESI-MS: m / z = 669.3 [M+H]+.

[0143] Separately, SZU-115 (291 mg, 0.5 mmol) and PyAOP (261 mg, 0.5 mmol) were dissolved in 3 mL of DMF. DIPEA (88 μL) was added and the mixture was stirred for 30 minutes. Then, BOC-PIP (93 mg, 0.5 mmol) was added and the mixture was reacted at room temperature for 3 hours. The reaction solution was freeze-dried and TFA / DCM mixed solvent (4 mL, 1:3 volume) was added and stirred for 1 hour. The solvent was removed by vacuum distillation. The compound PIP-115 (214 mg, yield 66%) was purified by HPLC. ESI-MS: m / z = 650.3 [M+H]+.

[0144] IBRU-2 (100 mg, 0.15 mmol) and PIP-115 (98 mg, 0.15 mmol) were dissolved in 3 mL of DMF-H2O (4:1 volume). A mixture of CuSO4·5H2O (5 mg) and sodium L-ascorbate (5 mg) was added, and the mixture was stirred at room temperature for 8 hours. The reaction solution was filtered, and the filtrate was freeze-dried. The compound IBRU-115 (77 mg, 39% yield) was purified by HPLC. ESI-MS: m / z = 659.8 [(M / 2) + H]+.

[0145] Compound IBRU-115 (70 mg, 0.053 mmol) was dissolved in 0.5 mL of DMF; Mal-PEG4-Val-Cit-PAB-PNP (46 mg, 0.053 mmol) was dissolved in 0.5 mL of DMF and slowly added dropwise to the IBRU-115 solution at room temperature. After the addition was complete, the reaction was continued at room temperature with shaking for 6 hours. The reaction solution was freeze-dried to remove the solvent, and compound K27 (67 mg, yield 62%) was obtained by preparative HPLC purification. ESI-MS: m / z = 684.3 [(M / 3) + H]+.

[0146] Synthesis of compound K28:

[0147]

[0148] Compound SZU-115 (1 mmol) and compound N3-PEG3-NHBOC (1 mmol) were dissolved in a mixture of CuSO4·5H2O (25 mg) and L-sodium ascorbate (25 mg) in 10 mL of DMF-H2O (4:1 volume, 10 mL). The mixture was stirred at room temperature for 8 hours. The reaction mixture was filtered, the solvent was removed by vacuum distillation, and compound SZU-1152 (396 mg, yield 44%, ESI-MS: m / z = 900.5 [M+H]+) was obtained by silica gel column chromatography. Compound SZU-1152 (300 mg, 0.33 mmol) and DMAP (40 mg) were dissolved in 5 mL of DMF, PyAOP (172 mg, 0.33 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Then, VC-PAB-OH (125 mg, 0.33 mmol) was added, and the mixture was stirred at room temperature for 6 hours. The reaction solution was freeze-dried, and the freeze-dried product was mixed with 10 mL of cold water. The pH was adjusted to 5 with dilute hydrochloric acid, and the solution was extracted with dichloromethane (5 x 5 mL). The extract was concentrated under reduced pressure to half, and TFA (5 mL) was added and stirred at room temperature for 1 hour. The solvent was removed by reduced pressure distillation, and the residue was purified by preparative HPLC to obtain compound SZU-1153 (146 mg, yield 38%), ESI-MS: m / z = 1161.6 [M+H]+. 1 mL of DMF solution of compound SZU-1153 (116 mg, 0.1 mmol) and DIPEA (10 μL) was added, and the mixture was shaken at room temperature for 3 hours. After freeze-drying and preparative purification by HPLC, compound SZU-1154 (100 mg, 69%) was obtained, ESI-MS: m / z = 724.8 [(M / 2)+H]+.

[0149] Take 0.2 mL of DMF solution containing compound NPC (19 mg, 0.062 mmol) and slowly add 1 mL of solution containing SZU-1154 (90 mg, 0.062 mmol). Shake and react overnight at room temperature. The reaction solution was lyophilized and purified by HPLC to obtain compound SZU-1155 (75 mg, 75% yield), ESI-MS: m / z = 807.4 [(M / 2) + H]+. Take 60 mg of compound SZU-1155 (0.037 mmol) and DMAP (5 mg) dissolved in 0.5 mL of DMF solution and slowly add 0.5 mL of DMF solution containing AZ7550 (18 mg, 0.037 mmol). Shake and react overnight at room temperature. The reaction solution was freeze-dried and purified by HPLC to obtain compound K28 (39.9 mg, yield 55%), ESI-MS: m / z = 980.5 [(M / 2) + H] +.

[0150] Synthesis of compound K28-1:

[0151]

[0152]

[0153] Compound ITA-ME (1 mmol), PyAOP, and DIPEA were condensed in DMF solvent with the addition of SZU-142 (1 mmol) to yield compound KJ-138 (ESI-MS: m / z = 457.2 [M+H]+). Similarly, the condensation of compound LYSYN-FMOC and VC-PAB-OH yielded compound LYSYN-VCPAB (ESI-MS: m / z = 517.3 [M+H]+). Further condensation of KJ-138 and LYSYN-VCPAB yielded compound KJ-139 (ESI-MS: m / z = 955.5 [M+H]+). A copper-catalyzed Click reaction of KJ-139 with compound N3-PEG3-NH2 yielded compound KJ-140 (ESI-MS: m / z = 1173.6 [M+H]+). Reacting equimolar amounts of compound KJ-140 and DBCO-NHS-Ester directly in DMF yields compound KJ-141 (ESI-MS: m / z = 730.9 [(M / 2)+H]+). Following the synthetic method for compound SZU-1155, compound KJ-142 (ESI-MS: m / z = 813.4 [(M / 2)+H]+) is obtained. Reacting compound KJ-142 and compound OTS514 in equimolar amounts in DMF at room temperature, followed by freeze-drying to remove DMF, and purification by HPLC yields compound K28-1 (ESI-MS: m / z = 925.9 [(M / 2)+H]+).

[0154] Synthesis of compound K29: Following the synthesis method of compound K2, replacing DBCO-PEG4-amine TFA salt with BP-22435 (Methyltetrazine-PEG4-amine HCl salt) yields compound K29. ESI-MS: m / z = 758.9 [(M / 2)+H]+.

[0155] Synthesis of compound K30: Following the synthesis method of compound K2, replacing DBCO-PEG4-amine TFA salt with TCO-PEG3-amine yields compound K30. ESI-MS: m / z = 749.4 [(M / 2)+H]+.

[0156] Synthesis of compound K31: Following the synthesis method of compound K28, replace compound DBCO-NHS-Ester with endo-BCN-PEG2-NHS ester and replace compound AZ7550 with OTS514 to obtain compound K31. ESI-MS: m / z = 944.0[(M / 2)+H]+.

[0157] Synthesis of compound K32:

[0158]

[0159]

[0160] Compound SZU-115 (1 mmol) and compound Azido-PEG3-Maleimide (1 mmol) were mixed in 10 mL of DMF-H2O (4:1 volume ratio), and CuSO4·5H2O (25 mg) and sodium L-ascorbate (25 mg) were added. The mixture was stirred at room temperature for 6 hours. After filtration, the filtrate was freeze-dried and purified by HPLC to obtain compound MM-PEG3-115 (600 mg, yield 63%), ESI-MS: m / z = 951.4 [M+H]+. Compound MM-PEG3-115 (190 mg, 0.2 mmol), compound DIPEA (35 μL), and PyAOP (105 mg) were mixed and dissolved in 2 mL of DMF and stirred evenly for 30 minutes. VC-PAB-OH (76 mg) was added and the mixture was stirred overnight at room temperature. The compound MM-VCPAB-115 (186 mg, yield 71%) was prepared by freeze drying and purified by HPLC. ESI-MS: m / z = 657.3 [(M / 2) + H] +.

[0161] Compound NPC (31 mg, 0.1 mmol) was dissolved in 0.5 mL of anhydrous DMF; MM-VCPAB-115 (132 mg, 0.1 mmol) and DMAP (12 mg) were dissolved together in 1 mL of DMF and slowly added to the DMF solution of NPC. After the addition was complete, the mixture was stirred overnight at room temperature. The solution was lyophilized and purified by silica gel column chromatography (ethyl acetate) to give compound MM-VC-PNP-115 (72 mg, yield 49%), ESI-MS: m / z = 739.3 [(M / 2) + H]+.

[0162] Compound MM-VC-PNP-115 (59 mg, 0.04 mmol) and DMAP (5 mg) were dissolved in 1 mL of dry DMF. MRTX1133 (24 mg, 0.04 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction solution was freeze-dried, and the residue was dissolved in a small amount of methanol. Compound K32 (45 mg, yield 58%) was purified by semi-preparative HPLC. ESI-MS: m / z = 970.0 [(M / 2) + H]+.

[0163] Synthesis of compound K33: Compound K33 was obtained by the same method as that used to synthesize K32. ESI-MS: m / z = 986.4 [(M / 2)+H]+.

