Camptothecin derivatives, antibody conjugated drugs thereof and uses thereof

Abstract

The application belongs to the field of drug conjugates, and provides a camptothecin derivative, an antibody conjugated drug thereof and use. The camptothecin derivative comprises a compound shown in formula (I) or a tautomer thereof, or a stereoisomer thereof, or a solvate thereof, or an isotopically labeled compound thereof, or a pharmaceutically acceptable salt or ester thereof. After structural optimization of the camptothecin derivative, the antibody conjugated drug of the camptothecin derivative shown in formula (III) is prepared, the metabolic stability of the drug is improved, tumor cells can be effectively killed, the therapeutic effect of the antibody drug conjugate is improved, the off-target risk is reduced, and the treatment window is increased.

Description

Technical Field

[0001] This invention belongs to the field of drug conjugates, specifically relating to a camptothecin derivative and its antibody-drug conjugate and its uses. Background Technology

[0002] Despite recent advances in immunotherapy and cell therapy, chemotherapy remains the most widely used strategy in cancer treatment. However, due to its low therapeutic index, conventional chemotherapeutic drugs, while exhibiting potent cytotoxicity against cancer cells, often show toxic effects on healthy tissues, significantly limiting their clinical efficacy. Therefore, developing efficient drug delivery systems with limited systemic toxicity for cancer treatment may be an effective strategy to address this problem. Thus, a novel concept, antibody-drug conjugates (ADCs), was conceived and developed. Typically, ADCs are formed by combining an antibody targeting a tumor-specific antigen or related antigen with several payloads via appropriate linkers.

[0003] Antibody-drug conjugates (ADCs) possess significant clinical therapeutic value due to their combined advantages of precise recognition and effective toxin killing by monoclonal antibodies. Toxins play a crucial role in determining the efficacy of ADCs, thus attracting widespread attention in this field. Topoisomerase I (TOPO-I) is an important ribozyme for genome stability and DNA cytoplasmic structure preservation; TOPO-I inhibitors may contribute to anti-tumor immunotherapy. The natural compound camptothecin (CPT) is a particularly attractive ADC payload, inducing apoptosis by forming a stable complex with TOPO-I and DNA. Therefore, developing a camptothecin derivative with a novel structure and constructing a superior antibody-drug conjugate based on it is of great significance for tumor treatment. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a camptothecin derivative, its antibody-drug conjugate, and its uses. This invention develops a class of camptothecin derivatives with unique structures that can serve as the warhead for antibody-drug conjugates, enabling the preparation of antibody-drug conjugates capable of effectively killing tumor cells. The antibody-drug conjugates of this invention further enhance the therapeutic efficacy of antibody-drug conjugates, reduce off-target risks, and increase the therapeutic window.

[0005] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a camptothecin derivative, the camptothecin derivative comprising a compound of formula (I) or its tautomer, stereoisomer, solvate, isotopically labeled compound, or pharmaceutically acceptable salt or ester thereof; ; R1 is selected from -OH, -SH, -COOH, or -NHRa ; R a Selected from hydrogen, deuterium, -C1-6 alkyl, -C2-6 alkenyl, -C2-6 ynyl, -C3-10 cycloalkyl, 4-10 heterocyclic, C6-10 aryl or 5-10 heteroaryl; R2 and R3 are each independently selected from hydrogen, deuterium, halogen, -CN, -C1-6 alkyl, -SR b -S(O)R b -S(O)2R b -OR b or -NR b R c Alternatively, R2, R3, and the carbon atom attached to them together form a C5-7 cycloalkyl group or a 5-7 membered heterocyclic group; R b and R c Each is independently selected from -hydrogen, -deuterium, -C1-6 alkyl, -C2-6 alkenyl, -C2-6 ynyl, -C3-10 cycloalkyl, -4-10 heterocyclic, -C6-10 aryl, and -5-10 heteroaryl; X is selected from =O, =S, or =Se; n is an integer selected from 0 to 5, for example, it can be 0, 1, 2, 3, 4 or 5, etc. E is absent, or E is selected from -C1-6 alkylene-, -C2-6 alkenyl-, -C1-4 alkylene-O-C1-6 alkylene-, -C1-4 alkylene-C3-8 cycloalkylene-, -C1-6 alkylene-NH-CO-, -C2-6 alkenyl-NH-CO-, -C1-4 alkylene-O-C1-6 alkylene-NH-CO-, or -C1-4 alkylene-C3-8 cycloalkylene-NH-CO-, wherein the carbon and nitrogen of the aforementioned groups are optionally separated by one or more R d Replace, R d It is selected from hydrogen, deuterium, halogen, -CN, -NO2, -OH, -COOH, -NH2, -C1-4 alkyl, -O-C1-4 alkyl-, -NH-C1-4 alkyl, -N-(C1-4 alkyl)2 or -C3-8 cycloalkyl.

[0006] Preferably, R1 is -OH.

[0007] Preferably, R2 is selected from -H, -F, -CN, -CH3 or -OCH3.

[0008] Preferably, R3 is selected from -H, -F, -CN, -CH3, -OCH3 or -SCH3.

[0009] Preferably, R2 and R3 are both selected from -F.

[0010] Preferably, X is 0.

[0011] Preferably, n=3.

[0012] Preferably, R d Selected from -C1-4 alkyl or -C3-6 cycloalkyl.

[0013] Preferably, R d It is selected from methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, or cyclopentyl.

[0014] Preferably, R d Selected from methyl.

[0015] Preferably, E is selected from , , .

[0016] Preferably, the compound is selected from the following structures: , or .

[0017] In a second aspect, the present invention provides a linker-drug, the linker-drug comprising a compound of formula (II) or its tautomer, stereoisomer, solvate, isotopically labeled compound, or pharmaceutically acceptable salt or ester thereof; ; E, R2, R3, X, and n are as defined in the first aspect; R'1 is selected from -O-, -S-, -COO- or -N(Ra)-, where Ra is as defined in the first aspect; T is a reactive group that can covalently link with specific functional groups on the antibody; Q represents a structure that covalently connects T and L; L is an optional linking group; S is a shorter linker or an optional self-igniting group, and L is covalently linked to R'1.

[0018] Preferably, the T is selected from the following structures: , or .

[0019] Preferably, Q is selected from the following structures: , , or ; Among them, w 1 w 2w 3 w 4 It is an integer from 1 to 18, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, where the dashed end is connected to T and the wavy end is connected to L.

[0020] Preferably, L is selected from a peptide chain consisting of 2-8 (e.g., 2, 3, 4, 5, 6, 7, or 8) amino acid residues, wherein the amino acids are one or more combinations of glycine, phenylalanine, glutamic acid, glutamine, serine, threonine, tyrosine, methionine, cysteine, valine, alanine, aspartic acid, asparagine, lysine, arginine, citrulline, and ornithine, or the following structures: , , or The amino acid has a Q-terminus attached to its nitrogen end and an S-terminus attached to its carbon end.

[0021] Preferably, S is selected from non-existent structures or the following structures: , or , where m is an integer from 1 to 18, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, etc.; where the dashed end is connected to L and the wavy end is connected to R'1.

[0022] Preferably, T is selected from the following structures: or .

[0023] Preferably, Q is selected from the following structures: or , where w 1 w 2 It is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, where the dashed end is connected to T and the wavy end is connected to L.

[0024] Preferably, L is selected from a peptide chain consisting of 2-6 (e.g., 2, 3, 4, 5 or 6) amino acid residues, wherein the amino acid is preferably glycine, phenylalanine, glutamic acid, glutamine, valine, alanine, lysine or citrulline, with Q attached to the nitrogen end and S attached to the carbon end.

[0025] Preferably, S is selected from non-existent structures or the following structures: , where m is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc., where the dashed end is connected to L and the wavy end is connected to R'1.

[0026] Preferably, T is selected from the following structures: .

[0027] Preferably, Q is selected from the following structures: , where w 1 It is an integer of 5, where the dashed end connects to T and the wavy end connects to L.

[0028] Preferably, L is selected from Gly-Gly-Phe-Gly-, wherein the nitrogen end of the amino acid is connected to the Q wavy line and the carbon end is connected to the S dashed line.

[0029] Preferably, S is selected from the following structures: , where m is an integer of 1, and the dashed end connects to L, and the wavy end connects to R'1.

[0030] Preferably, the linker-drug is selected from the following structures: or .

[0031] Thirdly, the present invention provides an antibody-drug conjugate comprising a compound of formula (III) or its tautomer, stereoisomer, solvate, isotopically labeled compound, or pharmaceutically acceptable salt or ester thereof. ; Wherein, Ab is an antibody or its antigen-binding fragment; T' is a linker that connects to an antibody or its antigen-binding fragment; r is an integer or decimal from 1 to 18, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, etc. Q, L, S, R'1, E, X, R2, R3 and n are as defined in the second aspect.

