Cyclic peptide toxin class drug-linkers, methods of making and using the same

Abstract

The application relates to the technical field of organic synthesis, in particular to a cyclic peptide toxin drug-linker as well as a preparation method and application thereof. The drug-linker is synthesized by coupling sites of PABC and aspartic acid in a nickel catalysis mode, the obtained drug-linker has higher activity, and the drug-linker shows strong inhibition on Hela cell lines and SKOV3 cell lines.

Description

Technical Field

[0001] This invention is a divisional application of application No. 202610320181.9, filed on March 17, 2026.

[0002] This invention relates to the field of organic synthesis technology, specifically to a cyclic peptide toxin drug-linker, its preparation method, and its application. Background Technology

[0003] The introduction of antibody-drug conjugates (ADCs) has significantly improved the precision of anticancer drug targeting while effectively reducing adverse reactions. ADCs mainly consist of monoclonal antibody mAbs, linkers, and cytotoxic drugs, combining the advantages of monoclonal antibody mAbs with the properties of cytotoxic drugs. This combination not only reduces drug-related side effects but also enhances therapeutic efficacy for various diseases. Currently, various antibody-drug conjugates (ADCs) are widely used in clinical practice.

[0004] One of the most important components of antibody-drug conjugates (ADCs) is the linker. Common linker types used in antibody-drug conjugates are cleavable and non-cleavable linkers. Cleavable linkers include pH-sensitive, redox-sensitive, enzyme-sensitive, and other types. Cleavable linkers can release the drug payload in specific environments and are therefore widely used in ADCs. Current methods link aspartic acid in the toxin to para-aminobenzylamine in the dipeptide VALINKER. Since the amide bond in the drug-loaded molecule is crucial for drug toxicity, and this key carboxyl group is central to maintaining the hydrogen bonds and active conformation within the toxin molecule, the formation of the amide bond irreversibly disrupts this hydrogen bond network, leading to changes in the toxin's three-dimensional structure and a significant reduction in activity. Even if successful targeted delivery is achieved, the released toxin is inactivated and unlikely to achieve the expected therapeutic effect. Summary of the Invention

[0005] To address the above problems, this invention provides a cyclic peptide toxin drug-linker, its preparation method, and its application.

[0006] The first aspect of this invention provides a cyclic peptide toxin drug-linker having any one of the following structural formulas: .

[0007] In this invention, asparagine is used instead of aspartic acid as the linking site. While retaining its original hydrogen bonding ability, the amide group of its side chain forms a carbamate bond through bridging with aminobenzyl alcohol, thereby obtaining three cyclic peptide toxin drug-linkers, achieving stable linkage, and finally releasing toxins with complete carboxyl structure and full biological activity at the target site through enzymatic decomposition.

[0008] A second aspect of this invention provides a method for preparing the aforementioned cyclic peptide toxin drug-linker, wherein the preparation process of compound 1 is as follows: The preparation process of compound 1 is as follows: compound 11a is mixed with p-aminobenzyl alcohol in a molar ratio of 1:3 to 1:5 and coupled under the action of a nickel catalyst to obtain compound 13a; wherein the amount of nickel catalyst is 10% to 20% of the molar mass of compound 11a; compound 13a is mixed with LINKER4 in a molar ratio of 1:1.1 to 1:1.5 and condensed to obtain compound 13b; compound 13b is mixed with m-chloroperoxybenzoic acid in a molar ratio of 1:1.1 to 1:1.2 and condensed to obtain compound 1. The structure of compound 11a is as follows: ; The structural formula of compound 13a is as follows: ; The structural formula of compound 13b is as follows: .

[0009] Specifically, the preparation process of compound 1 is as follows: LINKER4, 11a, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and an organic solvent are mixed and subjected to a condensation reaction and a deprotection reaction to obtain compound 13a. The molar ratio of LINKER4, 11a, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferably 1:1.1:1.1:1.1. The organic solvent is preferably dry dichloromethane. The condensation reaction and deprotection reaction are preferably carried out at room temperature (20℃~23℃) for 12h~48h, preferably 12h. Further, LINKER4 was dissolved in dry dichloromethane, and 11a, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added under ice bath conditions. The reaction was carried out at room temperature (20°C-23°C) for 12 h. The organic phase was washed with saturated sodium carbonate solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was dissolved in a dichloromethane / diethylamine solution, preferably with a dichloromethane mass fraction of 50%. The mixture was stirred at room temperature (20°C-23°C) for 1 h, and the solvent was removed under reduced pressure. The product was then separated by column chromatography to obtain compound 13b. Compound 13b was dissolved in a mixed solution of isopropanol and ethanol, with a concentration of 0.2 g / mL. m-chloroperoxybenzoic acid was added, and the mixture was reacted at room temperature (20°C-23°C) for 0.5 h. The solvent was removed under reduced pressure to obtain a crude product. The crude product was then separated by column chromatography to obtain compound 1.

[0010] In another preferred embodiment, the nickel catalyst is NiBr2.

[0011] In another preferred embodiment, the method for preparing the cyclic peptide toxin drug-linker includes the following steps: The preparation process of compound 2 is as follows: compound 11b and p-aminobenzyl alcohol are mixed in a molar ratio of 1:3 to 1:5 and coupled under the action of a nickel catalyst to obtain compound 14a; the amount of nickel catalyst is 10% to 20% of the molar mass of compound 11b; compound 14a is mixed with LINKER4 in a molar ratio of 1:1.1 to 1:1.3 and then condensed to obtain compound 14b; compound 14b is condensed with m-chloroperoxybenzoic acid in a molar ratio of 1:0.9 to 1:1.2 to obtain compound 2. The structural formula of compound 11b is as follows: ; The structural formula of compound 14a is as follows: ; The structural formula of compound 14b is as follows: .

[0012] Specifically, the preparation process of compound 2 is as follows: LINKER4 is dissolved in dry dichloromethane, and 11b, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are added under ice bath conditions of 0℃~4℃. The reaction is carried out at room temperature of 20℃~23℃ for 12 h. The organic phase is washed with saturated sodium carbonate solution, dried over anhydrous sodium sulfate, and the solvent is removed under reduced pressure to obtain a crude product. The crude product is dissolved in a dichloromethane / diethylamine solution, wherein the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%. The mixture is stirred at room temperature of 20℃~23℃ for 1 h, and the solvent is removed under reduced pressure. The product is then separated by column chromatography to obtain compound 14b. The molar ratio of LINKER4, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1. The 13b was dissolved in a mixed solution of isopropanol and ethanol, and the concentration of compound 13b in the resulting system was 0.2 g / mL. Then, m-chloroperoxybenzoic acid was added, and the mixture was reacted at room temperature (20°C to 23°C) for 0.5 h. The solvent was removed under reduced pressure to obtain a crude product. The crude product was then separated by column chromatography to obtain compound 2.