[0164] Synthesis of compound K34:

[0165]

[0166]

[0167] Compound KJ001 (120 mg, 0.5 mmol) and compound BBNB (143 mg, 0.5 mmol) were mixed and dissolved in 3 mL of DMF. K2CO3 (100 mg) was added and the mixture was stirred at room temperature for 8 hours. The reaction solution was filtered, and the solvent was removed by vacuum distillation of the filtrate. 5 mL of concentrated hydrochloric acid was added and the mixture was stirred at room temperature for 3 hours. The solution was neutralized to pH 8 with Na2CO3, and a solid product precipitated. The solid product was dried under vacuum to obtain KJ002 (123 mg, 75% yield). ESI-MS: m / z = 329.2 [M+H]+. Compound KJ002 (100 mg, 0.3 mmol) was dissolved in 2 mL of chloroform. Triethylamine (100 μL) was added, and the mixture was cooled to 0°C in an external bath. 0.5 mL of chloroform solution containing 25 μL of phosgene was slowly added. After the addition was complete, the mixture was stirred at room temperature overnight. After thorough washing with cold water (3 x 2 mL), chloroform was removed, and the mixture was dried over anhydrous sodium sulfate. The residue was filtered, and the solvent was removed by vacuum distillation to obtain compound KJ003 (71 mg, 64% yield), ESI-MS: m / z = 371.1 [M+H]+. Compound KJ003 (60 mg, 0.16 mmol) and compound KJ004 (36 mg, 0.16 mmol) were dissolved in 0.5 mL of DMF, and triethylamine (60 μL) was added. The mixture was reacted overnight at room temperature with shaking. The reaction solution was lyophilized, and the lyophilized product was purified by HPLC to obtain compound KJ005 (63 mg, 66% yield), ESI-MS: m / z = 596.1 [M+H]+. Compound KJ005 (60 mg) was mixed with 1 mL of concentrated hydrochloric acid and stirred overnight at room temperature. The reaction solution was neutralized to pH 4 with Na2CO3. The solid was filtered out, washed three times with pure water, and dried under vacuum to obtain compound KJ006 (51 mg, yield 89%). ESI-MS: m / z = 568.1 [M+H]+.

[0168] Following the synthetic method of compound 101-Lys-2, compound KJ007 (ESI-MS: m / z = 918.3 [M+H]+) can be obtained.

[0169] Following the synthetic method of compound 101-Lys-DBCO, compound KJ008 ((ESI-MS: m / z = 1201.5 [M+H]+) can be obtained.

[0170] The final step, following the synthesis method of compound K2, involves reacting KJ008 with MC-Val-Cit-PAB-PNP to obtain compound K34{ESI-MS: m / z = 900.4[(M / 2) + H] +}.

[0171] Synthesis of compound K35:

[0172]

[0173] Compound AZ7550 was dissolved in DMF, and an equimolar amount of anhydrous K2CO3 was added. An equimolar amount of BocNH-C6-BR was dissolved in an appropriate amount of DMF and slowly added to the DMF solution of AZ7550 at room temperature (about 1 hour). After the addition was complete, the reaction was continued at room temperature for 6 hours. The reaction solution was filtered and freeze-dried. The freeze-dried product was added to a 1:3 TFA / DCM mixed solvent and stirred at room temperature for 1 hour. The solvent was removed by vacuum distillation, and an appropriate amount of pure water was added to dissolve the product. The pH was adjusted to 9 with saturated sodium carbonate solution, and the product was extracted three times with an equal volume of dichloromethane. The extracts were combined, dried with anhydrous K2CO3, filtered, and the filtrate was distilled under reduced pressure to obtain a solid. The solid was dried under vacuum to obtain compound NH2-C6-7550 (ESI-MS: m / z = 585.4 [M+H]+).

[0174] Compound SZU-115 (120 mg, 0.2 mmol) and DIPEA (35 μL) were mixed in 2 mL of DMF. PyAOP (105 mg, 0.2 mmol) was added and the mixture was stirred for 0.5 hours. NH2-C6-7550 (117 mg, 0.2 mmol) was added and the reaction was continued at room temperature for 6 hours. After freeze-drying, compound KJ-7550 (144.7 mg, yield 63%) was obtained by HPLC purification. ESI-MS: m / z = 1148.6 [M+H]+.

[0175] Compound KJ-7550 (100 mg, 0.087 mmol) and compound N3-PEG3-NH2 (20 mg, 0.087 mmol) were mixed and dissolved in DMF-H2O (4:1 volume, 2 mL). CuSO4·5H2O (5 mg) and sodium L-ascorbate (5 mg) were added, and the mixture was stirred overnight at room temperature. The reaction solution was filtered and lyophilized. The lyophilized product was purified by HPLC to obtain a purified solution containing the product. The purified solution was concentrated under reduced pressure and lyophilized to obtain compound KJ-7551 (50 mg, yield 42%). ESI-MS: m / z = 683.9[(M / 2)+H]+.

[0176] Compound KJ-7551 (45 mg, 0.033 mmol) was dissolved in 0.5 mL of DMF, and DIPEA (10 μL) and DBCO-NHS (14 mg, 0.035 mmol) were added. The mixture was reacted with shaking at room temperature for 3 hours. The product solution was purified by semi-preparative HPLC and lyophilized to obtain compound K35 (38.8 mg, yield 71%). ESI-MS: m / z = 827.4 [(M / 2) + H]+.

[0177] The synthesis of compound K36 followed the synthetic route below, referring to the synthesis method and reaction conditions of compound K35, except that SZU-115 was replaced with 102-N3, yielding compound KJ-7552 (ESI-MS: m / z = 608.3 [(M / 2)+H]+); replacing N3-PEG3-MC with Propargyl-PEG4-amine yielded compound KJ-7553 (ESI-MS: m / z = 723.9 [(M / 2)+H]+); further reaction of KJ-7553 with DBCO-NHS yielded compound K36 (ESI-MS: m / z = 867.5 [(M / 2)+H]+).

[0178]

[0179]

[0180] The synthesis of compound K37 followed the synthetic route below, referring to the synthetic method and reaction conditions of compound K2, except that DBCO-PEG4-amine TFA salt was replaced with NH2-C6-7550, yielding compound KJ-7554 (ESI-MS: m / z = 570.3 [(M / 2)+H]+); replacing MC-Val-Cit-PAB-PNP with DBCO-PEG4-Val-Cit-PAB-PNP yielded compound K37 (ESI-MS: m / z = 693.7 [(M / 3)+H]+).

[0181]

[0182] Synthesis of compound K38:

[0183]

[0184] Compound AZ7550 (98 mg, 0.2 mmol) was dissolved in 0.5 mL of DMF; separately, compound Bromo-PEG3-azide (57 mg, 0.2 mmol) was dissolved in 1 mL of DMF and slowly added to the AZ7550 solution with stirring. After the addition was complete, the reaction was continued to be stirred overnight at room temperature. The reaction solution was lyophilized and purified by HPLC to obtain compound N3-PEG3-7550 (90.6 mg, yield 66%), ESI-MS: m / z = 687.4 [M+H]+.

[0185] Compound N3-PEG3-7550 (83 mg, 0.12 mmol) and compound GY100 (32 mg, 0.12 mmol) were mixed and dissolved in 2 mL of DMF-H2O (4:1 volume). CuSO4·5H2O (5 mg) and sodium L-ascorbate (5 mg) were added, and the mixture was stirred overnight at room temperature. The reaction solution was filtered, the filtrate was lyophilized, and the product solution was purified by HPLC. The product solution was lyophilized to give compound GY7550 (66.5 mg, yield 53%), ESI-MS: m / z = 948.5 [M+H]+.

[0186] Compound GY7550 (60 mg, 0.057 mmol) and DBU (23 μL) were dissolved in 1 mL of dry DMF, and NCO-C2-NHBOC (12 μL, 0.07 mmol) was added. The mixture was reacted overnight at room temperature. The reaction solution was lyophilized, and TFA / DCM (1:3 volume, 1 mL) was added. The mixture was stirred at room temperature for 1 hour. The solvent was removed by vacuum distillation, and the residue was purified by HPLC. The purified product was lyophilized to give compound GY7550-1 (33.5 mg, yield 52%). ESI-MS: m / z = 1034.5 [M+H]+.

[0187] Compound GY7550-1 (30 mg, 0.0265 mmol) and DBCO-PEG4-Val-Cit-PAB-PNP (30 mg, 0.0278 mmol) were dissolved in 1 mL of DMF. After stirring and mixing thoroughly, DMAP (6 mg) was added to form a reaction solution, which was then reacted overnight at room temperature with shaking. The solvent was removed by freezing the reaction solution, and the lyophilized product was purified by semi-preparative HPLC. The purified product solution was then lyophilized to give compound K38 (37 mg, yield 71%). ESI-MS: m / z = 987.5 [(M / 2) + H]+.

