Camptothecin derivative and preparation thereof

Abstract

Provided in the present invention are a camptothecin derivative and an antibody-drug conjugate thereof. The compound has a high activity, low toxicity and good affinity, and achieves a direct targeted killing effect on tumor cells with the antibody-drug conjugate. The antibody-drug conjugate containing the antibody exhibits a significant cell killing effect, and has a high tumor inhibition rate.

Description

Camptothecin derivative and preparation thereof TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a camptothecin derivative used as an antitumor drug and a preparation method thereof. The present application obtains a more optimal camptothecin antitumor drug through molecular structure modification, so as to be more suitable as an antibody conjugated drug. BACKGROUND

[0002] Camptothecin drugs are small molecule compounds with antitumor activity, and are known to exhibit antitumor effects by inhibiting DNA topoisomerase I. Many camptothecin drugs have been widely used in clinical practice, and the main indications are bone cancer, prostate cancer, breast cancer, pancreatic cancer, etc. Irinotecan, as a representative compound, is developed by Dainippon Sumitomo Pharma Co., Ltd. Irinotecan does not need to be activated by using enzymes, and has stronger topoisomerase I inhibitory activity and stronger cell killing activity in vitro against various cancer cells. In particular, it also shows effects on cancer cells showing resistance to SN-38 and the like through the expression of P-glycoprotein. The drug was used as a single chemotherapy drug in the early stage and was promoted to the third phase of clinical trials, and the main indications were bone cancer, prostate cancer, breast cancer, pancreatic cancer, etc. Because of the large side effects and narrow therapeutic window, the directly administered irinotecan ultimately failed to be successfully marketed.

[0003] The advantage of antibody-drug conjugate (ADC) drugs is to increase water solubility and improve targeting specificity. The specific antibody binds to the antigen, carries the drug to the target cells around the target cells, releases the drug near the target cells, effectively kills tumor cells, and reduces toxic side effects. In order to overcome the side effects of direct administration of irinotecan, Dainippon Sumitomo Pharma Co., Ltd. developed irinotecan as an ADC toxin, which targets the HER2 target point and uses a single antibody to connect 8 toxins. Camptothecin drugs have considerable application prospects in ADC drugs. Trastuzumab deruxtecan (trade name Enhertu), an antibody conjugated drug using irinotecan toxin, as the first camptothecin ADC drug to be marketed, has proven the drug-making ability and application prospects of such drugs in the ADC field. However, ADC drugs designed later using irinotecan as a toxin for other target points have safety problems, so the number of conjugated drugs can only be reduced, and the corresponding ADC drug treatment index is reduced.

[0004] In recent years, ADC drugs with camptothecin derivatives as warheads have attracted widespread attention, especially the listing of Enhertu and Sacituzumab govitecan. People have been committed to finding highly efficient and low-toxic camptothecin derivatives. So far, a series of semi-synthetic and synthetic camptothecin derivatives have appeared and entered the clinical application or clinical trial stage, such as hydroxycamptothecin (HCPT), irinotecan (CPT-11), topotecan (TPT), and 9-aminocamptothecin. ADC drugs combine the high efficiency of cytotoxic small molecules and the high selectivity of antibodies to specific tumor cells. There is still a need to develop highly efficient and low-toxic ADC drugs for more indications.

[0005] The technical problem to be solved by the present application is to explore and find more optimal anti-tumor camptothecin derivatives, reduce the toxicity of the compounds, improve the safety and effectiveness of the compounds in ADC drug applications, and obtain anti-tumor ADC drugs with excellent efficacy. SUMMARY

[0006] The present application aims to provide a camptothecin compound with low toxicity and good anti-tumor activity.

[0007] The specific technical solutions of the present application are as follows:

[0008] In a first aspect, the present application provides a compound represented by general formula I or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, hydrate, solvate, prodrug analog:

[0009] wherein Ra, R b are each independently selected from C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 3-8 cycloalkyl, -(CH2) n1 -OH, -(CH2) n1 -NR6R7, -(CH2) n1 -C(O)NR6R7, -(CH2) n1 -N(R6)C(O)R7;

[0010] In the above groups, n1, n2, n3 are each independently an integer from 0 to 6; R6, R7, R8 of each group are the same or different, and each is independently selected from hydrogen, C 1-6 alkyl, hydroxyl, C 1-6 haloalkyl;

[0011] Preferably, R a , Rb each independently selected from C 1-6 alkyl; further preferably methyl, ethyl.

[0012] In a preferred embodiment of the present application, the compound of general formula I or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, hydrate, solvate, prodrug analog, including but not limited to the following compounds:

[0013] The second aspect of the present application provides a linker-drug conjugate comprising a camptothecin derivative of formula II or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof or a mixture thereof:

[0014] wherein Ra, R b each independently selected from C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 3-8 cycloalkyl, -(CH2) n1 -OH, -(CH2) n1 -NR6R7, -(CH2) n1 -C(O)NR6R7, -(CH2) n1 -N(R6)C(O)R7;

[0015] In the above groups, each of n1, n2, n3 is independently an integer from 0 to 6; each of R6, R7, R8 is the same or different, and each is independently selected from hydrogen, C 1-6 alkyl, hydroxyl, C 1-6 haloalkyl;

[0016] Preferably, R a , R b each independently selected from C 1-6 alkyl; further preferably methyl, ethyl.

[0017] In a preferred embodiment of the present application, the linker-drug conjugate comprising a camptothecin derivative of general formula II or its pharmaceutically acceptable salt or solvate, or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, including but not limited to the following compounds:

[0018] In a third aspect, the present application provides an antibody-drug conjugate of Formula III or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0019] wherein Ra, Rb are as defined in the first aspect of the present application in Formula I; Ab is an antibody which can form a linkage through a heteroatom thereof to the Linking Unit, said antibody is selected from the group consisting of murine, chimeric, humanized, fully human, antibody fragments, bispecific antibodies and multispecific antibodies. The antibody, antibody fragment or antigen binding fragment thereof is selected from the group consisting of: an anti-EGFRvIII antibody, an anti-DLL-3 antibody, an anti-CD70 antibody, an anti-LIV-1 antibody, an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-HER3 (ErbB3) antibody, an anti-MUC1 / CD227 antibody, an anti-AXL antibody, an anti-CD166 antibody, an anti-B7-H3 (CD276) antibody, an anti-PTK7 / CCK4 antibody, an anti-PRLR antibody, an anti-EFNA4 antibody, an anti-5T4 antibody, an anti-NOTCH3 antibody, an anti-CD142 antibody, an anti-CA6 antibody, an anti-GPR20 antibody, an anti-CD174 antibody, an anti-CD71 antibody, an anti-EphA2 antibody, an anti-LYPD3 antibody, an anti-FGFR2 antibody, an anti-FGFR3 antibody, an anti-FRα antibody, an anti-CEACAMs antibody, an anti-GCC antibody, an anti-Integrin Av antibody, an anti-CAIX antibody, an anti-P-cadherin antibody, an anti-GD3 antibody, an anti-Cadherin 6 antibody, an anti-LAMP1 antibody, an anti-FLT3 antibody, an anti-BCMA antibody, an anti-CD79b antibody, an anti-CD19 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD74 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD37 antibody, an anti-CD138 antibody, an anti-CD352 antibody, an anti-CD25 antibody, or an anti-CD123 antibody.

[0020] Ab is further selected to be an antibody, antigen binding fragment thereof, or variant thereof to LIV-1 comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises a HCDR1 as set forth in SEQ ID NO: 5, a HCDR2 as set forth in SEQ ID NO: 6, and a HCDR3 as set forth in SEQ ID NO: 7; and the light chain variable region comprises a LCDR1 as set forth in SEQ ID NO: 38, a LCDR2 as set forth in SEQ ID NO: 39, and a LCDR3 as set forth in SEQ ID NO: 40.

[0021] Preferably, the LIV-1 antibody, antigen-binding fragment thereof, or variant thereof further comprises: a heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 63; and a light chain variable region amino acid sequence as set forth in SEQ ID NO: 71.

[0022] Preferably, the LIV-1 antibody, antigen-binding fragment thereof, or variant thereof further comprises a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from a heavy chain constant region of an IgGl, IgG2, IgG4 antibody or variant thereof; preferably comprises a human IgGl heavy chain constant region, more preferably the amino acid sequence of the human IgGl heavy chain constant region is as set forth in SEQ ID NO: 57, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 57. The antibody light chain constant region is selected from a light chain constant region of a kappa or lambda chain or variant thereof. Preferably a human kappa light chain constant region, more preferably the amino acid sequence of the human kappa light chain constant region is as set forth in SEQ ID NO: 58, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 58.

[0023] Preferably, the antigen-binding fragment is selected from the group consisting of: a Fab fragment, a Fab' fragment, a F(ab)2 fragment, a Fv fragment, and a ScFv.

[0024] wherein n is 7.5-8.0, preferably n is 8.0.

[0025] In one preferred embodiment of the present application, the antibody-drug conjugate of general formula III, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, comprises but is not limited to the following compound:

[0026] wherein Ab is an antibody, antibody fragment, or antigen-binding fragment thereof, the antibody is selected from a chimeric antibody, a humanized antibody, or a fully human antibody, as defined in general formula III; and n is 7.5-8.0, preferably n is 8.0.

[0027] In another embodiment of the present application, the antibody-drug conjugate of general formula III, or a pharmaceutically acceptable salt or solvate thereof, comprises but is not limited to the following structure:

[0028] wherein n is 7.5-8.0 in each formula, preferably n is 8.0; huH11-3-2-P1 in each formula represents an anti-LIV-1 antibody or antigen binding fragment thereof having a heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 63, and a light chain variable region amino acid sequence as set forth in SEQ ID NO: 71; a heavy chain constant region amino acid sequence as set forth in SEQ ID NO: 57, and a light chain constant region amino acid sequence as set forth in SEQ ID NO: 58; and S is a sulfur atom in each of the above formulas.

[0029] In a fourth aspect, the present application provides a method for preparing a compound of Formula I. The compounds of the present application can be prepared by a variety of means known to those skilled in the art. The present application can be synthesized using the methods described herein and methods of organic chemical synthesis or variations thereof understood by those skilled in the art. Preferably the methods include, but are not limited to, the following methods. The intermediate compounds used in the preparation process can be purchased or prepared according to the prior art.

[0030] In a preferred embodiment of the present application, a method for preparing a compound of Formula I, the synthetic route is as follows:

[0031] The compound of Formula I is obtained by condensation reaction of General Formula SM-1 and General Formula SM-2; wherein Ra, Rb are defined as in General Formula I of the first aspect of the present application.