[0013] In another preferred embodiment, the preparation process of compound 3 is as follows: Compound 11f was coupled with p-aminobenzyl alcohol in a molar ratio of 1:3 to 1:5 under the action of a nickel catalyst to obtain compound 15a; the mass of the nickel catalyst was 10% to 20% of the molar mass of compound 11f; compound 15a was condensed with LINKER4 in a molar ratio of 1:1.1 to 1:1.3 to obtain compound 3. The structural formula (1) of compound 11f is as follows: ; The structural formula of compound 15a is as follows: .

[0014] Specifically, the preparation process of compound 3 is as follows: LINKER4 is dissolved in dry dichloromethane, and 11f, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are added under ice bath conditions of 0℃~4℃, wherein the molar ratio of LINKER4, 11f, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1; the reaction is carried out at room temperature of 20℃~23℃ for 12 h, the organic phase is washed with saturated sodium carbonate solution, dried with anhydrous sodium sulfate, and the solvent is removed under reduced pressure to obtain a crude product. The crude product is dissolved in a dichloromethane / diethylamine solution, wherein the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%, and the mixture is stirred at room temperature of 20℃~23℃ for 1 h. The solvent is removed under reduced pressure, and the mixture is separated by column chromatography to obtain compound 3. The concentration of 7NH3 / MeOH is preferably 7 mol / L; the alcoholic solution of ammonia is preferably a methanolic solution of ammonia.

[0015] In another preferred embodiment, the LINKER4 has the following structural formula: .

[0016] In another preferred embodiment, the condensation reaction is carried out at a temperature of 20°C to 23°C for a time of 12h to 48h.

[0017] A third aspect of the present invention provides the use of the aforementioned cyclic peptide toxin drug-linker in the preparation of antibody-drug conjugates.

[0018] Compared with the prior art, the present invention has the following beneficial effects: In this invention, asparagine is used instead of aspartic acid as the linker site. Its side-chain amide group retains its original hydrogen bonding ability while forming a carbamate bond via bridging with p-aminobenzyl alcohol, thus yielding three cyclic peptide toxin drug-linkers. These cyclic peptide toxin drug-linkers achieve stable linkage while fully preserving the toxin's native active conformation. Ultimately, at the target site, enzymatic decomposition releases a toxin with a complete carboxyl structure and full biological activity, thereby resolving the fundamental contradiction of impaired drug efficacy during coupling at the molecular level and achieving highly efficient and precise targeted delivery. This invention strategically transfers the linker site from the key carboxyl group of the toxin to the structurally biomimetic amide group of asparagine and innovatively introduces the enzymatically cleavable bridging unit of carbamate-p-aminobenzyl alcohol, ultimately linking it to an antibody via maleimide. At the chemical level, this design fully preserves the intramolecular hydrogen bond network that maintains the activity of the toxin, ensuring the final release of the native toxin with complete biological activity. At the application level, it significantly expands the applicable range from limited non-critical carboxyl toxins to a broader spectrum of amide-based active molecules, providing a brand-new underlying technology platform for the development of efficient and universal next-generation ADCs. Attached Figure Description

[0019] Figure 1 Figures show the cell activity results of different compounds; A is the result of the HeLa cell line, and B is the result of the SKOV3 cell line.

[0020] Figure 2 This is the proton spectrum of LINKER1.

[0021] Figure 3 This is the carbon spectrum of LINKER1.

[0022] Figure 4 This is the mass spectrum of LINKER1.

[0023] Figure 5 This is the proton spectrum of LINKER2.

[0024] Figure 6 This is the carbon spectrum of LINKER2.

[0025] Figure 7 This is the mass spectrum of LINKER2.

[0026] Figure 8 This is the proton spectrum of LINKER3.

[0027] Figure 9 This is the carbon spectrum of LINKER3.

[0028] Figure 10 This is the mass spectrum of LINKER3.

[0029] Figure 11This is the proton spectrum of LINKER4.

[0030] Figure 12 This is the carbon spectrum of LINKER4.

[0031] Figure 13 This is the mass spectrum of LINKER4.

[0032] Figure 14 This is the proton spectrum of 13a.

[0033] Figure 15 This is the carbon spectrum of 13a.

[0034] Figure 16 This is the mass spectrum of 13a.

[0035] Figure 17 This is the proton spectrum of 13b.

[0036] Figure 18 This is the carbon spectrum of 13b.

[0037] Figure 19 The mass spectrum is for 13b.

[0038] Figure 20 This is the hydrogen spectrum of compound 1.

[0039] Figure 21 This is the carbon spectrum of compound 1.

[0040] Figure 22 This is the mass spectrum of compound 1.

[0041] Figure 23 This is the proton spectrum of 14a.

[0042] Figure 24 This is the carbon spectrum of 14a.

[0043] Figure 25 The mass spectrum is for 14a.

[0044] Figure 26 The spectrum is a 14b proton spectrum.

[0045] Figure 27 This is the carbon spectrum of 14b.

[0046] Figure 28 The mass spectrum is for 14b.

[0047] Figure 29 This is the hydrogen spectrum of compound 2.

[0048] Figure 30 This is the carbon spectrum of compound 2.

[0049] Figure 31 This is the mass spectrum of compound 2.

[0050] Figure 32 The hydrogen spectrum is 15a.

[0051] Figure 33 This is the carbon spectrum of 15a.

[0052] Figure 34 This is the mass spectrum of 15a.

[0053] Figure 35 This is the hydrogen spectrum of compound 3.

[0054] Figure 36 This is the carbon spectrum of compound 3.