[0188] Synthesis of compound K39:

[0189]

[0190] The condensation reaction of equimolar amounts of SZU-142 and itaconic acid monomethyl ester under the action of PyAOP and DIPEA yields compound SZU-275 (ESI-MS: m / z = 471.2 [M+H]+). Following the synthesis method of GY7550-1 and compound K38, compound K39 is obtained, with ESI-MS: m / z = 748.8 [(M / 2)+H]+.

[0191] Synthesis of compound K40:

[0192]

[0193]

[0194] Compound SZU-115 (111 mg, 0.19 mmol) and compound PyAOP (99 mg, 0.19 mmol) were dissolved in 3 mL of DMF. DIPEA (36 μL) was added, and the mixture was stirred for 30 minutes. Then, a 0.2 mL DMF solution of compound 92-61-9 (44 mg, 0.19 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction solution was freeze-dried, and compound 115-2 (95 mg, 63% yield) was purified by HPLC. ESI-MS: m / z = 791.3 [M+H]+.

[0195] Compound 115-2 (90 mg) and N3-PEG3-NH2 (25 mg) were dissolved in 2 mL of H2O-DMF (1:3). CuSO4·5H2O (10 mg) and sodium L-ascorbate (10 mg) were added, and the mixture was stirred for 8 hours. The reaction solution was filtered, the filtrate was freeze-dried, and purified by HPLC to obtain compound 115-3 (79 mg, yield 69%). ESI-MS: m / z = 1009.5 [M+H]+.

[0196] Compound 115-3 (75 mg, 0.075 mmol) was dissolved in 1 mL of N-methylpyrrolidone, and TCDI (13.5 mg) was added. The mixture was reacted overnight at room temperature. The reaction solution was lyophilized and purified by HPLC to give compound K40 (59 mg, 75% yield). ESI-MS: m / z = 1051.5 [M+H]+.

[0197] Synthesis of compound K41:

[0198] Following the synthetic method for compound K16, replacing KJ-008 with compound KJ-140 yields compound K41, ESI-MS: m / z = 775.4 [M+H]+. Compound KJ-140 was synthesized via a Click reaction of compound GY121 and N3-PEG3-MC (ESI-MS: m / z = 659.3 [M+H]+).

[0199]

[0200] Synthesis of compound K42:

[0201]

[0202] Compounds Exatecan and Gly-NHS were dissolved in an equimolar mixture in an appropriate amount of DMF. An equimolar amount of DIPEA was added and the mixture was stirred until the reaction was complete. An equimolar amount of piperidine was then added, and the mixture was stirred at room temperature for 3 hours. The solvent was removed by freeze-drying, and the remaining solution was separated by silica gel column chromatography (DCM / MeOH: 5:1) to obtain compound Glytecan (ESI-MS: m / z = 493.2 [M+H]+). Glytecan and DMAP were dissolved in an equimolar amount in DMSO. An equimolar amount of CMCL in DMSO solution was slowly added dropwise to the Glytecan solution, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was freeze-dryed, and the remaining solution was separated by silica gel column chromatography (DCM / MeOH: 5:1) to obtain compound Glytecan-1 (ESI-MS: m / z = 585.2 [M+H]+).

[0203] Compounds Glytecan-1 (50 mg, 0.085 mmol) and KJ-008 (68 mg, 0.085 mmol) were mixed and dissolved in 1.5 mL of DMF. Anhydrous K2CO3 (12 mg) was added, and the mixture was stirred at room temperature for 6 hours. The reaction solution was filtered, and the filtrate was freeze-dried. The freeze-dried product was purified by semi-preparative HPLC to obtain compound K42 (43 mg, yield 38%). ESI-MS: m / z = 671.8 [(M / 2) + H]+.

[0204] Synthesis of compound KJ-008:

[0205]

[0206] Compound Azido-PEG3-amine (110 mg, 0.5 mmol) and compound Maleimide-NHS ester (133 mg, 0.5 mmol) were mixed in 2 mL of DMF and stirred at room temperature for 30 minutes. Then, 0.5 mL of purified water, CuSO4·5H2O (13 mg), and sodium ascorbate (13 mg) were added to form the reaction mixture, and the mixture was stirred for 6 hours. The reaction solution was filtered to remove insoluble matter, and the filtrate was freeze-dried and purified by HPLC to obtain compound KJ-008 (288 mg, yield 73%), ESI-MS: m / z = 794.4 [M+H]+.

[0207] Synthesis of compound K43:

[0208]

[0209] Compound SZU-136 (213 mg, 0.5 mmol) and compound diethyl phosphorochloridate (87 mg, 0.5 mmol) were dissolved in 3 mL of chloroform, and DMAP (62 mg) was added and the mixture was stirred overnight. The reaction solution was washed three times with cold water (3 x 5 mL), the chloroform layer was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove chloroform. After standing, solid Phosp-136 (258 mg, yield 92%) was obtained. ESI-MS: m / z = 561.2 [M+H]+.

[0210] Compound Phosp-136 (101 mg, 0.18 mmol) and N3-PEG3-MC (67 mg, 0.18 mmol) were mixed and dissolved in 3 mL of DMF-H2O (4:1 volume). CuSO4·5H2O (12 mg) and sodium ascorbate (12 mg) were added, and the mixture was stirred and reacted at room temperature for 6 hours. The reaction solution was frozen, the lyophilized product was dissolved in a small amount of methanol to remove insoluble matter, and the filtrate was purified by HPLC to obtain compound K43 (94 mg, yield 56%), ESI-MS: m / z = 930.4 [M+H]+.

[0211] Synthesis of compound K44:

[0212]

[0213] A 2 mL chloroform solution of compound GY121 (145 mg, 0.5 mmol) was slowly added to a 1 mL chloroform solution of triphosgene (150 mg, 0.5 mmol). The reaction mixture was stirred at room temperature for 2 hours, followed by the addition of OMACR (65 mg, 0.5 mmol). The reaction mixture was then allowed to react overnight at room temperature. The reaction mixture was concentrated under reduced pressure and separated by silica gel column chromatography (dichloromethane-methanol 10:1) to give compound GY1211 (143 mg, yield 64%), ESI-MS: m / z = 446.2 [M+H]+.

[0214] Compound GY1211 (98 mg, 0.22 mmol) and compound N3-PEG4-N3 (64 mg) were dissolved in 5 mL of DMF-H2O (4:1 volume). CuSO4·5H2O (8 mg) and sodium L-ascorbate (8 mg) were added, and the mixture was stirred at room temperature for 6 hours. The mixture was filtered, the filtrate was freeze-dried, and purified by HPLC to obtain compound K44 (50 mg, yield 31%).

[0215] ESI-MS: m / z = 734.4 [M+H]+.

[0216] The synthesis of compound K45 followed the same route as compound K44, but with compound GY121 replaced by SZU-136, yielding compound K45. ESI-MS: m / z = 869.4 [M+H]+.

[0217]

[0218] Synthesis of compound K46:

[0219]

[0220] Compound KJ-7550 (100 mg, 0.087 mmol) and compound N3-PEG3-NCS (23 mg, 0.087 mmol) were dissolved in 2 mL of DMF-H2O (4:1 volume). A mixture of CuSO4·5H2O (5 mg) and L-sodium ascorbate (5 mg) was added and the mixture was stirred at room temperature for 12 hours. The reaction solution was filtered, the filtrate was lyophilized, and the lyophilized product was purified by HPLC to give compound K46 (85.8 mg, yield 70%), ESI-MS: m / z = 705.4 [(M / 2)+H]+.

[0221] Synthesis of compound K47:

[0222]

[0223] Equimolar amounts of compound KJ008 and phosgene were mixed in an appropriate amount of DMF, and two equivalent amounts of TEA were added. The mixture was stirred overnight at room temperature. The solvent was removed by freeze-drying, and the residue was purified by preparative liquid chromatography. The purified solution containing the product was collected and freeze-dried to obtain compound K47. ESI-MS: m / z

[0224] =622.2[(M / 2)+H]+.

[0225] Synthesis of compound K48:

[0226]

[0227] Following the synthesis method of KJ-7550, compound TCO-115 was obtained, ESI-MS: m / z = 908.5 [M+H]+; following the synthesis method of KJ-7551, compound K48 was obtained, ESI-MS: m / z = 1168.6 [M+H]+.

[0228] Synthesis of compound K49:

[0229]

[0230] Compound KJ-1b (100 mg, 0.3 mmol) and compound EB-ACRY (58 mg, 0.3 mmol) were dissolved in 1 mL of DMF, and anhydrous K2CO3 (100 mg) was added. The mixture was stirred at room temperature for 6 hours. The reaction solution was filtered, lyophilized, and purified by HPLC to obtain compound KJ-096 (70 mg, yield 52%), ESI-MS: m / z = 447.2 [M+H]+. Compound KJ-096 (65 mg) was added to 1 mL of concentrated hydrochloric acid and stirred overnight at room temperature. The mixture was concentrated under reduced pressure, and purified water was added. After stirring, the mixture was filtered to obtain compound KJ-097 hydrochloride (60 mg), ESI-MS: m / z = 419.2 [M+H]+. Compound KJ-097 hydrochloride (58 mg, 0.127 mmol) and PyAOP (70 mg, 0.134 mmol) were mixed in 1.5 mL of DMF, and DIPEA (88 μL) was added and stirred evenly for 30 min. DBCO-PEG4-amine TFA salt (67 mg, 0.128 mmol) was then added, and the mixture was stirred overnight at room temperature. The reaction solution was lyophilized, and the lyophilized product was purified by HPLC. The purified product solution was lyophilized to give compound K49 (81 mg, yield 69%), ESI-MS: m / z = 924.5 [M+H]+.