[0032] In a fifth aspect, the present application provides a method for preparing a linker-drug conjugate of Formula II comprising a camptothecin derivative, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof. The compounds of the present application can be prepared by a variety of means known to those skilled in the art. The present application can be synthesized using the methods described herein and methods of organic chemical synthesis or variations thereof understood by those skilled in the art. Preferably the methods include, but are not limited to, the following methods. The intermediate compounds used in the preparation process can be purchased or prepared according to the prior art.

[0033] In a preferred embodiment of the present application, a method for preparing a compound of Formula II, the synthetic route is as follows:

[0034] wherein Ra, Rb are defined as in General Formula II of the second aspect of the present application.

[0035] In a sixth aspect, the present application provides a method for preparing a Ligand- Drug Conjugate of Formula III or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer thereof or a mixture thereof or a pharmaceutically acceptable salt or solvate thereof. The compounds of the present application can be prepared by using various ways known to those skilled in the art. The present application can be synthesized using the methods described herein and the methods of organic chemistry synthesis or variations thereof understood by those skilled in the art. Preferably the methods include but are not limited to the following methods. The intermediate compounds used in the preparation process can be purchased or prepared according to the prior art.

[0036] In a preferred embodiment of the present application, a method for preparing a Ligand- Drug Conjugate of Formula III, the synthetic route is as follows:

[0037] After reduction of Ab, the Ligand-Drug Conjugate of Formula III is prepared by reacting with the linker-drug conjugate of camptothecin derivatives (Formula II), and the reducing agent is preferably TCEP-HCl; wherein Ab, Ra, Rb are as defined in the general Formula III of the third aspect of the present application; and n is 7.5-8.0, preferably n is 8.0.

[0038] In a seventh aspect, the present application provides a pharmaceutical composition comprising: a therapeutically effective amount of one or more of the compounds of Formula I or a hydrate, solvate, prodrug, stereoisomer or tautomer thereof, and optionally a pharmaceutically acceptable carrier, excipient, adjuvant, vehicle or diluent; or a pharmaceutical composition comprising: a therapeutically effective amount of the Ligand-Drug Conjugate of Formula III or a compound thereof, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0039] Preferably, the pharmaceutical composition is used for treating LIV-1 mediated diseases or conditions.

[0040] Preferably, the diseases or conditions include but are not limited to bladder cancer, breast cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumor, prostate cancer, colorectal cancer, glioma, mesothelioma.

[0041] The compounds of the present application can be used directly for the prophylaxis and treatment, or preferably in the form of pharmaceutical compositions. Although the active ingredients can be administered alone, it is preferred to present them in the form of pharmaceutical preparations or compositions. Thus, the present application provides a pharmaceutical preparation comprising a compound of the present application together with a pharmaceutically acceptable diluent, excipient or carrier (herein collectively referred to as "carrier" material). The pharmaceutical compositions of the present application can take the form of pharmaceutical preparations as described hereinafter.

[0042] The pharmaceutical preparations of the present application include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous (bolus or infusion), and intraarticular), inhalation (including fine particle powders or aerosols that can be generated by means of various types of metered dose pressurized aerosols), nebulizers or inhalers, rectal, intraperitoneal, and topical (including dermal, buccal, sublingual, and intraocular) administration, although the most suitable route in any given case will depend, for example, on the condition and state of the recipient.

[0043] Advantages of the present application: The present application provides a new camptothecin derivative, which has high activity and low toxicity, good affinity, realizes direct targeted killing effect on tumor cells with antibody drug conjugates, and the cell killing effect of the antibody drug conjugate containing the antibody is significant, and has a strong tumor inhibition rate.

[0044] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (including the examples) can be combined with each other to constitute a new or preferred technical scheme.

[0045] Terminology

[0046] The following lists definitions for various terms used to describe the present application. These definitions apply to the terms as they are used throughout the specification and claims, unless otherwise limited in specific instances either individually or as part of a larger group.

[0047] The term "alkyl" in the present application refers to a saturated straight-chain or branched-chain hydrocarbon group, which in certain embodiments contains 1 to 6 carbon atoms, C 1-6 Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl, n-hexyl, or the like.

[0048] The term "halo" in the present application refers to a group formed by the replacement of a hydrogen atom on a carbon atom with a halogen atom, wherein the halogen atom includes, but is not limited to, F, Cl, Br, I.

[0049] The term "alkoxy" in the present application refers to -O-alkyl, wherein the alkyl includes, but is not limited to, C 1-6 alkyl and C 3-6Cycloalkyl, specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropoxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and halogenated versions thereof.

[0050] An "effective amount" as used herein means an amount that is capable of achieving the desired therapeutic effect in the desired subject without causing undue negative effects, and the specific amount can be determined by those skilled in the art as needed.

[0051] "Treating" as used herein means a method of alleviating or abating a disease and its symptoms; and "preventing" as used herein means a method of reducing or eliminating the onset of symptoms or complications of a disease, condition, or disorder.

[0052] It should be understood that other terms not explained above but appearing in the present application should be defined according to the common understanding of those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1: NMR hydrogen spectrum of compound 1-A

[0054] Figure 2: NMR hydrogen spectrum of compound 1-B

[0055] Figure 3: NMR hydrogen spectrum of compound 2-A

[0056] Figure 4: NMR hydrogen spectrum of compound 2-B

[0057] Figure 5: NMR hydrogen spectrum of compound 3-A

[0058] Figure 6: NMR hydrogen spectrum of compound 3-B

[0059] Figure 7: NMR hydrogen spectrum of HY2019

[0060] Figure 8: NMR hydrogen spectrum of HY2020

[0061] Figure 9: NMR hydrogen spectrum of HY2021

[0062] Figure 10: NMR hydrogen spectrum of HY2022

[0063] Figure 11: NMR hydrogen spectrum of HY2023

[0064] Figure 12: NMR hydrogen spectrum of HY2024

[0065] Figure 13: ELISA binding assay of chimeric antibody to human LIV-1-his

[0066] Figure 14: ELISA binding assay of chimeric antibody to human LIV-1-his

[0067] Figure 15: ELISA binding assay of chimeric antibody to human LIV-1-his

[0068] Figure 16: ELISA binding assay of chimeric antibody to human LIV-1-his

[0069] Figure 17: FACS binding assay of chimeric antibody to 293F cells expressing human LIV-1

[0070] Figure 18: FACS binding assay of chimeric antibody to 293F cells expressing human LIV-1

[0071] Figure 19: FACS binding assay of chimeric antibody to 293F cells expressing human LIV-1

[0072] Figure 20: ELISA to identify the binding ability of PTM-removed humanized antibody to human LIV-1-his

[0073] Figure 21: ELISA to identify the binding ability of PTM-removed humanized antibody to human LIV-1-his

[0074] Figure 22: FACS to identify the binding ability of PTM-removed humanized antibody to 293F cells expressing human LIV-1

[0075] Figure 23: FACS to identify the binding ability of PTM-removed humanized antibody to 293F cells expressing monkey LIV-1

[0076] Figure 24: In vitro tumor cell killing activity of anti-LIV-1 antibody drug conjugate

[0077] Figure 25: In vitro tumor cell killing activity of anti-LIV-1 antibody drug conjugate

[0078] Figure 26: Average tumor volume change of mice in different groups on different days

[0079] Figure 27: Average tumor volume change of mice in different groups on different days

[0080] Figure 28: Body weight change of mice in different groups on different days

[0081] Figure 29: Body weight change of mice in different groups on different days DETAILED DESCRIPTION

[0082] The present application will be further described by the following examples. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the present application. Therefore, simple modifications to the present application under the premise of the present application are within the scope of the present application.

[0083] The structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS); specific compounds1 H-NMR see Example characterization data.

[0084] Known starting materials of the present application can be synthesized or purchased using or following procedures known in the art.

[0085] The control antibody used in the examples is hLIV22, and the antibody drug conjugate is Ladiratuzumab vedotin; Ladiratuzumab vedotin (also known as SGN-LIV1A, described herein as hLIV22-vcMMAE) is an LIV-1 -directed antibody-drug conjugate (ADC) produced by conjugating the humanized antibody hLIV22 to the monomethyl auristatin E (vcMMAE) with a valine-citrulline linker via a drug linker. hLIV22 is a humanized version of the mouse BR2-22a antibody, described in U.S. Patent No. 9,228,026 B2. Methods of making the hLIV22 antibody are also disclosed in U.S. Patent No. 9,228,026 B2. The light chain amino acid sequence of hLIV22 is provided herein as SEQ ID NO: 59, and the heavy chain amino acid sequence of hLIV22 is provided herein as SEQ ID NO: 60. Synthesis and conjugation of the drug linker vcMMAE (also known as 1006) is further described in U.S. Patent No. 9,228,026 B2 and U.S. Patent Publication No. 20050238649.

[0086] Preparation of compound 1-A of Example 1

[0087] First step: preparation of intermediate I

[0088] Compound SM-1 (15 g) was dissolved in 150 mL of methanol, formaldehyde (24.3 g) was added at 0 °C and stirred for half an hour, sodium cyanotrihydroxide (20.3 g) was added at 0 °C, and the reaction was carried out at room temperature for 2 hours. LCMS showed that the reaction was complete. The reaction liquid was extracted with ethyl acetate and water, the organic phase was combined, dried over anhydrous sodium sulfate, and passed through a column to obtain 10 g of yellow oil (intermediate I).

[0089] Second step: preparation of intermediate II

[0090] Intermediate I (0.24 g) was dissolved in a mixed solution of methanol (8 mL) and ethyl acetate (4 mL), Pd / C (50 mg) was added, a hydrogen balloon was covered, and vacuum replacement was performed three times. The reaction was stirred at room temperature for 4 h, filtered, washed, and rotary dried to obtain 128 mg of white solid (intermediate II).

[0091] Third step: preparation of compound 1-A

[0092] Intermediate II (45 mg) was dissolved in DMF (20 mL), SM-2 (176 mg) was added, under N2protection, the reaction solution was cooled in an ice bath, HATU (162 mg) was added, DIPEA (192 mg) was added, the reaction solution was stirred at room temperature for 1 h, 5 mL of water was added to quench the reaction, the reaction solution was extracted with ethyl acetate (10 mL x 3), the organic phase was combined and washed with saturated sodium chloride solution (5 mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated solution was purified by high performance liquid chromatography (C18 column, column type: Ultimate XB-C18, 10 um, 50 mm diameter, 250 mm length, mobile phase: 1‰ TFA aqueous solution + acetonitrile), the corresponding fractions were collected, and a yellow solid 40 mg (compound 1-A) was obtained after freeze-drying.