[0055] Figure 37 This is the mass spectrum of compound 3. Detailed Implementation

[0056] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0057] Linkers, a crucial component of antibody-drug conjugates (ADCs), play a vital role. Their chemical properties influence not only the pharmacokinetics and biodistribution of ADCs in plasma but also the type and nature of metabolites within tumor cells and the liver. A typical function of linkers is to increase the accumulation of metabolites in tumor cells, thereby enhancing therapeutic efficacy. Ideally, linkers are chemically stable in the bloodstream, releasing the drug only upon reaching the target cancer cells. Common linker types used in antibody-drug conjugates (ADCs) are briefly categorized here: cleavable linkers and non-cleavable linkers. Cleavable linkers include pH-sensitive, redox-sensitive, enzyme-sensitive, and other types. Cleavable linkers can release the drug payload in specific environments and are therefore widely used in ADCs. Unlike the chemically unstable linkers discussed so far, peptide linkers combine higher system stability with the ability to rapidly release the drug enzymatically in target cells. To avoid the formation of potentially less active metabolites, a self-destructive spacer is designed to spatially separate the drug from the enzymatic cleavage site. The p-aminobenzoyl group, denoted as PABC, is one of the most commonly used spacer groups, a bifunctional p-aminobenzyl alcohol group. This group links to a peptide via an amino group, forming an amide bond. Cytotoxic drugs containing amino groups are linked to this linker via carbamate functionality. Considering all factors, it was decided to retain the peptide-based linker chain Ala-Val and use a nickel-catalyzed approach to synthesize the drug linker by connecting the PABC and aspartic acid coupling sites.

[0058] English-Chinese abbreviation comparison: Fmoc-Ala-OH represents N-fluorenylmethoxycarbonyl-L-alanine, purchased from Saen Chemical Technology (Shanghai) Co., Ltd.; t BuOH represents tert-butanol, Boc2O represents di-tert-butyl dicarbonate, DCM represents dichloromethane; EtOAc represents ethyl acetate, purchased from Weilan Chemical Reagent Store in the High-tech Zone, HOBT represents 1-hydroxybenzotriazole, EDCI represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and TFA represents trifluoroacetic acid, purchased from Saen Chemical Technology (Shanghai) Co., Ltd.

[0059] Example 1: The preparation method of LINKER4 is as follows.

[0060] 1. Preparation of LINKER1.

[0061] Add 36.7 g, 0.1 mol of Fmoc-Ala-OH, 10.985 g, and 0.09 mol of DMAP to 200 mL of [a solution / liquid]. t The solution was added to BuOH solution. After stirring for 10 minutes, a solution of 0.1309 kg (0.6 mol) of Boc₂O in 150 mL of tBuOH was added, and the temperature was maintained while stirring continued for 40 minutes. Subsequently, the pressure was reduced to remove tBuOH, yielding a crude product. The crude product was dissolved in 100 mL of EtOAc, quenched with 2 × 100 mL of 0.5 N hydrochloric acid aqueous solution, and washed with 2 × 100 mL of saturated Na₂CO₃ aqueous solution. After separation of the organic layer, the product was dried over MgSO₄. Finally, the solvent was removed to obtain a white solid. The white solid was purified by silica gel column chromatography, eluting with CH₂Cl₂ and MeOH in a volume ratio of 96:4, to obtain a white solid LINKER1 with a yield of 11.6 g (0.8 mmol), representing 80%. 1HNMR (600MHz, CDCl3) δ7.68–7.65(m,2H),7.44–7.41(m,3H),7.38(ddd,J=6.5,3.8,.2Hz,1H),7.32(dd,J=10.3,4.6Hz,2H),7.16–7.13(m,2H),5. 69(ddd,J=17.6,10.3,7.6Hz,1H),5.06–5.02(m,1H),4.97–4.94(m,1H), 3.86–3.80(m,1H),2.95–2.88(m,1H),1.44(s,9H),1.10(d,J=6.8Hz,3H). 13CNMR(151MHz,DMSO)δ172.6,156.3,144.3,144.3,141.2,128.1,127.5, 125.7,125.7,120.6,80.8,66.0,55.3,50.4,47.2,40.6,28.1,26.8,17.4. HRMS(ESI)m / zcalculatedforC 22 H 25 NO4[M+Na]+: 390.1784; found: 390.1697. (Specific details are as follows...) Figures 2-4 As shown.

[0062] 2. Preparation of LINKER2.

[0063] The reaction equations for the preparation process are as follows: .

[0064] 13.9 g (17.9 mmol) of the monocyclic octapeptide LINKER1 was dissolved in 100 mL of dichloromethane. 100 mL of diethylamine was added, and the reaction mixture was stirred at 23°C for 1 hour until the complete reaction of LINKER1 was confirmed by thin-layer chromatography. The reaction was terminated with 100 mL of water, the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography with a CH2Cl2 / MeOH volume ratio of 96:4 to give 5.82 g (0.13 mmol) of a white solid, designated as dipeptide 4, in 84.5% yield. A solution of 5.82 g (9.95 mmol) of dipeptide 4, 6.41 g (21.81 mmol) of Fmoc-Val-OH, and 1.48 g (10.93 mmol) of HOBT in 200 mL of anhydrous dichloromethane was prepared at 0°C. 2.09 g (10.93 mmol) of EDCI was added fractionally. The reaction mixture was heated to 23°C and stirred for 12 hours. Dichloromethane was then concentrated to a minimum volume under reduced pressure. The residue was quenched with 0.5N hydrochloric acid aqueous solution and washed with saturated sodium carbonate aqueous solution. The organic layer was separated, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude protected tripeptide. This crude product was dissolved in 200 mL of dichloromethane containing 50% v / v diethylamine and stirred at 23°C for 1 hour to achieve deprotection of the 9-fluorenemethoxycarbonyl group. After complete reaction of the starting material, the reaction was quenched with 500 mL of water, monitored by thin-layer chromatography. The organic layer was separated, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate eluent of 20:80 to give 7.00 g (8.97 mmol) of a white solid, LINKER2, in 81% yield.

[0065] 1HNMR(600MHz,DMSO-d6)δ8.26(d,J=6.8Hz,1H),7.90(s,2H),7.76(s,2H),7.42(s,2H),7.37(s,1H),7.33(s,2H),4.29(s, 1H),4.21(s,2H),4.13(s,1H),3.91(s,1H),1.97(s,1H),1.39(s,9H),1.25(d,J=7.4Hz,3H),0.90(dd,J=19.7,6.8Hz,6H). 13CNMR(151MHz,DMSO)δ172.1,171.4,156.5,144.4,144.3,141.2,128.1,127.5 ,125.9,120.6,80.7,66.2,60.1,48.8,47.1,40.6,31.0,28.1,19.7,18.7,17.4. HRMS(ESI)m / zcalculatedforC27 H 34 N2O5S[M]+:466.2468;found:466.8085. (Specific details are as follows...) Figures 5-7 As shown.

[0066] 3. Preparation of LINKER3.

[0067] The reaction equation for the preparation process is as follows: .