[0231] Synthesis of compound K50: Following the synthetic route of compound K49, replacing compound KJ-1b with compound KJ-1a yields compounds K50 {ESI-MS: m / z = 910.5 [M+H]+}; KJ-098 {ESI-MS: m / z = 433.3 [M+H]+}; and KJ-099 {ESI-MS: m / z = 405.2 [M+H]+}.

[0232] Synthesis of compounds K51 and K52:

[0233]

[0234]

[0235] Following the synthetic method of GY7550-1 used in the synthesis of compound K38, the reaction of compound KJ-096 with compound NCO-C2-NHBOC yields compound KJ-100 {ESI-MS: m / z = 533.3 [M+H]+}; the reaction of compound KJ-100 with DBCO-PEG4-Val-Cit-PAB-PNP yields compound K51.

[0236] {ESI-MS: m / z=736.8[(M / 2)+H]+}.

[0237] Similarly, replacing compound KJ-096 with compound KJ-098 yields compound KJ-101 {ESI-MS: m / z = 519.3 [M+H]+}; and compound K52 {ESI-MS: m / z = 729.9 [(M / 2)+H]+}:

[0238]

[0239] Synthesis of compound K53:

[0240]

[0241]

[0242] Compound GY100 (110 mg, 0.42 mmol) and compound N3-PIP (95 mg, 0.42 mmol) were dissolved in 2 mL of DMF-H2O (4:1 volume), and CuSO4·5H2O (10 mg) and sodium L-ascorbate (10 mg) were added. The mixture was reacted with shaking at room temperature for 6 hours. The solvent was removed by vacuum distillation, and 4 mL of TFA-DCM (1:3 volume) was added and stirred at room temperature for 2 hours. The solvent was removed by vacuum distillation, and compound GY225 (112 mg, yield 69%) was purified by HPLC. ESI-MS: m / z = 388.2 [M+H]+.

[0243] Following the synthetic methods of compounds KJ-096 and KJ-097, compounds GY226 {ESI-MS: m / z = 500.3 [M+H]+} and GY227 {ESI-MS: m / z = 472.2 [M+H]+} were obtained. Following the synthetic method of compound K49, compound K53 {ESI-MS: m / z = 977.5 [M+H]+} was obtained.

[0244] Synthesis of compound K54:

[0245]

[0246] Similarly, referring to the synthetic methods of compounds GY225, KJ-096, and KJ-097, compounds GY228 {ESI-MS: m / z = 402.2 [M+H]+}, GY229 {ESI-MS: m / z = 514.3 [M+H]+}, and GY300 {ESI-MS: m / z = 486.2 [M+H]+} can be obtained. Referring to the synthetic method of compound K49, compound K54 {ESI-MS: m / z = 991.5 [M+H]+} can be obtained.

[0247] Synthesis of compound K55:

[0248]

[0249]

[0250] Compound HO-PEG3-DBCO (481 mg, 1 mmol) was dissolved in 5 mL of anhydrous THF, and CMO-Chloride (86 μL) was slowly added. The reaction was carried out at room temperature for 3 hours under nitrogen protection. The solvent was removed by vacuum distillation to give compound CMO-PEG3-DBCO (616 mg, 100% yield), ESI-MS: m / z = 617.2 [M+H]+. Compound GY229 (103 mg, 0.2 mmol) and compound CMO-PEG3-DBCO (123 mg, 0.2 mmol) were mixed and dissolved in 3 mL of dichloromethane, and the reaction was stirred overnight at room temperature. The solvent was removed by vacuum distillation, and the residue was separated by silica gel column chromatography (chloroform) to give compound K55 (155 mg, 71% yield), ESI-MS: m / z = 1050.5 [M+H]+.

[0251] The synthesis of compound K56 was performed following the same methods as compounds GY7550-1 and K38. Compound K56 was obtained from compound GY226 (ESI-MS: m / z = 763.4 [(M / 2)+H]+):

[0252]

[0253] Synthesis of compound K57:

[0254]

[0255]

[0256] Compound FUMA-MOP (83 mg, 0.4 mmol) was dissolved in 2 mL of DMF, and PyAOP (210 mg, 0.4 mmol) and DIPEA (70 μL) were added and stirred until homogeneous. KJ-1b (134 mg, 0.4 mmol) was then added; the mixture was stirred at room temperature for 3 hours. The solvent was removed by vacuum distillation, and the solution was purified by silica gel column chromatography (MeOH-DCM = 1:5) to give compound KJ-102 (142 mg, yield 68%), ESI-MS: m / z = 523.3 [M+H]+. Following the synthetic methods of compounds GY7550-1 and K38, compound K57 {ESI-MS: m / z = 774.9 [(M / 2)+H]+} was obtained.

[0257] The synthesis of compound K58 followed the same method as compound K57, except that compound KJ-1b was replaced with KJ-1a, yielding compound KJ-104 (ESI-MS: m / z = 509.2 [M+H]+; and compound K58 {(ESI-MS: m / z = 767.9 [(M / 2)+H]+}):

[0258]

[0259] Synthesis of compound K59:

[0260]

[0261] Compounds Pro-123 (119 mg, 0.5 mmol) and BroQui (164 mg, 0.5 mmol) were dissolved in 3 mL of DMF. K2CO3 (140 mg) was added and the mixture was stirred at room temperature for 10 hours. The reaction solution was filtered, and the filtrate was freeze-dried. 10 mL of concentrated hydrochloric acid was added to the freeze-dried product and the mixture was stirred at room temperature overnight. The pH was neutralized with sodium carbonate, and the precipitated solid was filtered and dissolved in methanol. Further HPLC purification yielded compound KJ-178 (107 mg, yield 58%), ESI-MS: m / z = 369.2 [M+H]+. Compound KJ-179 (ESI-MS: m / z = 557.2 [M+H]+) was synthesized according to the method of PMQ-2. Compound KJ-179 (84 mg, 0.15 mmol) and K2CO3 (28 mg) were mixed in 2 mL of DMF, and DBCO-PEG4-VA-PAB-Cl (138 mg, 0.15 mmol) was added. The mixture was stirred overnight at room temperature. After filtration, the filtrate was freeze-dried and purified by HPLC to obtain compound K59 (101.5 mg, yield 47%). ESI-MS: m / z = 720.8 [(M / 2) + H]+.

[0262] Synthesis of DBCO-PEG4-VA-PAB-Cl: Equimolar amounts of DBCO-PEG4-Val-Ala-PAB and chloromethyl chloroformate were reacted in dry DCM at room temperature for 10 hours. The solvent was removed by depressurized distillation to obtain the compound DBCO-PEG4-VA-PAB-Cl (ESI-MS: m / z = 920.4 [M+H]+).

[0263] The synthesis of compound K60 followed the method used for compound K59. Using SZU-103 as the starting material, compound K60 {ESI-MS: m / z = 756.8 [(M / 2) + H] +} was obtained.

[0264]

[0265] Synthesis of compound K61:

[0266]

[0267]

[0268] In a 2 mL H2O-DMF (1:4) solution containing compound GY121 (87 mg, 0.3 mmol) and N3-PEG3-COOEt (79 mg, 0.3 mmol), CuSO4·5H2O (10 mg) and sodium L-ascorbate (10 mg) were added, and the mixture was subjected to a Click reaction at room temperature for 6 hours. The mixture was filtered, the filtrate was freeze-dried, and compound GY1401 (111 mg, yield 67%) was purified by HPLC. ESI-MS: m / z = 551.3 [M+H]+. Compound GY1401 (100 mg, 0.18 mmol) and triphosgene (32 μL, 0.19 mmol) were dissolved in 3 mL of DMF. DMAP (22 mg) was added and the mixture was stirred at room temperature for 3 hours. Then, PZ-ACRY-PEG4-DBCO.TFA (142 mg, 0.18 mmol) and DMAP (33 mg) were added sequentially and the mixture was stirred at room temperature overnight. The reaction solution was freeze-dried, and the freeze-dried product was extracted with ethyl acetate (3 x 15 mL). The combined organic phases were separated and washed once with 30 mL of saturated sodium bicarbonate solution and once with saturated sodium chloride solution. The organic phase was separated and dried with anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by HPLC to give compound K61 (79 mg, yield 35%). ESI-MS: m / z = 626.8 [(M / 2) + H] +.