[0093] 1 H-NMR (400 MHz, DMSO-d6) δ: δ 7.57-7.49 (m, 2H), 5.69 (s, 1H), 5.51 (d, J = 16.3 Hz, 1H), 5.29 (d, J = 16.3 Hz, 1H), 5.22 (d, J = 18.6 Hz, 1H), 5.03 (dd, J = 18.8, 11.6 Hz, 1H), 4.13-3.98 (m, 1H), 3.96-3.88 (m, 2H), 3.00 (s, 7H), 2.44-2.25 (m, 6H), 2.04-1.75 (m, 2H), 0.97-0.92 (m, 3H);

[0094] Ms m / z (ESI): 552.2 [M+H]+.

[0095] Preparation of compound 1-B of Example 2

[0096] First step: preparation of intermediate I

[0097] Compound SM-1 (10 g) was dissolved in methanol (120 mL), 40% formaldehyde aqueous solution (8.1 g) was added at room temperature, the reaction solution was stirred at room temperature for 30 min, sodium cyanotrihydroxide (6.78 g) was added, the reaction solution was stirred at room temperature for 4 h, ethyl acetate (200 mL) was added to the reaction solution, the pH value of the solution was adjusted to 8 with saturated sodium bicarbonate solution, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to dryness. The crude product was column chromatographed to obtain 4.3 g of colorless oil (intermediate I).

[0098] Second step: preparation of intermediate II

[0099] The intermediate II was prepared according to the procedure described in Step 2 of Example 1.

[0100] Third Step: Preparation of Compound 1-B

[0101] The intermediate II was prepared according to the procedure described in Step 3 of Example 1.

[0102] 1 H-NMR (400 MHz, DMSO-d6) δ: δ 9.78 (s, 1H), 9.06 (s, 1H), 7.82 (d, J = 10.9 Hz, 1H), 7.32 (s, 1H), 6.54 (s, 1H), 5.63 (dd, J = 8.6, 4.6 Hz, 2H), 5.42 (s, 2H), 5.31 (d, J = 18.8 Hz, 1H), 5.21 (d, J = 18.8 Hz, 1H), 3.98 - 3.84 (m, 2H), 3.76 (s, 1H), 3.22 - 3.13 (m, 2H), 2.80 (s, 6H), 2.41 (s, 3H), 2.27 - 2.11 (m, 2H), 1.91 - 1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H);

[0103] Ms m / z (ESI): 551 [M+H]+.

[0104] Preparation of Compound 2-A of Example 3

[0105] First Step: Preparation of Intermediate I

[0106] The intermediate II was prepared according to the procedure described in Step 3 of Example 1.

[0107] Second Step: Preparation of Compound 2-A

[0108] Into a reaction vial was placed intermediate I (150 mg) and 1.5 mL of a sodium acetate / acetic acid aqueous solution. After the solution was clear, 2 drops of an aqueous acetaldehyde solution was added. After stirring for 1 min, sodium cyanotrihydrogenate (29.0 mg) was added. After stirring for 3 min, 1.0 mL of acetonitrile was added and stirring was continued for 5 min. The reaction solution was purified by reverse phase separation (mobile phase: water + acetonitrile) and lyophilized to give 100 mg of a white solid. The solid was further purified by high performance liquid chromatography (C18 column, column type: Ultimate XB-C18, 10 um, 50 mm diameter, 250 mm length, mobile phase: 1 ‰ TFA (water) + acetonitrile) and lyophilized to give 11.75 mg of a yellowish solid (compound 2-A).

[0109] 1H NMR (400 MHz, MeOD) δ 7.53 - 7.42 (m, 2H), 5.67 (s, 1H), 5.49 (d, J = 16.5 Hz, 1H), 5.22 (dd, J = 32.7, 17.6 Hz, 2H), 4.95 (d, J = 18.7 Hz, 1H), 4.00 (dd, J = 36.8, 9.2 Hz, 3H), 3.41 (s, 2H), 3.16 (s, 2H), 2.99 (s, 3H), 2.40 - 2.25 (m, 5H), 1.88 (q, J = 7.3 Hz, 2H), 1.39 (t, J = 6.8 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H);

[0110] Ms m / z (ESI): 566.2 [M+H]+.

[0111] Preparation of compound 2-B of Example 4

[0112] First step: preparation of intermediate I

[0113] Referring to the method of Example 1, step 3, SM-1 and SM-2 were condensed in the presence of HATU and DIPEA to give intermediate I.

[0114] Second step: preparation of compound 2-B

[0115] Intermediate I (80 mg) and 1 mL sodium acetate / acetic acid aqueous solution were placed in a reaction bottle, after the solution was clear, 2 drops of acetaldehyde aqueous solution was added, after stirring for 1 min, sodium cyanotrihydrogenate (15.5 mg) was added, after stirring for 3 min, 0.5 mL acetonitrile was added and stirring was continued for 5 min; the reaction solution was purified by high performance liquid chromatography (C18 column, column type: Ultimate XB-C18, 10 um, 50 mm diameter, 250 mm length, mobile phase: 1‰ TFA (water) + acetonitrile), freeze-dried, to obtain 11 mg of light yellow solid (compound 2-B).

[0116] 1H NMR (400 MHz, DMSO): δ 9.65 (d, J = 45.9 Hz, 1H), 9.17-9.00 (m, 1H), 7.84 (d, J = 11.0 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 5.79-5.55 (m, 2H), 5.43 (s, 2H), 5.34 (d, J = 18.7 Hz, 1H), 5.20 (d, J = 18.7 Hz, 1H), 4.02-3.83 (m, 3H), 3.26-3.09 (m, 4H), 2.85 (s, 3H), 2.42 (s, 3H), 2.30-2.21 (m, 1H), 2.18-2.09 (m, 1H), 1.93-1.77 (m, 2H), 1.23 (dd, J = 13.1, 6.5 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H);

[0117] Ms m / z (ESI): 565 [M+H]+.

[0118] Preparation of compound 3-A in Example 5

[0119] First step: preparation of intermediate I

[0120] Referring to the method of Example 1, step 3, SM-1 and SM-2 were subjected to condensation reaction under the action of HATU and DIPEA to prepare intermediate I.

[0121] Second step: preparation of compound 3-A

[0122] Intermediate I (35 mg) was dissolved in sodium acetate buffer solution (5 mL, pH = 5) and ethanol (5 mL), and acetaldehyde (38 mg), sodium cyanotrihydrogenate (54 mg) were added. The reaction was stirred at room temperature for 20 min, and LCMS was used to monitor the completion of the reaction. The reaction solution was purified by preparative high performance liquid chromatography (C18 column, column type: Ultimate XB-C18, 10 μm, 50 mm diameter, 250 mm length, using a mixture of water (containing 0.1% TFA) and acetonitrile with decreasing polarity as eluent), to obtain 10 mg of a light yellow solid (compound 3-A).

[0123] 1H NMR (400 MHz, DMSO): δ 9.60 (s, 1H), 9.13 (d, J = 8.6 Hz, 1H), 7.85 (d, J = 11.0 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.67 (d, J = 8.5 Hz, 1H), 5.53 (s, 1H), 5.43 (s, 2H), 5.35 (d, J = 19.0 Hz, 1H), 5.19 (d, J = 19.0 Hz, 1H), 3.87 (d, J = 34.6 Hz, 3H), 3.16 (dd, J = 38.4, 21.0 Hz, 5H), 2.43 (s, 3H), 2.35 - 2.09 (m, 2H), 1.86 (dt, J = 10.9, 6.8 Hz, 2H), 1.34 - 1.14 (m, 6H), 0.88 (t, J = 7.3 Hz, 3H);

[0124] LCMS: r.t = 0.804 min, m / z 579 (M+H) + .

[0125] Preparation of compound 3-B of Example 6

[0126] First step: preparation of intermediate I

[0127] Intermediate I was prepared by condensation reaction of SM-1 and SM-2 in the presence of HATU and DIPEA, according to the method of Example 1, step 3.

[0128] Second step: preparation of compound 3-B

[0129] Intermediate I (45 mg) was dissolved in sodium acetate buffer solution (5 mL) and ethanol (5 mL) with pH = 5, and then acetaldehyde (49 mg), sodium cyanotrihydrogenate (69 mg) were added. The reaction was stirred at room temperature for 20 min, and LCMS was used to monitor the reaction. The reaction solution was purified by preparative high performance liquid chromatography (C18 column, Durashell C18, 10 μm, 50 mm diameter, 260 mm length, using a mixture of water (containing 0.1% TFA) and acetonitrile with decreasing polarity as eluent), to obtain 12 mg of a light yellow solid (compound 3-B).

[0130] 1 H NMR (400 MHz, DMSO): δ 9.56 (s, 1H), 9.07 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 11.0 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 5.60 (s, 2H), 5.43 (s, 2H), 5.35 (d, J = 18.6 Hz, 1H), 5.19 (d, J = 18.7 Hz, 1H), 3.95 (s, 3H), 3.21 (d, J = 17.4 Hz, 5H), 2.42 (s, 3H), 2.23 (dd, J = 26.6, 21.7 Hz, 2H), 1.92 - 1.78 (m, 2H), 1.27 - 1.15 (m, 6H), 0.87 (t, J = 7.3 Hz, 3H);

[0131] LCMS: r.t = 0.799 min, m / z 579 (M+H) + .

[0132] Example 7 Preparation of Linker-Payload HY-2019

[0133] First step: preparation of intermediate I

[0134] SM-1 (15 g) was dissolved in 150 mL of methanol, and formaldehyde (24.3 g) was added at 0 °C and stirred for half an hour. Sodium cyanotrihydrogenate (20.3 g) was added at 0 °C, and the reaction was carried out at room temperature for 2 hours. LCMS showed that the reaction was complete. The reaction solution was extracted with ethyl acetate and water, and the combined organic phase was dried over anhydrous sodium sulfate and passed through a column to obtain 10 g of yellow oil.

[0135] Second step: preparation of intermediate 2

[0136] Intermediate 1 (8 g) and SM-2 (6.6 g) were dissolved in 80 mL of DCM, 20 mL of TFA was added, and the reaction was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction mixture was extracted with ethyl acetate and water, and the product was obtained as a yellow oil (420 mg of intermediate 2) by column chromatography.

[0137] Third step: Preparation of intermediate 3

[0138] Intermediate 2 (400 mg) was dissolved in 8 mL of DMF, 200 mg of palladium on carbon was added, and the reaction was stirred under hydrogen for 2 hours. LCMS showed the reaction was complete. The filtrate was collected by filtration, and the filtrate was used directly in the next step.