[0068] 7 g (8.5 mmol) of the monocyclic octapeptide LINKER2 was dissolved in 50 mL of dichloromethane, and 50 mL of diethylamine was added. The mixture was stirred at 23°C for 1 hour until TLC confirmed complete consumption of LINKER2. The reaction was terminated with 100 mL of water. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude product was purified by silica gel column chromatography using a CH2Cl2 / MeOH eluent system of 96:4 (v / v) to obtain the dipeptide. 3.95 g of the prepared dipeptide (4.85 mmol or more), 3.2 g of 3-maleimide propionic acid (10.4 mmol), and 0.7 g of HOBT (5.9 mmol) were dissolved in 100 mL of anhydrous dichloromethane and a solution was prepared at 0 °C. 1.09 g of EDCI (5.93 mmol) was added in portions at 0 °C, and the mixture was heated to 23 °C and stirred for 12 hours. The DCM was then concentrated to a minimum volume under vacuum, and the residue was treated with 0.5 N hydrochloric acid aqueous solution and washed with saturated Na₂CO₃ aqueous solution. The organic layer was separated, dried with anhydrous Na₂SO₄, and concentrated under reduced pressure to obtain the crude protected tripeptide. This crude product was dissolved in 200 mL of dichloromethane containing 50% v / v diethylamine and stirred at 23 °C for 1 hour to deprotect the 9-fluorenemethyloxycarbonyl group. After the starting material was completely consumed, the reaction was stopped by TLC monitoring with 500 mL of water. The organic layer was separated, dried with anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography with a petroleum ether / ethyl acetate volume ratio of 20:80. The residue was purified to give 3.00 g and 4.97 mmol of white solid LINKER3, with a yield of 81%.

[0069] 1HNMR (600MHz, DMSO-d6) δ8.11(s,1H),4.17(s,1H),2.98(s,1H),1.87(s,1H),1.40(s,9H),1.26(s,3H),0.89(s,3H),0.79(s,3H). 13CNMR (151MHz, DMSO) δ181.9,172.1,171.2,171.1,169.8,135.0,80.7,60.2,57.5,48.8,40.6,34.5,34.1,31.0,28.1,19.6,18.6,17.4,14.6. HRMS(ESI)m / zcalculatedforC 19 H 29 N3O6[M]+:395.2056;found:395.1757. (Specific details are as follows...) Figures 8-10 As shown.

[0070] 4. Preparation of LINKER4.

[0071] The reaction equation for the preparation process is as follows: .

[0072] 7.00 g (8.97 mmol) of LINKER3 was dissolved in 50 mL of TFA and stirred at 23 °C for 1 hour. Subsequently, the TFA was removed under vacuum to obtain the crude product. The crude product was subjected to silica gel column chromatography with a gradient elution of CH2Cl2 / MeOH, wherein the volume percentage of CH2Cl2 was gradually increased from 0% to 2%, yielding 5.63 g (12 mmol) of pure LINKER4, a white solid in 90% yield. 1HNMR(500MHz,DMSO-d6)δ12.48(s,1H),8.25(d,J=6.9Hz,1H),8.01(d,J=8.9Hz,1H),7.01(s,2H),4.17(d,J=7.2Hz,2H ),3.73–3.52(m,2H),2.44(dd,J=15.9,8.3Hz,2H),1.92(q,J=6.8Hz,1H),1.27(d,J=7.3Hz,3H),0.83(d,J=28.0Hz,6H). 13CNMR(151MHz,DMSO)δ174.4,171.2,171.2,169.8,135.0,59.4,57.7,52.0,49.1,47.9, 47.8,44.5,40.6,34.5,34.4,34.3,34.1,32.0,31.8,31.0,19.5,18.6,17.6,17.5,14.0. HRMS(ESI)m / zcalculatedforC 15 H 21 N3O6[M+Na]+: 362.1430; found: 362.1390. (Specific details are as follows...) Figures 11-13 As shown.

[0073] Example 2: Preparation of compound 1.

[0074] The reaction equation for the preparation of 13a is as follows: .

[0075] In a 20 mL dried Schlenk tube, 0.25 mmol of 11a, 0.5 mmol of K₂CO₃, 20 mol% of 1,10-o-phenanthroline monohydrate, 10 mol% of NiBr₂, and 1.0 mmol of 4-aminobenzyl were added. Then, 2 mL of toluene was added under a nitrogen atmosphere, and the mixture was heated at 130 °C for 48 hours. After cooling the reaction mixture to 23 °C, 3.0 mL of ethyl acetate was added, and the mixture was concentrated under reduced pressure. The residue was purified by column chromatography using a gradient elution with hexane and ethyl acetate to give the pure product, 13a. 1HNMR(600MHz,DMSO-d6)δ10.74(d,J=60.0Hz,1H),8.62(s,1H),8.32(d,J=7.8Hz,1H),8.11(d,J=29.6Hz,2H),7.88(d,J=24.2Hz,2H),7.72(d,J=6 1.3Hz,2H),7.55(s,1H),7.45(d,J=30.7Hz,1H),7.23(s,2H),7.17(s,3H) ,7.12(s,1H),7.04(s,1H),6.95(s,1H),5.75(s,2H),5.19(s,1H),4.84–4 .48(m,2H),4.31(d,J=64.2Hz,5H),4.03(dt,J=22.7,13.9Hz,1H),3.74( s,1H),3.40(s,1H),3.19(s,3H),2.63(d,J=15.1Hz,1H),2.44–2.17(m,3H ),2.03(d,J=48.0Hz,2H),1.90(s,2H),1.74(d,J=54.3Hz,3H),1.41(s,7H ),1.25(s,1H),1.17(d,J=6.7Hz,3H),1.05(s,1H),0.75(d,J=8.4Hz,3H).13CNMR(151MHz,DMSO)δ204.4,187.2,173.1,172.9,172.8,171.6,171.4,171.0,170.6,170.3,168.3,137.8,136.9 ,135.5,134.7,130.1,129.7,128.5,125.0,123.4,122.6,122.6,121.5,121.0,120.2,119.0,116.8,116.4,114.5,1 11.5,73.7,73.7,67.7,66.5,62.2,60.8,60.0,58.3,58.1,55.2,54.0,53.3,51.3,42.6,42.6,40.5,36.3,35.5,33 .8,31.3,29.5,28.1,25.8,25.3,22.7,21.5,20.2,19.0,17.7,15.7,15.3,14.4,13.8,13.3,11.5,11.5,11.2,10.4. HRMS(ESI)m / zcalculatedforC. 51 H 69 N 11 O 11 S[M]+:1311.4899;found:1311.5193. (Specific details are as follows...) Figures 14-16 As shown.