[0032] Preferably, T' is selected from , or The dashed line is connected to Q, and the wavy line is connected to Ab. t1 and t2 are each independent integers from 0 to 18, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0033] Preferably, t1 and t2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0034] Preferably, T' is , where t1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0035] Preferably, T' is .

[0036] Preferably, Ab is an antibody that specifically binds to HER2 or TROP2 or its antigen-binding fragment.

[0037] Preferably, Ab is trastuzumab or tadaboterub.

[0038] Preferably, r is an integer or decimal from 1 to 12, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0039] Preferably, r is an integer or decimal of 2-8, such as 2, 3, 4, 5, 6, 7 or 8, for example, an integer or decimal of 4-8, such as 4, 5, 6, 7 or 8, for example, an integer or decimal of 6-8, such as 6, 7 or 8, for example, an integer or decimal of 7-8 (e.g., about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8).

[0040] Preferably, the antibody-drug conjugate is selected from: , , , .

[0041] Fourthly, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate described in the third aspect.

[0042] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0043] Fifthly, the present invention provides the use of any one or a combination of at least two of the camptothecin derivatives described in the first aspect, the linker-drug described in the second aspect, the antibody-drug conjugate described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of an antitumor formulation.

[0044] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0045] Compared with the prior art, the present invention has the following beneficial effects: This invention develops a class of camptothecin derivatives with unique structures, which can be used as warheads for antibody-drug conjugates to prepare antibody-drug conjugates that can effectively kill tumor cells; the therapeutic effect of antibody-drug conjugates prepared based on the camptothecin derivatives is significantly improved, and the therapeutic window is increased. Attached Figure Description

[0046] Figure 1 The results show the cytotoxicity of compounds P1 and P2 on the NCI-N87 cell line.

[0047] Figure 2 The results show the cytotoxicity of compounds P1 and P2 on the SKBR-3 cell line.

[0048] Figure 3 The results show the cytotoxicity of compounds P1 and P2 on the HCC1954 cell line.

[0049] Figure 4 The results show the cytotoxicity of compounds P1 and P3 on the NCI-N87 cell line.

[0050] Figure 5 The results show the cytotoxicity of compounds P1 and P3 on the SKBR-3 cell line.

[0051] Figure 6 The results of cytotoxicity assays of compounds Trastuzumab-ADC-1, Trastuzumab-ADC-2, R-Trastuzumab-mc-GGFG-Dxd, Datopotamab-ADC-3, Datopotamab-ADC-4, and R-Datopotamab-mc-GGFG-Dxd on the NCI-N87 cell line are presented.

[0052] Figure 7 The results of cytotoxicity assays of compounds Trastuzumab-ADC-1, Trastuzumab-ADC-2, R-Trastuzumab-mc-GGFG-Dxd, Datopotamab-ADC-3, Datopotamab-ADC-4, and R-Datopotamab-mc-GGFG-Dxd on the SKBR-3 cell line are presented.

[0053] Figure 8 The results (tumor volume) show the tumor-suppressive proliferation activity of Tratuzumab-ADC-1 and R-Trastuzumab-mc-GGFG-Dxd in the CDX model of the mouse NCI-N87 cell line.

[0054] Figure 9The results of tumor suppressor proliferation activity assays (body weight) of Tratuzumab-ADC-1 and R-Trastuzumab-mc-GGFG-Dxd in the CDX model of mouse NCI-N87 cell line. Detailed Implementation

[0055] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments are merely illustrative while showing preferred embodiments of the invention, as those skilled in the art will appreciate various changes and modifications within the concept and scope of the invention from these detailed descriptions.

[0056] Terminology and definitions.

[0057] In this document, the singular forms introduced by “a,” “an,” and “the,” etc., include the plural meaning, unless otherwise stated. Furthermore, the terms “a,” “a or more,” and “at least one” are used interchangeably herein.

[0058] In this document, unless otherwise stated, whether the numerical value or range is preceded by "approximately", it encompasses a reasonable approximation range that would be understood by one of those skilled in the art, such as ±10%, ±5%, ±3%, ±2%, ±1%, or ±0.5% of the specified value.

[0059] One or more features in one embodiment of this invention may be combined with one or more features in another embodiment without departing from the concept and idea of ​​the invention.

[0060] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publicly available and patent documents cited herein are incorporated by reference and are suitable for various purposes. The cited documents are to be regarded as indicating the skill level of someone skilled in the art, but this should not be construed as an admission that they preceded this invention, which did so.

[0061] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0062] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0063] Example 1 Synthesis of toxins, synthesis of compound P1.

[0064] The synthetic route for compound P1 is shown below. Step 1: Synthesis of compound P1-1.

[0065] Ethynyltrimethylsilane (4.06 g, 41.3 mmol, 5.72 mL) was added to anhydrous tetrahydrofuran (50.0 mL). Butyllithium (2.50 M, 17.2 mL) was added with stirring and the mixture was stirred at -78 °C for 45 min; then a solution of (2S)-2-methyloxapropylcyclone (2.00 g, 34.4 mmol, 2.41 mL) in anhydrous tetrahydrofuran (50.0 mL) was added dropwise and the mixture was stirred at -78 °C for 15 min; then a solution of boron trifluoride diethyl ether (7.33 g, 51.7 mmol, 6.35 mL) was added dropwise and the mixture was stirred at -78 °C for 1 h. TLC (petroleum ether / ethyl acetate = 10:1, Rf = 0.25) showed that the reaction was complete. The reaction mixture was quenched by adding saturated ammonium chloride (100 mL) at 0 °C and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give a colorless oily (2S)-5-(trimethylsilyl)pent-4-yn-2-ol (4.90 g, 91.0% yield).

[0066] 1 H NMR (400MHz, CDCl3): δppm 3.90-4.01 (m, 1H), 2.33-2.48 (m, 2H), 1.26 (d, J = 6.1 Hz, 3H), 0.17 (s, 9H).

[0067] Step 2, Synthesis of compound P1-2.

[0068] (2S)-5-(trimethylsilyl)pent-4-yn-2-ol (4.90 g, 31.4 mmol) was added to anhydrous N,N-dimethylformamide (30.0 mL). Imidazole (2.77 g, 40.8 mmol) and tert-butyldiphenylchlorosilane (11.2 g, 40.8 mmol, 10.4 mL) were added with stirring at 0 °C. The resulting solution was heated to 25 °C and stirred for 12 hours. TLC (petroleum ether / ethyl acetate = 10:1, Rf = 0.65) showed that the reaction was complete. The reaction mixture was quenched by adding water (100 mL) at 25 °C and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1, Rf = 0.65) to give a colorless oily tert-butyldiphenyl{[(2S)-5-(trimethylsilyl)pent-4-yn-2-yl]oxo}silane (12.3 g, 99.4% yield).

[0069] 1 H NMR (400MHz, CDCl3): δppm 7.65-7.74 (m, 4H), 7.34-7.47 (m, 6H), 4.01(m, 1H), 2.28-2.47 (m, 2H), 1.17 (d, J = 6.0 Hz, 3H), 1.07 (s, 9H), 0.14 (s, 9H).

[0070] Step 3: Synthesis of compound P1-3.

[0071] 12.3 g (31.2 mmol) of tert-butyldiphenyl{[(2S)-5-(trimethylsilyl)pent-4-yn-2-yl]oxo}silane was added to methanol (50.0 mL). Potassium carbonate (6.46 g (46.7 mmol) was added with stirring. The resulting solution was stirred at 25 °C for 2 hours. TLC (petroleum ether / ethyl acetate = 10:1, Rf = 0.35) showed that the reaction was complete. The reaction mixture was quenched by adding water (100 mL) at 25 °C and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1, Rf = 0.35) to give colorless oily tert-butyl[(2S)-pent-4-yn-2-oxy]diphenylsilane (5.30 g, 52.7% yield).

[0072] 1H NMR (400MHz, CDCl3): δppm 7.48-7.60 (m, 4H), 7.19-7.34 (m, 6H), 3.85 (m, 1H), 2.08-2.27 (m, 2H), 1.07 (d, J = 6.4 Hz, 3H), 0.93 (s, 10H).

[0073] Step 4: Synthesis of compound P1-4.