[0269] Synthesis of compound PZ-ACRY-PEG4-DBCO.TFA:

[0270] Equimolar amounts of compounds BPC and MBA were subjected to a substitution reaction in DMF at room temperature. After the reaction was monitored by mass spectrometry, the solvent was removed by vacuum distillation, saturated sodium bicarbonate solution was added and stirred thoroughly, ethyl acetate was added to extract the product, the organic phase was washed with water and dried with anhydrous sodium sulfate, filtered, and the filtrate was distilled to dryness to obtain compound BMAPC. BMAPC was dissolved in methanol, and 3 equivalents of LiOH·H2O were added and stirred at room temperature until the reaction was complete. The solvent was removed by vacuum distillation, pure water was added, and the pH was adjusted to 6 with acetic acid, precipitating a solid compound. The solid compound was filtered and dried to obtain compound BPMAA (ESI-MS: m / z = 271.2 [M+H]+).

[0271] Compound BPMAA (81 mg, 0.3 mmol) and compound DBCO-PEG4-amine TFA salt (191 mg, 0.3 mmol) were dissolved in 3 mL of DMF. DIPEA (157 μL), NHS (38 mg, 0.33 mmol), and EDC·HCl (64 mg, 0.33 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was lyophilized, and 10 mL of TFA-DCM (1:3 volume) solvent was added to the lyophilized product. The mixture was stirred at room temperature for 2 hours, and the solvent was removed by vacuum distillation. The residue was purified by preparative HPLC to obtain the TFA salt of compound PZ-ACRY-PEG4-DBCO (104 mg, yield 44%). ESI-MS: m / z =

[0272] 676.3[M+H]+.

[0273] Synthetic route of compound K62:

[0274]

[0275]

[0276] Synthesis of compound BA514-ACID: Compound OTS514 (110 mg, 0.3 mmol) and compound MBA (54 mg, 0.3 mmol) were mixed and dissolved in 2 mL of DMF. Cs2CO3 (98 mg, 0.3 mmol) was added, and the mixture was stirred at room temperature. Mass spectrometry was used to monitor the reaction until completion. The reaction solution was freeze-dried, and the lyophilized product was purified by HPLC to obtain compound MBA514.TFA (58 mg, ESI-MS: m / z = 463.2 [M+H]+). MBA514.TFA (50 mg, 0.086 mmol) was dissolved in 1 mL of DMF, cooled to 5 °C, and DMAP (22 mg) was added. Separately, (Boc)2O (20 μL) was dissolved in 0.5 mL of DMF and slowly added to the MBA514 solution. The mixture was allowed to rise naturally to room temperature and stirred for 12 hours. The product was freeze-dried, and the freeze-dried product was purified by HPLC to obtain a product-containing separation solution. The product was then freeze-dried to obtain compound BOCMBA514 (28 mg), which was dissolved in 1 mL of methanol. 0.1 mL of LiOH·H2O (10 mg) aqueous solution was added, and the mixture was shaken at room temperature until the reaction was complete. The solvent was removed by vacuum distillation, and 1 mL of water was added. The pH was adjusted to 3 by dilute hydrochloric acid under cooling. The precipitated solid was centrifuged and filtered, and then dried under vacuum to obtain compound BA514-ACID (25 mg, ESI-MS: m / z = 549.2 [M+H]+).

[0277] Synthesis of compounds PZ-100 and P-Z225: Following the synthesis method of compound PZ-136, compound SZU-136 was replaced with GY100 to obtain compound PZ-100 (ESI-MS: m / z = ).

[0278] 388.2 [M+H]+). Following the synthesis method of GY225, the TFA salt of PZ-225 can be obtained (ESI-MS: m / z = 514.3 [M+H]+).

[0279] Synthesis of compound BP514-225: Compound BA514-ACID (100 mg, 0.18 mmol) and condensing agent PyAOP (100 mg, 0.19 mmol) were dissolved in 2.5 mL of DMF, and DIPEA (50 μL) was added and mixed thoroughly. Separately, a DMF solution (2.5 mL) of the TFA salt of compound PZ-225 (125 mg, 0.18 mmol) and DIPEA (50 μL) was slowly added to the above BA514-ACID solution. The reaction mixture was shaken and reacted overnight at room temperature. The reaction mixture was lyophilized, and 10 mL of TFA-DCM (1:3 volume) was added to the lyophilized product. The mixture was stirred naturally at room temperature for 1 hour. The solvent was removed by distillation under reduced pressure. The residue was purified by HPLC to obtain the TFA salt of compound BP514-225 (93 mg, yield 49%), ESI-MS: m / z = 945.4 [M+H]+.

[0280] Synthesis of compound K62: BP514-225.TFA (53 mg, 0.05 mmol) and DMAP (10 mg) were dissolved in 1 mL of DMF. A DMF solution (1 mL) of Mal-PEG4-Val-Cit-PAB-PNP (44 mg, 0.05 mmol) was slowly added dropwise to the BP514-225.TFA solution at room temperature. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction solution was lyophilized, and the lyophilized product was purified by HPLC to obtain the TFA salt of compound K62 (28 mg, yield 31%). ESI-MS: m / z = 838.5 [(M / 2) + H]+.

[0281] Synthesis of compound K63:

[0282]

[0283] Compound KJ-3b (95 mg, 0.25 mmol) was dissolved in 2 mL of anhydrous ethanol, and 0.2 mL of 30% H2O2 solution was added. The mixture was heated to 70 °C and reacted for 5 hours. Then, 0.2 mL of 30% H2O2 solution was added, and the reaction continued overnight. A small amount of 10% Pd / C was added, and the mixture was heated to 70 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by HPLC to obtain compound KJ-3b-O (57 mg, yield 58%), ESI-MS: m / z = 395.2 [M+H]+.

[0284] Referring to the synthesis methods of compounds GY7550-1 and K38, compound K63{ESI-MS: m / z=710.8[(M / 2)+H]+} can be obtained.

[0285] Synthesis of compound K64:

[0286]

[0287] OTS514 (182 mg, 0.5 mmol) was dissolved in 3 mL of DMF, and K2CO3 (70 mg) was added and stirred until homogeneous. Separately, bromohexyne (81 mg, 0.5 mmol) was dissolved in 1 mL of DMF and slowly added dropwise to the OTS514 solution under stirring at room temperature, allowing the reaction to proceed overnight. The reaction mixture was filtered, concentrated under reduced pressure, and purified by HPLC to obtain compound OTS5141 (147 mg, yield 66%), ESI-MS: m / z = 445.2 [M+H]+. Compound OTS5141 (120 mg, 0.27 mmol) was dissolved in 2 mL of H2O-DMF (3 mL, 1:3 volume ratio), and a mixture of GY155 (137 mg, 0.27 mmol), CuSO4·5H2O (8 g mg), and L-ascorbic acid sodium (8 mg) was added and stirred at room temperature for 6 hours. The reaction solution was filtered, and the filtrate was freeze-dried. The freeze-dried product was purified by HPLC to obtain compound GY191 (198 mg, yield 77%), ESI-MS: m / z = 950.4 [M+H]+. GY191 (190 mg, 0.2 mmol) was dissolved in 1 mL of DMF, and 50 mg of anhydrous K2CO3 was added and stirred until homogeneous. Separately, MBA (36 mg, 0.2 mmol) was dissolved in 2 mL of DMF and slowly added to the GY191 solution over 3 hours at room temperature. After the addition was complete, the mixture was stirred overnight at room temperature. The reaction solution was filtered, and the filtrate was freeze-dried. The freeze-dried product was dissolved in 3 mL of methanol, and LiOH·H₂O (50 mg) was added. The mixture was stirred at room temperature for 10 hours. Methanol was removed by vacuum distillation, and 5 mL of pure water was added. The pH was adjusted to 5 with dilute hydrochloric acid, stirred evenly, and then freeze-dried. The freeze-dried product was purified by HPLC to obtain compound ACID-191 hydrochloride (103 mg), ESI-MS: m / z = 1034.5 [M+H]+. ACID-191 hydrochloride (95 mg, 0.09 mmol) and PyAOP (52 mg, 0.1 mmol) were dissolved in 2 mL of DMF, and DIPEA (70 μL) was added. After stirring evenly, Boc hexamethylenediamine (20 μL) was added, and the mixture was stirred at room temperature overnight. After freeze-drying, 3 mL of TFA-DCM (1:3) solvent was added and stirred for 1 hour to remove Boc. The solvent was removed by vacuum evaporation, and the compound AMINE-191.2TFA salt (67 mg, yield 55%) was purified by HPLC. ESI-MS: m / z = 1132.6 [M+H]+.