[0076] The reaction equation for the preparation of 13b is as follows: .

[0077] 0.521 g (5 mmol) of 13a, 0.186 g (5.5 mmol) of LINKER4, and 0.068 g (5.5 mmol) of 1-hydroxybenzotriazole were dissolved in 20 mL of anhydrous dichloromethane and kept at 0°C under an inert atmosphere. 0.1 g (5.5 mmol) of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride was added to this solution in 20 mL of dry CH2Cl2, and the mixture was stirred at 0°C for 12 hours. After removing most of the CH2Cl2, the reaction solution was quenched with 0.5 N aqueous HCl and washed with a saturated sodium carbonate aqueous solution. The organic layer was then separated and dried over Na2SO4. The crude product was purified by silica gel column chromatography using a CH2Cl2 / MeOH elution system of 98.5:1.5 (v / v) to give 0.515 g (0.0048 mol) of 13b in 77.3% yield as a white solid. 13b was dissolved in 50 mL of dichloromethane containing 50% diethylamine by volume. The mixture was stirred at 23°C for 1 hour, monitored by TLC / HPLC, until the reaction was complete. The reaction was then stopped with 100 mL of deionized water. The organic phase was separated, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel column flash chromatography using CH₂Cl₂ and MeOH in a mobile phase of 98.5:1.5 (v / v). The purified fraction was concentrated to give 0.515 g (0.0048 mol) of a white solid 13b, with a yield of 77.3%.1HNMR(600MHz,DMSO-d6)δ11.22(s,1H),9.73(s,1H),8.61(s,1H),8.47(s,1H),8.35(s,1H),8.24(s,1H),8.13(d,J=6.7Hz,1H),8.05(d,J=7.2Hz,2H),7.99(d,J=8.4Hz,1H),7.92(d,J=10.0Hz,1H),7.61(d,J=7.8Hz,1H),7.48(d,J=8.3Hz,1H),7.25(d,J=8.0Hz,2H),7.10(d,J=7.7Hz,2H),7.00(d,J=5.6Hz,2H),5.36(s,1H),4.93(s,2H),4.48(s,1H),4.39(s,1H),4.30(s,1H),4.20(s,1H),4.13(s,1H),4.04(s,2H),3.84(s,1H),3.60(s,2H),3.48(d,J=19.4Hz,1H),3.38(s,1H),3.23(d,J=14.6Hz,1H),3.11(s,1H),2.93(s,4H),2.80(s,1H),2.69(s,1H),2.40(s,2H),2.25(s,1H),2.06(s,1H),2.01(s,3H),1.94(s,2H),1.58(s,1H),1.48(s,2H),1.33(t,J=10.5Hz,5H),1.24(s,3H),1.17(s,4H),0.91(d,J=6.6Hz,3H),0.83(s,12H)。13CNMR(151MHz,DMSO)δ173.1,171.3,171.3,171.2,171.0,171.0,170.9,170.9,170.6,170.3,168.3,136.9,135.0,132.6,129.5,127.6,125.0,122.6,121.0,119.6,119.1,116.4,111.5,72.8,62.5,59.6,58.4,58.3,54.0,53.9,53.3,51.1,49.5,42.0,41.9,40.5,40.4,39.1,38.7,36.3,35.5,34.5,34.2,33.8,31.7,30.7,30.4,29.5,27.0,25.8,25.3,21.5,20.9,19.6,18.6,18.4,16.2,15.7,15.4,11.5,11.5,11.2。HRMS(ESI)m / zcalculatedforC。66 H 88 N 14 O 16 S[M+H]+:1158.6233;found:1158.5366. (Specific details are as follows...) Figures 17-19 As shown. The reaction process for preparing compound 1 is as follows:

[0078] .

[0079] 0.515 g (0.0048 mol, 1 equivalent) of compound 13b was dissolved in methanol containing 7N ammonia solution (7 mL, 100 μL / mg concentration, 0.045 mol, 7 equivalents). The mixture was stirred at 23 °C for 3 hours until TLC confirmed complete reaction of the starting material. The reaction was extinguished with 20 mL of water and concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography with an elution system of CH2Cl2 and MeOH in a volume ratio of 80:20, yielding 0.407 g (0.36 mmol) of a white solid, compound 1, in 74% yield. 1HNMR(600MHz,DMSO-d6)δ11.23(s,1H),9.74(s,1H),8.52(d,J=46.8Hz,2H),8.35(s,2H),8.19(s,1H),8.14(d,J=7.1Hz,1H),8.10(d,J=8.0Hz,1H),8.06(d,J=9.8Hz,1H),8.01(s,1H),7.87(d,J=10.4Hz,1H),7.71(s,1H),7.61(d,J=8.0Hz,1H),7.47(t,J=10.2Hz,4H),7.32(s,1H),7.25(d,J=8.1Hz,1H),7.10(d,J=8.0Hz,2H),7.01(s,2H),5.76(s,1H),5.33(s,1H),5.18(s,1H),5.01–4.93(m,1H),4.71(s,1H),4.50(d,J=9.8Hz,1H),4.39(s,2H),4.31(s,1H),4.21(t,J=8.9Hz,1H),4.12(t,J=7.5Hz,1H),3.85–3.80(m,1H),3.74(d,J=11.2Hz,1H),3.61(d,J=6.8Hz,2H),3.40(d,J=17.7Hz,2H),3.24(d,J=14.3Hz,2H),3.09(d,J=14.9Hz,1H),2.91(d,J=7.2Hz,4H),2.81(d,J=7.9Hz,1H),2.62(s,1H),2.40(s,1H),2.25(s,3H),2.02–1.94(m,3H),1.84(d,J=11.4Hz,1H),1.58(s,1H),1.47(s,2H),1.32(dd,J=19.5,7.0Hz,6H),1.24(s,5H),1.17(t,J=7.2Hz,4H),0.90–0.80(m,13H)。 13CNMR(151MHz,DMSO)δ173.1,171.3,171.2,171.0,171.0,171.0,170.9,170.6,170.3,168.3,136.9 ,135.0,132.6,129.5,127.6,124.9,122.6,121.0,119.6,119.1,116.4,111.5,72.8,62.5,59.6,58 .4,58.3,54.0,53.9,53.3,51.1,49.5,42.0,41.9,40.5,40.4,39.1,38.7,36.3,35.5,34.5,34.2,3 3.7,30.7,30.4,27.0,25.8,25.3,21.5,20.9,19.6,18.6,18.4,16.2,15.7,15.3,11.5,11.5,11.2. HRMS(ESI)m / zcalculatedforC 64 H 86 N 14 O15S[M+H]+:1322.6188,found:1322.3711. (Details are as follows...) Figures 20-22 As shown.