[0074] 2-Amino-4,5-difluorobenzoic acid (3.00 g, 17.3 mmol) was dissolved in acetonitrile (60.0 mL), followed by the addition of triethylamine (3.51 g, 34.7 mmol, 4.82 mL), 4-dimethylaminopyridine (212 mg, 1.73 mmol), and BOC anhydride (5.29 g, 24.3 mmol, 5.57 mL). The mixture was stirred at 25 °C for 3 hours, and TLC (petroleum ether / ethyl acetate = 3 / 1, R0) was performed. f =0.40) indicates the reaction is complete. The reaction mixture was diluted with water (100 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was then analyzed by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 3 / 1, R...). f =0.40) The residue was purified to give 2-{[(tert-butoxy)carbonyl]amino}-4,5-difluorobenzoic acid (2.91 g, 61.3% yield) as a yellow solid.

[0075] 1 H NMR (400 MHz, DMSO- d 6 ): δppm 10.56 (s, 1H), 8.26 (dd, J = 13.9, 7.5Hz, 1H), 7.91 (dd, J = 11.2, 9.2 Hz, 1H), 1.49 (s, 9H).

[0076] Step 5: Synthesis of compound P1-5.

[0077] A solution of 2-{[(tert-butoxy)carbonyl]amino}-4,5-difluorobenzoic acid (2.50 g, 9.15 mmol) and methoxy(methyl)amine hydrochloride (1.78 g, 18.3 mmol) in N,N-dimethylformamide (40.0 mL) was prepared with O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphonium salt (HATU) (6.96 g, 18.3 mmol) and N,N-diisopropylethylamine (DIEA) (3.55 g, 27.4 mmol, 4.78 mL). The mixture was stirred at 25 °C for 1 hour, and LC-MS showed that the starting materials were consumed. The reaction solution was diluted with water (100 mL) and extracted with ethyl acetate (60.0 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue, which was then purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 91 / 9). A yellow oily tert-butyl N-{4,5-difluoro-2-[methoxy(methyl)aminocarbonyl]phenyl}aminomethyl ester (2.46 g, 85.0% yield) was obtained.

[0078] LCMS (ESI, m / z): 316.12 [M+H-55] + : 261.1.

[0079] Step 6: Synthesis of compound P1-6.

[0080] 4.08 g (12.7 mmol) of tert-butyl[(2S)-pentan-4-ynyl-2-oxy]diphenylsilane was dissolved in tetrahydrofuran (20.0 mL). The solution was cooled to -78 °C using a dry ice-ethyl acetate bath. 5.06 mL (2.5 M) of n-butyllithium was slowly added dropwise, and the mixture was stirred at -78 °C for 1 hour. Then, a solution of tert-butylN-{4,5-difluoro-2-[methoxy(methyl)aminocarbonyl]phenyl}aminomethyl ester (1.00 g, 3.16 mmol) in tetrahydrofuran (20.0 mL) was added. The dry ice bath was removed, and the temperature was allowed to rise naturally to room temperature. TLC (petroleum ether / ethyl acetate = 10 / 1, R f =0.55) indicates that the starting material has been consumed. The reaction solution was diluted with water (80.0 mL) and extracted with ethyl acetate (20.0 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue, which was then analyzed by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1, R...). f =0.55) The residue was purified to give yellow oily tert-butyl N-{2-[(5S)-5-[(tert-butyldiphenylsilyl)oxo]hex-2-ynyl]-4,5-difluorophenyl}aminomethyl ester (1.73 g, 94.5% yield).

[0081] 1 H NMR (400 MHz, CDCl3): δppm 10.76 (s, 1H), 8.44 (dd, J = 13.5, 7.4Hz, 1H), 8.05 (dd, J = 10.8, 8.7 Hz, 1H), 7.70 (m, 6H), 7.36-7.46 (m, 9H), 2.60 (dd, J = 5.3, 2.6 Hz, 2H), 1.54 (s, 9H), 1.26-1.31 (m, 5H), 1.08 (s, 9H), 1.07 (s, 3H).

[0082] Step 7: Synthesis of compound P1-7.

[0083] Tert-butyl N-{2-[(5S)-5-[(tert-butyldiphenylsilyl)oxo]hex-2-ynyl]-4,5-difluorophenyl}aminomethyl ester (1.73 g, 2.94 mmol) and 2,6-dimethylpyridine (158 mg, 1.47 mmol) were dissolved in toluene (20.0 mL). Wet palladium on carbon (188 mg, 176 μmol, 10% w / w) was added under a nitrogen atmosphere, followed by three purgings with hydrogen. The reaction was carried out at 25 °C under a hydrogen atmosphere (15 psi) for 1 hour. LC-MS showed that the starting material was completely consumed. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was analyzed by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1, R...). f =0.60) The residue was purified to give colorless oily tert-butyl N-{2-[(5S)-5-[(tert-butyldiphenylsilyl)oxo]hexanoyl]-4,5-difluorophenyl}aminomethyl ester (1.70 g, 99.3% yield).

[0084] LSMS (ESI, m / z): 581.28 [M+Na] + : 604.2.

[0085] Step 8: Synthesis of compound P1-8.

[0086] Tert-butyl N-{2-[(5S)-5-[(tert-butyldiphenylsilyl)oxo]hexanoyl]-4,5-difluorophenyl}aminomethyl ester (1.70 g, 2.92 mmol) was dissolved in dichloromethane (20.0 mL), and then trifluoroacetic acid (4.61 g, 40.4 mmol, 3.00 mL) was added. The mixture was stirred at 25 °C for 2 hours, and the reaction solution was concentrated under reduced pressure to obtain a residue. The residue was dissolved in dichloromethane (20.0 mL) and washed with saturated NaHCO3 solution (30.0 mL). The organic phase was concentrated under reduced pressure to give a yellow oily (5S)-1-(2-amino-4,5-difluorophenyl)-5-[(tert-butyldiphenylsilyl)oxo]hexane-1-one (1.29 g, 91.6% yield).

[0087] LSMS (ESI, m / z): 481.22 [M+H] + : 482.2.

[0088] Step 9: Synthesis of compound P1-9.

[0089] To a solution of (5S)-1-(2-amino-4,5-difluorophenyl)-5-[(tert-butyldiphenylsilyl)oxo]hexane-1-one (1.29 g, 2.68 mmol) and (4S)-4-ethyl-4-hydroxy-1H,3H,4H,6H,7H,8H,10H-pyrano[3,4-f]indoleazine-3,6,10-trione (705 mg, 2.68 mmol) in toluene (40.0 mL), 2-methylphenol (1.74 g, 16.1 mmol) and p-toluenesulfonic acid (PTSA) (231 mg, 1.34 mmol) were added, and the mixture was stirred at 100 °C for 12 hours. LC-MS showed that the starting material was completely consumed. The reaction solution was concentrated under reduced pressure to obtain the residue, which was then analyzed by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 1 / 1, R...). f =0.40) Purification of the residue yielded a pink solid (19S)-10-[(4S)-4-[(tert-butyldiphenylsilyl)oxo]pentyl]-19-ethyl-6,7-difluoro-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20 ] Twenty-one carbon-1(21),2,4,6,8,10,15(20)-heptaen-14,18-dione (450 mg, 23.7% yield).

[0090] LCMS (ESI, m / z): 708.28 [M+H]+ : 709.2.

[0091] Step 10: Synthesis of compound P1.

[0092] (19S)-10-[(4S)-4-[(tert-butyldiphenylsilyl)oxo]pentyl]-19-ethyl-6,7-difluoro-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20 150 mg, 212 μmol of 1-carbo-1(21),2,4,6,8,10,15(20)-heptaen-14,18-dione was dissolved in acetonitrile (6.00 mL), and then fluoroboric acid (186 mg, 846 μmol, 132 μL, 40% purity) was added. The mixture was stirred at 25 °C for 3 hours, and LCMS showed that the feed was completely consumed. The reaction solution was concentrated under reduced pressure to obtain the residue, which was then purified by Prep-HPLC (CD04-Welch Ultimate C18 150×25×7μm column, wavelength: UV 220nm & 254nm; column temperature: 30℃; mobile phase: 0.1% TFA, water: acetonitrile, elution gradient: 27-57%, elution time: 10 min). The obtained product was then freeze-dried to obtain a white solid (19S)-19-ethyl-6,7-difluoro-19-hydroxy-10-[(4S)-4-hydroxypentyl]-17-oxa-3,13-diazapentacyclo[11.8.0.0]. 2 , 11 .0 4 , 9 .0 15 , 20 ] 21-C-1(21),2,4,6,8,10,15(20)-Heptadene-14,18-dione (16.1 mg, 16.1% yield, 99.6% purity).

[0093] LCMS (ESI, m / z): 470.17 [M+H] + : 471.3.