[0288]

[0289]

[0290] Following the synthesis reaction of ACID-191 hydrochloride and Boc hexamethylenediamine, equimolar reactions of AMINE-191.2TFA salt and SZU-160 yield compound 160-191 {ESI-MS: m / z = 857 [(M / 2) + H]} +}

[0291]

[0292] Compounds 160-191 and Propargyl-PEG3-amine were subjected to a standard click reaction in a DMF-H2O (3:1) system catalyzed by copper sulfate and sodium L-ascorbate. The resulting compound AMINE-64 was purified by HPLC. (ESI-MS: m / z = 950 [(M / 2) + H]) +}

[0293]

[0294] The TFA salt of compound AMINE-64 (106 mg, 0.05 mmol) was dissolved in 1 mL of DMF, and CS2 (20 mg) and TEA (35 μL) were added. The mixture was stirred overnight at room temperature. TsCl (p-toluenesulfonyl chloride, 10 mg) was added, and the reaction was continued at room temperature until completion (MS monitoring). The reaction solution was filtered, and the filtrate was lyophilized and purified by HPLC to obtain the TFA salt of compound K64 (43 mg, yield 42%). ESI-MS: m / z = 972.2 [(M / 2) + H] + .

[0295] Synthesis of compound K65: Following the synthetic route of compound K24, compound BP-22468 was replaced with compound SZU-191 {ESI-MS: m / z = 1042.5 [M+H]}. +}, thus obtaining compound K65 {ESI-MS: m / z = 774.8 [(M / 2) + H] +},

[0296]

[0297] Synthesis of compound K66:

[0298]

[0299]

[0300] Compounds N3-FMOC-LYS (119 mg, 0.3 mmol), NHS (38 mg, 0.33 mmol), and EDCI (63 mg) were dissolved sequentially in dry DMF (3 mL). The mixture was stirred at room temperature for 1 hour, followed by the addition of GY191 (285 mg, 0.3 mmol). The reaction was then continued overnight at room temperature. The reaction solution was lyophilized, and the lyophilized product was purified by silica gel column chromatography (DCM / MeOH = 5:1) to obtain compound 191-1 (247 mg, yield 62%), ESI-MS: m / z = 664.3 [(M / 2) + H]. + .

[0301] Compound 191-1 (200 mg, 0.15 mmol) and compound Propargyl-PEG4-Maleimide (60 mg, 0.15 mmol) were mixed and dissolved in 5 mL of DMF / H2O (3:1 volume). C was then added. u The mixture of SO4·5H2O (10 mg) and L-ascorbic acid sodium (10 mg) was stirred at room temperature for 3 hours. The reaction solution was filtered, the filtrate was lyophilized, and 3 mL of DMF and piperidine (0.5 mL) were added to the lyophilized product. The mixture was stirred at room temperature overnight. The reaction solution was lyophilized again, and the lyophilized product was purified by HPLC to give compound 191-2 (124 mg, yield 55%), ESI-MS: m / z = 751.4 [(M / 2)+H] + .

[0302] Compound 191-2 (100 mg, 0.067 mmol) was dissolved in 2 mL of DMF, and TEA (0.1 mL) and Allyl-NCS (10 μL) were added. The mixture was shaken and reacted overnight at room temperature. The reaction mixture was lyophilized, and the lyophilized product was purified by HPLC to obtain compound K66 (51.5 mg, yield 48%), ESI-MS: m / z = 801.0 [(M / 2) + H]. + .

[0303] Synthesis of compound K67:

[0304]

[0305]

[0306] Compound SZU-136 (425 mg, 1 mmol) was dissolved in 10 mL of THF-DCM (1:1, v / v). DMAP (60 mg) was added and stirred until dissolved. Fmoc-gly-cl (316 mg, 1 mmol) was then added, and the reaction was allowed to proceed overnight at room temperature. 1 mL of piperidine was added to the reaction mixture, and the reaction was continued at room temperature with stirring for 3 hours. The solvent was removed by vacuum distillation, and the residue was separated by silica gel column chromatography (MeOH / DCM = 1:5, v / v) to give compound GLY-136 (265 mg, 55% yield). ESI-MS: m / z = 482.2 [M+H] + Compound GLY136 (240 mg, 0.5 mmol) was dissolved in DMSO (3 mL), and 0.2 mL of triethylamine (TEA) and 50 mg of PROP-NCS were added. The mixture was stirred at room temperature for 6 hours. The reaction solution was lyophilized, and the lyophilized product was separated by silica gel column chromatography (MeOH / DCM = 1:5, v / v) to give compound PROP-136 (215 mg, yield 74%), ESI-MS: m / z = 581.2 [M+H]. + .

[0307] Compound N3-PEG3-NH2 (66 mg, 0.3 mmol) and compound Mal-NHS (80 mg, 0.3 mmol) were dissolved in 3 mL of DMF-H2O (2:1, v / v). After stirring at room temperature for 1 hour, PROP-136 (175 mg, 0.3 mmol), CuSO4·5H2O (10 mg), and L-sodium ascorbate (10 mg) were added. The resulting mixture was stirred at room temperature overnight. The reaction solution was filtered, the filtrate was lyophilized, and the lyophilized product was purified by preparative HPLC. The purified solution was concentrated under reduced pressure, and the concentrate was lyophilized to give compound K67 (111 mg, yield 39%). ESI-MS: m / z = 950.4 [M+H] + .

[0308] Example 2. In the following examples, the PD-L1 antibody (Atezolizumab, CAS number: 1380723-44-3, sequence reference: https: / / go.drugbank.com / drugs / DB11595) was used.

[0309] https: / / www.genome.jp / dbget-bin / www_bget?dr:D10773)

[0310] Purchased from MedChemExpress; purified by dialysis.

[0311] The steps for preparing antibody samples for detection are as follows:

[0312] Take 20 μg of the sample to be tested into an EP tube, add 2 μL of GlycoBuffer 2 (10X), and then add ultrapure water to make the final volume of the system 20 μL. Dilute the above sample 10 times (final concentration 0.1 mg / mL) and test the sample according to the detection parameters below.

[0313] The antibody detection parameters and mass spectrometry conditions are as follows:

[0314] (a) Mass spectrometry conditions:

[0315] 1. Instruments and equipment

[0316]

[0317] 2. Reagents, test solutions

[0318] Acetonitrile NA LC-MS Merck Formic acid F298780 LC-MS aladdin Purified water NA NA Watsons

[0319] 3. Liquid Chromatography Conditions

[0320] (1) Mobile phase gradient

[0321] 1min 80％ 5％ 8min 20％ 80％ 12min 20％ 80％ 12.1min 80％ 5％

[0322] (2) Detection parameters

[0323] Detection wavelength <![CDATA[ NA ]]> Flow rate <![CDATA[ 0.3 mL / min]]> Injection volume <![CDATA[ 10μL ]]> Column temperature <![CDATA[ 60℃ ]]> Collection time <![CDATA[ 12 min]]> Washout mode Gradient elution

[0324] 4. Mass spectrometry conditions

[0325]

[0326]

[0327] (II) Molecular weight and mass spectrometry analysis of naked antibodies and conjugated antibodies, taking PD-L1 antibody Atezolizumab as an example:

[0328] Take 20 μg of the antibody sample to be tested into an EP tube, add 2 μL of GlycoBuffer 2 (10X), and then add ultrapure water to make the final volume of the system 20 μL. Dilute the above sample 10 times (final concentration 0.1 mg / mL) and test the sample according to the instrument detection parameters mentioned above. The molecular weight of Atezolizumab was determined to be 144590.5 by mass spectrometry deconvolution detection. The molecular weight mass spectrometry analysis method for the conjugated antibody is the same as that for the naked antibody.

[0329] (III) Examples of maleimide group coupling

[0330] Conjugation methods, exemplified by compound K3 and antibody PD-L1:

[0331]

[0332] Dissolve PD-L1 antibody (Atezolizumab, 90 mg) in 20 mL of DPBS (20 mg / mL) and adjust the pH to 7.0 with 5 mM EDTA solution. Add 12 equivalents of TCEP solution (using 5 mM TCEP aqueous solution) and reduce at room temperature for 3 hours. After purification by filtration through a 30 kDa Vivaspin ultrafiltration centrifugal concentrator, directly replace the medium in 20 mL of 4.2 mM histidine solution.

[0333] The histidine solution (5 mL) of the above-mentioned reducing antibody was adjusted to pH 7.5-8 using Tris buffer; 150 μL of N,N-dimethylacetamide solution containing 12 mg of compound K3 was added. This reaction mixture was shaken at 20°C for 6 hours. After purification by ultrafiltration using a 30 DK Vivaspin centrifugal concentrator, the medium was changed to obtain a histidine solution of Atezolizumab conjugated with K3 (Atezolizumab-K3, PD-L1-K3) at 4.2 mM. Molecular weight and degree of conjugation (DAR) mass spectrometry analysis was performed. The average molecular weight was determined to be 151606 by SEC-QTOF-MS, and the average DAR was 4.12.

[0334] Similarly, other K-series compounds, such as PD-L1-KX, coupled with maleimide groups and Atezolizumab, were also studied:

[0335]

[0336] The PD-L1 antibody is Atezolizumab, where X represents the ordinal number of compound K and n represents the DAR value. The detected DAR values ​​are between 3.5 and 4.5, as shown in Table 1.

[0337] Table 1.