[0080] Example 3: Preparation of compound 2.

[0081] The reaction equation for the preparation of 14a is as follows: .

[0082] In a 20 mL dried Schlenk tube, 0.25 mmol of 11b, 0.5 mmol of K₂CO₃, 20 mol% of 1,10-phenanthroline monohydrate, 10 mol% of NiBr₂, and 1.0 mmol of 4-aminobenzyl alcohol were added. Then, under a nitrogen atmosphere, 2 mL of toluene was added, and the mixture was heated to 130°C and reacted for 48 hours. After cooling the reaction mixture to room temperature (23°C), 3.0 mL of ethyl acetate was added, and the mixture was concentrated under reduced pressure. The residue was purified by column chromatography using a gradient elution with n-hexane and ethyl acetate to give pure product 14a. 1HNMR(600MHz,DMSO-d6)δ11.23(s,1H),8.65(d,J=36.4Hz,2H),8.46(t,J=6.8Hz,2H),8.36(s,1H),8.24(s,1H),8.05(d,J=10.0Hz,2H),7.99(d,J=8.9Hz,1H),7.94(d,J=18.3Hz,1H),7.61(d,J=8.0Hz,1H),7.50(s,1H),7.45(s,1H),7.25(d,J=8.1Hz,1H),7.11(d,J=7.4Hz,1H),7.01(t,J=7.4Hz,1H),5.36(s,1H),4.94(d,J=13.1Hz,2H),4.53–4.45(m,1H),4.39(dd,J=18.5,8.9Hz,1H),4.30(dd,J=1.7,7.0Hz,1H),4.20(t,J=9.0Hz,1H),4.04(s,2H),3.84(s,1H),3.67–3.56(m,3H),3.47(d,J=16.3Hz,2H),3.22(d,J=14.2Hz,2H),3.12(dd,J=17.4,8.0Hz,3H),2.91(d,J=17.7Hz,4H),2.79(d,J=11.0Hz,1H),2.74(s,1H),2.66(d,J=45.3Hz,1H),2.43–2.36(m,2H),2.06(s,1H),2.01(s,3H),1.94(s,1H),1.58(s,1H),1.49(s,2H),1.38(s,1H),1.34(d,J=7.0Hz,3H),1.26(d,J=6.2Hz,13H),1.22–1.19(m,1H),1.17(t,J=7.5Hz,2H),1.12(d,J=7.1Hz,1H),0.90(d,J=6.8Hz,3H),0.85–0.80(m,9H)。13CNMR(151MHz,DMSO)δ173.1,172.9,172.8,171.4,171.0,170.6,170.3,168.3,136.9,1 30.1,129.7,125.0,122.6,121.5,120.2,119.0,116.8,116.4,114.5,111.5,73.7,66.5, 62.2, 60.0, 58.3, 55.2, 54.0, 53.3, 51.3, 42.6, 42.6, 40.5, 36.3, 35.5, 33.8, 31.3, 29.5, 28.1, 25.8, 25.3, 22.7, 21.5, 20.2, 19.0, 16.2, 15.7, 15.3, 13.3, 11.5, 11.5, 11.2, 10.4. HRMS(ESI) m / z calculated for C51H69N11O12S[M]+: 1059.4848.; found: 1059.2975. Details are as follows. Figures 23-25 As shown.

[0083] The reaction equation for the preparation of 14b is as follows: .

[0084] In a 20 mL dried Schlenk tube, 0.25 mmol of 14a, 0.5 mmol of K₂CO₃, 20 mol% of 1,10-phenanthroline monohydrate, 10 mol% of NiBr₂, and 1.0 mmol of 4-aminobenzyl alcohol were added. Then, 2 mL of toluene was added under a nitrogen atmosphere, and the mixture was heated to 130°C for 48 hours. After cooling the reaction mixture to room temperature, 3.0 mL of ethyl acetate was added, and the mixture was concentrated under reduced pressure. The residue was purified by column chromatography using n-hexane and ethyl acetate as eluents to obtain the purified product, 14b. 1H NMR(600MHz,DMSO-d6)δ11.23(s,1H),8.65(d,J=36.4Hz,2H),8.46(t,J=6.8Hz,2H),8.36(s,1H),8.24(s,1H), 8.05(d,J=10.0Hz,2H),7.99(d,J=8.9Hz,1H),7.94(d,J=18.3Hz,1H),7.61(d,J=8.0Hz,1H),7.50(s,1H),7.45( s,1H),7.25(d,J=8.1Hz,1H),7.11(d,J=7.4Hz,1H),7.01(t,J=7.4Hz,1H),5.36(s,1H),4.94(d,J=13.1Hz,2H), 4.53–4.45(m,1H),4.39(dd,J=18.5,8.9Hz,1H),4.30(dd,J=11.7,7.0Hz,1H),4.20(t,J=9.0Hz,1H),4.04(s,2H ),3.84(s,1H),3.67–3.56(m,3H),3.47(d,J=16.3Hz,2H),3.22(d,J=14.2Hz,2H),3.12(dd,J=17.4,8.0Hz,3H) ,2.91(d,J=17.7Hz,4H),2.79(d,J=11.0Hz,1H),2.74(s,1H),2.66(d,J=45.3Hz,1H),2.43–2.36(m,2H),2.06(s ,1H),2.01(s,3H),1.94(s,1H),1.58(s,1H),1.49(s,2H),1.38(s,1H),1.34(d,J=7.0Hz,3H),1.26(d,J=6.2Hz, 13H),1.22–1.19(m,1H),1.17(t,J=7.5Hz,2H),1.12(d,J=7.1Hz,1H),0.90(d,J=6.8Hz,3H),0.85–0.80(m,9H).13CNMR(151MHz,DMSO)δ173.1,172.9,172.8,171.4,171.0,170.6,170.3,168.3,136.9,1 30.1,129.7,125.0,122.6,121.5,120.2,119.0,116.8,116.4,114.5,111.5,73.7,66.5, 62.2,60.0,58.3,55.2,54.0,53.3,51.3,42.6,42.6,40.5,36.3,35.5,33.8,31.3,29.5,28.1,25.8,25.3,22.7,21.5,20.2,19.0,16.2,15.7,15.3,13.3,11.5,11.5,11.2,10.4. HRMS(ESI)m / z calculated for C51H69N11O12S[M]+:1059.4848.;found:1059.2975. (Details follow.) Figures 26-28 As shown.