[0094] 1 H NMR (400 MHz, DMSO- d6): δppm 8.34 (dd, J = 12.1, 8.9 Hz, 1H), 8.18(dd, J = 11.4, 8.2 Hz, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.43 (s, 2H), 5.29(s, 2H), 4.42 (d, J = 4.8 Hz, 1H), 3.65 (dt, J = 11.4, 5.7 Hz, 1H), 3.17 (t,J = 7.2 Hz, 2H), 1.80-1.92 (m, 2H), 1.63-1.78 (m, 2H), 1.45-1.56 (m, 2H),1.05 (d, J = 6.1 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H).

[0095] Example 2 Synthesis of toxins, synthesis of compound P2.

[0096] The synthetic route for compound P2 is shown below. Step 1: Synthesis of compound P2-1.

[0097] Ethynyltrimethylsilane (4.06 g, 41.3 mmol, 5.72 mL) was added to anhydrous tetrahydrofuran (50.0 mL). Butyllithium (2.50 M, 17.2 mL) was added with stirring and the mixture was stirred at -78 °C for 45 min; then a solution of (2R)-5-(trimethylsilyl)pent-4-yn-2-ol (2.00 g, 34.4 mmol, 2.41 mL) in anhydrous tetrahydrofuran (50.0 mL) was added dropwise and the mixture was stirred at -78 °C for 15 min; then a solution of boron trifluoride diethyl ether (7.33 g, 51.7 mmol, 6.35 mL) was added dropwise and the mixture was stirred at -78 °C for 1 h. TLC (petroleum ether / ethyl acetate = 10:1, R f =0.25) indicates the reaction is complete. The reaction mixture was quenched by adding saturated ammonium chloride (100 mL) solution at 0 °C and extracted with ethyl acetate 300 mL (100 mL × 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give a colorless oily (2R)-5-(trimethylsilyl)pent-4-yn-2-ol (4.63 g, 86.0% yield).

[0098] 1H NMR (400MHz, CDCl3): δppm 3.89-3.99 (m, 1H), 2.30-2.48 (m, 2H), 1.25-1.28 (m, 3H), 0.16 (s, 9H).

[0099] Step 2, Synthesis of compound P2-2.

[0100] (2R)-5-(trimethylsilyl)pent-4-yn-2-ol (4.63 g, 29.6 mmol) was added to anhydrous N,N-dimethylformamide (30.0 mL). Imidazole (2.62 g, 38.5 mmol) and tert-butyldiphenylchlorosilane (10.6 g, 38.5 mmol, 9.86 mL) were added with stirring at 0 °C. The resulting solution was heated to 25 °C and stirred for 12 hours. TLC (petroleum ether / ethyl acetate = 10:1, R f =0.65) indicates the reaction is complete. The reaction mixture was quenched by adding water (100 mL) at 25 °C and extracted with 300 mL of ethyl acetate (100 mL × 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give a colorless oily tert-butyldiphenyl{[(2R)-5-(trimethylsilyl)pent-4-yn-2-yl]oxo}silane (11.6 g, 99.2% yield).

[0101] 1 H NMR (400MHz, CDCl3): δppm 7.51-7.61 (m, 4H), 7.22-7.32 (m, 6H), 3.82-3.92 (m, 1H), 2.15-2.32 (m, 2H), 1.03 (d, J = 6.0 Hz, 3H), 0.93 (s, 9H),0.00 (s, 9H).

[0102] Step 3: Synthesis of compound P2-3.

[0103] 11.6 g (29.4 mmol) of tert-butyldiphenyl{[(2R)-5-(trimethylsilyl)pent-4-yn-2-yl]oxo}silane was added to methanol (50.0 mL). Potassium carbonate (6.09 g, 44.1 mmol) was added with stirring. The resulting solution was stirred at 25 °C for 2 hours. TLC (petroleum ether / ethyl acetate = 10:1, R f=0.35) indicates that the reaction is complete. The reaction mixture was quenched by adding water (100 mL) at 25 °C and extracted with 300 mL of ethyl acetate (100 mL × 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give a colorless oily tert-butyl[(2R)-pentan-4-yn-2-oxy]diphenylsilane (6.30 g, 66.5% yield).

[0104] 1 H NMR (400MHz, CDCl3): δppm 7.66-7.72 (m, 4H), 7.36-7.47 (m, 6H), 3.89-4.06 (m, 1H), 2.22-2.42 (m, 2H), 1.22 (d, J = 6.0 Hz, 3H), 1.04-1.09 (m,9H).

[0105] Step 4: Synthesis of compound P2-4.

[0106] 4.08 g (12.7 mmol) of tert-butyl[(2R)-pentan-4-ynyl-2-oxy]diphenylsilane was added to anhydrous tetrahydrofuran (20.0 mL). While stirring, 5.06 mL (2.50 M) of n-butyllithium was added, and the mixture was stirred at -78 °C for 1 hour. Then, a solution of 1.00 g (3.16 mmol) of P1-5 in anhydrous tetrahydrofuran (20.0 mL) was added dropwise. The resulting solution was slowly heated to 25 °C and stirred for 1 hour. LC-MS showed the reaction was complete. TLC (petroleum ether / ethyl acetate = 10:1, R f =0.55) indicates that the reaction is complete. The reaction mixture was quenched by adding water (50.0 mL) at 0 °C and extracted with 150 mL of ethyl acetate (50.0 mL × 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give a colorless oily tert-butyl N-{2-[(5R)-5-[(tert-butyldiphenylsilyl)oxo]hex-2-ynyl]-4,5-difluorophenyl}aminomethyl ester (1.60 g, 87.6% yield).

[0107] LCMS (ESI, m / z): 577.25 [M+H] + : 578.2.

[0108] 1H NMR (400MHz, CDCl3): δppm 10.68 (s, 1H), 8.35 (dd, J = 13.5, 7.4Hz, 1H), 7.97 (dd, J = 10.8, 8.7 Hz, 1H), 7.57-7.67 (m, 4H), 7.25-7.42 (m,6H), 3.89-4.13 (m, 1H), 2.46-2.58 (m, 2H), 1.45 (s, 9H), 1.21 (d, J = 6.1 Hz,3H), 1.00 (s, 9H).

[0109] Step 5: Synthesis of compound P2-5.

[0110] A mixture of tert-butyl N-{2-[(5R)-5-[(tert-butyldiphenylsilyl)oxo]hexanoyl]-4,5-difluorophenyl}aminomethyl ester (1.60 g, 2.77 mmol) and 2,6-dimethylpyridine (148 mg, 1.38 mmol) in toluene (20.0 mL) was added to dry Pd / C (589 mg, 10% w / w), and stirred at 25 °C for 1 hour under a hydrogen atmosphere (15 psi). LCMS showed the reaction was complete. TLC (petroleum ether / ethyl acetate = 10:1, Rf = 0.55) showed the reaction was complete. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a colorless oily tert-butyl N-{2-[(5R)-5-[(tert-butyldiphenylsilyl)oxo]hexanoyl]-4,5-difluorophenyl}aminomethyl ester (1.83 g). The crude product can be used directly in the next step without purification.

[0111] LCMS (ESI, m / z): 581.28 [M+Na] + : 604.2.

[0112] 1H NMR (400MHz, CDCl3): δppm 11.02 (br s, 1H), 8.46 (dd, J = 13.7, 7.7Hz, 1H), 7.64-7.73 (m, 4H), 7.54 (dd, J = 11.1, 8.6 Hz, 1H), 7.34-7.46 (m,6H), 3.83-3.94 (m, 1H), 2.72 (t, J = 7.3 Hz, 2H), 1.65-1.76 (m, 2H), 1.54 (s,9H), 1.44-1.52 (m, 2H), 1.13 (d, J = 6.1 Hz, 3H), 1.06 (s, 9H).

[0113] Step 6: Synthesis of compound P2-6.

[0114] 1.83 g (3.15 mmol) of tert-butyl N-{2-[(5R)-5-[(tert-butyldiphenylsilyl)oxo]hexanoyl]-4,5-difluorophenyl}aminomethyl ester was added to dichloromethane (20.0 mL). Trifluoroacetic acid (2.00 mL) was added with stirring. The resulting solution was stirred at 25 °C for 2 hours. LC-MS showed the reaction was complete. TLC (petroleum ether / ethyl acetate = 10:1, R f =0.4) indicates the reaction is complete. The reaction mixture was concentrated under reduced pressure to remove trifluoroacetic acid, the system was adjusted to alkalinity with an aqueous sodium bicarbonate solution, and extracted with 90.0 mL (30.0 mL × 3) of ethyl acetate. The combined organic layers were washed with saturated brine (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give a colorless oily (5R)-1-(2-amino-4,5-difluorophenyl)-5-[(tert-butyldiphenylsilyl)oxo]hexane-1-one (1.42 g, 93.7% yield).

[0115] LCMS (ESI, m / z): 481.22 [M+H] + : 482.3.