[0139] Fourth step: Preparation of intermediate 4

[0140] To the filtrate from the previous step, SM-3 (218 mg) and COMU (192 mg) and DIEA (159 mg) were added, and the reaction was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The product was obtained as a yellow solid (300 mg) by column chromatography.

[0141] Fifth step: Preparation of intermediate 5

[0142] Intermediate 4 (300 mg) was dissolved in 4 mL of DMF, 0.5 mL of diethanolamine was added, and the reaction was stirred for 2 hours. LCMS showed the reaction was complete. The product was obtained as a yellow solid (200 mg) by reverse phase column chromatography.

[0143] Sixth step: Preparation of HY-2019

[0144] Intermediate 5 (100 mg) and SM-4 (74 mg) were dissolved in 4 mL of DMF, COMU (74 mg) and DIEA (60 mg) were added, and the reaction was stirred for 2 hours. LCMS showed the reaction was complete. The product was obtained as a yellow solid (8 mg of HY-2019).

[0145] Example 8: Preparation of Linker-Payload HY-2020

[0146] First step: Preparation of intermediate I

[0147] SM-1 (10 g) was dissolved in 120 mL of methanol, 40% formaldehyde aqueous solution (8.1 g) was added at room temperature and stirred for half an hour, sodium cyanotrihydrogenate (6.78 g) was added, and the reaction was carried out at room temperature for 4 hours. To the reaction solution, EA (200 mL) was added, and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a colorless oil 4.3 g.

[0148] Second step: Preparation of intermediate 2

[0149] Intermediate 1 (2.4 g) and SM-2 (2.0 g) were dissolved in 48 mL of dichloromethane, 5 mL of TFA was added under ice bath, and the reaction was carried out at room temperature for 1 hour. To the reaction solution, DCM (100 mL) was added, cooled under ice bath, and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The organic phase was separated, washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a light yellow sticky crude product. The crude product was columned to obtain a colorless gum 480 mg.

[0150] Third step: Preparation of intermediate 3

[0151] Intermediate 2 (480 mg) was dissolved in 12 mL of a mixed solution of methanol and ethyl acetate (V 甲醇 :V 乙酸乙酯 = 2:1), 50 mg of palladium-carbon was added, a hydrogen balloon was covered, and vacuum replacement was performed three times. The reaction was stirred at room temperature for 4 hours, filtered, washed, and rotary evaporated to obtain a white solid 420 mg.

[0152] Fourth step: Preparation of intermediate 4

[0153] Intermediate 4 (400 mg) was dissolved in DMF (15 mL), SM-3 (476.5 mg) was added, and N2 protection was performed. The reaction was cooled under ice bath, HATU (442.6 mg) was added, DIPEA (523.27 mg) was added, and the reaction was stirred at room temperature for 1 hour. The reaction was cooled under ice bath, water (80 mL) was added to the reaction solution, and a solid was precipitated. The solid was collected by filtration, and water (20 mL) and acetonitrile (5 mL) were added. The mixture was freeze-dried to obtain a gray solid 900 mg.

[0154] Fifth step: Preparation of intermediate 5

[0155] Intermediate 4 (500 mg) was dissolved in 12 mL of DMF, triethylenediamine (195 mg) was added at room temperature, and the reaction was stirred at room temperature for 3 hours. The reaction solution was directly passed through a reverse phase, and the system was 0.1% TFA / acetonitrile. The reaction was freeze-dried to obtain a yellow solid 380 mg.

[0156] Step 6: Preparation of HY-2020

[0157] Intermediate 5 (100 mg) was dissolved in 4 mL of DMF, SM-4 (74.2 mg) was added, N2 protection, ice bath, HATU (77.7 mg) was added, DIPEA (91.4 mg) was added, stirred at room temperature for 1 h, the reaction solution was directly sent to preparation, the system was 0.1% formic acid / acetonitrile, freeze-dried, and white solid 60 mg (HY-2020) was obtained.

[0158] Example 9 Preparation of Linker-Payload HY-2021

[0159] Step 1: Preparation of Intermediate 1

[0160] Compound SM-1 (10.0 g) was dissolved in acetic acid-sodium acetate buffer (150 mL), and an aqueous acetaldehyde solution (40%, 9.5 g) was added at room temperature. Stirring was performed at room temperature for 5 min, and sodium cyanotrihydroxyborate (2.7 g) was added. Stirring was performed at room temperature overnight, and then ethyl acetate (100 mL*3) was added to the reaction solution. Neutralization was performed with saturated sodium bicarbonate solution to pH ~ 8, and the organic phase was separated. Rotary evaporation was performed to obtain white solid crude product, and the product was obtained by slurry in ethyl acetate to obtain white solid 5.0 g.

[0161] Step 2: Preparation of Intermediate 2

[0162] Intermediate 1 (3.0 g) was dissolved in MeOH (45 mL), and an aqueous formaldehyde solution (40%, 3.27 g) was added at room temperature. Stirring was performed at room temperature for 1 h, and sodium cyanotrihydroxyborate (0.844 g) was added. Stirring was performed at room temperature for 1 h, and LCMS showed that the reaction was complete. The reaction solution was quenched into NaHCO3, and ethyl acetate (50 mL*3) was added to the reaction solution. The organic phase was washed with saturated sodium chloride solution (100 mL) once, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain light yellow oil 2.5 g.

[0163] Step 3: Preparation of Intermediate 3

[0164] Intermediate 2 (2.45 g) and SM-2 (1.90 g) were dissolved in DCM (38 mL), and TFA (3.05 mL) was added under ice bath. After addition, stirring was performed at room temperature for 1 h. The reaction solution was quenched into saturated sodium bicarbonate solution and neutralized to pH ~ 8. Ethyl acetate (150 mL*2) was added, and the organic phase was separated. Washing was performed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain light yellow viscous crude product. Column chromatography was performed on the crude product, and the eluent was DCM-DCM / MeOH=50 / 1 to obtain colorless gum 500 mg.

[0165] Fourth Step: Preparation of Intermediate 4

[0166] Intermediate 3 (500 mg) was dissolved in a mixture of MeOH (7.5 mL) and ethyl acetate (2.5 mL), Pd / C (150 mg) was added, a hydrogen balloon was covered, and vacuum was replaced for 3 times. After stirring at room temperature for 4 h, filtration, washing, and rotary evaporation, a white solid was obtained (400 mg).

[0167] Fifth Step: Preparation of Intermediate 5

[0168] Intermediate 4 (400 mg) was dissolved in DMF (12 mL), SM-3 (467 mg) was added, and N2 protection was performed. After cooling in an ice bath, HATU (400 mg) was added, DIPEA (340 mg) was added, and stirring was performed at room temperature for 1 h. LCMS detection showed that the reaction was complete, and the reaction solution was directly subjected to reversed phase (CAN in water 60-70% to obtain the target product). After freeze-drying, a white solid was obtained (250 mg).

[0169] Sixth Step: Preparation of Intermediate 6

[0170] Intermediate 5 (250 mg) was dissolved in DMF (5 mL), and triethylenediamine (160 mg) was added at room temperature. After stirring at room temperature overnight, the reaction solution was directly subjected to reversed phase (0.1% TFA / acetonitrile), and freeze-drying was performed to obtain a yellow solid (150 mg).

[0171] Seventh Step: Preparation of HY-2021

[0172] Intermediate 6 (150.0 mg) was dissolved in DMF (4 mL), and SM-4 (86 mg) was added. After N2 protection and addition of HATU (86 mg) and DIPEA (98 mg) under ice bath, stirring was performed at room temperature for 1 h. The reaction solution was directly subjected to preparation (0.1% formic acid / acetonitrile), and freeze-drying was performed to obtain a white solid (40 mg).

[0173] Preparation of Linker-Payload HY-2022

[0174] First Step: Preparation of Intermediate 1

[0175] Dissolve compound SM-1 (10.0 g) in acetic acid-sodium acetate buffer (150 mL), add aqueous acetaldehyde (40%, 9.5 g) at room temperature, stir for 5 min at room temperature, add NaBH3CN (2.7 g), stir overnight at room temperature; add ethyl acetate (200 mL) to the reaction solution, neutralize to pH ~ 8 with saturated sodium bicarbonate solution, separate the organic phase, and spin dry to obtain a white solid crude product; purify the crude product by reverse phase with pure water / acetonitrile, and freeze dry to obtain a white solid 4.3 g.

[0176] Second step: preparation of intermediate 2

[0177] Dissolve intermediate 1 (4.3 g) in MeOH (43 mL), add aqueous formaldehyde (40%, 4.74 g) at room temperature, stir for 1 h at room temperature; add compound NaBH3CN (1.24 g), stir for 1 h at room temperature; spin off the formaldehyde, add DCM (200 mL), water (100 mL), and separate the organic phase; wash the organic phase with saturated sodium chloride solution (100 mL) once, dry over anhydrous sodium sulfate, filter, and spin dry to obtain a light yellow oil 4.1 g.

[0178] Third step: preparation of intermediate 3

[0179] Dissolve intermediate 2 (3.1 g) and SM-2 (2.4 g) in DCM (60 mL), add TFA (4.8 mL) in an ice bath; after addition, stir for 1 h at room temperature; add DCM (100 mL) to the reaction solution, cool in an ice bath, neutralize to pH ~ 8 with saturated sodium bicarbonate solution, separate the organic phase, wash with saturated sodium chloride solution (50 mL), dry over anhydrous sodium sulfate, filter, and spin dry to obtain a light yellow sticky crude product; purify the crude product by column chromatography with DCM-DCM / MeOH = 50 / 1 as eluent to obtain a colorless gum 580 mg.

[0180] Fourth step: preparation of intermediate 4

[0181] Dissolve intermediate 3 (580 mg) in MeOH (8 mL), ethyl acetate (2 mL), add Pd / C (90 mg), cover with a hydrogen balloon, replace 3 times under vacuum, stir for 4 h at room temperature, filter, wash, and spin dry to obtain a white solid 480 mg.

[0182] Fifth step: preparation of intermediate 5

[0183] Intermediate 4 (480 mg) was dissolved in DMF (14 mL), HM-582_10 (816.7 mg) was added; N2 protection, ice bath cooling, HATU (701.6 mg) was added, DIPEA (596.1 mg) was added, stirring at room temperature for 1 h; the reaction solution was cooled in an ice bath, water (80 mL) was added to the reaction solution, a solid was precipitated, filtration, and the solid was collected; water (20 mL), acetonitrile (5 mL) were added, and freeze-drying was performed to obtain 800 mg of a gray solid.