[0085] The reaction equation for the preparation of compound 2 is as follows: .

[0086] 0.587 g (0.0055 mol) of compound 14a was dissolved in 7N NH3 / MeOH in methanol and stirred at 23°C for 3 hours until the starting material was confirmed to have completely reacted by TLC. The reaction was quenched with 20 mL of water and concentrated under reduced pressure. The crude product was purified by silica gel flash column chromatography, eluting with CH2Cl2 and MeOH in a volume ratio of 80:20, to give 0.46 g (0.41 mol) of compound 2, a white solid in 74% yield. 1H NMR (600MHz, DMSO) δ11.94,9.84,8.52,8.43,8.42,8.38,8.35,8.32,8.27,8.26, 8.20,8.19,8.01,7.92,7.88,7.87,7.80,7.79,7.70,7.48,7.46,7.44,7.41,7.34 ,7.33,7.31,7.18,7.17,7.16,7.11,7.00,5.39,5.34,5.16,5.15,5.07,5.04,4.96,4.95,4.70,4.67,4.45,4.43,4.37,4.29,4.29,4.18,4.17,4.15,3.92,3.90,3 .88,3.79,3.78,3.73,3.71,3.62,3.60,3.43,3.41,3.40,3.38,3.25,3.24,3.23,3.22,2.97,2.96,2.94,2.93,2.91,2.90,2.89,2.88,2.75,2.74,2.71,2.62,2.4 2,2.25,2.22,2.21,2.03,2.02,2.00,1.93,1.83,1.58,1.47,1.36,1.34,1.32,1.31,1.27,1.25,1.24,1.17,1.16,1.15,0.92,0.91,0.90,0.87,0.86,0.83,0.82.13CNMR(151MHz,DMSO)δ173.2,172.9,172.9,172.8,171.5,171.5,171.5,171.3,171.2,171.1,14 1.2,137.0,136.7,135.1,134.9,130.5,130.2,129.6,129.2,128.1,127.5,127.1,124.9,122.7,1 21.0,120.4,119.1,117.2,117.1,116.4,111.1,69.1,59.6,58.3,56.2,53.8,51.2,41.9,36.2,29.5,29.5,29.4,29.3,29.1,29.0,27.0,25.8,25.3,22.5,16.3,16.2,15.6,15.3,11.6,11.4,11.3. HRMS(ESI)m / z calculated for C64H86N14O16S[M]+:1338.6067,found:1338.3477. (Details as follows.) Figures 29-31 As shown.

[0087] Example 4: Preparation of compound 3.

[0088] The reaction equation for the preparation process of 15a is as follows: 0.583 g (5.6 mmol, 1.12 equivalents) of 11f,L, 0.21 g (6.16 mmol, 1.23 equivalents) of INKER4, and 0.076 g (6.16 mmol, 1.23 equivalents) of 1-hydroxybenzotriazole were dissolved in 20 mL of anhydrous dichloromethane and the reaction was carried out at 0°C under an inert atmosphere. 0.112 g (6.16 mmol, 1.23 equivalents) of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride was added to this solution, and the mixture was stirred at 0°C for 12 hours in 20 mL of dry CH2Cl2. After most of the CH2Cl2 was removed under reduced pressure, the reaction solution was quenched with 2 × 100 mL of 0.5 N water-soluble HCl and washed with 2 × 100 mL of saturated Na2CO3 aqueous solution. The organic layer was then separated and dried with Na2SO4. The crude product was purified by silica gel column chromatography using CH₂Cl₂ and MeOH in a volume ratio of 98.5:1.5 to give 0.577 g (0.0054 mol) of a white solid 15a, with a yield of 77.3% and a melting point of 138–139°C. The crude protected 15a was dissolved in 50 mL of a solution of diethylamine and CH₂Cl₂ in a volume ratio of 50%. The mixture was stirred at 23°C for 1 hour, and the reaction was monitored by TLC / H. The mixture was stirred at 23°C for 1 hour, and monitored by TLC / HPLC until the starting material was observed to be completely consumed. The reaction was terminated with 100 mL of deionized water. The organic phase was separated, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using CH₂Cl₂ and MeOH in a volume ratio of 98.5:1.5. The concentrated and purified fraction yielded 0.515 g and 0.0048 mol of a white solid 15a, with a yield of 77.3%.1H NMR (600 MHz, DMSO-d6) δ 11.64 (s, 1H), 8.87 (s, 1H), 8.54 (s, 1H), 8.34–8.24 (m, 3H), 7.99–7.72 (m, 4H), 7.66 (d, J = 8.1 Hz, 1H), 7.42 (s, 3H), 7.20 (s, 1H), 7.10 (d, J = 6.9 Hz, 1H), 6.98 (s, 1H), 5.33 (s, 1H), 5.22 (d, J = 7.3 Hz, 1H), 5.02 (s, 1H), 4.90 (s, 1H), 4.85 (d, J = 10.4 Hz, 1H), 4.76 (s, 1H), 4.73–4.57 (m, 1H), 4.30 (d, J = 12.4 Hz, 4H), 4.08 (d, J = 12.1 Hz, 3H), 3.94–3.87 (m, 1H), 3.70 (s, 2H), 3.61 (s, 1H), 3.49 (d, J = 17.0 Hz, 4H), 3.27 (s, 4H), 3.10 (s, 2H), 2.95 (d, J = 13.4 Hz, 3H), 2.76 (s, 1H), 2.65–2.52 (m, 3H), 2.41 (s, 1H), 2.25 (s, 2H), 2.13 (s, 2H), 2.02 (s, 3H), 1.99 (s, 3H), 1.95 (s, 3H), 1.89 (d, J = 18.9 Hz, 1H), 1.55 (s, 2H), 1.24 (s, 3H), 1.17 (d, J = 7.3 Hz, 1H), 1.11 (s, 1H), 0.93 (d, J = 7.1 Hz, 2H), 0.83 (s, 3H), 0.79 (s, 3H). 13C NMR (151 MHz, DMSO) δ 172.5, 172.2, 171.7, 171.1, 170.8, 170.7, 170.6, 170.3, 170.2, 170.1, 168.4, 167.5, 13,5.5, 134.8, 132.8, 127.7, 125.0, 124.0, 121.8, 119.8, 117.9, 112.8, 111.3, 72.8, 71.6, 63.3, 62.4, 59.6, 53.3, 53.1, 52.8, 51.2, 50.4, 43.5, 42.6, 41.8, 41.5, 40.5, 35.1, 35.0, 29.6, 25.7, 21.4, 21.2, 21.0, 15.2, 11.6, 11.5, 11.2. HRMS (ESI) m / z calculated for C68H88N14O20S [M + H]+: 1399.6062; found: 1399.1431. Specifically as follows. [[ID=!]] Figures 32-34 As shown.