[0116] 1H NMR (400MHz, CDCl3): δppm 7.67-7.77 (m, 5H), 7.34-7.49 (m, 8H), 6.42 (dd, J = 12.1, 6.7 Hz, 1H), 3.89 (m, 1H), 2.67 (t, J = 7.4 Hz, 2H), 1.70 (m, 2H), 1.46-1.57 (m, 2H), 1.12 (d, J = 6.1 Hz, 3H), 1.07 (s, 9H).

[0117] Step 7: Synthesis of compound P2-7.

[0118] To a mixture of (5R)-1-(2-amino-4,5-difluorophenyl)-5-[(tert-butyldiphenylsilyl)oxo]hexane-1-one (400 mg, 830 μmol) and (4S)-4-ethyl-4-hydroxy-1H,3H,4H,6H,7H,8H,10H-pyrano[3,4-f]indoleazine-3,6,10-trione (219 mg, 830 μmol) in toluene (20.0 mL), 4-methylbenzenesulfonic acid monohydrate (71.5 mg, 415 μmol) and 2-methylphenol (539 mg, 4.98 mmol) were added, and the mixture was stirred at 100 °C for 2 hours. LC-MS showed the reaction was complete. TLC (petroleum ether / ethyl acetate = 1:1, R f =0.4) indicates the reaction is complete. The filtrate was directly concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) to give a colorless oily (19S)-10-[(4R)-4-[(tert-butyldiphenylsilyl)oxo]pentyl]-19-ethyl-6,7-difluoro-19-hydroxy-17-oxa-3,13-diazapentane[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20 ] Twenty-one carbon-1(21),2,4,6,8,10,15(20)-heptaene-14,18-dione (260 mg, 44.2% yield).

[0119] LCMS (ESI, m / z): 708.28 [M+H] + : 709.4.

[0120] Step 8: Synthesis of compound P2.

[0121] (19S)-10-[(4R)-4-[(tert-butyldiphenylsilyl)oxo]pentyl]-19-ethyl-6,7-difluoro-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20 50.0 mg, 70.5 μmol of 1(21),2,4,6,8,10,15(20)-heptaen-14,18-dione was added to acetonitrile (2.50 mL). Fluoroboric acid (155 mg, 705 μmol, 110 μL, 40% purity) was added with stirring. The resulting solution was stirred at 25 °C for 1 hour. LC-MS showed that the reaction was complete. The filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by Prep-HPLC (CD04-Welch Ultimate C18 150×25×7μm column, wavelength: UV 220nm & 254nm; column temperature: 30℃; mobile phase: 0.1% TFA, water: acetonitrile, elution gradient: 24-54%, elution time: 14 min), and the resulting product was then freeze-dried to give compound P2 as a white solid (15.0 mg, 44.6% yield, 98.7% purity).

[0122] LCMS (ESI, m / z): 470.17 [M+H] + : 470.9.

[0123] 1 H NMR (400MHz, DMSO- d 6): δppm 8.35 (dd, J = 11.4, 9.2 Hz, 1H), 8.14-8.24 (m, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 5.43 (s, 2H), 5.30 (br s, 2H), 4.41(d, J 1.05 (d, J = 6.1 Hz,3H), 0.87 (t, J = 7.3 Hz, 3H).

[0124] Example 3 Synthesis of toxins, synthesis of compound 3.

[0125] The synthetic route for compound P3 is shown below. Step 1: Synthesis of compound P3-1.

[0126] To (19S)-10-(chloromethyl)-19-ethyl-6,7-difluoro-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20 2,4,6,8,10,15(20)-heptaen-14,18-dione (2.00 g, 9.73 mmol) and (4S)-4-ethyl-4-hydroxy-1H,3H,4H,6H,7H,8H,10H-pyrano[3,4-f]indoleazine-3,6,10-trione (2.56 g, 9.73 mmol) were added to a solution of toluene (20.0 mL) with 2-methylphenol (6.31 g, 58.4 mmol) and p-toluenesulfonic acid pyridine (PPTS) (611.18 mg, 2.43 mmol) and stirred at 100 °C for 12 hours. LCMS showed that the starting materials were completely consumed. The reaction solution was filtered, and the resulting filter cake was washed three times with toluene (5.00 mL × 3). The solution was then concentrated under reduced pressure to obtain a yellow solid (19S)-10-(chloromethyl)-19-ethyl-6,7-difluoro-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.0]. 2 , 11 .0 4 , 9 .0 15 , 20 ] 21-C-1(21),2,4,6,8,10,15(20)-Heptadene-14,18-dione (3.01 g, 71.2% yield).

[0127] LCMS (ESI, m / z): 432.07 [M+H] + : 433.0.

[0128] Step 2, Synthesis of compound P3-2.

[0129] To (19S)-10-(chloromethyl)-19-ethyl-6,7-difluoro-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .04 , 9 .0 15 , 20 Twenty-one carbon-1(21),2,4,6,8,10,15(20)-heptaen-14,18-dione (2.00 g, 4.62 mmol) and (3E)-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2-yl)but-3-en-2-ol (1.96 g, 9.24 mmol) in 1,4-dioxane (30.0 mL) and water (3.00 mL). Potassium carbonate (1.92 g, 13.9 mmol), tris(dibenzylacetone)dipalladium (Pd2(dba)3) (846 mg, 924 μmol), and 1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxo-6-phosphatamidoadaramane (811 mg, 2.77 mmol) were added to the solution. The mixture was substituted three times with N2 and stirred at 100 °C under a nitrogen atmosphere for 3 hours. LC-MS showed that the starting material was completely consumed. The reaction solution was diluted with water (60.0 mL) and extracted with ethyl acetate (20.0 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow oily (19S)-19-ethyl-6,7-difluoro-19-hydroxy-10-[(2E)-4-hydroxy-4-methylpent-2-en-1-yl]-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20 ] 3.01 g of 21-carbon-1(21),2,4,6,8,10,15(20)-heptaen-14,18-dione (71.2% yield) was used directly for the next step.

[0130] LCMS (ESI, m / z): 482.17 [M+H] + : 483.1.

[0131] Step 3: Synthesis of compound P3.

[0132] The compound (19S)-19-ethyl-6,7-difluoro-19-hydroxy-10-[(2E)-4-hydroxy-4-methylpent-2-en-1-yl]-17-oxa-3,13-diazapentacyclo[11.8.0.0] was used. 2 , 11 .0 4 , 9 .0 15 , 20200 mg (415 μmol) of 2-1(21),2,4,6,8,10,15(20)-heptaen-14,18-dione was dissolved in tetrahydrofuran (4.00 mL). Wet palladium on carbon (40.0 mg, 41.5 μmol, 10% w / w) was added under a nitrogen atmosphere, followed by three exposures to hydrogen. The mixture was stirred at 0 °C for 1 hour under a hydrogen atmosphere (15 psi). LC-MS showed that the starting material was completely consumed. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by Prep-HPLC (CD24-XPT C18 150×25×7μm column, wavelength: UV 220nm & 254nm; column temperature: 30℃; mobile phase: 0.1% TFA, water: acetonitrile, elution gradient: 22-52%, elution time: 11 min), and the resulting product was then freeze-dried to give a yellow solid compound P3 (5.55 mg, 2.59% yield, purity 93.7%).

[0133] LCMS (ESI, m / z): 484.18 [M+H] + : 485.3.

[0134] 1 H NMR (400 MHz, DMSO- d 6 ): δppm 8.36 (dd, J = 12.0, 8.8 Hz, 1H), 8.21 (dd, J = 11.5, 8.3 Hz, 1H), 7.33 (s, 1H), 6.40-6.71 (m, 1H), 5.44 (s, 2H), 5.32 (s, 2H), 3.18 (t, J = 7.3 Hz, 2H), 1.83-1.91 (m, 2H), 1.69-1.77 (m, 2H), 1.54-1.59 (m, 2H), 1.04-1.08 (m, 6H), 0.88 (t, J = 7.2 Hz, 3H).

[0135] The reference toxin Dxd was purchased from MedChemExpress, lot number: 268112, catalog number: HY-13631D.

[0136] Control toxin Exatecan: Purchased from: Wuyan Pharmaceutical Technology (Shanghai) Co., Ltd., batch number: WY052-S3A-250101, product number: WY052-S3Aa.

[0137] Example 4 Synthesis of PL 1 of linker-toxin The synthetic route for linker-toxin PL1 is shown below. Step 1: Synthesis of compound PL 1-1.