[0338]

[0339] (iv) An example of antibody-coupled with an azide compound (using compound K23 as an example):

[0340]

[0341]

[0342] The activated ester DBCO-acid, DBCO-ESTER (20 eq), was mixed with the PD-L1 antibody Atezolizumab (1 eq) in pure water containing 10% DMSO and reacted with shaking at 10°C for 12 hours. Mass spectrometry was used to detect the complete antibody reaction. Small molecules were removed from the reaction mixture using a 10 kDa molecular filter, and the conjugate compound PD-L1-DBCO was eluted with DPBS solution, yielding a degree of conjugation of 4. 10% DMSO and 1.5 equivalents of compound K23 were added to the DPBS solution of the conjugate antibody PD-L1-DBCO, and the mixture was reacted with shaking at 25°C for 12 hours. Small molecules were removed using a 20 kDa molecular filter. The antibody was dissolved in DPBS to obtain a DPBS solution of the conjugate antibody compound PD-L1-K23. SEC-QTOF-MS determined the average molecular weight to be 152203, and the average degree of conjugation to be 4.

[0343] Using the same route and method as that used to prepare PD-L1-K23, conjugates of compounds K11, K21, K44, and K45 coupled with PD-L1 antibodies were obtained (DAR=4): PD-L1-K11 (average molecular weight 150549.3), PD-L1-K21 (average molecular weight 152693.1), PD-L1-K44 (average molecular weight 147541.6), and PD-L1-K45 (average molecular weight 148066.1).

[0344]

[0345] General formula II represents PD-L1-K11, PD-L1-K21, PD-L1-K23, PD-L1-K44, and PD-L1-K45, where the PD-L1 antibody is Atezolizumab.

[0346] (V) Example of antibody conjugation using DBCO as the conjugating group (taking compound K57 as an example): Referring to the preparation method of PD-L1-K23, replace compound DBCO-ESTER with Azido-PEG3-NHS ester, and replace compound K23 with compound K57 to obtain the antibody-conjugated product PD-L1-K57 (average molecular weight measured by SEC-QTOF-MS: 153825.3, conjugation ratio DAR = 4):

[0347]

[0348] Similarly, using the same route and method as for preparing PD-L1-K57, conjugates (DAR=4) of compounds K28, K28-1, K31, K35, K36, K37, K38, K39, K49, K50, K51, K52, K53, K54, K55, K56, K57, K58, K59, K60, K61, and K63 conjugates of PD-L1 antibodies can be obtained: PD-L1-K28, PD-L1-K28-1, PD-L1-K31, PD-L1-K35, and PD-L1-K57. K36,PD-L1-K37,PD-L1-K38,PD-L1-K39,PD-L1-K49,PD-L1-K50,PD-L1-K51,PD-L1-K52,PD-L1-K53,PD-L1-K54, PD-L1-K55, PD-L1-K56, PD-L1-K57, PD-L1-K58, PD-L1-K59, PD-L1-K60, PD-L1-K61, PD-L1-K63, PD-L1-K65, see Table 2:

[0349]

[0350] The PD-L1 antibody is Atezolizumab.

[0351] Table 2.

[0352] molecular weight 153348.5 152912.0 153056.3 152123.4 152443.7 153825.3 153404.6 Coupler PD-L1-K39 PD-L1-K49 PD-L1-K50 PD-L1-K51 PD-L1-K52 PD-L1-K53 PD-L1-K54 molecular weight 151494.1 149204.0 149147.9 151398.4 151342.3 149416.1 149472.2 Coupler PD-L1-K55 PD-L1-K56 PD-L1-K57 PD-L1-K58 PD-L1-K59 PD-L1-K60 PD-L1-K61 molecular weight 151098.0 151610.5 151702.6 151646.5 151277.9 151561.5 150517.3 Coupler PD-L1-K63 PD-L1-K65 molecular weight 151190.1 151698.5

[0353] (vi) Examples of antibodies conjugated with compounds using carboxylic acid as the coupling group (taking compound K4 as an example):

[0354]

[0355]

[0356] PD-L1 antibody (Atezolizumab, 100 mg) was dissolved in 20 mL of PBS (PD-L1 monoclonal antibody solution). A mixture of 6 mg of EDC·HCl and 44 mg of K4 was added to 2 mL of anhydrous DMSO solution of compound NHS (N-hydroxysuccinimide, 3 mg), and stirred at room temperature for 1 hour. This mixture was then added to the PD-L1 monoclonal antibody solution for conjugation. 5 μL of DIPEA was added to the reaction mixture at 10°C, and the mixture was stirred overnight at 25°C. The mixture was then ultrafiltered using a 30 kDa MWCO centrifuge tube to replace the antibody with purified water, and then freeze-dried to obtain the antibody-conjugated K4 product.

[0357] PD-L1-K4: 71 mg. The average molecular weight was 150,689 as determined by SEC-QTOF-MS, and the coupling ratio DAR was 3.89.

[0358] Similarly, using the same route and method as for preparing PD-L1-K4, conjugates of compounds K5, K9, and K12 conjugated with PD-L1 antibodies can be obtained: PD-L1-K5 (average molecular weight 152202, conjugation ratio DAR = 3.97), PD-L1-K9 (average molecular weight 148620.8, conjugation ratio DAR = 3.78), and PD-L1-K12 (average molecular weight 149520.4, conjugation ratio DAR = 4.01).

[0359] The PD-L1 antibody is Atezolizumab.

[0360] (vii) Examples of antibodies conjugated with compounds using isothiocyanate as the coupling group (taking compound K46 as an example):

[0361]

[0362] PD-L1 antibody (Atezolizumab, 100 mg) and 5 μL of TEA were dissolved in 20 mL of PBS (PD-L1 monoclonal antibody solution). 0.5 mL of DMSO solution containing compound K46 (20 mg, 20 molar volume) was added to the PD-L1 monoclonal antibody solution, and the mixture was stirred at 25°C for 6 hours. The mixture was then subjected to ultrafiltration using a 30 kDa MWCO centrifuge tube, and the antibody was replaced with purified water. The product, PD-L1-K46 (102 mg), was obtained by freeze-drying. The average molecular weight was determined to be 153042 by SEC-QTOF-MS, and the conjugation ratio (DAR) was 5.99.

[0363] Similarly, using the same route and method as for preparing PD-L1-K46, conjugates of compounds K40, K47, K48, and K64 conjugated with PD-L1 antibodies can be obtained: PD-L1-K40 (average molecular weight 150855.6, conjugation ratio DAR = 5.96), PD-L1-K47 (average molecular weight 152026.8, conjugation ratio DAR = 5.98), PD-L1-K48 (average molecular weight 151612.5, conjugation ratio DAR = 6.01), and PD-L1-K64 (average molecular weight 156108.3, conjugation ratio DAR = 5.93).

[0364] The PD-L1 antibody is Atezolizumab.

[0365] Example 3. ELISA detection method for compound immune cell activating factor (IFN-γ)

[0366] The immunomodulatory activity of each compound was expressed by its ability to induce the production of the immune cytokine IFN-γ by human peripheral monocytes (PBMCs), and their EC50 values ​​are shown in Table 3.

[0367] Preparation of human peripheral monocytes (PBMCs):

[0368] 1) Collect peripheral blood from healthy volunteers into heparin anticoagulant tubes, take 5 mL of fresh blood, and add PBS to dilute the blood at a 1:1 ratio (generally, the blood collection tube contains 2 mL + 2 mL).

[0369] 2) Take 5 mL of lymphocyte separation fluid into a 15 mL sterile centrifuge tube for later use.

[0370] 3) Draw up diluted blood and slowly add it along the test tube wall 1 cm above the separating liquid, so that the diluted blood overlaps on the separating liquid and forms a clear interface with the separating liquid.

[0371] 4) Centrifuge at room temperature, 2000 rpm, for 20 minutes. At this point, five layers will form in the centrifuge tube: the top layer is plasma, and the layer between the plasma layer and the lymphocyte separation medium is a white membrane-like layer of lymphocytes.

[0372] 5) Carefully aspirate the mononuclear cell layer in the middle, which is a white membrane, and try to remove all the mononuclear cells. Wash twice with more than 5 volumes of PBS, centrifuging at 1500 rpm for 10 min each time.

[0373] 6) Remove the supernatant, add 1 mL of 1640 culture medium and mix well. Take a small amount for cell counting.

[0374] General method:

[0375] The prepared PBMCs were seeded into 96-well plates (Costar, 3894), with 2 x 10⁵ cells per well. Ten uniformly distributed concentrations (0.0001–10 μM, RPMI-1460 medium containing 0.1% DMSO) of each compound from Table 3 were added to the wells containing the cells. The cells were then incubated at 37°C for 24 hours in a 5% CO₂ incubator. The IFN-γ concentration in the cell culture supernatant was measured using the Human-IFN-γ kit (Cisbio) according to the kit's instructions, and the EC50 values ​​of each compound were calculated (see Table 3).

[0376] Table 3.