[0089] The reaction equation for the preparation of compound 3 is as follows: 0.577 g (0.0054 mol, 1.12 equivalents) of compound 15a was dissolved in 7N NH3 / MeOH in methanol and stirred at 23°C for 3 hours until the starting material was confirmed to be completely consumed by TLC. The reaction mixture was chelated with 20 mL of water and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with an elution system of CH2Cl2 and MeOH in a volume ratio of 80:20 to give 0.456 g (0.40 mmol) of a white solid 15b, which is compound 3, in 74% yield.1HNMR(600MHz,DMSO-d6)δ11.65(s,1H),8.85–8.79(m,1H),8.58(d,J=3.9Hz,1H),8.35(d,J=42.7Hz,1H),8.24(d,J=10.4Hz,2H),7.92(t,J=9.1Hz,2H),7.83(d,J=9.4Hz,1H),7.71(s,2H),7.66(d,J=8.0Hz,1H),7.51–7.47(m,2H),7.46(d,J=3.3Hz,2H),7.38(s,1H),7.31(s,2H),7.20(d,J=7.6Hz,1H),7.10(d,J=7.7Hz,1H),5.29–5.22(m,2H),5.04–4.99(m,1H),4.87(dd,J=10.3,3.8Hz,1H),4.74–4.66(m,2H),4.38(s,1H),4.31–4.28(m,2H),4.24–4.19(m,1H),4.06(dd,J=12.5,5.2Hz,1H),3.94–3.89(m,1H),3.85(d,J=11.4Hz,1H),3.78(d,J=6.0Hz,1H),3.70(dd,J=8.0,3.9Hz,1H),3.46(dd,J=17.5,8.3Hz,4H),3.24(dd,J=15.3,7.2Hz,2H),3.13(t,J=13.3Hz,2H),3.00–2.94(m,2H),2.90(d,J=7.2Hz,1H),2.77(d,J=15.4Hz,1H),2.61–2.56(m,1H),2.47–2.37(m,1H),2.25(s,1H),2.14–2.10(m,1H),2.03(d,J=6.9Hz,3H),1.96(s,3H),1.93–1.87(m,1H),1.56(d,J=6.7Hz,2H),1.43–1.36(m,1H),1.24(s,3H),1.20(s,2H),1.11(dd,J=13.5,7.7Hz,1H),0.91(d,J=7.1Hz,3H),0.83(d,J=7.5Hz,3H),0.79(d,J=6.7Hz,3H)。13CNMR(151MHz,DMSO)δ172.4,172.2,171.8,171.6,170.7,170.7,170.6,170. 5,170.1,168.3,167.5,137.9,136.6,132.8,129.6,129.5,128.1,127.7,123.6 ,119.9,112.8,111.3,71.7,69.0,63.3,62.3,59.7,59.5,56.4,53.0,52.9,51.3,50.4,42.8,41.7,35.0,34.8,29.9,25.7,21.2,21.0,15.2,11.7,11.4,11.2. HRMS(ESI)m / z calculated for C62H82N14O17S[M]+:1326.5703,found:1326.1031. Details are as follows. Figures 35-37 As shown.

[0090] The cell viability of the cells prepared above was tested.

[0091] Compounds 2 and 3 were selected for cell activity assays, and their cytotoxicity was tested to explore the relationship between payload and drug-linker activity. Compound 2 was designated O-3, compound 3 P-3, and compound 1 N-3. Compounds 11a (without LINKER4) were designated I-4 and 11b (without LINKER4) as controls. Results are as follows: Figure 1 As shown, due to IC N-3 50 The value must be greater than 50 µM, which is much higher than that of the other compounds. Therefore, the broken line in the figure only shows the results for other compounds. The IC50 value for N-3 is... 50 The values ​​are displayed below the horizontal axis in the graph. From Figure 1 It can be seen that compound 2 has an IC50 value in both the HeLa and SKOV3 cell lines. 50 The values ​​were highest at concentrations of 1.832 µM and 5.813 µM, respectively, demonstrating significant antitumor activity. Meanwhile, compound 3 showed high IC50 values ​​in HeLa and SKOV3 cell lines. 50 The values ​​were highest at concentrations of 1.712 µM and 1.939 µM, respectively. For I-4 as the effective payload, the introduction of the linker had a relatively small effect on drug-linker activity. Compared to compound 2, compound 3 showed stronger activity, indicating that compounds containing linkers have higher activity. Compound 3 exhibited strong inhibitory effects on HeLa and SKOV3 cell lines, with IC50 values ​​of [missing value]. 50 The values ​​were 1.712 µM and 1.939 µM, respectively. The IC50 values ​​of M-4 against HeLa and SKOV3 cell lines were... 50The values ​​were 1.912 µM and 1.830 µM, respectively. N-3 and its load H-4 showed similar activities, with IC50 > 50 µM. Therefore, the introduction of the linker had little impact on the overall activity of the load.

[0092] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cyclic peptide toxin drug-linker, characterized in that, The structural formula is as follows: 。 2. A method for preparing the cyclic peptide toxin drug-linker according to claim 1, characterized in that, Includes the following steps: The preparation process of compound 3 is as follows: Step 1: Compound 11f and p-aminobenzyl alcohol are coupled in a molar ratio of 1:3 to 5 under the action of a nickel catalyst to obtain compound 15a; the mass of the nickel catalyst is 10% to 20% of the molar mass of compound 11f. Step 2: Compound 15a and LINKER4 are subjected to a condensation reaction at a molar ratio of 1:1.1~1.3 to obtain compound 3; The structural formula of compound 11f is as follows: ； The structural formula of compound 15a is as follows: ； The nickel catalyst is NiBr2.

3. The method for preparing the cyclic peptide toxin drug-linker according to claim 2, characterized in that, The structure of LINKER4 is as follows: 。 4. The method for preparing the cyclic peptide toxin drug-linker according to claim 2, characterized in that, The condensation reaction is carried out at a temperature of 20℃~23℃ for 12h~48h.

5. The use of the cyclic peptide toxin drug-linker of claim 1 in the preparation of antibody-drug conjugates.

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