[0138] 4-Methylbenzene-1-sulfonic acid (6.73 mg, 39.1 μmol) was added to anhydrous tetrahydrofuran (2.00 mL) of [2-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)acetamido]methyl acetate (144 mg, 391 μmol), and stirred at 0 °C for 30 minutes. Then (19S)-19-ethyl-6,7-difluoro-19-hydroxy-10-[(4S)-4-hydroxypentyl]-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20 [C21-1(21),2,4,6,8,10,15(20)-heptaen-14,18-dione (92.0 mg, 195 μmol). The resulting solution was heated to 25 °C and stirred for 1.5 hours. LCMS showed 25% of the starting material and 26% of the product. The reaction mixture was concentrated under reduced pressure to give the residue.] The residue was purified by Prep-HPLC (CD24-XPT 150×25×7μm column, wavelength: UV 220nm & 254nm; column temperature: 30℃); mobile phase: 0.1% TFA, water: acetonitrile, elution gradient: 43-63%, elution time: 14 min). The resulting product was then freeze-dried to obtain a yellow solid (9H-fluorene-9-yl)methyl N-{[({[(2S)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxane-17-oxa-3,13-diazapentacyclo[11.8.0.0]). 2 , 11 .0 4 , 9 .0 15 , 20 [C21-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]pentane-2-yl]oxo}methyl)aminocarbonyl]methyl}aminomethyl ester (40.0 mg, 23.3% yield).

[0139] LCMS (ESI, m / z): 778.28 [M+H] + : 779.3.

[0140] Step 2, Synthesis of compound PL 1-2.

[0141] To (9H-fluorene-9-yl)methyl N-{[({[(2S)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxane-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20 Piperidine (98.4 mg, 1.16 mmol) was added to a mixture of N,N-dimethylformamide (4.00 mL) of 2-carbon-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]pentane-2-yl]oxo}methyl)aminocarbonyl]methyl}aminomethyl ester (45.0 mg, 57.7 μmol). The mixture was stirred at 0 °C for 30 min. LCMS showed that the reaction was complete. The reaction mixture was washed three times with methyl tert-butyl ether / petroleum ether (1 v / 20 v, 5.00 mL × 3) solution, and the lower organic phase was concentrated under reduced pressure to give a yellow solid 2-amino-N-({[(2S)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxane-17-oxa-3,13-diazapentacyclo[11.8.0.0]). 2 , 11 .0 4 , 9 .0 15 , 20 [C21-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]pentane-2-yl]oxo}methyl)acetate (35.0 mg, crude product), the crude product is used directly in the next step.

[0142] LCMS (ESI, m / z): 556.21 [M+H] + : 557.3.

[0143] Step 3: Synthesis of compound PL1.

[0144] At 0°C, 2-amino-N-({[(2S)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxane-17-oxa-3,13-diazapentacyclo[11.8.0.0]) 2 , 11 .0 4 , 9 .0 15 , 20The mixture of N,N-dimethylformamide (2.00 mL) of 2-C-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]pentan-2-yl]oxo}methyl)acetamide (35.0 mg, crude) was added to (2S)-2-(2-{2-[6-(2,5-dioxane-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino]acetamido}acetamido)-3-phenylpropionic acid (29.7 mg, 62.9 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (35.8 mg, 94.3 μmol) and N,N-diisopropylethylamine (DIEA) (24.4 mg, 188 μmol), and the mixture was stirred at 0 °C for 1 hour. LCMS showed the reaction was complete. The mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by Prep-HPLC (CD24-XPT C18 150×25×7μm column, wavelength: UV 220nm & 254nm; column temperature: 30℃; mobile phase: 0.1% TFA, water: acetonitrile, elution gradient: 30-50%, elution time: 14 min), and then the product was freeze-dried to give a yellow solid 6-(2,5-dioxonyl-2,5-dihydro-1H-pyrrole-1-yl)-N-{[({[(1S)-1-({[({[(2S)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxonyl-17-oxa-3,13-diazapentacyclo[11.8.0.0]). 2 , 11 .0 4 , 9 .0 15 , 20 [Typocarbon-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]pentane-2-yl]oxo}methyl)aminocarbonyl]methyl}aminocarbonyl)-2-phenylethyl]aminocarbonyl}methyl)aminocarbonyl]methyl}hexamamide (11.3 mg, 17.4% yield).

[0145] LCMS (ESI, m / z): 1010.4 [M+H] + : 1011.6.

[0146] 1 H NMR (400 MHz, DMSO- d 6) δ ppm 8.41 - 8.33 (m, 2H), 8.26 - 8.16 (m, 2H), 8.09 - 8.02 (m, 2H), 7.98 (t, J= 5.3 Hz, 1H), 7.32 (s, 1H), 7.22 - 7.14 (m,5H), 6.98 (s, 2H), 5.43 (s, 2H), 5.32 (s, 2H), 4.60 - 4.51 (m, 2H), 4.48 - 4.42(m, 1H), 3.69 - 3.65 (m, 7H), 3.35 (t, J = 7.1 Hz, 2H), 3.18 (t, J = 7.3 Hz, 2H), 3.02 - 2.95 (m, 1H), 2.79 - 2.71 (m, 1H), 2.09 (t, J = 7.4 Hz, 2H), 1.90 -1.82 (m, 2H), 1.70 - 1.55 (m, 4H), 1.52 - 1.40 (m, 5H), 1.22 - 1.15 (m, 2H), 1.07(d, J = 6.4 Hz, 3H), 0.87 (t, J = 7.6 Hz, 3H).

[0147] Example 5 Synthesis of linker-toxin PL2.

[0148] The synthetic route for linker-toxin PL2 is shown below. Step 1: Synthesis of compound PL 2-1.

[0149] 4-Methylbenzene-1-sulfonic acid (PTSA) (22.7 mg, 131 μmol) was added to anhydrous tetrahydrofuran (6.00 mL) of [2-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)acetamido]methyl acetate (485 mg, 1.32 mmol) and stirred at 0 °C for 30 minutes. Then (19S)-19-ethyl-6,7-difluoro-19-hydroxy-10-[(4R)-4-hydroxypentyl]-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20[298 mg, 658 μmol] Twentidocosa-1(21),2,4,6,8,10,15(20)-heptaen-14,18-dione. The resulting solution was heated to 25 °C and stirred for 1.5 h. LCMS showed 21% of the starting material and 27% of the product. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by Prep-HPLC (CD24-XPT C18 150×25×7μm column, wavelength: UV 220nm & 254nm; column temperature: 30℃; mobile phase: 0.1% TFA, water: acetonitrile, elution gradient: 43-63%, elution time: 14 min), and then the product was freeze-dried to obtain a yellow solid (9H-fluorene-9-yl)methyl N-{[({[(2R)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxane-17-oxa-3,13-diazapentacyclo[11.8.0.0]). 2 , 11 .0 4 , 9 .0 15 , 20 [C21-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]pentane-2-yl]oxo}methyl)aminocarbonyl]methyl}aminomethyl ester (60.0 mg, 10.9% yield).

[0150] LCMS (ESI, m / z): 778.28 [M+H] + : 779.3.

[0151] Step 2, Synthesis of compound PL 2-2.

[0152] To (9H-fluorene-9-yl)methyl N-{[({[(2R)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxane-17-oxa-3,13-diazapentacyclo[11.8.0.0] 2 , 11 .0 4 , 9 .0 15 , 20Piperidine (131 mg, 1.54 mmol) was added to a mixture of N,N-dimethylformamide (6.00 mL) and 2-carbamate (61 mg, 77.0 μmol). The mixture was stirred at 0 °C for 30 min. LCMS showed that the reaction was complete. The reaction mixture was washed three times with a mixture of methyl tert-butyl ether / petroleum ether (1 v / 20 v, 10 mL × 3). The lower organic phase was concentrated under reduced pressure to give a yellow solid 2-amino-N-({[(2R)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxane-17-oxa-3,13-diazapentacyclo[11.8.0.0]). 2 , 11 .0 4 , 9 .0 15 , 20 [21),2,4,6,8,10,15(20)-heptaen-10-yl]pentane-2-yl]oxo}methyl)acetate (45.0 mg, crude product), the crude product is used directly in the next step.

[0153] LCMS (ESI, m / z): 556.21 [M+H] + : 557.2.

[0154] Step 3: Synthesis of compound PL 2.