[0184] Step 6: Preparation of intermediate 6

[0185] Intermediate 5 (400 mg) was dissolved in DMF (8 mL), triethylenediamine (154.4 mg) was added at room temperature, and stirring was performed at room temperature overnight; the reaction solution was directly subjected to reverse phase, the system was 0.1% TFA / acetonitrile, and freeze-drying was performed to obtain 280 mg of a yellow solid.

[0186] Step 7: Preparation of HY-2022

[0187] Intermediate 6 (100.0 mg) was dissolved in DMF (4 mL), SM-4 (47.6 mg) was added, N2 protection, HATU (49.8 mg) was added under ice bath, DIPEA (58.6 mg) was added, stirring was performed at room temperature for 1 h; the reaction solution was directly subjected to preparation, the system was 0.1% formic acid / acetonitrile, and freeze-drying was performed to obtain 27 mg of a white solid.

[0188] Example 11 Preparation of Linker-Payload HY-2023

[0189] Step 1: Preparation of intermediate 1

[0190] SM-1 (8 g) was dissolved in a mixed solution of water / methanol (5:3), acetaldehyde (34.4 mL) was added, and finally NaBH3CN (6.5 g) was added, and the reaction was performed at room temperature for 2 h; the reaction solution was extracted with DCM, and the organic phase was dried and then subjected to silica gel column chromatography, PE:EA=10:1~5:1, and rotary evaporation was performed to obtain 5.8 g of colorless oil and 2.3 g of yellowish oil.

[0191] Step 2: Preparation of intermediate 2

[0192] Intermediate 1 (4 g), SM-2 (2.9 g) were dissolved in 80 mL of DCM, TFA was added at 0°C, and after the addition was completed, the reaction was allowed to recover to room temperature for 2 h; the reaction solution was rotary evaporated, dissolved in DCM, and subjected to silica gel column chromatography, PE:EA=1:4, and rotary evaporation was performed to obtain 890 mg of colorless oil.

[0193] Step 3: Preparation of Intermediate 3

[0194] Intermediate 2 (840 mg) was dissolved in 16 mL of MeOH / EA = 1 / 1 mixed solvent, Pd / C was added, and the hydrogen balloon was covered. The gas was exchanged for 3 times, and stirred at room temperature for 2 h. The reaction solution was filtered to remove Pd / C, and the mother liquor was rotary evaporated to obtain 600 mg of gray oil.

[0195] Step 4: Preparation of Intermediate 4

[0196] Intermediate 3 (480 mg,) and HATU (465 mg) were dissolved in 5 mL of DMF at 0°C, stirred for 15 min, then SM-3 (597 mg) and 2,6-dimethylpyridine (377 mg) were added, and the reaction was maintained at 0°C for 4 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phase was dried and then stirred with silica gel column. The column was eluted with DCM:MeOH = 20:1 to obtain 190 mg of light yellow solid.

[0197] Step 5: Preparation of Intermediate 5

[0198] Intermediate 4 (190 mg) and triethylenediamine (240 mg) were dissolved in 2 mL of DMF and reacted at room temperature for 3 h. The reaction solution was directly purified by reverse phase column, H2O (0.1% TFA):CAN (80%:20%), and freeze-dried to obtain 80 mg of yellow solid.

[0199] Step 7: Preparation of HY-2023

[0200] Intermediate 5 (70 mg) and SM-4 (56 mg) were dissolved in 3.5 mL of DMF at 0°C, HATU (56 mg) and DIEA (28 mg) were added at 0°C, and the reaction was maintained at room temperature for 2 h. The reaction solution was directly purified by reverse phase preparation, and freeze-dried to obtain 31 mg of yellow powder.

[0201] Example 12 Preparation of Linker-Payload HY-2024

[0202] Step 1: Preparation of Intermediate 1

[0203] In N2 environment, SM-1 (10 g) was dissolved in MeOH (1 L), acetaldehyde (47.54 g) was added, the reaction solution was stirred at room temperature for 1 h, NaBH3CN (8.14 g) was added to the reaction solution in batches. The reaction solution was reacted at 0 °C for 1 h; LCMS showed that the reaction was completed, the reaction solution was concentrated to obtain a crude product, the crude product was purified by silica gel column (PE:EtOAc = 5:1) to obtain white oil 8 g.

[0204] Second step: preparation of intermediate 2

[0205] In N2 environment, intermediate 1 (6.82 g) and SM-2 (5 g) were dissolved in DCM (60 mL), TFA (4.8 mL) was slowly added to the reaction solution, the reaction solution was stirred at 0 °C to room temperature for 1 h; LCMS showed that the reaction was completed, the reaction solution was slowly added to NaHCO3, DCM was extracted, and the crude product was concentrated to obtain a colorless oil 1 g.

[0206] Third step: preparation of intermediate 3

[0207] In H2 environment, intermediate 2 (1 g) was dissolved in DMF (10 mL), Pd / C (0.55 g) was added, the hydrogen balloon was replaced for 3 times, the reaction solution was stirred at room temperature overnight, LCMS showed that the reaction was completed, the reaction solution was filtered to remove Pd / C, and the crude product was concentrated to obtain a colorless oil 0.5 g.

[0208] Fourth step: preparation of intermediate 4

[0209] In N2 environment, intermediate 3 (0.5 g) and SM-3 (0.9 g) were dissolved in DMF (10 mL), HATU (0.77 g) and DIPEA (0.43 g) were slowly added; the reaction solution was reacted at 0 °C to room temperature for 2 h; LCMS showed that the reaction was completed, water was added to the reaction solution, ethyl acetate was extracted, the organic phase was washed with saturated brine, and the crude product was concentrated to obtain a gray solid product 200 mg.

[0210] Fifth step: preparation of intermediate 5

[0211] Intermediate 5 (200 mg) was dissolved in DMF (1 mL), 1,3-diazabicyclo[1.1.1] pentane (63.22 mg) was added, the reaction solution was stirred at room temperature for 4 h, LCMS showed that the reaction was completed, the reaction was directly reversed to obtain a white solid product 80 mg.

[0212] Step 7: Preparation of HY-2023

[0213] Intermediate 5 (80 mg) was dissolved in DMF (1 ml) with SM-4 (56.8 mg), HATU (45.2 mg) and DIPEA (26.8 mg) was added. The reaction was stirred at room temperature for 1 h. LCMS showed the reaction was complete. The reaction was purified to give 10 mg of white solid.

[0214] Preparation of conjugated drug in Example 13

[0215] The amino acid sequences of the variable region and constant region of the heavy and light chains of the huH11-3-2-P1 antibody in this example are shown in Table 10 below. In the following examples, the humanized antibody huH11-3-2-P1 was further synthesized with Linker-Payload to prepare antibody drug conjugates.

[0216] The huH11-3-2-P1 antibody solution (weight of solution: 5.4 kg; corresponding to 120 g of antibody) was placed in a glass reaction vessel, and further 0.01 mol / L acetate buffer solution (4.3 L, pH 5.5) was added thereto. To this solution, 0.5 ml / L of an aqueous EDTA solution (8.3 mL; 5 equivalents per antibody) was added, and then 0.3 mol / L of a disodium hydrogen phosphate aqueous solution was added thereto to adjust the pH to 7.3. Under stirring at 35 to 39°C, 0.010 mol / L of a tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (390 mL; 4.7 equivalents per antibody molecule) was added thereto, and the resultant was stirred for 2 hours at an internal temperature of 35 to 39°C to reduce the interchain disulfide bond of the antibody. The obtained reaction solution was allowed to cool, and over 20 minutes, a compound Linker-Payload (8.2 g; 9.7 equivalents per antibody molecule) dissolved in 80% dimethyl sulfoxide aqueous solution (900 mL) was added thereto under stirring at an internal temperature of 13 to 17°C, and the resultant was stirred for 1 hour at the same temperature to bind the compound Linker-Payload to the antibody. Next, 0.1 mol / L of an N-ethylthio cysteine aqueous solution (210 mL; 25 equivalents per antibody molecule) was added thereto, and the resultant was further stirred for 1 hour at the same temperature to quench the excess portion of the compound Linker-Payload, and then the pH was adjusted to 5.0 using 10% acetic acid aqueous solution. The resultant antibody drug conjugate was designated as LNF2108-HY2019, LNF2108-HY2020, LNF2108-HY2021, LNF2108-HY2022, LNF2108-HY2023, LNF2108-HY2024, respectively, and after completion of the preparation, the DAR value of the prepared ADC sample was analyzed to be about 8.

[0217] LNF2108-vcMMAE process: huH11-3-2-P1 antibody solution (weight of solution: 5.4 kg; corresponding to 120 g of antibody) was placed in a glass reaction vessel, and further 0.01 mol / L acetate buffer solution (4.3 L, pH 5.5) was added thereto. To this solution, 0.5 ml / L of EDTA aqueous solution (8.3 mL; 5 equivalents per antibody) was added, and then 0.3 mol / L disodium hydrogen phosphate aqueous solution was added thereto to adjust the pH to 7.3. Under stirring at 35°C to 39°C, 0.010 mol / L tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (207 mL; 2.5 equivalents per antibody molecule) was added thereto, and the resulting product was stirred at an internal temperature of 35°C to 39°C for 2 hours to reduce the interchain disulfide bond of the antibody. The obtained reaction solution was allowed to cool, and over 20 minutes, compound Linker-Payload (6.32 g; 6.0 equivalents per antibody molecule) dissolved in 80% dimethyl sulfoxide aqueous solution (900 mL) was added thereto under stirring at an internal temperature of 13°C to 17°C, and the resulting product was stirred at the same temperature for 1 hour to bind the compound Linker-Payload to the antibody. Next, 0.1 mol / L N-ethylmaleimide aqueous solution (210 mL; 25 equivalents per antibody molecule) was added thereto, and the resulting product was further stirred at the same temperature for 1 hour to quench the excess portion of the compound vcMMAE, and then the pH was adjusted to 5.0 using 10% acetic acid aqueous solution. The resulting antibody drug conjugate was named LNF2108-vcMMAE, and the DAR value of the prepared ADC sample was about 4 after the preparation was completed.

[0218] Preparation of antibody

[0219] Preparation of LIV1-FC protein

[0220] The human Liv1 sequence was found from Uniprot, numbered Q13433, and the amino acid sequence is shown as SEQ ID NO: 1; the first extracellular region is amino acids 29-325, and the amino acid sequence is shown as SEQ ID NO: 2. The Linker and IgG4-Fc sequence (the Linker and IgG4-Fc amino acid sequence is shown as SEQ ID NO: 3) was added to the C-terminus of the LIV1 extracellular region sequence; the signal peptide and Kozak sequence were added to the N-terminus of the protein sequence, and after sequence optimization by Nanjing Kingsriver Biotechnology Co., Ltd., it was cloned into the PCDNA3.4 vector, transiently transfected 293F cells, and the LIV-1-FC protein was obtained, and the amino acid sequence is shown as SEQ ID NO: 4.