[0377]

[0378]

[0379]

[0380] Pretreatment notes for compound samples before immune cell activating factor assay:

[0381] Note a: Enzyme metabolism method

[0382] Human liver microsomal enzyme kit (Model MVT-1.0, Beijing Huizhi Taikang Pharmaceutical Technology Co., Ltd.):

[0383] 1) Thaw all components of the kit in an ice bath and place on ice for later use; 2) Mix the components to be tested according to the set concentration ratio and pre-incubate at 37°C for 5 min; 3) Aliquot 195 μL / tube of the above mixture into centrifuge tubes, incubate in a 37°C water bath, add 5 μL of liver microsomes to each sample, mix by aspiration and retraction 3 times, and start the metabolic reaction under 37°C water bath conditions; 4) At the 2-hour time point, add the stop solution to the incubation system to terminate the reaction; Filter the filtrate through a 20K molecular membrane, and then test each component according to the general method to determine the EC50 value.

[0384] Note b: Glutathione reduction method

[0385] Take a PBS buffer solution with pH 6, mix it according to the set concentration ratio, add an equimolar amount of glutathione, stir at 37°C for 24 hours under nitrogen protection, and then test each component according to the general method to determine the EC50 value.

[0386] Note c: Enzyme lysis method

[0387] Each compound was mixed with Cathepsin-B enzyme (10 μg, Cat.#:10483-H08H, SinoBiological) in PBS at the set concentration ratio and incubated at room temperature for 3 hours. The filtrate was filtered through a 20K molecular membrane, and each component was tested according to a general method to determine the EC50 value.

[0388] Note d: Hydrolysis method

[0389] Take an acetic acid / sodium acetate buffer solution with pH 5, mix it according to the set concentration ratio, stir at 37°C for 72 hours, and then test each component according to the general method to determine the EC50 value.

[0390] Note e: Phosphodiesterase method

[0391] Each compound was mixed with phosphodiesterase I (10 μg, Cat.#: P4506, Sigma-Aldrich) in PBS at the set concentration ratio and incubated at room temperature for 3 hours. The filtrate was filtered through a molecular membrane at 20 K, and each component was tested according to a general method to determine the EC50 value.

[0392] Example 4. Validation of the antitumor effect of the representative K-series compound conjugate antibody (PD-L1-KX: X represents the serial number of the K-series compound) in this invention.

[0393] Example of establishing a mouse colorectal cancer tumor model and tumor suppression experiments:

[0394] MC38-hPD-L1 cells (a humanized MC38 colon cancer cell line expressing human PD-L1 protein, from Nanmo Biotechnology) in logarithmic growth phase were collected by digestion and centrifugation. After washing twice with PBS, the cells were counted, and the cell concentration was adjusted to 2.3 × 10⁻⁶. 5 Cells / mL. The mice were 6-week-old SPF-grade humanized C57BL / 6 mice. The hair on the right back of the mice was shaved clean with a shaver. 100 μL of cell suspension was drawn up with a syringe, and after removing air bubbles, it was injected subcutaneously into the back of the mice. Drug administration began when the tumor diameter reached between 4 and 5 mm.

[0395] Evaluation of the therapeutic effect and antitumor effect of the compound in tumor-bearing mice: Tumor-bearing mice (BALB / c) were randomly divided into three groups: Control group, Atezolizumab antibody group (PD-L1 antibody group), and PD-L1-KX conjugate group (Atezolizumab conjugate compound KX), with 5 mice in each group. Injectable solutions were prepared according to the dosage (solvent composition: 5% DMSO; +40% PEG300; +5% Tween 80; +50% PBS). The dosage was 3 mg / kg for the PD-L1 antibody group and 4 mg / kg for the PD-L1-KX conjugate group, administered intraperitoneally, 100 μL each time. Administration was performed on days 8 and 12 after tumor implantation, for a total of two injections. Tumor volume was measured with calipers every three days at the start and after administration, and the survival status of the mice was recorded. Tumor volume was calculated as: 0.5 × a × b. 2 Where a is the major axis and b is the minor axis. When the tumor diameter of a mouse reaches 2 cm, it should be euthanized by cervical dislocation according to animal ethics, and the tumor tissue should be removed to calculate the tumor inhibition rate {(Control group tumor volume – Coupled group tumor volume / Control group tumor volume) X100%}. The results are shown in Table 4.

[0396] The K-series compounds of this invention, conjugated with PD-L1 antibodies, significantly enhance the anti-tumor effect targeting PD-L1 (tumor volume inhibition rate against mouse colon cancer MC38-hPD-L1 cells, compared to 67% for Atezolizumab), as shown in Table 3 below (where PD-L1-KX represents the product of each K-series compound conjugated with Atezolizumab in formulas I-IV, and X represents the serial number of the K-series compound):

[0397] Table 4.

[0398] Tumor inhibition rate % 100 79 95 92 93 PD-L1-KX PD-L1-K6 PD-L1-K7 PD-L1-K8 PD-L1-K9 PD-L1-K10 Tumor inhibition rate % 94 83 91 78 82 PD-L1-KX PD-L1-K11 PD-L1-K12 PD-L1-K13 PD-L1-K14 PD-L1-K15 Tumor inhibition rate % 86 88 73 82 83 PD-L1-KX PD-L1-K16 PD-L1-K17 PD-L1-K18 PD-L1-K19 PD-L1-K20 Tumor inhibition rate % 93 81 79 83 89 PD-L1-KX PD-L1-K21 PD-L1-K22 PD-L1-K23 PD-L1-K24 PD-L1-K25 Tumor inhibition rate % 81 91 77 73 77 PD-L1-KX PD-L1-K26 PD-L1-K27 PD-L1-K28 PD-L1-K28-1 PD-L1-K29 Tumor inhibition rate % 79 96 95 91 75 PD-L1-KX PD-L1-K30 PD-L1-K31 PD-L1-K32 PD-L1-K33 PD-L1-K34 Tumor inhibition rate % 81 91 73 76 82 PD-L1-KX PD-L1-K35 PD-L1-K36 PD-L1-K37 PD-L1-K38 PD-L1-K39 Tumor inhibition rate % 91 93 89 84 76 PD-L1-KX PD-L1-K40 PD-L1-K41 PD-L1-K42 PD-L1-K43 PD-L1-K44 Tumor inhibition rate % 87 91 80 72 71 PD-L1-KX PD-L1-K45 PD-L1-K46 PD-L1-K47 PD-L1-K48 PD-L1-K49 Tumor inhibition rate % 78 92 79 83 79 PD-L1-KX PD-L1-K50 PD-L1-K51 PD-L1-K52 PD-L1-K53 PD-L1-K54 Tumor inhibition rate % 98 79 77 84 73 PD-L1-KX PD-L1-K55 PD-L1-K56 PD-L1-K57 PD-L1-K58 PD-L1-K59 Tumor inhibition rate % 74 89 87 75 95 PD-L1-KX PD-L1-K60 PD-L1-K61 PD-L1-K62 PD-L1-K63 PD-L1-K64 Tumor inhibition rate % 93 89 87 75 93 PD-L1-KX PD-L1-K65 PD-L1-K66 PD-L1-K67 Tumor inhibition rate % 88 91 97

[0399] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An immunostimulatory small molecule compound conjugated with an antibody, characterized in that, The general formula of the immunoactivating small molecule compound of the conjugated antibody is shown below: ， The linking functional groups are maleimide groups, carboxyl groups, and isothiocyanates; the linking chain is an alkyl chain; the KX-body is the main part of the immune-activating small molecule KX; the antibody is a PD-L1 antibody, specifically atezolizumab; and the immune-activating small molecule compound is one of the following: ， ， ， ， ， ， , ， ， ， ， , ， , ， ， ， ， ， ， ， ， ， ， ， , ， , , , , , , , ， , ， ， 。 2. The immunoactivating small molecule compound conjugated antibody of claim 1, wherein, The linking functional group is a maleimide group, and the immunoactivating small molecule compound of the conjugated antibody is: ， KX is an immune-activating small molecule compound, n is the DAR value, and the DAR value is between 3.5 and 4.

5.

3. The immunoactivating small molecule compound conjugated antibody of claim 1, wherein, The linking functional group is a carboxyl group, and the immunoactivating small molecule compound of the conjugated antibody is: ， The general formula IV represents the following conjugated antibodies: PD-L1-K4, PD-L1-K5, PD-L1-K9, and PD-L1-K12.

4. The immunoactivating small molecule compound conjugated antibody of claim 1, wherein, The linking functional group is an isothiocyanate group, and the immunoactivating small molecule compound of the conjugated antibody is: , Wherein, general formula V is the following conjugated antibodies: PD-L1-K46, PD-L1-K47, PD-L1-K48, PD-L1-K64.

5. An immunoactivating small molecule compound conjugated to an antibody, characterized in that, The antibody is a PD-L1 antibody, specifically atezolizumab, and the immune-activating small molecule compound is one of the following: ， , ， ， , ， , ， ， ， , , , , ， , , , ， ， , , , , , ， , ， 。 6. The use of the immunomodulatory small molecule compound of the conjugate antibody according to any one of claims 1-5 in the preparation of an anti-colon cancer drug.

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