[0155] At 0°C, 2-amino-N-({[(2R)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxane-17-oxa-3,13-diazapentacyclo[11.8.0.0]) 2 , 11 .0 4 , 9 .0 15 , 20A mixture of N,N-dimethylformamide (3.00 mL) of 2-carbon-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]pentane-2-yl]oxo}methyl)acetamide (45.0 mg, 80.8 μmol) was added to (2S)-2-(2-{2-[6-(2,5-dioxane-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino]acetamido}acetamido)-3-phenylpropionic acid (38.2 mg, 80.8 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (46.1 mg, 121 μmol) and N,N-diisopropylethylamine (DIEA) (31.3 mg, 242 μmol), and the mixture was stirred at 0 °C for 1 hour. LCMS showed the reaction was complete. The mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by Prep-HPLC (CD24-XPT C18 150×25×7μm column, wavelength: UV 220nm & 254nm; column temperature: 30℃; mobile phase: 0.1% TFA, water: acetonitrile, elution gradient: 30-50%, elution time: 14 min), and then the product was freeze-dried to give a white solid 6-(2,5-dioxonyl-2,5-dihydro-1H-pyrrole-1-yl)-N-{[({[(1S)-1-({[({[(2R)-5-[(19S)-19-ethyl-6,7-difluoro-19-hydroxy-14,18-dioxonyl-17-oxa-3,13-diazapentacyclo[11.8.0.0]). 2 , 11 .0 4 , 9 .0 15 , 20 [21),2,4,6,8,10,15(20)-heptaen-10-yl]pentane-2-yl]oxo}methyl)aminocarbonyl]methyl}aminocarbonyl)-2-phenylethyl]aminocarbonyl}methyl)aminocarbonyl]methyl}hexamamide (29.8 mg, 35.7% yield).

[0156] LCMS (ESI, m / z): 1010.4 [M+H] + : 1011.7.

[0157] 1 H NMR (400 MHz, DMSO- d 6) δ ppm 8.42 - 8.33 (m, 2H), 8.26 - 8.15 (m, 2H), 8.09 - 8.02 (m, 2H), 7.98 (t, J= 5.8 Hz, 1H), 7.32 (s, 1H), 7.22 - 7.12 (m,5H), 6.98 (s, 2H), 5.42 (s, 2H), 5.31 (s, 2H), 4.55 (t, J = 7.0 Hz, 2H), 4.47 (dt, J = 4.8, 8.8 Hz, 1H), 3.68 - 3.56 (m, 7H), 3.35 (t, J = 7.1 Hz, 2H), 3.18 (s, 2H), 3.03 - 2.96 (m, 1H), 2.79 - 2.71 (m, 1H), 2.09 (t, J = 7.3 Hz, 2H), 1.92 -1.82 (m, 2H), 1.72 - 1.55 (m, 4H), 1.52 - 1.39 (m, 5H), 1.17 (t, J = 7.6 Hz, 2H), 1.06 (d, J = 6.0 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0158] Comparative Example 1 mc-GGFG-Dxd, source: Changzhou WuXi AppTec Helian Biotechnology Co., Ltd., batch number: XDC-CZ-20240123, catalog number: EC21907-18-P1. Example 6 Synthesis and detection of antibody-drug conjugates Preparation of the antibody-drug conjugate trastuzumab-ADC-1. The antibody-drug conjugate trastuzumab-ADC-1 was prepared by a one-pot reaction, and the specific steps are as follows.

[0159] (1) Add TCEP (0.3 mM) to a solution of trastuzumab (Herceptin) (prepared according to the published corresponding protein sequence, light chain as shown in SEQ ID NO:1, heavy chain as shown in SEQ ID NO:2, by standard methods for preparing monoclonal antibodies, containing 0.03 mM trastuzumab, 40 mM sodium phosphate buffer, 2 mM EDTA, pH 7), and allow the reaction mixture to react at 37°C for 2 h.

[0160] SEQ ID NO:1: DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0161] SEQ ID NO:2: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0162] (2) Add PL1 (0.45 mM) dissolved in DMSO (commercially available from Aldrich Sigma) to the product of step (1) and allow the reaction mixture to react at 22°C for 1 hour.

[0163] (3) The reaction mixture was purified using a desalting column (type: 40K, 0.5 mL, REF: 87766, Lot#SJ251704, manufacturer: Thermo) to obtain the target trastuzumab-ADC-1.

[0164] The conjugation method for trastuzumab-ADC-2 is the same as that for trastuzumab-ADC-1.

[0165] Datopotamab-ADC-3 and Datopotamab-ADC-4 are conjugated using DAR4 technology (prepared according to the scheme in patent WO2020 / 164561, with the light chain as shown in SEQ ID NO:3 and the heavy chain as shown in SEQ ID NO:4).

[0166] SEQ ID NO:3: DIQMTQSPSSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0167] SEQ ID NO:4: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0168] Comparative Example 2 Synthesis of R-Trastuzumab-mc-GGFG-Dxd The control ADC was prepared by a one-pot reaction, and the specific steps are as follows: (1) Add TCEP (0.3 mM) to a solution of trastuzumab (prepared according to the published corresponding protein sequence by standard methods for preparing monoclonal antibodies, containing 0.03 mM trastuzumab, 40 mM sodium phosphate buffer, 2 mM EDTA, pH 7), and allow the reaction mixture to react at 37°C for 2 h.

[0169] (2) Add a linker-toxin (MC-GGFG-Dxd, 0.45 mM) dissolved in DMSO (commercially available from Aldrich Sigma) to the product of step (1), and allow the reaction mixture to react at 22°C for 1 hour.

[0170] (3) The reaction mixture was purified using a desalting column (type: 40K, 0.5 mL, REF: 87766, Lot#SJ251704, manufacturer: Thermo) to obtain the control R-Trastuzumab-mc-GGFG-Dxd.

[0171] The R-Datopotamab-mc-GGFG-Dxd coupling utilizes DAR4 technology (prepared according to the scheme in patent WO2020 / 164561).

[0172] Example 7 DAR value detection of antibody-drug conjugates.

[0173] The distribution of each component of the completely reduced drug was analyzed using LC-MS (Agilent PLRP-S 1000A column, 8 µm, 50 × 2.1 mm, flow rate: 0.6 mL / min, ambient temperature) to detect the drug-to-antibody ratio (DAR). The injection volume was 5 μg, with mobile phase solvent A being a 0.5% aqueous solution of TFA and mobile phase solvent B being a 0.5% TFA acetonitrile solution. Substances loaded with different drugs were eluted using a sequential fractional gradient elution. The results are shown in Table 1.

[0174] Table 1 The above results indicate that the PL molecule to be tested can be coupled with different antibodies using different conjugation methods to efficiently generate ADC molecules with the target DAR value.

[0175] Test Example 1 Detection of the inhibitory activity of antibody-drug conjugates on in vitro cell activity.

[0176] Dilute the antibody-drug conjugates (ADCs) to be tested (9 concentration gradients) with test medium (containing 10% FBS). Digest tumor cells using trypsin according to standard methods, collect cell counts, and resuspend in test medium (containing 10% FBS). Add the diluted cells to 96-well plates, incubate overnight, and then add the diluted ADCs. After 4–6 days, add 50 μL of CellTiter-Glo® reagent to each well and incubate at room temperature for 10 minutes to ensure cell lysis and ATP release. Record the luminescence values ​​(RLU, Relative Light Units) using a microplate reader (manufacturer: Molecular Devices, model: SpectraMax M5e) to evaluate the inhibitory effect of the analyte on cell proliferation.

[0177] The brand product number and batch number information for Dxd and Exatecan are shown below.

[0178] The reference toxin Dxd was purchased from MedChemExpress, lot number: 268112, catalog number: HY-13631D.

[0179] Control toxin Exatecan: Purchased from: Wuyan Pharmaceutical Technology (Shanghai) Co., Ltd., batch number: WY052-S3A-250101, product number: WY052-S3Aa.

[0180] The sources of tumor cells are shown in Table 2.

[0181] Table 2 All cell culture conditions in Table 2 are based on the methods provided by ATCC.

[0182] The results of the toxin test are shown in Table 3 and Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 As shown.

[0183] Table 3 From Table 3 and Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 As can be seen from the data, P1 has similar cytotoxicity to Exa and is superior to Dxd, P2 is weaker than Exa, and P3 has similar cytotoxicity to Dxd.

[0184] The results of the antibody-drug conjugate experiments are shown in Table 4 and Figure 6 , Figure 7 As shown.

[0185] Table 4 The above results indicate that both Trastuzumab-ADC-1 and Datopotamab-ADC-3 use P1 as a cytotoxin, and their in vitro tumor proliferation inhibition activities are similar compared to the two control ADCs that use Dxd as the toxin; Trastuzumab-ADC-2 and Datopotamab-ADC-4 both use P2 as a cytotoxin, and their in vitro tumor proliferation inhibition activities are weaker compared to the two control ADCs that use Dxd as the toxin.

[0186] Test Example 2 In vivo activity assay for antibody-drug conjugates.

[0187] Efficacy test of Trastuzumab-ADC-1 on NCI-N87 xenograft tumor.

[0188] 1. Experimental materials.

[0189] Test compound: Trastuzumab-ADC-1, R-Trastuzumab-mc-GGFG-Dxd.