[0221] Screening of LIV1 monoclonal antibody

[0222] The mice were immunized by 5 six to eight-week-old female BALB / c mice. The LIV1-FC was mixed with complete Freund's adjuvant at a volume ratio of 1:1 by using a single-channel emulsifier for 30 minutes. The mice were subcutaneously injected for the first immunization, and the immunization dose was 100 μg per mouse, and 100 μL was injected per mouse. The second immunization was performed 14 days after the first immunization. The LIV1-FC antigen protein was mixed with incomplete Freund's adjuvant at a volume ratio of 1:1 by using a single-channel emulsifier for 30 minutes. The mice were subcutaneously injected for the second immunization, and the immunization dose was 50 μg per mouse. The same operation was performed for the third immunization 14 days later. Seven days after the third immunization, the tail blood was collected, and the antibody titer was measured by ELISA. The mice with the best titer were selected for subsequent cell fusion experiments.

[0223] Three days before cell fusion, the mice were shocked with 100 μg of LIV-1-Fc protein for cell fusion. After the mice were executed by breaking the neck, they were soaked in 70% alcohol for 5 minutes for disinfection. The mouse abdomen was cut open with scissors, and the mouse spleen was removed. The mouse spleen was washed twice with serum-free DMEM and gently crushed with a syringe core and passed through a 70 μm filter. The cells were transferred to a 50 mL centrifuge tube, 30 mL of DMEM was added, and centrifugation was performed at 300 g for 5 minutes. After the supernatant was discarded, 20 mL of serum-free DMEM was used for resuspension and cell counting. The prepared SP2 / 0 cells and mouse spleen cells were mixed at a ratio of 1:5 to 1:10, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. After resuspension with 15 mL of electrofusion buffer and washing twice, the cell density was adjusted to 1-2 x 10 7 After fusion, HAT medium was added, and 3 x 104 cells per well were plated. After 7 days of culture at 37°C, the cells were replaced, and samples were taken for detection after 10 days.

[0224] The LIV-1-His (ACRO, LV1-H5223) protein was coated on a 96-well ELISA plate at a concentration of 5 μg / mL and an amount of 100 μL / well. The 96-well ELISA plate was coated at 4°C overnight. The next day, the coating solution was discarded, and 5% skimmed milk powder was added at 200 μL / well. After 2 hours of blocking at 37°C, the plate was washed with PBST for 3 times. The hybridoma cell culture supernatant was added at 50 μL, and incubated at 37°C for 1 hour. After washing with PBST for 3 times, the secondary antibody goat anti-mouse IgG-HRP was added and incubated at 37°C for 1 hour. After washing with PBST for 3 times, the color was developed for 10 minutes, and the color development was terminated. The absorbance value at 450 nm was detected by an enzyme-labeled instrument.

[0225] Flow cytometry was used to detect the binding ability of the hybridoma cell culture supernatant to LIV-1 expressed by 293F cells. 293F-LIV-1 cells were collected, and the cells were plated at 105 cells per well in a 96-well plate and washed once with PBS. 100 μL of the hybridoma cell culture supernatant was incubated with the cells at 37°C for 1 h. After incubation, the cells were centrifuged at 300 g and the supernatant was gently aspirated. The cells were washed twice with PBS at the same speed. 100 μL of DPBS with a fluorescent secondary antibody was added to each well, and the mixture was incubated at 37°C for 1 h. After the reaction, the cells were centrifuged and washed twice with PBS. Finally, 200 μL of PBS was added to each well to resuspend the cells, and flow cytometry was used for detection. Nine hybridoma cell strains with the best binding activity were finally screened, and were named 2D2, 6A6, 13F9, 18H10, 23D7, 23D9, 28F1, 29A7, and 33E11.

[0226] Monoclonal antibody cloning and antibody preparation

[0227] The hybridoma cells screened by ELISA and flow cytometry were cultured by cloning. The positive clones obtained were counted, and the cell density was adjusted to 10-20 cells / mL. The diluted cells were plated in a 96-well cell culture plate containing HT (Hypoxanthine (5 mM) and Thymidine (0.8 mM)) at 100 μL per well. After the cells grew for 10 days, the monoclonal cells were observed under a microscope, and the obtained cells were subjected to ELISA and FACS detection according to the method of Example 2. The positive monoclonal cells screened were transferred to a 24-well cell culture plate for culture. When the cells covered the bottom of the cell culture well, they were again transferred to a 6-well plate, and gradually expanded to a T125 flask for culture. After 2 days, the cell supernatant was collected, purified, and the obtained antibody was evaluated by the same method. Monoclonal cell strains with good binding activity were screened, including 2D2-D12, 6A6-A5, 13F9-C9, 18H10-E5, 18H10-F4, 23D7-D7, 23D7-E8, 23D9-G4, 28F1-C3, 29A7-H2, and 33E11-A7.

[0228] Sequencing of antibody variable region genes

[0229] RNA extraction was performed according to the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Catalog No: 9767), and then PrimeScript TMThe RNA was reverse transcribed into cDNA according to the instructions of the RT-PCR Kit (TAKARA, Catalog No.: RR014A). The reverse transcribed cDNA sample was added into a PCR tube in the following order: 6 μL of water, 2 μL of cDNA, 1 μL of heavy chain primer and 1 μL of light chain primer, 10 μL of PCR enzyme mix, and the PCR reaction was performed with the following settings: 95 °C for 5 min, 35 cycles of (95 °C for 30 s, 55 °C for 15 s, 72 °C for 30 s), and 72 °C for 10 min. The heavy chain and light chain PCR products were identified by agarose gel electrophoresis after the PCR reaction. The amplification products were ligated into PMD 20T vectors and entrusted to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. The heavy and light chain antibody fragment amino acid sequences and antibody names are shown in Tables 1-3.

[0230] Table 1 Amino acid sequence number (SEQ ID NO:) of the heavy chain variable region of the mouse-derived antibody

[0231] Table 2 Amino acid sequence number (SEQ ID NO:) of the light chain variable region of the mouse-derived antibody

[0232] Table 3 Amino acid sequence number of the mouse-derived antibody

[0233] Preparation of chimeric antibodies

[0234] The obtained mouse-derived antibody heavy chain variable region sequence was connected to the constant region of human IgG1 antibody; the mouse-derived antibody light chain variable region was connected to the human κ light chain constant region. The amino acid sequence of the human heavy chain constant region is shown in SEQ ID NO: 57, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 58. The signal peptide sequence and Kozak sequence were added before the antibody amino acid sequence, and the DNA sequence was optimized and synthesized by Jinser Biological Technology Co., Ltd. After the sequence synthesis, it was cloned into the pCDNA3.4 vector (Invitrogen), and the plasmid was extracted and transfected into 293F cells. The 293F cells were cultured to the logarithmic growth phase, and the density was adjusted to 3 × 106 / mL with Expi 293F expression medium. OPTI MEM medium 6 mL was added with 40 μg of light chain plasmid and 40 μg of heavy chain plasmid, and OPTIMEM medium 6 mL was added with ExpiFectamine TM293 transfection reagent 320 μL. The transfection reagent was added to the DNA mixture, mixed well and allowed to stand for 15 min, then added to the cells. Incubate at 37°C, 8% CO2, 100 r / min for 20 h, then add Enhancer I 600 μL and Enhancer II 6 mL. After 5 days of incubation, the sample was collected and purified, and the obtained chimeric antibodies were named Chi-1, Chi-2, Chi-3, Chi-4, Chi-5, Chi-6, Chi-7, and Chi-8, respectively. The positive control antibody was Ladiratuzumab vedotin (hLIV22) (WHO Drug Information, Vol. 31, No. 2, 2017), and the antibody heavy chain sequence is shown in SEQ ID NO: 59, and the light chain sequence is shown in SEQ ID NO: 60. The same method was used to transiently transfect 293F cells.

[0235] Purification of chimeric antibodies

[0236] First, prepare the Protein A affinity column, and equilibrate the column with PBS (pH 7.4); centrifuge (4500 r / min, 10 min) and filter the cell culture supernatant through a 0.45 μm filter, then wash the column with PBS (pH 7.4) until the OD450 value is close to zero; elute with 50 mmol / L, pH 3.5 glycine-hydrochloric acid buffer (50 mmol / L glycine solution, adjusted to pH 3.5 with 1 mol / L hydrochloric acid), collect the eluate in the peak area, adjust to pH 6.0, obtain the purified antibody, determine the concentration, and store at -20°C for future use.

[0237] ELISA to identify the binding ability of chimeric antibodies to human LIV-1-his protein

[0238] Human LIV-1-his (ACRO, LV1-H5223) protein was diluted to 2 μg / mL and plated in 96-well plates, 100 μL per well, coated at 4°C overnight, the next day after the supernatant was discarded, 300 μL of 2% skim milk was used to block at 37°C for 2 h, after the blocking solution was discarded, the plate was washed 3 times with PBST. Chimeric antibodies were diluted to 20 μg / mL, 4-fold dilution of 12 gradients; the gradient-diluted antibodies were added to the ELISA plate with human LIV-1-His protein at a volume of 200 μL / well, incubated at 37°C for 1 h, washed 3 times with PBST, added 100 μL of goat anti-human IgG-FC secondary antibody, reacted at 37°C for 1 h, washed 3 times with PBST again, then added 100 μL of TMB color developing solution and developed for 10 min, after the reaction was completed, 100 μL of 1M hydrochloric acid solution was added to each well to stop the reaction, and the data was read and recorded at 450 nm, the data was fitted by four parameters, the results are shown in Table 4, Figures 13-16, and the EC50 value was calculated. Chimeric antibodies Chi-1, Chi-2, Chi-3, Chi-6 and Chi-8 have good binding capacity to human LIV-1-his.

[0239] Table 4 Binding EC50 of chimeric antibodies to human LIV-1-his

[0240] Flow cytometry binding of chimeric antibodies to 293F cells expressing human LIV-1 protein

[0241] Logarithmic growth phase 293F cells expressing human LIV-1 were taken, centrifuged at 300g for 5 min, after the supernatant was discarded, the cells were resuspended with 5 mL of DPBS, and the cell density was adjusted to 2 x 106 / mL. The cells were inoculated in a U-bottom 96-well plate at a volume of 50 μL / well. The sample protein was adjusted to a concentration of 5 μg / mL, and 7 gradients were diluted according to a 3-fold gradient, added to the U-shaped 96-well plate at a volume of 50 μL / well, and reacted at 37°C for 1 h. After centrifugation at 300g for 5 min, the supernatant was discarded and the cells were washed twice with 200 μL of DBS. 100 μL of DPBS with fluorescent secondary antibody was added to each well, and reacted at 37°C for 1 h. After the reaction was completed, the cells were centrifuged and washed twice with DPBS. Finally, 200 μL of DPBS was added to each well to resuspend the cells, and the data was analyzed by flow cytometry. The data was fitted by four parameters, the results are shown in Table 5, Figures 17-19. Except for chimeric antibodies Chi-5 and Chi-8, the other chimeric antibodies have good binding to human LIV-1 expressed on 293F cells.

[0242] Table 5 Binding EC50 of chimeric antibodies to human LIV-1 expressed on 293F cells

[0243] Affinity determination of chimeric antibodies

[0244] The affinity of the monoclonal antibody to the antigen LIV-1-His protein (ACRO, item number: LV1-H5223) was determined using Biacore (T200). The ultrapure water was filtered with a 0.22 μm filter membrane to prepare the HBS-EP buffer; the chimeric antibody stock solution was diluted to 6 μg / mL with the HBS-EP buffer as the ligand; the LIV-1-his protein was diluted 8 times with the HBS-EP buffer from 100 nM starting, and one zero point was added as the analyte; an appropriate amount of glycine 1.5 was taken as the regeneration liquid; the ligand, analyte, regeneration liquid, and HBS-EP buffer were placed on the sample tray; the program was set: ligand flow rate 10 μL / min, 20 s; analyte 30 μL / min, binding time 100 s, dissociation time 600 s; regeneration liquid 30 μL / min, 30 s. The program was started. The Biacore T200 Control Software collected the SPR signal and saved it, and then the Biacore T200 Evaluation analysis software was used for data processing. The affinity kinetic curve was fitted according to the 1:1 Langmuir binding model, and the KD value was calculated as shown in Table 6.

[0245] Table 6 Affinity determination of chimeric antibodies

[0246] Flow cytometry determination of endocytosis effect of chimeric antibodies

[0247] RAJI cells were collected and adjusted to a cell density of 2 x 106 / mL with PBS. The chimeric antibody was adjusted to a concentration of 12 μg / mL and 0.8 μg / mL, and 100 μL was added to each well of a U-shaped 96-well plate and mixed well, and incubated at 4°C for 1 h. After the reaction was completed, the cells were washed twice with PBST, resuspended with 200 μL of PBS, and one plate was placed at 4°C and the other plate was placed at 37°C. After standing for 4 h, centrifugation was performed at 2000 r / min for 3 min and the cells were washed twice with PBST, 100 μL of 1:400 diluted APC secondary antibody was added to each well, and incubated at 4°C for 1 h. After washing twice with PBS, the cells were loaded onto the instrument and the data was analyzed. As shown in Table 7, the endocytosis effect of chimeric antibody Chi-1 4h RAJI cells was significant.

[0248] Table 7 Endocytosis determination results of chimeric antibodies

[0249] Humanization of chimeric antibodies

[0250] The humanization of mouse-derived antibodies was performed by the CDR grafting method, see US patents 4816567; 5225539; 5530101; 5585089; 5693762 and 6180370, and was performed according to the following method.

[0251] The light chain and heavy chain variable region sequences of the chimeric antibody Chi-1 were aligned with the human immunoglobulin gene database on the NCBI website (http: / / www.ncbi.nlm.nih.gov / igblast / ). The human IGVH and IGVK with the highest homology to antibody Chi-1 and also with high expression and also used by other drugs were selected as the framework for humanization. The selected light chain germline acceptor sequence of antibody Chi-1 is human GKV1-16*01, and the selected heavy chain germline acceptor sequence is human IGHV1-46*01. The variable region (VH+VL) of antibody Chi-1 was homologously modeled, and the amino acids within 5 angstroms of the variable region or some conserved amino acid residues were selected and compared with the mouse-derived antibody to obtain humanized antibody heavy chains VH1, VH2, VH3, VH4, VH5, and humanized antibody light chain variable regions VL1, VL2, VL3, VL4, VL5, the sequences of which are shown in Table 8 below.

[0252] Table 8 Amino acid sequences of heavy and light chain variable regions of humanized antibody Chi-1 after point mutation

[0253] Transient expression of humanized antibodies

[0254] The humanized heavy chain and light chain variable region sequences were connected to the constant region of human IgG1 antibody, the heavy chain constant region amino acid sequence is shown in SEQ ID NO: 57, and the light chain variable region sequence was added with the kappa light chain constant region, the light chain constant region amino acid sequence is shown in SEQ ID NO: 58. A signal peptide sequence and a Kozak sequence were added before the antibody amino acid sequence, and the DNA sequence was optimized and synthesized by Kings River Biotechnology Co., Ltd. After sequence synthesis, it was cloned into the pCDNA3.4 vector (Invitrogen) and the plasmid was extracted. Five humanized heavy chain plasmids and five humanized light chain plasmids were obtained. The heavy chain plasmid and the light chain plasmid were combined orthogonally, transfected into 293F cells, and transiently expressed to obtain 25 humanized antibodies.

[0255] Affinity determination of humanized antibodies

[0256] The affinity of the monoclonal antibody to the antigen LIV-1-His was determined using Biacore (T200). The ultrapure water was filtered through a 0.22 μm filter membrane to prepare the HBS-EP buffer; the LIV-1 monoclonal antibody stock solution was diluted to 6 μg / mL with the HBS-EP buffer as the ligand; the LIV-1-His protein was diluted 8 times from 100 nM starting, 2 times, and then added with a zero point with the HBS-EP buffer as the analyte; an appropriate amount of glycine 1.5 was taken as the regeneration liquid; the ligand, analyte, regeneration liquid, and HBS-EP buffer were placed on the sample tray; the program was set: ligand flow rate 10 μL / min, 20 s; analyte 30 μL / min, binding time 100 s, dissociation time 600 s; regeneration liquid 30 μL / min, 30 s. The program was started. The Biacore T200 Control Software collected the SPR signal and saved it, and then the data were processed using the Biacore T200 Evaluation analysis software. The affinity kinetic curve was fitted according to the 1:1 Langmuir binding model, and the KD value was calculated. The affinities of huH11-3-2, huH11-3-3, and huH11-4-3 were more prominent than those of the control antibodies.

[0257] Table 9 Affinity determination of the humanized antibody

[0258] PTM removal of the humanized antibody

[0259] The CDR region of the light chain variable region VL2 of the humanized antibody huH11-3-2 was mutated after analyzing the number of mutations of the combined amino acids, the stability of the antibody, and the affinity determination. The heavy chain variable region VH3 remained unchanged. The amino acid C55 at the 55th position of VL2-CDR2 was replaced with F55 to remove the extra cysteine. For the NS deamidation site of VL2-CDR3, mutations were made to NA, AS, and NE, respectively. The obtained antibody light chain variable region after mutation was named VL2-P1, VL2-P5, and VL2-P8. The light chain after PTM removal was combined with the antibody heavy chain, and the 293F cells were transiently transfected for antibody expression to obtain the antibodies huH11-3-2-P1, huH11-3-2-P5, and huH11-3-2-P8.

[0260] Table 10 LCDRs and VL amino acid sequences of the humanized antibody after PTM removal

[0261] Affinity determination of the antibody after LIV1 humanization and PTM site removal

[0262] The affinity of the monoclonal antibody to the antigen LIV-1-His was determined using Biacore (T200). The ultrapure water was filtered with a 0.22 μm filter membrane to prepare the HBS-EP buffer; the LIV-1 monoclonal antibody stock solution was diluted to 6 μg / mL with the HBS-EP buffer as the ligand; the LIV-1-His protein was diluted 8 times with the HBS-EP buffer from 100 nM starting, and one zero point was added as the analyte; an appropriate amount of glycine 1.5 was taken as the regeneration liquid; the ligand, analyte, regeneration liquid, and HBS-EP buffer were placed on the sample tray; the program was set: ligand flow rate 10 μL / min, 20 s; analyte 30 μL / min, binding time 100 s, dissociation time 600 s; regeneration liquid 30 μL / min, 30 s. The program was started. The Biacore T200 Control Software collected the SPR signal and saved it, and then the Biacore T200 Evaluation analysis software was used for data processing. The affinity kinetic curve was fitted according to the 1:1 Langmuir binding model, and the KD value was calculated, as shown in Table 11. These data show that humanization does not significantly affect the binding affinity of huH11-3-2-P1, huH11-3-2-P5, and huH11-3-2-P8 to LIV-1.

[0263] Table 11 Affinity determination of huH11-3-2-P1, huH11-3-2-P5, and huH11-3-2-P8

[0264] ELISA identification of the binding ability of PTM-removed humanized antibodies to human LIV-1-his protein

[0265] Human LIV-1-His protein was diluted to 2 μg / mL and plated in 96-well plates, 100 μL per well, coated overnight at 4°C, the next day after the supernatant was discarded, 300 μL of 2% skim milk was added to block at 37°C for 2 h, after the blocking solution was discarded, the plates were washed 3 times with PBST. The antibody was diluted to 40 μg / mL, 8 gradients of 5-fold dilutions were prepared; the gradient-diluted antibody was added to the ELISA plate with human LIV-1-His protein at a volume of 200 μL / well, incubated at 37°C for 1 h, washed 3 times with PBST, 100 μL of goat anti-human IgG-FC secondary antibody was added, reacted at 37°C for 1 h, washed 3 times with PBST again, then 100 μL of TMB color developing solution was added to develop color for 10 min, after the reaction was completed, 100 μL of 1M hydrochloric acid solution was added to each well to stop the reaction, and the data was read and recorded at 450 nm. The data was fitted by four-parameter fitting, the results are shown in Table 12, Figure 20, Figure 21, and the results show that huH11-3-2-P1, huH11-3-2-P5, and huH11-3-2-P8 have significant human LIV-1-his protein binding ability.

[0266] Table 12 Determination of humanized antibody binding ability to human LIV-1-his protein

[0267] FACS detection of LIV-1 humanized antibody after removal of PTM site

[0268] Logarithmic growth phase 293F cells expressing human / monkey LIV-1 were taken, centrifuged at 300 g for 5 min, the supernatant was discarded, the cells were resuspended with DPBS, and the cell density was adjusted to 2 x 106 / mL, and the cells were inoculated into a U-bottom 96-well plate at a volume of 50 μL / well. The sample protein was taken, the protein concentration was adjusted to 5 μg / mL, and 3-fold gradient dilution was performed, a total of 11 concentration points, added to the U-shaped 96-well plate at a volume of 50 μL / well, reacted at 37°C for 1 h, centrifuged at 300 g for 5 min, the supernatant was discarded and washed twice with 200 μL of DBS, 100 μL of DPBS with fluorescent secondary antibody was added to each well, reacted at 37°C for 1 h, after the reaction was completed, centrifuged and washed twice with DPBS, finally 200 μL of DPBS was added to each well to resuspend the cells, and the data was analyzed by flow cytometry, four-parameter fitting was performed, and the results are shown in Table 13, Figure 22, Figure 23. The results show that huH11-3-2-P1, huH11-3-2-P5, and huH11-3-2-P8 also have significant binding ability to human / monkey LIV-1-293F cells.