[0190] Experimental cells: NCI-N87 cells, purchased from ATCC.

[0191] Experimental animals: Balb / c nu nude mice, purchased from Shanghai Lingchang Biotechnology Company Limited.

[0192] 2. Experimental design.

[0193] 2.1. Cell treatment.

[0194] NCI-N87 cells were cultured in 1640 medium containing 10% FBS in 15cm diameter dishes. When 80-90% confluence was achieved, the cells were digested with trypsin-EDTA, washed twice with PBS, centrifuged, and resuspended in pre-chilled PBS. Cells were counted using a cell counter, and the PBS was diluted to a concentration of 5 × 10⁻⁶ cells / mL. 7 / mL.

[0195] 2.2. Tumor cell transplantation.

[0196] Balb / c nu mice were acclimatized to the laboratory environment for 2-5 days, and then subcutaneously inoculated with NCI-N87 cells at a cell density of 5 × 10⁶ cells.6 / animal, inoculated with 0.2 mL (containing 50% Matrigel), until the tumor grows to 150-200 mm 3 At that time, the experiment was conducted.

[0197] 2.3. Animal drug administration and testing.

[0198] The tumor-bearing nude mice enrolled were administered the drugs according to the regimens in Table 5.

[0199] Table 5 2.4. Tumor volume and body weight measurement: Tumor volume and body weight were measured twice a week, and TGI was calculated.

[0200] 3. Experimental results.

[0201] The experimental results are shown in Table 6 (NCI-N87 xenograft model data) and Figure 8 All the ADCs of the present invention showed an inhibitory effect on tumor growth, and no significant weight loss or drug toxicity was observed in any group of animals during the administration period.

[0202] Table 6 Note: ** in the table indicates that the P < 0.01 between the test sample group and the blank sample (DPBS) group.

[0203] The above results indicate that the Tratuzumab-ADC-1 molecule has better tumor-suppressing activity in vivo than the positive R-Trastuzumab-mc-GGFG-Dxd.

[0204] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A camptothecin derivative, characterized in that, The camptothecin derivatives include compounds of formula (I) or their tautomers, stereoisomers, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts or esters thereof; ； R1 is selected from -OH, -SH, -COOH, or -NHR a ; R a Selected from hydrogen, deuterium, -C1-6 alkyl, -C2-6 alkenyl, -C2-6 ynyl, -C3-10 cycloalkyl, 4-10 heterocyclic, C6-10 aryl or 5-10 heteroaryl; R2 and R3 are each independently selected from hydrogen, deuterium, halogen, -CN, -C1-6 alkyl, -SR b -S(O)R b -S(O)2R b -OR b or -NR b R c Alternatively, R2, R3, and the carbon atom attached to them together form a C5-7 cycloalkyl group or a 5-7 membered heterocyclic group; R b and R c Each is independently selected from -hydrogen, -deuterium, -C1-6 alkyl, -C2-6 alkenyl, -C2-6 ynyl, -C3-10 cycloalkyl, -4-10 heterocyclic, -C6-10 aryl, and -5-10 heteroaryl; X is selected from =O, =S, or =Se; n is an integer selected from 0 to 5; E is absent, or E is selected from -C1-6 alkylene-, -C2-6 alkenyl-, -C1-4 alkylene-O-C1-6 alkylene-, -C1-4 alkylene-C3-8 cycloalkylene-, -C1-6 alkylene-NH-CO-, -C2-6 alkenyl-NH-CO-, -C1-4 alkylene-O-C1-6 alkylene-NH-CO-, or -C1-4 alkylene-C3-8 cycloalkylene-NH-CO-, wherein the carbon and nitrogen of the aforementioned groups are optionally separated by one or more R. d Replace, R d It is selected from hydrogen, deuterium, halogen, -CN, -NO2, -OH, -COOH, -NH2, -C1-4 alkyl, -O-C1-4 alkyl-, -NH-C1-4 alkyl, -N-(C1-4 alkyl)2 or -C3-8 cycloalkyl.

2. The camptothecin derivative according to claim 1, characterized in that, R1 is -OH; Preferably, R2 is selected from -H, -F, -CN, -CH3, or -OCH3-SCH3; Preferably, R3 is selected from -H, -F, -CN, -CH3, -OCH3 or -SCH3; Preferably, R2 and R3 are both selected from -F; Preferably, X is 0; Preferably, n=3; Preferably, R d Selected from -C1-4 alkyl or -C3-6 cycloalkyl; Preferably, R d Selected from methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, or cyclopentyl; Preferably, R d Selected from methyl; Preferably, E is selected from , , ; Preferably, the compound is selected from the following structures: , or .

3. A linker-drug, characterized in that, The linker-drug comprises a compound of formula (II) or its tautomer, stereoisomer, solvate, isotopically labeled compound, or pharmaceutically acceptable salt or ester; ； E, R2, R3, X, and n are as defined in claim 1 or 2; R'1 is selected from -O-, -S-, -COO- or -N(Ra)-, where Ra is as defined in claim 1 or 2; T is a reactive group that can covalently link with specific functional groups on the antibody; Q represents a structure that covalently connects T and L; L is an optional linking group; S is a shorter linker or an optional self-igniting group, and L is covalently linked to R'1.

4. The linker-drug according to claim 3, characterized in that, The T is selected from the following structures: , or ; Preferably, Q is selected from the following structures: , , or ; Among them, w 1 w 2 w 3 w 4 The integers are from 1 to 18, where the dashed end connects to T and the wavy end connects to L; Preferably, L is selected from a peptide chain consisting of 2-8 amino acid residues, wherein the amino acids are one or more combinations of glycine, phenylalanine, glutamic acid, glutamine, serine, threonine, tyrosine, methionine, cysteine, valine, alanine, aspartic acid, asparagine, lysine, arginine, citrulline, and ornithine, or the following structures: , , or In this structure, the nitrogen terminus of the amino acid is connected to Q, and the carbon terminus is connected to S. Preferably, S is selected from non-existent structures or the following structures: , or , where m is an integer from 1 to 18; where the dashed end connects to L, and the wavy end connects to R'1; Preferably, T is selected from the following structures: or ; Preferably, Q is selected from the following structures: or , where w 1 w 2 The integers are from 1 to 10, where the dashed end connects to T and the wavy end connects to L; Preferably, L is selected from a peptide chain consisting of 2-6 amino acid residues, wherein the amino acid is preferably glycine, phenylalanine, glutamic acid, glutamine, valine, alanine, lysine or citrulline, and the amino acid is connected to Q at the nitrogen end and S at the carbon end. Preferably, S is selected from non-existent structures or the following structures: , where m is an integer from 1 to 10, where the dashed end connects to L and the wavy end connects to R'1; Preferably, T is selected from the following structures: ; Preferably, Q is selected from the following structures: , where w 1 The integer is 5, where the dashed end connects to T and the wavy end connects to L; Preferably, L is selected from Gly-Gly-Phe-Gly-, wherein the nitrogen end of the amino acid is connected to the Q wavy line and the carbon end is connected to the S dashed line. Preferably, S is selected from the following structures: , where m is an integer of 1, and the dashed end connects to L, and the wavy end connects to R'1.

5. The linker-drug according to claim 3 or 4, characterized in that, The linker-drug is selected from the following structures: or .

6. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate includes the compound of formula (III) or its tautomer, stereoisomer, solvate, isotopically labeled compound, or pharmaceutically acceptable salt or ester; ； Wherein, Ab is an antibody or its antigen-binding fragment; T' is a linker that connects to an antibody or its antigen-binding fragment; r is an integer or decimal from 1 to 18; Q, L, S, R'1, E, X, R2, R3, and n are as defined in any one of claims 3-5.

7. The antibody-drug conjugate according to claim 6, characterized in that, T' is selected from , or In this context, the dashed line is connected to Q, the wavy line is connected to Ab, and t1 and t2 are each independent integers from 0 to 18. Preferably, t1 and t2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Preferably, T' is , where t1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Preferably, T' is ; Preferably, the Ab is an antibody that specifically binds to HER2 or TROP2 or its antigen-binding fragment; Preferably, Ab is trastuzumab or dedabrotuzumab; Preferably, r is an integer or decimal from 1 to 12; Preferably, r is an integer or decimal between 2 and 8.

8. The antibody-drug conjugate according to claim 6 or 7, characterized in that, The antibody-drug conjugate is selected from: ， ， ， 。 9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody-drug conjugate according to any one of claims 6-8; Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

10. Use of any one or a combination of at least two of the camptothecin derivative of claim 1 or 2, the linker-drug of any one of claims 3-5, the antibody-drug conjugate of any one of claims 6-8, or the pharmaceutical composition of claim 9 in the preparation of an antitumor formulation.

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