[0269] Table 13 Determination of humanized antibody binding ability to human / monkey LIV-1-293F cells

[0270] Example 15 Killing test of camptothecin derivatives on tumor cells

[0271] Test of camptothecin derivatives on killing activity of tumor cells in vitro by taking MCF-7 cells

[0272] Experimental procedure: trypsinize cells, collect and centrifuge, resuspend cells with MEM medium containing 10% FBS, count with trypan blue, adjust cell density to 1*10^4 cells / ml, add 100 μL per well to 96-well cell culture plates; dilute standard DXD and samples with MEM medium containing 10% FBS, prepare concentration of 30 μM, dilute 9 concentration points by 10 times, set another 0 concentration point as control, mix 50 μL per well with cells in the culture plate, seal with PBS, incubate at 37°C / 5% CO2 cell incubator for 5 days; after incubation, add 50 μL detection reagent per well, avoid light for 3 min, detect RLU value with microplate reader;

[0273] Data processing: killing rate (%) = (RLU value of control well-RLU value of experimental well) / RLU value of control well*100, make four-parameter equation with log value of concentration and cell killing rate, calculate EC50 value of standard and sample; corresponding test results are as follows in Table 14:

[0274] Table 14 in vitro tumor killing activity

[0275] Example 16 Killing activity of antibody drug conjugate on tumor cells in vitro

[0276] Antibody drug conjugate (ADC) against LIV-1 can effectively kill tumor cells expressing LIV-1, we selected PC-3 tumor cell line expressing LIV-1 as target cells to evaluate the killing activity of ADC on tumor cells in vitro.

[0277] Prepare samples: dilute the test product with 10% FBS+DMEM medium to a concentration of 3.03 μM, dilute 3 gradients by 3 times, then continue to dilute 5 gradients by 5 times, add a 0 concentration point, a total of 9 concentration points. 50 μl per well, three replicates; collect PC-3 cells by trypsinization, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells with 10% FBS+DMEM medium and count to adjust the cell density to 2*104 / ml, 100ul of cells per well are plated in 96-well plates; incubate in a 37°C CO2 incubator for 5 days, after incubation, add 50 μl CTG per well to detect cell viability, fit the four-parameter curve to calculate the killing EC50 value, the results are shown in Table 15, Figure 24, Figure 25.

[0278] Table 15 in vitro tumor killing activity of LIV-1 ADC

[0279] Example 17 Anti-LIV-1 conjugated antibody in vivo efficacy experiment

[0280] In vivo efficacy study of antibody drug conjugate on male NOG mice human prostate cancer cell PC-3 subcutaneous xenograft model

[0281] The male NOG mice were used to establish human prostate cancer cell PC-3 subcutaneous xenograft model to investigate the therapeutic effect of antibody drug conjugate on the model. The human prostate cancer cell PC-3 used in this experiment was cultured in DMEM high glucose medium added with 10% FBS in a 37℃ incubator containing 5% CO2. The cells were continuously cultured for ten generations before the mice were inoculated. PC-3 cells were collected (the cell concentration was adjusted to 1x108 / mL) and mixed with Matrigel at a volume ratio of 1:1, 0.1 mL per mouse was inoculated subcutaneously in the right flank of male NOG mice, and the inoculation amount of PC-3 cells for each mouse was 5x106. When the average tumor volume of the mice reached about 180 mm3, the mice were randomly divided into groups according to the tumor volume and body weight, respectively: PBS, LNF2108-vcMMAE, LNF2108-DXD, LNF2108-HY2019, LNF2108-HY2020, LNF2108-HY2021, LNF2108-HY2022, LNF2108-HY2023, LNF2108-HY2024, HLIV22-vcMMAE, 6 mice in each group. The mice were injected with drugs through the tail vein at a dose of 10 mg / kg on the day of grouping (defined as D0 day), and the whole experimental period was only given once, and the tumor volume and body weight were measured twice a week. The maximum diameter (D) and minimum diameter (d) of the tumor were measured with an electronic vernier caliper, and the tumor volume (mm3) was calculated using the following formula: [Dxd2] / 2, and the tumor growth inhibition rate TGI (%) of each drug administration group was calculated according to the formula: (1-drug administration group average volume / control group average volume) x 100%.

[0282] The tumor inhibition rate is shown in Table 16; the tumor volume is shown in Figures 26 and 27, and the mouse body weight change is shown in Figures 28 and 29. The results show that each alternative molecule of LNF2108 project has a significant anti-tumor effect.

[0283] Table 16 Anti-tumor activity of LIV1 ADC

Claims

A compound of general formula I or a stereoisomer, geometric isomer, tautomer, pharmaceutical salt, hydrate, solvate, or prodrug analog thereof: in, Ra、R b Selected independently from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 3-8 Cycloalkyl, -(CH2) n1 -OH, -(CH2) n1 -NR6R7、-(CH2) n1 -C(O)NR6R7、-(CH2) n1 -N(R6)C(O)R7; In the above groups, n1, n2, and n3 are each an integer from 0 to 6; R6, R7, and R8 of each group may be the same or different, and are each independently selected from hydrogen, C, and C. 1-6 Alkyl, hydroxyl, C 1-6 Halogenated alkyl groups. The compound according to claim 1, characterized in that, Ra and Rb are each independently selected from C1-6 alkyl groups. The compound according to claim 1, characterized in that, Ra and Rb are independently selected from methyl and ethyl groups, respectively. The compound according to claim 1, characterized in that, The compound shown in Formula I is a compound with any of the following structures: Or its stereoisomers, geometric isomers, tautomers, pharmaceutical salts, hydrates, solvates, or prodrug analogs. A compound of general formula II comprising a linker-drug conjugate of a camptothecin derivative of formula I or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof: Among them Ra, R b Selected independently from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 3-8 Cycloalkyl, -(CH2) n1 -OH, -(CH2) n1 -NR6R7、-(CH2) n1 -C(O)NR6R7、-(CH2) n1 -N(R6)C(O)R7; In the above groups, n1, n2, and n3 are each an integer from 0 to 6; R6, R7, and R8 of each group may be the same or different, and are each independently selected from hydrogen, C, and C. 1-6 Alkyl, hydroxyl, C 1-6 Halogenated alkyl groups. The compound according to claim 5 is characterized in that, Ra and Rb are each independently selected from C1-6 alkyl groups. The compound according to claim 5 is characterized in that, Ra and Rb are independently selected from methyl and ethyl groups, respectively. [Amended according to Rule 26, 14.07.2025] The linker-drug conjugate comprising camptothecin derivatives according to claim 5 is characterized in that, The compound shown in Formula II is a compound with any of the following structures: An antibody-drug conjugate comprising Formula II, as shown in Formula III, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof: in, Ra and Rb are independently selected from C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C3-8 cycloalkyl, -(CH2)n1-OH, -(CH2)n1-NR6R7, -(CH2)n1-C(O)NR6R7, and -(CH2)n1-N(R6)C(O)R7; n1, n2, and n3 are each independently integers from 0 to 6; R6, R7, and R8 of each group may be the same or different, and are each independently selected from hydrogen, C 1-6 Alkyl, hydroxyl, C 1-6 Halogenated alkyl; preferably, R a R b Selected independently from C 1-6 Alkyl; more preferably methyl or ethyl; Ab is an antibody that can form a linking bond between its heteroatoms and linking units. The antibody is selected from murine antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies. The antibodies, antibody fragments, or antigen-binding fragments thereof are not limited to being selected from: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-CD70 antibody, anti-LIV-1 antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, and anti-Integrin. Antibody Av, anti-CAIX, anti-P-cadherin, anti-GD3, anti-Cadherin 6, anti-LAMP1, anti-FLT3, anti-BCMA, anti-CD79b, anti-CD19, anti-CD33, anti-CD56, anti-CD74, anti-CD22, anti-CD30, anti-CD37, anti-CD138, anti-CD352, anti-CD25, or anti-CD123; where n is 7.5-8.

0. The antibody-drug conjugate according to claim 9 is characterized in that, Ab is an antibody against LIV-1, its antigen-binding fragment, or a variant thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 5, HCDR2 as shown in SEQ ID NO: 6, and HCDR3 as shown in SEQ ID NO: 7; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 38, LCDR2 as shown in SEQ ID NO: 39, and LCDR3 as shown in SEQ ID NO:

40. The antibody-drug conjugate according to claim 9 is characterized in that, Ab is an LIV-1 antibody, its antigen-binding fragment or a variant thereof, which further comprises: the heavy chain variable region amino acid sequence as shown in SEQ ID NO: 63; and the light chain variable region amino acid sequence as shown in SEQ ID NO:

71. The antibody-drug conjugate according to claim 9 is characterized in that, The ligand-drug conjugate of general formula III or its pharmaceutically acceptable salt or solvate, or its tautomers, mesosomes, racemates, enantiomers, or diastereomers, include, but are not limited to, the following compounds: The Ab mentioned therein is an antibody, an antibody fragment, or its antigen-binding fragment, and the antibody is selected from chimeric antibodies, humanized antibodies, or fully human antibodies, specifically as defined in general formula III; in each formula, n is 7.5-8.

0. The antibody-drug conjugate according to claim 9 is characterized in that, The compound represented by Formula III has any of the following structures: In each of the formulas, n is 7.5-8.0; huH11-3-2-P1 represents an anti-LIV-1 antibody or its antigen-binding fragment, which has the heavy chain variable region amino acid sequence as shown in SEQ ID NO:63, and the light chain variable region amino acid sequence as shown in SEQ ID NO:71; the heavy chain constant region amino acid sequence as shown in SEQ ID NO:57, and the light chain constant region amino acid sequence as shown in SEQ ID NO:58; S in the above formulas is the sulfur atom in the thiol residue formed after the interchain disulfide bond on huH11-3-2-P1 is opened. A pharmaceutical composition comprising a therapeutically effective dose of the compound as claimed in any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Use of the compound as claimed in any one of claims 1 to 13 in the preparation of a medicament for treating LIV-1 mediated diseases or conditions.

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